Laser drilling technique for creating nanoscale holes

A method of forming extremely small pores in a substrate that is used, for example, in patch clamp applications is provided that employs an energy absorbing material beyond a back side of the substrate to allow a laser to be focused adjacent the exit side of the substrate so as to generate a pore through the substrate and can also form a crater in the back side of the substrate and in which the pore may propagate from the crater in a drilling direction that can oppose a laser transmission direction.

CROSS REFERENCE TO RELATED APPLICATION

BACKGROUND OF THE INVENTION

The present invention relates to electrophysiology and “patch clamping” for investigating ionic and molecular transport through cellular membranes via ion channels and, in particular, to a substrate providing a set of nano to microscale pores that may be readily sealed to cellular membranes. Ion channel investigation using patch clamps often plays an important role in drug discovery and preliminary drug screening or evaluation, for example, by providing a model that shows an effect of a drug on ion channels. Doing so can be useful for either avoiding adverse effects or for creating a positive therapeutic effect for the treatment of ion channel related diseases.

Drug screening can require a large number of ion channel measurements. Accordingly, in current practice, planar patch clamps are preferable because they allow parallelization of multiple samples on a substrate, often referred to as a wafer, chip, or well-plate, and facilitate measurement automation. Each sample has a cell or cell wall that is positioned so that an ion channel in the cell or cell wall is aligned with a pore at the sample site. The cell or cell wall is sealed to the patch clamp substrate in a manner that allows a small amount of electrical current to be used in performing ion channel investigations, typically by way of an extremely high resistance seal between the patch clamp substrate and the cell wall (a gigaohm seal or gigaseal). Gigaohm seals achieved using on-chip patch clamp procedures usually have electrical resistance values of about 1 gigaohm, with resistance values of up to about 5 gigaohms being achieved in some relatively rare instances.

Planar patch clamp substrates can be made from, for example, silicon, Teflon®, PDMS (polydimethylsiloxane), PSG (phosphosilicate glass), or glass. While such materials prove suitable for many planar patch clamp implementations, a single crystal quartz (quartz) material can be a particularly desirable for making planar patch clamp substrates. Quartz exhibits particularly high electrical insulating properties and is piezoelectric. Its unique electrical characteristics allow it to be used as a patch clamp substrate by providing very low levels of background noise while performing ion channel investigations. Furthermore, quartz exhibits particularly good mechanical characteristics such as, for example, good hardness, thermal stability, and chemical stability characteristics. Despite a general recognition of quartz's suitability for use as a patch clamp substrate, many of its desirable characteristics, such as hardness, make fabricating (micromachining) the pores in a quartz substrate rather difficult and/or time consuming.

Traditionally, micromachining of quartz is performed using a combination of lithography and reactive ion etching (RIE). However, RIE techniques require multiple steps and are relatively slow processes.

Another method of micromachining quartz is by way of direct laser beam ablation. During direct laser beam ablation, a high power density, short pulse width femtosecond laser beam is irradiated directly onto quartz. The nonlinear interaction between the ultrafast laser pulses and quartz, which has a band gap of about 9 eV, results in a cyclic multiphoton absorption and electron excitation between the ground and excited states. During this process, the initial excited electrons induce an avalanche ionization and generate a microplasma which ablates the quartz. However, since quartz has a wide band gap, this approach is also slow and is limited in terms of pore diameter and material thickness that can be achieved.

Recently, numerous advances have been made in micromachining of pores in non-quartz substrates, for example, by utilizing nanosecond lasers, such as excimer lasers instead of femtosecond lasers. Excimer lasers, which emit ultraviolet (UV) light, have been successfully implemented in relatively fast drilling procedures in non-quartz materials. However, quartz has excellent optical transmission over a large spectrum, from UV to infrared (IR), whereby it is transparent to light(s) in this spectrum. Since quartz is transparent to and therefore substantially unaffected by UV light(s), it has been widely accepted that excimer lasers are not usable for micromachining quartz.

Furthermore, although various patch clamping and other techniques have been developed and, at least to some extent, standardized for successfully modeling and investigating ion channel function voltage-sensitive (or voltage-gated) ion channels, in-depth investigation of yet other types of ion channels, such as mechanosensitive ion channels, remains at least somewhat frustrating and/or impracticable. Accordingly, numerous molecular mechanisms and their functionalities within mechanosensitive ion channels remain unknown, whereby cellular responses to mechanical stimuli remain some of the least understood of the known sensory mechanisms.

SUMMARY OF THE INVENTION

The present invention provides an improved technique for the generation of nanoscale-sized pores, for example, pores having diameters of nearly 200 nm, using a laser through a substrate that is substantially transparent to the laser's emitted wavelength and therefore tends to be unaffected by the laser using previously known practices. In the technique, the substrate is backed by an energy absorbing material that has relatively high coefficients of thermal expansion and UV absorption. The laser light is transmitted through the substrate and focused adjacent a back side of the substrate, for example, at or immediately in front of or behind an interface defined between the substrate and energy-absorbing material. Doing so can increase a temperature of the energy-absorbing material which, in turn, heat the substrate from beyond its back side. Heating the energy-absorbing material in this manner increases the temperature of the substrate to an extent that melts the substrate, and correspondingly facilitates melting-type formation of small diameter smooth pores and can also lead to formation of a crater(s) at the back side of the substrate. According to some aspects of the invention, the crater can be formed by a shock wave that results from thermal expansion of the energy absorbing material, but in any event, is formed by ablation or other thermal related removal of material from the back side of the substrate. The pore and/or crater are therefore created by a sandwich drilling or sandwich drilling-like technique that can give the pore and/or crater, of the quartz chip, surface characteristics that are similar to surface characteristics of pipettes that have been fire-polished with open flame procedures. In other words, the pore(s) and/or crater(s) formed according to aspects of the present invention have surfaces that are substantially smooth, their surface irregularities having been removed or attenuated by surface tension induced flowing of melted material in such irregularities which tends to smooth the same.

Specifically then, the present invention provides a method of creating nanoscale holes by using steps including creating a multi-layered assembly that comprises a substrate material and an energy absorbing material. The substrate material receives the nanoscale hole and defines a front side and an opposing back side. The energy absorbing material is adjacent the back side of the substrate material. A laser is applied through the multi-layered assembly, by initially passing through the substrate material and being focused at and absorbed by the energy absorbing material, increasing a temperature of the back side of the substrate material to a greater extent than an increase in temperature of the front side of the substrate material. Continued application of such laser stimulus to the energy absorbing material may correspondingly heat and/or pressurize the back side of the substrate, which can initiate a drilling of or establishing a pore through the substrate.

It is therefore an object of at least one embodiment of the invention to utilize a laser to drill a pore in a substrate by indirectly heating the substrate by focusing the laser at or near an interface between the substrate and an energy absorbing material. This allows for laser micromachining of a material that might otherwise be transparent to light of a wavelength emitted by the laser.

The method can further include a step of producing a crater at the back side of the substrate material which, in some embodiments, can be produced by a shock wave that increases both a temperature and a pressure at an interface defined between the back side of the substrate material and the energy absorbing material. The crater formation can be an initiating step for drilling of the pore and the pore, in some embodiments, is drilled in a drilling direction that opposes a direction of laser transmission through the substrate.

It is thus an object of at least one embodiment of the invention to provide a method for forming craters in back sides of substrates and/or drilling pores from the crater toward the front side of the substrate.

The substrate material may be a single crystal quartz material and the laser can be a UV emitting excimer laser or other lasers, e.g., CO2 lasers, and/or others.

It is therefore an object of at least one embodiment of the invention to generate pores and/or craters in a single crystal quartz wafer or chip and may be a further object of at least one embodiment of the invention to perform laser micromachining on a substrate that is substantially transparent to a wavelength of light that is emitted by the laser.

The energy absorbing material may be a liquid media, for example, an ultraviolet radiation absorbing organic liquid such as acetone and/or fluorescence immersion oil. Such liquid media can have a thermal expansion coefficient of at least about 700×10−6 K−1and an ultraviolet absorption coefficient of at least about 1.46 cm−1, for example, an ultraviolet absorption coefficient of between about 1.46 cm−1to 1.65 cm−1.

It is thus an object of at least one embodiment of the invention to provide a liquid medium for use as an energy absorbing material that can be placed under the substrate for performing a laser induce backside etching of the back side of the substrate. The backside etching may be performed on a quartz chip that has been pre-etched or thinned from an initially thick substrate. The initially thick substrate may be about 100-500 microns thick and can be either laser or wet etched, for example, with a buffered oxide etchant (BOE) or hydrofluoric (HF) etchant, in a pre-thinning procedure, down to about 20-50 microns in thickness before the nanopore laser drilling is initiated, which may improve suction and reduce series resistance.

The pore can have a smaller diameter than the crater which it intersects, and the pore diameter can be generally constant along its length or a major portion thereof, and a crater sidewall can include an undercut or groove extending thereinto.

It is thus an object of at least one embodiment of the invention to provide a patch clamp chip that has a smooth pore that opens into a relatively larger diameter crater. This configuration can provide suitable structure to which a cell can be mounted.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Referring now toFIG. 1, the present invention may use an ArF excimer laser10having collimating and focusing optics11to direct a narrow collimated beam12of light along an axis15toward a back surface of a sandwich-like, multi-layered assembly14that is held on a mechanical stage16. The laser may, for example, have a wavelength range of about 155 nm to about 195 nm and is preferably operated to emit a beam12of light having a wavelength of about 193 nm. In other embodiments, laser10may be yet other types of lasers, such as CO2 lasers, and/or others, depending on, for example, a desired wavelength that is selected based on particular characteristics of components within the multi-layered assembly14, in other words, how the multi-layered assembly14reacts to exposure to light having such wavelength(s), and/or other factors.

The laser10may include a variable attenuating mirror for controlling how much of the laser beam is to be transmitted, and a stencil metal mask with an adjustable aperture that allows production of different laser beam shapes and sizes. Laser10may further include a stage16that can be controlled by an automated controller18of the type well known in the art for providing control signals22to the laser10, controlling its output power in a series of pulses as will be described and providing control signals22to actuator motors24providing x-y control of the stage16.

Referring now toFIGS. 2 and 3, the multi-layered assembly14may include a substrate26, an underlying energy absorbing material34, and a backer plate32that supports the energy absorbing material34and substrate26. The energy absorbing material34cooperates with the substrate26to perform a one-step micromachining process in which the laser10drills through the substrate26, forming small diameter smooth holes therethrough, while causing a nominal amount of surface roughness to the substrate26. For example, a synergistic relationship between the energy absorbing material34and the substrate26allow for using the laser10to perform a one-step micromachining process to create holes or pores that have diameters as small as 200 nm, while resulting in an end-of-procedure surface roughness of merely tens of nanometers as measured from the respective surface(s) of the substrate26. Furthermore, submicrometer holes, bores, or pores may be formed in the substrate26along with crater-shaped depressions that are formed in a surface of the substrate that faces a direction of back from the substrate. Such relationships between the components of multi-layered assembly14, and techniques for forming such hole, pores, and craters in the one-step micromachining procedure, are discussed in greater detail elsewhere herein.

Still referring toFIGS. 2 and 3, within the multi-layered assembly14, substrate26can be, for example, a clear solid material that is transparent to the UV light emitted by laser10(FIG. 1), preferably a single crystal quartz chip or wafer. A suitable such single crystal quartz wafer for use as substrate26is the 350 μm thick quartz wafer that is readily available from the University Wafer Company of South Boston, Mass.

A front surface of the upper substrate26may have a series of depressions or wells28formed at regular x-y grid locations29. The wells28provide a thinned portion30at the locations29measured along axis15having a thickness of 100 to 1000 g and may be molded, ground, or etched in the substrate26. The diameter of the wells28may be relatively large, for example, 5.0 mm, and serve simply to permit a generally thicker substrate26in regions outside of the locations29for structural convenience. Referring specifically toFIG. 2, the substrate26can define a single, unitary structure with multiple wells28formed therein. However, it is also contemplated that substrate26can be an assembly of multiple, relatively smaller, individual quartz wafers or chips, each having only a single well28formed therein.

Referring yet further toFIGS. 2 and 3, in some embodiments, backer plate32is positioned as a lowermost component of the multi-layered assembly14and can serve as, for example, a structural base or part of a structural frame thereto. Backer plate32may be a supportive slide that is incorporated into the multi-layered assembly14, underlying and being spaced vertically below the substrate26. Optional spacer31is one suitable structure that can be used to establish such vertical spacing between the substrate26and backer plate32. The spacer31can be formed out of, for example, polydimethylsiloxane (PDMS). The PDMS may be cast on the rear surface of the substrate26through a mold produced using integrated circuit techniques to provide precisely controlled spacer thickness or may be spun-coated and selectively removed except at the edges of the substrate26. Regardless of the particular manner in which the PDMS is incorporated into the multi-layered assembly14, it is preferably configured to form a chamber defined by the PDMS at its outer perimeter and defining a space between the substrate26and backer plate32.

The space between the substrate26and the backer plate32is filled with energy absorbing material34which can be a liquid media and, in some embodiments, a UV absorbing organic liquid. The energy absorbing material34has thermal expansion and/or UV absorption coefficients that are large enough to heat and/or squeeze the substrate26to an extent that facilitates, catalyzes, or initiates laser drilling of the substrate26. The energy absorbing material34can have a thermal expansion coefficient of at least about 700×10−6K−1and a UV absorption coefficient of at least about 1.46 cm−1, preferably being within a range of between about 1.46 cm−1to 1.65 cm−1. Suitable UV absorbing organic liquids for use as energy absorbing material34include, for example, acetone and fluorescence immersion oil, along with other suitable materials that may increase temperature when exposed to UV radiation to an extent that may correspondingly heat the substrate material, interfacing therewith, so as to melt the substrate material. Examples of other suitable energy absorbing materials34include, but are not limited to, pyrene, naphthalene, and toluene, and/or other materials.

Still referring toFIGS. 2 and 3, the particular amount of volume of energy absorbing material34is selected based on the intended configuration of multi-layered assembly14, and laser drilling techniques that are implemented, and intended end-use configuration and characteristics of substrate26for its use in a patch clamping setup. In some embodiments, a relatively small amount of energy absorbing material34is used by sandwiching a thin layer of the energy absorbing material34between backer plate32and substrate26, without incorporating spacers31. In other embodiments, such as the one illustrated inFIG. 2, a relatively greater volume of energy absorbing material34is provided, with the particular volume being a function of the space between the backer plate32and substrate26as dictated by spacer31.

Regardless of whether the energy absorbing material34is implemented as a thin layer that is tightly sandwiched between the substrate26and a backer plate32substantially adjacent and below the substrate26, or implemented as a thicker layer that fills a larger space between the substrate26and backer plate32as dictated by spacer31, the energy absorbing material34and substrate26are preferably in a face-to-face or an abutting relationship with respect to each other. In such configuration, the substrate26and energy absorbing material34define an interface35therebetween. Interface35allows the energy absorbing material34to transmit heat and/or pressure to the substrate26with relatively little energy loss in so doing.

Referring now toFIGS. 1 and 4, the laser10may be positioned above a first location29and pulsed by the controller18to produce a series of controlled light pulses40of laser beam12that is focused at the interface35or substantially adjacent or proximate thereto. The light pulses40are directed toward a front side46, passing through an outer surface47thereof. Since substrate26is substantially transparent to UV light, the light pulses40pass through the entire thickness of the substrate26, leaving the substrate26through an outer surface49of a back side48of the substrate26. At this point, the substrate26is substantially unheated or otherwise affected by the light pulses40, at least compared to the temperature increase, thermal expansion, and/or other responses of the energy absorbing material to the stimulus of the light pulses40.

Still referring toFIGS. 1 and 4, the laser10indirectly heats the substrate26by primarily heating the energy absorbing material34which, in turn, secondarily heats the substrate26at its back side48or from below. In particular, the light pulses40that are focused at the interface35, heat the energy absorbing material34and therefore also the outer surface49of the back side48of substrate26to establish rapid increases temperature and pressure at the interface35. This correspondingly leads to rapid thermal expansion of the energy absorbing material34and/or exit side48of substrate26that can lead to an ablation of material from the exit side48or, in some embodiments, create a shock wave52at the interface35. In any event, a one-step emission of the laser10may be used for wholly fabricating a crater53and/or pore54in the substrate26, explained in greater detail elsewhere herein. The particular configuration and characteristics of the crater53and/or pore54are influenced by, amongst other things, the particular setup of the laser10and its output qualities, described in greater detail elsewhere herein, whereby the craters53and/or pores54are give desired sizes, shapes, and/or other characteristics by selecting a corresponding setup and output qualities of the laser10.

Referring now toFIGS. 5-8, shows an embodiment of crater53that can be a disc-shaped depression extending into the back side48of the substrate. Crater53has a relatively flat bottom wall102that can have a circular perimeter shape and extend generally parallel to the outer surface49of back side48. Seen best in the SEM (scanning electron microscope) image ofFIG. 8, crater53of this embodiment includes a sidewall105that may taper slightly inwardly toward the bottom wall102. An upper portion of sidewall105, in its orientation ofFIG. 8, transitions into the outer surface49of back side58by way of an arcuate surface that may have been at least partially smoothed during its formation. The smoothing is analogous to the smoothing achieved on an exponentially larger scale with open flame fire polishing, for example, while fire polishing a pipette or some other structure that is exponentially larger than the crater53and/or pore54.

Intuitively, overall dimensions of the crater53are functions of dimensions and characteristics of the sidewall105. Stated another way, crater53defines a crater depth which corresponds to a height dimension of the sidewall105. A crater width or diameter is defined by a (greatest) distance measured between facing surfaces of the sidewall105. As can be extrapolated from the size scale provided inFIG. 8, crater53may have a crater diameter of about 40 μm and a crater depth of about 10 μm.

In some embodiments, along at least part of the perimeter of crater53, an undercut110extends radially into the sidewall53, between the sidewall and the bottom wall102. Such undercut110may define a groove as an interlocking structure into which portions of a cell can flow under certain conditions, allowing parts of the cell's membrane to engage against, for example, a projecting shoulder defined between the undercut110and sidewall105.

Referring now toFIGS. 7 and 8, after completion of the laser micromachining of substrate26, a smoothed annular edge or shoulder can define an opening between the crater53and a pore54, such opening being labeled as a pore-crater opening153. Like various portions of the crater53, the pore-crater opening153can be fire polished and smoothed so as to eliminate substantially all surface irregularities. Seen best inFIG. 8, the pore-crater opening153maybe located in the middle of bottom wall102of the crater.

The pore54extends between the pore-crater opening153and a pore-surface opening147that opens into the pore54from the outer surface47of the front side46of substrate26. Accordingly, in embodiments of substrate26that include a crater53formed thereinto, pore54extends between the crater53and the front side46. In embodiments of substrate that do not include a crater53, the pore54extends between the front and exit sides46,48, in other words, through the entire thickness dimension of the substrate26. Regardless of the particular end-use configuration of substrate26, pore54has a substantially constant diameter along at least a major portion of its length. Accordingly, the pore-surface and pore-crater openings147,153may define opening diameters that are about the same size, for example with the larger of the two openings being no more than about 50% greater than the smaller of the two openings. As another example, the pore54can define minimum diameter and maximum diameter segments along its length, with the maximum diameter segment being no more than about 50% greater in magnitude than the smaller diameter segment.

Referring toFIGS. 4-8, a crater53and/or pore54may be formed into substrate26of the multi-layered stack14in the following way. The multi-layered stack14is assembled and the laser10is set up to based on intended characteristics of the crater53and/or54, such as pore diameter and/or others. As discussed above, light pulses40are focused away from the front side46of the substrate26and toward its interface35with the energy absorbing material34. Doing so causes a thermal expansion and also pressure increase of the affected material(s) within the multi-layered stack14, which may be largely the energy absorbing material34at this early stage. Correspondingly, the interface35and the back side48of the substrate26may be secondarily affected by the changes occurring within the energy absorbing material34. Such secondary affects may be an indirect heating characteristic of laser10, by way of the substrates'26intimate interaction with the energy absorbing material34.

For example, by focusing the light pulses proximate the interface35, increasing temperature and pressure of the energy absorbing material34can be transmitted to the substrate26, establishing a localized zone of increasing temperature and pressure of the back side48nearest the point of focus of the light pulses40. This may cause temperature and pressure differentials between the front and exit sides46,48of the substrate26but in any event will increase temperature and pressure at the back side48. When such values increase enough, a crater53and/or pore54can be established by way of this a one-step micromachining procedure.

Referring now toFIGS. 5-7, although the Applicant does not wish to be bound by a particular theory, it is contemplated that the formation of crater53may be an initiator of the drilling of pore54. In such embodiments, once crater53is formed, the near molten material of bottom wall102is more receptive to accepting energy from or, in other words, is less transparent to the UV from laser10than material in a room temperature at-rest substrate26. Accordingly, due to the preheating of bottom wall102during establishment of the crater53, the light pulses40are able to further ablate material at their point of interaction with the preheated bottom wall102, allowing the light pulses40to pierce therethrough and begin formation of the columnar pore54.

Referring now toFIGS. 6 and 7, while the emission of light pulses40continues, so does the ablation or melting away of more material at the particular location which the light pulses40pass through the material of the substrate (FIG. 6). In this regard, the pore54may propagate upwardly toward the front side46of the substrate so as to define a drilling direction “D” that opposes the direction of the light pulses40passing through the substrate26. In yet other embodiments, the pore54is not formed in the drilling direction “D” but instead, the drilling direction can extend in the same direction as the light pulses40passing through the substrate26, whereby the pore54drilling may originate at the outer surface47of the front side46. Although the terms “front” and “back” in describing various portions of the substrate26have been implemented in a convenient sense, it is fully contemplated that the by, for example, inverting the arrangement of the components of stack14, or positioning the laser10on the opposing side of the stack14, such terms might then assume generally opposite means. In other words, regardless of the particular orientation of the stack14and/or relative positions between the stack14and laser10, in preferred embodiments, the drilling direction “D” extends from the interface35between the energy absorbing and substrate materials34,26, toward the side or outer surface of the substrate26that opposes the energy absorbing material. That is, the drilling direction “D” typically extends away from the location where the shock wave52occurred, whether such direction is the same as or opposite to the direction of laser10emission.

Regardless of how the pore54is created in a particular embodiment, the pore characteristics such as, for example, pore diameter, may be controlled or manipulated at least to some extent by adjusting the set up and controls of the laser10. For some crater53and/or pore54formation procedures, the laser output power may be fixed at 5 W and a variable attenuator of the laser10can be set to allow about 70%, for example, 73% of total beam transmission, and operated for 2500 pulses at a 100 Hz repetition rate for the light pulses40. Yet other set ups are contemplated, again, based on the intended characteristics of the crater53and/or pore54that are being formed.

For example, Table 1 below illustrates how pore diameter can be influenced by changing transmission rate or repetition rate of the emitted light pulses40, from 50 Hz pulses to 100 Hz pulses during their emission from the laser10. To establish such data of Table 1, laser energy was fixed at 50 mJ and six drilling schemes (Recipes) were tested to show various combinations of the two transmission rates, and the corresponding influence on pore diameter.

TABLE IDrilling Recipes for Two Different TransmissionRates and Their ResultsPore DiameterPore Diameter50 Hz100 Hz(μm) at 83%(μm) at 87%RecipePulsesPulsesTransmission RateTransmission Rate1300000.216a3.841250020000.6214.1683100040000.3424.4554150030000.3954.5115200025001.5945.2706200040003.2105.421aNote:This approaches the laser wavelength limit of 193 nm.

Referring generally toFIGS. 8-11, the different configurations of these different embodiments show that, similar to the data in Table 1, influencing output characteristics of laser10may be used to form craters53and/or pores54that have different features, configurations, and/or other characteristics, as desired. Namely, using (relatively) higher transmission rates for laser10, for example, transmission rates of greater than about 80%, produces craters53with flat bottom walls102, like those seen inFIGS. 8-10. Using (relatively) lower transmission rates for laser10, for example, transmission rates that are less than about 80%, produces craters53that have tapering sidewalls and substantially no discernable bottom wall, like that seen inFIG. 11.

Specifically regarding the embodiments ofFIGS. 8-10, of the embodiments of craters53that have flat bottom walls102or semispherical configurations, crater depth and sidewall configuration can also be controlled by the transmission rate or power of laser10. Of the three different embodiments shown inFIGS. 8-10, the embodiment of crater53ofFIG. 10has the most shallow crater depth and also has a semicircular or concave-up, arcuate transition between the bottom wall102and the sidewall105. The crater53ofFIG. 10that was formed with a transmission rate of laser10of about 83%.

Again comparing the embodiments ofFIGS. 8-10, the crater53ofFIG. 9has a deeper crater depth than that ofFIG. 10but is shallower than that ofFIG. 8. The crater53ofFIG. 9has a similar semicircular or concave-up, configuration to that seen inFIG. 10, with a somewhat flatter bottom wall102when compared thereto. The crater53ofFIG. 9was formed with a transmission rate that is between 83% and 87% which was used in making craters53ofFIGS. 10 and 8, respectively. The crater ofFIG. 8has been previously discussed and, when compared to the craters53ofFIGS. 9 and 10, has the deepest crater depth and the flattest bottom wall102, deviating the most from a semicircular cross-sectional configuration. As discussed elsewhere in greater detail, the crater53ofFIG. 8includes an undercut110that is defined between the bottom and sidewalls102and105.

Referring now toFIG. 11, this embodiment of crater53is made by setting the laser10to a relatively low transmission rate, for example, a rate of about 74%. This sub-80% transmission rate forms a crater53that appears trumpet-like or arcuately tapering in cross-section, such that the sidewall105tapers conically down to where it connects to the pore54. The sidewall105can also, in some related embodiments, have scales or patterned discontinuities across surfaces thereof.

Referring now generally toFIGS. 12-13, regardless of the particular configuration of crater53and pore54, after the substrate26has been laser micromachined into a usable wafer or chip having a crater53and/or pore54, it can be implemented into a suitable investigative tool or instrument, depending on the particular intended end-use research or study that will be performed. It has already been shown that the craters53of the invention can provide gigaohm seals that are not only satisfactory in performance, but may be notably superior when compared to currently known techniques. For example, whereas known methods of glass chip production yield about 60% of produced units that can achieve 1.0-1.5 gigaohm seals, and a substantially lower percentage that can achieve about 5.0 gigaohm seals, preliminary mockup productions of glass chips using the inventive procedure(s) have already successfully produced a substantial percentage of produced units that have achieved 7.0 or greater gigaohm seals. In view of such promising preliminary results, it is fully contemplated and expected that the inventive methods disclosed herein are fully capable of producing chips that achieve or approach 15 gigaohm sealing capabilities.

Referring specifically now toFIG. 12, in some embodiments, the substrate26can be used in a planar patch clamp apparatus to investigate ion channel performance. As one example of such investigation, the substrate26, shown having the same orientation as seen inFIG. 8and therefore an inverted orientation with respect to that shown inFIG. 7, may receive a cell60within the crater53to expose a portion of the cell wall62to be accessible through the pore54. A light suction applied by a pump67from the front side46may adhere the cell wall62to the surface of crater53with a 5 to 30 gigaohm resistance between a solution64on the side of the substrate26holding the cell60and a solution66on the side of the substrate26opposite solution64. The application of suction may correspondingly also pull a portion of the cell wall62into the undercut110in a manner that enhances the seal of the cell60to the substrate26by way of the mechanical interlocking relationship therebetween. Although the cell60is shown inFIG. 12as having its membrane or cell wall62ruptured over the pore54, it is, of course, contemplated that the cell wall62remains intact for various other types of studies or investigations.

A sharp suction applied by a pump67at the outer surface47of the front side46or other means may be used to provide electrical connection to the interior of the cell60by a sensitive electrical detector70permitting measurement of electrical differences between the exterior and interior of the cell60through an electrode72communicating with the interior of the cell60referenced to solution64outside the cell60.

Referring now toFIG. 13, since preferred embodiments of substrate26are made from a single crystal quartz material, such embodiments may be incorporated into an apparatus to investigate mechanosensitive ion channel performance and function. The apparatus ofFIG. 13is largely analogous to that ofFIG. 12, only being configured to piezoelectrically actuate, stress, or otherwise stimulate the cell wall62so as to measure, by way of detector70, gating responses of the particular ion channel that cooperates with the pore54.

Still referring toFIG. 13, a bather200can be provided that acts as an enclosure, retaining the solution64therein. Ends of the substrate26extend through opposing sides of the barrier200. A power source210provides electrical stimulus for stimulating the piezoelectric behaviors of the substrate. A controller220sends and controls an electrical signal to the substrate, through conductors225that lead to the substrate. The actual connection(s) of the conductors225to the substrate26can be accomplished with suitable terminals. For example, terminals230and235are attached to the opposing outer surfaces46and47at a first end of the substrate26, appearing as a left end inFIG. 13, and connected to the controller220by a first pair of conductors225. Terminals240and245are attached to the opposing outer surfaces46and47at a second end of the substrate26, appearing as a right end inFIG. 13, and connected to the controller220by a second pair of conductors225.

Referring yet further toFIG. 13, in such an embodiment, the controller220may place the cell wall62under compressive and/or tensile stresses along multiple axes of movement or actuation. Depending on the particular cut of the crystal, controller220may establish a voltage across the thickness of the substrate26, specifically by establishing a voltage between the upper terminals235,245and the lower terminals230,240. Depending on the polarity of the signal, doing so will cause the substrate26to compress or elongate with respect to its thickness dimension which correspondingly compresses or stretches the cell wall62in such direction. Here too, depending on the particular cut of the crystal, controller220may establish a voltage across the length of the substrate26, specifically by establishing a voltage between the left end terminals230,235and the right end terminals240,245. Again depending on the polarity of the signal, doing so will cause the substrate26to compress or elongate, only this time with respect to its length dimension, compressing or stretching the cell wall62in a corresponding manner. While doing so, the detector70, senses notable gating and/or other responses of the particular ion channel or other portions of the cell60, depending on the particular configuration of detector70.

Referring yet further toFIG. 13, regardless of the particular configuration of the cell membrane investigating apparatus and its setup and controls, the substrate26according to the invention provides an “on-chip” piezoelectric system that may be used as a suitable alternative for imposing mechanical deformations to a membrane which to date has been primarily studied with pipettes and conventional patch clamping. Substrate26can be configured and implemented specifically for ones of, e.g., (i) lower frequency actuation of the ion channels, (ii) higher frequency mechanical probing, as well as (iii) static stress modulation of the membrane, as desired based on the particular investigation being performed.