Assay timed by electrical resistance change and test strip

Assays for liquid analytes are performed on a bibulous matrix containing dried reagents which produce a visibly detectable reaction product. Application of liquid sample to the bibulous matrix is detected by measuring a drop in resistance across the matrix. A preferred test article for performing the method includes the matrix and a pair of spaced-apart electrodes in electrical contact with a reaction zone on the matrix. The test article is used in combination with a detection unit having means for probing the electrodes to determine when electrical resistance in the matrix has decreased. The assay methods and apparatus are particularly useful for performing enzyme assays where signal developed as a function of time is critical.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates generally to methods and apparatus for 
detecting analytes in liquid samples. More particularly, the present 
invention relates to assays for detecting enzymes and components of enzyme 
pathways where both the time and temperature of the assay are controlled. 
Enzymes and enzymatic pathways play an important role in medicine and are 
the subject of many clinical tests. Examples of tests for single enzymes 
include tests for amylase, creatine kinase, alanine aminotransferase, 
aspartate aminotransferase, streptokinase, and thrombin. Examples of tests 
for enzymatic pathways include the prothrombin time test, and the 
activated partial thromboplastin time test. These later tests measure the 
enzymatic pathways involved in the extrinsic and intrinsic blood 
coagulation systems. 
Tests involving enzymatic reactions tend to be technically demanding. 
Enzymes and enzymatic pathways are typically assayed by measuring the rate 
at which the enzyme or enzymatic pathway in question converts a particular 
enzymatic substrate into its product. Such rate measurements require 
precise test timing since timing errors are directly translated into 
errors in the calculated amount of enzyme or component in an enzyme 
pathway. 
Temperature control is also critical since most enzymes have reaction rates 
that change dramatically as a function of temperature. Typically, higher 
temperatures produce a higher reaction rate, and lower temperatures 
produce a lower reaction rate. Enzymatic pathways, consisting of a number 
of temperature sensitive enzymatic steps, are often extremely temperature 
sensitive as a result of the cascade effect. 
Because of these technical demands, most enzyme and enzymatic pathway tests 
have tended to be complex, with performance generally limited to clinical 
laboratories. While such centralized testing may be adequate for 
infrequent, or routine medical conditions, visiting a doctor's office or a 
clinic on a regular basis for frequent or emergency medical tests is less 
acceptable. Thus there exists a need for convenient and simple tests, that 
can be performed by unskilled users for the measurement of enzymes and 
enzyme pathways. 
A variety of simplified "test-strip" assays have been developed to allow 
semi-skilled and unskilled users to detect analytes, such as pregnancy 
hormones, cholesterol, and glucose in urine, blood, and other patient 
samples. These test strip assays are most useful with non-enzymatic 
analytes where detection does not vary with minor fluctuations in test 
time or temperature. As previously discussed, enzymatic reactions are less 
tolerant, and require more precise control over these variables, generally 
rendering them unsuitable for use in the home or other non-clinical 
environment. 
one such test strip for performing blood glucose analysis, available from 
LifeScan Inc., Milpitas, Calif., relies on applying a drop of blood to a 
polyamide membrane having impregnated reagents which produce a chromogenic 
reaction in response to the glucose level in applied blood. Simplified 
low-cost tests such as this are often referred to as "home tests", to 
designate the fact that they have achieved a price and simplicity level 
that would allow widespread adoption in non-professional settings. 
For these reasons, it would be desirable to provide simplified assays, test 
articles, and test systems for detecting problematic analytes, such as 
enzymes and components of enzyme pathways in a variety of patient samples, 
such as blood, urine, and the like. In particular, the test articles and 
test systems should permit simplified assay protocols, preferably allowing 
for an automatic timing cycle which is initiated as soon as a sample is 
applied to a test article. The test articles and test systems should 
optionally also facilitate providing precise temperature control of a test 
region on the article, preferably without the need to enclose the test 
article in a heated chamber or other structure which limits the user 
access. The assays, test articles, and test systems should be readily 
usable with small sample volumes, particularly with small blood volumes 
such as a single blood drop. The test articles should further inhibit loss 
of the patient sample from the test article by evaporation or other means, 
particularly when using very small sample voluanes. The test article and 
test system should still further provide for monitoring of the presence of 
sample within the test article and be able to warn the user when excessive 
amounts of sample have been lost. Such test articles and test systems 
should be both easy to manufacture and easy to use, preferably being 
producible at relatively low costs. 
2. Description of the Background Art 
Assay devices which detect the presence of an analyte based on the 
enzymatic conversion of a substrate to a visible or detectable product 
within an absorptive matrix are described in U.S. Pat. Nos. 5,059,525; 
5,059,394; 4,256,693; 4,935,346; 3,791,933; and 3,663,374. Analytical 
apparatus having means for detecting sample application are described in 
U.S. Pat. Nos. 5,049,487 and 4,420,566. The '487 patent describes a timing 
circuit which is triggered by detecting a change in reflectance caused by 
wetting of a porous matrix. The '566 patent describes the measurement of 
light absorbance to confirm that a liquid sample has been applied to a 
slide prior to analysis. Systems for controlling the temperature of 
analytical test substrates are described in U.S. Pat. Nos. 4,720,372; 
4,219,529; and 4,038,030. Analytical test substrates comprising ion 
selective electrodes are described in U.S. Pat. Nos. 4,171,246 and 
4,053,381. 
SUMMARY OF THE INVENTION 
According to the present invention, apparatus and assays are provided for 
performing timed assays, particularly timed enzymatic assays under 
temperature control. The apparatus includes both a test article which 
receives a liquid sample being tested and a detection unit which receives 
the test article and optically determines a change in the test article 
resulting from presence of analyte in the sample over time. The observed 
change can thus be related to the presence (and usually amount) of analyte 
present in the sample. Such an apparatus permits performance of simplified 
assay protocols and formats where the application of sample to the test 
article present in the detection unit initiates a timing cycle and where 
the observed changes in the test article can then be detected as a 
function of time relative to the application of sample. 
The test article comprises a bibulous matrix having one or more dried 
reagents present therein. The reagents are selected to produce a 
detectable signal in the presence of an analyte in a liquid sample applied 
to the matrix. A pair of spaced-apart electrodes are disposed on either 
side of a target location on the matrix so that application of a liquid 
sample to the target location will lower electrical resistance between the 
electrodes. In this way, initial sample application can be detected by 
monitoring the resistance across the electrodes, with a lowering of the 
resistance initiating a timing cycle in the detection unit. Additionally, 
a subsequent rise in electrical resistance between the electrodes may 
indicate that sample has evaporated or otherwise been lost from the target 
location which can be a particular problem with relatively lengthy test 
protocols, particularly when employing small sample volumes. 
The test article can further be designed to reduce evaporative loss of 
liquid sample. The spaced-apart electrodes will preferably be configured 
to leave only a narrow gap therebetween, and the remaining portions of the 
matrix surface(s) will be covered by other materials. Usually, at least a 
portion of the remaining covering will be transparent to permit optical 
assessment of the matrix of the test article during assay protocols as 
described in more detail hereinafter. With such a design, only the narrow 
gap between electrodes (which defines a target location for receiving 
sample) will remain uncovered, thus permitting liquid sample application 
but preventing significant evaporation from the bibulous matrix 
thereafter. 
A preferred construction for the test article of the present invention will 
comprise a relatively thin membrane defining the bibulous matrix, a pair 
of spaced-apart metal foil strips defining the electrodes and covering 
substantially the entire top surface of the membrane, and a transparent 
layer or support, such as a clear plastic strip, covering the bottom 
surface of the membrane. Such a test article permits sample application on 
the top of the membrane through the gap between adjacent electrodes and 
further permits optical viewing of the membrane through the transparent 
bottom. 
The detection unit of the present invention will include a support stage 
for receiving the test article so that a reaction zone on the test article 
is disposed at a viewing location within the detection unit. The detection 
unit further includes means for measuring the electrical resistance across 
the test article when the test article is in place on the support stage. 
Usually, the resistance detecting means will include a pair of plates or 
probes which contact the bibulous matrix on either side of the reaction 
zone (which is located at or near the target location), preferably 
comprising a pair of plates which contact the electrodes described above. 
The detection unit, however, could be designed to function with test 
articles which do not include discrete electrodes. In particular, the 
detection unit could be designed to directly probe spaced-apart locations 
on the membrane surface. While such a design will generally be less 
preferred, it is considered to be within the broad scope of the present 
invention. 
The detection unit will further comprise a heater for heating the test 
article approximate the reaction zone. In the exemplary embodiment, the 
heater will include heated plates which contact the metal foil electrodes 
on either side of the target location on the test article. The metal foil 
electrodes are thus able to transfer heat from the heated metal plates of 
the detection unit directly to the target location and reaction zone 
without blocking or obscuring the target zone for a sample application. 
The detection unit still further includes optical viewing means for 
detecting an optical change in the reaction zone, typically through the 
transparent layer of the test article. In exemplary embodiments, the 
optical viewing means comprises a light source which directs light against 
the reaction zone and an optical detector which detects light emitted or 
reflected from the reaction zone. 
According to the method of the present invention, a volume of a liquid 
sample is applied to the target location on a bibulous matrix, where the 
bibulous matrix comprises one or more dried reagents which in the presence 
of analyte initiate time-dependent production of a detectable signal. The 
precise time the sample is applied can be determined by measuring a change 
in electrical resistance across the target location on the matrix. 
Production of the detectable signal is then measured at one or more times 
after the resistance change is first detected. In this way, the 
timed-dependant production of the detectable signal can be carefully 
monitored over time and accurately related back to the presence in amount 
of analyte in the liquid sample.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS 
Assays according to the present invention are useful for detecting a wide 
variety of soluble analytes in virtually any type of biological or other 
sample which is liquid or which can be liquified. The methods and 
apparatus will find their greatest use with patient specimens, such as 
blood, serum, plasma, urine, cerebral fluid, spinal fluid, ocular lens 
liquid (tears), saliva, sputum, semen, cervical mucus, scrapings, swab 
samples, and the like, but might also find use with food, environmental, 
industrial, and other samples where substances can be detected using 
enzymatic, immunological, and similar techniques. The methods and 
apparatus of the present invention are particularly useful for detecting 
analytes in very small liquid samples, typically having a volume in the 
range from about 3 .mu.l to 50 .mu.l, usually from 5 .mu.l to 30 .mu.l, 
but will also be suitable for use with much larger samples. In particular, 
the methods and apparatus of the present invention will be useful for 
analyzing very small blood samples, typically comprising a single drop of 
blood, employing non-skilled or semi-skilled personnel, frequently being 
self-administered. 
The methods and apparatus of the present invention are also particularly 
suited for perfoming enzymatic assays within a bibulous matrix where an 
analyte in a sample applied to the matrix initiates, modulates, or 
otherwise affects the enzymatic conversion of a substrate (initially dried 
within the matrix) into an optically detectable product, such as a 
colored, luminescent, or fluorescent product. The present invention 
provides for very accurate timing of such enzymatic reactions based on 
initiation of a timing cycle at the moment the liquid sample is initially 
applied to the test article. The test article and detection unit can 
further provide for accurate temperature control of the test article, 
further assuring accuracy of the test result. 
Assays according to the present invention provide for sensing of sample 
addition to a test article by measuring electrical resistance across a 
portion of the bibulous matrix to which the sample is applied. As the 
bibulous matrix and all reagents present therein are initially in a dry 
state, the resistance of the bibulous matrix prior to sample addition will 
be relatively high. Application of a liquid sample will immediately lower 
the electrical resistance across that portion of the matrix to which it is 
applied, thus providing a marker or trigger for initiation of a timing 
cycle. In the case of enzymatic reactions, build-up of the optically 
detectable product will take some time and is usually highly dependent on 
the amount of analyte present within the sample (as well as other factors 
such as temperature). By providing control circuitry within or in 
combination with the detection unit, the amount of optically detectable 
product accumulating within the test article over time can be precisely 
determined. Based on one or more such data points, usually a plurality of 
such data points, the amount of analyte initially present in the sample 
can then be accurately calculated. 
The detection unit of the present invention will comprise means for 
monitoring and measuring resistance across that portion of the test 
article to which liquid sample is to be applied. The resistance detecting 
circuitry will be connected to timing circuitry, which in turn will be 
connected to a suitable calculating means. Conveniently, the detection 
unit may include an interface for connecting to a digital controller, 
usually a microprocessor, which may be in the form of a general purpose 
personal computer. In this way, much of the timing and analytical function 
of the system of the present invention can be performed within the digital 
controller, with the specialized detection and sample conditioning 
functions being performed by the specialized apparatus of the present 
invention. 
Referring now to FIG. 1, an exemplary test article 10 constructed in 
accordance with the principles of the present invention will be described. 
The test article 10 includes a bibulous matrix structure 12, typically in 
the form of a flat membrane; a support structure 14, typically in the form 
of a transparent strip; and an electrode structure 16, typically 
comprising a pair of spaced-apart electrodes 18 separated by a gap 20. The 
membrane 12, support structure 14, and electrode structure 16 will be 
laminated together, for example by adhesive layers 22 and 24, as 
illustrated. The adhesive layers 22 and 24 will include central apertures 
26 and 28 which permit viewing of the membrane 12 through the support 
structure 14 and application of sample to the membrane through the gap 20 
in electrode structure 16. Preferably, the electrode structure 16 will 
include additional slots 30 formed in the electrodes 18, which slots 
define a target location for the application of liquid sample on the test 
article 10. 
The bibulous matrix 12 will be composed of a material which can absorb 
liquid and which can contain, in dried form, the reagent(s) necessary for 
performing a desired assay. A wide variety of bibulous matrix materials 
might be used, including paper, methyl cellulose, porous polymers, and the 
like. The dimensions of bibulous matrix 12 should be such that at least a 
portion of the matrix can be saturated with liquid sample to both 
solubilize the necessary reagent(s) and to permit transport of the 
detectable reaction product to the lower side of the matrix so that it 
will be visible through the support structure 14. 
In the preferred embodiment where small samples of blood are being 
analyzed, the bibulous matrix 12 will be a porous membrane structure 
composed of a hydrophilic (bibulous), non-swellable polymeric matrix 
material having pore dimensions which permit entry of blood plasma and 
proteins while excluding blood cells, particularly red blood cells 
(erythrocytes). The membrane should be composed of a single, continuous 
polymeric material with a foam-like structure consisting of a torturous 
network of channels having widths on the order of microns (.mu.m). The 
torturous network of channels is "densely packed" in that the "void 
volume" occupied by the empty space of the channels is an appreciable 
percentage of the total membrane volume, typically 10% or greater. Since 
all reaction chemistry, and subsequent signal generation, takes place in 
the void volume, a high void volume is desirable for producing a strong 
signal. A torturous network of channels is desired over straight and 
direct pores, (such as the short, direct pores obtained with nucleopore 
membranes), as longer average channel lengths tend to produce an 
increasing isolation between the zone of the membrane where reaction 
chemistry is occurring, and the excess sample remaining on the surface of 
the membrane. This helps to render the system less sensitive to variations 
in applied sample volume. 
In the specific case of blood coagulation assays, the porous membrane 
structure 12 will be impregnated with reagents necessary to induce 
coagulation in blood plasma which enters the interior of the porous matrix 
and to produce a detectable signal as an indication of the coagulation 
capability of the blood. It is particularly critical to the present 
invention that the polymeric matrix material of the porous membrane 12 be 
substantially free from interference with the coagulation pathway which is 
being induced. In particular, the polymeric matrix material should be free 
from surface effects, interactions, and artifacts which might induce 
coagulation or inactivate components such as enzymes, of the initiated 
pathway. Unintended initiation of a coagulation pathway could lead to 
false positive determinations while enzyme inactivation could lead to 
false negative determinations. It is therefore important that the 
polymeric matrix material have no promoting or diminishing effect on the 
coagulation reactions occurring within the membrane. Criteria can be for 
determining if a membrane is acceptable for use in coagulation testing are 
set forth in detail in copending application Ser. No. 07/874,667, the full 
disclosure of which is incorporated herein by reference. A particularly 
preferred polymeric matrix material for performing blood coagulation 
assays is a 0.45 .mu.m asymmetric polysulfone membrane material available 
from Filterite-Memtec, 9690 Deeveco Road, Suite 7, Timonium, Md. 21093, 
Catalog No. BTS-25. 
The region of bibulous matrix 12 which is beneath the sample application 
target location slots 30 will be the reaction zone. It is in this region 
that the matrix 12 is first wetted and where the actual chemical reaction 
which results in production of a detectable reaction product occurs. When 
used with very small samples, as described above, the reaction zone will 
typically be relatively small, frequently being 1 cm in diameter or less, 
often being less than 0.5 cm. The remaining regions of the matrix 12 which 
are not wetted by the sample will not undergo a chemical reaction and will 
not accumulate visible reaction product. 
Chemical reagents necessary for performing an assay according to the 
present invention will be impregnated within the bibulous matrix 12 and 
will be reconstituted by application of the liquid sample thereto. For the 
preferred enzymatic assays of the present invention, the reagents will 
include an enzyme substrate which is converted into an optically 
detectable product, typically a fluorescent, luminescent, or colored 
product, as a result of interaction with an enzyme. The enzyme may be the 
desired analyte or related to the analyte, or the enzyme may be added to 
the liquid sample and the production of detectable product by the enzyme 
modulated or otherwise affected by presence of analyte in the sample. The 
substrate may be a natural enzyme substrate which produces a natural 
detectable product, e.g. in the case of peroxidases, oxidases, hydrolases, 
and the like, or may be a synthetic substrate comprising a substrate 
group, such as a polysaccharide or peptide, which is cleavably linked to a 
reporter molecule, such as a chromogenic, chemiluminescent, or fluorogenic 
molecule. The presence or activity of the enzyme in the sample results in 
cleavage of the linker, causing a change in the optical characteristics of 
the reporter molecule. A variety of useful substrates are described in 
Haughland, Molecular Probes Handbook of Fluorescent Probes in Research 
Chemicals, Molecular Probes, Inc., Eugene, Oreg., the full disclosure of 
which is incorporated herein by reference. 
In the exemplary case of blood coagulation assays, necessary reagents 
include a coagulation initiator which initiates a preselected event or 
stage in either an extrinsic or intrinsic coagulation pathway and a 
substrate which is activated by a component which is produced in a 
subsequent stage of the coagulation pathway. A buffer will also be 
provided to maintain the test pH within a range compatible with the 
coagulation pathway, and optional reagents include flow control agents 
which decrease the chromatographic separation of the various test 
components as blood plasma enters the membrane, cofactors which sustain or 
enhance the chemical reactions of the coagulation pathway, stability 
enhancers, and pigments which enhance the optical characteristics of the 
test article. Typically, these reagents will be combined in one or more 
aqueous solution(s) which are applied to all or a portion of the polymeric 
matrix material. The matrix material may then be dried or lyophilized (and 
optionally mounted on the handle 14) to form a test article having the 
reagents non-covalently adsorbed therein. In some cases, it may be 
possible to covalently attach at least some of the reagents, although 
covalent attachment will usually not be necessary. Particular coagulation 
inhibitors, substrates, buffers, coagulation cofactors, and fluid control 
agents are set forth in application Ser. No. 07/874,667, the full 
disclosure of which has previously been incorporated herein by reference. 
The support structure 14 may take a variety of forms. The support structure 
14 is intended primarily to act as a physical support for the remaining 
components of the test article 10 and should be optically transmissive, 
preferably being completely transparent at the light wavelengths of 
interest to the assay protocol. In a preferred aspect of the present 
invention, the support structure 14 will also act as a moisture barrier in 
preventing loss of sample from the bibulous membrane 12. Suitable support 
structures 14 may be composed of transparent plastics, such as 
polystyrene, which is sufficiently thick and rigid to serve as a handle to 
permit manipulation of the test article 10 in the method steps of the 
present invention. Polystyrene strips having a length in the range from 
about 2 cm to 10 cm, a width in the range from about 0.5 cm to 2 cm, and a 
thickness in the range from about 0.1 mm to 0.5 mm have been found to be 
acceptable. 
The electrode structure 16, is intended primarily to facilitate measurement 
of the electrical resistance across the reaction zone of bibulous matrix 
12. It will be appreciated that the methods of the present invention could 
utilize electrical resistance probes which contact the bibulous matrix 
directly, that is without the need for providing intermediate contacting 
electrodes. The preferred test article 10 of the present invention, 
however, provides a suitable electrode structure in order to facilitate 
electrical resistance measurement using the detection unit, as described 
in more detail hereinafter. 
The electrode structure 16 preferably also serves as a cover for the 
bibulous matrix 12 to inhibit evaporative and other losses of sample fluid 
therefrom. Thus, the electrode structure 16 will generally cover a 
majority of the exposed surface of the bibulous matrix 12, typically 
leaving only the small gap 20 and slots 30 therebetween. 
Conveniently, both electrode halves 18 of the electrode structure 16 will 
be composed entirely of a conductive material, usually a metal foil. Use 
of the metal foil also acts to enhance heat transfer to the reaction zone 
of bibulous matrix 12, as described in more detail in connection with the 
detection unit hereinafter. It will be appreciated, however, that the 
individual electrodes 18 need not be composed entirely of electrically 
conductive material, and in fact only need to define a discrete conductive 
path from the bibulous matrix 12 to a location which can be probed or 
connected by the detection unit. 
It should be further appreciated that the test articles of the present 
invention are not limited to structures having the electrode 16 and 
support structure 14 sandwiched about a bibulous matrix 12. Test articles 
according to the present invention require only that a pair of 
spaced-apart electrodes be provided on either side of a sample target 
location on the bibulous matrix, where the electrodes facilitate 
interconnection with a electrical resistance measuring mechanism, such as 
that provided by the detection unit of the present invention. 
The adhesive layers 22, 24 may be composed of any suitable material, 
typically being double sided tape, such as that available from 3M 
Corporation, Minneapolis, Minn.. 
Referring now to FIG. 2, a detection unit 32 is schematically illustrated. 
The detection unit 32 includes plates 34 which contact the electrodes 18 
of the test article 10 when the test article has been inserted into the 
detection unit. The contact plates 34 act as probes in measuring 
electrical resistance across the reaction zone in matrix 12 beneath the 
target location slots 30. Plates 34 will be connected to conventional 
electrical resistance monitoring circuitry in order to provide an output 
suitable for a digital control unit (not illustrated) or microprocessor. 
In a preferred aspect of the present invention, plates 34 will be heated, 
typically by conventional electrical resistance heaters. For example, the 
plates 34 may have heating coils on the surface thereof or embedded 
therein. The heating coils, in turn, could be connected to a conventional 
power supply 35, with heat being controlled by conventional control 
circuitry or by a separate computer or other digital controller. Heat 
provided by plates 34 will be transferred by metallic electrode plates 18 
to the matrix 12 to an area at least partially overlapping with the 
reaction zone. 
Detection unit 32 further includes a system 40 for optically viewing the 
reaction zone when the test article 10 is in place within the unit. This 
optical system may monitor reflectance, fluorescence, or luminescence. In 
this example, a fluorescence system 40 is illustrated. This fluoresence 
system 40, as illustrated, includes a light source 42 and a notch filter 
44 which together provide a light beam 46 which falls on the reaction zone 
of test article 10 through the transparent support structure 14. Reflected 
or emitted light 48 passes through a second notch filter 50, with filtered 
light being detected by photodetector 52. In the exemplary case of blood 
coagulation assays, detection will usually be based on fluorescence, where 
the light beam 46 is provided at an excitation wavelength and the light 
beam 48 is emitted at a known emission wavelength. 
In summary, an assay according to the present invention may be performed by 
applying a liquid sample through the target location 30 on the electrode 
structure 16 of the test article 10. The liquid sample will flow through 
the gap 20 and other slots in the target location into a reaction zone 
within bibulous matrix 12. The presence of analyte in the sample will 
affect or modulate the production of a detectable reaction product within 
the matrix 12, eventually providing an optically detectable change on the 
lower surface of the matrix. The production of optically active reaction 
products may be observed by directing a light beam 46 through the 
transparent support 14 onto the reaction zone of membrane 12. The 
fluorescent material at present, will emit light at a fluorescent 
wavelength which is eventually detected by a photodetector 52. The amount 
of signal produced over time will depend on the amount of analyte present 
in the sample. 
FIG. 3 illustrates the changes in resistance and optical characteristics 
that typically take place during the course of an assay. Before the 
application of a fluid sample, the resistance across the electrode gap is 
extremely high. By contrast, the optical signal (here shown as 
fluorescence) is low. Upon application of sample at time zero, there is an 
immediate drop in resistance. By contrast, there is no corresponding 
change in the optical signal until appreciable amounts of the enzyme 
substrate have been converted to detectable product within the reaction 
membrane. As time continues, the resistance across the electrode junction 
on the reagent strip may increase, due to drying of the strip. As 
previously discussed, the system may choose to reject a particular sample 
as being insufficiently moist if the resistance measurement becomes too 
high. 
The following examples are offered by way of illustration, not by way of 
limitation. 
EXPERIMENTAL 
General Methodology 
Instrument: 
Observations were performed using a prototype instrument. The instrument 
optics included a Siemens BPW-34B photodetector mounted below a 550 
nanometer filter with a 25 nanometer bandwidth (S25-550-A, Corion 
Corporation, Holliston, Mass.). The specimens were illuminated by a 
Mini-Maglite.sup.TM LM3A001 light bulb (Mag Instrument Inc., Ontario, CA) 
with output filtered through a 500 nanometer filter with a 25 nanometer 
bandwidth (Corion Corporation, S25-500-A). The output from the 
photodetector was amplified by an instrumentation amplifier (described on 
page 89 of the IC Users Casebook, 1988, by Joseph Car, Howard Samms & 
Company), digitized by a 12 bit analog to digital converter, and recorded 
on an IBM compatible personal computer. Temperature control was achieved 
by placing reagent strip 10 in a heated reagent stage 50, as shown in FIG. 
4. The stage 50 included an upper heater and a lower heater with test 
strip 10 in the middle. A slot 52 was provided into the upper stage to 
facilitate insertion and removal of the strip 10 into the stage, and to 
provide easy sample access from the top. The stage was heated by eight, 
200 ohm 1/4 watt resistors connected in parallel. For the lower heater, 
four resistors were mounted on a thin circuit board 54 and were used to 
heat a 0.015" thick aluminum plate 56, with a 0.5" diameter hole 58 as an 
optics aperture. For the upper heater, four resistors were mounted on a 
circuit board 60 and used to heat two separate, and electrically isolated, 
0.015" thick aluminum plates 62. Circuit board 60 additionally had a 0.45" 
by 1.5" opening 52 in it to allow easy application of sample to the 
reagent strip's aperture 30. 
As shown in both FIG. 2 and FIG. 4, aluminum plates 62 on the stage's upper 
heater made a tight contact with the opposite sides of the test strip's 
foil surface electrodes. The stage's relatively thick aluminum plates 
acted to evenly distribute the heat emanating from the resistors, and 
helped insure good heat flow to the test strip's foil surface. The tight 
contact between the meter's aluminum plates, and the test strip's foil 
electrodes, was also used to form an electrical circuit that was used to 
detect the state of the strip's fluid detection sensor. 
The temperature was regulated by monitoring the stage via an Acculex RTDR-2 
temperature sensitive resistor (Keithley MetraByte Instruments Corp., 
Taunton Mass.). The electronics used to construct the fluid sensor and the 
temperature sensor are standard resistance monitoring circuits described 
on pages 55 and 62 of the Dascon-1 Manual, copyright 1983, by Metrabyte 
Corporation, Taunton, Mass. Unless otherwise noted, temperatures were 
maintained at 37.degree. C. by a feedback control program monitored by an 
IBM compatible personal computer. The computer system controlled a 
switching circuit that energized the heater, using a 6 volt power source, 
whenever temperatures dropped below 37.degree. C. In use, reagent strips 
were allowed to pre-equilibrate to 37.degree. C. for a minimum of 60 
seconds before sample was applied. 
Preparation of Boc-Val-Pro-Arg-Rhodamine 110: 
Five grams of Boc-Val-Pro-OH (catalog A-2480) were purchased from Sachem 
Bioscience, Inc., Philadelphia Pa.. This was conjugated onto (CBZ-Arg) 
2-Rhodamine-110 to produce (Boc-Val-Pro-Arg) 2-Rhodamine 110 following the 
methods of Mangel, et. al. in U.S. Pat. No. 4,557,862 and U.S. Pat. No. 
4,640,893. This formed the enzyme substrate used to detect thrombin 
production in the exemplary prothrombin time assay disclosed here. 
Preparation of photo-etched electrode apertures: 
A etching pattern was prepared, consisting of a repeated series of the 
following configuration: one 0.625" long, 0.015" wide electrode gap, as 
shown as 20 on FIG. 1, and two 0.185" long, 0.007 wide fluid apertures 
shown as 30 on FIG. 1, arranged in a cross configuration on the center of 
the electrode gap, with each slit separated by an angle of 60.degree. from 
the other slits. This pattern was etched onto I mil thick aluminum foil by 
Accutech, Inc., San Fernando, Calif. using conventional photo-etching 
techniques. The apertures were trimmed to a square 0.500".times.0.500" 
configuration, with the aperture centered in the middle of this square. 
Preparation of coated membrane: 
Coated membranes were prepared generally as described in copending 
application Ser. No. 07/874,667, now abandoned. In a 20 ml vial, using a 
small magnetic stirrer, the following were combined: 6 ml 0.2M HEPES pH 
7.4; 2 ml H2O; 1 ml 100 mM CaC12; and 500 mg Sigma poly vinyl alcohol 
P-8136; and were stirred for about 2 hours until the PVA had totally 
dissolved. 
One gram of Sigma protease-free bovine serum albumin A-3292 was added, and 
the mixture was allowed to stir for about 20 minutes until it had totally 
dissolved. A solution of 4.5x concentrated Dade-C thromboplastin (Baxter 
B4216-20, Baxter Healthcare Corporation, Miami, Fla.), was prepared by 
adding 880 .mu.l H2O to nominal 4 ml containers of thromboplastin. One ml 
of the concentrated (4.5x) Dade-C thromboplastin was added to the dip and 
was stirred for 10 minutes. One ml of a concentrated (4 mg/ml) solution of 
fluorescent thrombin substrate dissolved in 50% isopropanol, 50% H20 was 
then added, and the resulting mixture stirred for 10 minutes. 
Membrane dip: 
The large pore side (dull side) of a BTS-25 0.45 .mu.m asymmetric 
polysulfone membrane (Memtec/Filterite Corporation, Timonium Md.) was 
rapidly coated with fresh dip, and the excess dip was gently squeegeed 
off. The coated membrane was immediately dried in a mechanical convection 
oven at 50.degree. C. for 15 minutes. The membrane was then stored with 
desiccant under cool (4.degree. C.) conditions until use. 
Preparation of membrane strips: 
The reagent's support layer 14, FIG. 1, was composed of 10 mil thick 
transparent styrene. The various layers were held together by 0.5" wide 3M 
415 double sided adhesive tape, made by the 3M Corporation. To 
hand-assemble the strips, two preliminary assemblies were first created. 
The first assembly consisted of a series of 0.25" diameter holes spaced 
0.500" apart, pre-punched in the double sided adhesive tape. This adhesive 
tape 24, FIG. 1, was then applied to a length of foil containing a series 
of repeating apertures, each 0.500" apart. Excess foil, used to keep the 
repeating aperture structures intact in the absence of the adhesive tape, 
was then trimmed away, producing an "aperture tape" assembly with adhesive 
on one side, and a repeating series of electrode apertures on the other 
side. This was kept flat to keep the delicate electrode aperture 
structures intact. 
A second preliminary assembly was created consisting of a strip of 
transparent styrene laminated with 3M 415 tape and a length of 0.5" wide 
treated reaction membrane. To avoid migration of adhesive into the 
reaction membrane, and it's possible deleterious effects on storage, a 
series of 0.25" holes were punched every 0.500" in the sections of the 
tape that were immediately below the "reaction zone" on the final strip 
22, FIG. 1. Thus the coated membrane in the reaction zone did not come 
into immediate contact with any materials except the metallic foil 
aperture covering. The two preliminary assemblies were then aligned with 
the aid of a light box, and laminated together. The repeating subunits on 
the final assembly were then cut into individual strips for testing. 
This reagent, and the meter described previously, were then used to perform 
the following experiments. 
Experiment 1: 
Resistance drop upon addition of biological fluids: Reagent strips were 
prepared according to the above methods. The resistance across the strips 
was monitored as a function of time. Before addition of blood, the 
resistance across the electrode junction was effectively infinite (&gt;100 
mega ohms). Upon addition of blood, the resistance immediately dropped to 
about 30 kilo ohms. 
Experiment 2: 
Detection of insufficient sample by resistance readings. Reagent stripe 
prepared according to above methods were each given 1.0, 2.0, 3.0, and 4.0 
.mu.l of whole blood. After six minutes of reaction time, the respective 
resistance measurements of the various strips were: 
______________________________________ 
Sample Size Resistance 
______________________________________ 
1.0 .mu.l 2.5 mega ohms 
2.0 .mu.l 400K kilo ohms 
3.0 .mu.l 60K kilo ohms 
4.0 .mu.l 40K kilo ohms 
______________________________________ 
Insufficient sample thus could be detected by a substantial increase in 
resistance. 
Experiment 3: 
Effect of metal foil on reaction kinetics at low humidity: Strips were 
prepared omitting the metallic foil, and studied with Sigma coagulation 
control I, II and III plasma in a low ambient humidity environment and 
manual initiation of test timing. In the absence of the surface covering, 
lower fluorescence intensity developed, and the reaction kinetics for 
level II and level III (prolonged and very prolonged PT time controls) was 
significantly prolonged. 
Experiment 4: 
Effect of a hole in the transparent reagent support on reaction kinetics at 
low humidity: Strips were prepared containing the metallic foil, but 
additionally with a 0.25" diameter hole placed in the transparent support 
immediately below the reaction zone on the membrane. These were studied 
with coagulation control I, II and III plasma (C-7916, C-8916 and C-9916, 
Sigma Chemical Company, St. Louis, Mo.) in a low ambient humidity 
environment. In the absence of the surface covering, lower fluorescence 
intensity developed, and the reaction kinetics for level II and level III 
(prolonged and very prolonged PT time controls) was significantly 
prolonged. 
Experiment 5: 
Effect of metallic foil cover on temperature equilibration of reagent: 
Before they are applied to the meter, the test samples may be at a variety 
of different initial temperatures (typically 15.degree.-30.degree. C.). To 
give accurate results, the test article must equilibrate these different 
samples to the same reaction temperature (typically 37.degree. C.) as 
rapidly as possible. This experiment measured the relative effectiveness 
of a metallic foil cover, versus a non-metallic plastic cover, at rapidly 
equilibrating different samples. 
In this experiment, a series of test articles were made up with either 1 
mil thick aluminum covers, or 5 mil thick transparent styrene covers. 
These were placed into a meter stage and equilibrated to 37.degree. C. 10 
.mu.l drops of water (at 4.degree. C.) were then applied, and the 
temperatures monitored by a non-contact infrared thermometer (C-1600 
meter, Linear Laboratories Company, Fremont, Calif.). Both the aluminum 
foil and the transparent styrene were painted flat black (Model 2529 
marker, Testor Corp., Rockford Ill.), to provide equivalent conditions for 
the infrared beam. The resulting temperatures were: 
______________________________________ 
Time after application 
(sec.) Foil temp. 
Plastic temp. 
______________________________________ 
1 33 20 
5 35 23 
10 37 26 
15 37 28 
30 37 33 
45 37 37 
______________________________________ 
The results show that the foil covering equilibrated the sample to the 
desired temperature more quickly than did the plastic covering. 
Experiment 6: 
Performing fluorescence assays without a meter hatch: In the following 
experiment, the meter was programmed to take fluorescence measurements by 
turning its light source on briefly every 15 seconds, and recording the 
fluorescence value. Immediately before the meter's own light source was 
turned on, however, the meter also recorded a "background" value. This 
background value was then subtracted from each fluorescence measurement. 
It was found that when a non-light transmissive covering with a narrow slit 
was used, such as the metal foil arrangement discussed previously, the 
system was remarkably insensitive to the presence or absence of external 
light--e.g. normal room lighting, or indirect sunlight. This is attributed 
to a combination of factors, including the great attenuation of ambient 
light by the metal foil covering on the strip, the high fluorescence 
efficiency and amounts of the Rhodamine 110 fluorophore used in this 
experiment, and the background subtracting algorithm used here. 
Experiment 7: 
Clinical study: Venous blood from 27 patients, including 20 patients being 
treated with Coumadin, was drawn and stored in citrate. 10 .mu.l samples 
of whole blood from each patient were then run on the device as described 
above. Fluorescence measurements were taken every 15 seconds for six 
minutes. The time required for the fluorescence intensity to reach half 
maximum (T50%)was plotted versus the reference prothrombin times as 
determined on a MLA Electra 750 coagulation meter. A regression analysis 
was done using Microsoft Excel 4.0 software. 
A best fit polynomial to relate the T50% levels to the reference instrument 
was found to be prothrombin time (corrected)=1.93+0.079* T50%. The 
correlation coefficient (R.sup.2) was 0.92. The results from this 
experiment are shown in FIG. 5. The results show that this configuration 
can give good clinical results. 
Although the foregoing invention has been described in some detail by way 
of illustration and example, for purposes of clarity of understanding, it 
will be obvious that certain changes and modifications may be practiced 
within the scope of the appended claims.