Apparatus and method for the collection of analytes on a dermal patch

The invention is an improved method and apparatus for collecting analytes on a dermal patch, where the patch controls the ionization state of the analyte. This can be accomplished by delivering electricity to the patch or by a buffer or other means for controlling the pH of fluids entering the patch. Analytes in perspiration can be concentrated on the patch without the occurrence of significant back-diffusion of the analytes.

FIELD OF THE INVENTION 
The present invention relates to an improved method and apparatus for 
collecting analytes on a dermal patch. 
BACKGROUND OF THE INVENTION 
A. Chemical Analysis of Body Fluids 
The determination of a patient's physiological status is frequently 
assisted by a chemical analysis of a body fluid of that patient. Such an 
analysis normally determines the existence and/or concentration of a 
chemical species in the body fluid of the patient as an indication of that 
patient's condition. Increasingly, such analyses are being used to 
determine the presence of a drug of addiction or a metabolite of such a 
drug in the body fluid of an individual. 
Many analytes of interest can be detected in urine, which is readily 
available from a subject and can be collected non-invasively. For these 
reasons, the primary method for detecting drugs of abuse today is urine 
analysis. 
Blood, however, is also frequently analyzed for the presence of drugs of 
addiction as well as a wide variety of other analytes. Blood collection 
is, however, inherently invasive, and carries the risk of infection 
associated with any invasive procedure. Blood testing must also be 
conducted at a physician's office or at another facility equipped to 
analyze blood, which reduces the convenience of blood tests and increases 
their cost. In addition, testing a blood sample can only reveal 
information about chemicals or metabolites that are present in the blood 
of the subject at the time the sample is taken, and cannot detect the 
presence of such analytes over a period of time. 
Perspiration can also be collected in order to analyze a chemical species 
present in the body. The non-invasive manner in which it can be collected 
renders perspiration suitable for use outside of a physician's office. In 
addition, a variety of molecules which are expressed in perspiration can 
be analyzed. 
B. Diagnostic Kits for Collecting Perspiration 
A variety of diagnostic kits for monitoring an analyte in sweat have been 
developed. For example, U.S. Pat. No. 3,552,929 to Fields, et al. 
discloses a band-aid-type test patch suited for determining the chloride 
ion concentration in perspiration as a method of diagnosing cystic 
fibrosis. The apparatus disclosed in Fields comprises an absorptive sweat 
collecting pad with an impermeable overlying layer for the purpose of 
preventing evaporation. When the absorptive pad is saturated, the patch is 
removed from the skin and exposed to a series of strips impregnated with 
incremental quantities of silver chromate or silver nitrate, the color of 
which undergoes a well known change upon conversion to the chloride salt. 
U.S. Pat. No. 4,706,676 to Peck discloses a dermal collection device which 
comprises a binder to prevent reverse migration of an analyte, a liquid 
transfer medium which permits transfer of an analyte from the dermal 
surface to the binder, and an occlusive cover across the top of the liquid 
transfer medium and binder. Peck also discloses the application of such a 
dermal collection patch to detect various environmental chemicals to which 
humans are exposed. After the dermal collection device has been worn on a 
patient's skin for a period of time, the patch is removed for analysis, 
which involves the chemical separation of the bound substance of interest 
from the binding reservoir and thereafter undertaking qualitative and/or 
quantitative measurement of the substance of interest by conventional 
laboratory techniques. 
Another quantitative sweat collection patch is disclosed in U.S. Pat. No. 
4,756,314 to Eckenhoff. This patch uses a diffusion rate-limited membrane 
as a means to maintain a constant flow of fluid into the patch. The patch 
comprises an impermeable outer boundary structure, and is therefore an 
occlusive patch. 
However, prior art diagnostic test patches are generally only useful for 
determining the presence of analytes which are present in sweat in 
relatively high concentrations, such as halide ions. In addition, the 
occlusive outer layer-type devices of the prior art are susceptible to the 
problem of back diffusion of perspiration and/or the analytes contained 
therein, including changes in the skin's transport characteristics, both 
outward (Brebner, D. F., J. Physiol., 175:295-302 (1964)) and inward 
(Feldmann, R. J., Arch. Dermat., 91:61-666(1965)). The maintenance of this 
aqueous state also fosters bacterial colonization. Thus, there remains a 
need in many diverse applications for an improved method and apparatus for 
the non-invasive determination of the presence or concentration of an 
analyte in a body fluid such as perspiration. 
SUMMARY OF THE INVENTION 
In one aspect, the present invention comprises a method of collecting and 
detecting an analyte contained in the perspiration of a subject mammal 
while minimizing the back-diffusion of the analyte into that subject 
mammal. In this method, the analyte preferably has a pK.sub.a within the 
range of about 7.2 to 10.0 and has a plurality of ionization states, 
wherein the analyte exists in an ionized form in at least one such 
ionization state and in a nonionized form in another such ionization 
state. This method comprises the steps of: 
a. placing a dermal patch on the outer surface of the skin of the subject 
mammal, wherein the patch comprises an absorbent material capable of 
containing perspiration of the mammal; 
b. passing perspiration through the skin of the mammal into the absorbent 
material, thereby passing the analyte into the patch, if the analyte is 
present in the mammal's perspiration; 
c. controlling the ionization state of the analyte in the patch so that the 
ratio of the amount of the analyte in the patch in the ionized form to the 
amount of the analyte therein in the nonionized form is greater than 1000; 
and thereafter 
d. detecting the analyte in the patch. 
In one embodiment of the foregoing method, the controlling step comprises 
controlling the pH of the outer surface of the skin of the subject 
underneath the patch so that the pH of the outer surface of the subject's 
skin is maintained within a selected range, the range being calculated to 
at least substantially prevent the back-diffusion of the analyte from the 
patch. Preferably, the pH of the outer surface of the skin of the subject 
underneath the patch is maintained below about 7.0, and more preferably 
below about 5.0. In this embodiment, the controlling step can comprise 
providing a buffer in fluid contact with the outer surface of the skin of 
the subject, the buffer being designed to maintain the pH of the surface 
of the skin of the subject within the selected range. The controlling step 
can also, in an alternative embodiment, comprise the delivery of 
electricity to the patch or to the surface of the subject mammal's skin. 
Preferably, the controlling step comprises producing a ratio of the amount 
of the ionized form of the analyte in the patch to the amount of the 
nonionized form of the analyte in the patch that is greater than 5000. 
Other embodiments of this aspect of the present invention are also 
contemplated. In one alternative embodiment of the foregoing method, water 
is permitted to escape from the patch, thereby concentrating the analyte 
in the patch. In another embodiment, the method can additionally comprise 
the step of determining the amount of the analyte present in the patch. In 
yet another embodiment of this method, water is prevented from escaping 
from the patch during the passing step. 
Another aspect of the present invention comprises a dermal patch to be worn 
on the skin of a subject mammal for detecting an analyte in the subject's 
perspiration, wherein the analyte has a pK.sub.a within the range of about 
7.2 to 10.0 and can have a plurality of ionization states, including at 
least one ionization state where the analyte is in an ionized form and at 
least one ionization state where the analyte is in a nonionized form. A 
patch according to this aspect of the present invention comprises: 
an absorbent material in fluid communication with the outer surface of the 
skin of the subject for collecting perspiration which passes through the 
skin and into the patch; and 
means for controlling the ionization state of the analyte such that the 
ratio of the analyte in the ionized form to the nonionized form thereof 
within the patch is greater than 1000. 
In one embodiment, the means for controlling the ionization state in the 
patch comprises a means for controlling the pH of the outer surface of the 
skin of the subject beneath the absorbent material of the patch so that 
the pH is maintained within a selected range, the range being calculated 
to cause the analyte to concentrate on the patch and not back-diffuse. In 
this embodiment, the means for controlling the pH of the outer surface of 
the skin of the subject comprises a buffer in fluid contact with the 
surface of the skin of the subject. Alternatively, the means for 
controlling the ionization state can comprise a means for delivering 
electricity to the outer surface of the skin of the subject beneath the 
absorbent material of the patch. Such a means for delivering electricity 
can comprise, for example, an iontophoresis device. 
In this aspect of the present invention, the patch can be configured to 
permit water to escape from the patch, so as to concentrate the analyte in 
the patch. Alternatively, the patch can be configured to prevent water 
from escaping from the patch. 
Another aspect of the present invention consists of a dermal patch to be 
worn on the skin of a subject mammal for detecting an analyte in the 
subject's perspiration, wherein the patch comprises: 
an absorbent material in fluid communication with the outer surface of the 
skin of the subject for collecting perspiration which passes through the 
skin and into the patch; and 
a buffer in the absorbent material, wherein the buffer is capable of 
maintaining a pH within a selected range, the range being calculated to 
cause the analyte to concentrate in the patch. 
In yet another aspect, the present invention comprises a dermal patch to be 
worn on the skin of a subject mammal for detecting an analyte in the 
subject's perspiration, wherein the patch comprises: 
an absorbent material in fluid communication with the outer surface of the 
skin of the subject for collecting perspiration which passes through the 
skin and into the patch; and 
a source of electricity that delivers electricity to the patch or to the 
outer surface of the skin adjacent the patch. 
A further aspect of the present invention comprises a method of collecting 
and detecting an analyte contained in the interstitial fluid of a subject 
mammal while minimizing the back-diffusion of the analyte into the subject 
mammal, wherein this method comprises the steps of: 
a. placing a dermal patch on the outer surface of the skin of the mammal, 
the patch comprising an absorbent material capable of containing 
perspiration of the mammal; 
b. passing perspiration through the skin of the subject mammal into the 
absorbent material, thereby passing the analyte into the patch, if the 
analyte is present in perspiration of the subject mammal, wherein the 
ratio of the amount of the analyte obtained in the patch to the amount of 
the analyte in the interstitial fluid of the subject mammal is greater 
than 10; and thereafter 
c. detecting the analyte in the patch. 
In a preferred embodiment of this method, the ratio of the amount of the 
analyte obtained in the patch to the amount of the analyte in the 
interstitial fluid of the subject mammal is greater than 100. In another 
embodiment, water is prevented from escaping from the patch during the 
passing step. 
Another aspect of the present invention comprises a dermal patch to be worn 
on the skin of a subject mammal for detecting an analyte in the subject's 
interstitial fluid, wherein the patch comprises: 
an absorbent material in fluid communication with the outer surface of the 
skin of the subject for collecting perspiration which passes through the 
skin and into the patch; and 
means for obtaining a ratio of the amount of the analyte in the patch to 
the amount of the analyte in the interstitial fluid of the subject of 
greater than 10. 
In a preferred embodiment of this aspect, the means for obtaining comprises 
means for obtaining a ratio of the amount of the analyte in the patch to 
the amount of the analyte in the interstitial fluid of greater than 100. 
In one embodiment, the means for obtaining can comprise means for 
controlling the pH of the outer surface of the skin of the subject beneath 
the absorbent material of the patch so that the pH is maintained within a 
selected range, the range being calculated to cause the analyte to 
concentrate on the patch and not back-diffuse. Such a means for 
controlling the pH of the outer surface of the skin of the subject can 
comprise, for example, a buffer in fluid contact with the surface of the 
skin of the subject. In a further embodiment, the means for obtaining 
comprises means for delivering electricity to the outer surface of the 
skin of the subject beneath the absorbent material of the patch. In this 
embodiment, the means for delivering electricity can comprise an 
iontophoresis device. 
In another embodiment of this aspect of the present invention, the patch is 
configured to permit water to escape from the patch, so as to concentrate 
the analyte in the patch. Alternatively, the patch can be configured to 
prevent water from escaping from the patch.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
I. Dermal Patches for Detecting Analytes 
A. Non-Occlusive Dermal Patches 
Referring to FIG. 1, there is disclosed a dermal patch 10 according to one 
embodiment of the present invention, illustrated as being secured to the 
surface of the skin 12 of a subject. As will be appreciated by one of 
skill in the art, the patch of the present invention may be used for 
veterinary purposes as well as on humans. In addition, the patch can be 
used in more diverse applications such as in agriculture or any other 
environment where a chemical species is to be detected in a fluid. The 
preferred use, however, is for determination of preselected chemical 
species or analyte in sweat (perspiration), and the ensuing discussion is 
principally directed to that use. 
Moisture expressed from the skin 12 within the perimeter of the test patch 
10 first accumulates in a concentration zone 14 beneath the first side of 
a gas permeable filter or layer 16 which is in fluid communication with 
the skin 12. The concentration zone 14 preferably contains an absorbent 
material, such as a fluid permeable medium 20 which may be cotton gauze or 
other commonly available fluid permeable material. For example, a layer of 
any of a variety of known fiber webs such as knitted fabrics, or non-woven 
rayon or cellulose fibers may be used. Filtration Sciences #39 is a 
particularly preferred fluid-permeable medium for use as a concentration 
zone in the present invention. In a preferred embodiment, the absorbent 
material contains binders, such as antibodies, for specifically binding 
analytes of interest to the absorbent material of the patch. As used 
herein, the term "absorbent material" designates any fluid permeable 
material capable of collecting or holding analytes contained in 
perspiration. Preferably, such a material is also able to concentrate such 
analytes on the patch. 
The term "fluid permeable" is used herein to describe a material which will 
permit the passage of the liquid phase of fluids expressed from the skin 
and which will also allow the passage of the vapor phase of such fluids. A 
fluid permeable filter or layer will thus allow the passage of water in 
both the liquid and vapor phases. "Water" is used herein to denote both 
the liquid and vapor phases of water unless reference is specifically made 
to a particular phase. 
Moisture from perspiration accumulates in the interfiber spaces of the 
medium 20. Under the influence of body heat which is readily conducted 
from the surface of the skin through the liquid phase, the liquid water 
component of the perspiration will tend to volatilize. Such volatilized 
water can thereby pass through the gas permeable filter or layer 16, which 
is located on the side of the medium 20 distal of the skin 12, and leave 
the patch 10. 
As previously discussed, the patch 10 is provided with a gas permeable 
filter 16. The term "gas permeable" is used to describe a material which 
permits the passage of gases, including the vapor phase of fluids 
expressed from the skin, but substantially retains the fluid phase within 
the patch. Any of a variety of suitable commercially available 
microfiltration membrane filters may be used for this purpose, such as the 
Gore-Tex 0.45 micron Teflon filter manufactured by W. L. Gore & 
Associates, Inc. (Elkton, Md.). 
Adjacent a second side of the gas permeable filter 16 is a discharge zone 
18. As previously discussed, the gas permeable filter 16 retains the fluid 
phase but permits escape of the vapor phase of the fluid component in 
perspiration. Thus, the vapor component, which primarily consists of 
vaporized water, continuously escapes through the gas permeable filter 16 
exiting the second side thereof into discharge zone 18, which is in 
communication with the atmosphere. In an alternative embodiment, not 
separately illustrated, the gas permeable filter 16 is replaced by a fluid 
permeable membrane which permits passage of the liquid phase. In this 
embodiment, liquid, or a combination of vapor and liquid, will be 
permitted to escape from the patch. Any of a variety of fluid permeable 
filters are commercially available which can be used to form a fluid 
permeable filter used in this embodiment of the present invention. A 
preferred fluid permeable filter is constructed from James River Paper 
Drape. 
A flexible, gas permeable outer layer 22 is preferably disposed adjacent 
the second side of filter 16 in the discharge zone 18. This layer serves 
to protect the filter 16 against physical damage such as abrasion, and can 
also serve as a barrier for preventing chemical contamination of the 
filter material from the outside. Outer layer 22 may comprise any of a 
variety of commercially available gas permeable tapes and films which are 
known to one of skill in the art. Outer layer 22 may also be distinct from 
or integral with tape 26, discussed below. Alternatively, depending upon 
the intended application of the patch, outer layer 22 may be deleted 
altogether, where it does not appear that abrasion or external 
contamination will deleteriously affect the patch 10, or where the gas 
permeable layer 16 is made from a material which is itself resistant to 
abrasion and/or external contamination, thus obviating the need for the 
outer layer 22. 
The patch 10 illustrated in FIG. 1 is secured to the surface of the skin by 
means of a peripheral band of tape 26. Preferably, the tape 26 will extend 
around all sides of the patch 10. For example, an annular ring of tape can 
be die punched for use with a circular patch, or the center of a 
rectangular piece of tape can be removed to expose outer layer 22 or 
filter 16 of a rectangular patch (see FIGS. 1 and 3, respectively). 
Alternatively, outer layer 22 and tape 26 can be deleted altogether and 
layers 16 and 20 can be secured to the surface of the skin by a bandage or 
through the use of an adhesive. One such method would be to capture layers 
16 and 20 under a bandage or wrapping surrounding the arm or the leg. In 
this case, the gases and/or fluids are permitted to escape through layers 
16 and 20 and into the bandage, where they may either collect or from 
which they are dissipated into the environment. 
A large variety of hypoallergenic or other suitable tapes are well known in 
the art, which may be adapted for use with the patch 10 of the present 
invention. Different tapes or adhesives may be desirable depending upon 
the intended use of the test kit, based upon their ability to adhere to 
the skin during different conditions such as daytime wearing under 
clothing, during sleep, during profuse sweating for prolonged periods or 
during showers. It has been determined that the most desirable tapes 
include multiple perforations which prevent sweat from building up 
underneath the tape and eventually compromising the integrity of the 
adhesive. Preferably, a tape, such as Dermiclear marketed by Johnson & 
Johnson, is used. More preferably, the tape comprises a layer of 3M 1625 
Tegaderm wound dressing available from the 3M Company (St. Paul, Minn.). 
Any of a wide variety of means for securing the patch 10 to the skin 12 may 
be utilized. For example, the tape 26 can be eliminated and gauze layer 20 
provided with a lower adhesive layer to perform the same function. One 
such adhesive membrane is the MN-100 adhesive membrane manufactured by 
Memtec of Minnetonka, Minn. This membrane is fluid permeable so that it 
passes fluid as would the gauze layer 20, yet has one adhesive side so 
that it will stick to the skin. Alternatively, outer protective layer 22 
can comprise an annular flange 23, extending radially outwardly beyond the 
outer edges of filter 16 and gauze 20 (see FIG. 2a). The lower surface of 
the flange 23 is then provided with a suitable adhesive. 
The surface temperature of human skin varies regionally. However, it is 
generally within the range of from about 86.degree. F. to about 90.degree. 
F. at rest, and can rise to much higher temperatures under conditions of 
strenuous exertion. At those temperatures, a number of chemical species of 
interest for the purpose of the present invention, such as creatine kinase 
or a high or low density lipoprotein, have a sufficiently low vapor 
pressure that volatilization is not a significant factor in the efficiency 
of the concentration function. At the same time, the substantial aqueous 
component will have a sufficiently high vapor pressure that it will tend 
to volatilize, thereby concentrating the less volatile fractions. However, 
in some applications the chemical species of interest will have a high 
enough vapor pressure, even at the resting temperature of human skin or 
the temperature of another surface to which a patch of the present 
invention is applied, such that escape of the vapor phase through the gas 
permeable filter 16 of the analyte of interest will disadvantageously 
impair the efficacy of the test patch. For these analytes, a modified 
patch must be used. 
B. Dermal Patches for Detecting Volatile Analytes 
Referring to FIGS. 2 and 2a, there is disclosed a modified patch 11 
according to the present invention for use in detecting an analyte having 
a propensity to escape through the gas permeable filter 16 as a vapor 
under ordinary use conditions. The test patch 11 comprises a concentration 
zone 14 defined on its inner boundary by the skin 12 to which the patch 11 
is secured. The outer boundary of the concentration zone 14 is defined by 
the gas permeable filter or layer 16, which separates the concentration 
zone 14 from the discharge zone 18. Disposed in the concentration zone 14, 
and adjacent the gas permeable filter 16, is a binder layer 31 for binding 
and preventing the escape of molecules of the volatile analyte. The binder 
layer 31 is separated from the gauze layer 20 by a porous layer 28, which 
may compromise any of a variety of films for retaining the binder layer 31 
yet permitting passage of fluid. 
In the embodiment illustrated in FIG. 2a, perspiration will pool in the 
interfiber spaces of the gauze 20, and will percolate through porous layer 
28 into the binder layer 31. In that layer, a chemically active or 
biochemically active binder material will act to selectively bind the 
volatile analyte, thereby preventing it from escaping as a vapor through 
gas permeable filter 16. As discussed in connection with the embodiment 
illustrated in FIG. 1, it is also possible to replace the gas permeable 
filter 16 with a fluid permeable layer, where the presence of fluid on the 
outside of the test patch would not be undesirable. 
The binder layer 31 may comprise any of a variety of binders depending upon 
the nature of the volatile analyte to be determined. For example, the 
binder may chemically bind with the analyte or adsorb the analyte to be 
determined. Thus, when the analyte being collected is ethanol, the binder 
layer advantageously contains activated charcoal. In addition, the binder 
layer may comprise a specific binding partner of the analyte to be 
determined, such as a polyclonal or monoclonal antibody or an antigen 
matched to a specific antibody desired to be measured in the perspiration. 
The patch 11 is additionally provided with tape 26 or another means for 
securing the patch to the skin of a subject, as has been detailed in 
connection with the embodiment illustrated in FIG. 1. Patch 11 is 
illustrated, however, as having a unitary outer layer 22 extending beyond 
the perimeter of the underlying layers to form an annular flange 23, which 
is provided with an adhesive on its lower surface. As discussed in 
connection with the embodiment of FIG. 1, outer protective layer 22 
permits the escape of water vapor yet protects the filter material from 
chemical contamination from the outside. As also discussed above, gas 
permeable layer 16 can also in some cases function as the outer layer 22. 
C. Dermal Patches Having a Microbead Layer 
Referring to FIGS. 3 and 3a, there is disclosed a further embodiment of the 
test patch of the present invention wherein an inner porous layer 28 and 
an outer porous layer 30 define a space for containing a microbead layer 
32. The microbeads of such a microbead layer 32 can desirably have 
attached thereto a capture reagent, such as antibodies or other means for 
binding analytes of interest. The inner layer 28 and outer layer 30 
preferably comprise the same material, which may be any suitable material 
for providing an unrestricted flow of fluid through the patch while 
trapping the microbeads in between. One suitable material for porous 
layers 28, 30 is the fluid permeable and microporous film known by the 
name Ultipor (nylon 6) and manufactured by Pall Corporation in Glen Cove, 
N.Y. Additional manufacturers of suitable nylon filtration membranes 
include Micron Separations, Inc. of Westborough, Mass., and Cuno of 
Meridan, Conn. Porous layers 28, 30 may also be comprised of materials 
other than nylon, such as polycarbonate, modified polyvinylchloride and 
polysulfone. 
The gauze, the inner and outer porous layers and the adhesive tape in all 
embodiments can be cut to size with conventional dies. The gauze 20 and 
the inner porous layer 28 can be fixed to the adhesive ring 26 with 
conventional adhesives, such as those used on the adhesive surface itself. 
Alternatively, they could be heated or ultrasonically bonded together. The 
proper amount of microbeads can then be placed on top of the inner porous 
layer, after which the outer porous surface is attached by similar means. 
Typically, in a one-inch diameter patch, from about 0.05 grams to about 1 
gram of microbeads will be used, and preferably from about 0.1 to about 
0.4 grams will be used. The inner and outer porous surfaces may have to be 
staked or spot-welded together in some pattern, as will be appreciated by 
one of skill in the art, to prevent the microbeads from collecting in one 
area. 
The free adhesive surface is optimally covered by pull-away paper (not 
illustrated) with adequate space to be gripped with one's fingers. The 
patch is packaged in a paper or plastic pouch similar to that used in 
conventional band-aid packaging. The assembled unit could be terminally 
sterilized or pasteurized prior to sale. Alternatively, the package could 
comprise a vapor barrier such as a metallic foil or, mylar and even 
include oxygen or moisture absorbent means such as a small packet of any 
of a variety of known desiccants, such as silica gel, calcium chloride, 
calcium carbonate, phosphorous pentoxide or others as will be appreciated 
by one of skill in the art. 
The total thickness of microbead layer 32 can be varied considerably. 
However, if a color change is to be used to detect an analyte and the such 
color change is to be brought about by immersing the patch in appropriate 
reagent baths, layer 32 is preferably no more than about 3 mm thick since 
color changes occurring at immobilized sites on thicker layers would not 
likely be observable. More preferably, the microbead layer is between 
about 1 mm and about 2 mm thick. If such color change analysis is not 
performed, the microbead layer 32 can alternatively be torn open, 
releasing loose microbeads which can be used to conduct chemical analysis 
for detecting the presence of an analyte bound to the microbeads by 
conventional means, such as in a cuvette. 
Optimally, the diameter of the beads in microbead layer 32 will be at least 
about one order of magnitude larger than the diameter of the pores in 
inner porous layer 28 and outer porous layer 30. For example, the beads 
contained in microbead layer 32 may have diameters within the range of 
from about 5 to 50 microns, and preferably within the range of from about 
5 to about 10 microns. If 10-micron diameter beads are utilized in the 
microbead layer 32, for example, inner porous layer 28 and outer porous 
layer 30 will optimally comprise a median pore size of approximately 1 
micron. 
The microbead layer 32 may comprise any of a variety of known materials 
including polystyrene, latex, and glass. Beads sized from approximately 
0.05 micron to 100 micron which are suitable for the present application 
are available from Polysciences of Warrington, Pa. 
Microbead layer 32 serves as the support for an immobilized specific 
binding partner for the analyte to be determined. Thus, a molecule with a 
high chemical affinity for a specific component in the fluid to be 
analyzed will be immobilized to the microbeads in microbead layer 32. 
D. Dermal Patches Having an Impermeable Outer Layer 
Referring to FIG. 5, there is disclosed a further embodiment of the present 
invention, particularly suited for use under conditions in which profuse 
sweating is not present, such as in passive insensible perspiration, 
wherein the test patch is provided with an impermeable outer layer 42. In 
order to minimize any back diffusion of fluid into the skin, an absorptive 
layer 44 is provided to form a reservoir for drawing moisture away from 
the surface of the skin and through support 46 to which is bound a 
specific binding partner for at least one analyte to be determined. Layer 
44 may include chemical means for binding or collecting moisture such as a 
desiccant, as has been previously discussed, which is suitable for use in 
proximity to the skin. The patch may be further provided with an 
underlying porous layer 48 to separate support 46 from the surface of the 
skin, and the patch is provided with any of the means for attachment to 
the skin as have been previously discussed. 
E. Dermal Patches which Minimize Lateral Diffusion of Sweat in a Patch 
Referring to FIG. 6, there is disclosed a modified patch 13 according to 
the present invention, in which all intervening layers between the skin 12 
and the binder layer 30 have been deleted. By disposing the binder layer 
(i.e., the layer having a specific binding partner for an analyte to be 
determined) directly adjacent the skin, lateral diffusion of sweat 
throughout the binder layer 30 is minimized. The proximity of the binder 
layer 30 to the skin 12 allows the output of each duct of the sweat glands 
to contact or be in fluid communication with a relatively small area of 
the binder layer 30. For a variety of reasons which will be apparent to 
one of skill in the art, it may also be desired to mount a microporous 
membrane, preferably a fluid permeable membrane 50 atop the binder layer 
30. 
The evaporative capacity of the binder layer 30 and the fluid permeable 
membrane 50 is preferably sufficient relative to the output capacity of 
the individual sweat ducts, to minimize lateral diffusion of sweat away 
from the immediate area of the duct. This embodiment has special 
application for monitoring the chemical composition of insensible 
perspiration and/or non-exercise perspiration, in instances where output 
from the sweat glands is limited. Due to the magnification effect detailed 
infra, the present embodiment is also particularly suited for monitoring 
low concentration analytes. 
By limiting the suppressive characteristics of moisture or water on the 
skin, through the use of materials having a maximal evaporative capacity, 
the instant embodiment allows increase of the through-put rate of sweat in 
the patch by maximizing sweat gland output. Nadel and Stolwijk, J. Applied 
Physiology, 35(5): 689-694 (1973), disclose that sweat gland activity is 
suppressed by water lying on the skin, finding a difference in whole body 
sweat rate of 40% between wet and dry skin. Mitchell and Hamilton, 
Biological Chemistry, 178: 345-361 (1948), found that loss of water and 
solutes in insensible perspiration presumably stops whenever the surface 
of the skin is covered with a film of water. Brebner and Kerslake, J. 
Physiology, 175: 295-302 (1964), postulate that the reason for this 
phenomenon is that water in contact with the skin causes the epidermal 
cells of the skin to swell and thus block the sweat ducts. 
The ability of the present invention to produce a positive response based 
upon the presence of relatively low concentrations of analyte is 
particularly advantageous in view of the fact that, during active 
exercise, a 1/4" diameter area of skin provides approximately 35 
microliters of sweat per hour, whereas a similar diameter area of skin 
produces sweat at a non-exercise rate of only about 3.2 microliters per 
hour. The present embodiment is further advantageous as not requiring the 
user to exercise, but only to wear the patch for an equal or typically 
longer period during rest or at normal activity levels. 
Thus, homogeneous diffusion of sweat throughout the binder layer is 
preferably minimized when using the instant invention in conjunction with 
insensible and/or non-exercise perspiration and/or a determination of 
minute amounts of analyte contained within perspiration. The minimized 
lateral diffusion of perspiration throughout the binder layer 30, 
according to the present invention, provides a more concentrated 
collection of sweat at each sweat duct, thereby providing a greater amount 
of selected analyte to be determined at that area. 
F. Dermal Patches Having Multiple Test Zones 
Referring to FIG. 7, there is shown a modified binder layer 52 for a patch 
according to the present invention, wherein two or more distinct zones are 
provided on the binder layer 52. The use of a reference zone or of several 
distinct test zones is contemplated for both the single layer patch 
discussed in connection with FIG. 6, as well as the embodiments discussed 
in connection with FIGS. 1-3a and 5. The multi-zone binder layer 52 may 
also be used for certain embodiments to be discussed hereinafter in 
connection with FIGS. 6-10 when specific binding chemistry is used. 
One or more of the zones, such as determination zone 60 (FIG. 7), is used 
to test for an analyte of interest within sweat, as detailed previously. 
One or more of the remaining zones, such as reference zone 61, is used as 
a reference indicator. 
Reference zone 61 performs a variety of functions, depending upon the 
desired application of the test patch. For example, reference zone 61 can 
be provided with color change chemistry as discussed previously to provide 
the wearer with an indication that the patch has been worn for long enough 
that a sufficient sample volume has traversed the patch to provide a 
meaningful test for the analyte of choice. For this purpose, reference 
zone 61 is provided with affinity chemistry for a preselected reference 
substituent such as lgG, albumin or any other sweat component which is 
reliably present. Preferably, the selected reference substituent is one 
which provides a reasonably accurate measurement of the volume of sweat 
put through the system. 
This use of the reference zone 61 may be facilitated by first determining 
the rough concentration ratio of a reference substituent such as albumin 
to the analyte to be determined and providing the patch with color change 
chemistry which provides a visual indication of the presence of the 
reference substituent only well after the elution of the analyte to be 
determined has exceeded the lower limits of detection. Reference 
substituents such as albumin will typically be present in significantly 
greater quantities than the analyte. Thus, in order to accomplish the 
objective of indicating passage of a sufficient sample volume, the 
"sensitivity" of the patch for the reference substituent is preferably 
lower than for the analyte. This can be achieved by using a 
proportionately lower amount of a specific binding partner for the 
reference substituent than for the analyte, other dilutions in the assay, 
or simply selecting a less abundant reference substituent. Selection of a 
suitable reference substituent and concentration determinations can be 
readily made through simple experimentation by one of skill in the art. 
G. Use of Dermal Patches Having Multiple Test Zones to Prevent Tampering 
Alternatively, and particularly useful in assays for drugs of abuse and 
their metabolites, a reference zone 61 (FIG. 7) can provide an indication 
that the skin patch was actually worn by the desired patient, parolee or 
other subject. One inherent limitation in a test in which a subject 
desires a negative result is the possibility that the subject will simply 
remove the patch after administration and replace it just prior to 
reexamination. This possibility gives rise to the ability of the wearer to 
ensure false negative results. 
However, by provision of a reference zone 61 to detect a known component in 
sweat, the test results will reveal test patches that have not been worn 
for the test period. Reference zone 61 thus provides a method of 
preventing false negative evaluations due to tampering or removal of the 
test patch. 
A reference zone 61 to detect a known component in sweat may also be 
provided as a positive control zone to ensure the discovery of false 
negative test results due to degradation of reagents or other components 
of the patch. In non drug-of-abuse screens, the indication produced within 
the reference zone 61 will preferably be a visible color change by a 
chemical or antibody/antigen colorimetric interaction occurring or 
becoming apparent to the wearer when a predetermined amount of the 
reference analyte has passed through the interaction area. 
Optionally, a reference zone 61 may be provided as a negative control zone 
to enable the discovery of false positive results. A preferred negative 
control zone will have an immobilized specific binding partner for an 
analyte known to be absent in human sweat. The analyte's specific binding 
partner must be known to not cross react with components present in human 
sweat. An example of an appropriate analyte is bacteriophage T4 coat 
protein. 
In yet a further embodiment of the present invention (not illustrated) two 
or more analyte determination zones 60 are provided in a single test 
patch. The use of multiple test zones is particularly useful in 
applications such as a drug of abuse screen where testing for any one or 
more of a wide variety of analytes may be desired. For example, a single 
test patch may be used to screen for any of a plurality of drugs of abuse, 
such as THC, Phencyclidine morphine and Methadone. A positive result for 
any of the drugs on the screen may provide sufficient proof of an offense 
such as a violation of parole, or can be used to signal the need for more 
quantitative follow up investigations. Used as an initial screening tool, 
the present invention offers the advantages of being non-invasive, and 
much less expensive than conventional quantitative analyses. For these 
reasons, a screening test patch as disclosed herein is particularly suited 
for initial screening of large populations such as parolees, inmates, 
military personnel or others where monitoring is desired. 
The analyte determination zone 60 and analyte reference zone 61 may be 
physically separated on the patch, such as in concentric circles or 
discrete zones, as illustrated in FIG. 7, or in the case of only two or 
three analytes, interspersed throughout. In the latter case, positive 
results of different determinations would be indicated by the appearance 
of different colors. 
II. Placement of Dermal Patches 
Although a patch of the present invention can be used to collect analytes 
contained in any of a variety of body fluids, perspiration is the desired 
fluid to be collected due to its dependable supply and its similarity to 
blood, albeit with lower analyte concentrations. Although components found 
in saliva could also be collected with a patch of the present invention, 
saliva is often contaminated with molecules not expressed by the body, 
such as foodstuffs. Therefore, in a preferred embodiment, the patches of 
the present invention are placed on the skin surface of a subject. 
A. Characteristics of Sweat Glands and Perspiration 
Sweat glands are classified to be either of two types. Eccrine type sweat 
glands function primarily to regulate body temperature through their 
relationship to evaporative heat loss. It is the eccrine type sweat gland 
that provides the sweat associated with exercise and is therefore the 
source of perspiration of interest for many applications of the patch of 
the present invention. Apocrine type sweat glands are larger secreting 
elements which are localized only in relatively isolated areas of the body 
such as the axilla, pubic and mammary areas. 
Sato and Fusako, American J. Physiology, 245(2): 203-208 (1983), estimate 
that the diameter of the duct of the sweat gland is approximately 40 
microns. According to Scheupoein and Blank, Physiological Review, 51(4): 
702-747 (1971), the average density of sweat glands on the skin surface is 
approximately 250 per square centimeter. Thus, the total surface area of 
sweat gland ducts of the skin represent 1/318 of the total surface area of 
the patch of the instant invention. The visible result on a test patch of 
the present invention when, for example, using known ELISA technology to 
determine a low concentration analyte, is the appearance of a number of 
tiny color changes on the binder or absorptive layer associated with the 
output of specific ducts. If significant lateral diffusion of sweat is 
permitted prior to contact with the immobilized binding partner, the color 
change is frequently too diffuse to detect with the naked eye. 
Although the etiology of perspiration is relatively complex, it is known to 
be caused by both mental states such as mental exercise and emotional 
stress; thermal stress, as the sedentary body's response to temperature 
control; and exercise stress as the physically active body's response to 
temperature control. 
In addition to the foregoing distinctions, perspiration can be either 
insensible or sensible. Insensible sweat appears to be caused by water 
diffusion through dermal and epidermal layers. Its purpose appears to be 
not related to thermal regulation at all, but to aid in such things as the 
improvement of mechanical interaction between the skin and surfaces to 
facilitate grip. Further complexities arise with regard to the spatial 
distribution of sweat glands and the flow rates of the various types of 
perspiration. Specialized areas of the palms and soles of the feet sweat 
continuously, although at a very low rate. The rate of insensible 
perspiration is dependent upon the position of the particular area in 
question relative to the heart. For example, elevating a limb over the 
heart decreases the insensible perspiration rate in that limb. 
At temperatures of about 31 .degree. C. in a resting human adult, 
insensible perspiration proceeds at a rate of between about 6-10 grams per 
square meter per hour from the skin of the arm, leg and trunk, up to about 
100 grams per square meter per hour for palmer, planter and facial skin. 
The latter three areas jointly account for approximately 42% of the total 
water loss from the body due to insensible perspiration. Such insensible 
perspiration first begins on the dorsal surfaces of the foot and spreads 
to higher places on the body as the temperature increases. One reported 
study determined that the average water loss due to insensible 
perspiration for a body surface area of 1.75 square meters ranged from 381 
ml, 526 ml and 695 ml per day at ambient temperatures of 22.degree. C., 
27.degree. C. and 30.degree. C., respectively. 
In contrast to insensible perspiration which does not appear to be 
associated with a particular surface element of the skin, sensible 
perspiration has been associated with the eccrine gland. The number of 
actively secreting eccrine glands varies among individuals and depends 
upon the part of the body observed and the type of sweat response created. 
Maximum gland density varies from between about 200 per square centimeter 
on the forearm to over 400 per square centimeter on the thenar eminence. 
The appearance of sensible sweat begins at either when the skin temperature 
exceeds about 94.degree. F. or the rectal temperature exceeds about 
0.2.degree. F. over normal core temperature. Maximum rates of sweat volume 
loss can be as high as 2 liters per hour in average subjects and can be as 
high as 4 liters per hour for brief periods. Sensible perspiration begins 
in the distal parts of the lower extremities and progresses upward as the 
environmental temperature is elevated. Thus, the dorsum of the foot begins 
to sweat long before the chest. The pattern of sensible sweat response 
also shifts from one region of the body to another as the thermal stress 
increases. Under mild thermal stress, sweating is present mainly in the 
lower extremities. As the thermal stress further increases, sweating 
spreads to the trunk. Due to its large surface area, the trunk becomes the 
dominant water loss surface. Eventually, extremely high rates are found in 
the trunk while rates in the lower extremities may actually decline. The 
forehead can produce extremely high sweat rates but is among the last 
areas to sweat in response to thermal stress. 
B. Placement of Dermal Patches 
Although a patch of the present invention can be worn at any practical 
location on the body, preferable locations for the patch include the skin 
on the sole of the foot and areas on the chest, back, and biceps. The 
patch is able to be worn in confidence in these areas, and these areas are 
not covered with excessive hair, so that the patch may be secured with 
conventional adhesive tapes. 
The patch can advantageously be located on different regions of the body 
depending upon a variety of factors. It is well known that the quantity of 
perspiration generated is a function of both the location on the body, as 
well as the physical activity during and immediately preceding collection. 
This is due to both different densities of sweat glands on different 
regions of the body, as well as to certain regulatory functions of those 
glands. 
Other desirable placement locations for the patches of the present 
invention will depend on the conditions under which it is desired to 
detect analytes. Using the parameters described above and other known 
factors, one of skill in the art will understand how to choose a desirable 
location on the body of a subject on which to place a patch. 
III. Chemical Species Detectible with a Dermal Patch 
A large variety of chemical species which are detectable in blood are also 
present in sweat, although typically in a much lesser concentration. Early 
investigation into the composition of perspiration centered on 
electrolytes, including sodium, chloride, calcium and potassium. Extreme 
individual variation was found among individuals, and the electrolyte 
composition also differed depending upon whether the sweat was stimulated 
by thermal, mental or other etiology. 
Further research has identified numerous additional components in sweat, 
including both electrolytes and more complex biological molecules. Some 
illustrative chemical species which have been identified in sweat are 
identified in Table I below: 
TABLE 1 
______________________________________ 
Chemical Components of Sweat 
______________________________________ 
diphtheria antitoxin sulfates 
ascorbic acid iodine 
thiamine iron 
riboflavin fluorine 
nicotinic acid bromine 
amino acids bismuth 
ethanol lactic acid 
antipyrine pyruvate glucose 
creatinine nitrogen 
C-14 methylurea ammonia 
C-14 acetamide uric acid 
C-14 urea nicotine 
thiourea morphine 
paraaminohippuric acid 
sulfanilamide 
mannitol sucrose atabrin 
lactate methadone 
sodium chloride phencyclidine 
potassium aminopyrine 
calcium sulfaguanidine 
magnesium sulfadiacine 
phosphorous amphetamines 
manganese benzoylecgonine 
theophylline phenobarbital 
parathion androgen steroids 
tetrahydrocannabinol phencyclidine 
insulin phenytoin 
cimetidine carbamazepine 
dimethylacetamide 
______________________________________ 
Any of the entries in Table I for which affinity chemistry can be developed 
can be an appropriate subject of a test patch according to the present 
invention. Since most of the components listed in Table I are 
non-volatile, they will be trapped in the concentration zone 14 of the 
patch 10 illustrated in FIG. 1a, or on the binder layer 30 of FIG. 6. 
However, some components, most notably ethanol, would volatilize under the 
influence of body heat, thereby enabling escape in the vapor phase through 
the test patch. Where the analyte to be determined is ethanol or another 
volatile component, a patch of the present invention may be modified as 
described in connection with the embodiment illustrated in FIG. 2 to 
contain specific binding partners for the analyte. 
In one preferred embodiment, the analyte to be determined in perspiration 
is the enzyme creatine kinase MB (CK-MB) which is expressed from the 
cardiac muscle during myocardial infarction and other cardiac distress. A 
monoclonal antibody raised against CK-MB can be immobilized to the 
microbeads in accordance with any of a variety of conventional methods, 
such as the cyanogen bromide technique described in Pharmacia product 
literature (Pharmacia, Inc., Piscataway, N.J.). 
The antibody which is to be used for the purpose of complexing with CK-MB 
may be immobilized on any of a variety of supports known in the art. For 
example, anti-CK-MB antibody may be bound to polysaccharide polymers using 
the process described in U.S. Pat. No. 3,645,852. Alternatively, the 
antibody may be bound to supports comprising filter paper, or plastic 
beads made from polyethylene, polystyrene, polypropylene or other suitable 
material as desired. Preferably, the support will take the form of a 
multiplicity of microbeads which can conveniently be formed into microbead 
layer 32, illustrated in FIG. 3a. 
As an alternative to a microbead support layer, the specific binding 
partner could be immobilized directly to the inner porous layer 20 or 28 
on FIG. 3a, to the underside of filter 16 of FIG. 1a, or to appropriate 
absorbent materials used in any of the embodiments of the dermal patch. In 
this manner, the need for microbead layer 32 could be eliminated entirely. 
Fluid permeable membranes which are specifically designed for binding 
antibody proteins are commercially available, such as Zetapor from Cuno, 
and Protrans, available from ICN in Costa Mesa, Calif. 
The monoclonal antibodies useful in the present invention can be produced 
and isolated by processes which are well known in the art, such as those 
discussed by Milstein and Kohler, reported in Nature, 256: 495-497 (1975). 
In particular, Jackson describes a method of producing anti-CK-MM (an 
indicator of the status of skeletal muscles) and anti-CK-MB antibodies in 
Clin. Chem., 30/7: 1157-1162 (1984)). 
Alternatively, the components of a commercially available diagnostic kit 
can be utilized which incorporate the CK-MM enzyme chemically bound to a 
bead support. A suitable kit marketed as the Isomune-Ck Diagnostic Kit by 
Roche of Nutley, N.J., is one commercially available candidate. This kit 
includes a goat antisera to human CK-MM and donkey anti-goat antibody 
covalently bound to styrene beads. A mixture would produce an immobilized 
conjugate having a specific affinity for human CK-MM. A more direct and 
less expensive procedure, however, would be to immobilize the anti-CK-MM 
monoclonal antibody directly to the microbead support in accordance with 
methods now well known in the art. 
IV. Detecting Analytes 
A. Using Color Change Chemistry to Detect Analytes 
Any of a number of methods known to the art can be used to detect an 
analyte collected on a patch of the present invention. One such method 
involves the use of color change chemistry to visualize the presence of an 
analyte on a patch. In this embodiment, after the test patch has been worn 
for a suitable period of time, it can be removed by the wearer (in 
non-drug screen tests) and developed to produce a visible indicium of the 
test result. Such a test patch can be marketed together with a developer 
packet such as packet 34 shown in FIG. 4 which contains known developer 
reagents for the immunoassay. The reagent packet 34 comprises a container 
36 having a removably secured top 38. A flap 40 on the top 38 of the 
reagent packet facilitates gripping the top 38 and peeling away from 
container 36 to reveal the reagent contained therein. As an example, a 
protein electrophoresis stain such as Coomassie brilliant blue or amido 
black 10b, can be bound to purified analyte contained in the reagent 
packet 34. When a test patch is immersed in the packet 34, any antibodies 
on the test patch that are unbound by analyte in the perspiration will 
become occupied by stained purified analyte in the packet 34. There will 
thus be an inverse relationship between the amount of stain absorbed by 
the patch and the amount of enzyme passed through the patch. In this 
embodiment, the user would place the patch in the fluid of the packet 34, 
wait for some period of time such as 30 seconds or more, rinse the patch 
under tap water and relate the resultant color of the patch to the 
presence of the enzyme. A color comparison chart and control zone on the 
patch having no bound antibody may be provided to aid in this 
interpretation. 
Alternatively, the user could support the test patch on an open vessel, 
such as a small jar or vial, or empty container similar in design to 
reagent packet 34 securing the adhesive border of the patch to the rim of 
the vessel, and then pour contents of packet 34 on top of the test patch. 
Gravity would assist the transport of the contents of packet 34 through 
the test patch to maximize the efficiency of the stain/binding reaction, 
and to facilitate visualization of the color change. 
The system could readily be designed so that the user performs the 
interpretation of the concentration of the analyte not in the patch at all 
but by observing the packet contents once the contents have traversed the 
patch. This method would be similar to conventional ELISA assay methods 
where the packet contents contain enzyme conjugates which will react to 
specific enzyme substrates. The enzyme substrates would be added to the 
packet contents after those contents transversed the test patch. 
If the perspiration contained molecules of interest, they would bind to the 
specific immobilized binding partner on the patch. If this occurred, 
enzyme conjugates in the packet would pass freely across the test patch 
and enzymatically modify the enzyme substrate producing a controlled color 
change in the solution in the packet. If the perspiration contained the 
desired molecules of interest, enzyme conjugates would then be bound in 
transit across the patch and would be unavailable to cause color change in 
the substrate solution. Other immunoassay schemes can be readily adapted 
for use in the present invention by one of skill in the art. 
A variety of well known immunoassay schemes for visualizing the presence of 
an analyte of interest are well known in the art, and need not be detailed 
here. However, the optimal immunoassay scheme is generally one which is 
simple and requires the fewest steps. For many types of assays, it will be 
desirable for the wearer to obtain rapid results such as a color change to 
demonstrate a positive or negative result with as few steps as possible. 
On the other hand, drug of abuse screens are more likely to be evaluated 
by clinical staff instead of by the test subject, and there is less 
concern for a "user friendly" product. 
For example, in a patch of the present invention designed for determining 
both the presence of CK-MM and CK-MB enzyme, the immobilized specific 
binding partner for each of those enzymes will be segregated to separate 
regions of the test patch. In this manner, if an enzyme-linked immunoassay 
system is utilized, a common enzyme and a common substrate could be used. 
Alternatively, a different color can be used to express the presence of 
different analytes. 
B. Detecting a Metabolite of an Analyte Collected on a Patch 
One problem which has been encountered in detecting analytes contained in 
patches, especially when such analytes are drugs of abuse, is that many 
conventional systems for performing drug testing do not test for the 
analytes which are collected on a patch but rather for the metabolites of 
such analytes. This is because the analytes themselves are not expressed 
in some body fluids. For example, cocaine is present in perspiration but 
not in urine. Therefore, urine is not tested for the presence of the 
cocaine molecule itself but rather for the presence of the major urine 
metabolite of cocaine in man, benzoylecgonine ("BE"), in order to detect 
cocaine use by a subject. 
Currently, the primary method for the diagnosis of drug abuse is by urine 
analysis. Many conventional diagnostic systems, therefore, are designed to 
screen for drug analytes (or their metabolites) in urine. For example, 
numerous companies have developed very sophisticated automated systems to 
quantify cocaine metabolites in urine. Such systems are highly sensitive 
to the presence of the major cocaine metabolite in human urine, 
benzoylecgonine or BE. However, since the cocaine molecule itself is not 
present in urine, many of these systems, such as the SYVA EMIT system 
(Palo Alto, Calif.) and Roche RIA system (Nutley, N.J.), are virtually 
blind to the cocaine molecule itself. 
In order to take advantage of conventional diagnostic systems that perform 
drug abuse testing by urinalysis, it is important that the drug contents 
of a patch of the present invention be measurable by such diagnostic 
systems. Unfortunately, most of the kits on the market which test for the 
presence of analytes such as cocaine are designed to detect metabolites of 
such molecules rather than the analytes themselves. In order to utilize 
such diagnostic systems to test for a desired analyte, therefore, the 
contents of a patch must be chemically modified. 
In accordance with another aspect of the present invention, therefore, an 
analyte contained in a patch which is not detectable by conventional 
diagnostic systems, particularly systems for performing urinalysis, is 
chemically modified so that it can be detected by such systems. In this 
aspect, an analyte passed through the skin of a subject in perspiration is 
collected on an absorbent material in the patch. The analyte can then be 
chemically modified and detected while still in the absorbent layer or 
while bound to a microbead in a microbead layer. Alternatively, the 
analyte can be freed from the absorbent material, such as through chemical 
elution or by dissolving the absorbent material, in order to allow the 
analyte to be detected by a conventional diagnostic system. The analyte is 
then chemically modified so that it can be detected in such a diagnostic 
system. 
As long as the analyte and the metabolite of that analyte which is detected 
by a diagnostic system are known and a means of converting the analyte 
into its metabolite is known, it is within the knowledge of one of skill 
in the art to chemically modify such an analyte so that it can be 
detected. Thus, any such analyte contained in a patch of the present 
invention can be tested using conventional diagnostic systems. However, an 
example of how to chemically modify a particular analyte commonly tested 
for, cocaine, will be detailed below. 
Cocaine is metabolized in the body by either pH changes or cholinesterase 
enzymes. Cocaine is unstable at pH values higher than 7, and thus can be 
converted to BE either through exposure to high pH or to cholinesterase 
enzymes. Therefore, in order to chemically modify the cocaine on a patch 
and convert it to BE in order to make it detectable by conventional 
urinalysis, cocaine molecules can be extracted from the patch and then 
exposed to a solution at pH 11 at room temperature for 10 minutes or more. 
Following this modification step, the patch extract is returned to a 
neutral pH and then analyzed with conventional diagnostic systems. As is 
obvious to one of skill in the art, other methods of hydrolyzing the ester 
linkages of the cocaine molecule in order to produce BE, such as through 
the use of enzymes, can also be performed in order to prepare an extract 
of a patch of the present invention so that it can be detected by 
conventional diagnostic systems. 
C. Eluting Analytes from Dermal Patches 
Another difficulty encountered in detecting analytes that are contained in 
perspiration and collected on a patch is that, unless color change 
chemistry is used to detect such analytes, these analytes usually have to 
be removed from the patch in order to detect them. Removing the analytes 
normally involves chemically eluting them from the patch, which is both 
labor intensive and time consuming. 
Therefore, in yet another aspect of the present invention, a patch is 
provided in which the absorbent material of the patch on which analytes 
are collected is dissolvable. When such absorbent material is dissolved, 
the analytes contained therein are made available for detection through 
further diagnostic procedures. As in other embodiments of a patch of the 
present invention, a patch incorporating a dissolvable absorbent material 
is placed in fluid communication with the skin of a subject in order to 
collect analytes contained in perspiration. Such a patch also preferably 
contains a gas permeable layer between the absorbent material and the 
outside of the patch in order to allow the fluids expressed through the 
skin in perspiration to escape to the outside of the patch in their vapor 
phase. 
The analytes of interest that are collected on the absorbent material are 
preferably able to withstand the chemical treatment which results in the 
dissolution of the absorbent material. Thus, the dissolution of the 
absorbent material will not affect the analysis of the analytes contained 
in the absorbent material. One of skill in the art will be able to 
recognize whether a particular analyte will be chemically changed by a 
particular chemical treatment used to dissolve the absorbent material. If 
one of skill in the art would be unsure as to whether a particular analyte 
would withstand such chemical treatment, it is a matter of routine 
experimentation to treat a sample of the analyte under the conditions of 
the chemical treatment and then determine whether the analyte has been 
chemically altered. 
In another embodiment, the chemical treatment of the absorbent material 
converts an analyte of interest contained in the absorbent material into a 
detectable metabolite or into some other detectable species. For example, 
the treatment of cocaine with a strong base converts it into BE, a common 
cocaine metabolite found in urine. The same strong base can also be used 
to dissolve an absorption disk made from a material sensitive to strong 
bases. In this embodiment, the dissolving of the absorbent material does 
not interfere with the analysis of the analyte contained in the absorbent 
material, but instead actually allows the analyte to be analyzed. 
An absorbent material for use in this aspect of the present invention can 
be made from any of a variety of materials which can be chemically 
dissolved. For example, a number of materials are variously susceptible to 
chemical attack and dissolution by acids and/or bases. Among these 
materials are Nylon 6/6 (sold as Vydyne 909 by Monsanto Co., St. Louis, 
Mo.), Phenolic (sold as Polychem 102 by Budd Co.), Polyester (PBT) (sold 
as Celanex 3300-2 by Celanese Plastics), and polyurethane (TP) (sold as 
Pellethane 2363-55D by The Upjohn Co.). To dissolve any of these 
materials, an appropriately strong acid or base is added the material, as 
is known to those of skill in the art. 
Absorbent materials can also comprise a woven protein web, such as a web 
made from protein fibers approximately 0.03 inches thick. Such fibers are 
disclosed by Baumgartner, J. Forensic Sciences, 34: 1433-1453 (1989)). 
Another dissolvable material which can be used as the absorbent material in 
the patch of the present invention is polystyrene. In this embodiment, 
solvents of polystyrene can be used to dissolve such absorbent material. 
Such solvents include chlorinated and aromatic hydrocarbons, esters, 
ketones, essential oils of high terpene content and turpentine. Specific 
examples of such solvents include cyclohexanone, dichloroethylene, and 
methylenedichloride. 
Materials and solvents other than those listed above, of course, can also 
be used in this aspect of the present invention. The foregoing materials 
and solvents are therefore exemplary of this aspect of the present 
invention and not intended to be limiting. 
V. Quantitative Determination of an Analyte in Perspiration 
A. Dermal Patches for the Quantitative Determination of an Analyte 
In another aspect of the present invention, the amount of an analyte that 
is present in a given volume of a subject's perspiration can be 
discovered. An embodiment of this aspect of the present invention is 
illustrated in FIG. 11. In this embodiment, a fluid permeable support 
layer 120 is in fluid communication with the skin 12 of a subject mammal, 
such as a human, and is located between the skin 12 of the subject and an 
absorptive layer 130 made of an absorptive material. 
In the embodiment illustrated in FIG. 11, the support layer 120 also 
comprises a rate-limiting structure which limits the passage of 
perspiration from the skin 12 to the absorptive layer 130 to a rate lower 
than the rate of insensible perspiration of the subject. The insensible 
rate of perspiration is the rate of continuous perspiration of a subject 
which occurs without regard to the regulation of the temperature of the 
subject and which is not normally noticed by that subject. For humans, the 
rate of insensible perspiration of sweat glands in the arm, leg or trunk 
is approximately 6-10 ml/m*m*hr (Randall, W. C., Am. J. Phys. Med., 32: 
292 (1953)). Since the rate of perspiration of the subject will almost 
always be equal to or greater than the rate of passage of such 
perspiration through the rate-limited support layer 120, the rate of 
perspiration passing into the absorptive layer 130 can be kept 
approximately constant. 
The rate-limited support layer 120 can be made from any material which can 
control the rate of diffusion of the components of perspiration. For 
example, diffusion can be controlled by a membrane. The rate of diffusion 
of any particular membrane is related to physical characteristics of the 
membrane such as its molecular composition, thickness, and, in the case of 
a porous type of membrane, its pore size. One example of a porous type of 
membrane which can be used as a rate-limited structure in this embodiment 
of the present invention is a polyester-supported polycarbonate 
microporous membrane, such as that manufactured by Nuclepore (Menlo Park, 
Calif.). The pore density, pore size and thickness of the membrane can be 
adjusted to provide the necessary limited fluid transport rate for this 
application. Another example of a porous membrane is nylon 6,6, such as 
that manufactured by Pall Corp. (Glencove, N.Y.). 
An alternative to using a porous type rate-limited membrane is to use a 
rate-limiting structure comprising a dialysis or osmotic non-porous 
membrane. Such membranes have the advantage of having molecular weight 
specificity, which may increase analyte sensitivity. For example, if one 
were interested in collecting a therapeutic drug or its metabolites in the 
absorptive layer 130 and these analytes had a molecular weight of 1000 
Dalton, one could choose a dialysis membrane which would pass only 
molecules which are smaller than 2000 Dalton in size. Larger molecules 
would be excluded from passing into the absorptive layer 130. By limiting 
the molecules which pass into the absorptive layer 130, interference by 
other components in perspiration in the laboratory analysis of the analyte 
in the absorptive layer 130 is minimized. Although the support layer 130 
of this embodiment of the invention has been described as comprising a 
rate-limited structure, one of skill in the art will recognize that the 
support layer 130 and the rate-limited structure can be two separate 
membranes or structures in fluid communication with each other. 
The absorptive layer 130 is located distally of the support layer 120 so 
that said support layer 120 is between the subject's skin 12 and the 
absorptive layer when the patch is being worn. The absorptive layer can be 
made from any number of absorbent materials. If passive absorption of an 
analyte is adequate to capture that analyte on the patch, then a layer of 
medical grade paper such as Filtration Sciences medical grade paper 
(FS#39) will suffice. If active absorption is required then substances 
such as monoclonal antibodies specifically tailored for high affinity to 
the analyte can be chemically coupled to the absorptive layer 130 in order 
to concentrate the analyte on the absorptive material, as previously 
described. 
In this embodiment of the invention, a gas permeable layer 140, which in a 
preferred embodiment is also an outer protective layer, is located 
distally from the skin 12 of the subject on the side of the absorptive 
layer 130 opposite that which borders the support layer 120. The gas 
permeable, outer protective layer 140 can be made, for example, from 1625 
Tegaderm wound dressing made by the 3M Company (St. Paul, Minn.). In a 
preferred embodiment, the gas permeable layer 140 extends beyond the areas 
of skin 12 covered by the support layer 120 and the absorptive layer 130 
when the dermal patch 110 is applied to the skin 12 of a subject. In this 
way, the support layer 120 and absorptive layer 130 are protected from 
external abrasion and wear. 
A means for attaching the patch to the skin of a subject is also preferably 
applied to a portion of the outer protective layer 140 which extends 
beyond the support layer 120 and the absorptive layer 130. Most commonly, 
the means for attaching is an adhesive composition. For example, in a 
patch 110 in which the outer protective layer 140 (excluding that portion 
to which an adhesive is applied) is approximately 14 cm.sup.2, an adhesive 
can be applied to an area of approximately 1 cm around the outer perimeter 
of the outer protective layer 140 on the side of the outer protective 
layer 140 in contact with the subject's skin in order to attach the patch 
110 to the skin 12 of a subject. 
In a more preferred embodiment, a pooling area 150 is formed between the 
outer protective layer 140 and the subject's skin 12 when the patch is 
worn on the subject's skin 12. Such a pooling area 150 can be formed, for 
example, by an area 152 of the outer protective layer 140 which extends 
beyond the support layer 120 and the absorptive layer 130 and to which no 
adhesive is applied. Such a pooling area 150 collects the excess 
perspiration that is not diffused across the support layer 120 and allows 
it to dissipate into the environment across the outer protective layer 
140. By providing such a pooling area, the back-diffusion of the 
components of perspiration across the skin 12 is minimized, since excess 
perspiration which is unable to pass into the absorptive layer 130 is 
shunted into the pooling area 150. Since the rate of flow of perspiration 
into the absorptive layer 130 is controlled by the rate-limiting structure 
of the support layer 120, the absorbent material of the absorptive layer 
130 is in fluid communication with the pooling area 150 only through the 
support layer 120. 
This pooling area is unattached to the subject's skin, and provides a 
sufficient amount of space to accommodate extra perspiration which does 
not pass across the rate-limited structure of the support layer 120. For 
example, during times of heavy exercise, the rate of perspiration of the 
subject might rise well beyond the rate at which perspiration can be 
passed into the absorptive layer 130. During such times of heavy 
perspiration, the pooling area 150 acts as a "shunt" to divert 
perspiration away from the support layer 120. The volatile components of 
such perspiration then evaporate through the gas permeable layer 140. In 
this way, the back-diffusion of perspiration and the buildup of bacteria 
under the rate-limited structure of the support layer 120 can be avoided 
or at least mitigated. 
B. Using Dermal Patches to Determine the Amount of an Analyte in 
Perspiration 
In order to determine the length of time a patch has been worn, the amount 
of a reference analyte contained in a certain volume of perspiration of a 
subject must first be determined. This analyte must be present in an 
approximately constant amount in a given volume of perspiration for the 
period of time that the patch is worn by a subject. Once such an analyte 
and its concentration in perspiration is known, the amount of time a patch 
is worn can be determined because the rate at which perspiration passes 
into the absorptive layer is held approximately constant by the 
rate-limited structure. Since the rate of passage of perspiration is known 
and the amount of the reference analyte in a given volume is known, once 
the total amount of the analyte in absorptive layer is known the amount of 
time the patch has been worn by a subject can be determined. 
The volume of perspiration concentrated on a patch can also be determined 
through the use of this embodiment of the present invention. The 
rate-limited structure of the support layer 120 in this embodiment is 
preferably designed to allow the passage of perspiration to the absorptive 
layer 140 at a rate lower than the minimal rate of passage of perspiration 
through the skin, thereby assuring a relatively constant rate of flow of 
perspiration into the absorptive layer 140. The total volume of 
perspiration concentrated on the absorption disk is thus directly related 
to and can be determined by the duration of wear. 
In order to quantitatively determine the amount of an analyte contained in 
a given volume of a subject's perspiration, a patch having a rate-limited 
structure as described above is first placed on the skin of a subject, 
preferably a mammal. Perspiration is then passed across this rate-limited 
structure at a known rate. For example, if the rate at which perspiration 
is allowed to pass across the rate-limited structure is equal to or less 
than the insensible rate of perspiration of the subject, perspiration will 
pass into the absorbent material at approximately a constant rate. After a 
sufficient test period of time has elapsed to allow a detectable amount of 
the analyte to be tested for to pass into the absorbent material, the 
patch is removed from the skin of the subject. When the patch is removed, 
the amount of time between the placement of the patch on the skin of the 
subject and the removal of the patch is recorded. 
In order to then determine the total volume of perspiration which has 
passed into the absorptive layer 140 and concentrated analytes there, the 
rate of flow of perspiration into the absorptive layer 140 (as determined 
by the rate at which perspiration passes across the rate-limited structure 
of the support layer 120) is first multiplied by the amount of time the 
patch has been worn. This figure indicates the volume of perspiration 
which has passed through the support layer 120 and into the absorptive 
layer 130. The total quantity of analyte in the absorptive layer is then 
determined. By dividing the total amount of analyte present by the total 
volume of perspiration which has passed into the absorptive layer 130, the 
average amount of the analyte in a given volume of the subject's 
perspiration can be determined. 
The above described aspect of the present invention is thereby suited to be 
used in many areas of diagnostics where quantitative information about a 
particular analyte is necessary. For example, this invention can be used 
to monitor therapeutic drug administration, determine the nutritional 
adequacy of a subject's diet, or explore hormonal imbalances in a 
particular subject. 
VI. Increasing Analyte Concentration and Controlling Back-Diffusion in a 
Dermal Patch 
A. The Problem of Back-Diffusion 
An analyte which has passed through the skin in perspiration is usually 
removed from the exterior surface of the skin through washing or through 
various natural processes. Thus, such an analyte will not normally 
accumulate on the skin's surface. However, analytes which pass into a 
dermal patch can become more highly concentrated than they normally would 
on the surface of the skin. If an analyte does become concentrated on a 
dermal patch, it becomes possible for that analyte to diffuse back through 
the skin of the subject wearing the patch, a phenomenon which has been 
termed "back-diffusion". 
Previous reports in the literature suggest that an analyte will 
back-diffuse after the concentration of the analyte on a dermal patch 
rises above the concentration of the analyte in the sweat or interstitial 
fluid of a subject. In fact, a mathematical model has even been generated 
to elucidate the pharmacokinetics of back-diffusion (Peck, Carl C., et 
al., "Continuous Transepidermal Drug Collection: Basis for Use in 
Assessing Drug Intake and Pharmacokinetics", J. Pharmacokinetics and 
Biopharmacology, 9:41-58 (1981)). This model suggests that back-diffusion 
will occur when an analyte is concentrated on a dermal patch, and that 
such back-diffusion must be prevented in order to accurately quantitate 
the amount of an analyte which passes into a dermal patch. Thus, many 
prior art references suggest using specific binding chemistry to prevent 
back-diffusion. 
B. Back-Diffusion and Dermal Patches of the Present Invention 
It is one of the surprising discoveries of the present invention, however, 
that such specific binding chemistry is not necessary to prevent 
back-diffusion. This discovery was first made during the experiment 
illustrated in FIG. 12, in which a patch without any specific binding 
chemistry was placed on the skin of a subject who had ingested cocaine. In 
this test, the concentrations of cocaine and cocaine metabolites found on 
the patch were charted for approximately 200 hours following the subject's 
ingestion of cocaine. The results of this test showed that the 
concentration of cocaine on the dermal patch rose immediately during the 
first hours of the test, and thereafter stayed at approximately the same 
level for the remaining 200 hours. Thus, the patch was able to concentrate 
analytes over a period of almost 200 hours without exhibiting significant 
back-diffusion. 
Furthermore, during that 200-hour time period, the concentration of cocaine 
in the subject's system was decreasing, as shown in FIG. 12 by the 
declining concentration of BE in the subject's urine. Thus, the dermal 
patch used in this test was able to maintain a concentration of cocaine 
that was higher than that in the subject's system, again demonstrating 
that significant back-diffusion was prevented. These results were 
unexpected in light of the teachings of the prior art, which would have 
led one of skill in the art to expect to observe back-diffusion during 
this test. 
It is believed that the surprising results of this test were due to the 
ionization states of the analyte of interest collected on the patch, in 
this case cocaine. In the experiment illustrated in FIG. 12, the 
ionization states of the cocaine molecules collected on the dermal patch 
used in that experiment were affected by the pH of the dermal patch and 
the pH of the exterior surface of the skin underneath the patch relative 
to the pH of the body fluids beneath the surface of the skin, as well as 
by the pK.sub.a of cocaine. It is the interaction of these pH and pK.sub.a 
values which affects the ionization state of an analyte. 
The occurrence of back-diffusion can be substantially prevented by 
controlling the ionization state of an analyte being collected on a dermal 
patch. For example, the pH of a dermal patch can be controlled in order to 
also control the pH of the surface of the skin beneath that patch. Once 
analytes pass through the area of skin underneath such a dermal patch 
having a controlled pH, they will become ionized and thus substantially 
unable to back-diffuse. Thus, after determining the pK.sub.a of an analyte 
of interest, standard pharmacokinetic equations can be used to determine 
pH values at which that analyte will become ionized once it passes to the 
surface of the skin of a subject. In this way, a particular analyte of 
interest can be collected on a dermal patch without the risk of subsequent 
back-diffusion. 
We believe that one reason that the ionized form of various analytes do not 
back-diffuse is that these ionized analyzed can attach themselves to 
larger molecules that are too large to be capable of back-diffusion. 
C. The Effect of Occlusion on Back-Diffusion 
In order to evaluate this model for controlling back-diffusion, the pH of 
skin underneath a non-occlusive patch such as that used in the experiment 
illustrated in FIG. 12 was next determined by conducting a further test. 
In this test, patches were constructed which had 1/2" long pieces of 
litmus paper between the absorptive layers and the Tegaderm outer layers 
of each of the patches, and which further had 1/2" long pieces of litmus 
paper between the skin and the absorptive layers of these patches. Such 
patches were placed on the chests (below the diaphragm) and biceps of each 
of three male volunteers for seven days. The colors of the pieces of 
litmus paper in each patch were monitored while these patches were worn. 
The results of this experiment indicated that in all three volunteers the 
pH of both the volunteers' skin and the absorptive layers of each of the 
patches reached only between about 4.5 and 5.0. Further, this pH was 
reached and thereafter maintained in each case within 24 hours. Human skin 
normally has a pH of about 4.4. Thus, the application of a non-occlusive 
patch does not appear to significantly change the skin's pH. 
By comparison, occlusion of the skin can bring about a much greater change 
in the skin's pH. In a study done on the effects of occluding skin, the 
skin of ten subjects was wrapped with plastic film (Saran brand plastic 
wrap) for approximately five days. The results of this test showed that 
the pH of the skin of these subjects shifted gradually over the course of 
the test from 4.38 before occlusion to 7.05 on the fourth day of occlusion 
(Aly, Raza, et al., "Effect of Prolonged Occlusion on the Microbial Flora, 
pH, Carbon-dioxide and Trans-Epidermal Water Loss on Human Skin," Journal 
of Investigative Dermatology, 71:378-381 (1978)). 
As discussed in further detail below, a rise in pH values such as that 
observed in the occlusion tests performed by Aly can significantly affect 
the amount of back-diffusion from a dermal patch. Prior art patches, which 
are occlusive in nature, appear to have experienced problems with 
back-diffusion due to an unintended and undiscovered shift in the pH of 
the skin below such patches. By contrast, the non-occlusive nature of the 
dermal patches of the present invention results in only a small change in 
the pH of the surface of the skin under such patches. Since the skin is 
naturally slightly acidic, the maintenance of a relatively acid pH will 
prevent back-diffusion problems. The detrimental effects of a rise in pH 
on analyte absorption can thus be obviated by using the patches of the 
present invention. 
D. Controlling Back-Diffusion 
The transport of many substances across the skin, including the 
back-diffusion of analytes, is believed to occur by means of passive 
diffusion across the stratum cornium, a structure which has a high lipid 
content (Orland, 1992). Passive diffusion across a lipid barrier normally 
occurs only if the substance in question is non-ionized, because ionized 
molecules cannot cross such a barrier (Labaune, J. P., "Handbook of 
Pharmacokinetics," 1989, pp. 18-25). In order to control back-diffusion, 
therefore, the pH of the surface of the skin below a dermal patch can be 
controlled, such as with a buffer, so that analytes which pass through the 
stratum cornium become ionized once they reach the surface of the skin, 
thereby losing their ability to pass back through the stratum cornium. 
Alternatively, back-diffusion can be prevented by ionization of analytes in 
other ways known to those having ordinary skill in the art. For example, 
analytes can be ionized by electricity, such as by iontophoresis. Devices 
such as the Phoresor II.TM. (made by Iomed, Inc., Salt Lake City, Utah) 
can be outfitted to ionize analytes collected on patches. These devices, 
which were originally designed to deliver drugs by means of electrodes 
attached to the skin, can be adapted to deliver electricity to a patch or 
to the skin adjacent the patch. However, other methods can be used to 
deliver electricity to the patch or skin, such as by simply attaching a 
pair of electrodes connected to a battery or other source of electricity. 
It is believed that an analyte which has passed into a patch can also be 
bound onto a patch with an antibody and simultaneously ionized by means of 
an ionized molecule that is also bound to the antibody. 
The degree of ionization of a molecule is easily determined if its pK.sub.a 
and the pH of its environment is known. The general Henderson-Hasselbach 
equation for a weak base shows: 
EQU pH=pK.sub.a +log (C.sub.nonion /C.sub.ion) 
Where: 
C.sub.ion is the concentration of the ionized molecule; and 
C.sub.nonion is the concentration of the non-ionized molecule. 
The concentration of an ionized molecule on either side of a lipid barrier, 
such as the skin barrier, can be found by extending the above equation for 
a weak acid molecule: 
##EQU1## 
or for a weak base: 
##EQU2## 
Where: CB is the total concentration of the molecule in the interstitial 
fluid of a subject; 
CP is the total concentration of the molecule in the patch; 
pHB is the pH of the interstitial fluid; 
pHP is the pH of the patch; 
By using these pharmacokinetic equations, for any given analyte and 
subject, a pH can be selected for a patch which will cause the number of 
molecules of an analyte in the patch to be larger than the number of 
molecules of that analyte present in the interstitial fluid of the subject 
wearing the patch. When using a patch with such a selected pH, non-ionized 
molecules which pass through the stratum cornium will tend to be ionized 
when they reach the patch, thereby preventing the back-diffusion of those 
molecules. 
In practice, when collecting an analyte of interest on a dermal patch 
according to one preferred method of the present invention, a pH value or 
a range of pH values is first selected according to the equations above. 
The absorbent material of the patch is then preferably maintained at the 
selected pH or range of pH values in order to concentrate the analyte on 
the patch. As the patch is worn, the non-ionized form of the analyte in 
the interstitial fluid of a subject will naturally diffuse across the 
stratum cornium of a subject's skin in order to try and reach equilibrium 
with the non-ionized form of the analyte on the surface of the subject's 
skin. Once on the exterior surface of the skin, the non-ionized analyte 
molecule will be ionized due to the selected pH of the patch, thus 
preventing the analyte molecule from back-diffusing. In addition, after 
the non-ionized molecule becomes ionized, the concentration of non-ionized 
analyte molecules on the surface of the skin will be decreased and thereby 
cause more nonionized analyte molecules on the interior side of the skin 
barrier to cross to the exterior side in order to try to reestablish an 
equilibrium concentration of non-ionized analyte molecules on each side of 
the skin barrier. 
As discussed above, in prior art occlusive patches, the pH of the patch 
quickly approaches 7.05. This severely limits the ratio of analyte in the 
patch to analyte in the interstitial fluid. It is preferable, when using 
both occlusive and non-occlusive patches of this embodiment of the 
invention, to provide a ratio of analyte in the patch to analyte in the 
interstitial fluid of over 10, more preferably over 100. In one preferred 
embodiment, as illustrated below, such ratios can be provided by 
maintaining the pH of the patch below a given level. 
An example of this method of collecting analytes on a dermal patch using a 
selected pH is outlined below. When using a non-occlusive patch, such as 
is described herein, the pH of the surface of the skin of a subject will 
remain at about 5.0. Perspiration as well as plasma and interstitial fluid 
all have a pH of about 7.2 (Orland, 1992). Using the equations above, it 
can be determined that when detecting the analyte cocaine, which is a weak 
base and has a pK.sub.a of about 8.7, the selected pH of 5.0 for the patch 
will drive the ratio of cocaine molecules on the exterior surface of the 
skin to that in the interstitial fluid to over 100. The number of ionized 
cocaine molecules on the exterior surface of the subject's skin compared 
to the number of non-ionized molecules is also much higher. Thus: 
##EQU3## 
Applying the foregoing equations to prior art patches, in which the pH 
would quickly approach 7.05, the result would be CP=CB.times.1.4. Thus, by 
maintaining a patch pH of 5.0, a 110-fold increase in the ratio of 
concentration of analyte in the patch to concentration of analyte in the 
interstitial fluid can be obtained. 
It can be seen that for weak base analytes such as cocaine, the higher skin 
pH observed under occlusive-type dermal patches will allow back-diffusion 
to occur. Thus, when it is advantageous to use an occlusive-type patch, 
such as when extra protection from the environment is desired, the pH of 
the skin under such an occlusive patch should be controlled in order to 
prevent back-diffusion. In these applications, a buffer can be used to 
control the pH of the surface of the skin below the patch. A buffer of any 
specified pH can be generated by controlling the ratio of acid to base in 
a mixture containing an acid and a base, such as a mixture of acetic acid 
and NaOH. Such a mixture can be made more basic by increasing the 
concentration of base, in this case NaOH, or can instead be made more 
acidic by increasing the concentration of acid. A buffer of this kind can 
maintain a desired pH in the patch and on the surface of the skin under 
the patch when the patch is worn. Thus, even if an occlusive dermal patch 
is used to collect an analyte, by controlling the pH of the patch the 
problem of back-diffusion can be virtually eliminated. 
A particular application of the present invention is the prevention of the 
back-diffusion of a drug of abuse which has been collected on a dermal 
patch of the present invention. Table 2 below lists the pK.sub.a 's of the 
major drugs of abuse, all of which are weak bases (Wilson, J., Abused 
Drugs, a Laboratory Pocket Guide, AACC Press, 1990). 
TABLE 2 
______________________________________ 
Drug pK.sub.a 
______________________________________ 
Heroin 7.6 
Methamphetamine 9.9 
Amphetamine 9.8 
Morphine 8.1 
Phencyclidine 8.5 
Cocaine 8.7 
______________________________________ 
The pK.sub.a for most of such analytes of interest is in the range of 7.2 
to 10.0. The pK.sub.a values of an analyte of interest can be used in 
connection with the general Henderson-Hasselbach equation given above in 
order to determine the ratio of the ionized form of any given analyte to 
its nonionized form in the patch. For analytes in this range of pK.sub.a 
's in prior art patches, in which the pH of the patch quickly approaches 
7.05, the ratio of C.sub.ion to C.sub.nonion will vary from approximately 
1.4 to approximately 891. As an example, for cocaine, which has a pk.sub.a 
of 8.7, this ratio will be 45 in a prior art occlusive patch. In contrast, 
using a patch of the present invention in which the pH of the patch is 
buffered to 5.0, the ratio will be 5012. In preferred embodiments of the 
present invention for both occlusive and nonocclusive patches, the ratio 
of ionized forms of the analyte to nonionized forms of the analyte in the 
patch will be over 1000, and more preferably over 5000. 
As in the case of cocaine, using the equations described above it can be 
shown that a pH of about 5.0 on the surface of a subject's skin below a 
patch will cause the above-listed drugs of abuse to collect on the patch. 
Thus, for example, a non-occlusive patch of the present invention will 
concentrate the above analytes without the problem of back-diffusion. 
In addition to solving the back-diffusion problems of prior art dermal 
patches, the present discovery also makes it possible to improve the 
ability of a dermal patch to concentrate an analyte. This can likewise be 
accomplished by adjusting the pH of a patch and the surface of the skin 
below the patch. By determining the pK.sub.a of an analyte of interest and 
using the equations above, an appropriate pH for the patch can be selected 
such that the equilibrium concentration of the ionized form of the analyte 
is much greater than the equilibrium concentration of the non-ionized form 
of the analyte. When non-ionized analytes then pass across the skin and 
into a patch having a pH selected in this way, they will be ionized, thus 
driving the further diffusion of non-ionized analyte molecules into the 
patch. Determining the pK.sub.a of an analyte of interest, if it is not 
already known, is within the knowledge of one of skill in the art, and 
thus requires only routine experimentation. 
The present discovery further suggests a method of quantitatively 
determining the amount of an analyte which passes through the body of a 
subject. Such a method first involves the placement of a dermal patch on a 
subject. The back-diffusion of analyte molecules collected on this patch 
is controlled in this method, such as by selecting an appropriate buffer 
for the patch. After a specified period of time, the patch is removed from 
the subject's skin and the amount of time the patch was worn is recorded. 
The amount of an analyte which has passed into the patch is then 
determined. By preventing the back-diffusion of an analyte, the amount of 
analyte collected on the patch over the specified period of time will more 
closely reflect the amount of the analyte which passed through the 
subject's system over that period of time. 
VII. Prevention of Tampering with Dermal Patches 
In some uses of the present dermal patches, it is advantageous to provide a 
means for indicating whether a wearer has removed a patch during the 
examination period, particularly in situations where a wearer has an 
incentive to make sure that the patch produces a specific result. For 
example, if it is desired to determine whether a wearer has ingested a 
drug of abuse, safeguards are desirably provided to prevent tampering with 
the dermal patch. 
A. Dermal Patches with Radial Slits 
One embodiment of a patch for preventing tampering is illustrated in FIG. 
8. in this embodiment, the patch 62 is secured to the skin 64 with an 
adhesive member 65. The adhesive member 65 is preferably constructed of a 
material that is strong enough to hold the patch 62 to the skin 64, but 
that is relatively easily torn such as during removal of the patch from 
the skin. A suitable material for use in this preferred embodiment is 
Tegaderm 1625, manufactured by Minnesota, Mining, and Manufacturing Corp. 
of St. Paul, Minn. Other companies, including Avery and Johnson & Johnson, 
manufacture similar suitable materials; the Johnson & Johnson product 
being sold under the trademark "Bioclusive." It has been found, however, 
that with sufficient patience, a wearer could remove an adhesive member of 
this type and replace it without leaving any visible indication that the 
adhesive member has been removed. Therefore, in the particularly preferred 
embodiment shown, the adhesive member 65 has stress razors 66 in the form 
of a plurality of radial slits around its outer perimeter. The stress 
razors 66 can be arranged in any of a wide variety of configurations and 
densities and accrue the advantage of tearing upon removal, as will be 
apparent to one of skill in the art.f 
In the embodiment illustrated in FIG. 8, the radial slits 66 extend 
approximately 0.05 inches in length from the outer edge toward the center 
of the patch 62. The slits 66 may be arranged with any of a variety of 
regular or irregular spacings therebetween, and, in the preferred 
embodiment are preferably spaced approximately every 0.10 inches around 
the perimeter of the patch 62. The adhesive force of the material of the 
adhesive member 65 is preferably more than the force needed to tear the 
adhesive member at the stress razors 66, so that if the patch 62 is 
removed, the material of the adhesive member is torn. Thus, when a patch 
of this preferred embodiment is worn, a torn adhesive member serves as an 
indication that the wearer has likely tampered with the patch. Of course, 
the weakening of the adhesive member 65 may be accomplished by providing 
perforations rather than slits and the slits or perforations may be 
oriented in directions other than radially. 
During storage prior to use, it is desirable to cover the adhesive member 
to prevent it from sticking to any surface; otherwise the stress razors 66 
could become torn prior to use. Accordingly, in the preferred embodiment 
shown in FIG. 8, the patch is provided with an inner cover 69 to protect 
the adhesive member 65. The inner cover 69 is removed to expose the 
adhesive member 65 prior to application of the patch 62 to a subject's 
skin. Any of a variety of non-adherent materials known to those of skill 
in the art may be used for the inner cover 69, such as those commonly used 
to cover adhesive bandages. 
The patch 62 is virtually impossible to remove and replace without showing 
visible signs of tampering. Thus, any analytes in sweat produced from skin 
under the concentration zone 14 during the time the patch is worn should 
be present in the patch. 
However, a particularly shrewd subject desiring to produce false negative 
results could obtain additional test patches. This shrewd subject would 
obtain false negative results by removing the initially applied test patch 
and replacing the test patch just prior to the time the patch is to be 
removed for assay. In order to ensure that the patch removed from the 
subject is the same patch which was initially applied to the subject, an 
identifying marker which is difficult to reproduce can be incorporated 
into the patch. For example, a bar code identification strip 67, similar 
to the bar codes used at supermarket check out stands can be incorporated 
into the patch, preferably just below the adhesive member 65. For best 
results in protecting against replacement of the patch, it is important 
that the identifying marker not be easily removed and replaced without 
providing an indication that the patch has been tampered with. 
In a preferred embodiment, the patch 62 has a filter 68 between the outer 
layer 65 and concentration zone 14, as described above in connection with 
FIGS. 1-3a. In a particularly preferred embodiment, the filter is a fluid 
permeable filter formed from a James River Paper Drape. 
The preferred adhesive members of the embodiment shown in FIG. 8, made from 
adhesive materials, such as Tegaderm, which are relatively weak in 
strength, have generally been designed for hospital patients who are not 
expected to perspire at high rates. Therefore, the moisture vapor 
transmission rate (MVTR) of these materials is relatively low. For 
example, the MVTR of Tegaderm is approximately 810 g/m*m*day. However, an 
active person may perspire at instantaneous rates as high as 26000 
g/m*m*day. Consequently, an active person may put out more sweat than 
these adhesive members can transmit to the atmosphere. If this sweat 
accumulates for any significant period of time, channels may be formed 
between the skin 64 and the adhesive member 65, allowing sweat to exit 
between the adhesive member and the skin, rather than be absorbed by the 
patch 62. 
B. Dermal Patches with Pinhole Perforations 
In accordance with a further embodiment of the present invention for 
preventing tampering, illustrated in FIG. 9, there is provided a patch 70 
having an adhesive member 72 which allows excessive sweat to be freely 
transmitted to the outside through pinhole perforations 73. The pinhole 
perforations may be distributed throughout a wide band 75 extending from 
the outer perimeter of the adhesive member to a narrow band 77 surrounding 
the test region 821 of the patch 70. 
Sweat produced beneath test region 81, over which there are no pinhole 
perforations 73, will be absorbed by the test region and will not be 
transmitted to the outside. The test region 81 includes the area of the 
patch 70 directly under the concentration zone 14 of the patch as well as 
the area immediately outside this zone. The narrow band 77 outside the 
concentration zone 14 of the patch has no pinhole perforations 73, and 
substantially restricts sweat forming underneath the test region 81 from 
communicating with the wide band 75 where sweat is transmitted to the 
outside. 
The width of the narrow band 77, is preferably between 0.025 and 0.250 
inches, more preferably between 0.05 and 0.125 inches. Narrow band widths 
less than the preferred width are not expected to keep contact with the 
skin, whereas narrow band widths greater than the preferred width may 
allow sweat channels to form, creating a path for sweat forming within the 
test region 81 to communicate with the outside. 
C. Use of Soluble Markers to Prevent Tampering 
A wearer of the patch in screenings for drugs of abuse would be expected to 
be rather creative in circumventing the protections of the patch. For 
example, a creative wearer could try to wash out the concentrated sweat 
components from the patch while the patch remains on the wearer's skin. 
Such washing could be attempted using a needle and syringe, such as those 
commonly used by intravenous drug abusers for drug injection. For those 
patches employing specific binding chemistry, attempted elution of the 
concentrated components using water would likely prove unsuccessful. Even 
for those patches not employing specific binding chemistry for the analyte 
being tested, elution with water alone would be difficult, requiring 
substantial volumes of water without triggering the detection of tampering 
through the removal of the patch from the skin. However, certain analytes 
could successfully be at least partially eluted using other solvents. 
Thus, in order to detect tampering with the patch through elution of the 
patch's contents using water or other solvents, a known amount of a marker 
which is readily soluble in either aqueous or non-aqueous solvents, can be 
added to the concentration zone during manufacture of the patch. The 
marker should be easily quantifiable. The marker should also be soluble in 
either aqueous or non-aqueous solvents depending on the likely route of 
elution of the analyte. Additionally, the marker should be suitable for 
prolonged skin contact and not be readily absorbed by the skin. A variety 
of dyes used in the production of makeup have these suitable 
characteristics. Oil red N (catalogue number 29,849-2) sold by Aldrich 
Chemical Corp. of Milwaukee, Wis. is a suitable lipid soluble dye. DG01 
red and DH60 yellow, both available from Virginia Dare Extract Co. of 
Brooklyn, N.Y. are suitable water soluble dyes. These water soluble dyes 
can be easily quantitated by elution from the patch followed by measuring 
optical density at 6500 nm for the red or 5800 nm for the yellow dye. The 
quantity of dye remaining can be compared with the range of the amount of 
dye found to be remaining in patches worn continuously without tampering 
for the same length of time. 
Non-visible markers could also be used to prevent the wearer of the patch 
from obtaining feedback regarding the extent of marker remaining in the 
patch. A colorless protein could be used for this purpose. A protein 
should be chosen that is easily identified in the lab, and also not be 
expected in human sweat. For example, Bovine gamma globulins, such as 
those sold by Sigma Chemical Co. of St. Louis, Mo., could also be used as 
a marker. The presence of these markers can be easily ascertained using 
Bovine IgG RID kit, available from ICN of Costa Mesa, Calif. 
Thus, when a suitable marker is employed within the patch, when the patch 
is analyzed for the particular analyte being tested, the patch can also be 
analyzed for the presence of the marker. For visible markers, such as 
makeup dyes, the presence of the marker may be analyzed by simply viewing 
the patch. For non-visible markers, the non-visible marker can be assayed 
along with the analyte. A significant decrease in the amount of marker 
present would be an indication of tampering through elution of the patch 
with a solvent. 
D. Use of Adulterants to Prevent Tampering 
A further method of tampering with the patch would be to add an adulterant 
to the patch which interferes with the assay chemistry. Numerous materials 
have been used to adulterate urine tests for drugs of abuse. The most 
commonly used, and generally most effective method of producing a false 
negative result in a urine test is to dilute the urine by ingestion of 
excessive amounts of fluids. Advantageously, this approach would not 
likely be successful in producing false negative results in the sweat 
collection patch of the present invention because interstitial 
concentration of drug metabolites is less likely to be influenced by 
ingestion of fluids. 
However, the addition of certain adulterants to the patch may interfere 
with the analysis chemistry. For example, acids and bases are known to 
interfere with assays for many drug metabolites by altering the 
metabolites' molecular structure. Additionally, many household products, 
such as detergents, ammonia, ascorbic acid (Vitamin C), and drain openers 
have been used to interfere with urine assays. These products produce 
extremes of pH or changes in other chemical parameters, and would be 
expected to result in trauma to the skin if used in connection with tests 
using the patch of the present invention. This trauma could be noted by 
the technician removing the patch. 
However, weak acids and bases, as well as eye drops sold under the 
trademark "Visine," are also known to interfere with a variety of assays 
for drug metabolites in urinalysis. However, these materials would not be 
expected to produce skin trauma. Thus, the use of these materials or other 
compounds interfering with an assay that do not cause skin trauma might go 
unnoticed by the technician removing the patch if the fluid contents of 
the material have had time to evaporate across the outer layer of the 
patch. However, "Visine" and most other adulterants would be expected to 
contain ionic materials. 
Thus, in order to detect the use of an adulterant, test strips can be 
incorporated into the patch which will detect the presence of various 
ionic materials or of extremes of pH. Litmus paper, such as Hydrion pH 
test paper, available from Baxter Scientific Products, is well known as an 
indicator of variances of pH. Accordingly, a short piece, for example 1 cm 
by 1/2 cm, of litmus paper could be incorporated into the patch to detect 
the various household products identified above which are known to be 
highly acidic or basic. 
Many test strips are also known for detecting the presence of ionic 
materials. For example Baxter Scientific Products supplies test strips 
from a variety of manufacturers for the detection of each of the following 
ions: aluminum, ammonium, chromate, cobalt, copper, ion, nickel, nitrate, 
peroxide, sulphite, tin, and calcium. In addition, test strips sold under 
the name "Qantab" are available from Baxter Scientific Products which 
identify the presence of chlorine ions. Other test strips available from 
the same supplier show glucose, protein, and ketones. Most of these test 
strips are read by simply comparing the color of the strips with a color 
chart included with the strips. Thus, the test strips provide a simple 
method of identifying the introduction of any of a variety of adulterant 
materials. 
In order to detect adulterants, such as "Visine," which contain ionic 
materials not known to the person performing the test, the tester must 
first assay the adulterant using a variety of test strips for ions to 
ascertain which ions are present in the materials. Once the appropriate 
ions are detected, the test strips corresponding to those ions can be 
incorporated into the patch in order to provide an indication that the 
adulterant has been added to the patch. 
Curiously, any particular adulterant might produce false negative results 
in some assays and false positive results in others. For each assay, the 
common adulterants which could be used to produce false negative results 
could be identified by testing the assays with the addition of small 
amounts of these known materials. Test strips could then be included which 
would detect the addition of these adulterants. 
In a preferred embodiment, the test strip or strips are placed facing the 
skin, where the strips are not visible to the wearer. The wearer is 
thereby not provided any feedback which aids the wearer in deception. 
E. Use of a Light Attenuation Layer to Prevent Tampering 
Many biological compounds are known to be affected by various spectral 
bands of light energy. For example, urine samples for analysis of LSD must 
be kept from exposure to strong light. Schwartz, Arch. Inter. Med. 148: 
2407-12 (1988). Further examples of compounds which require protection 
from light include cocaine hydrochloride, Martindale Extra Pharmacopoeia, 
29th Ed., p. 1213, and morphine sulphate, Id., p. 1310. It is expected 
that these and other compounds may be affected by exposure to light while 
being concentrated in the collection patch as well. 
Many analytes to be determined by a patch of the present invention may 
require collection and storage in the patch for prolonged periods of time 
(up to several weeks). These analytes are, therefore, exposed to 
substantial quantities of photoradiation. This quantity of photoradiation 
may be substantially greater than during a urine assay for the same or 
similar analyte. Also, many analytes have peculiarly high sensitivity to 
light. Thus, for analytes of peculiarly high photosensitivity or for those 
requiring prolonged collection and storage, it is particularly important 
to shield photosensitive analytes from light during prolonged storage in 
the patch. 
Accordingly, in still another embodiment of the present invention, 
illustrated in FIG. 10, there is provided a test patch 90 having a light 
attenuation layer 92 between the outer adhesive layer 65 and the 
concentration zone 14. In FIG. 10, the adhesive layer 65, is shown having 
stress razors 66, however, this feature is to be understood as being 
optional in this embodiment of the invention. 
The attenuation layer 92 is provided in order to attenuate the transmission 
of light into the concentration zone 14 where the biological compound of 
interest is being collected and stored. The layer 92 should be 
substantially impervious to the transmission of photoradiation, yet should 
also allow relatively unrestricted passage of the aqueous components of 
sweat to the outer adhesive layer 65. The layer 92 should be of sufficient 
porosity that diffusion of the aqueous components of sweat occurs at least 
as rapidly as sweat normally accumulates in the patch. 
Because light of many wavelengths is capable of degrading the various 
biological compounds which may be of interest, the layer 92 should have 
optical properties which attenuate light throughout a wide spectrum. 
Attenuation can be achieved by either reflection or absorption of incoming 
light. Reflection may be achieved through, for example, the use of any of 
a variety of metallic surfaces. When used in accordance with certain 
preferred embodiments of the present invention, the attenuation layer 92 
should allow passage of aqueous components of sweat. In order to provide a 
reflective layer with the suitable permeability, thin metallic foil with 
small holes can be provided. For example, aluminum foil, commercially 
available from many sources including Reynolds Aluminum Co., could be 
perforated with a plurality of small holes. 
Absorptive attenuation layers can be provided through the use of a black 
surface. Preferably, these surfaces would continue to allow permeability 
of aqueous components of sweat. It is important that any dye or 
pigmentation in the attenuation layer 92 not bleed when exposed to the 
aqueous components of sweat and also that it not interfere with any 
binding chemistry or in the analysis of the analyte. Any of a variety of 
thin black papers having these properties are commercially available and 
are suitable for use as in the attenuation layer. For example, black 
Deltaware cellulose membrane filters available from Baxter Scientific 
Products have been found to be especially useful for use as an attenuation 
layer. This product is available in a variety of porosities; more open 
pores are preferred. Thus, in the preferred embodiment, 0.6 micron black 
Deltaware filters are provided. 
In an alternative to the provision of an attenuation layer (not shown), the 
adhesive layer 65 can be made to attenuate light, either through 
absorption or reflection. As an example of an absorptive adhesive layer, 
black colorant, such as fine carbon black powder, could be incorporated 
into the extrusion of the adhesive sheet. 
VIII. Determining Allergic Sensitivity with a Dermal Patch 
A. Dermal Allergic Reactions 
In a further aspect of the present invention, a patch can be used to 
determine whether a subject is allergic to a particular allergen. 
Allergens include various forms of pollen, dust, animal skin and fur, 
chemicals such as insecticides or food additives, and foods. The presence 
of an allergen on the skin of an individual sensitive to that allergen 
causes an immune system reaction, known as an allergic reaction, in that 
individual. 
Certain components of the immune system involved in provoking an allergic 
reaction, such as IgE, complement, and various immune cells, are believed 
to be able to migrate in the dermis. Components of the immune system also 
circulate in the blood supplying the skin, and as part of an allergic 
reaction to an allergen on the skin the permeability of the blood vessels 
supplying the skin is increased. Immune components of the blood are 
thereby also believed to participate in a dermal allergic reaction. Thus, 
the presence of an allergen on the skin results in the migration and 
concentration of immune components of the body on the surface of the skin 
where the allergen is present. 
B. Using Dermal Patches to Determine Allergic Sensitivity 
A subject, preferably a mammal, can be tested for its sensitivity to an 
allergen by contacting an allergen to the skin of the subject and then 
detecting any immune components which pass through the skin of the subject 
and onto a patch of the present invention. In this embodiment, a patch is 
used which contains an allergen in fluid communication with the skin of 
the subject when the patch is worn on the skin of the subject. For 
example, the allergen can be contained in the absorbent material of the 
patch. 
In a preferred embodiment, an agent is also present in the patch in fluid 
communication with the skin of a wearer of the patch. The agent is one 
capable of increasing the permeability of the capillaries in the subject's 
dermis. Such an agent can thus increase the permeability of the 
capillaries in the dermis beneath the patch and facilitate the flow of 
immune components to the site of the allergen. 
To determine whether a subject is allergic to a particular allergen, a 
patch of the present invention which additionally includes an allergen is 
placed on the surface of the skin of the subject. In this embodiment, when 
perspiration reaches the patch, the allergen is in fluid communication 
with the skin of the subject and contacts the skin so as to cause an 
allergic reaction in the subject, if the subject is sensitive to the 
allergen. The patch will then be able to collect bodily components on the 
absorbent material of the patch which are associated with an allergic 
reaction, such as immune system components, which migrate to the location 
of the allergen. Once such components have accumulated in the absorbent 
material, the patch is removed, and the presence of such components is 
detected. If such allergic reaction-associated components are present on 
the patch, this is indicative that the subject is allergic to the allergen 
tested. 
Alternatively, the skin of the subject can be exposed to an allergen in any 
other way, such as simply by placing a sample of the allergen on the skin 
of the subject. Perspiration and other components expressed through the 
skin can then be accumulated in a patch of the present invention located 
proximate to the area of the skin of the subject which was exposed to the 
allergen. If an analyte indicative of an allergic reaction is then 
detected in the perspiration accumulated on the patch, the subject can be 
diagnosed as being allergic to the allergen. 
The patch used in this embodiment of the present invention can be any of 
the types previously described. Preferably, a specific binding partner 
capable of binding and concentrating particular bodily components 
indicative of an allergic reaction are included in the absorptive layer 
(or concentration zone) of this aspect of the present invention. 
As an example of the present embodiment of the invention, an antigen such 
as pollen can be placed in the absorptive layer of the patch so that when 
perspiration penetrates the absorptive layer and brings moisture to that 
layer, the allergen can migrate through the absorptive layer to the lower 
surface of the patch in contact with the skin and provoke an allergic 
reaction, if the subject is prone to develop an allergic reaction to the 
allergen. Alternatively, the allergen can be placed directly on the lower 
surface of the patch so that it immediately comes into contact with the 
skin of a subject wearing the patch. 
After an immune response is triggered in a subject who is allergic to the 
allergen, components involved in the response will increase in 
concentration in the vicinity of the patch, since it is the site of the 
allergen. As sensible and insensible perspiration pass through the skin 
and into the patch, the immune components which pass through the skin with 
such perspiration concentrate on the absorptive layer of the patch. 
Agents which increase capillary permeability in the dermis immediately 
beneath the patch are preferably included in the patch. Molecules 
circulating in the capillaries beneath the skin can thereby be made to 
diffuse into the interstitial space of the skin and from there into 
perspiration. Such perspiration can then carry these molecules into the 
patch so that they can be detected. 
The following examples describe only specific applications of the present 
invention. 
EXAMPLE 1 
Preparation of Monoclonal Antibodies to CK-MB for Use on a Test Patch 
In accordance with one known process for preparing monoclonal antibodies, 
mice such as Balb/c female mice or other mouse strains or even other 
suitable animals such as rats or rabbits are immunized with an amount of 
the CK-MB enzyme to initiate an immune response. The enzyme dosage and 
immunization schedule for producing useful quantities of suitable 
splenocytes can be readily determined depending on the animal strain used. 
The size and spacing of doses of CK-MB or other antigen are of prime 
importance in the antibody response. Fortunately, a wide range of antigen 
doses commonly affords immunity against harmful agents. Thus, a small dose 
of antigen is usually sufficient to initiate an antibody response, i.e., 
microgram quantities of proteins are frequently adequate. However, a 
minimum dosage for initiating an immune response does typically exist, 
although doses of antigen below the minimum dose necessary to initiate an 
antibody response will usually maintain antibody production which is 
already in process. For example, an initial immunization with 
approximately 50 .mu.g of the enzyme may be followed by a 
hyperimmunization series of five injections. 
When certain compounds which are themselves not necessarily antigenic are 
mixed with an antigen, enhanced antibody production against the antigen 
occurs, as evidenced by the appearance of large amounts of antibody in the 
serum, a prolonged period of antibody production, and a response to lower 
doses of antigen. Such substances are called "adjuvants" and include 
Freund's incomplete and complete adjuvants and alum gels. Thus, a given 
dose of antigen is usually more effective when injected subcutaneously 
with an adjuvant or when injected as repeated small aliquots than when 
administered intravenously. 
Typically, the adjuvants of Freund are preferred. The original "complete" 
Freund's adjuvant mixture consists of mineral oil, waxes and killed 
tubercle bacilli. Antigen is added to the adjuvant mixture in an aqueous 
phase to form a water-in-oil emulsion in which each water droplet is 
surrounded by a continuous oil phase containing tubercle bacilli. The 
mixture is commonly injected subcutaneously into experimental animals. 
Injection stimulates a marked granulomatous reaction with lesions 
consisting largely of collections of histiocytes, epithelioid cells and 
lymphocytes. The local lymph node shows a small increase in plasma cells. 
Following the immunization with a primary dose of a soluble protein 
antigen, specific antibodies normally first appear in the serum after a 
few days and then increase in number until about the second week. 
Thereafter, the number of serum antibodies slowly declines over a period 
of weeks to months. 
The first serum antibodies to appear after antigenization are IgM 
antibodies. These are usually followed by the appearance of IgG 
antibodies. Later, as antibody serum levels increase, IgM antibodies 
disappear, probably as a result of specific feedback suppression of IgG 
antibodies. 
After the "primary response" to a protein has passed, a second dose of the 
same antigen given months or even years later usually elicits an intense 
and accelerated "specific secondary response" in which serum antibody 
usually begins to rise within two or three days of exposure. The serum 
levels of antibody in a secondary response may reach as high as 10 mg per 
mi. 
The animal is subsequently sacrificed and cells taken from its spleen are 
suspended in an appropriate medium and fused with myeloma cells, such as 
those obtainable from the murine cell line Sp2/O-Ag14. The result is 
hybrid cells, referred to as "hybridomas," which are capable of 
reproduction in vitro and which produce a mixture of antibodies specific 
to each of the various recognizable sites on the CK-MB enzyme. 
The myeloma cell line selected should be compatible with the spleen cells, 
and optimally should be a cell line of the same species as the spleen 
cells. Although the murine cell line Sp2/O-Ag14 has been found to be 
effective for use with mouse spleen cells, other myeloma cell lines can 
alternatively be used. See, for example, Nature, 276: 269-270 (1978). 
The myeloma cell line used should preferably be of the so-called "drug 
resistant" type, so that any unfused myeloma cells will not survive in a 
selective medium, while hybrid cells will survive. A variety of drug 
resistant myelomas are known. 
The mixture of unfused spleen cells, unfused myeloma cells and fused cells 
are diluted and cultured in a selective medium which will not support the 
growth of the unfused myeloma cells for a time sufficient to allow death 
of all unfused cells. A drug resistant unfused myeloma cell line will not 
survive more than a few days in a selective medium such as HAT 
(hypoxanthine, aminopterin and thymidine). Hence, the unfused myeloma 
cells perish. Since the unfused spleen cells are nonmalignant, they have 
only a finite number of generations until they fail to reproduce. The 
fused cells, on the other hand, continue to reproduce because they possess 
the malignant quality contributed by the myeloma parent and the enzyme 
necessary to survive in the selected medium contributed by the spleen cell 
parent. 
The supernatant from each of a plurality of hybridoma containing wells is 
evaluated for the presence of antibody to a specific site unique to the 
CK-MB enzyme structure. Hybridomas are then selected producing the desired 
antibody to that specific site. This selection may be, for example, by 
limiting dilution, in which the volume of diluent is statistically 
calculated to isolate a certain number of cells (e.g., 1 to 4) in each 
separate well of a microliter plate. In this way, individual hybridomas 
may be isolated for further cloning. 
Once the desired hybridoma has been selected, it can be injected into host 
animals of the same species as those used to prepare the hybridoma, 
preferably syngeneic or semi-syngeneic animals. Injection of the hybridoma 
will result in the formation of antibody producing tumors in the host 
after a suitable incubation time, resulting in a very high concentration 
of the desired antibody in the blood stream and in the peritoneal exudate 
of the host. Although the hosts have normal antibodies in their blood and 
exudate, the concentration of these normal antibodies is only about 5% of 
the concentration of the desired monoclonal antibody. The monoclonal 
antibody may then be isolated in accordance with techniques known in the 
art. 
EXAMPLE 2 
Preparation of Microbead Test Patch 
One specific application of the present invention is the dual determination 
of skeletal muscle and cardiac muscle status as a result of exercise. A 
dermal patch is constructed in accordance with the embodiment illustrated 
at FIGS. 3 and 3a. The gauze layer is prepared by cutting a circular patch 
having an approximately 1-inch diameter from a Johnson & Johnson non-stick 
gauze pad. The inner and outer porous layers are next prepared by cutting 
two circular patches of Ultipor (nylon 6), from Pall Corporation in Glen 
Cove, N.Y. Ultipor membrane is both fluid permeable and microporous, and a 
membrane is selected having, for example, a 1 micron rating. The microbead 
layer is prepared by covalently bonding monoclonal antibody raised against 
CK-MB to a multiplicity of polystyrene beads having a mean particle size 
of at least about 10 microns. 
The patch is assembled by distributing approximately 0.2 gram of microbeads 
across the surface of one of the porous layers. The second porous layer is 
thereafter disposed adjacent the microbeads, and the gauze layer is next 
placed on top of the second porous layer. At this point, the patch is 
upside-down. The peripheral edges of each of the first and second porous 
layers and the gauze layers are secured together by conventional 
heat-sealing techniques. Thereafter, the subassembly is turned over and an 
annular torus of adhesive tape having approximately a 2-inch outside 
diameter and slightly less than a 1-inch inside diameter is secured 
thereto to produce a finished patch. 
EXAMPLE 3 
Cardiac Muscle Status Test 
The patch of Example 1 is then secured to the chest of a healthy 40-year 
old male and worn throughout a 36-mile (130-minute) bicycle ride. Upon 
removal of the patch following the ride, the test patch is immersed in a 
first solution containing an excess of enzyme labeled anti-CK-MB for 
approximately 30 minutes, to permit. conjugation of labeled antibody with 
immobilized analyte. The patch is then rinsed under tap water to remove 
unbound labeled antibody and immersed in a second solution containing a 
substrate for the bound enzyme label, which undergoes a color change when 
acted upon by the enzyme. Appearance of color through the top porous layer 
indicates the presence of CK-MB, and possible cardiac injury. Comparison 
to a color chart permits rough quantification. 
EXAMPLE 4 
Test for Use of Marijuana 
THC polyclonal antibody from sheep (available from Biogenesis, Bournmouth, 
England) is diluted 1:100 in PBS (pH 7.5). The antibodies are bound to 
Gelman 0.45.mu. (SU-450) Ultrabind Supported Membrane, following the 
protocol in Gelman Original Equipment Manufacturer application P.N. 
31,084. The membranes are air dried. Disks, 3/8 inch in diameter, are cut 
from the coated Gelman membranes. These 3/8 inch disks are mounted at the 
center of a 1/4 inch diameter hole cut in the center of a one inch 
diameter circle of Tegaderm 1625 Transparent Dressing (available from 
Minnesota Mining and Manufacturing, St. Paul, Minn.). 
Three mounted membranes are secured to the chest of a subject who then 
smokes a marijuana cigarette. Three mounted membranes are also secured to 
a subject who has never used marijuana in any form and who agrees not to 
use it for the next seven days. The membranes remain in place until they 
are removed, seven days later. Each of the removed membranes is flushed 
five times with 300 .mu.l of 0.2% Tween 20 in PBS. The membranes are 
incubated for 30 minutes in 100 .mu.l of E-Z Screen Cannabinoid enzyme 
conjugate from the EoZ Screen Test Kit (available from Environmental 
Diagnostics, Inc., Burlington, N.C.). 
After incubation, each membrane is flushed three times with 300 .mu.l of 
0.2% Tween 20 in PBS, followed by three flushes with PBS alone. The 
membranes are then incubated in TMB Membrane Peroxide Substrate (available 
from Kirkegaard & Perry Labs, Gaithersburg, Md.) for 10 minutes. A light 
blue background appears in all six membranes. White dots appear over the 
background on the three membranes taken from the subject who smoked a 
marijuana cigarette, indicating sweat gland output of sweat containing THC 
derivatives. No white dots appear on the three membranes taken from the 
subject who has never used marijuana. 
EXAMPLE 5 
Positive Control Patch 
Mouse anti-human IgG, Fc monoclonal antibody (available from ICN, Costa 
Mesa, Calif.) is diluted 1:100 in PBS (pH 7.5). The antibodies are bound 
to Gelman 0.45.mu. (SU-450) Ultrabind Supported Membrane, following the 
protocol in Gelman Original Equipment Manufacturer application P.N. 
31,084. The membranes are air dried. Disks, 3/8 inch in diameter, are cut 
from the coated Gelman membranes. These 3/8 inch disks are centered and 
mounted on a 1/4 inch diameter hole cut in the center of a one inch 
diameter circle of Tegaderm 1625 Transparent Dressing. 
Three mounted membranes are secured to the chest of five human subjects. 
The membranes remain in place until they are removed, seven days later. 
Each of the removed membranes is flushed five times with 300 .mu.l of 0.2% 
Tween 20 in PBS. The membranes are incubated for 30 minutes in 100 .mu.l 
of Horseradish peroxidase enzyme conjugated to goat anti-human IgG, Fc 
polyclonal antibody (available from ICN, Costa Mesa, Calif.) diluted 
1:1000 in PBS. 
After incubation, each membrane is flushed three times with 300 .mu.l of 
0.2% Tween 20 in PBS, followed by three flushes with PBS alone. The 
membranes are then incubated in TMB Membrane Peroxide Substrate (available 
from Kirkegaard & Perry Labs, Gaithersburg, Md.) for 10 minutes. Blue dots 
corresponding to individual sweat ducts appear over the background on all 
of the membranes, indicating that the chemistry of the patches is 
operative by their detection of the IgG expected in the sweat of all 
subjects. 
EXAMPLE 6 
Chemical Modification of Cocaine Collected on a Patch 
Absorption disks, 3/8 inch in diameter, are cut from Gelman membranes 
(Gelman 0.45.mu. (SU-450) Ultrabind Supported Membranes). These 3/8 inch 
disks are mounted at the center of a 1/4 inch diameter hole cut in the 
center of a one inch diameter circle of Tegaderm 1625 Transparent Dressing 
(available from Minnesota Mining and Manufacturing, St. Paul, Minn.) to 
form a patch. 
Three of such patches are secured to the chest of a subject who then 
ingests cocaine. Three patches are also secured to a subject who has never 
used cocaine in any form and who agrees not to use it for the next seven 
days. The patches remain in place until they are removed seven days later 
from each subject. 
The cocaine molecules and other components present in the membranes of each 
patch are then eluted from the membranes by soaking each of the membranes 
in a synthetic urine matrix for 30 to 60 minutes at room temperature with 
mechanical agitation to form an analyte solution. Following elution, the 
analyte solutions derived from each of the patches are brought to a pH of 
11 by the addition of NaOH to each of the solutions. The solutions are 
reacted for 20 minutes at pH 11 and at room temperature, after which the 
solutions are neutralized with HCl. 
Each solution is then subjected to diagnostic analysis with the Roche RIA 
system (Nutley, N.J.) for detecting the metabolite of cocaine BE. The 
subject who ingested cocaine tests positive for the cocaine metabolite BE, 
while the subject who did not consume cocaine over the test period does 
not test positive for BE. 
EXAMPLE 7 
Preparation and Use of a Dissolvable Absorption Disk 
Nylon 6/6 fibers (Vydyne 909 from Monsanto Co.) are formed into an 
absorbent gauze. Disks approximately 3/8 inch in diameter are cut from 
such gauze and are then mounted at the center of a 1/4 inch diameter hole 
cut in the center of a one inch diameter circle of Tegaderm 1625 
Transparent Dressing (available from Minnesota Mining and Manufacturing, 
St. Paul, Minn.) to form a patch. Such a patch is then applied to a 
subject. The subject is directed to ingest cocaine, and a quantity of 
perspiration is then allowed to accumulate on the patch. 
When a sufficient period of time has passed for a detectable amount of 
cocaine to accumulate on the patch, the patch is removed from the subject 
and placed in an insoluble container. A base capable of dissolving the 
Nylon 6/6 fibers is then poured over the patch. Once the nylon absorption 
disk is dissolved, the undissolved components of the patch are removed 
from the container. Since cocaine is converted into benzoylecgonine (BE) 
in the presence of a base, the cocaine contained in the disk is 
metabolized to BE when the disk is dissolved. 
The solution of the dissolved nylon, BE, and the other remaining components 
of the used absorption disk are next neutralized. This solution is then 
analyzed using a Roche RIA system (Nutley, N.J.). The BE in the solution 
is detected and the amount of BE concentrated in the absorption disk is 
determined. 
EXAMPLE 8 
Quantitative Determination of a Component of Perspiration 
To determine how much of an analyte is contained in a given volume of 
sweat, a patch is first constructed having a support layer made from a 
polyester-supported polycarbonate microporous membrane, manufactured by 
Nuclepore (Menlo Park, Calif.). Over this is placed an absorbent material 
such as Filtration Sciences medical grade paper (FS#39) for accumulating 
and concentrating perspiration. The surface area of the layer of absorbent 
material should be the same as or smaller than that of the support layer 
so that when placed on a subject's skin, only the support layer is in 
contact with the subject's skin. Over this layer is then placed an outer 
protective layer made of 1625 Tegaderm wound dressing made by the 3M 
Company (St. Paul, Minn.). This outer layer is of a larger surface area 
than either the support layer or the absorbent material and covers both of 
these layers. The outer layer separates the absorbent material from the 
outside of the patch and helps prevent perspiration from entering the 
absorbent layer except through the support layer. The outer perimeter of 
the outer layer has an adhesive on the side of the outer layer that faces 
the skin of a subject when the patch is applied to the skin of such a 
subject in order to secure the patch. 
Such a patch is next placed on the skin of a subject whose perspiration is 
to be tested for the presence of theophylline. The subject wears the patch 
for 7 days, during which time perspiration passes through the support 
layer at a rate of less than 6 grams/m.sup.2 /hour. After this the patch 
is removed and subjected to analysis to determine the amount of 
theophylline contained in the patch. 
To determine the volume of sweat that has passed into the absorbent 
material of the patch, the rate at which perspiration passed into the 
absorbent material is multiplied by the amount of time the patch was worn, 
i.e., 7 days. The amount of theophylline contained in the patch is then 
determined. These numbers are then related in order to determine the 
amount of analyte contained in a given volume of perspiration by dividing 
the amount of the analyte in the patch by the volume of perspiration which 
passed through the support layer into the absorbent material. 
EXAMPLE 9 
Preparation and Use of a Dermal Patch to Determine the Sensitivity of a 
Subject to an Allergen 
In order to determine whether an individual is allergic to cat hair, a 
preparation containing cat hair is first placed on the lower surface of a 
disk 3/8 inch in diameter made of Filtration Sciences medical grade paper 
(FS#39). The upper surface of the disk is mounted at the center of a 1/4 
inch diameter hole cut in the center of a one inch diameter circle of 
Tegaderm 1625 Transparent Dressing (available from Minnesota Mining and 
Manufacturing, St. Paul, Minn.). The patch is then placed on the surface 
of the skin of a human subject for approximately 3 days in order to 
accumulate perspiration on the disk and form a concentrate. The disk is 
then removed and analyzed to detect IgA against cat hair. The presence of 
IgA against cat hair indicates that the subject has expressed an allergic 
reaction to the cat hair antigen. 
Although this invention has been described in terms of certain preferred 
embodiments and assay schemes, other embodiments and assays that are 
apparent to those of ordinary skill in the art are also within the scope 
of this invention. Accordingly, the scope of the invention is intended to 
be defined only by reference to the appended claims. 
EXAMPLE 10 
Constructing a Dermal Patch which Inhibits Back-Diffusion 
Absorption disks, 3/8 inch in diameter, are first cut from Gelman membranes 
(Gelman 0.45.mu. (SU-450) Ultrabind Supported Membranes). These absorption 
disks are next soaked in a buffer of 0.1M acetic acid at a pH of 5.0, and 
the disk is allowed to dry. The 3/8 inch disks are then mounted at the 
center of a 1/4 inch diameter hole cut in the center of a one inch 
diameter circle of Tegaderm 1625 Transparent Dressing (available from 
Minnesota Mining and Manufacturing, St. Paul, Minn.) to form a patch. The 
buffer-soaked absorption disk could also be mounted onto the Tegaderm 
dressing while still wet. 
EXAMPLE 11 
Preventing the Back-Diffusion of a Drug of Abuse 
A patch is constructed according to Example 10. The buffer is added to the 
absorptive layer (absorption disk) of the patch in order to keep the pH of 
the patch and the surface of a subject's skin below the patch in the range 
of 4.5-5.0. When the patch is placed on the skin of the subject who has 
ingested one of the drugs of abuse listed in Table 2 (above), the patch 
concentrates the particular drug ingested, without any substantial 
back-diffusion. 
EXAMPLE 12 
Quantitatively Determining the Amount of an Analyte Present in a Subject 
A patch is constructed according to the method of Example 10 with an 
absorption disk having a surface area of 10 cm.sup.2. This patch is then 
placed on the biceps of a subject's arm. The subject, weighing 168 pounds, 
is given 126 mg of cocaine, which is subsequently nasally ingested. The 
patch is worn for approximately 200 hours, and the subject is not allowed 
to ingest any more cocaine. After 200 hours the patch is removed in order 
to determine how much cocaine has been collected. In this experiment, 
approximately 500 ng (0.5 mg) of cocaine is recovered on the patch.