Patent Publication Number: US-2011060270-A1

Title: Microporation of Tissue for Delivery Of Bioactive Agents

Description:
BACKGROUND OF THE INVENTION 
     This invention relates generally to the field of transmembrane delivery of drugs or bioactive molecules to an organism. More particularly, this invention relates to a minimally invasive to non-invasive method of increasing the permeability of the skin, mucosal membrane or outer layer of a plant through microporation of this biological membrane, which can be combined with sonic, electromagnetic, and thermal energy, chemical permeation enhancers, pressure, and the like for selectively enhancing flux rate of bioactive molecules into the organism and, once in the organism, into selected regions of the tissues therein. 
     The stratum corneum is chiefly responsible for the well known barrier properties of skin. Thus, it is this layer that presents the greatest barrier to transdermal flux of drugs or other molecules into the body and of analytes out of the body. The stratum corneum, the outer horny layer of the skin, is a complex structure of compact keratinized cell remnants separated by lipid domains. Compared to the oral or gastric mucosa, the stratum corneum is much less permeable to molecules either external or internal to the body. The stratum corneum is formed from keratinocytes, which comprise the majority of epidermal cells, that lose their nuclei and become corneocytes. These dead cells comprise the stratum corneum, which has a thickness of only about 10-30 μm and, as noted above, is a very resistant waterproof membrane that protects the body from invasion by exterior substances and the outward migration of fluids and dissolved molecules. The stratum corneum is continuously renewed by shedding of corneum cells during desquamination and the formation of new corneum cells by the keratinization process. 
     Underlying the stratum corneum is the viable cell layer of the epidermis and the dermis, or connective tissue layer. These layers together make up the skin. Microporation of these underlying layers (the viable cell layer and dermis) has not previously been used but may enhance transdermal flux. Deep to the dermis are the underlying structures of the body, including fat, muscle, bone, etc. 
     Microporation of the mucous membrane has not been used previously. The mucous membrane generally lacks a stratum corneum. The most superficial layer is the to epithelial layer which consists of numerous layers of viable cells. Deep to the epithelial layer is the lamina propria, or connective tissue layer. 
     Microporation of plants has been previously limited to select applications in individual cells in laboratory settings. Plant organisms generally have tough outer layers to provide resistance to the elements and disease. Microporation of this tough outer layer of plants enables the delivery of substances useful for introduction into the plant such as for conferring the desired trait to the plant or for production of a desired substance. For example, a plant may be treated such that each cell of the plant expresses a particular and useful peptide such as a hormone or human insulin. 
     The flux of a drug or analyte across the biological membrane can be increased by changing either the resistance (the diffusion coefficient) or the driving force (the gradient for diffusion). Flux may be enhanced by the use of so-called penetration or chemical enhancers. Chemical enhancers are well known in the art and a more detailed description will follow. 
     Another method of increasing the permeability of skin to drugs is iontophoresis. Iontophoresis involves the application of an external electric field and topical delivery of an ionized form of drug or an un-ionized drug carried with the water flux associated with ion transport (electro-osmosis). While permeation enhancement with iontophoresis has been effective, control of drug delivery and irreversible skin damage are problems associated with the technique. 
     Sonic energy has also been used to enhance permeability of the skin and synthetic membranes to drugs and other molecules. Ultrasound has been defined as mechanical pressure waves with frequencies above 20 kHz, H. Lutz et al.,  Manual of Ultrasound  3-12 (1984). Sonic energy is generated by vibrating a piezoelectric crystal or other electromechanical element by passing an alternating current through the material, R. Brucks et al., 6  Pharm. Res.  697 (1989). The use of sonic energy to increase the permeability of the skin to drug molecules has been termed sonophoresis or phonophoresis. 
     Although it has been acknowledged that enhancing permeability of the skin should theoretically make it possible to transport molecules from inside the body through the skin to outside the body for collection or monitoring, practicable methods have not been disclosed. U.S. Pat. No. 5,139,023 to Stanley et al. discloses an apparatus and method for noninvasive blood glucose monitoring. In this invention, chemical permeation enhancers are used to increase the permeability of mucosal tissue or skin to glucose. Glucose then passively diffuses through the mucosal tissue or skin and is captured in a receiving medium. The amount of glucose in the receiving medium is measured and correlated to determine the blood glucose level. However, as taught in Stanley et al., this method is much more efficient when used on mucosal tissue, such as buccal tissue, which results in detectable amounts of glucose being collected in the receiving medium after a lag time of about 10-20 minutes. However, the method taught by Stanley et al. results in an extremely long lag time, ranging from 2 to 24 hours depending on the chemical enhancer composition used, before detectable amounts of glucose can be detected diffusing through human skin (heat-separated epidermis) in vitro. These long lag times may be attributed to the length of time required for the chemical permeation enhancers to passively diffuse through the skin and to enhance the permeability of the barrier stratum corneum, as well as the length of time required for the glucose to passively diffuse out through the skin. Thus, Stanley et al. clearly does not teach a method for transporting blood glucose or other analytes non-invasively through the skin in a manner that allows for rapid monitoring, as is required for blood glucose monitoring of diabetic patients and for many other body analytes such as blood electrolytes. 
     While the use of sonic energy for drug delivery is known, results have been largely disappointing in that enhancement of permeability has been relatively low. There is no consensus on the efficacy of sonic energy for increasing drug flux across the skin. While some studies report the success of sonophoresis, J. Davick et al., 68  Phys. Ther.  1672 (1988); J. Griffin et al., 47  Phys. Ther.  594 (1967); J. Griffin &amp; J. Touchstone, 42  Am. J. Phys. Med.  77 (1963); J. Griffin et al., 44  Am. J. Phys Med.  20 (1965); D. Levy et al., 83  J. Clin. Invest.  2074); D. Bommannan et al., 9  Pharm. Res.  559 (1992), others have obtained negative results, H. Benson et al., 69  Phys Ther.  113 (1988); J. McElnay et al., 20  Br. J. Clin. Pharmacol.  4221 (1985); H. Pratzel et al., 13  J. Rheumatol  1122 (1986). Systems in which rodent skin were employed showed the most promising results, whereas systems in which human skin was employed have generally shown disappointing results. It is well known to those skilled in the art that rodent skin is much more permeable than human skin, and consequently the above results do not teach one skilled in the art how to effectively utilize sonophoresis as applied to transdermal delivery and/or monitoring through human skin. 
     A significant improvement in the use of ultrasonic energy in the monitoring of analytes and also in the delivery of drugs to the body is disclosed and claimed in application Ser. No. 08/152,442 filed Nov. 15, 1993, now U.S. Pat. No. 5,458,140, and Ser. No. 08/152,174 filed Dec. 8, 1993, now U.S. Pat. No. 5,445,611, both of which are incorporated herein by reference. In these inventions, the transdermal sampling of an analyte or the transdermal delivery of drugs, is accomplished through the use of sonic energy that is modulated in intensity, phase, or frequency or a combination of these parameters coupled with the use of chemical permeation enhancers. Also disclosed is the use of sonic energy, optionally with modulations of frequency, intensity, and/or phase, to controllably push and/or pump molecules through the stratum corneum via perforations introduced by needle puncture, hydraulic jet, laser, electroporation, or other methods. 
     The formation of micropores (i.e. microporation) in the stratum corneum to enhance the delivery of drugs has been the subject of various studies and has resulted in the issuance of patents for such techniques. 
     Jacques et al., 88  J. Invest. Dermatol.  88-93 (1987), teaches a method of administering a drug by ablating the stratum corneum of a region of the skin using pulsed laser light of wavelength, pulse length, pulse energy, pulse number, and pulse repetition rate sufficient to ablate the stratum corneum without significantly damaging the underlying epidermis and then applying the drug to the region of ablation. This work resulted in the issuance of U.S. Pat. No. 4,775,361 to Jacques et al. The ablation of skin through the use of ultraviolet-laser irradiation was earlier reported by Lane et al., 121  Arch. Dermatol.  609-617 (1985). Jacques et al. is restricted to use of few wavelengths of light and expensive lasers. 
     Tankovich, U.S. Pat. No. 5,165,418 (hereinafter, “Tankovich &#39;418”), discloses a method of obtaining a blood sample by irradiating human or animal skin with one or more laser pulses of sufficient energy to cause the vaporization of skin tissue so as to produce a hole in the skin extending through the epidermis and to sever at least one blood vessel, causing a quantity of blood to be expelled through the hole such that it can be collected. Tankovich &#39;418 thus is inadequate for noninvasive or minimally invasive permeabilization of the stratum corneum such that a drug can be delivered to the body or an analyte from the body can be analyzed. 
     Tankovich et al., U.S. Pat. No. 5,423,803 (hereinafter, “Tankovich &#39;803”) discloses a method of laser removal of superficial epidermal skin cells in human skin for cosmetic applications. The method comprises applying a light-absorbing “contaminant” to the outer layers of the epidermis and forcing some of this contaminant into or through the intercellular spaces in the stratum corneum, and illuminating the infiltrated skin with pulses of laser light of sufficient intensity that the amount of energy absorbed by the contaminant will cause the contaminant to explode with sufficient energy to tear off some of the epidermal skin cells. Tankovich &#39;803 further teaches that there should be high absorption of energy by the contaminant at the wavelength of the laser beam, that the laser beam must be a pulsed beam of less than 1 μs duration, that the contaminant must be forced into or through the upper layers of the epidermis, and that the contaminant must explode with sufficient energy to tear off epidermal cells upon absorption of the laser energy. This invention also fails to disclose or suggest a method of drug delivery or analyte collection. 
     Raven et al., WO 92/00106, describes a method of selectively removing unhealthy tissue from a body by administering to a selected tissue a compound that is highly absorbent of infrared radiation of wavelength 750-860 nm and irradiating the region with corresponding infrared radiation at a power sufficient to cause thermal vaporization of the tissue to which the compound was administered but insufficient to cause vaporization of tissue to which the compound had not been administered. The absorbent compound should be soluble in water or serum, such as indocyanine green, chlorophyll, porphyrins, heme-containing compounds, or compounds containing a polyene structure, and power levels are in the range of 50-1000 W/cm 2  or even higher. 
     Konig et al., DD 259351, teaches a process for thermal treatment of tumor tissue that comprises depositing a medium in the tumor tissue that absorbs radiation in the red and/or near red infrared spectral region, and irradiating the infiltrated tissue with an appropriate wavelength of laser light. Absorbing media can include methylene blue, reduced porphyrin or its aggregates, and phthalocyanine blue. Methylene blue, which strongly absorbs at 600-700 nm, and a krypton laser emitting at 647 and 676 nm are exemplified. The power level should be at least 200 mW/cm 2 . 
     It has been shown that by stripping the stratum corneum from a small area of the skin with repeated application and removal of cellophane tape to the same location one can easily collect arbitrary quantities of interstitial fluid, which can then be assayed for a number of analytes of interest. Similarly, the ‘tape-stripped’ skin has also been shown to be permeable to the transdermal delivery of compounds into the body. Unfortunately, ‘tape-stripping’ leaves a open sore which takes weeks to heal, and for this, as well as other reasons, is not considered as an acceptable practice for enhancing transcutaneous transport in wide applications. 
     As discussed above, it has been shown that pulsed lasers, such as the excimer laser operating at 193 nm, the erbium laser operating near 2.9 μm or the CO, laser operating at 10.2 μm, can be used to effectively ablate small holes in the human stratum corneum. These laser ablation techniques offer the potential for a selective and potentially non-traumatic method for opening a delivery and/or sampling hole through the stratum corneum. However, due to the prohibitively high costs associated with these light sources, there have been no commercial products developed based on this concept. The presently disclosed invention; by defining a method for directly conducting thermal energy into or through the biological membrane with very tightly defined spatial and temporal resolution, makes it possible to produce the desired micro-ablation of the biological membrane very low cost energy sources. 
     In view of the foregoing problems and/or deficiencies, the development of a method for safely enhancing the permeability of the biological membrane for minimally invasive or noninvasive monitoring of body analytes in a more rapid time frame would be a significant advancement in the art. It would be another significant advancement in the art to provide a method of minimally invasively or non-invasively enhancing the transmembrane flux rate of a drug into a selected area of an organism. 
     Significant advancements in the delivery of drugs and other compounds are being made through the use of various techniques that increase the permeability of a biological membrane, such as the skin or mucosal membrane. Even more promising advances have been made through techniques for creating micropores, as disclosed in the aforementioned applications. 
     Nevertheless, it is desirable to improve upon these technologies by forming micropores at selected depths in the biological membrane and to deliver both small and large compounds, in terms of molecular weight and size, through the micropores into the body. 
     BRIEF SUMMARY OF THE INVENTION 
     This invention provides a method for enhancing the transmembrane flux rate of a permeant into a selected site of an organism comprising the steps of enhancing the permeability of said selected site of the organism to said permeant by means of (a) porating a biological membrane at said selected site by means that form a micropore in said biological membrane, thereby reducing the barrier properties of said biological membrane to the flux of said permeant and (b) contacting the porated selected site with a composition comprising an effective amount of said permeant, whereby the transmembrane flux rate of said permeant into the organism is enhanced. 
     This invention further provides the method of enhancing the transmembrane flux rate further comprising applying to said site of said organism an enhancer to increase the flux of said permeant into said organism. The invention also provides the method wherein said enhancer comprises sonic energy, and more specifically, wherein the said sonic energy is applied to said site at a frequency in the range of about 10 Hz to 1000 MHz, and wherein said sonic energy is modulated by means of a member selected from the group consisting of frequency modulation, amplitude modulation, phase modulation, and combinations thereof. Alternatively, the said enhancer comprises an electromagnetic field, and, more specifically, iontophoresis or a magnetic field, or a mechanical force, chemical enhancer, or thermal enhancer. Additionally, the invention further provides a method wherein any of the methods of sonic, electromagnetic, mechanical, thermal, or chemical enhancement may be applied in any combination thereof to increase the transmembrane flux rate of said permeant into or through said micropore. 
     This invention also provides a method of further enhancing the transmembrane flux rate with an enhancer, wherein said enhancers at said site are applied so as to increase the flux rate of the permeant into tissues surrounding the micropore. The said enhancer can comprise sonic energy. Furthermore, the said sonic energy is applied to said site at a frequency in the range of about 10 Hz to 1000 MHz, wherein said sonic energy is modulated by means of a member selected from the group consisting of frequency modulation, amplitude modulation, phase modulation, and combinations thereof. Alternatively, the said enhancer comprises sonic or thermal energy, electroporation, iontophoresis, chemical enhancers, mechanical force, or a magnetic field, or any combination thereof. 
     The invention further includes the method of enhancing the transmembrane flux rate of a permeant further comprising applying to said site of said organism an enhancer, wherein any of the methods of methods of sonic or thermal energy, electroporation, iontophoresis, chemical enhancers, mechanical force, or a magnetic field may be applied in any combination thereof further comprising the method of combining sonic or thermal energy, electroporation, iontophoresis, chemical enhancers, mechanical force, or a magnetic field to increase the flux rate of the permeant into tissues surrounding the micropore. 
     The invention also includes the method of further enhancing the transmembrane flux rate within and beneath the outer layer wherein said porating of said biological membrane in said site is accomplished by means selected from the group consisting of (a) ablating the biological membrane by contacting said site, up to about 1000 μm across, of said biological membrane with a heat source such that a micropore is formed in said biological membrane at said site; (b) puncturing said biological membrane with a micro-lancet calibrated to form a micropore of up to about 1000 μm in diameter; (c) ablating the biological membrane by a beam of sonic energy onto said biological membrane up to about 1000 μm in diameter; (d) hydraulically puncturing said biological membrane with a high pressure jet of fluid to form a micropore of up to about 1000 μm in diameter and (e) puncturing said biological membrane with short pulses of electricity to form a micropore of up to about 1000 μm in diameter. Further, the invention includes the method wherein said porating is accomplished by contacting said site, up to about 1000 μm across, with a heat source to conductively transfer an effective amount of thermal energy to said site such that the temperature of some of the water and other vaporizable substances in said site is elevated above their vaporization point creating a micropore to a selected depth in the biological membrane at said site or wherein said porating is accomplished by contacting said site, up to about 1000 μm across, with a heat source to conductively transfer an effective amount of thermal energy to said site such that the temperature of some of the tissue at said site is elevated to the point where thermal decomposition occurs creating a micropore to a selected depth in the biological membrane at said site. Additionally, the invention includes the method of porating said biological membrane in said site further comprising treating at least said site with an effective amount of a substance that exhibits sufficient absorption over the emission range of a pulsed light source and focusing the output of a series of pulses from said pulsed light source onto said substance such that said substance is heated sufficiently to conductively transfer an effective amount of thermal energy to said biological membrane to elevate the temperature to thereby create a micropore. The invention also includes the method wherein said pulsed light source emits at a wavelength that is not significantly absorbed by said biological membrane. The invention further provides the method wherein said pulsed light source is a laser diode emitting in the range of about 630 to 1550 nm, wherein said pulsed light source is a laser diode pumped optical parametric oscillator emitting in the range of about 700 and 3000 nm, wherein said pulsed light source is a member selected from the group consisting of arc lamps, incandescent lamps, and light emitting diodes. The invention also includes the method further comprising providing a sensing system for determining when the micropore in the biological membrane has reached the desired dimensions, including width, length, and depth, and, further, wherein said sensing system comprises light collection means for receiving light reflected from said site and focusing said reflected light on a detector for receiving said light and sending a signal to a controller wherein said signal indicates a quality of said light, and a controller coupled to said detector and to said light source for receiving said signal and for shutting off said light source when a preselected signal is received, or, alternatively, an electrical impedance measuring system which can detect the changes in the impedance of the biological membrane at different depths into the organism as the micropore is formed. 
     The invention also provides the method of enhancing the transmembrane flux rate within and beneath the outer layer further comprising cooling said site and adjacent tissues such that said site and adjacent tissues are in a cooled condition. The said cooling means comprises a Peltier device. 
     The invention also includes the method of enhancing the transmembrane flux within and beneath the outer layer further comprising, prior to porating said site, illuminating at least said site with light such that said site is sterilized. 
     This invention also includes the method of enhancing the transmembrane flux within and beneath the outer layer further comprising contacting said site with a solid element, wherein said solid element functions as a heat source to conductively transfer an effective amount of thermal energy to said biological membrane to elevate the temperature to thereby create a micropore. Further, said heat source is constructed to modulate the temperature of said site to greater than 100° C. within about 10 nanoseconds to 50 milliseconds and then returning the temperature of said site to approximately ambient temperature within about 1 millisecond to 50 milliseconds and wherein a cycle of raising the temperature and returning to ambient temperature is repeated one or more times effective for porating the biological membrane to the desired depth. The invention further includes the method of using a heat source wherein said returning to approximately ambient temperature of said site is carried out by withdrawing said heat source from contact with said site and wherein the modulation parameters are selected to reduce sensation to the animal subject. 
     The invention includes the method for enhancing transmembrane flux rates using a heat source and sensing system further comprising providing means for monitoring electrical impedance between said solid element and said organism through said site and adjacent tissues and means for advancing the position of said solid element such that as said poration occurs with a concomitant change in impedance, said advancing means advances the solid element such that the solid element is in contact with said site during heating of the solid element, until the selected impedance is obtained. Further, the invention includes this method further comprising means for withdrawing said solid element from contact with said site wherein said monitoring means is capable of detecting a change in impedance associated with contacting a selected layer underlying the surface of said site and sending a signal to said withdrawing means to withdrawn said solid element from contact with said site. 
     The method of enhancing the transmembrane flux rate using a solid element wherein said solid element is heated by delivering an electrical current through an ohmic heating element and, further, wherein said solid element is formed such that it contains an electrically conductive component and the temperature of said solid element is modulated by passing a modulated electrical current through said conductive element. Additionally, the invention includes the method wherein said solid element is positioned in a modulatable magnetic field wherein energizing the magnetic field produces electrical eddy currents sufficient to heat the solid element. 
     The invention also includes the method of enhancing the transmembrane flux rate wherein said porating is accomplished by puncturing said site with a micro-lancet calibrated to form a micropore of up to about 1000 μm in diameter, by a beam of sonic energy directed onto said site to form a micropore of up to about 1000 μm in diameter, by hydraulically puncturing said biological membrane with a high pressure jet of fluid to form a micropore of up to about 1000 μm in diameter, or, alternatively, by puncturing said biological membrane with short pulses of electricity to form a micropore of up to about 1000 μm. in diameter. 
     The invention further comprises the method of enhancing the transmembrane flux rate of a permeant wherein said permeant comprises a nucleic acid. More specifically, the invention includes the method wherein said nucleic acid comprises DNA or wherein the nucleic acid comprises RNA. 
     The invention further includes the method of enhancing the transmembrane flux rate of a permeant wherein the micropore in the biological membrane extends into a portion of the outer layer of the biological membrane ranging from 1 to 30 microns in depth, extends through the outer layer of the biological membrane ranging from 10 to 200 microns in depth, extends into the connective tissue layer of the biological membrane ranging from 100 to 5000 microns in depth, or extends through the connective tissue layer of the biological membrane ranging from 1000 to 10000 microns in depth. 
     The invention further includes the method of enhancing the transmembrane flux rate of a permeant, wherein the micropore penetrates the biological membrane to a depth determined to facilitate desired activity of the selected permeant. 
     The invention further includes the method of enhancing the transmembrane flux rate of a permeant wherein the permeant comprises a polypeptide, including wherein the polypeptide is a protein or a peptide, and further including wherein the peptide comprises insulin or a releasing factor; a carbohydrate, including wherein the carbohydrate comprises a heparin; an analgesic, including wherein the analgesic comprises an opiate; a vaccine; or a steroid. 
     The invention further includes the method of enhancing the transmembrane flux rate of a permeant wherein the permeant is associated with a carrier. The invention further includes the method wherein the carrier comprises liposomes; lipid complexes; microparticles; or polyethylene glycol compounds. More specifically, the invention further includes the method wherein the permeant is a vaccine in combination with the method wherein the permcant is associated with a carrier. 
     The invention further includes the method of enhancing the transmembrane flux rate of a permeant wherein the permcant comprises a substance which has the ability to change its detectable response to a stimulus when in the proximity of an analyte present in the organism. 
     An object of the invention is to provide a method for controlling transmembrane flux rates of drugs or other molecules into the body and, if desired, into the bloodstream through minute perforations in the biological membrane, including stratum corneum or other layers of the skin or in the mucosa or outer layers of a plant. 
     It is still another object of the invention to provide a method of delivering drugs into the body through micropores in the biological membrane in combination with sonic energy, permeation enhancers, pressure gradients, electromagnetic energy, thermal energy, and the like. 
     An object of the invention is to minimize the barrier properties of the biological membrane using poration to controllably collect analytes from within the body through perforations in the biological membrane to enable the monitoring of these analytes. 
     It is also an object of the invention to provide a method of monitoring selected analytes in the body through micropores in the biological membrane in combination with sonic energy, permeation enhancers, pressure gradients, electromagnetic energy, mechanical energy, thermal energy, and the like. 
     These and other objects may be accomplished by providing a method for monitoring the concentration of an analyte in an individual&#39;s body comprising the steps of enhancing the permeability of the biological membrane of a selected area of the individual&#39;s body surface to the analyte by means of 
     (a) porating the biological membrane of the selected area by means that form a micropore in the biological membrane optionally without causing serious damage to the underlying tissues, thereby reducing the barrier properties of the biological membrane to the withdrawal of the analyte; 
     (b) collecting a selected amount of the analyte; and 
     (c) quantitating the analyte collected. 
     In one preferred embodiment, the method further comprises applying sonic energy to the porated selected area at a frequency in the range of about 5 kHz to 100 MHz, wherein the sonic energy is modulated by means of a member selected from the group consisting of frequency modulation, amplitude modulation, phase modulation, and combinations thereof. In another preferred embodiment, the method comprises contacting the selected area of the individual&#39;s body with a chemical enhancer with the application of electromagnetic, thermal, mechanical, or sonic energy to further enhance analyte withdrawal. 
     Porating of the biological membrane is accomplished by means selected from the group consisting of (a) ablating the biological membrane by contacting a selected area, up to about 1000 μm across, of the biological membrane with a heat source such that the temperature of tissue-bound water and other vaporizable substances in the selected area is elevated above the vaporization point of the water and other vaporizable substances thereby removing the biological membrane in the selected area; (b) puncturing the biological membrane with a micro-lancet calibrated to form a micropore of up to about 1000 μm in diameter; (c) ablating the biological membrane by focusing a tightly focused beam of sonic energy onto the stratum corneum; (d) hydraulically puncturing the biological membrane with a high pressure jet of fluid to form a micropore of up to about 1000 μm in diameter and (e) puncturing the biological membrane with short pulses of electricity to form a micropore of up to about 1000 μm in diameter. 
     One preferred embodiment of thermally ablating the biological membrane comprises treating at least the selected area with an effective amount of a dye that exhibits strong absorption over the emission range of a pulsed light source and focusing the output of a series of pulses from the pulsed light source onto the dye such that the dye is heated sufficiently to conductively transfer heat to the stratum corneum to elevate the temperature of tissue-bound water and other vaporizable substances in the selected area above the vaporization point of the water and other vaporizable substances. Preferably, the pulsed light source emits at a wavelength that is not significantly absorbed by skin. For example, the pulsed light source can be a laser diode emitting in the range of about 630 to 1550 nm, a laser diode pumped optical parametric oscillator emitting in the range of about 700 and 3000 nm, or a member selected from the group consisting of arc lamps, incandescent lamps, and light emitting diodes. A sensing system for determining when the barrier properties of the stratum corneum have been surmounted can also be provided. One preferred sensing system comprises light collection means for receiving light reflected from the selected area and focusing the reflected light on a photodiode, a photodiode for receiving the focused light and sending a signal to a controller wherein the signal indicates a quality of the reflected light, and a controller coupled to the photodiode and to the pulsed light source for receiving the signal and for shutting off the pulsed light source when a preselected signal is received. 
     In another preferred embodiment, the method further comprises cooling the selected area of biological membrane and adjacent tissues with cooling means such that said selected area and adjacent tissues are in a selected cooled, steady state, condition prior to, during, and/or after poration. 
     In still another preferred embodiment, the method comprises ablating the biological membrane such that interstitial fluid exudes from the micropores, collecting the interstitial fluid, and analyzing the analyte in the collected interstitial fluid. After the interstitial fluid is collected, the micropore can be sealed by applying an effective amount of energy from the laser diode or other light source such that interstitial fluid remaining in the micropore is caused to coagulate. Preferably, vacuum is applied to the porated selected area to enhance collection of interstitial fluid. 
     In yet another preferred embodiment, the method comprises, prior to porating the biological membrane, illuminating at least the selected area with light such that the selected area illuminated with the light is sterilized. 
     Another preferred method of porating the biological membrane comprises contacting the selected area with a solid element such that the temperature of the selected area is raised from ambient temperature to greater than 100° C. within about 10 nanoseconds to 50 ms and then returning the temperature of the selected area to approximately ambient skin temperature within about 1 to 50 ms, wherein this cycle of raising the temperature and returning to approximately ambient temperature is repeated a number of time effective for reducing the barrier properties of the biological membrane. Preferably, the step of returning to approximately ambient temperature is carried out by withdrawing the solid element from contact with the biological membrane. It is also preferred to provide means for monitoring electrical impedance between the solid element and the body through the selected area of biological membrane and adjacent tissues and means for advancing the position of the solid element such that as the ablation occurs with a concomitant reduction in resistance, the advancing means advances the solid element such that the solid element is in contact with the biological membrane during heating of the solid element. Further, it is also preferred to provide means for withdrawing the solid element from contact with the biological membrane, wherein the monitoring means is capable of detecting a change in impedance associated with contacting a layer underlying the biological membrane or a layer thereof and sending a signal to the withdrawing means to withdrawn the solid element from contact with the biological membrane. The solid element can be heated by an ohmic heating element, can have a current loop having a high resistance point wherein the temperature of the high resistance point is modulated by passing a modulated electrical current through said current loop to effect the heating, or can be positioned in a modulatable alternating magnetic field of an excitation coil such that energizing the excitation coil with alternating current produces eddy currents sufficient to heat the solid element by internal ohmic losses. 
     A method for enhancing the transmembrane flux rate of an active permeant into a selected area of a body comprising the steps of enhancing the permeability of the biological membrane layer of the selected area of the body surface to the active permeant by means of 
     (a) porating the biological membrane of the selected area by means that form a micropore in the biological membrane optionally without causing serious damage to the underlying tissues and thereby reducing the barrier properties of the biological membrane to the flux of the active permeant; and 
     (b) contacting the porated selected area with a composition comprising an effective amount of the permeant such that the flux of the permeant into the body is enhanced. 
     In a preferred embodiment, the method further comprises applying energy to the porated selected area for a time and at an intensity and a frequency effective to create a fluid streaming effect and thereby enhance the transmembrane flux rate of the permeant into the body. 
     A method is also provided for applying a tattoo to a selected area of skin on an individual&#39;s body surface comprising the steps of: 
     (a) porating the stratum corneum of the selected area by means that form a micropore in the stratum corneum optionally without causing serious damage to the underlying tissues and thereby reduce the barrier properties of the stratum corneum to the flux of a permeant; and 
     (b) contacting the porated selected area with a composition comprising an effective amount of a tattooing ink as a permeant such that the flux of said ink into the body is enhanced. 
     A method is still further provided for reducing a temporal delay in diffusion of an analyte from blood of an individual to said individual&#39;s interstitial fluid in a selected area of biological membrane comprising applying means for cooling to said selected area of skin. 
     A method is yet further provided for reducing evaporation of interstitial fluid and the vapor pressure thereof, wherein said interstitial fluid is being collected from a micropore in a selected area of the biological membrane of an individual, comprising applying means for cooling to said selected area of biological membrane. 
     In accordance with still further embodiments, the present invention is directed to a method for delivering bioactive agents into the body through micropores formed at selected depths in a biological membrane, such as the skin, or mucous membrane or outer layer of a plant. The method involves porating an outer layer of the biological membrane through any of the poration techniques known in the art, but to a sufficient and desired depth into or through the biological membrane, and contacting the porated site with an effective quantity of the bioactive agent of low or high molecular weight and size. This process can be enhanced by applying further permeation enhancement measures either before, during or after the bioactive agent is delivered. For example, sonic energy, iontophoresis, magnetic fields, electroporation, chemical permeation enhancer, osmotic pressure and atmospheric pressure measures may be applied to the porated site to enhance the permeability of layers beneath the outer layer of the biological membrane. 
    
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS 
         FIG. 1  shows a schematic representation of a system for delivering laser diode light and monitoring the progress of poration. 
         FIG. 2  shows a schematic representation of a closed-loop feedback system for monitoring poration. 
         FIG. 3A  shows a schematic representation of an optical poration system comprising a cooling device. 
         FIG. 3B  shows a top view of a schematic representation of an illustrative cooling device according to  FIG. 3B . 
         FIG. 4  shows a schematic representation of an ohmic heating device with a mechanical actuator. 
         FIG. 5  shows a schematic representation of a high resistance current loop heating device. 
         FIG. 6  shows a schematic representation of a device for modulating heating using inductive heating. 
         FIG. 7  shows a schematic representation of a closed loop impedance monitor using changes in impedance to determine the extent of poration. 
         FIGS. 8A-D  show cross sections of human skin treated with copper phthalocyanine and then subjected, respectively, to 0, 1, 5, and 50 pulses of 810 nm light with an energy density of 4000 J/cm 2  for a pulse period of 20 ms. 
         FIGS. 9-11  show graphic representations of temperature distribution during simulated thermal poration events using optical poration. 
         FIGS. 12 and 13  show graphic representations of temperature as a function of time in the stratum corneum and viable epidermis, respectively, during simulated thermal poration events using optical poration. 
         FIGS. 14-16  show graphic representations of temperature distribution, temperature as a function of time in the stratum corneum, and temperature as a function of time in the viable epidermis, respectively, during simulated thermal poration events using optical poration wherein the tissue was cooled prior to poration. 
         FIGS. 17-19  show graphic representations of temperature distribution, temperature as a function of time in the stratum corneum, and temperature as a function of time in the viable epidermis, respectively, during simulated thermal poration events wherein the tissue was heated with a hot wire. 
         FIGS. 20-22  show graphic representations of temperature distribution, temperature as a function of time in the stratum corneum, and temperature as a function of time in the viable epidermis, respectively, during simulated thermal poration events wherein the tissue was heated with a hot wire and the tissue was cooled prior to poration. 
         FIGS. 23 and 24  show graphic representations of temperature distribution and temperature as a function of time in the stratum corneum, respectively, during simulated thermal poration events wherein the tissue is heated optically according to the operating parameters of Tankovich &#39;803. 
         FIG. 25  shows a graphic representation of interstitial fluid (ISF;) and, blood (*) glucose levels as a function of time. 
         FIG. 26  shows a scatter plot representation of the difference term between the ISF glucose and the blood glucose data of  FIG. 25 . 
         FIG. 27  shows a histogram of the relative deviation of the ISF to the blood glucose levels from  FIG. 25 . 
         FIG. 28  shows a cross section of an illustrative delivery apparatus for delivering a drug to a selected area on an individual&#39;s skin. 
         FIGS. 29A-C  show graphic representations of areas of skin affected by delivery of lidocaine to selected areas where the stratum corneum is porated ( FIGS. 29A-B ) or not porated ( FIG. 29C ). 
         FIG. 30  shows a plot comparing the amount of interstitial fluid harvested from micropores with suction alone ( ) and with a combination of suction and ultrasound (*). 
         FIGS. 31 ,  32 , and  33  show a perspective view of an ultrasonic transducer/vacuum apparatus for harvesting interstitial fluid, a cross section view of the same apparatus, and cross sectional schematic view of the same apparatus, respectively. 
         FIGS. 34A-B  show a top view of a handheld ultrasonic transducer and a side view of the spatulate end thereof, respectively. 
         FIG. 35  is a graphical representation showing the enhancing effects of microporation in the transdermal delivery of testosterone. 
     
    
    
     DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT 
     It is to be understood that this invention is not limited to the particular configurations, process steps, and materials disclosed herein as such configurations, process steps, and materials may vary somewhat. It is also to be understood that the terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof. 
     It must be noted that, as used herein the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a method for delivery of “a drug” includes reference to delivery of a mixture of two or more drugs, reference to “an analyte” includes reference to one or more of such analytes, and reference to “a permeation enhancer” includes reference to a mixture of two or more permeation enhancers or techniques such as a combination of ultrasound and electroporation. 
     Thus, as used herein, the singular form may be used interchangeably with the plural form, and vice versa, i.e.: “layer” could mean layers or “layers” could mean layer. 
     As used herein, “including” or “includes” or the like means including, without limitation. 
     In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below. 
     As used herein “organism” means the entire animal or plant being acted upon by the methods described herein. 
     As used herein, “poration,” “microporation,” or any such similar term means the formation of a small hole or pore to a desired depth in or through the biological membrane, such as skin or mucous membrane, or the outer layer of an organism to lessen the barrier properties of this biological membrane to the passage of analytes from below the surface for analysis or the passage of permeants or drugs into the body for selected purposes, or for certain medical or surgical procedures. The microporation process referred to herein is distinguished from the openings formed by electroporation principally by the minimum dimensions of the micropores which shall be no smaller than 1 micron across and at least 1 micron in depth, whereas the openings formed with electroporation are typically only a few nanometers in any dimension. Nevertheless, electroporation is useful to facilitate uptake of selected permeants by the targeted tissues beneath the outer layers of an organism after the permeant has passed through the micropores into these deeper layers of tissue. Preferably the hole or micropore will be no larger than about 1 mm in diameter, and more preferably no larger than about 300 μm in diameter, and will extend to a selected depth, as described hereinafter. 
     As used herein, “micropore” or “pore” means an opening formed by the microporation method. 
     As used herein “ablation” means the controlled removal of material which may include cells or other components comprising some portion of a biological membrane or tissue caused by any of the following: kinetic energy released when some or all of the vaporizable components of such material have been heated to the point that vaporization occurs and the resulting rapid expansion of volume due to this phase change causes this material, and possibly some adjacent material, to be removed from the ablation site; thermal, mechanical, or sonic decomposition of some or all off the tissue at the poration site. 
     As used herein ablation of a tissue or puncture of a tissue may be achieved utilizing the same energy source. 
     As used herein, “tissue” means any component of an organism including but not limited to, cells, biological membranes, bone, collagen, fluids and the like comprising to some portion of the organism. 
     As used herein, “sonic” or “acoustic” are interchangeable and cover the frequency space from 0.01 Hz and up. 
     As used herein, “ultrasonic” describes a subset of sonic comprising frequencies greater or equal to 20,000 Hz with no upper limit. 
     As used herein “puncture” or “micro-puncture” means the use of mechanical, hydraulic, sonic, electromagnetic, or thermal means to perforate wholly or partially a biological membrane such as the skin or mucosal layers of an animal or the outer tissue layers of a plant. 
     To the extent that “ablation” and “puncture” accomplish the same purpose of poration, i.e. the creating a hole or pore in the biological membrane optionally without significant damage to the underlying tissues, these terms may be used interchangeably. 
     As used herein, “penetration enhancement” or “permeation enhancement” means an increase in the permeability by utilization of a permeation enhancer of a biological membrane such as the skin or mucosal or buccal membrane or a plant&#39;s outer layer of tissue to a bioactive agent, drug, analyte, dye, stain, microparticle, microsphere, compound, or other chemical formulation (also called “permeant”), i.e., so as to increase the rate at which a bioactive agent, drug, analyte, stain, micro-particle, microsphere, compound, or other chemical formulation permeates the biological membrane and facilitates the withdrawal of analytes out through the biological membrane or the delivery of substances through the biological membrane and into the underlying tissues. The enhanced permeation effected through the use of such enhancers can be observed, for example, by observing diffusion of a dye, as a permeant, through animal or human skin using a diffusion apparatus. 
     As used herein, “penetration enhancer,” “permeation enhancer,” “enhancer,” and the like includes all substances and techniques that increase the flux of a permeant, analyte, or other molecule across the skin, and is limited only by functionality. In other words, all cell envelope disordering compounds and solvents and physical techniques such as electroporation, iontophoresis, magnetic fields, sonic energy, thermal energy, or mechanical pressure or manipulation such as a local massaging of the site and any chemical enhancement agents are intended to be included. 
     As used herein “chemical enhancer” means a substance that increases the flux of a permeant or analyte or other substance across a biological membrane and is limited only by function. 
     As used herein, “dye,” “stain,” and the like shall be used interchangeably and refer to a biologically suitable chromophore that exhibits suitable absorption over some or all of the emission range of a pulsed light source used to ablate tissues to form micropores therein. 
     As used herein, “transdermal” or “percutaneous” or “transmembrane” or “transmucosal” or “transbuccal” means passage of a permeant into or through the biological membrane or tissue to achieve effective therapeutic blood levels or tissue levels of a drug, or the passage of a molecule present in the body (“analyte”) out through the biological membrane or tissue so that the analyte molecule may be collected on the outside of the body. 
     As used herein, the term “bioactive agent,” “permeant,” “drug,” or “pharmacologically active agent” or “deliverable substance” or any other similar term means any chemical or biological material or compound suitable for delivery by the methods previously known in the art and/or by the methods taught in the present invention, that induces a desired effect, such as a biological or pharmacological effect, which may include but is not limited to (1) having a prophylactic effect on the organism and preventing an undesired biological effect such as preventing an infection, (2) alleviating a condition caused by a disease, for example, alleviating pain or inflammation caused as a result of disease, (3) either alleviating, reducing, or completely eliminating the disease from the organism, and/or (4) the placement within the viable tissue layers of the organism of a compound or formulation which can react, optionally in a reversible manner, to changes in the concentration of a particular analyte and in so doing cause a detectable shift in this compound or formulation&#39;s measurable response to the application of energy to this area which may be electromagnetic, mechanical or acoustic. The effect may be local, such as providing for a local anesthetic effect, or it may be systemic. This invention is not drawn to novel permeants or to new classes of active agents other than by virtue of the microporation technique, although substances not typically being used for transdermal, transmucosal, transmembrane or transbuccal delivery may now be useable. Rather it is directed to the mode of delivery of bioactive agents or permeants that exist in the art or that may later be established as active agents and that are suitable for delivery by the present invention. 
     Such substances include broad classes of compounds normally delivered into the organism, including through body surfaces and membranes, including skin as well as by injection, including needle, hydraulic, or hypervelocity methods. In general, this includes but is not limited to: Polypeptides, including proteins and peptides (e.g., insulin); releasing factors, including Luteinizing Hormone Releasing Hormone (LHRH); carbohydrates (e.g., heparin); nucleic acids; vaccines; and pharmacologically active agents such as antiinfectives such as antibiotics and antiviral agents; analgesics and analgesic combinations; anorexics; antihelminthics; antiarthritics; antiasthmatic agents; anticonvulsants; antidepressants; antidiabetic agents; antidiarrheals; antihistamines; antiinflammatory agents; antimigraine preparations; antinauseants; antineoplastics; antiparkinsonism drugs; antipruritics; antipsychotics; antipyretics; antispasmodics; anticholinergics; sympathomimetics; xanthine derivatives; cardiovascular preparations including potassium and calcium channel blockers, beta-blockers, alpha-blockers, and antiarrhythmics; antihypertensives; diuretics and antidiuretics; vasodilators including general coronary, peripheral and cerebral; central nervous system stimulants; vasoconstrictors; cough and cold preparations, including decongestants; hormones such as estradiol, testosterone, progesterone and other steroids and derivatives and analogs, including corticosteroids; hypnotics; immunosuppressives; muscle relaxants; parasympatholytics; psychostimulants; sedatives; and tranquilizers. By the method of the present invention, both ionized and nonionized permeants may be delivered, as can permeants of any molecular weight including substances with molecular weights ranging from less than 50 Daltons to greater than 1,000,000 Daltons. 
     As used herein, an “effective” amount of a permeant means a sufficient amount of a compound to provide the desired local or systemic effect and performance at a reasonable benefit/risk ratio attending any treatment. An “effective” amount of an enhancer as used herein means an amount selected so as to provide the desired increase in tissue permeability and the desired depth of penetration, rate of administration, and amount of permeant delivered. 
     As used herein, “carriers” or “vehicles” refer to carrier materials without significant pharmacological activity at the quantities used that are suitable for administration with other permeants, and include any such materials known in the art, e.g., any liquid, gel, solvent, liquid diluent, solubilizer, microspheres, liposomes, microparticles, lipid complexes, or the like, that is sufficiently nontoxic at the quantities employed and does not interact with the drug to be administered in a deleterious manner. Examples of suitable carriers for use herein include water, buffers, mineral oil, silicone, inorganic or organic gels, aqueous emulsions, liquid sugars, lipids, microparticles, waxes, petroleum jelly, and a variety of other oils and polymeric materials. 
     As used herein, a “biological membrane” means a tissue material present within a living organism that separates one area of the organism from another and, in many instances, that separates the organism from its outer environment. Skin and mucous and buccal membranes are thus included as well as the outer layers of a plant. Also, the walls of a cell or a blood vessel would be included within this definition. 
     As used herein, “mucous membrane” or “mucosa” refers to the epithelial linings of the mouth, nasopharynx, throat, respiratory tract, urogenital tract, anus, eye, gut and all to other surfaces accessible via an endoscopic device such as the bladder, colon, lung, blood vessels, heart and the like. 
     As used herein, the “buccal membrane” includes the mucous membrane of the mouth. 
     As used herein, “outer layer” and “connective-tissue layer” are parts of the biological membrane and have the following meanings. “Outer layer” means all or part of the epidermis of the skin or the epithelial lining of the mucous membrane or the outer layer of a plant. The most superficial portion of the animal epidermis is the stratum corneum, as is well known in the art. The deeper portion of the epidermis is called, for simplicity, the “viable cell layer” hereinafter. Beneath the outer layer is the “connective tissue layer.” The connective tissue layer means the dermis in the skin or the lamina propria in the mucous membrane or other underlying tissues in plants or animals. 
     As used herein, “organism” or “individual” or “subject” or “body” refers to any of a human, animal, or plant to which the present invention may be applied. 
     As used herein, “analyte” means any chemical or biological material or compound suitable for passage through a biological membrane by the technology taught in this present invention, or by technology previously known in the art, of which an individual might want to know the concentration or activity inside the body. Glucose is a specific example of an analyte because it is a sugar suitable for passage through the skin, and individuals, for example those having diabetes, might want to know their blood glucose levels. Other examples of analytes include, but are not limited to, such compounds as sodium, potassium, bilirubin, urea, ammonia, calcium, lead, iron, lithium, salicylates, antibodies, hormones, or an exogenously delivered substance and the like. 
     As used herein, “into” or “in” a biological membrane or layer thereof includes penetration in or through only one or more layers (e.g., all or part of the stratum corneum or the entire outer layer of the skin or portion thereof). 
     As used herein, “through” a biological membrane or layer thereof means through the entire depth of the biological membrane or layer thereof. 
     As used herein, “transdermal flux rate” is the rate of passage of any analyte out through the skin of a subject or the rate of passage of any bioactive agent, drug, pharmacologically active agent, dye, particle or pigment in and through the skin separating the organism from its outer environment. “Transmucosal flux rate” and “transbuccal flux rate” refer to such passage through mucosa and buccal membranes and “transmembrane flux rate” refers to such passage through any biological membrane. 
     As used herein, “transdermal,” “transmucosal,” “transbuccal” and “transmembrane” may be used interchangeably as appropriate within the context of their use. 
     As used herein, the terms “intensity amplitude,” “intensity,” and “amplitude” are used synonymously and refer to the amount of energy being produced by a sonic, thermal, mechanical or electromagnetic energy system. 
     As used herein, “frequency modulation” or “sweep” means a continuous, graded or stepped variation in the frequency of a sonic, thermal, mechanical or electromagnetic energy in a given time period. A frequency modulation is a graded or stepped variation in frequency in a given time period, for example 5.4-5.76 MHz in 1 sec., or 5-10 MHz in 0.1 sec., or 10-5 MHz in 0.1 sec., or any other frequency range or time period that is appropriate to a specific application. A complex modulation can include varying both the frequency and intensity simultaneously. For example, FIGS. 4A and 4B of U.S. Pat. No. 5,458,140 could, respectively, represent amplitude and frequency modulations being applied simultaneously to a single sonic energy transducer. 
     As used herein, “amplitude modulation” means a continuous, graded or stepped variation in the amplitude or intensity of a sonic, thermal, mechanical or electromagnetic energy in a given time period. 
     As used herein “phase modulation” means the timing of a sonic, thermal, mechanical or electromagnetic energy or signal has been changed relative to its initial state. An example is shown in FIG. 4C of U.S. Pat. No. 5,458,140. The frequency and amplitude of the signal can remain the same. A phase modulation can be implemented with a variable delay such as to selectively retard or advance the signal temporarily in reference to its previous state, or to another signal. 
     As used herein “signal,” or “energy” may be used synonymously. The sonic, thermal, mechanical or electromagnetic energy, in its various applications such as with frequency, intensity or phase modulation, or combinations thereof and the use of chemical enhancers combined with sonic, thermal, mechanical or electromagnetic energy, as described herein, can vary over a frequency range of between about 0.01 Hz to 1000 MHz, with a range of between about 0.1 Hz and 30 MHz being preferred. 
     As used herein, “non-invasive” means not requiring the entry of a needle, catheter, or other invasive instrument into a part of the subject including the skin or a mucous membrane. 
     As used herein, “minimally invasive and “non-invasive” are synonymous. 
     As used herein, “microparticles” or “microspheres” or “nanoparticles” or “nanospheres” or “liposomes” or “lipid complexes” may be used interchangeably. 
     Means for Poration of the Biological Membrane 
     The formation of a micropore in the biological membrane can be accomplished by various state of the art means as well as certain means disclosed herein that are improvements thereof. While the following techniques and examples are made with respect to porating the biological membrane, it should be understood that the improvements described herein also apply to porating the mucous or buccal membrane or the outer layers of a plant. 
     The use of laser ablation as described by Jacques et al. in U.S. Pat. No. 4,775,361 and by Lane et al., supra, certainly provide one means for ablating the stratum corneum using an excimer laser. At 193 nm wavelength, and 14 ns pulse width, it was found that about 0.24 to 2.8 μm of stratum corneum could be removed by each laser pulse at radiant exposure of between about 70 and 480 mJ/cm 2 . As the pulse energy increases, more tissue is removed from the stratum corneum and fewer pulses are required for complete poration of this layer. The lower threshold of radiant exposure that must be absorbed by the stratum corneum within the limit of the thermal relaxation time to cause suitable micro-explosions that result in tissue ablation is about 70 mJ/cm 2  within a 50 millisecond (ms) time. In other words, a total of 70 mJ/cm 2  must be delivered within a 50 ms window. This can be done in a single pulse of 70 mJ/cm 2  or in 10 pulses of 7 mJ/cm 2 , or with a continuous illumination of 1.4 watts/cm 2  during the 50 ms time. The upper limit of radiant exposure is that which will ablate the stratum corneum without damage to underlying tissue and can be empirically determined from the light source, wavelength of light, and other variables that are within the experience and knowledge of one skilled in this art. 
     By “delivery”, in the context of the application of energy, is meant that the stated amount of energy is absorbed by the tissue to be ablated. At the excimer laser wavelength of 193 nm, essentially 100% absorption occurs within the first 1 or 2 μm of stratum corneum tissue. Assuming the stratum corneum is about 20 μm thick, at longer wavelengths, such as 670 nm, only about 5% of incident light is absorbed within the 20 μm layer. This means that about 95% of the high power beam passes into the tissues underlying the stratum corneum where it will likely cause significant damage. In the context of delivery of a bioactive agent, the term means providing the bioactive agent to the desired location. 
     The ideal is to use only as much power as is necessary to perforate the biological membrane or other selected skin, mucosal, or tissue layers without causing bleeding, thermal, or other unacceptable damage to underlying and adjacent tissues from which analytes are to be extracted or permeants delivered. 
     It would be beneficial to use sources of energy more economical than energy from excimer lasers. Excimer lasers, which emit light at wavelengths in the far UV region, are much more expensive to operate and maintain than, for example, diode lasers that emit light at wavelengths in visible and IR regions (600 to 1800 nm). However, at the longer wavelengths, the biological membrane becomes increasingly more transparent and absorption occurs primarily in the underlying tissues. 
     The present invention facilitates a rapid and minimally traumatic method of eliminating the barrier function of the biological membrane to facilitate the transmembrane transport of substances into the body when applied topically or to access the analytes within the body for analysis. The method utilizes a procedure which begins with the contact application of a small area heat source to the targeted area of the biological membrane. 
     The heat source must have several important properties, as will now be described. First, the heat source must be sized such that contact with the biological membrane is confined to a small area, typically about 1 to 1000 μm in diameter. Second, it must have the capability to modulate the temperature of the biological membrane at the contact point from ambient surface temperature to greater than the vaporization point of a sufficient amount of the components within the biological membrane and then return to approximately ambient temperature with cycle times to minimize collateral damage to viable tissues and trauma to the subject. This modulation can be created electronically, mechanically, or chemically. 
     Additionally, for selected applications, an inherent depth limiting feature of the microporation process can be facilitated if the heat source has both a small enough thermal mass and limited energy source to elevate its temperature such that when it is placed in contact with tissues with more than 30% water content, the thermal dispersion in these tissues is sufficient to limit the maximum temperature of the heat source to less than 100 C. This feature effectively stops the thermal vaporization process once the heat probe had penetrated through the stratum corneum into or through the lower layers of the epidermis. 
     However, if one utilizes a heat probe which can continue to deliver sufficient energy into or through the hydrated viable tissue layers beneath the outer layer of the biological membrane, the poration process can continue into the body to a selected depth, penetrating through deeper layers including, e.g., in the case of the skin, through the epidermis, the dermis, and into the subcutaneous layers below if desired. The concern when a system is designed to create a micropore extending some distance into or through the viable tissues beneath the stratum corneum, mucosal or buccal membranes is principally how to minimize damage to the adjacent tissue and the sensation to the subject during the poration process. Experimentally, we have shown that if the heat probe used is a solid, electrically or optically heated element, with the active heated probe tip physically defined to be no more than a few hundred microns across and protruding up to a few millimeters from the supporting base, that a single pulse, or multiple pulses of current can deliver enough thermal energy into or through the tissue to allow the ablation to penetrate as deep as the physical design allows, for example, until the support base acts as a component to limit the extent of the penetration into or through the tissue, essentially restricting the depth to which the heat probe can penetrate into a micropore to contact fresh, unporated tissue. If the electrical and thermal properties of said heat probe, when it is in contact with the tissues, allow the energy pulse to modulate the temperature of said probe rapidly enough, this type of deep tissue poration can be accomplished with essentially no pain to the subject. Experiments have shown that if the required amount of thermal energy is delivered to the probe within less than roughly 20 milliseconds, that the procedure is painless. Conversely, if the energy pulse must be extended beyond roughly 20 milliseconds, the sensation to the subject increases rapidly and non-linearly as the pulse width is extended. 
     An electrically heated probe design which supports this type of selected depth poration can be built by bending a 50 to 150 micron diameter tungsten wire into a sharp kink, forming a close to 180 degree bend with a minimal internal radius at this point. This miniature ‘V’ shaped piece of wire can then be mounted such that the point of the ‘V’ extends some distance out from a support piece which has copper electrodes deposited upon it. The distance to which the wire extends out from the support will define the maximum penetration distance into or through the tissue when the wire is heated. Each leg of the tungsten ‘V’ will be attached to one of the electrodes on the support carrier which in turn can be connected to the current pulsing circuit. When the current is delivered to the wire in an appropriately controlled fashion, the wire will rapidly heat up to the desired temperature to effect the thermal ablation process in a single pulse or in multiple pulses of current. By monitoring the dynamic impedance of the probe and knowing the coefficient of resistance versus temperature of the tungsten element, closed loop control of the temperature of the contact point can easily be established. Also, by dynamically monitoring the impedance through the body from the contact point of the probe and a second electrode placed some distance away, the depth of the pore can be estimated based on the different impedance properties of the tissue as one penetrates deeper into the body. 
     An optically heated probe design which supports this type of selected depth poration can be built by taking an optical fiber and placing on one end a tip comprised of a solid cap or coating. A light source such as a laser diode will be coupled into the other end of the fiber. The side of tip facing the fiber must have a high enough absorption coefficient over the range of wavelengths emitted by the light source that when the photons reach the end of the fiber and strike this face, some of them will be absorbed and subsequently cause the tip to heat up. The specific design of this tip, fiber and source assembly may vary widely, however fibers with gross diameters of 50 to 1000 microns across are common place items today and sources emitting up to thousands of watts of optical energy are similarly common place. The tip forming the actual heat probe can be fabricated from a high melting point material, such as tungsten and attached to the fiber by machining it to allow the insertion of the fiber into a cylindrical bore at the fiber end. If the distal end of the tip has been fabricated to limit the thermal diffusion away from this tip and back up the supporting cylinder attaching the tip to the fiber within the time frame of the optical pulse widths used, the photons incident upon this tip will elevate the temperature rapidly on both the fiber side and the contact side which is placed against the tissues surface. The positioning of the fiber/tip assembly onto the tissue surface, can be accomplished with a simple mechanism designed to hold the tip against the surface under some spring tension such that as the tissue beneath it is ablated, the tip itself will advance into the tissue. This allows the thermal ablation process to continue into or through the tissue as far as one desires. An additional feature of this optically heated probe design is that by monitoring the black body radiated energy from the heated tip that is collected by the fiber, a very simple closed loop control of the tip temperature can be effected. Also, as described earlier, by dynamically monitoring the impedance through the body from the contact point of the probe and a second electrode placed some distance away, the depth of the pore can be determined based on the different impedance properties of the tissue as one penetrates deeper into the body. The relationship between pulse width and sensation for this design is essentially the same as for the electrically heated probe described earlier. 
     Impedance can be used to determine the depth of a pore made by any means. It can be used as an input to a control system for making pores of selected depth. The impedance measured may be the complex impedance measured at a frequency selected to highlight the impedance properties of the selected tissues in a selected organism. 
     An additional feature of this invention is the large increase in efficiency which can be gained by combining the poration of the outer layers of the biological membrane with other permeation enhancement techniques which can now be optimized to function on the various barriers to effective delivery of the desired compound into or through the internal spaces it needs to go to be bio-effective. In particular, if one is delivering a DNA compound either naked, fragmented, encapsulated or coupled to another agent, it is often desired to get the DNA into the living cells without killing the cell to allow the desired uptake and subsequent performance of the therapy. It is well know in the art that electroporation, iontophoresis, and ultrasound can cause openings to form, temporarily, in the cell membranes and other internal tissue membranes. By having breached the stratum corneum or mucosal layer or outer layer of a plant and if desired the epidermis and dermis or deeper into a plant, electroporation, iontophoresis, magnetic fields, and sonic energy can now be used with parameters that can be tailored to act selectively on these underlying tissue barriers. For example, for any electromagnetic or sonic energy enhancement means, the specific action of the enhancement can be designed to focus on any part of the pore, e.g., on the bottom of the pore by the design of the focusing means employed such as the design of the electrodes, sonic and magnetic field forming devices and the like. Alternatively, the enhancer can be focused more generally on the entire pore or the area surrounding the pore. In the case of electroporation, where pulses exceeding 50 to 150 volts are routinely used to electroporate the stratum corneum or mucosal layer, in the environment we present, pulses of only a few volts can be sufficient to electroporate the cell, capillary or other membranes within the targeted tissue. This is principally due to the dramatic reduction in the number of insulating layers present between the electrodes once the outer surface of the biological membrane has been opened. Similarly, iontophoresis can be shown to be effective to modulate the flux of a fluid media containing the DNA through the micropores with very small amounts of current due to the dramatic reduction in the physical impedance to fluid flow through these porated layers. 
     Whereas ultrasound has previously been used to accelerate the permeation of the stratum corneum or mucosal layer, by eliminating this barrier via the micropores, we have created the opportunity to utilize sonic energy to permeabilize the cell, capillary or other structures within the targeted tissue. As in the cases of electroporation and iontophoresis, we have demonstrated that the sonic energy levels needed to effect a notable improvement in the trans-membrane flux of a substance are much lower than when stratum corneum or mucosal layers are left intact. The mode of operation of all of these active methods, electroporation, iontophoresis, magnetic fields, mechanical forces or ultrasound, when applied solely or in combination, after the poration of biological membrane has been effected is most similar to the parameters typically used in in vitro applications where single cell membranes are being opened up for the delivery of a substance. 
     With the heat source placed in contact with the surface of the biological membrane, it is cycled through a series of one or more modulations of temperature from an initial point of ambient temperature to a peak temperature in excess of 123° C. and back to ambient surface temperature. To minimize or eliminate the animal&#39;s sensory perception of the microporation process, these pulses are limited in duration, and the interpulse spacing is long enough to allow cooling of the viable tissue layers in the biological membrane, and most particularly the innervated tissues, to achieve a mean temperature within the innervated tissues of less than about 45 C. These parameters are based on the thermal time constants of the human skin&#39;s viable epidermal tissues (roughly 30-80 ms) located between the heat probe and the innervated tissue in the underlying dermis. The result of this application of pulsed thermal energy is that enough energy is conducted into or through the stratum corneum within the tiny target spot that the local temperature of this volume of tissue is elevated sufficiently higher than the vaporization point of the tissue-bound water content in the stratum corneum. As the temperature increases above 100 C, the water content of the stratum corneum (typically 5% to 15%) within this localized spot, is induced to vaporize and expand very rapidly, causing a vapor-driven removal of those corneocytes in the stratum corneum located in proximity to this vaporization event. U.S. Pat. No. 4,775,361 teaches that a stratum corneum temperature of 123° C. represents a threshold at which this type of flash vaporization occurs. As subsequent pulses of thermal energy are applied, additional layers of the stratum corneum are removed until a micropore is formed through the stratum corneum down to the next layer of the epidermis, the stratum lucidum. By limiting the duration of the heat pulse to less than one thermal time constant of the epidermis and allowing any heat energy conducted into or through the epidermis to dissipate for a sufficiently long enough time, the elevation in temperature of the viable layers of the epidermis is minimal. This allows the entire microporation process to take place without any sensation to the subject and no damage to the underlying and surrounding tissues. If the heat probe can achieve temperatures greater than 300 degrees C. some of the poration may be due to the direct thermal decomposition of the tissue. 
     The present invention comprises a method for painlessly, or with little sensation, creating microscopic holes, i.e. micropores, from about 1 to 1000 μm across, in a biological membrane of an organism. The key to successfully implementing this method is the creation of an appropriate thermal energy source, or heat probe, which is held in contact with the biological membrane. The principle technical challenge in fabricating an appropriate heat probe is designing a device that has the desired contact with the biological membrane and that can be thermally modulated at a sufficiently high frequency. 
     It is possible to fabricate an appropriate heat probe by contacting the biological membrane with a suitable light-absorbing compound, such as a dye or stain, or any thin film or substance selected because of its ability to absorb light at the wavelength emitted by a selected light source. In this instance, the selected light source may be a laser diode emitting at a wavelength which would not normally be absorbed by the biological membrane. By focusing the light source to a small spot on the surface of the topical layer of the dye, stain, thin film or substance the targeted area can be temperature modulated by varying the intensity of the light flux focused on it. It is possible to utilize the energy from laser sources emitting at a longer wavelength than an excimer laser by first topically applying to the stratum corneum a suitable light-absorbing compound, such as a dye, stain, thin film or substance selected because of its ability to absorb light at the wavelength emitted by the laser source. The same concept can be applied at any wavelength and one must only choose an appropriate dye or stain and optical wavelength. One need only look to any reference manual to find which suitable dyes and wavelength of the maximum absorbance of that dye. One such reference is Green,  The Sigma - Aldrich Handbook of Stains, Dyes and Indicators , Aldrich Chemical Company, Inc. Milwaukee, Wis. (1991). For example, copper phthalocyanine (Pigment Blue 15; CPC) absorbs at about 800 nm; copper phthalocyanine tetrasulfonic acid (Acid Blue 249) absorbs at about 610 nm; and Indocyanine Green absorbs at about 775 nm; and Cryptocyanine absorbs at about 703 nm. CPC is particularly well suited for this embodiment for the following reasons: it is a very stable and inert compound, already approved by the FDA for use as a dye in implantable sutures; it absorbs very strongly at wavelengths from 750 nm to 950 nm, which coincide well with numerous low cost, solid state emitters such as laser diodes and LEDs, and in addition, this area of optical bandwidth is similarly not absorbed directly by the skin tissues in any significant amount; CPC has a very high vaporization point (&gt;550C in a vacuum) and goes directly from a solid phase to a vapor phase with no liquid phase; CPC has a relatively low thermal diffusivity constant, allowing the light energy focused on it to selectively heat only that area directly in the focal point with very little lateral spreading of the ‘hot-spot’ into the surrounding CPC thereby assisting in the spatial definition of the contact heat-probe. 
     The purpose of this disclosure is not to make an exhaustive listing of suitable dyes, stains, films or substances because such may be easily ascertained by one skilled in the art from data readily available. 
     The same is true for any desired particular pulsed light source. For example, this method may be implemented with a mechanically shuttered, focused incandescent lamp as the pulsed light source. Various catalogs and sales literature show numerous lasers operating in the near UV, visible and near IR range. Representative lasers are Hammamatsu Photonic Systems Model PLP-02 which operates at a power output of 2×10 −8  J, at a wavelength of 415 nm; Hammamatsu Photonic Systems Model PLP-05 which operates at a power output of 15 J, at a wavelength of 685 nm; SDL, Inc., SDL-3250 Series pulsed laser which operates at a power output of 2×10 6  J at a wavelength of about 800-810 nm; SDL, Inc., Model SDL-8630 which operates at a power output of 500 mW at a wavelength of about 670 nm; Uniphase Laser Model AR-081-15000 which operates at a power output of 15,000 mW at a wavelength of 790-830 nm; Toshiba America Electronic Model TOLD9150 which operates at a power output of 30 mW at a wavelength of 690 nm; and LiCONIX, Model Diolite 800-50 which operates at a power 50 mW at a wavelength of 780 nm. 
     For purposes of the present invention a pulsed laser light source can emit radiation over a wide range of wavelengths ranging from between about 100 nm to 12,000 nm. Excimer lasers typically will emit over a range of between about 100 to 400 nm. Commercial excimer lasers are currently available with wavelengths in the range of about 193 nm to 350 nm. Preferably a laser diode will have an emission range of between about 380 to 1550 nm. A frequency doubled laser diode will have an emission range of between about 190 and 775 nm. Longer wavelengths ranging from between about 1300 and 3000 nm may be utilized using a laser diode pumped optical parametric oscillator. It is expected, given the amount of research taking place on laser technology, that these ranges will expand with time. 
     Delivered or absorbed energy need not be obtained from a laser as any source of light, whether it is from a laser, a short arc lamp such as a xenon flashlamp, an incandescent lamp, a light-emitting diode (LED), the sun, or any other source may be used. Thus, the particular instrument used for delivering electromagnetic radiation is less important than the wavelength and energy associated therewith. Any suitable instrument capable of delivering the necessary energy at suitable wavelengths, i.e. in the range of about 100 nm to about 12,000 nm, can be considered within the scope of the invention. The essential feature is that the energy must be absorbed by the light-absorbing compound to cause localized heating thereof, followed by conduction of sufficient heat to the tissue to be ablated within the time frame allowed. 
     In one illustrative embodiment, the heat probe itself is formed from a thin layer, preferably about 5 to 1000 μm thick, of a solid, non-biologically active substance placed in contact with a selected area of an individual&#39;s skin that is large enough to cover the site where a micropore is to be created. The specific formulation of the chemical compound is chosen such that it exhibits high absorption over the spectral range of a light source selected for providing energy to the light-absorbing compound. The probe can be, for example, a sheet of a solid compound, a film treated or coated with or containing a suitable light absorbing compound, or a direct application of the light-absorbing compound to the skin as a precipitate or as a suspension in a carrier. Regardless of the configuration of the light-absorbing heat probe, it must exhibit a low enough lateral thermal diffusion coefficient such that any local elevations of temperature will remain sufficiently spatially defined and the dominant mode of heat loss will preferably be via direct conduction into biological membrane through the point of contact between the skin and the probe. 
     The required temperature modulation of the probe can be achieved by focusing a light source onto the probe layer and modulating the intensity of this light source. If the energy absorbed within the illuminated area is sufficiently high, it will cause the probe layer heat up. The amount of energy delivered, and subsequently both the rate of heating and peak temperature of the probe layer at the focal point, can be easily modulated by varying the pulse width and peak power of the light source. In this embodiment, it is only the small volume of probe layer heated up by the focused, incident optical energy that forms the heat probe, additional material of this probe layer which may have been applied over a larger area then the actual poration site is incidental. By using a solid phase light-absorbing compound with a relatively high melting point, such as copper phthalocyanine (CPC), which remains in its solid phase up to a temperature of greater than 550 C, the heat probe can be quickly brought up to a temperature of several hundred degrees C., and still remain in contact with the biological membrane, allowing this thermal energy to be conducted into or through the stratum corneum. In addition, this embodiment comprises choosing a light source with an emission spectrum where very little energy would normally be absorbed in the tissues of the biological membrane. 
     Once the targeted area has the light-absorbing probe layer placed in contact to it, the heat probe is formed when the light source is activated with the focal waist of the beam positioned to be coincident with the surface of the treated area. The energy density of light at the focal waist and the amount of absorption taking place within the light-absorbing compound are set to be sufficient to bring the temperature of the light-absorbing compound, within the area of the small spot defined by the focus of the light source, to greater than 123° C. within a few milliseconds. As the temperature of the heat probe rises, conduction into or through the biological membrane delivers energy into or through these tissues, elevating the local temperature of the biological membrane. When enough energy has been delivered into or through this small area of biological membrane to cause the local temperature to be elevated above the boiling point of some of the water and other vaporizable components contained in these tissues, a flash vaporization of this material takes place, removing some portion of the biological membrane at this location and forming a micropore. 
     By turning the light source on and off, the temperature of the heat probe can be rapidly modulated and the selective ablation of these tissues can be achieved, allowing a very precisely dimensioned hole to be created, which can selectively penetrate only through the first 10 to 30 microns of the biological membrane, or can be made deeper. 
     An additional feature of this embodiment is that by choosing a light source that would normally have very little energy absorbed by the biological membrane or underlying tissues, and by designing the focusing and delivery optics to have a sufficiently high numerical aperture, the small amount of delivered light that does not happen to get absorbed in the heat probe itself, quickly diverges as it penetrates deep into the body. Since there is very little absorption at the delivered wavelengths, essentially no energy is delivered to the biological membrane directly from the light source. This three dimensional dilution of coupled energy in the tissues due to beam divergence and the low level of absorption in the untreated tissue results in a completely benign interaction between the light beam and the tissues, with no damage being done thereby. 
     In one preferred embodiment of the invention, a laser diode is used as the light source with an emission wavelength of 800±30 nm. A heat-probe can be formed by topical application of a transparent adhesive tape that has been treated on the adhesive side with a 0.5 cm spot formed from a deposit of finely ground copper phthalocyanine (CPC). The CPC exhibits extremely high absorption coefficients in the 800 nm spectral range, typically absorbing more than 95% of the radiant energy from a laser diode. 
       FIG. 1  shows a system  10  for delivering light from such a laser diode to a selected area of an individual&#39;s biological membrane and for monitoring the progress of the poration process. The system comprises a laser diode  14  coupled to a controller  18 , which controls the intensity, duration, and spacing of the light pulses. The laser diode emits a beam  22  that is directed to a collection lens or lenses  26 , which focuses the beam onto a mirror  30 . The beam is then reflected by the mirror to an objective lens or lenses  34 , which focuses the beam at a preselected point  38 . This preselected point corresponds with the plane of an xyz stage  42  and the objective hole  46  thereof, such that a selected area of an individual&#39;s biological membrane can be irradiated. The xyz stage is connected to the controller such that the position of the xyz stage can be controlled. The system also comprises a monitoring system comprising a CCD camera  50  coupled to a monitor  54  The CCD camera is confocally aligned with the objective lens such that the progress of the poration process can be monitored visually on the monitor. 
     In another illustrative embodiment of the invention, a system of sensing photodiodes and collection optics that have been confocally aligned with the ablation light source is provided.  FIG. 2  shows a sensor system  60  for use in this embodiment. The system comprises a light source  64  for emitting a beam of light  68 , which is directed through a delivery optics system  72  that focuses the beam at a preselected point  76 , such as the surface of an individual&#39;s skin  80 . A portion of the light contacting the skin is reflected, and other light is emitted from the irradiated area. A portion of this reflected and emitted light passes through a filter  84  and then through a collection optics system  88 , which focuses the light on a photodiode  92 . A controller  96  is coupled to both the laser diode and the photodiode for, respectively, controlling the output of the laser diode and detecting the light that reaches the photodiode. Only selected portions of the spectrum emitted from the skin pass through the filter. By analyzing the shifts in the reflected and emitted light from the targeted area, the system has the ability to detect when the stratum corneum has been breached, and this feedback is then used to control the light source, deactivating the pulses of light when the microporation of the stratum corneum is achieved. By employing this type of active closed loop feedback system, a self regulating, universally applicable device is obtained that produces uniformly dimensioned micropores in the stratum corneum, with minimal power requirements, regardless of variations from one individual to the next. 
     In another illustrative embodiment, a cooling device is incorporated into the system interface to the skin.  FIG. 3A  shows an illustrative schematic representation thereof. In this system  100 , a light source  104  (coupled to a controller  106 ) emits a beam of light  108 , which passes through and is focused by a delivery optics system  112 . The beam is focused by the delivery optics system to a preselected point  116 , such as a selected area of an individual&#39;s skin  120 . A cooling device  124 , such as a Peltier device or other means of chilling, contacts the skin to cool the surface thereof. In a preferred embodiment of the cooling device  124  ( FIG. 3B ), there is a central hole  128  through which the beam of focused light passes to contact the skin. Referring again to  FIG. 3A , a heat sink  132  is also preferably placed in contact with the cooling device. By providing a cooling device with a small hole in its center coincident with the focus of the light, the tissues in the general area where the poration is to be created may be cooled to 5° C. to 10° C. This cooling allows a greater safety margin for the system to operate in that the potential sensations to the user and the possibility of any collateral damage to the epidermis directly below the poration site are reduced significantly from non-cooled embodiment. Moreover, for monitoring applications, cooling minimizes evaporation of interstitial fluid and can also provide advantageous physical properties, such as decreased surface tension of such interstitial fluid. Still further, cooling the tissue is known to cause a localized increase in blood flow in such cooled tissue, thus promoting diffusion of analytes from the blood into the to interstitial fluid and promoting diffusion of delivered permeants away from the pore site or into the tissue underlying the pore. 
     The method can also be applied for other micro-surgery techniques wherein the light-absorbing compound/heat-probe is applied to the area to be ablated and then the light source is used to selectively modulate the temperature of the probe at the selected target site, affecting the tissues via the vaporization-ablation process produced. 
     A further feature of the invention is to use the light source to help seal the micropore after its usefulness has passed. Specifically, in the case of monitoring for an internal analyte, a micropore is created and some amount of interstitial fluid is extracted through this opening. After a sufficient amount of interstitial fluid had been collected, the light source is reactivated at a reduced power level to facilitate rapid clotting or coagulation of the interstitial fluid within the micropore. By forcing the coagulation or clotting of the fluid in the pore, this opening in the body is effectively sealed, thus reducing the risk of infection. Also, the use of the light source itself for both the formation of the micropore and the sealing thereof is an inherently sterile procedure, with no physical penetration into the body by any device or apparatus. Further, the thermal shock induced by the light energy kills any microbes that may happen to be present at the ablation site. 
     This concept of optical sterilization can be extended to include an additional step in the process wherein the light source is first applied in an unfocused manner, covering the target area with an illuminated area that extends 100 μm or more beyond the actual size of the micropore to be produced. By selecting the area over which the unfocused beam is to be applied, the flux density can be correspondingly reduced to a level well below the ablation threshold but high enough to effectively sterilize the surface of the skin. After a sufficiently long exposure of the larger area, either in one continuous step or in a series of pulses, to the sterilizing beam, the system is then configured into the sharply focused ablation mode and the optical microporation process begins. 
     Another illustrative embodiment of the invention is to create the required heat probe from a solid element, such as a small diameter wire. As in the previously described embodiment, the contacting surface of the heat probe must be able to have its temperature modulated from ambient biological membrane temperatures to temperatures greater than 123° C., within the required time allowed of, preferably, between about 1 microsecond to 50 milliseconds at the high temperature (on-time) and at least about 1 to 50 ms at the low temperature (off-time). In particular, being able to modulate the temperature up to greater than 150° C. for an “on” time of around 5 ms and an off time of 50 ms produces very effective thermal ablation with little or no sensation to the individual. 
     Several methods for modulating the temperatures of the solid element heat probe contact area may be successfully implemented. For example, a short length of wire may be brought up to the desired high temperature by an external heating element such as an ohmic heating element used in the tip of a soldering iron.  FIG. 4  shows an ohmic heating device  140  with a mechanical actuator. The ohmic heating device comprises an ohmic heat source  144  coupled to a solid element heat probe  148 . The ohmic heat source is also coupled through an insulating mount  152  to a mechanical modulation device  156 , such as a solenoid. In this configuration, a steady state condition can be reached wherein the tip of the solid element probe will stabilize at some equilibrium temperature defined by the physical parameters of the structure, i.e., the temperature of the ohmic heat source, the length and diameter of the solid element, the temperature of the air surrounding the solid element, and the material of which the solid element is comprised. Once the desired temperature is achieved, the modulation of the temperature of the selected area of an organism&#39;s biological membrane  160  is effected directly via the mechanical modulation device to alternatively place the hot tip of the wire in contact with the biological membrane for, preferably, a 5 ms on-time and then withdraw it into the air for, preferably, a 50 ms off-time. 
     Another illustrative example ( FIG. 5 ), shows a device  170  comprising a current source  174  coupled to a controller  178 . The current source is coupled to a current loop  182  comprising a solid element  186  formed into a structure such that it presents a high resistance point. Preferably, the solid element is held on a mount  190 , and an insulator  194  separates different parts of the current loop. The desired modulation of temperature is then achieved by merely modulating the current through the solid element. If the thermal mass of the solid element is appropriately sized and the heat sinking provided by the electrodes connecting it to the current source is sufficient, the warm-up and cool-down times of the solid element can be achieved in a few milliseconds. Contacting the solid element with a selected area of biological membrane  198  heats the biological membrane to achieve the selected ablation. 
     In  FIG. 6  there is shown still another illustrative example of porating the biological membrane with a solid element heat probe. In this system  200 , the solid element  204  can be positioned within a modulatable alternating magnetic field formed by a coil of wire  208 , the excitation coil. By energizing the alternating current in the excitation coil by means of a controller  212  coupled thereto, eddy currents can be induced in the solid element heat probe of sufficient intensity that it will be heated up directly via the internal ohmic losses. This is essentially a miniature version of an inductive heating system commonly used for heat treating the tips of tools or inducing out-gassing from the electrodes in vacuum or flash tubes. The advantage of the inductive heating method is that the energy delivered into the solid element heat probe can be closely controlled and modulated easily via the electronic control of the excitation coil with no direct electrical connection to the heat probe itself. If the thermal mass of the solid element heat probe and the thermal mass of the biological membrane in contact with the tip of the probe are known, controlling the inductive energy delivered can allow precise control of the temperature at the contact point  216  with the biological membrane  220 . Because the biological membrane tissue is essentially non-magnetic at the lower frequencies at which inductive heating can be achieved, if appropriately selected frequencies are used in the excitation coil, then this alternating electromagnetic field will have no effect on the organism&#39;s tissues. 
     If a mechanically controlled contact modulation is employed, an additional feature may be realized by incorporating a simple closed loop control system wherein the electrical impedance between the probe tip and the subject&#39;s skin is monitored. In this manner, the probe can be brought into contact with the subject&#39;s skin, indicated by the step-wise reduction in resistance once contact is made, and then held there for the desired “on-time,” after which it can be withdrawn. Several types of linear actuators are suitable for this form of closed loop control, such as a voice-coil mechanism, a simple solenoid, a rotary system with a cam or bell-crank, and the like. The advantage is that as the thermal ablation progresses, the position of the thermal probe tip can be similarly advanced into the biological membrane, always ensuring good a contact to facilitate the efficient transfer of the required thermal energy. Also, for poration of skin, the change in the conductivity properties of the stratum corneum and the epidermis can be used to provide an elegant closed loop verification that the poration process is complete, i.e., when the resistance indicates that the epidermis has been reached, it is time to stop the poration process. Similar changes in impedance can be used to control the depth of penetration to other layers as well. 
       FIG. 7  shows an illustrative example of such a closed loop impedance monitor. In this system  230 , there is an ohmic heat source  234  coupled to a wire heat probe  238 . The heat source is mounted through an insulating mount  242  on a mechanical modulator  246 . A controller  250  is coupled to the wire and to the skin  254 , wherein the controller detects changes in impedance in the selected area  258  of skin, and when a predetermined level is obtained the controller stops the poration process. 
     Along the same line as hydraulic poration means are microlancets adapted to just penetrate the stratum corneum for purposes of administering a permeant, such as a drug, through the pore formed or to withdraw an analyte through the pore for analysis. Such a device is considered to be “minimally invasive” as compared to devices and/or techniques which are non-invasive. The use of micro-lancets that penetrate below the stratum corneum for withdrawing blood are well known. Such devices are commercially available from manufacturers such as Becton-Dickinson and Lifescan and can be utilized in the present invention by controlling the depth of penetration. As an example of a micro-lancet device for collecting body fluids, reference is made to Erickson et al., International Published PCT Application WO 95/10223 (published 20 Apr. 1995). This application shows a device for penetration into or through the dermal layer of the skin, without penetration into subcutaneous tissues, to collect body fluids for monitoring, such as for blood glucose levels. 
     Poration of a biological membrane can also be accomplished using sonic means. Sonic-poration is a variation of the optical means described above except that, instead of using a light source, a very tightly focused beam of sonic energy is delivered to the area of the stratum corneum to be ablated. The same levels of energy are required, i.e. a threshold of 70 mJ/cm 2 /50 ms still must be absorbed. The same pulsed focused ultrasonic transducers as described in parent application Ser. Nos. 08/152,442 (now U.S. Pat. No. 5,458,140) and 08/152,174 (now U.S. Pat. No. 5,445,611) can be utilized to deliver the required energy densities for ablation as are used in the delivery of sonic energy which is modulated in intensity, phase, or frequency or a combination of these parameters for the transdermal sampling of an analyte or the transdermal delivery of drugs. This has the advantage of allowing use of the same transducer to push a drug through the stratum corneum or pull a body fluid to the surface for analysis to be used to first create a micropore. 
     Additionally, electroporation or short bursts or pulses of electrical current can be delivered to the stratum corneum with sufficient energy to form micropores. Electroporation is known in the art for producing pores in biological membranes and electroporation instruments are commercially available. Thus, a person of skill in this art can select an instrument and conditions for use thereof without undue experimentation according to the guidelines provided herein. 
     The micropores produced in the biological membrane by the methods of the present invention allow high flux rates of a variety of molecular weight therapeutic compounds to be delivered transmembranely. In addition, these non-traumatic microscopic openings into the body allow access to various analytes within the body, which can be assayed to determine their internal concentrations. 
     Example 1 
     In this example, skin samples were prepared as follows. Epidermal membrane was separated from human cadaver whole skin by the heat-separation method of Klingman and Christopher, 88  Arch. Dermatol.  702 (1963), involving the exposure of the full thickness skin to a temperature of 60° C. for 60 seconds, after which time the stratum corneum and part of the epidermis (epidermal membrane) were gently peeled from the dermis. 
     Example 2 
     Heat separated stratum corneum samples prepared according to the procedure of Example 1 were cut into 1 cm 2  sections. These small samples were than attached to a glass cover slide by placing them on the slide and applying an pressure sensitive adhesive backed disk with a 6 mm hole in the center over the skin sample. The samples were then ready for experimental testing. In some instances the skin samples were hydrated by allowing them to soak for several hours in a neutral buffered phosphate solution or pure water. 
     As a test of these untreated skin samples, the outputs of several different infrared laser diodes, emitting at roughly 810, 905, 1480 and 1550 nanometers were applied to the sample. The delivery optics were designed to produce a focal waist 25 μm across with a final objective have a numerical aperture of 0.4. The total power delivered to the focal point was measured to be between 50 and 200 milliwatts for the 810 and 1480 nm laser diodes, which were capable of operating in a continuous wave (CW) fashion. The 905 and 1550 nm laser diodes were designed to produce high peak power pulses roughly 10 to 200 nanoseconds long at repetition rates up to 5000 Hz. For the pulsed lasers the peak power levels were measured to be 45 watts at 905 nm and 3.5 watts at 1550 nm. 
     Under these operating conditions, there was no apparent effect on the skin samples from any of the lasers. The targeted area was illuminated continuously for 60 seconds and then examined microscopically, revealing no visible effects. In addition, the sample was placed in a modified Franz cell, typically used to test transdermal delivery systems based on chemical permeation enhancers, and the conductivity from one side of the membrane to the other was measured both before and after the irradiation by the laser and showed no change. Based on these tests which were run on skin samples from four different donors, it was concluded that at these wavelengths the coupling of the optical energy into or through the skin tissue was so small that no effects are detectable. 
     Example 3 
     To evaluate the potential sensation to a living subject when illuminated with optical energy under the conditions of Example 2, six volunteers were used and the output of each laser source was applied to their fingertips, forearms, and the backs of their hands. In the cases of the 810, 905 and 1550 nm lasers, the subject was unable to sense when the laser was turned on or off. In the case of the 1480 nm laser, there was a some sensation during the illumination by the 1480 nm laser operating at 70 mW CW, and a short while later a tiny blister was formed under the skin due to the absorption of the 1480 nm radiation by one of the water absorption bands. Apparently the amount of energy absorbed was sufficient to induce the formation of the blister, but was not enough to cause the ablative removal of the stratum corneum. Also, the absorption of the 1480 nm light occurred predominantly in the deeper, fully hydrated (85% to 90% water content) tissues of the epidermis and dermis, not the relatively dry (10% to 15% water content) tissue of the stratum corneum. 
     Example 4 
     Having demonstrated the lack of effect on the skin in its natural state (Example 3), a series of chemical compounds was evaluated for effectiveness in absorbing the light energy and then transferring this absorbed energy, via conduction, into or through the targeted tissue of the stratum corneum. Compounds tested included India ink; “SHARPIE” brand indelible black, blue, and red marking pens; methylene blue; fuschian red; epolite #67, an absorbing compound developed for molding into polycarbonate lenses for protected laser goggles; tincture of iodine; iodine-polyvinylpyrrolidone complex (“BETADINE”); copper phthalocyanine; and printers ink. 
     Using both of the CW laser diodes described in Example 2, positive ablation results were observed on the in vitro samples of heat-separated stratum corneum prepared according to Example 1 when using all of these products, however some performed better than others. In particular the copper phthalocyanine (CPC) and the epolite #67 were some of the most effective. One probable reason for the superior performance of the CPC is its high boiling point of greater the 500 C and the fact that it maintains its solid phase up to this temperature. 
     Example 5 
     As copper phthalocyanine has already been approved by the FDA for use in implantable sutures, and is listed in the Merck index as a rather benign and stabile molecule in regard to human biocompatability, the next step taken was to combine the topical application of the CPC and the focused light source to the skin of healthy human volunteers. A suspension of finely ground CPC in isopropyl alcohol was prepared. The method of application used was to shake the solution and then apply a small drop at the target site. As the alcohol evaporated, a fine and uniform coating of the solid phase CPC was then left on the surface of the skin. 
     The apparatus show in  FIG. 1  was then applied to the site, wherein the CPC had been topically coated onto the skin, by placing the selected area of the individual&#39;s skin against a reference plate. The reference plate consists of a thin glass window roughly 3 cm×3 cm, with a 4 mm hole in the center. The CPC covered area was then positioned such that it was within the central hole. A confocal video microscope ( FIG. 1 ) was then used to bring the surface of the skin into sharp focus. Positioning the skin to achieve the sharpest focus on the video system also positioned it such that the focal point of the laser system was coincident with the surface of the skin. The operator then activated the pulses of laser light while watching the effects at the target site on the video monitor. The amount of penetration was estimated visually by the operator by gauging the amount of defocusing of the laser spot in the micropore as the depth of the micropore increased, and this can be dynamically corrected by the operator, essentially following the ablated surface down into the tissues by moving the position of the camera/laser source along the “z” axis, into the skin. At the point when the stratum corneum had been removed down to the epidermis, the appearance of the base of the hole changed noticeably, becoming much wetter and shinier. Upon seeing this change, the operator deactivated the laser. In many instances, depending on the state of hydration of the subject as well as other physiological conditions, a dramatic outflow of interstitial fluid occurred in response to the barrier function of the stratum corneum being removed over this small area. The video system was used to record this visual record of the accessibility of interstitial fluid at the poration site. 
     Example 6 
     The procedure of Example 5 was followed except that the CPC was applied to a transparent adhesive tape, which was then caused to adhere to a selected site on the skin of an individual. The results were substantially similar to those of Example 5. 
     Example 7 
     Histology experiments were performed on cadaver skin according to methods well known in the art to determine ablation threshold parameters for given dye mixtures and collateral damage information. The top surface of the skin sample was treated with a solution of copper phthalocyanine (CPC) in alcohol. After the alcohol evaporated, a topical layer of solid phase CPC was distributed over the skin surface with a mean thickness of 10 to 20 μm.  FIG. 8A  shows a cross-section of full thickness skin prior to the laser application, wherein the CPC layer  270 , stratum corneum  274 , and underlying epidermal layers  278  are shown.  FIG. 8B  shows the sample after a single pulse of 810 nm light was applied to an 80 um diameter circle with an energy density of 4000 J/cm2, for a pulse period of 20 ms. It is noteworthy that there was still a significant amount of CPC present on the surface of the stratum corneum even in the middle of the ablated crater  282 . It should also be noted that laboratory measurements indicate that only about 10% of the light energy incident on the CPC is actually absorbed, with the other 90% being reflected or backscattered. Thus the effective energy flux being delivered to the dye layer which could cause the 
     desired heating is only about 400 J/cm2. 8C shows the sample after 5 pulses of 810 nm light were applied, wherein the stratum corneum barrier was removed with no damage to the underlying tissue. These results are a good representation of the “ideal” optically modulated thermal ablation performance.  FIG. 8D  shows the sample after 50 pulses were applied. Damaged tissue  286  was present in the epidermal layers due to carbonization of non ablated tissue and thermal denaturing of the underlying tissue.  FIGS. 8A-8C  show separations between the stratum corneum and the underlying epidermal layers due to an artifact of dehydration, freezing, and preparations for imaging. 
     Example 8 
     To examine the details of the thermal ablation mechanism, a mathematical model of the skin tissues was constructed upon which various different embodiments of the thermal ablation method could be tried. This model computes the temperature distribution in a layered semi-infinite medium with a specified heat flux input locally on the surface and heat removal from the surface some distance away, i.e. convection is applied between the two. The axisymmetric, time-dependent diffusion equation is solved in cylindrical coordinates using the alternating-direction-implicit (ADI) method. (Note: Constant Temp. B.C. is applied on lower boundary to serve as z-&gt;inf; and zero radial heat flux is applied on max radial boundary to serve as r-&gt;inf). The layers are parallel to the surface and are defined as: (1) dye; (2) stratum corneum; (3) underlying epidermis; and (4) dermis. The depth into the semi-infinite medium and thermal properties, density (rho), specific heat (c), and conductivity (k) must be specified for each layer. 
     First, a heat-transfer coefficient, h, on the skin is computed based on the “steady,” “1-D,” temperature distribution determined by the ambient air temperature, skin surface temperature, and dermis temperature. It is assumed that there is no dye present and provides “h” on the skin surface. The program then allows one to use this “h” on the dye layer surface or input another desired “h” for the dye surface. Next, the “steady” temperature distribution is computed throughout all layers (including the dye layer) using the specified “h” at the dye surface. This temperature distribution is the initial condition for the time-dependent heating problem. This constitutes the “m-file” initial.m. The program then solves for the time-dependent temperature distribution by marching in time, computing and displaying the temperature field at each step. 
     Each embodiment of the method described herein, for which empirical data have been collected, has been modeled for at least one set of operational parameters, showing how stratum corneum ablation can be achieved in a precise and controllable fashion. The output of the simulations is presented graphically in two different formats: (1) a cross-sectional view of the skin showing the different tissue layers with three isotherms plotted on top of this view which define three critical temperature thresholds, and (2) two different temperature-vs-time plots, one for the point in the middle of the stratum corneum directly beneath the target site, and the second for the point at the boundary of the viable cell layers of the epidermis and the underside of the stratum corneum. These plots show how the temperature at each point varies with time as the heat pulses are applied as if one could implant a microscopic thermocouple into the tissues. In addition, the application of this model allows investigation of the parametric limits within which the method can be employed to set the outer limits for two important aspects of the methods performance. First, general cases are presented cases that define the envelope within which the method can be employed without causing pain or undesired tissue damage. 
     For any given heat source, as described in the several different embodiments of the invention, there is a point at which the effect on the subject&#39;s skin tissues becomes non-optimal in that the subject perceives a pain sensation, or that the viable cells in the underlying epidermis and/or dermis sustain temperatures, which if maintained for a long enough duration, will render damage to these tissues. Accordingly, a test simulation was run using the optically heated topical copper phthalocyanine (CPC) dye embodiment as a baseline method to establish how the thermal time constants of the different skin tissue layers essentially define a window within which the method can be employed without pain or damage to adjacent tissue layers. 
       FIGS. 9 and 10  show schematic cross-sectional views of the skin and the topical dye layer. In each figure, three distinct isotherms are displayed: (1) 123 C, the point at which vaporization of the water in the tissue produces an ablation of the tissue; (2) 70 C, the point at which viable cells will be damaged if this temperature is maintained for several seconds; and (3) 45 C, the average point at which a sensation of pain will be perceived by the subject. This pain threshold is described in several basic physiology texts, but experience shows this threshold to be somewhat subjective. In fact, in repeated tests on the same individual, different poration sites within a few millimeters of each other can show significantly different amounts of sensation, possibly due to the proximity to a nerve ending in relationship to the poration site. 
     The dimensions on the graphs show the different layers of the dye and skin, as measured in m, with flat boundaries defining them. Whereas the actual skin tissues have much more convoluted boundaries, in a mean sense for the dimensions involved, the model provides a good approximation of the thermal gradients present in the actual tissues. The dimensions used in this, and all subsequent simulations, for the thicknesses of the CPC dye layer and the various skin layers are as follows: dye, 10 m; stratum corneum, 30 m; underlying epidermis, 70 m; and dermis, 100 m. 
     Additional conditions imposed on the model for this particular simulation are shown in the following tables: 
     
       
         
           
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Initial Conditions for Finite Difference Thermal Model 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Ambient Air Temperature 
                 Ta = 20 C. 
               
               
                   
                 Skin Surface Temperature 
                 Ts = 30 C. 
               
               
                   
                 Dermis Temperature 
                 Td = 37 C. 
               
               
                   
                 Dye Vaporization Temperature 
                 Tvap = 550 C. 
               
               
                   
                 S.C. Vaporization Temperature 
                 Tc1 = 123 C. 
               
               
                   
                 Tissue Damage Temperature 
                 Tc2 = 70 C. 
               
               
                   
                 “Pain” Temperature 
                 Tc3 = 45 C. 
               
               
                   
                 Radius of Irradiated Area 
                 R hot  = 30 m 
               
               
                   
                 Energy Density Applied 
                 FLUX = 400 Joules/cm 2   
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
             
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                 Parameter 
                 Dye 
                 S.C. 
                 Epidermis 
                 Dermis 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Thermal 
                 0.00046 
                 .00123 
                 0.00421 
                 0.00421 
               
               
                   
                 Conductivity 
               
               
                   
                 Density 
                 0.67 
                 1.28 
                 1.09 
                 1.09 
               
               
                   
                 Specific Heat 
                 0.8 
                 1.88 
                 3.35 
                 3.35 
               
               
                   
                   
               
            
           
         
       
     
     When these simulations are run, the following conservative assumptions are imposed: 
     1. While some portion of the stratum corneum may be shown as having a temperature already exceeded the ablation threshold for thermal vaporization of the water content, this event is not modeled, and the subsequent loss of heat energy in the tissues due to this vaporization is not factored into the simulation. This will cause a slight elevation in the temperatures shown in the underlying tissues from that point on in the simulation run. 
     2. Similarly, when some portion of the copper phthalocyanine (CPC) dye layer is shown to have reached its vaporization point of 550° C., this event is not modeled, but the temperature is merely hard-limited to this level. This will also cause a slight elevation of the subsequent temperatures in the underlying layers as the simulation progresses. 
     Even with these simplifications used in the model, the correlation between the predicted performance and the empirically observed performance based on both clinical studies and histological studies on donor tissue samples is remarkable. The key data to note in  FIGS. 9 and 10  are the length of time which the heat pulse is applied, and the location of the three different threshold temperatures displayed by the isotherms. 
     In  FIG. 9 , with a pulse length of 21 milliseconds, the 70° C. isotherm just crosses the boundary separating the stratum corneum and the viable cell layers in the epidermis. In in vitro studies on donor skin samples under these conditions, fifty pulses of thermal energy delivered 50 milliseconds apart cause detectable damage to this top layer of living cells (see  FIG. 8D ). However, it was also shown in the in vitro studies that five pulses of heat energy at these same operating parameters, did not produce any significant damage to these tissues. It seems reasonable that even though the nominal damage threshold may have been exceeded, at least in a transient sense, this temperature must be maintained for some cumulative period of time to actually cause any damage to the cells. Nevertheless, the basic information presented by the simulation is that if one keeps the “on-time” of the heat pulse to less than 20 milliseconds with the flux density of 400 Joules/cm 2 , then no damage to the living cells in the underlying epidermis will be sustained, even though the ablation threshold isotherm has been moved well into or through the stratum corneum. In other words, by using a low flux density thermal energy source, modulated such that the “on time” is suitably short, ablation of the stratum corneum can be achieved without any damage to the adjacent cells in the underlying epidermis (see  FIG. 8C ). This is possible in large part due to the significantly different thermal diffusivities of these two tissues layers. That is, the stratum corneum, containing only about 10% to 20% water content, has a much lower thermal conductivity constant, 0.00123 J/(S*cm*K), than the 0.00421 J/(S*cm*K) of the epidermis. This allows the temperature to build up in the stratum corneum, while maintaining a tight spatial definition, to the point at which ablation will occur. 
     In  FIG. 10 , the same simulation scenario started in the damage threshold critical point run illustrated in  FIG. 9  is carried out farther in time. By leaving the heat pulse on for 58 milliseconds at the same flux density of 400 Joules/cm 2  within the 60 μm diameter circle of dye being heated, the pain sensory isotherm at 45° C. just enters the innervated layer of skin comprised by the dermis. In addition, the damage threshold isotherm moves significantly farther into the epidermal layer than where it was shown to be in  FIG. 9 . Relating this simulation to the numerous clinical studies conducted with this method, an excellent verification of the model&#39;s accuracy is obtained in that the model shows almost exactly the duration of ‘on-time’ that the heat probe can be applied to the skin before the individual feels it. In clinical tests, a controllable pulse generator was used to set the “on-time” and “off-time” of a series of light pulses applied to the topical layer of copper phthalocyanine (CPC) dye on the skin. While maintaining a constant “off-time” of 80 milliseconds, the “on-time” was gradually increased until the subject reported a mild “pain” sensation. Without exception, all of the subjects involved in these studies, reported the first “pain” at an “on-time” of between 45 and 60 milliseconds, very close to that predicted by the model. In addition, the site-to-site variability mentioned previously as regards the sensation of “pain” was noted in these clinical studies. Accordingly, what is reported as “pain” is the point at which the first unambiguous sensation is noticeable. At one site this may be reported as pain, whereas at an adjacent site the same subject may report this as merely “noticeable.” 
     One element of this clinical research is the realization that even at the same site, a non-uniform pulse-train of heat pulses may work with the subject&#39;s psycho-physiological neuro-perception to cause a genuine reduction in perceived sensation. For example, a series of shorter length heat pulses can be used to saturate the neurons in the area, momentarily depleting the neuro-transmitters available at this synaptic junction and therefore limiting the ability to send a “pain” message. This then allows a longer pulse following these short pulses to be less noticeable than if it were applied at the beginning of the sequence. Accordingly, a series of experiments was conducted with some arbitrarily created pulse trains, and the results were consistent with this hypothesis. An analogy for this situation might be found in the perception when one first steps into a very hot bath that is painful at first, but quickly becomes tolerable as one acclimates to the heat sensation. 
     Example 9 
     An object of this invention is to achieve a painless, micro-poration of the stratum corneum without causing any significant damage to the adjacent viable tissues. As described in the simulation illustrated in Example 8 and  FIGS. 9-10 , a boundary appears to exist for any given flux density of thermal energy within the ablation target spot within which the micro-poration can be achieved in just such a painless and non-traumatic manner. Both the in vivo and in vitro studies have shown that this is the case, and this has permitted development through empirical methods of some operational parameters that appear to work very well. The following set of simulations shows how the method works when these specific parameters are used. 
     In the first case, a pulse train of ten pulses, 10 milliseconds “on-time” separated by 10 milliseconds “off-time” is applied to the CPC-covered skin.  FIG. 11  shows the final temperature distribution in the skin tissues immediately after this pulse train has ended. As can be seen, the isotherms representing the three critical temperature thresholds show that stratum corneum ablation has been achieved, with no sensation present in the dermal layer nerves and very little cross-over of the damage threshold into or through the viable cells of the underlying epidermis. As mentioned previously, it appears that to actually do permanent cell damage, the epidermal cells must not only be heated up to a certain point, but they also must be held at this temperature for some period of time, generally thought to be about five seconds.  FIGS. 12 and 13  show the temperature of the stratum corneum and the viable epidermis, respectively, as a function of time, showing heating during the “on-time” and cooling during the “off-time” for the entire ten cycles. Relating this simulation to the in vivo studies conducted, note that in better than 90% of the poration attempts with the system parameters set to match the simulation, effective poration of the stratum corneum was achieved without pain to the subject, and in subsequent microscopic examination of the poration site several days later, no noticeable damage to the tissues was apparent. The in vitro studies conducted on whole thickness donor skin samples were also consistent with the model&#39;s prediction of behavior. 
     Example 10 
     In conducting both the empirical in vivo studies, and these simulations, it appears that prechilling of the skin aids in optimizing the micro-poration process for reducing the probability of pain or damage to adjacent tissues. In practice, this can easily be achieved using a simple cold-plate placed against the skin prior to the poration process. For example, applying a Peltier cooled plate to the 1 cm diameter circle surrounding the poration target site, with the plate held at roughly 5° C. for a few seconds, significantly reduces the temperature of the tissues. A schematic illustration of an experimental device used for this purpose in the laboratory is shown in  FIGS. 3A-B . By applying exactly the same ten-cycle pulse train as used in the run illustrated in Example 9, one can see, by comparing  FIG. 11  to  FIG. 14 ,  FIG. 12  to  FIG. 15 , and  FIG. 13  to  FIG. 16 , how much improvement can be made in the control of the temperature penetration into or through the skin tissues. Once again, the relatively low thermal diffusivity and specific heat of the stratum corneum as compared to the epidermis and dermis is advantageous. Once cooled, the highly hydrated tissues of the epidermis and dermis require a much larger thermal energy input to elevate their temperatures, whereas the stratum corneum, with its relatively dry makeup, can quickly be heated up to the ablation threshold. 
     Example 11 
     Once the basic thermal conduction mechanism of delivering the energy into or through the skin tissues underlying the effective painless ablation and micro-poration of the stratum corneum is understood, several different specific methods to achieve the required rapid temperature modulations of the contact point can be conceived, such as the hot wire embodiments illustrated in  FIGS. 4-7 . 
     A basic embodiment, as described herein, uses an Ohmic heating element ( FIG. 4 ), such as the tip of a small cordless soldering iron, with a suitably sized, relatively non-reactive, wire wrapped around it with a short amount of the wire left to protrude away from the body of the heater. When electricity is applied with a constant current source, the heater will come up to some temperature and within a few seconds, achieve a steady state with the convection losses to the surrounding air. Similarly, the wire, which is a part of this thermal system, will reach a steady state such that the very tip of the wire can be raised to almost any arbitrary temperature, up to roughly 1000° C. with these types of components. The tip can be sized to give exactly the dimension micropore desired. 
     In the laboratory, tungsten wires with a diameter of 80 μm attached to the replaceable tip of a “WAHL” cordless soldering iron with approximately 2 mm of wire protruding from the tip have been utilized. With a thermocouple, the temperature of the tip has been measured at its steady state, and it has been noted that by varying the constant current settings, steady state temperatures of greater than 700° C. can easily be reached. To achieve the desired modulation, a low mass, fast response electromechanical actuator was coupled to the tip such that the position of the wire could be translated linearly more than 2 mm at up to a 200 Hz rate. Then, by mounting the entire apparatus on a precision stage, this vibrating tip could very controllably be brought into contact with the skin surface in a manner where it was only in contact for less than 10 milliseconds at a time, the “on-time,” while an “off-time” of arbitrarily long periods could be achieved by setting the pulse generator accordingly. These in vivo studies showed that the poration could actually be achieved before the subject being porated even knew that the tip of the wire was being brought into contact with the skin. 
     To compare the performance of this embodiment to the optically heated topical CPC dye embodiment, the following simulations were run according to the procedure of Example 8. Essentially, by only varying the initial conditions, the hot wire embodiment can be run with the identical simulation code. Because the contact with the wire occurs essentially instantly, there is no time dependent build-up of heat in the CPC dye layer and when the wire is physically removed from contact with the skin, there is a no residual heat still left on the surface as there is with the heated CPC dye layer. Also, as the wire itself defines the area targeted for ablation/micro-poration, there should be no lateral diffusion of thermal energy prior to its application to the stratum corneum. The comparative performances of the “hot-wire” embodiment are shown in  FIGS. 17-19 . 
     Example 12 
     In this example, the procedure of Example 11 was followed except that the skin was pre-cooled according to the procedure of Example 10. Similarly, pre-cooling the target site yields similarly positive results with the “hot-wire” embodiment. The results of the pre-cooled simulation of the “hot-wire” approach are shown in  FIGS. 20-22 . 
     Example 13 
     As discussed in the background introduction of this disclosure, the Tankovich &#39;803 patent appears at first glance to be similar to the presently claimed invention. In this example, the simulation model was set up with the operating parameters specified in Tankovich &#39;803, i.e. a pulse width of 1 s and a power level of 40,000,000 W/cm 2 .  FIGS. 23 and 24  show that under these conditions no portion of the stratum corneum reaches the threshold for flash vaporization of water, 123 C, and thus no ablation/microporation of the stratum corneum occurs. In practice, applying this type of high peak power, short duration pulse to the topical dye layer merely vaporizes the dye off of the surface of the skin with no effect on the skin. This example, thus, demonstrates that the conditions specified by Tankovich &#39;803 are inoperative in the presently claimed invention. 
     Example 14 
     In this example, interstitial fluid obtained after porating the skin according to the procedure of Example 6 was collected and analyzed to determine the glucose concentration thereof. Data were obtained on four non-diabetic subjects and six type I diabetic subjects undergoing a glucose load test. Subject&#39;s ages ranged from 27 to 43. The goal of the study was to examine the utility of the method for painlessly harvesting enough interstitial fluid (ISF) from the subjects to allow the ISF samples to be assayed for glucose content, and then compare these concentrations to the glucose level presenting in the subject&#39;s whole blood. 
     All subjects had both the blood and ISF glucose assays performed with the “ELITE” system from Miles-Bayer. All ten subjects underwent identical measurement protocols, with adjustments being made regarding the glucose load and insulin shot for those subjects with insulin dependent diabetes. 
     The basic design of the study was to recruit a modest number of volunteers, some with diabetes and some without diabetes, from which a series of sample pairs of ISF and whole blood were drawn every 3 to 5 minutes throughout the 3 to 4 hour duration of the study period. Both the blood and the ISF samples were assayed for glucose and the statistical relationship between the blood glucose levels and the interstitial fluid determined. To examine the hypothesized temporal lag of the ISF glucose levels as compared to the whole blood glucose levels, the study subjects were induced to exhibit a significant and dynamic change in their glucose levels. This was accomplished by having each subject fast for 12 hours prior to beginning the test and then giving the subject a glucose load after his or her baseline glucose levels have been established via a set of three fasting blood and ISF glucose levels. After the baseline levels had been established, the subjects were given a glucose load in the form of sweet juice based on the following guidelines:
         i. For the control subjects, the glucose load was calculated based on a 0.75 gram glucose per pound of body weight.   ii. For the subjects with insulin dependent diabetes the glucose load was 50 grams of glucose. In addition, immediately after taking the glucose load the diabetic subjects will self inject their normal morning dose of fast acting insulin. In the case where the diabetic subject presents with fasting glucose levels above 300 mg/dL, they were asked to give themselves their insulin injection first, and the glucose load was provided after their blood glucose levels have dropped to below 120 mg/dL.       

     Each subject recruited was first given a complete description of the study in the “Informed Consent” document which they were required to understand and sign before they were officially enrolled into the program. Upon acceptance, they completed a medical history questionnaire. The detailed clinical procedure implemented was: 
     (a) Subject fasted from 9:00 p.m. the night before the study visit, consuming only water. No caffeine, cigarettes, fruit juice were allowed during this period. 
     (b) Subject arrived at the testing facility by 9:00 a.m. the next day. 
     (c) Subject was seated in a reclining chair provided for the subject to relax in throughout the study procedure. 
     (d) Both whole blood and ISF samples were taken at three to five minute intervals beginning upon the subject&#39;s arrival and continuing for the next three to four hours. The duration over which the data were collected was based on when the subject&#39;s blood glucose levels had returned to the normal range and stabilized after the glucose load. The ISF samples were harvested using the optical poration, ISF pumping method, described in more detail below. Each ISF sample was roughly 5 μL by volume to ensure a good fill of the ELITE test strip. The blood samples were obtained via a conventional finger prick lancet. Both the ISF and the blood samples were immediately assayed for glucose with the ELITE home glucometer system from Miles-Bayer. To improve the estimate of the ‘true’ blood glucose levels, two separate ELITE assays were be done on each finger stick sample. 
     (e) To facilitate the continued collection of the ISF from the same site through-out the entire data collection phase for a given individual, a 5 by 5 matrix of twenty five micropores was created on the subject&#39;s upper forearm, each micropore being between 50 and 80 μm across and spaced 300 μm apart. A 30 μm diameter teflon disk with a 6 mm hole in the center was attached to the subject&#39;s forearm with a pressure sensitive adhesive and positioned such that the 6 mm center hole was located over the 5 by 5 matrix of micropores. This attachment allowed a convenient method by which a small suction hose could be connected, applying a mild vacuum (10 to 12 inches of Hg) to the porated area to induce the ISF to flow out of the body through the micropores. The top of the teflon disk was fitted with a clear glass window allowing the operator to directly view the micro-porated skin beneath it. When a 5 μL bead of ISF was formed on the surface of the skin, it could easily be ascertained by visually monitoring the site through this window. This level of vacuum created a nominal pressure gradient of around 5 pounds/square inch (PSI). Without the micropores, no ISF whatsoever could be drawn from the subject&#39;s body using only the mild vacuum. 
     (f) After the first three sample pairs have been drawn, the subject was given a glucose load in the form of highly sweetened orange juice. The amount of glucose given was 0.75 grams per pound of body weight for the nondiabetic subjects and 50 grams for the diabetic subjects. The diabetic subjects also self administered a shot of fast acting insulin, (regular) with the dosage appropriately calculated, based on this 50 gram level of glucose concurrent with the ingestion of the glucose load. With the normal 1.5 to 2.5 hour lag between receiving an insulin shot and the maximum effect of the shot, the diabetic subjects were expected to exhibit an upwards excursion of their blood glucose levels ranging up to 300 mg/dL and then dropping rapidly back into the normal range as the insulin takes effect. The nondiabetic subjects were expected to exhibit the standard glucose tolerance test profiles, typically showing a peak in blood glucose levels between 150 mg/dL and 220 mg/dL from 45 minutes to 90 minutes after administering the glucose load, and then a rapid drop back to their normal baseline levels over the next hour or so. 
     (g) Following the administration of the glucose load or glucose load and insulin shot, the subjects had samples drawn, simultaneously, of ISF and finger prick whole blood at five minute intervals for the next three to four hours. The sampling was terminated when the blood glucose levels in three successive samples indicate that the subject&#39;s glucose had stabilized. 
     Upon examination of the data, several features were apparent. In particular, for any specific batch of ELITE test strips, there exist a distinct shift in the output shown on the glucometer in mg/dL glucose as compared to the level indicated on the blood. An elevated reading would be expected due to the lack of hematocrit in the ISF and to the normal differences in the electrolyte concentrations between the ISF and whole blood. Regardless of the underlying reasons for this shift in output, it was determined via comparison to a reference assay that the true ISF glucose levels are linearly related to the values produced by the ELITE system, with the scaling coefficients constant for any specific batch of ELITE strips. Consequently, for the comparison of the ISF glucose levels versus the whole blood measurements, first order linear correction was applied to the ISF data as follows: 
       ISF glucose =0.606*ISF ELITE +19.5. 
     This scaling of the output of the ELITE glucometer when used to measure ISF glucose levels, allows one to examine, over the entire data set, the error terms associated with using ISF to estimate blood glucose levels. Of course, even with no linear scaling whatsoever, the correlations between the ISF glucose values and the blood glucose levels are the same as the scaled version. 
     Based on the majority of the published body of literature on the subject of ISF glucose as well as preliminary data, it was originally expected that a 15 to 20 minute lag between the ISF glucose levels and the those presented in the whole blood from a finger stick would be observed. This is not what the data showed when analyzed. Specifically, when each individual&#39;s data set is analyzed to determine the time shift required to achieve the maximum correlation between the ISF glucose levels and the blood glucose levels it was discovered that the worst case time lag for this set of subjects was only 13 minutes and the average time lag was only 6.2 minutes, with several subjects showing a temporal tracking that was almost instantaneous (about 1 minute). 
     Based on the minimal amount of lag observed in this data set, the graph shown in  FIG. 25  presents all ten of the glucose load tests, concatenated one after another on an extended time scale. The data are presented with no time shifting whatsoever, showing the high level of tracking between the ISF and blood glucose levels the entire clinical data set being dealt with in exactly the same manner. If the entire data set is shifted as a whole to find the best temporal tracking estimate, the correlation between the ISF and blood glucose levels peaks with a delay of two (2) minutes at an r value of r=0.97. This is only a trivial improvement from the unshifted correlation of r=0.964. Therefore, for the remainder of the analysis the ISF values are treated with no time shift imposed on them. That is, each set of blood and ISF glucose levels is dealt with as simultaneously collected data pairs. 
     After the unshifted Elite ISF readings had been scaled to reflect the proportional glucose present in the ISF, it was possible to examine the error associated with these data. The simplest method for this is to assume that the average of the two ELITE finger-stick blood glucose readings is in fact the absolutely correct value, and then to merely compare the scaled ISF values to these mean blood glucose values. These data are as follows: Standard Deviation Blood-ISF, 13.4 mg/dL; Coefficient of Variance of ISF, 9.7%; Standard Deviation of the Two Elites, 8.3 mg/dL; and Coefficient of Variance of Blood (Miles), 6%. 
     As these data show, the blood based measurement already contains an error term. Indeed, the manufacturer&#39;s published performance data indicates that the ELITE system has a nominal Coefficient of Variance (CV) of between 5% and 7%, depending on the glucose levels and the amount of hematocrit in the blood. 
     An additional look at the difference term between the ISF glucose and the blood glucose is shown in the form of a scatter plot in  FIG. 26 . In this figure, the upper and lower bounds of the 90% confidence interval are also displayed for reference. It is interesting to note that with only two exceptions, all of the data in the range of blood glucose levels below 100 mg/dL fall within these 90% confidence interval error bars. This is important as the consequences of missing a trend towards hypoglycemia would be very significant to the diabetic user. That is, it would be much better to under-predict glucose levels in the 40 to 120 mg/dL than to over predict them. 
     Essentially, if one assumes that the basic assay error when the ELITE system is used on ISF is comparable to the assay error associated with the ELITE&#39;s use on whole blood, then the Deviation of the ISF glucose from the blood glucose can be described as: 
       ISF deviation =[(ISF actual ) 2 +(ISF actual ) 2 ] 1/2 . 
     Applying this equation to the values shown above, one can solve for the estimated ‘true’ value of the ISF error term: 
       ISF actual =[(ISF deviation ) 2 −(Blood actual ) 2 ] 1/2 .
 
     Or, solving the equation, 
       ISF actual =[(13.4) 2 −(8.3) 2 ] 1/2 =10.5 mg/dl.
 
     A histogram of the relative deviation of the ISF to the blood glucose levels is shown in  FIG. 27 . 
     Drug Delivery Through Pores in the Biological Membrane 
     The present invention also includes a method for the delivery of drugs, including drugs currently delivered transmembrane, through micropores in the stratum corneum or other biological membrane. In one illustrative embodiment, the delivery is achieved by placing the solution in a reservoir over the poration site. In another illustrative embodiment, a pressure gradient is used to further enhance the delivery. In still another illustrative embodiment, sonic energy is used with or without a pressure gradient to further enhance the delivery. The sonic energy can be operated according to traditional transdermal parameters or by utilizing acoustic streaming effects, which will be described momentarily, to push the delivery solution through the porated biological membrane. 
     Example 15 
     This example shows the use of stratum corneum poration for the delivery of lidocaine, a topical analgesic. The lidocaine solution also contained a chemical permeation enhancer formulation designed to enhance its passive diffusion across the stratum corneum. A drawing of an illustrative delivery apparatus  300  is shown in  FIG. 28 , wherein the apparatus comprises a housing  304  enclosing a reservoir  308  for holding a drug-containing solution  312 . The top portion of the housing comprises an ultrasonic transducer  316  for providing sonic energy to aid in transporting the drug-containing solution through micropores  320  in the stratum corneum  324 . A port  328  in the ultrasonic transducer permits application of pressure thereto for further aiding in transporting the drug-containing solution through the micropores in the stratum corneum. The delivery apparatus is applied to a selected area of an individual&#39;s skin such that it is positioned over at least one, and preferably a plurality, of micropores. An adhesive layer  332  attached to a lower portion of the housing permits the apparatus to adhere to the skin such that the drug-containing solution in the reservoir is in liquid communication with the micropores. Delivery of the drug-through the micropores results in transport into the underlying epidermis  336  and dermis  340 . 
     Five subjects were tested for the effectiveness of drug delivery using poration together with ultrasound. The experiment used two sites on the subjects left forearm about three inches apart, equally spaced between the thumb and upper arm. The site near the thumb will be referred to as site  1  the site furthest from the thumb will be referred to as site  2 . Site  1  was used as a control where the lidocaine and enhancer solution was applied using an identical delivery apparatus  300 , but without any micro-poration of the stratum corneum or sonic energy. Site  2  was porated with 24 holes spaced 0.8 millimeters apart in a grid contained within a 1 cm diameter circle. The micropores in Site  2  were generated according to the procedure of Example 6. Lidocaine and low level ultrasound were applied. Ultrasound applications were made with a custom manufactured Zevex ultrasonic transducer assembly set in burst mode with 0.4 Volts peak to peak input with 1000 count bursts occurring at 10 Hz with a 65.4 kHz fundamental frequency, i.e., a pulse modulated signal with the transducer energized for 15 millisecond bursts, and then turned off for the next 85 milliseconds. The measured output of the amplifier to the transducer was 0.090 watts RMS. 
     After application of the lidocaine, sensation measurements were made by rubbing a 30 gauge wire across the test site. Experiments were executed on both sites, Site  1  for 10 to 12 minute duration and Site  2  for two 5 minute duration intervals applied serially to the same site. Both sites were assessed for numbness using a scale of 10 to 0, where 10 indicated no numbness and 0 indicated complete numbness as reported by the test subjects. The following summary of results is for all 5 subjects. 
     The control site, site  1 , presented little to no numbness (scale 7 to 10) at 10 to 12 minutes. At approximately 20 minutes some numbness (scale 3) was observed at site  1  as the solution completely permeated the stratum corneum. Site  1  was cleaned at the completion of the lidocaine application. Site  2  presented nearly complete numbness (scale 0 to 1) in the 1 cm circle containing the porations. Outside the 1 cm diameter circle the numbness fell off almost linearly to 1 at a 2.5 cm diameter circle with no numbness outside the 2.5 cm diameter circle. Assessment of site  2  after the second application resulted in a slightly larger totally numb circle of about 1.2 cm diameter with numbness falling off linearly to 1 in an irregular oval pattern with a diameter of 2 to 2.5 cm perpendicular to the forearm and a diameter of 2 to 6 cm parallel to the forearm. Outside the area no numbness was noted. A graphic representation of illustrative results obtained on a typical subject is shown in  FIGS. 29A-C .  FIGS. 29A and 29B  show the results obtained at Site  2  (porated) after 5 and 10 minutes, respectively.  FIG. 29C  shows the results obtained at Site  1  (control with no poration). 
     Sonic Energy and Enhancers for Enhancing Transdermal Flux 
     The physics of sonic energy fields created by sonic transducers can be utilized in a method by which sonic frequency can be modulated to improve on flux rates achieved by other methods. As shown in FIG. 1 of U.S. Pat. No. 5,445,611, hereby incorporated herein by reference, the energy distribution of an sonic transducer can be divided into near and far fields. The near field, characterized by length N, is the zone from the first energy minimum to the last energy maximum. The zone distal to the last maximum is the far field. The near (N) field pattern is dominated by a large number of closely spaced local pressure peaks and nulls. The length of the near field zone, N, is a function of the frequency, size, and shape of the transducer face, and the speed of sound in the medium through which the ultrasound travels. For a single transducer, intensity variations within its normal operating range do not affect the nature of the sonic energy distribution other than in a linear fashion. However, for a system with multiple transducers, all being modulated in both frequency and amplitude, the relative intensities of separate transducers do affect the energy distribution in the sonic medium, regardless of whether it is skin or another medium. 
     By changing the frequency of the sonic energy by a modest amount, for example in the range of about 1 to 20%, the pattern of peaks and nulls remains relatively constant, but the length N of the near field zone changes in direct proportion to the frequency. Major changes the frequency, say a factor of 2 or more, will most likely produce a different set of resonances or vibrational modes in the transducer, causing a significantly and unpredictably different near field energy pattern. Thus, with a modest change in the sonic frequency, the complex pattern of peaks and nulls is compressed or expanded in an accordion-like manner. By selecting the direction of frequency modulation, the direction of shift of these local pressure peaks can be controlled. By applying sonic energy at the surface of the skin, selective modulation of the sonic frequency controls movement of these local pressure peaks through the skin either toward the interior of the body or toward the surface of the body. A frequency modulation from high to low drives the pressure peaks into the body, whereas a frequency modulation from low to high pulls the pressure peaks from within the body toward the surface and through the skin to the outside of the body. 
     Assuming typical parameters for this application of, for example, a 1.27 cm diameter sonic transducer and a nominal operating frequency of 10 MHz and an acoustic impedance similar to that of water, a frequency modulation of 1 MHz produces a movement of about 2.5 mm of the peaks and nulls of the near field energy pattern in the vicinity of the stratum corneum. From the perspective of transdermal and/or transmucosal withdrawal of analytes, this degree of action provides access to the area well below the stratum corneum and even the epidermis, dermis, and other tissues beneath it. For any given transducer, there may be an optimal range of frequencies within which this frequency modulation is most effective. 
     The flux of a drug or analyte across the skin can also be increased by changing either the resistance (the diffusion coefficient) or the driving force (the gradient for diffusion). Flux can be enhanced by the use of so-called penetration or chemical enhancers. 
     Chemical enhancers are comprised of two primary categories of components, i.e., cell-envelope disordering compounds and solvents or binary systems containing both cell-envelope disordering compounds and solvents. 
     Cell envelope disordering compounds are known in the art as being useful in topical pharmaceutical preparations and function also in analyte withdrawal through the skin. These compounds are thought to assist in skin penetration by disordering the lipid structure of the stratum corneum cell-envelopes. A comprehensive list of these compounds is described in European Patent Application 43,738, published Jun. 13, 1982, which is incorporated herein by reference. It is believed that any cell envelope disordering compound is useful for purposes of this invention. 
     Suitable solvents include water; diols, such as propylene glycol and glycerol; mono-alcohols, such as ethanol, propanol, and higher alcohols; DMSO; dimethylformamide; N,N-dimethylacetamide; 2-pyrrolidone; N-(2-hydroxyethyl)pyrrolidone, N-methylpyrrolidone, 1-dodecylazacycloheptan-2-one and other n-substituted-alkyl-azacycloalkyl-2-ones (azones) and the like. 
     U.S. Pat. No. 4,537,776, Cooper, issued Aug. 27, 1985, contains an excellent summary of prior art and background information detailing the use of certain binary systems for permeant enhancement. Because of the completeness of that disclosure, the information and terminology utilized therein are incorporated herein by reference. 
     Similarly, European Patent Application 43,738, referred to above, teaches using selected diols as solvents along with a broad category of cell-envelope disordering compounds for delivery of lipophilic pharmacologically-active compounds. Because of the detail in disclosing the cell-envelope disordering compounds and the diols, this disclosure of European Patent Application 43,738 is also incorporated herein by reference. 
     A binary system for enhancing metoclopramide penetration is disclosed in UK Patent Application GB 2,153,223 A, published Aug. 21, 1985, and consists of a monovalent alcohol ester of a C8-32 aliphatic monocarboxylic acid (unsaturated and/or branched if C18-32) or a C6-24 aliphatic monoalcohol (unsaturated and/or branched if C14-24) and an N-cyclic compound such as 2-pyrrolidone, N-methylpyrrolidone and the like. 
     Combinations of enhancers consisting of diethylene glycol monoethyl or monomethyl ether with propylene glycol monolaurate and methyl laurate are disclosed in U.S. Pat. No. 4,973,468 as enhancing the transdermal delivery of steroids such as progesterons and estrogens. A dual enhancer consisting of glycerol monolaurate and ethanol for the transdermal delivery of drugs is shown in U.S. Pat. No. 4,820,720. U.S. Pat. No. 5,006,342 lists numerous enhancers for transdermal drug administration consisting of fatty acid esters or fatty alcohol ethers of C 2  to C 4  alkanediols, where each fatty acid/alcohol portion of the ester/ether is of about 8 to 22 carbon atoms. U.S. Pat. No. 4,863,970 shows penetration-enhancing compositions for topical application comprising an active permeant contained in a penetration-enhancing vehicle containing specified amounts of one or more cell-envelope disordering compounds such as oleic acid, oleyl alcohol, and glycerol esters of oleic acid; a C 2  or C 3  alkanol and an inert diluent such as water. 
     Other chemical enhancers, not necessarily associated with binary systems include DMSO or aqueous solutions of DMSO such as taught in Herschler, U.S. Pat. No. 3,551,554; Herschler, U.S. Pat. No. 3,711,602; and Herschler, U.S. Pat. No. 3,711,606, and the azones (n-substituted-alkyl-azacycloalkyl-2-ones) such as noted in Cooper, U.S. Pat. No. 4,557,943. 
     Some chemical enhancer systems may possess negative side effects such as toxicity and skin irritation. U.S. Pat. No. 4,855,298 discloses compositions for reducing skin irritation caused by chemical enhancer containing compositions having skin irritation properties with an amount of glycerin sufficient to provide an anti-irritating effect. 
     Because the combination of microporation of the stratum corneum and the application of sonic energy accompanied by the use of chemical enhancers can result in an improved rate of analyte withdrawal or permeant delivery through the stratum corneum, the specific carrier vehicle and particularly the chemical enhancer utilized can be selected from a long list of prior art vehicles some of which are mentioned above and incorporated herein by reference. To specifically detail or enumerate that which is readily available in the art is not thought necessary. The invention is not drawn to the use of chemical enhancers per se and it is believed that all chemical enhancers, useful in the delivery of drugs through the skin, will function with dyes in optical microporation and also with sonic energy in effecting measurable withdrawal of analytes from beneath and through the skin surface or the delivery of permeants or drugs through the skin surface. 
     Example 16 
     Modulated sonic energy and chemical enhancers were tested for their ability to control transdermal flux on human cadaver skin samples. In these tests, the epidermal membrane had been separated from the human cadaver whole skin by the heat-separation method of Example 1. The epidermal membrane was cut and placed between two halves of the permeation cell with the stratum corneum facing either the upper (donor) compartment or lower (receiver) compartment. Modified Franz cells were used to hold the epidermis, as shown in FIG. 2 of U.S. Pat. No. 5,445,611. Each Franz cell consists of an upper chamber and a lower chamber held together with one or more clamps. The lower chamber has a sampling port through which materials can be added or removed. A sample of stratum corneum is held between the upper and lower chambers when they are clamped together. The upper chamber of each Franz cell is modified to allow an ultrasound transducer to be positioned within 1 cm of the stratum corneum membrane. Methylene blue solution was used as an indicator molecule to assess the permeation of the stratum corneum. A visual record of the process and results of each experiment was obtained in a time stamped magnetic tape format with a video camera and video cassette recorder (not shown). Additionally, samples were withdrawn for measurement with an absorption spectrometer to quantitate the amount of dye which had traversed the stratum corneum membrane during an experiment. Chemical enhancers suitable for use could vary over a wide range of solvents and/or cell envelope disordering compounds as noted above. The specific enhancer utilized was: ethanol/glycerol/water/glycerol monooleate/methyl laurate in 50/30/15/2.5/2.5 volume ratios. The system for producing and controlling the sonic energy included a programmable 0-30 MHz arbitrary waveform generator (Stanford Research Systems Model DS345), a 20 watt 0-30 MHz amplifier, and two unfocused ultrasound immersion transducers having peak resonances at 15 and 25 MHz, respectively. Six cells were prepared simultaneously for testing of stratum corneum samples from the same donor. Once the stratum corneum samples were installed, they were allowed to hydrate with distilled water for at least 6 hours before any tests were done. 
     Example 17 
     Effects of Sonic Energy Without Chemical Enhancers 
     As stated above in Example 16, the heat-separated epidermis was placed in the Franz cells with the epidermal side facing up, and the stratum corneum side facing down, unless noted otherwise. The lower chambers were filled with distilled water, whereas the upper chambers were filled with concentrated methylene blue solution in distilled water. 
     Heat Separated Epidermis: Immediately after filling the upper chambers with methylene blue solution, sonic energy was applied to one of the cells with the transducer fully immersed. This orientation would correspond, for example, to having the transducer on the opposite side of a fold of skin, or causing the sonic energy to be reflected off a reflector plate similarly positioned and being used to “push” analyte out of the other side of the fold into a collection device. The sonic energy setting was initially set at the nominal operating frequency of 25 MHz with an intensity equivalent to a 20 volt peak-to-peak (P-P) input wave form. This corresponds to roughly a 1 watt of average input power to the transducer and similarly, assuming the manufacturer&#39;s nominal value for conversion efficiency of 1% for this particular transducer, a sonic output power of around 0.01 watts over the 0.78 cm 2  surface of the active area or a sonic intensity of 0.13 watts/cm 2 . Three other control cells had no sonic energy applied to them. After 5 minutes the sonic energy was turned off. No visual indication of dye flux across the stratum corneum was observed during this interval in any of the cells, indicating levels less than approximately 0.0015% (v/v) of dye solution in 2 ml of receiver medium. 
     Testing of these same 3 control cells and 1 experimental cell was continued as follows. The intensity of sonic energy was increased to the maximum possible output available from the driving equipment of a 70 volt peak-to-peak input 12 watts average power input or (0.13 watts/cm 2 ) of sonic output intensity. Also, the frequency was set to modulate or sweep from 30 MHz to 10 MHz. This 20 MHz sweep was performed ten times per second, i.e., a sweep rate of 10 Hz. At these input power levels, it was necessary to monitor the sonic energy transducer to avoid overheating. A contact thermocouple was applied to the body of the transducer and power was cycled on and off to maintain maximum temperature of the transducer under 42 C. After about 30 minutes of cycling maximum power at about a 50% duty cycle of 1 minute on and 1 minute off, there was still no visually detectable permeation of the stratum corneum by the methylene blue dye. 
     A cooling water jacket was then attached to the sonic energy transducer to permit extended excitation at the maximum energy level. Using the same 3 controls and 1 experimental cell, sonic energy was applied at maximum power for 12 hours to the experimental cell. During this time the temperature of the fluid in the upper chamber rose to only 35 C, only slightly above the approximately 31° C. normal temperature of the stratum corneum in vivo. No visual evidence of dye flux through the stratum corneum was apparent in any of the four cells after 12 hrs. of sonic energy applied as described above. 
     Example 18 
     Effects of Sonic Energy Without Chemical Enhancers 
     Perforated Stratum Corneum: Six cells were prepared as described above in Example 16. The clamps holding the upper and lower chambers of the Franz cells were tightened greater than the extent required to normally seal the upper compartment from the lower compartment, and to the extent to artificially introduce perforations and “pinholes” into the heat-separated epidermal samples. When dye solution was added to the upper chamber of each cell, there were immediate visual indications of leakage of dye into the lower chambers through the perforations formed in the stratum corneum. Upon application of sonic energy to cells in which the stratum corneum was so perforated with small “pinholes,” a rapid increase in the transport of fluid through a pinhole in the stratum corneum was observed. The rate of transport of the indicator dye molecules was directly related to whether the sonic energy was applied or not. That is, application of the sonic energy caused an immediate (lag time approximately &lt;0.1 second) pulse of the indicator molecules through the pinholes in the stratum corneum. This pulse of indicator molecules ceased immediately upon turning off of the sonic energy (a shutoff lag of approximately &lt;0.1 second). The pulse could be repeated as described. 
     Example 19 
     Effects of Sonic Energy and Chemical Enhancers 
     Two different chemical enhancer formulations were used. Chemical Enhancer One or CE1 was an admixture of ethanol/glycerol/water/glycerol monooleate/methyl laurate in a 50/30/15/2.5/2.5 volume ratio. These are components generally regarded as safe, i.e. GRAS, by the FDA for use as pharmaceutical excipients. Chemical Enhancer Two or CE2 is an experimental formulation shown to be very effective in enhancing transdermal drug delivery, but generally considered too irritating for long term transdermal delivery applications. CE2 contained ethanol/glycerol/water/lauradone/methyl laurate in the volume ratios 50/30/15/2.5/2.5. Lauradone is the lauryl (dodecyl)ester of 2-pyrrolidone-5-carboxylic acid (“PCA”) and is also referred to as lauryl PCA. 
     Six Franz cells were set up as before (Example 16) except that the heat separated epidermis was installed with the epidermal layer down, i.e., stratum corneum side facing up. Hydration was established by exposing each sample to distilled water overnight. To begin the experiment, the distilled water in the lower chambers was replaced with methylene blue dye solution in all six cells. The upper chambers were filled with distilled water and the cells were observed for about 30 minutes confirming no passage of dye to ensure that no pinhole perforations were present in any of the cells. When none were found, the distilled water in the upper chambers was removed from four of the cells. The other two cells served as distilled water controls. The upper chambers of two of the experimental cells were then filled with CE1 and the other two experimental cells were filled with CE2. 
     Sonic energy was immediately applied to one of the two CE2 cells. A 25 MHz transducer was used with the frequency sweeping every 0.1 second from 10 MHz to 30 MHz at maximum intensity of 0.13 watts/cm 2 . After 10-15 minutes of sonic energy applied at a 50% duty cycle, dye flux was visually detected. No dye flux was detected in the other five cells. 
     Sonic energy was then applied to one of the two cells containing CE1 at the same settings. Dye began to appear in the upper chamber within 5 minutes. Thus, sonic energy together with a chemical enhancer significantly increased the transdermal flux rate of a marker dye through the stratum corneum, as well as reduced the lag time. 
     Example 20 
     Effects of Sonic Energy and Chemical Enhancers 
     Formulations of the two chemical enhancers, CE1 and CE2, were prepared minus the glycerin and these new formulations, designated CE1MG and CE2MG, were tested as before. Water was substituted for glycerin so that the proportions of the other components remained unchanged. Three cells were prepared in modified Franz cells with the epidermal side of the heat separated epidermis samples facing toward the upper side of the chambers. These samples were then hydrated in distilled water for 8 hours. After the hydration step, the distilled water in the lower chambers was replaced with either CE1MG or CE2MG and the upper chamber was filled with the dye solution. Sonic energy was applied to each of the three cells sequentially. 
     Upon application of pulsed, frequency-modulated sonic energy for a total duration of less than 10 minutes, a significant increase in permeability of the stratum corneum samples was observed. The permeability of the stratum corneum was altered relatively uniformly across the area exposed to both the chemical enhancer and sonic energy. No “pinhole” perforations through which the dye could traverse the stratum corneum were observed. The transdermal flux rate was instantly controllable by turning the sonic energy on or off. Turning the sonic energy off appeared to instantly reduce the transdermal flux rate such that no dye was visibly being actively transported through the skin sample; presumably the rate was reduced to that of passive diffusion. Turning the sonic energy on again instantly resumed the high level flux rate. The modulated mode appeared to provide a regular pulsatile increase in the transdermal flux rate at the modulated rate. When the sonic energy was set to a constant frequency, the maximum increase in transdermal flux rate for this configuration seemed to occur at around 27 MHz. 
     Having obtained the same results with all three samples, the cells were then drained of all fluids and flushed with distilled water on both sides of the stratum corneum. The lower chambers were then immediately filled with distilled water and the upper chambers were refilled with dye solution. The cells were observed for 30 minutes. No holes in the stratum corneum samples were observed and no large amount of dye was detected in the lower chambers. A small amount of dye became visible in the lower chambers, probably due to the dye and enhancer trapped in the skin samples from their previous exposures. After an additional 12 hours, the amount of dye detected was still very small. 
     Example 21 
     Effects of Sonic Energy and Chemical Enhancers 
     Perforated Stratum Corneum: Three cells were prepared with heat-separated epidermis samples with the epidermal side facing toward the upper side of the chamber from the same donor as in Example 16. The samples were hydrated for 8 hours and then the distilled water in the lower chambers was replaced with either CE1MG or CE2MG. The upper chambers were then filled with dye solution. Pinhole perforations in the stratum corneum samples permitted dye to leak through the stratum corneum samples into the underlying enhancer containing chambers. Sonic energy was applied. Immediately upon application of the sonic energy, the dye molecules were rapidly pushed through the pores. As shown above, the rapid flux of the dye through the pores was directly and immediately correlated with the application of the sonic energy. 
     Example 22 
     Effects of Sonic Energy and Chemical Enhancers 
     A low cost sonic energy transducer, TDK #NB-58S-01 (TDK Corp.), was tested for its capability to enhance transdermal flux rates. The peak response of this transducer was determined to be about 5.4 MHz with other local peaks occurring at about 7 MHz, 9 MHz, 12.4 MHz, and 16 MHz. 
     This TDK transducer was then tested at 5.4 MHz for its ability to enhance transdermal flux rate in conjunction with CE1MG. Three cells were set up with the epidermal side facing the lower chamber, then the skin samples were hydrated for 8 hrs. The dye solution was placed in the lower chamber. The transducer was placed in the upper chamber immersed in CE1MG. Using swept frequencies from 5.3 to 5.6 MHz as the sonic energy excitation, significant quantities of dye moved through the stratum corneum and were detected in the collection well of the cell in 5 minutes. Local heating occurred, with the transducer reaching a temperature of 48 C. In a control using CE1MG without sonic energy, a 24 hour exposure yielded less dye in the collection well than the 5 minute exposure with sonic energy. 
     This example demonstrates that a low cost, low frequency sonic energy transducer can strikingly affect transdermal flux rate when used in conjunction with an appropriate chemical enhancer. Although higher frequency sonic energy will theoretically concentrate more energy in the stratum corneum, when used with a chemical enhancer, the lower frequency modulated sonic energy can accelerate the transdermal flux rate to make the technology useful and practical. 
     Example 23 
     Demonstration of molecule migration across human skin: Tests with the TDK transducer and CE1MG described above were repeated at about 12.4 MHz, one of the highest local resonant peaks for the transducer, with a frequency sweep at a 2 Hz rate from 12.5 to 12.8 MHz and an sonic energy density less than 0.1 W/cm 2 . The epidermal side of the heat-separated epidermis was facing down, the dye solution was in the lower chamber, and the enhancer solution and the sonic energy were placed in the upper chamber. Within 5 minutes a significant amount of dye had moved across the stratum corneum into the collection well. Ohmic heating in the transducer was significantly less than with the same transducer being driven at 5.4 MHz, causing an increase in temperature of the chemical enhancer to only about 33 C. 
     Even at these low efficiency levels, the results obtained with CE1MG and sonic energy from the TDK transducer were remarkable in the monitoring direction. FIGS. 3A and 3B of U.S. Pat. No. 5,445,611 show plots of data obtained from three separate cells with the transdermal flux rate measured in the monitoring direction. Even at the 5 minute time point, readily measurable quantities of the dye were present in the chemical enhancer on the outside of the stratum corneum, indicating transport from the epidermal side through the stratum corneum to the “outside” area of the skin sample. 
     To optimize the use of the sonic energy or the sonic energy/chemical enhancer approach for collecting and monitoring analytes from the body, means for assaying the amount of analyte of interest are required. An assay system that takes multiple readings while the unit is in the process of withdrawing analytes by sonic energy with or without chemical enhancers makes it unnecessary to standardize across a broad population base and normalize for different skin characteristics and flux rates. By plotting two or more data points in time as the analyte concentration in the collection system is increasing, a curve-fitting algorithm can be applied to determine the parameters describing the curve relating analyte withdrawal or flux rate to the point at which equilibrium is reached, thereby establishing the measure of the interval concentration. The general form of this curve is invariant from one individual to another; only the parameters change. Once these parameters are established, solving for the steady state solution (i.e., time equals infinity) of this function, i.e., when full equilibrium is established, provides the concentration of the analyte within the body. Thus, this approach permits measurements to be made to the desired level of accuracy in the same amount of time for all members of a population regardless of individual variations in skin permeability. 
     Several existing detection techniques currently exist that are adaptable for this application. See, D. A. Christensen, in 1648 Proceedings of Fiber Optic, Medical and Fluorescent Sensors and Applications 223-26 (1992). One method involves the use of a pair of optical fibers that are positioned close together in an approximately parallel manner. One of the fibers is a source fiber, through which light energy is conducted. The other fiber is a detection fiber connected to a photosensitive diode. When light is conducted through the source fiber, a portion of the light energy, the evanescent wave, is present at the surface of the fiber and a portion of this light energy is collected by the detection fiber. The detection fiber conducts the captured evanescent wave energy to the photosensitive diode which measures it. The fibers are treated with a binder to attract and bind the analyte that is to be measured. As analyte molecules bind to the surface (such as the analyte glucose binding to immobilized lectins such as concanavalin A, or to immobilized anti-glucose antibodies) the amount of evanescent wave coupling between the two fibers is changed and the amount of energy captured by the detection fiber and measured by the diode is changed as well. Several measurements of detected evanescent wave energy over short periods of time support a rapid determination of the parameters describing the equilibrium curve, thus making possible calculation of the concentration of the analyte within the body. The experimental results showing measurable flux within 5 minutes (FIGS. 3A and 3B of U.S. Pat. No. 5,445,611) with this system suggest sufficient data for an accurate final reading are collected within 5 minutes. 
     In its most basic embodiment, a device that can be utilized for the application of sonic energy and collection of analyte comprises an absorbent pad, either of natural or synthetic material, which serves as a reservoir for the chemical enhancer, if used, and for receiving the analyte from the skin surface. The pad or reservoir is held in place, either passively or aided by appropriate fastening means, such as a strap or adhesive tape, on the selected area of skin surface. 
     An sonic energy transducer is positioned such that the pad or reservoir is between the skin surface and the transducer, and held in place by appropriate means. A power supply is coupled to the transducer and activated by switch means or any other suitable mechanism. The transducer is activated to deliver sonic energy modulated in frequency, phase or intensity, as desired, to deliver the chemical enhancer, if used, from the reservoir through the skin surface followed by collection of the analyte from the skin surface into the reservoir. After the desired fixed or variable time period, the transducer is deactivated. The pad or reservoir, now containing the analyte of interest, can be removed to quantitate the analyte, for example, by a laboratory utilizing any number of conventional chemical analyses, or by a portable device. Alternately, the mechanism for quantitating the analyte can be build into the device used for collection of the analyte, either as an integral portion of the device or as an attachment. Devices for monitoring an analyte are described in U.S. Pat. No. 5,458,140, which is incorporated herein by reference. 
     Example 24 
     An alternate method for detection of an analyte, such as glucose, following the sample collection through the porated skin surface as described above, can be achieved through the use of enzymatic means. Several enzymatic methods exist for the measurement of glucose in a biological sample. One method involves oxidizing glucose in the sample with glucose oxidase to generate gluconolactone and hydrogen peroxide. In the presence of a colorless chromogen, the hydrogen peroxide is then converted by peroxidase to water and a colored product. 
     Glucose Oxidase 
     Glucose Gluconolactone+H 2 O 2    
     2 H 2 O 2 +chromogen H 2 O+colored product 
     The intensity of the colored product will be proportional to the amount of glucose in the fluid. This color can be determined through the use of conventional absorbance or reflectance methods. By calibration with known concentrations of glucose, the amount of color can be used to determine the concentration of glucose in the collected analyte. By testing to determine the relationship, one can calculate the concentration of glucose in the blood of the subject. This information can then be used in the same way that the information obtained from a blood glucose test from a finger puncture is used. Results can be available within five to ten minutes. 
     Example 25 
     Any system using a visual display or readout of glucose concentration will indicate to a diagnostician or patient the need for administration of insulin or other appropriate medication. In critical care or other situations where constant monitoring is desired and corrective action needs to be taken almost concurrently, the display may be connected with appropriate signal means which triggers the administration of insulin or other medication in an appropriate manner. For example, there are insulin pumps which are implanted into the peritoneum or other body cavity which can be activated in response to external or internal stimuli. Alternatively, utilizing the enhanced transdermal flux rates possible with micro-poration of the stratum corneum and other techniques described in this invention, an insulin delivery system could be implemented transdermally, with control of the flux rates modulated by the signal from the glucose sensing system. In this manner a complete biomedical control system can be available which not only monitors and/or diagnoses a medical need but simultaneously provides corrective action. 
     Biomedical control systems of a similar nature could be provided in other situations such as maintaining correct electrolyte balances or administering analgesics in response to a measured analyte parameter such as prostaglandins. 
     Example 26 
     Similar to audible sound, sonic waves can undergo reflection, refraction, and absorption when they encounter another medium with dissimilar properties [D. Bommannan et al., 9  Pharm. Res.  559 (1992)]. Reflectors or lenses may be used to focus or otherwise control the distribution of sonic energy in a tissue of interest. For many locations on the human body, a fold of flesh can be found to support this system. For example, an earlobe is a convenient location which would allow use of a reflector or lens to assist in exerting directional control (e.g., “pushing” of analytes or permeants through the porated stratum corneum) similar to what is realized by changing sonic frequency and intensity. 
     Example 27 
     Multiple sonic energy transducers may be used to selectively direct the direction of transdermal flux through porated stratum corneum either into the body or from the body. A fold of skin such as an earlobe allow transducers to be located on either side of the fold. The transducers may be energized selectively or in a phased fashion to enhance transdermal flux in the desired direction. An array of transducers or an acoustic circuit may be constructed to use phased array concepts, similar to those developed for radar and microwave communications systems, to direct and focus the sonic energy into the area of interest. 
     Example 28 
     In this example, the procedure of Example 19 is followed with the exception that the heat-separated epidermis samples are first treated with an excimer laser (e.g. model EMG/200 of Lambda Physik; 193 nm wavelength, 14 ns pulse width) to ablate the stratum corneum according to the procedure described in U.S. Pat. No. 4,775,361, hereby incorporated by reference. 
     Example 29 
     In this example, the procedure of Example 19 is followed with the exception that the heat-separated epidermis samples are first treated with 1,1′-diethyl-4,4′-carbocyanine iodide (Aldrich,  max =703 nm) and then a total of 70 mJ/cm 2 /50 ms is delivered to the dye-treated sample with a model TOLD9150 diode laser (Toshiba America Electronic, 30 mW at 690 nm) to ablate the stratum corneum. 
     Example 30 
     In this example, the procedure of Example 29 is followed with the exception that the dye is indocyanine green (Sigma cat. no. 1-2633;  max =775 nm) and the laser is a model Diolite 800-50 (LiCONiX, 50 mW at 780 nm). 
     Example 31 
     In this example, the procedure of Example 29 is followed with the exception that the dye is methylene blue and the laser is a model SDL-8630 (SDL Inc.; 500 mW at 670 nm). 
     Example 32 
     In this example, the procedure of Example 29 is followed with the exception that the dye is contained in a solution comprising a permeation enhancer, e.g. CE1. 
     Example 33 
     In this example, the procedure of Example 29 is followed with the exception that the dye and enhancer-containing solution are delivered to the stratum corneum with the aid of exposure to ultrasound. 
     Example 34 
     In this example, the procedure of Example 31 is followed with the exception that the pulsed light source is a short arc lamp emitting over the broad range of 400 to 1100 nm but having a bandpass filter placed in the system to limit the output to the wavelength region of about 650 to 700 nm. 
     Example 35 
     In this example, the procedure of Example 19 is followed with the exception that the heat-separated epidermis samples are first punctured with a microlancet (Becton Dickinson) calibrated to produce a micropore in the stratum corneum without reaching the underlying tissue. 
     Example 36 
     In this example, the procedure of Example 19 is followed with the exception that the heat-separated epidermis samples are first treated with focused sonic energy in the range of 70-480 mJ/cm 2 /50 ms to ablate the stratum corneum. 
     Example 37 
     In this example, the procedure of Example 19 is followed with the exception that the stratum corneum is first punctured hydraulically with a high pressure jet of fluid to form a micropore of up to about 100 μm diameter. 
     Example 38 
     In this example, the procedure of Example 19 is followed with the exception that the stratum corneum is first punctured with short pulses of electricity to form a micropore of up to about 100 μm diameter. 
     Example 39 
     Acoustic Streaming 
     A new mechanism and application of sonic energy in the delivering of therapeutic substances into the body and/or harvesting fluids from within the body into an external reservoir through micro-porations formed in the biological membrane will now be described. An additional aspect of this invention is the utilization of sonic energy to create an acoustic streaming effect on the fluids flowing around and between the intact cells in the viable tissues beneath the outer layer of an organism, such as the epidermis and dermis of the human skin. Acoustic streaming is a well documented mode by which sonic energy can interact with a fluid medium. Nyborg, Physical Acoustics Principles and Methods, p. 265-331, Vol II-Part B, Academic Press, 1965. The first theoretical analysis of acoustic streaming phenomenon was given by Rayleigh (1884, 1945). In an extensive treatment of the subject, Longuet-Higgins (1953-1960) has given a result applicable to two dimensional flow that results in the near vicinity of any vibrating cylindrical surface. A three dimensional approximation for an arbitrary surface was developed by Nyborg (1958). As described by Fairbanks et al., 1975 Ultrasonics Symposium Proceedings, IEEE Cat. #75, CHO 994-4SU, sonic energy, and the acoustic streaming phenomenon can be of great utility in accelerating the flux of a fluid through a porous medium, showing measurable increases in the flux rates by up to 50 times that possible passively or with only pressure gradients being applied. 
     All previous transdermal delivery or extraction efforts utilizing ultrasound have focused on methods of interaction between the sonic energy and the skin tissues designed to permeabilize the stratum corneum layer. The exact mode of interaction involved has been hypothesized to be due exclusively to the local elevation of the temperature in the SC layer, and the resultant melting of the lipid domains in the intercellular spaces between the corneocytes. Srinivasan et al. Other researchers have suggested that micro-cavitations and or shearing of the structures in the stratum corneum opens up channels through which fluids may flow more readily. In general, the design of the sonic systems for the enhancement of transdermal flux rates has been based on the early realization that the application of an existing therapeutic ultrasound unit designed to produce a “deep-heating” effect on the subject, when used in conjunction with a topical application of a gelled or liquid preparation containing the drug to be delivered into the body, could produce a quantifiable increase in the flux rate of the drug into the body. In the context of the method taught herein to create micropores in this biological membrane, the use of sonic energy may now be thought of in a totally new and different sense than the classically defined concepts of sonophoresis. 
     Based on the experimental discovery mentioned in U.S. Pat. Nos. 5,458,140 and 5,445,611 that when a small hole existed or was created in the stratum corneum (SC) in the Franz cells used in the in vitro studies, that the application of an appropriately driven ultrasonic transducer to the fluid reservoir on either side of the porated SC sample, an “acoustic streaming” event could be generated wherein large flux rates of fluid where capable of being pumped through this porated membrane. 
     With the method taught herein to create the controlled micro-porations in the biological membrane in the organism, the application of the fluid streaming mode of sonic/fluid interaction to the induction of fluid into or out of the organism may now be practically explored. For example, clinical studies have shown that by making a series of four 80 μm diameter micropores in a 400 μm square, and then applying a mild (10 to 12 inches of Hg) suction to this area, an average of about 1 μl of interstitial fluid can be induced to leave the body for external collection in an external chamber. By adding a small, low power sonic transducer to this system, configured such that it actively generates inwardly converging concentric circular pressure waves in the 2 to 6 mm of tissue surrounding the poration site, it has been demonstrated that this ISF flux rate can be increased by 50%. 
     By relieving ourselves of the desire to create some form of direct absorption of sonic energy in the skin tissues (as required to generate heating), frequencies of sonic energy can be determined for which the skin tissues are virtually transparent, that is at the very low frequency region of 1 kHz to 500 KHz. Even at some of the lowest frequencies tested, significant acoustic streaming effects could be observed by using a micro-scope to watch an in vivo test wherein the subject&#39;s skin was micro-porated and ISF was induced to exit the body an pool on the surface of the skin. Energizing the sonic transducer showed dramatic visual indications of the amount of acoustic streaming as small pieces of particulate matter were carried along with the ISF as it swirled about. Typical magnitude of motion exhibited can be described as follows: for a 3 mm diameter circular pool of ISF on the surface of the skin, a single visual particle could be seen to be completing roughly 3 complete orbits per second. This equates to a linear fluid velocity of more than 2.5 mm/second. All of this action was demonstrated with sonic power levels into the tissues of less than 100 mW/cm2. 
     While one can easily view the top surface of the skin, and the fluidic activity thereon, assessing what is taking place dynamically within the skin tissue layers in response to the coupling into these tissues of sonic energy is much more difficult. One can assume, that if such large fluid velocities (e.g. &gt;2.5 mm/S) may be so easily induced on the surface, then some noticeable increase in the fluid flow in the intercellular channels present in the viable dermal tissues could also be realized in response to this sonic energy input. Currently, an increase in harvested ISF through a given set of microporations when a low frequency sonic energy was applied to the area in a circle surrounding the poration sites has been quantified. In this experiment, an ISF harvesting technique based solely on a mild suction (10 to 12 inches of HG) was alternated with using the exact same apparatus, but with the sonic transducer engaged. Over a series of 10 two-minute harvesting periods, five with mere suction and five with both suction and sonic energy active, it was observed that by activating the sonic source roughly 50% more ISF was collectable in the same time period. These data are shown in  FIG. 30 . This increase in ISF flux rate was realized with no reported increase in sensation from the test subject due to the sonic energy. The apparatus used for this experiment is illustrated in  FIGS. 31-33 . The transducer assembly in  FIGS. 31-33  is comprised of a thick walled cylinder of piezo-electric material, with an internal diameter of roughly 8 mm and a wall thickness of 4 mm. The cylinder has been polarized such that when an electrical field is applied across the metallized surfaces of the outer diameter and inner diameter, the thickness of the wall of the cylinder expands or contracts in response to the field polarity. In practice, this configuration results in a device which rapidly squeezes the tissue which has been suctioned into the central hole, causing an inward radial acoustic streaming effect on those fluids present in these tissues. This inward acoustic streaming is responsible for bringing more ISF to the location of the micro-porations in the center of the hole, where it can leave the body for external collection. 
     A similar device shown in  FIG. 34A-B  was built and tested and produced similar initial results. In the  FIG. 34A-B  version, an ultrasonic transducer built by Zevex, Inc. Salt Lake City, Utah, was modified by having a spatulate extension added to the sonic horn. A 4 mm hole was placed in the 0.5 mm thick spatulate end of this extension. When activated, the principle motion is longitudinal along the length of the spatula, resulting in essentially a rapid back and forth motion. The physical perturbation of the metallic spatula caused by the placement of the 4 mm hole, results in a very active, but chaotic, large displacement behavior at this point. In use, the skin of the subject was suctioned up into this hole, and the sonic energy was then conducted into the skin in a fashion similar to that illustrated in  FIG. 33 . 
     The novel aspect of this new application of ultrasound lies in the following basic areas: 
     1. The function of the sonic energy is no longer needed to be focused on permeabilizing the SC barrier membrane as taught by Langer, Kost, Bommannan and others. 
     2. A much lower frequency system can be utilized which has very little absorption in the skin tissues, yet can still create the fluidic streaming phenomenon desired within the intercellular passageways between the epidermal cells which contain the interstitial fluid. 
     3. The mode of interaction with the tissues and fluids therein, is the so-called “streaming” mode, recognized in the sonic literature as a unique and different mode than the classical vibrational interactions capable of shearing cell membranes and accelerating the passive diffusion process. 
     By optimizing the geometric configuration, frequency, power and modulations applied to the sonic transducer, it has been shown that significant increases in the fluid flux through the porated skin sites can be achieved. The optimization of these parameters is designed to exploit the non-linearities governing the fluid flow relationships in this microscopically scaled environment. Using frequencies under 200 kHz, large fluidic effects can be observed, without any detectable heating or other negative tissue interactions. The sonic power levels required to produce these measurable effects are very low, with average power levels typically under 100 milliwatts/cm2. 
     Therefore, the above examples are but representative of systems which may be employed in the utilization of sonic energy or sonic energy and chemical enhancers in the collection and quantification of analytes for diagnostic purposes and for the transmembrane delivery of permeants. The invention is directed to the discovery that the poration of the biological membrane followed by the proper use of sonic energy, particularly when accompanied with the use of chemical enhancers, enables the noninvasive or minimally invasive transmembrane determination of analytes or delivery of permeants. However, the invention is not limited only to the specific illustrations. There are numerous poration techniques and enhancer systems, some of which may function better than another, for detection and withdrawn of certain analytes or delivery of permeants through the stratum corneum. However, within the guidelines presented herein, a certain amount of experimentation to obtain optimal poration, enhancers, or optimal time, intensity and frequency of applied sonic energy, as well as modulation of frequency, amplitude and phase of applied sonic energy can be readily carried out by those skilled in the art. Therefore, the invention is limited in scope only by the following claims and functional equivalents thereof. 
     Further Advancements and Improvements 
     Advancements and improvements to the microporation techniques have been made, particularly suitable for, though not limited to, delivery applications. One advancement is to porate, using any one of the aforementioned microporation techniques, to a selected depth into or through biological membranes, including the skin, the mucous membrane, or plant outer layer, particularly for delivery of a drug or bioactive agent into the body. Another advancement is to deliver bioactive agents into the organism through micropores formed in the biological membrane. Still another advancement is to apply permeation enhancement measures before, during, or after microporation, so as to increase the permeability of layers within the microporated skin or mucosa when delivering substances, such as drugs or bioactive agents, thereinto or therethrough. 
     The micropore formed in the biological membrane may extend to a selected depth. A micropore extending into the epidermis may penetrate only the stratum corneum or selected depths into the viable cell layer or underlying connective tissue layer. Similarly, if formed in the mucous membrane, the micropore may penetrate only the superficial part of the epithelial layer or selected depths into the epithelial lining or underlying lamina propria and into tissue beneath. The micropore depth in either case can extend through the entire depth of the biological membrane. 
     As an example for microporating to a selected depth, if one utilizes a heat probe which can continue to deliver sufficient energy into or through the fully hydrated viable cell layers beneath the stratum corneum, the poration process can continue into the body to selected depths, penetrating through the epidermis, the dermis, and into or through the subcutaneous layers below if desired. The concern when a system is designed to create a micropore extending some distance into or through the viable tissues in the epidermis or dermis, or the epithelial lining or lamina propria, is how to minimize damage to the adjacent tissue and the sensation to the subject during the poration process. 
     Experimentally, we have shown that if the heat probe used is a solid, electrically or optically heated element, with the active heated probe tip physically defined to be no more than a few hundred microns across and protruding up to a few millimeters from the supporting base, that a single pulse, or multiple pulses of current can deliver enough thermal energy into the tissue to allow the ablation to penetrate as deep as the physical design allows, that is, until the support base limits the extent of the penetration into or through the tissue. If the electrical and thermal properties of said heat probe, when it is in contact with the tissues, allow the energy pulse to modulate the temperature of said probe rapidly enough, this type of deep tissue poration can be accomplished with essentially no pain to the subject. Experiments have shown that if the required amount of thermal energy is delivered to the probe within less than roughly 20 milliseconds (20-50 msec), that the procedure is painless. Conversely, if the energy pulse must be extended beyond roughly 20 milliseconds (20-50 msec), the sensation to the subject increases rapidly and non-linearly as the pulse width is extended. 
     Similarly, an electrically heated probe design which supports this type of selected deep poration can be built by bending a 50 to 150 micron diameter tungsten wire into a sharp kink, forming a close to 180 degree bend with a minimal internal radius at this point. This miniature ‘V’ shaped piece of wire can then be mounted such that this ‘V’ extends some distance out from a support piece which has copper electrodes deposited upon it. The distance to which the wire extends out from the support will define the maximum penetration distance into the tissue when the wire is heated. Each end of the tungsten ‘V’ will be attached to one of the electrodes on the support carrier which in turn can be connected to the current pulsing circuit. When the current is delivered to the wire in an appropriately controlled fashion, the wire will rapidly heat up to the desired temperature to effect the thermal ablation process in a single pulse or in multiple pulses of current. By monitoring the dynamic impedance of the probe and knowing the coefficient of resistance versus temperature of the tungsten element, closed loop control of the temperature of the contact point can easily be established. Also, by dynamically monitoring the impedance through the body from the contact point of the probe and a second electrode placed some distance away, the depth of the pore can be determined based on the different impedance properties of the tissue as one penetrates deeper into the body. Once the impedance properties of a selected tissue of a selected organism have been routinely determined, this parameter can be used to determine the pore depth and can be used in a control system to control pore depth. 
     Likewise, one embodiment of an optically heated probe design which supports this type of selected depth poration can be built by taking an optical fiber and placing on one end a tip comprised of a solid cap or coating. A light source such as a laser diode will be coupled into the other end of the fiber. The side of tip facing the fiber must have a high enough absorption coefficient over the range of wavelengths emitted by the light source that when the photons reach the end of the fiber and strike this face, some of them will be absorbed and subsequently cause the tip to heat up. The specific design of this tip, fiber and source assembly may vary widely, however fibers with gross diameters of 50 to 1000 microns across are common place items today and sources emitting up to thousands of watts of optical energy are similarly common place. The tip forming the actual heat probe can be fabricated from a high melting point material, such as tungsten and attached to the fiber by machining it to allow the insertion of the fiber into a cylindrical bore at the fiber end. If the distal end of the tip has been fabricated to limit the thermal diffusion away from this tip and back up the supporting cylinder attaching the tip to the fiber within the time frame of the optical pulse widths used, the photons incident upon this tip will elevate the temperature rapidly on both the fiber side and the contact side which is placed against the tissues surface. The positioning of the fiber/tip assembly onto the tissue surface, can be accomplished with a simple mechanism designed to hold the tip against the surface under some spring tension such that as the tissue beneath it is ablated, the tip itself will advance into the tissue. This allows the thermal ablation process to continue into or through the tissue as far as one desires. An additional feature of this optically heated probe design is that by monitoring the black body radiated energy from the heated tip that is collected by the fiber, a very simple closed loop control of the tip temperature can be effected. Also, as described earlier, by dynamically monitoring the impedance through the body from the contact point of the probe and a second electrode placed some distance away, the depth of the pore can be estimated based on the different impedance properties of the tissue as one penetrates deeper into the body. The relationship between pulse width and sensation for this design is essentially the same as for the electrically heated probe described earlier. 
     For example, some vaccine applications are known to be most effective if delivered into the dermal layer so as to be in proximity to the Langerhan&#39;s or dendritic cells or other cells important for this immune response. This would imply a poration depth designed to pass through the epidermis, which in most cases would be roughly 180 microns to 250 microns deep. 
     As another example, when delivering some proteins and peptides, it is desirable to minimize the immune response to the permeant at the site of the administration and at the same time bypass the protease active zones in the skin tissues. In this case an even deeper pore may be desired, going as deep as 300 microns into the skin. 
     Alternatively, it may be desirable to leave a minimally thick layer of intact stratum corneum to minimize rapid initial uptake of a permeant and to provide some retention of the stratum corneum&#39;s barrier function to provide for a controlled release over a longer period of time. 
     An additional feature of this invention is the large increase in efficiency which can be gained by combining the poration of the layers of the biological membrane with other permeation enhancement techniques which can now be optimized to function on the various barriers to effect delivery of the desired compound into the internal spaces as necessary for bio-effectivity. In particular, if one is delivering a nucleic acid compound either naked, fragmented, encapsulated or coupled to another agent, it is often desired to get the nucleic acid into the living cells without killing the cell to allow the desired uptake and subsequent performance of the therapy. The application of electroporation, iontophoresis, magnetic fields and thermal and sonic energy can cause openings to form, temporarily, in the cell membranes and other internal tissues. Because we have shown how to breach the stratum corneum or epithelial layer of the mucosal membrane or the outer layer of a plant, and if desired the epidermis and dermis or deeper into a plant, electroporation, iontophoresis, magnetic fields and thermal and sonic energy can now be used with parameters that can be tailored to act selectively on these underlying tissue barriers and permeabilize the cell, capillary or other membranes within the targeted tissue. Electroporation, iontophoresis, magnetic fields, and thermal and sonic energy were previously inapplicable for this use. 
     In the case of electroporation, where pulses exceeding 50 to 150 volts are routinely used to electroporate the stratum corneum or outer layer of the mucosal membrane or outer layer of a plant, in the environment we present, pulses of only a few volts or less are sufficient to electroporate the cell, capillary or other membranes within the targeted tissue. This is principally due to the dramatic reduction in the number of insulating layers present between the electrodes once the skin, mucosal layer, or outer layer of a plant has been opened. 
     Similarly, iontophoresis can be shown to be effective to modulate the flux of a fluid media containing the nucleic acid through the micropores with very small amounts of current due to the dramatic reduction in the physical impedance to fluid flow through these porated layers. 
     In the case of sonic energy, whereas classically sonic energy has been used to accelerate the permeation of the stratum corneum or mucosal layer, by eliminating this barrier, sonic energy can now be used to permeabilize the cell, capillary or other membranes within the targeted tissue. As in the cases of electroporation and iontophoresis, we have demonstrated that the sonic energy levels needed to effect a notable improvement in the transmembrane flux of a substance are much lower than when skin or mucosal layers are left intact. Other permeation enhancement measures involve changing the osmotic pressure or physical pressure at the microporated site, for example applying a mild pneumatic pressure to the permeant reservoir to force a particular fluid flow into the organism through the micropores 
     The mode of operation of all of these active methods, electroporation, iontophoresis, magnetic or thermal or sonic energy, when applied solely or in combination, after the poration of the skin or mucosal layer or the outer layer of a plant has been effected, has the advantage of being able to use parameters typically used in in vitro applications where single cell membranes are opened up for the delivery of a substance. Examples of these parameters are well known in the literature. For example, Sambvrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989. 
     The micropores produced in the biological membrane by the methods of the present invention allow high flux rates of large (as well as small) molecular weight therapeutic compounds to be delivered transdermally or transmucosally or transmembrane. In addition, these non-traumatic microscopic openings into the body allow access to various analytes within the body, which can be assayed to determine their internal concentrations. 
     Delivery of Bioactive Agents 
     Still another advancement of the present invention involves the use of poration of the biological membrane for the delivery of a bioactive agent, e.g., polypeptides, including proteins and peptides (e.g., insulin); releasing factors; including LHRH; carbohydrates (e.g., heparin); nucleic acids; vaccines; and pharmacologically active agents such as antiinfectives such as antibiotics and antiviral agents; analgesics and analgesic combinations; anorexics; antihelminthics; antiarthritics; antiasthmatic agents; anticonvulsants; antidepressants; antidiabetic agents; antidiarrheals; antihistamines; antiinflammatory agents; antimigraine preparations; antinauseants; antineoplastics; antiparkinsonism drugs; antipruritics; antipsychotics; antipyretics; antispasmodics; anticholinergics; sympathomimetics; xanthine derivatives; cardiovascular preparations including potassium and calcium channel blockers, beta-blockers, alpha-blockers, and antiarrhythmics; antihypertensives; diuretics and antidiuretics; vasodilators including general coronary, peripheral and cerebral; central nervous system stimulants; vasoconstrictors; cough and cold preparations, including decongestants; hormones such as estradiol, testosterone, progesterone and other steroids and derivatives and analogs, including corticosteroids; hypnotics; immunosuppressives; muscle relaxants; parasympatholytics; psychostimulants; sedatives; and tranquilizers. By the method of the present invention, both ionized and nonionized drugs may be delivered, as can drugs of either high, medium or low molecular weight. 
     Delivery of DNA and/or RNA can be used to achieve expression of a polypeptide, stimulate an immune response, or to inhibit expression of a polypeptide through the use of an “antisense” nucleic acid, especially an antisense RNA. The term “polypeptide” is used herein without any particular intended size limitation, unless a particular size is otherwise stated, and includes peptides of any length including proteins. Typical of polypeptides that can be expressed are those selected from the group consisting of oxytocin, vasopressin, adrenocorticotrophic hormone, epidermal growth factor, prolactin, luteinizing hormone releasing hormone, growth hormone, growth hormone releasing factor, insulin-like growth factors, insulin, erythropoietin, obesity protein such as leptin, somatostatin, glucagon, glucagon-like insulinotropic factors, parathyroid hormone, interferon, gastrin, interleukin-2 and other interleukins and lymphokines, tetragastrin, pentagastrin, urogastroine, secretin, calcitonin, enkephalins, endorphins, angiotensins, renin, bradykinin, bacitracins, polymixins, colistins, tyrocidin, gramicidines, and synthetic analogues, modifications and pharmacologically active fragments thereof, monoclonal antibodies and vaccines. This group is not to be considered limiting; the only limitation to the peptide or protein drug that may be expressed is one of functionality. Delivery of DNA and/or RNA is useful in gene therapy, vaccination, and any therapeutic situation in which a nucleic acid or a polypeptide should be administered in vivo. E.g., U.S. Pat. No. 5,580,859, hereby incorporated by reference. 
     One illustrative embodiment of the invention is a method for obtaining long term administration of a polypeptide comprising porating the biological membrane and then delivering a DNA encoding the polypeptide through the pores in the biological membrane, whereby cells of the tissue take up the DNA and produce the polypeptide for at least one month, and more preferably at least 6 months. Another illustrative embodiment of the invention is a method for obtaining transitory expression of a polypeptide comprising porating the biological membrane and then delivering an RNA or DNA encoding the polypeptide through the pores of the biological membrane, whereby cells of the tissue (e.g., the skin, mucous membrane, capillaries, or underlying tissue) take up the RNA or DNA and produce the polypeptide for less than about 20 days, usually less than about 10 days, and often less than about 3-5 days. The cells which take up the RNA or DNA could include the cells of the biological membrane, the underlying tissue or other target tissue reached by way of the capillaries. 
     The DNA and/or RNA can be naked nucleic acid optionally in a carrier or vehicle, and/or can be contained within microspheres, liposomes and/or associated with transfection-facilitating proteins, microparticles, lipid complexes, viral particles, charged or neutral lipids, carbohydrates, calcium phosphate or other precipitating agents, and/or other substances for stabilizing the nucleic acid. The nucleic acid can be contained in a viral vector that either integrates into the chromosome or is nonintegrating, in a plasmid, or as a naked polynucleotide. The nucleic acid can encode a polypeptide, or alternatively can code for an antisense RNA, for example for inhibiting translation of a selected polypeptide in a cell. When the nucleic acid is DNA, it can be a DNA sequence that is itself non-replicating, but is inserted into a plasmid wherein the plasmid further comprises a replicator. The DNA may also contain a transcriptional promoter, such as the CMV IEP promoter, which is functional in humans. The DNA can also encode a polymerase for transcribing the DNA. In one preferred embodiment, the DNA codes for both a polypeptide and a polymerase for transcribing the DNA. The DNA can be delivered together with the polymerase or with mRNA coding therefor, which mRNA is translated in the cell. In this embodiment, the DNA is preferably a plasmid, and the polymerase is preferably a phage polymerase, such as the T7 polymerase, wherein the T7 polymerase gene should include a T7 promoter. 
     The method can be used to treat a disease associated with a deficiency or absence or mutation of a specific polypeptide. In accordance with another aspect of the invention, the method provides for immunizing an individual, wherein such individual can be a human or an animal, comprising delivering a DNA and/or RNA to the individual wherein the DNA and/or RNA codes for an immunogenic translation product that elicits an immune response against the immunogen. The method can be used to elicit a humoral immune response, a cellular immune response, or a mixture thereof. 
     Example 40 
     This illustrative example shows the preparation and delivery of an mRNA. 
     In general, it should be apparent that, in practicing the invention, a suitable plasmid for in vitro transcription of mRNA can be readily constructed by those of ordinary skill in the art with a virtually unlimited number of cDNAs. Such plasmids can advantageously comprise a promoter for a selected RNA polymerase, followed by a 5′ untranslated region, a 3′ untranslated region, and a template for a polyadenylate tract. There should be a unique restriction site between these 5′ and 3′ untranslated regions to facilitate the insertion of any selected cDNA into the plasmid. Then, after cloning the plasmid containing the selected gene, the plasmid is linearized by digestion in the polyadenylation region and is transcribed in vitro to form mRNA transcripts. These transcripts are preferably provided with a 5′ cap. Alternatively, a 5′ untranslated sequence such as EMC can be used, which does not require a 5′ cap. 
     The readily available SP6 cloning vector, pSP64T, provides 5′ and 3′ flanking regions from the  Xenopus -globin gene, an efficiently translated mRNA. Any cDNA containing an initiation codon can be introduced into this plasmid, and mRNA can be prepared from the resulting template DNA. This particular plasmid can be digested with BglII to insert any selected cDNA coding for a polypeptide of interest. Although good results can be obtained with pSP64T when linearized and then transcribed with SP6 RNA polymerase, it is preferable to use the  Xenopus -globin flanking sequences of pSP64T with the phage T7 RNA polymerase. This is accomplished by purifying an approximately 150 by HindIII/EcoRI fragment from pSP64T and inserting it into a linearized approximately 2.9 kb HindIII/EcoRI fragment of pIBI131 (commercially available from International Biotechnologies, Inc., New Haven, Conn.) with T4 ligase. The resulting plasmid, pXBG, is adapted to receive any gene of interest at a unique BglII site situated between the two  Xenopus -globin sequences and for transcription of the selected gene with T7 polymerase. 
     A convenient marker gene for demonstrating in vivo expression of exogenous polynucleotides is chloramphenicol acetyltransferase, CAT. The CAT gene from the small BamHI/HindIII fragment of pSV2-CAT (ATCC No. 37155) and the BglII-digested pXBG are both incubated with the Klenow fragment of  E. coli  DNA polymerase to generate blunt ends, and then are ligated with T4 DNA ligase to form pSP-CAT. This plasmid is then digested with PstI and HindIII and the small fragment, comprising the CAT gene between the 5′ and 3′-globin flanking sequences of pSP64T. The T7 promoter-containing plasmid pIBI131 is also digested with PstI and HindIII, and the long fragment is purified. This fragment is then ligated to the CAT gene containing fragment with T4 DNA ligase to form the plasmid pT7CAT-An. 
     The pT7CAT-An plasmid DNA is purified according to methods well known in the art, e.g. U.S. Pat. No. 5,580,859. The resulting purified plasmid DNA is then linearized downstream of the polyadenylate region with an excess of PstI, and the resulting linearized DNA is then purified and transcribed in vitro according to the method of Example 5 of U.S. Pat. No. 5,580,859. The resulting mRNA is then purified according to the method of Example 5 of U.S. Pat. No. 5,580,859, which is sufficiently pure for delivery according the present invention. 
     The purified mRNA is delivered by porating a selected site on an individual according to the microporation procedures with selected pore depth which optimizes bioactivity and delivering an effective amount of mRNA to such site such that the mRNA passes through the skin or mucous membrane into the underlying tissue, where the mRNA is taken up by the cells. This delivery through the porated stratum corneum or mucous membrane can be aided with sonic energy and/or use sonic energy according to the procedure of Example 15 and/or with electroporation to enhance cellular uptake, and/or with a pressure differential for inducing flux through the pores in the skin or mucous membrane. Moreover, delivery can be aided by placing the mRNA is a carrier solution, such as a positively charged lipid complex or liposome, for enhancing the diffusion of the mRNA through the pores into the body or for facilitating uptake of the mRNA into cells. 
     Example 41 
     This example shows immunization of an individual with mRNA encoding the gp120 protein of HIV. The mRNA is prepared according to the procedure of Example 40 except the gene for gp120 (pIIIenv3-1 from the AIDS Research and Reagent Program, National Institute of Allergy and Infectious Disease, Rockville, Md.) is inserted into the plasmid pXBG of Example 40. The mRNA containing the gp120 gene is delivered according to the procedure of Example 40. 
     Example 42 
     This example shows immunization of an individual with DNA encoding the gp120 protein of HIV. The gp120 gene is inserted into a recombinant adenovirus according to the procedure of P. Muzzin et al., Correction of Obesity and Diabetes in Genetically Obese Mice by Leptin Gene Therapy, 93 Proc. Nat&#39;l Acad. Sci. USA 14804-14808 (1996); G. Chen et al., Disappearance of Body Fat in Normal Rats Induced by Adenovirus-mediated Leptin Gene Therapy, 93 Proc. Nat&#39;l Acad. Sci. USA 14795-99 (1996), hereby incorporated by reference. The resulting DNA is delivered according to the procedure of Example 41. 
     Example 43 
     In this example, the procedure of Example 42 is followed except that DNA encoding glycoprotein D of HSV-2 is substituted for the DNA encoding gp120 protein and additionally is combined with an effective amount of the glycoprotein D. 
     Example 44 
     In this example, a nucleic acid encoding the obesity protein leptin, such as a human leptin or a rat leptin cDNA, C. Guoxun et al., Disappearance of Body Fat in Normal Rats Induced by Adenovirus-mediated Leptin, 93 Proc. Nat&#39;l Acad. Sci. USA 14795-99 (1996), or a mouse leptin cDNA, P. Muzzin et al., Correction of Obesity and Diabetes in Genetically Obese Mice by Leptin Gene Therapy, 93 Proc. Nat&#39;l Acad. Sci. USA 14804-14808 (1996), both of which are hereby incorporated by reference, is delivered in an appropriate plasmid vector. The mammalian expression vector, pEUK-C1 (Clontech, Palo Alto, Calif.) is designed for transient expression of cloned genes. This vector is a 4.9 kb plasmid comprising a pBR322 origin of replication and an ampicillin resistance marker for propagation in bacteria, and also comprising the SV40 origin of replication, SV40 late promoter, and SV40 late polyadenylation signal for replication and expression of a selected gene in a mammalian cell. Located between the SV40 late promoter and SV40 late polyadenylation signal is a multiple cloning site (MCS) of unique XhoI, XbaI, SmaI, SacI, and BamHI restriction sites. DNA fragments cloned into the MCS are transcribed into RNA from the SV40 late promoter and are translated from the first ATG codon in the cloned fragments. Transcripts of cloned DNA are spliced and polyadenylated using the SV40 VPI processing signals. The leptin gene is cloned into the MCS of pEUK-C1 using techniques well known in the art, e.g. J. Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., 1989), hereby incorporated by reference. The resulting plasmid is delivered to a human or animal individual after poration of the skin or mucosal membrane according to the procedure described above in the previous examples. 
     Example 45 
     Delivery of Heparin. Heparins are useful therapeutic substances wherein the maintenance of a basal level equivalent to an intravenous infusion of roughly 1000 to 5000 IU per hour, subcutaneous injections twice daily of 5000-1000 IU of heparin, or 1500-6000 IU of low molecular weight heparins is a typical clinical dosage. Normally, heparin would not be considered a good candidate for a transdermal delivery system because of its relatively high resistance to crossing the skin due mainly to the molecular weight, 5000 to 30000 Da, of the substance. With the microporation techniques disclosed herein, a significant flux rate of heparin was easily achieved when a sufficient quantity of heparin, such as from a delivery reservoir attached to the skin surface where the micropores were placed, was administered. A heparin solution was applied to skin porated to a depth of approximately 100 μm, allowing either passive diffusion or coupled with iontophoresis (about 1 mA/cm 2 ) that was applied for a sufficient period of time to transport the heparin through the micropores into the underlying tissues. Evidence of delivery of heparin was observed by increased capillary dilation and permeability as evidenced by microscopic examination of the in vivo site for both the passive and iontophoretically enhanced delivery. In addition to showing a significant heparin flux using passive diffusion as the main driving force, heparin, being a highly charged compound, is a natural candidate for the coupling of an electrical field with the micropores to allow for an actively controllable flux rate and higher flux rates than possible through the same number of micropores than is possible with the passive diffusion method. An experiment was conducted wherein a site on the volar forearm of a healthy male volunteer was prepared by creating a matrix of 36 micropores within a 1 square cm area. A small reservoir containing a sodium heparin solution and the negative electrode for an iontophoretic system was attached to the site. The positive electrode was attached to the subject&#39;s skin some distance away using a hydrogel electrode obtained from Iomed, a commercial supplier of iontophoretic systems. The system was run for ten minutes at 0.2 milliamperes per square cm. After this period, microscopic examination of the site showed direct evidence of the delivery of heparin from the vasodilation of the capillaries and when a suction force was applied to extract a sample of interstitial fluid from the micropores, enough red blood cells exited the capillaries under this force to tint the collected ISF pink, indicating increased vaso-permeability in the area. Furthermore, when placed aside to see if the red cells would clot, no clotting took place, indicating the anticlotting effect of the heparin present in the tissues at work. 
     Example 46 
     Delivery of Insulin: Insulin, like many compounds normally present in the healthy individual, is a polypeptide which must be maintained in individuals, such as diabetics who need exogenous insulin, at both a basal level and be given in a pulsatile bolus fashion in response to meals and the subject&#39;s activity levels. Currently this is achieved via subcutaneous injections of fast acting and slow acting formulations. Because of the molecular weight of insulin, typically ˜6000, it is not able to be delivered at clinically useful levels with traditional transdermal or transmucosal methods. However, by opening the micropores through the barrier layers of the skin or mucosa, a clear path is provided allowing the delivery of the insulin into the viable tissues wherein the interstitial fluid present in these tissues will allow diffusion (including osmotically driven) of the insulin to and into the lymph system and capillary bed, delivering clinically useful amounts. A concentrated insulin solution containing 3500 IU/ml of recombinant human insulin purchased from Boehringer-Mannhein Co., was applied in a reservoir to a porated area of the subject&#39;s skin on the volar forearm covering 4 square cm. The healthy, 44 year old, male, non-diabetic, subject fasted for 14 hours prior to the start of the experiment. Intravenous and finger stick blood samples were drawn periodically prior to and after the delivery phase began and assayed for glucose, insulin and C-peptide. The finger stick blood glucose data showed a significant and rapid depression of the subject&#39;s glucose levels after approximately 4 hours, dropping from 100 mg/dl at the start to 67 mg/dl over a ten minute cycle and then returning to 100 in an additional ten minutes, hypothesized to be due to the subject&#39;s counter-regulatory system engaging and compensating for the delivered insulin. A repeat of this procedure with the addition of ultrasound operating at 44 khz, and 0.2 watts/square cm indicated a more rapid delivery of the insulin as evidenced by the subject&#39;s glucose levels which dropped from 109 mg/dl to 78 mg/dl less than 30 minutes after the delivery began. As in the case of example 45, for heparin delivery, a low current iontophoretic system can be coupled with the micropores to facilitate a greater flux rate and provide the ability to modulate this flux rate by varying the current, allowing a delivery on demand type of system to be built. Previous work with insulin has typically shown that relatively high iontophoretic currents are required to overcome the strong barrier properties of the intact stratum corneum. By porating the stratum corneum or mucosa, and optionally setting the poration parameters to make a deeper pore into or through the targeted biological membrane, a lower current density is required to produce the desired insulin flux rates. 
     Similarly, for uncharged or lower-charged insulin formulations, an active flux enhancement through the micropores can be effected by coupling a sonic field or sonophoresis, which may include frequencies normally described as ultrasonic, to help push the insulin into the tissues. An additional feature of the sonic field is its ability to enhance the permeability of the various barriers within the viable tissues letting the insulin reach a larger volume of tissue over which the desired absorption into the blood stream can take place. Modulating the sonic energy has been shown to be very effective in modulating the total flux of a compound through the micropores into or through the deeper tissues, providing a second means of developing a bolus delivery system. 
     The exact pathways of absorption of insulin when given as a subcutaneous injection are still a subject of some debate. One of the reasons this is still unclear is the widely varying levels of bio-availability demonstrated within a population, or even the same subject, on an injection-by-injection basis. One hypothesized pathway is the direct absorption through the capillaries and into the blood stream. A method for enhancing this process is to couple electroporation with the surface poration, where the electroporation has been specifically optimized to work in the region of the capillary endothelial membranes, creating temporarily, a large number of openings to enhance this direct absorption. As with the iontophoresis and sonophoresis described previously, the total voltage amplitude levels of the electroporation system required to effect this type of electroporation within these tissue layers beneath the outer surface are often lower than needed to penetrate through an intact outer surface due to the reduction of the bulk impedance of the outer layer of the biological membrane. 
     Example 47 
     Delivery of microparticles: The use of liposomes, lipid complexes, microspheres including nanospheres, PEGellated compounds (compounds combined with polyethylene glycol) and other microparticles as part of a drug delivery system is well developed for many different specific applications. In particular, when dealing with a compound which is easily broken down by the endogenous components in the body&#39;s tissues such as protease, nuclease, or carbohydrase enzymes in the skin, tissues, the macrophages or other cells present in the blood stream or lymph, increases in bio-availability and/or sustained release can frequently be realized by utilizing one of these techniques. Currently, once one has applied one of these techniques, the formulation is generally delivered via some type of injection. The present invention, by creating micropores through a biological membrane (e.g., the skin or a mucous membrane) and into the body to a selected depth, allows this type of microparticle to be delivered through the skin or mucosa. As described in the insulin example above, microporation, electroporation, iontophoresis, sonic energy, enhancers, as well as mechanical stimulation of the site such as pressure or massage may be combined in any combination to enhance the delivery and/or uptake of a specific formulation. In the case of some engineered microparticles, the pores may have an optimal to depth designed to bypass certain biologically active zones or place the particle within the zone of choice. For some microparticle delivery systems, the energy incident upon the particles after they have been delivered into or through the tissues beneath the surface may be used to trigger the accelerated release of the active compound, thereby allowing the external control of the flux rate of the therapeutic substance. 
     Example 48 
     Microparticles for implantable analyte monitoring: Another application of microparticles is to deliver a particle not as a therapeutic agent but as a carrier of a probe compound which could be interrogated non-invasively, for example, via electro-magnetic radiation from an external reader system to obtain information regarding the levels of a specific analyte in the body. One example is to incorporate in a porous microsphere a glucose specific fluorophore compound which, depending on the levels of glucose present in the surrounding tissues, would alter its fluorescent response in either amplitude, wavelength, or fluorescent lifetime. If the fluorophore was designed to be active with an excitation wavelength ranging from 700 nm to 1500 nm, a low cost infrared light source such as an LED or laser diode could be used to stimulate its fluorescent response, which would similarly be in this range of from 700 nm to 1500 nm. At these wavelengths, the skin and mucosal tissues absorb very little and would therefore allow a simple system to be built along these lines. 
     Glucose is one candidate analyte, for which experimental lifetime fluorescence probes have been developed and incorporated into subcutaneously inserted polymer implants which have been successfully interrogated through the skin with optical stimulation and detection methods. It would merely require the reformulation of these experimental implants into suitably sized microparticles to allow the delivery into or through the viable tissue layers via the micropores. However, any analyte could be targeted, and the method of interrogating the delivered microparticles could be via magnetic or electric field rather than optical energy. 
     Example 49 
     Delivery of a Vaccine 
     A bacterial, viral, toxoid or mixed vaccine is prepared as a solid, liquid, suspension, or gel as required. This formulation could include any one or combination of peptides, proteins, carbohydrates, DNA, RNA, entire microorganisms, adjuvants, carriers and the like. A selected site of an individual is porated (skin or mucous membrane) according to the procedures described above in Example 45 and the vaccine is applied to the porated site. The depth of the micropores may depend on the type of vaccine delivered. This delivery can be aided with electroporation, iontophoresis, magnetic or sonic energy, enhancers, as well as mechanical stimulation of the site such as pressure or massage according to the procedures described above and/or use electroporation, iontophoresis, magnetic or sonic energy, enhancers, as well as mechanical stimulation of the site such as pressure or massage to enhance cellular uptake. Additional or reinforcing doses can be delivered in the same manner to achieve immunization of the individual. 
     Example 50 
     Delivery of Testosterone: A commercially available testosterone patch, the Androderm® patch from TheraTech, Inc., was used in a set of experiments to evaluate the benefits of microporation as it applies to the delivery of this permeant. A hypergonadic male subject went off Androderm therapy for two days, after which a series of venous blood samples were drawn during the subsequent 24 hour period to establish this subject&#39;s baseline levels of testosterone. Two 2.5 mg Androderm patches were then installed as recommended by the manufacturer and a similar set of venous blood sample were drawn to measure the testosterone levels when the only transdermal flux enhancement method being used was the chemical permeation enhancers contained in the patch. After two more days of a washout period, two 2.5 Androderm patches were then similarly installed, but prior to the installation, the skin surface at the target sites was porated with 72 micropores per site, each pore measuring approximately 80 μm in width and 300 μm in length and extending to a depth of 80 to 120 μm. For the porated delivery phase a similar set of venous blood sample were drawn to measure the testosterone. The data from all three of these twenty four hour periods is shown in the  FIG. 35  titled ‘Effects of Microporation on Transdermal Testosterone Delivery’. A noteworthy feature of these data is that when the microporations are present, the testosterone levels in the subjects blood elevate much more rapidly, essentially preceding the rising edge of the un-porated cycle by more than four hours. Looking at the slope of and area under the curve we can calculate that more than a three-fold flux rate enhancement took place due to the microporations during the first four hours. 
     Example 51 
     Delivery of Alprostadil: Alprostadil, or PGE1, is a prostaglandin used therapeutically to treat male erectile dysfunction via it&#39;s vasodilator behavior. The standard delivery mode for this drug is a direct injection into the base of the penis or via a suppository inserted into the urethra. A set of experiments were conducted with two healthy male volunteers. Each subject had a site of 1 square cm on the base of the penis shaft prepared by porating 12 to 36 micropores on this area, with the thermal poration parameters set to create pores roughly 100 microns deep as measured from the surface of the skin. A concentrated solution of alprostadil was placed in a small reservoir patch placed on the poration site, an ultrasonic transducer was then placed on the top of the reservoir and activated and the subject&#39;s erectile and other clinical responses were recorded on video tape. Both subjects developed a significant amount of engorgement of the penis, estimated as achieving 70% of more of a full erection at the dose applied. In addition, a malar flush response to the systemic levels of the drug delivered was observed. Over a 30 to 60 minute delivery period, both subjects developed a profound malar flush extending from the face, neck, chest and arms. Both the erectile response and the malar flush provide evidence of the delivery of a clinically active amount of the drug, a well know vasodilator. 
     Example 52 
     Delivery of Interferon: Interferons are proteins of approximately 17-22,000 molecular weight, that are administered clinically to treat a variety of disease states, such as viral infections (e.g., hepatitis B and C), immune diseases (such as multiple sclerosis), and cancers (e.g., hairy cell leukemia). Due to their protein nature, interferons must currently be administered by injection, as they cannot be given orally and are too large for traditional transdermal or transmucosal delivery methods. To demonstrate delivery of an interferon via the microporation technique, a 100 microliter aliquot of alpha-interferon solution containing interferon with a specific activity of 100 million international units of interferon per mg dissolved in 1 ml of delivery solution, is applied to a 1 square cm area of porated skin, porated to a depth of 150-180 μm, thus falling short of the capillary bed, on the thigh of a healthy human subject. Trials are run using either purely passive diffusion and with the application of sonic energy to the region at sufficient amplitude, frequency, and modulation thereof to accelerate the migration of the interferon through pores into or through the underlying tissues without causing deleterious heating of the interferon solution. Venous blood draws are taken at various time intervals for both trials, and are assayed for interferon levels using radio-immunoassay and bioassay. Interferon is detected in the serum over the 4 hour time period monitored. The interferon levels for the sonically enhanced delivery experiment are detected sooner than for the passive experiment. In another experiment, the interferon is administered in dry powder form directly to the micropores in the porated area of the skin. Interferon is detected in the serum using the same techniques as described above. In another test, the interferon solution is applied in a gel with or without a backing film to the porated tissue of the buccal mucosa. Venous blood is drawn and assayed for interferon levels. Interferon is detected in the serum over the 3 hour time period monitored. In another experiment, the interferon is incorporated into a tablet containing a bio-erodable matrix; with a mucoadherent polymer matrix that to provided contact of the tablet over the area of buccal mucosa that is porated. Interferon is detected in the serum using the same techniques as described above. 
     Example 53 
     Delivery of morphine: A solution of morphine is applied to a porated area on the volar forearm of the human subjects. A positive pressure gradient is used to provide a basal delivery rate of the morphine into the body, as determined by assay of venous blood draws at appropriate time intervals for the presence of morphine. A basal level of morphine of approximately 3-6 ng/ml is achieved. Upon demand, an additional pressure bolus is applied to result in a spike in the delivery of the morphine. The additional pressure bolus is achieved in one test by use of ultrasound; or in another experiment by the use of a pressure spike. This type of delivery, in which a basal level of the morphine is continuously applied, with spikes in morphine delivery periodically upon demand, is useful in treating chronic and breakthrough pain. 
     Example 54 
     Delivery of a disease resistant DNA into a plant: The seeds of a selected corn plant are microporated. The seeds are placed in a solution of a permeant formulation containing DNA that encodes disease resistance proteins. Sonic energy is used, optionally, to enhance the delivery of the DNA into the corn seeds. The seeds are germinated and grown to maturity. The resulting seeds of the mature corn plants now carry the disease resistant gene. 
     Example 55 
     Delivery of DNA into a plant: The seeds of a sugar beet are microporated. The seeds are placed in a solution of a permeant formulation containing DNA that encodes human growth hormone. Electroporation, iontophoresis, sonic energy, enhancers, as well as mechanical stimulation of the site such as pressure may be used to enhance the delivery of the DNA into the seeds. The seeds are germinated and grown to maturity. The resulting mature beet plant can now be harvested and the human growth hormone extracted for subsequent purification and clinical use. 
     Example 56 
     An experiment was conducted wherein fluorescent dextran particles, MW approximately 10,000 Daltons, were applied in an aqueous solution by means of a reservoir patch over a one square cm of skin on the volar forearm of a human subject where 36 micropores extending approximately 80 μm in depth were formed. The reservoir patch was left in place for 5 minutes. The porated site and surrounding area were imaged with a fluorescent video microscope to evaluate the penetration of the permeant into the tissue. The fluorescence showed that within 5 minutes significant permeation of dextran occurred more than 2 mm away from the nearest micropore. The video assay system used 10 minutes later showed further diffusion so that the fluorescent flush extended 10 mm from the pores. This experiment gives clear evidence that this technique allows delivery of permeants with molecular weights of 10,000.