Volume independent diagnostic device

A process and device for quantitative determination of analyte concentrations in liquid samples. The device includes one or more bibulous matrices constructed and arranged to essentially eliminate sample volume sensitivity from analyte determinations. In accordance with the present invention, the device includes one or more test reagent-treated bibulous matrices covered at least partially by an impermeable coating or film. A liquid sample is applied to the uncovered portion of the bibulous matrix such that the liquid sample is metered into the bibulous matrix by the impermeable coating or film. The sample chromatographs through the bibulous matrix until the matrix is saturated with liquid. The process is essentially sample volume independent, providing more uniform and accurate quantitative analyte determinations.

FIELD OF THE INVENTION 
The present invention relates to a process and device for quantitatively 
determining analyte concentrations in liquids. More particularly, the 
present invention relates to an improved method of quantitatively 
determining the concentration of specific analytes in liquids, whereby 
assay sensitivity to the amount of test sample applied to the device is 
essentially eliminated. Overall, more accurate and more reproducible 
analyte determinations result. 
BACKGROUND OF THE INVENTION 
Presently, there are numerous test devices available to simply and rapidly 
test liquids for the presence or absence of a particular analyte. For 
example, in regards to body fluids, tests are available to detect glucose, 
uric acid or protein in urine and to detect glucose, potassium ion or 
cholesterol in blood. Historically, most of the available diagnostic test 
devices have been sensitive to the volume of the test sample, as well as 
to the concentration of the particular analyte of interest. 
The medical profession has provided an impetus to growth in this field by 
requiring analyte determinations that yield accurate and reproducible 
results and that can be performed quickly and cheaply. Such test methods 
are especially desirable in small or private medical offices, where fast 
and accurate results are required, but the volume of assay samples is low 
enough to preclude the investment in expensive diagnostic devices. In 
addition, the medical profession requires simple and essentially foolproof 
diagnostic tests, that are performable by relatively untrained personnel, 
due to the expense of having highly qualified personnel perform these 
routine assays. 
As a result, several test methods have been developed that are inexpensive, 
fast, and easy to perform. Among the most widely used diagnostic devices 
are the "dip-and-read" type devices. These devices are widely used in the 
chemical analysis of biological fluids. For example, numerous 
physiological functions can be monitored merely by dipping a reagent strip 
device into a sample of body fluid, such as urine, and observing a 
detectable response, such as a change in color or a change in the amount 
of light reflected from or absorbed by the test device. 
Many of the "dip-and-read" test devices for detecting body fluid components 
are capable of making quantitative or at least semiquantitative 
measurements. Thus, by measuring the response after a predetermined time, 
an analyst can obtain not only a positive indication of the presence of a 
particular analyte in a test sample, but also an estimate of how much of 
the analyte is present. Such test devices provide the physician with a 
facile diagnostic tool as well as the ability to gauge the extent of 
disease or of bodily malfunction. 
Test devices such as these usually comprise one or more bibulous matrices, 
such as absorbent paper, having incorporated therein a particular test 
reagent that produces a detectable response, e.g., a color change, in the 
presence of a specific test sample analyte. Depending on the test reagent 
system incorporated into a particular bibulous matrix, the test devices 
can detect the presence of glucose, ketone bodies, cholesterol, 
triglycerides, bilirubin, urobilinogen, occult blood, nitrite, protein, 
urea, potassium, and other substances. A specific change in the intensity 
of color observed within a specific time range after contacting the test 
device with a sample, is indicative of the presence of a particular 
analyte and of the concentration of the analyte in the sample. 
It is customary for reagent test devices to contain more than one test 
reagent-containing bibulous matrix, such that each test reagent-containing 
bibulous matrix is capable of detecting a particular analyte in a liquid 
test sample. For example, a diagnostic device could contain a test 
reagent-containing bibulous matrix responsive to glucose in urine and 
another bibulous matrix responsive to ketones, like acetoacetate, such 
that the second bibulous matrix is spaced from, but adjacent to, the 
glucose-responsive bibulous matrix. One diagnostic test device for urine 
contains eight adjacent test reagent-containing bibulous matrices 
providing analytical measurement of pH, protein, glucose, ketones, 
bilirubin, occult blood, nitrite, and urobilinogen. 
For some assays, such as those performed on whole blood, it has been found 
that the normal method of simply dipping the diagnostic device into the 
liquid sample cannot be used. For such assays the amount or volume of the 
test sample contacting the test-reagent containing diagnostic device is 
very critical. For example, dry reagent methods for testing whole blood or 
serum require the application of specific test sample volumes and the use 
of sophisticated filtering and separating techniques to obtain accurate 
results. 
Therefore, in order to achieve accurate and reproducible results, a very 
precise amount of sample must contact the test-reagent containing bibulous 
matrix each time an assay is performed. For these assays in particular, 
the development of a volume independent diagnostic device, wherein a 
precise and reproducible amount of test sample contacts the test 
reagent-containing bibulous matrix each time an assay is performed, would 
be extremely advantageous. Such a device would overcome the problems of 
inaccurate and inconsistent results due to differences in the amount of 
test sample contacting the test reagent in the bibulous matrix. The method 
and device of the present invention is primarily directed at providing a 
constant sample loading of test sample per unit volume of a test 
reagent-containing bibulous matrix, and as a result, a truly volume 
independent diagnostic device. 
Other considerations also arise in developing a process and device for 
testing liquids for a specific analyte. One important consideration is the 
gross sample size needed to perform the analyte determination. For 
instance, in testing whole blood, an ideal process includes withdrawing a 
whole blood sample in "noninvasive" amounts, such as a pin prick drop, 
and immediately depositing the undiluted whole blood sample on the 
diagnostic device. 
Another consideration is the degree of sophistication of the technician 
performing the assay. It is often desirable to have relatively untrained 
personnel carry out routine assays and obtain accurate quantitative 
results. In these situations, it is important that the assay method 
contain a minimum of manipulative steps, be free of possible interferences 
or contamination and provide for easy measurement. For instance, among the 
several possible manipulative steps, testing the incorrect sample or 
applying the incorrect amount of sample to the diagnostic device are the 
most probable areas of assay error. 
Therefore, a need exists for a process and device for rapidly and 
accurately testing a small volume of liquid for a particular analyte, 
wherein accurate and reproducible analyte concentrations are obtained 
independent of sample size. Such a method and device for determining 
analyte concentrations in liquids would allow medical personnel to carry 
out analyte assays on a more routine and more confident basis. 
The "dip-and-read" method for testing urine samples has enjoyed great 
success due to the ease, speed and low cost of testing liquid samples. 
However, substantial work is still being performed in this area as 
diagnostic device uses are demanding more accurate test methods, for more 
analytes, on smaller liquid test samples. Diagnostic device users are 
especially eager to reduce the possibility of test inaccuracy, usually by 
making the test method simpler and less operator dependent. The ideal way 
to reduce operator dependence is to eliminate the need to dilute the 
liquid test sample and to eliminate the need to introduce a precise sample 
volume to the diagnostic device. It is to the latter objective that the 
method and device of the present invention is directed. 
Indicative of the work conducted in this field is U.S. Pat. No. 3,798,004 
to Zerachia et al, disclosing a semiquantitative method for determining 
analyte concentrations with a laminated device including a 
reagent-impregnated matrix placed between a pair of liquid impervious 
members. The analyte-containing sample contacts the reagent-impregnated 
matrix along the matrix-exposed edge of the test device. As the 
analyte-containing sample progresses inwardly towards the center of the 
matrix, the analyte reacts with the reagent impregnated in the matrix to 
provide a visible color pattern. The depth of the inward penetration of 
the color pattern is measured to determine the concentration of the 
analyte in the test solution. The amount of test sample absorbed by the 
matrix is limited by the capacity of the matrix to hold liquids, so a 
semiquantitative determination of an analyte is possible. 
Similarly, Morison in U.S. Pat. No. 3,620,677 describes an indicating 
device including an impervious material encasing a reagent-treated 
capillary material, such that at least some of the capillary material is 
exposed. The analyte-containing sample is applied to the capillary 
material, and, as the sample chromatographs through the reagent-treated 
capillary material, a chromogenic reaction occurs. After complete analyte 
reaction, the chromogenic reaction ceases. Therefore the point that the 
color formation ends gives a reading of the approximate analyte 
concentration. The method disclosed in the Morison patent is volume 
dependent, as the amount of analyte, and therefore the degree of the 
chromogenic reaction, increases with sample volume. 
Nussbaum in U.S. Pat. No. 3,810,739 discloses a reagent-impregnated paper 
encased in a plastic covering, so arranged such that a test sample can be 
introduced only through a single opening. A chromogenic reaction occurs 
within the device, and is observed through the translucent plastic used to 
encase the reagent-impregnated paper. The method and device of the 
Nussbaum patent are directed to qualitatively determining the presence or 
absence of a particular chemical or bacterial constituent of a solid or 
liquid sample. The device is constructed to retard sample evaporation 
during periods of long reaction incubation, especially at high 
temperatures. As a result, the volume or weight of test sample is an 
unimportant variable in the method disclosed in the Nussbaum patent. 
U.S. Pat. No. 4,069,017 to Wu et al discloses contacting adjacent matrices 
in order to provide uniform distribution of a test sample from a first, 
untreated matrix to a second, reagent-impregnated matrix. Although the 
device does reduce volume dependence, the configuration of the matrices is 
specifically designed to provide a uniform bilirubin distribution to the 
reagent-impregnated matrix for uniform binding and chromogenic reaction. 
Kondo et al U.S. Pat. No. 4,256,693 discloses a multilayered device 
including a layer to remove insoluble constituents, a waterproof layer 
with an opening, a porous spreading layer and a reagent layer, in that 
order. This device delivers a less-than-saturating volume of test sample 
from the spreading layer to the reagent layer as a method to ensure even 
sample distribution. Accordingly, the spreading layer does not and could 
not act as a barrier layer. 
U.K. Pat. No. 2,090,549 discloses an analytical device for metering a 
precise quantity of blood utilizing a metering channel. Capillary action 
draws only a certain amount of blood into the metering channel for 
distribution through a filter layer to a layer impregnated with a reagent. 
U.S. Pat. No. 4,647,430 discloses a volume independent test device wherein 
a reagent-impregnated matrix is completely covered by a microporous film. 
A liquid sample penetrates the film until the matrix is saturated, 
resulting in a constant loading of sample per unit area. 
SUMMARY OF THE INVENTION 
In brief, the present invention is directed to a process and device for 
quantitatively determining analyte concentrations in liquids, whereby 
assay sensitivity to sample volume is essentially eliminated. It has been 
found that the method and device of the present invention unexpectedly 
give accurate and reproducible analyte determinations from small, but 
variable, test sample volumes by consistently metering a precise amount of 
the test sample to an assay area of the device. 
According to the method of the present invention, an excess sample amount 
of the analyte-containing liquid is applied to a diagnostic device 
comprising a first bibulous matrix that is adjacent to, and in contact 
with, a second bibulous matrix. The second bibulous matrix has been 
treated with a reagent suitable for detecting a specific analyte. In 
addition, the reagent-treated second bibulous matrix and a portion of the 
untreated first matrix are covered with a liquid-impermeable coating or 
film. The impermeable coating or film serves to assist in metering the 
liquid sample into the first and second bibulous matrices and to act as a 
barrier to prohibit the test sample from directly contacting the 
reagent-treated bibulous matrix. From an area of liquid test sample 
addition, the sample is directed to the reagent-treated area of the second 
bibulous matrix by the wicking action of the matrix until the matrices are 
saturated with liquid. 
The method and device of the present invention are ideally suited for 
quantitative analyte determinations that are sensitive to test sample 
volume. By the method of the present invention, the liquid-impermeable 
coating or film meters the test sample into the bibulous matrices. The 
liquid-impermeable coating or film covering the reagent-impregnated 
bibulous matrix also prohibits excess test sample from entering the assay 
area of the device. The liquid test sample is directed to the test 
reagent-treated assay area of the bibulous matrix by the wicking action of 
the bibulous matrix from the area of sample application. The 
reagent-treated assay area of the matrices absorbs liquid test sample only 
up to the point of matrix saturation, thereby providing a specific volume 
of liquid test sample for analyte determination from an initially variable 
volume of applied liquid test sample. 
More particularly, in accordance with the present invention, the device 
includes one or more hydrophilic bibulous matrices securely adhered to a 
hydrophobic substrate. At least one of the bibulous matrices, or, in 
another embodiment, at least a portion of the single bibulous matrix, is 
treated with a sufficient amount of a test reagent suitable to test for a 
specific analyte. As used herein, the expression "test reagent" is defined 
as a chemical or mixture of chemicals causing an observable or detectable 
reaction when contacted with the substance being detected. The test 
reagent-treated bibulous matrix, or test-reagent treated portion of the 
bibulous matrix, is laminated with a liquid-impermeable coating or film to 
act as a barrier and prohibit the test sample from directly contacting the 
test reagent-treated matrix. The excess test sample is applied to the 
uncovered portion of the bibulous matrix or matrices. The bibulous matrix 
or matrices absorbs the test sample, and the sample is metered by 
capillary action to the test reagent-treated matrix or matrices located 
beneath the liquid-impermeable coating or film. After the test sample 
saturates the bibulous matrices, no further liquid sample enters the 
aatrices, thereby making the method and device essentially volume 
independent. If the technician inadvertently applies some of the liquid 
test sample to the top surface of the liquid-impervious coating or film, 
after the liquid test sample saturates the bibulous matrix or matrices, 
any excess liquid test sample is wiped away from the top surface of the 
coating or film before quantitative analyte determination. 
In accordance with an important feature of the present invention, the test 
sample passes chromatographically to an assay area of the same, or an 
adjacent, bibulous matrix, containing a suitable test reagent, to perform 
the assay of interest. The test sample reacts or interacts with the test 
reagent in the assay region to produce a detectable change in the assay 
region, such as a color change, to chromogenically indicate the presence 
or absence of a particular analyte and/or to allow quantitative 
determination of the particular analyte. 
Therefore, the present invention is directed to a method and device for 
rapidly and effectively determining the presence and concentration of a 
particular analyte, independent of the test sample volume applied to the 
device More particularly, in accordance with another important feature of 
the present invention, one or more test reagent-treated, hydrophilic 
bibulous matrices are so arranged such that a precise and reproducible 
volume of a liquid test sample is metered to the test-reagent treated 
bibulous matrix for qualitative or quantitative determination of a 
particular analyte. 
According to the method of the present invention, an excess amount of test 
sample is deposited on the area of the bibulous matrix that is not covered 
by the liquid-impervious coating or film. The test sample, metered into 
the bibulous matrices by the liquid-impervious coating or film, passes 
chromatographically through the bibulous matrix to the assay area of the 
same bibulous matrix, or to the assay area of a second, adjacent bibulous 
matrix. In either case, the assay area has been previously treated to 
include a test reagent such that the particular analyte of interest can be 
detected immediately without any further manipulative steps such as 
dilution. In addition, covering the assay area of the bibulous matrices 
with a liquid-impermeable coating or film precludes immediate contact of 
the test sample with the test reagent-treated matrix. Therefore, a precise 
amount of test sample, essentially independent of the initial volume of 
test sample applied to the test device, is metered to the assay area and 
the excess test sample does not contaminate the assay area. 
In general, the amount of test sample applied to the exposed surface of the 
bibulous matrix is unimportant. The test sample deposited on the exposed 
area of the bibulous matrix is metered to the test reagent-treated matrix 
until the bibulous matrix is saturated with liquid. After matrix 
saturation, any liquid test sample contacting the top of the 
liquid-impermeable coating or film is removed before quantitative analyte 
determination. The excess test sample is prevented from reacting with, or 
contaminating, the test reagent in the test reagent-treated bibulous 
matrix by the liquid-impermeable film or coating. 
According to the method of the present invention, the liquid-impermeable 
film or coating and the bibulous matrix or matrices allow an excess amount 
of test sample to be applied to the diagnostic device. The exposed 
bibulous matrix will chromatograph, or meter, a precise amount of test 
sample to the assay area. The amount of test sample introduced into the 
assay area will depend upon the size and absorptivity of the particular 
bibulous matrix of the device. Accordingly, the resulting diagnostic 
device achieves constant loading of test sample per unit volume of 
bibulous matrix independent of the amount of sample applied to the 
diagnostic device, thereby producing a volume independent diagnostic 
device. 
The method and device of the present invention are ideally suited for 
performing a broad range of volume sensitive analyte determinations that 
are conducted primarily on bibulous matrices such as paper. These analyte 
determinations include assays for triglycerides, galactose, glucose, 
potassium ions, AST, cholesterol, creatinine, ALT, phenobarbital, 
bilirubin, theophylline, urea, dye samples and other immunochemical 
assays. 
Therefore, it is an object of the present invention to provide a method and 
device for determining analyte concentrations in liquid samples quickly, 
effectively and accurately. 
It is also an object of the present invention to provide a method and 
device for the rapid, convenient and effective analysis of analyte 
concentrations in small liquid samples. 
Another object of the present invention is to provide a method and device 
for determining analyte concentrations in liquids that is independent of 
sample volume applied to the device. 
Another object of the present invention is to provide a method and device 
that delivers a constant and reproducible amount of analyte-containing 
liquid to an assay area of the device, independent of the amount of excess 
sample applied to the device. 
Another object of the present invention is to provide a method and device 
to determine analyte concentrations in small liquid samples with a minimum 
of manipulative steps. 
Another object of the present invention is to provide a method and device 
to test small liquid samples for analytes wherein the nature and the 
amount of the analytes are not altered by the device prior to contacting 
the test-reagent treated bibulous matrix.

DETAILED DESCRIPTION OF THE INVENTION 
In accordance with the present invention, accurate and reproducible 
quantitative analyte determinations can be performed on liquid samples, 
such that the assay results are essentially independent of the test sample 
volume applied to the diagnostic device. According to the method and 
device of the present invention, a small liquid sample, such as a pinprick 
amount, is sufficient for quantitative analyte determinations, as opposed 
to the relatively large volume test samples required in the typical 
dip-and-test method of analyte determination. Unexpectedly, the device of 
the present invention, after the introduction of an excess amount of the 
test sample, meters a precise and constant amount of test sample to a test 
reagent-treated bibulous matrix. The test sample then is assayed for a 
particular analyte within minutes without any additional manipulative 
steps 
Surprisingly, the process and device of the present invention provide 
rapid, economical and accurate quantitative analyte determinations on 
liquid test samples without having to add a predetermined and precise 
amount of test sample to the diagnostic device. Overall, the process and 
device of the present invention are ideally suited for routine analyte 
determinations in small laboratories or private physician offices, wherein 
the number of assays may be relatively low, but accurate results are still 
required within a short time period. 
As will become apparent, the method and device of the present invention are 
especially suited for analyte determinations utilizing chromogenic or 
other visual responses to test for the presence, absence or concentration 
of various analytes in liquid test samples For quantitative analyte 
determinations, it is of primary importance that a known, constant amount 
of test sample reach the assay area of the diagnostic device in order for 
the chromogenic reaction to be detected and accurately measured. For 
example, presently used diagnostic test devices are not only sensitive to 
the concentration of the analyte of interest, but are also sensitive to 
the volume of test sample applied to the diagnostic device. Table I 
illustrates the affect of sample volume size upon various quantitative 
analyte determinations made on whole blood. For example, for each 1% 
change in sample volume, the quantitative assay of AST will change by 
0.3%. 
TABLE I 
______________________________________ 
VOLUME DEPENDENCY OF ANALYTE 
DETERMINATIONS 
Volume Dependence 
(% Change in Assay for a 
Analyte 1% Change in Sample Volume) 
______________________________________ 
AST 0.3 
Creatinine 0 to 0.5 
Cholesterol 1.3 
Triglycerides 
1.0 
Potassium 0.1 
Dye Samples 1.0 
______________________________________ 
In accordance with the present invention, it has been found that sample 
volume independence in analyte determinations of liquid samples is 
attained by metering the liquid test sample into one or more bibulous 
matrices. The test sample, as it is metered to the bibulous matrix, 
chromatographs through the bibulous matrix by wicking action until the 
matrix is saturated with test sample. After the bibulous matrix is 
saturated by the liquid, no further test sample enters the matrix. 
According to the method and device of present invention, the bibulous 
matrices absorb liquid only up to the point of matrix saturation. Thus, 
since no liquid enters the bibulous matrix after saturation, the device of 
the present invention is essentially volume independent, giving accurate 
and reproducible analyte determinations regardless of the volume of test 
sample applied to the device. 
Generally, the bibulous matrix of the present invention can be any 
hydrophilic, absorbent matrix that is amenable to treatment with a test 
reagent. The bibulous matrix also should permit the test sample to 
uniformly chromatograph through the matrix by wicking action at such a 
rate as to allow rapid analyte determinations. In addition, the bibulous 
matrix should not contaminate the test sample by test sample extraction of 
components of the bibulous matrix, by removing test sample constituents 
through chemical or physical interactions, or by appreciably altering the 
test sample in a way to make the subsequent analyte assays inconclusive, 
inaccurate or doubtful. 
The bibulous matrix of the present invention is a hydrophilic material, 
possessing the above-mentioned characteristics, that allows the test 
sample to move chromatographically, in response to capillary forces, 
through the matrix. The test sample migrates essentially unchanged through 
the bibulous matrix to an assay area of the device and is retained by the 
bibulous matrix. 
The bibulous matrix can be any hydrophilic material that allows the test 
sample to pass through the matrix to contact the assay area for analysis. 
Suitable bibulous matrices include hydrophilic inorganic powders, such as 
silica gel, alumina, diatomaceous earth and the like; sponge materials; 
glass fibers; argillaceous substances, cloth, hydrophilic natural 
polymeric materials, particularly cellulosic material, like cellulosic 
beads, and especially fiber-containing papers such as filter paper or 
chromatographic paper, synthetic or modified naturally-occurring polymers, 
such as nitrocellulose, cellulose acetate, polyvinyl chloride, 
polyacrylamide, polyacrylates, polyurethanes, crosslinked dextran, 
agarose, and other such crosslinked and noncrosslinked water-insoluble 
hydrophilic polymers. Hydrophobic substances, such as a hard, porous 
plastic, are not suitable for use as the bibulous matrix of the present 
invention. 
The bibulous matrices included in a device of the present invention can 
have different physical characteristics and can be of different chemical 
compositions or a mixture of chemical compositions. The matrices can also 
vary in regards to smoothness and roughness combined with hardness and 
softness. However, in every instance, the bibulous matrix must include a 
hydrophilic material. Regardless of the exact composition of the bibulous 
matrix, the primary considerations are absorbency, wicking action and 
transmittal of substantially unaltered test samples. 
In accordance with an important feature of the present invention, the 
hydrophilic, bibulous matrix includes a cellulosic material, such as 
paper, and preferably a fiber-containing paper, such as filter paper. 
Filter paper possesses all of the qualities required of a bibulous matrix 
of the present invention, plus the advantages of abundant supply, 
favorable economics, and a variety of suitable grades. Such paper has been 
found to be extremely satisfactory for use as a matrix material for 
suspending, transmitting and positioning both the test reagent and the 
test sample. Filter paper has been found to have particular utility in 
retaining the testing reagent, and in chromatographing the test sample by 
wicking action to the assay area. As known to those skilled in the art of 
basic chemistry, filter paper can be obtained in a variety of thicknesses 
and porosities. Since the method of the present invention requires the 
test sample to be metered into the bibulous matrix and subsequently 
chromatographed to the assay area of the bibulous matrix, it is well 
within the experimental techniques used by those skilled in the art of 
preparing test devices to determine the proper balance between bibulous 
matrix size, thickness and porosity in relation to concentration of test 
reagent. 
To achieve the full advantage of the present invention, the bibulous matrix 
is in the form of a pad, having dimensions of, for example, approximately 
0.25 cm by 0.5 cm to 0.5 cm by 1 cm. A pad of these dimensions allows an 
excess amount of test sample, applied at one end of the pad, to 
chromatograph through the pad to the assay area of the device within a 
reasonably short time. Increasing the size of the bibulous matrix 
substantially increases the time of analyte determination, and also 
requires a larger test sample. 
In one embodiment of the present invention, the first bibulous matrix, 
after appropriate sizing, e.g., 0.5 cm.times.0.5 cm, is secured to a 
transparent or opaque, hydrophobic plastic handle. Then, immediately 
adjacent to and in contact with the first bibulous matrix, a second 
bibulous matrix, containing the test reagent necessary to assay for a 
particular analyte, is secured to the hydrophobic handle. The 
liquid-impervious coating or film then is laminated over the second 
bibulous matrix and, optionally, over a portion of the first bibulous 
matrix. 
In accordance with an important feature of the present invention, the 
liquid impervious film or coating allows only that volume of liquid test 
sample sufficient to saturate the matrices to enter the bibulous matrices. 
The liquid impervious film or coating prohibits excess liquid test sample 
from entering the bibulous matrices, and thereby making the method and 
device volume independent. It has been found that by applying an excess 
amount of liquid test sample to a bibulous matrix of a diagnostic device 
having two contacting, adjacent bibulous matrices, lacking a covering 
layer of liquid impervious film or coating, results in an excess amount of 
liquid test sample entering the second bibulous matrix. The excess sample 
amount was observed as a free liquid collecting on the top surface of the 
second bibulous matrix. According to the method and device of the present 
invention, and as will be discussed more fully hereinafter, the liquid 
impermeable film or coating covering the two bibulous matrices prevents 
any excess liquid test sample from entering the second bibulous matrix. 
At the present time, reagent-strip test formats utilize a test-reagent 
treated bibulous matrix, whereby a fixed volume of test sample is applied 
to the reagent test strip, and the reflectance is measured at a fixed time 
or series of times. In using this format, if the volume of test sample 
varies, the quantitative determination of analyte varies. Therefore, to 
illustrate the new and unexpected results of the method and device of the 
present invention, several methods for reducing analyte determination 
dependence upon test sample volume were compared. More specifically, the 
methods included applying test samples to each of the following test 
devices: 
0. The standard control format, utilizing a single test reagent-treated 
bibulous matrix, with the test sample applied directly to the test area. 
1. The film cover format, wherein an untreated bibulous matrix is placed 
adjacent to and in contact with a second test reagent-treated bibulous 
matrix. The test-reagent treated matrix is covered completely by a clear, 
liquid impervious coating or film. The test sample is applied to the 
uncovered and untreated bibulous matrix and chromatographs through the 
untreated bibulous matrix to the reagent-treated bibulous matrix. After 
saturation, no further sample enters the bibulous matrices and any excess 
sample then is removed. 
2. The touch off format, wherein a single test-reagent treated bibulous 
matrix is utilized as in control format 0. A liquid test sample is applied 
to the single test-reagent treated bibulous matrix, then excess sample is 
removed from the bibulous matrix by contacting the matrix with a dry piece 
of filter paper and allowing the excess sample to wick from the bibulous 
matrix. 
3. Blot lightly format, wherein a test sample is applied to a single 
test-reagent treated bibulous matrix (format 0), then the excess sample is 
removed by blotting lightly with a dry piece of filter paper. 
4. Blot heavily format, wherein a test sample is applied to a single 
test-reagent treated bibulous matrix (format 0), then the excess sample is 
removed by blotting heavily with a dry piece of filter paper. 
5. Adjacent dry pad format, wherein an untreated bibulous matrix is placed 
adjacent to and in contact with a second test reagent-treated bibulous 
matrix A test sample is applied to the test reagent-treated bibulous 
matrix, and the untreated bibulous matrix wicks off the excess test sample 
from the test reagent-treated bibulous matrix. 
6. Adjacent dry pad with bridge format, wherein an untreated bibulous 
matrix is placed next to a second test-reagent treated bibulous matrix, 
but separated from the second matrix by a distance of 1 to 2 mm. A thin 
bridge of tissue paper connects the two bibulous matrices. The test sample 
is applied to the test reagent-treated bibulous matrix, and excess sample 
can wick to the untreated bibulous matrix through the tissue paper bridge. 
7. Apply to adjacent pad format, wherein an untreated bibulous matrix is 
placed adjacent to and in contact with a second test-reagent treated 
bibulous matrix as in format 5, however, the test sample is applied to the 
untreated bibulous matrix. 
8. Apply to adjacent pad with bridge format, wherein the test device is 
prepared as in format 6, however, the test sample is applied to the 
untreated bibulous matrix and wicks through the tissue paper bridge to the 
test reagent-treated bibulous matrix. 
9. Dip reagent format, utilizing the standard control format (format 0), 
except the test device is dipped into the test sample, as opposed to 
applying the test sample to the test device. 
The volume dependence of the above-described test devices was determined by 
applying dye solutions to test devices having untreated filter paper as 
the bibulous matrices and/or by applying analyte calibrator solutions to 
test devices having bibulous matrices treated with the appropriate test 
reagent for that particular analyte solution. Individual test results were 
determined by taking a reflectance measurement with a reflectance 
photometer at a suitable time and wavelength for that particular analyte 
determination. The reflectance, as taken from the reflectance scale of 
zero to one, was incorporated into the Kubelka-Munk function: 
EQU K/S=(1-R).sup.2 /2R, 
wherein K is the absorption coefficient, S is the scattering coefficient 
and R is reflectance. In FIGS. 1 through 20, the K/S values were plotted 
against the volume of liquid test sample applied to the test device. 
Generally, it can be stated that as reflectance decreases, the K/S value 
increases. 
For example, FIG. 1 shows three graphs of K/S values versus sample volume. 
Each graph shows the effect of adding increased dye sample to a diagnostic 
device having untreated bibulous matrices made of filter paper and 
arranged in the standard control format (0). Three separate dye solutions, 
having dye concentrations of 1.times.10.sup.-5 M, 2.times.10.sup.-5 M and 
3.times.10.sup.-5 M, were used. The graphs plotted in FIG. 1 illustrate 
the data tabulated in Example 1. In Example 1, each test was run in 
duplicate, using two instruments. The dye solutions were applied to 
untreated filter paper matrices, and the reflectance was measured at a 
wavelength of 630 nm (nanometers). The tabulated K/S values are given as 
duplicate pairs of the average K/S values for three replicate trials. The 
standard deviation over the replicate trials is also tabulated. Examples 2 
through 28 include similarly conducted tests, with similarly tabulated 
test results. 
EXAMPLE 1 
Dye Solution Applied to Untreated Filter Paper--Standard Control Format (0) 
FIG. 1: Tests performed in duplicate, using two instruments, two 
0.5.times.0.5 cm filter paper matrices, wavelength--630 nm. 
__________________________________________________________________________ 
VOLUME OF DYE 
CONCENTRATION OF DYE SOLUTION 
SOLUTION APPLIED 
1 .times. 10.sup.-5 M 
2 .times. 10.sup.-5 M 
3 .times. 10.sup.-5 M 
TO TEST DEVICE 
K/S 
STD. DEVIATION 
K/S 
STD. DEVIATION 
K/S 
STD. DEVIATION 
__________________________________________________________________________ 
20 uL 0.208 
0.003 0.465 
0.011 0.689 
0.046 
0.198 
0.006 0.443 
0.017 0.670 
0.023 
30 uL 0.417 
0.022 1.043 
0.053 1.619 
0.142 
0.381 
0.011 0.936 
0.070 1.620 
0.255 
40 uL 0.542 
0.005 1.559 
0.028 2.754 
0.061 
0.540 
0.012 1.633 
0.098 2.896 
0.201 
__________________________________________________________________________ 
EXAMPLE 2 
Cholesterol Solution Applied to Treated Filter Paper--Standard Control 
Format (0) 
FIG. 2: Data obtained as in Example 1, using appropriate commercial test 
reagent and wavelength--600 nm. 
__________________________________________________________________________ 
VOLUME OF CHOLESTEROL 
CONCENTRATION OF CHOLESTEROL SOLUTION 
SOLUTION APPLIED LOW MEDIUM HIGH 
TO TEST DEVICE K/S STD. DEVIATION 
K/S STD. DEVIATION 
K/S STD. 
__________________________________________________________________________ 
DEVIATION 
20 uL 0.450 
0.006 0.663 
0.003 0.910 
0.025 
30 uL 0.656 
0.017 0.993 
0.024 1.173 
0.079 
40 uL 0.768 
0.033 1.067 
0.039 1.205 
0.051 
__________________________________________________________________________ 
EXAMPLE 3 
Triglyceride Solution Applied to Treated Filter paper--Standard Control 
Format (0) 
FIG. 3: Data obtained as in Example 1, using appropriate commercial test 
reagent and wavelength--580 nm. 
__________________________________________________________________________ 
VOLUME OF TRIGLYCERIDE 
CONCENTRATION OF TRIGLYCERIDE SOLUTION 
SOLUTION APPLIED LOW MEDIUM HIGH 
TO TEST DEVICE K/S STD. DEVIATION 
K/S STD. DEVIATION 
K/S STD. 
__________________________________________________________________________ 
DEVIATION 
20 uL 0.301 
0.007 0.723 
0.013 1.146 
0.042 
30 uL 0.404 
0.020 1.286 
0.025 2.176 
0.080 
40 uL 0.512 
0.007 1.742 
0.016 3.132 
0.070 
__________________________________________________________________________ 
EXAMPLE 4 
Potassium Ion Solution Applied to Treated Filter Paper--Standard Control 
Format (0) 
FIG. 4: Data obtained as in Example 1, using appropirate commercial test 
regent and wavelength--640 nm. 
__________________________________________________________________________ 
VOLUME OF POTASSIUM 
CONCENTRATION OF POTASSIUM ION SOLUTION 
ION SOLUTION APPLIED 
LOW MEDIUM HIGH 
TO TEST DEVICE K/S 
STD. DEVIATION 
K/S 
STD. DEVIATION 
K/S 
STD. DEVIATION 
__________________________________________________________________________ 
20 uL 0.325 
0.007 0.450 
0.012 0.603 
0.009 
30 uL 0.378 
0.005 0.475 
0.017 0.653 
0.014 
40 uL 0.380 
0.016 0.501 
0.008 0.693 
0.017 
__________________________________________________________________________ 
EXAMPLE 5 
Glucose Solution Applied to Treated Filter Paper--Standard Control Format 
(0) 
FIG. 5: data obtained as in Example 1, using appropriate commercial test 
reagent and wavelength--b 620 nm. 
__________________________________________________________________________ 
VOLUME OF GLUCOSE 
CONCENTRATION OF GLUCOSE SOLUTION 
SOLUTION APPLIED 
LOW MEDIUM HIGH 
TO TEST DEVICE 
K/S 
STD. DEVIATION 
K/S 
STD. DEVIATION 
K/S 
STD. DEVIATION 
__________________________________________________________________________ 
20 uL 0.134 
0.002 0.329 
0.006 0.537 
0.035 
30 uL 0.203 
0.004 0.654 
0.018 1.096 
0.011 
40 uL 0.264 
0.003 1.006 
0.029 1.882 
0.045 
__________________________________________________________________________ 
As shown in FIGS. 1 through 5, the dependence upon sample volume for test 
samples applied directly to devices having the standard control format (0) 
is quite large. The volume dependence is seen in the large slope of the 
graphed functions in FIGS. 1 through 5. Quantitatively, it has been found 
that the standard control format (0) gives a change of calculated 
concentration of analyte of approximately 1% for each 1% variation in 
sample volume. This large change in apparent analyte concentration shows a 
relatively large dependence upon sample volume, thereby necessitating the 
application of a precisely-measured test sample volume to test devices 
having the configuration of format (0). 
However, by applying varying volumes of a dye solution to a device of the 
present invention, the film cover format (1), no volume dependence in 
regard to applied test sample size is shown. FIG. 6 illustrates the K/S 
vs. test-sample volume data of Example 6. The slopes of the graphs shown 
in FIG. 6 are unexpectedly lower than the slopes of graphs of FIGS. 1 
through 5 and, even more surprisingly the slopes of the graphs in FIG. 6 
approach zero, thereby approaching a volume dependence of zero for a 
device having the film cover format (1). 
EXAMPLE 6 
Dye Solution Applied to Untreated Filter Paper--Film Cover Format (1) 
FIG. 6: Tests performed in duplicate, using two instruments, two 
0.5.times.1.0 cm. filter paper matrices, wavelength--630 nm. 
__________________________________________________________________________ 
VOLUME OF DYE 
CONCENTRATION OF DYE SOLUTION 
SOLUTION APPLIED 
1 .times. 10.sup.-5 M 
2 .times. 10.sup.-5 M 
3 .times. 10.sup.-5 M 
TO TEST DEVICE 
K/S 
STD. DEVIATION 
K/S 
STD. DEVIATION 
K/S 
STD. DEVIATION 
__________________________________________________________________________ 
40 uL 0.270 
0.004 0.473 
0.015 0.639 
0.022 
0.260 
0.018 0.473 
0.018 0.626 
0.044 
60 uL 0.266 
0.012 0.476 
0.023 0.617 
0.024 
0.269 
0.005 0.475 
0.007 0.608 
0.035 
80 uL 0.258 
0.004 0.444 
0.006 0.607 
0.033 
0.272 
0.013 0.472 
0.013 0.660 
0.034 
__________________________________________________________________________ 
EXAMPLE 7 
Cholesterol Solution Applied to Treated Filter Paper--Film Cover Format (1) 
Data for Examples 7 and 8 was obtained as in Example 6, using the film 
cover format (1) and the appropiate test ereagent and wavelength for the 
particular analyte. Drops of the test sample were applied to the test 
device, however the volume of the drops of test sample applied to the test 
device was not quantitatively measured. 
__________________________________________________________________________ 
VOLUME OF CHOLESTEROL 
CONCENTRATION OF CHOLESTEROL SOLUTION 
SOLUTION APPLIED LOW MEDIUM HIGH 
TO TEST DEVICE K/S STD. DEVIATION 
K/S STD. DEVIATION 
K/S STD. 
__________________________________________________________________________ 
DEVIATION 
Not Quanti- 0.442 
0.012 0.629 
0.018 0.795 
0.011 
tatively 0.454 
0.022 0.699 
0.030 0.855 
0.042 
Measured 
__________________________________________________________________________ 
EXAMPLE 8 
Triglyceride Solution Applied to Treated Filter Paper--Film Cover Format 
(1) 
__________________________________________________________________________ 
VOLUME OF TRIGLYCERIDE 
CONCENTRATION OF TRIGLYCERIDE SOLUTION 
SOLUTION APPLIED LOW MEDIUM HIGH 
TO TEST DEVICE K/S STD. DEVIATION 
K/S STD. DEVIATION 
K/S STD. 
__________________________________________________________________________ 
DEVIATION 
Not Quanti- 0.371 
0.013 0.549 
0.027 0.860 
0.047 
tatively 0.384 
0.005 0.618 
0.030 0.888 
0.042 
Measured 
__________________________________________________________________________ 
In comparing the standard deviation values for the tests of Examples 1 
through 5 to the standard deviation values for the tests of Examples 6 
through 8, it is shown that the precision of the tests not precisely 
controlling the volume of test sample applied to the diagnostic device 
(Ex. 6-8) compare favorably to the tests performed utilizing the standard 
control format (0) and having a precise volume of test sample applied to 
the test device (Ex. 1-5). 
EXAMPLE 9 
Cholesterol Solution Applied to Treated Filter Paper--Touchoff Format (2) 
FIG. 7: Data obtained as in Example 2. 
__________________________________________________________________________ 
VOLUME OF CHOLESTEROL 
CONCENTRATION OF CHOLESTEROL SOLUTION 
SOLUTION APPLIED LOW MEDIUM HIGH 
TO TEST DEVICE K/S STD. DEVIATION 
K/S STD. DEVIATION 
K/S STD. 
__________________________________________________________________________ 
DEVIATION 
30 uL 0.429 
0.018 0.633 
0.033 0.846 
0.058 
40 uL 0.438 
0.007 0.639 
0.012 0.876 
0.009 
__________________________________________________________________________ 
EXAMPLE 10 
Triglyceride Solution Applied to Treated Filter Paper--Touchoff Format (2) 
FIG. 8: Data obtained as in Example 3. 
__________________________________________________________________________ 
VOLUME OF TRIGLYCERIDE 
CONCENTRATION OF TRIGLYCERIDE SOLUTION 
SOLUTION APPLIED LOW MEDIUM HIGH 
TO TEST DEVICE K/S STD. DEVIATION 
K/S STD. DEVIATION 
K/S STD. 
__________________________________________________________________________ 
DEVIATION 
30 uL 0.277 
0.017 0.647 
0.017 1.054 
0.033 
40 uL 0.285 
0.005 0.673 
0.027 1.005 
0.029 
__________________________________________________________________________ 
EXAMPLE 11 
Potassium Ion Solution Applied to Treated Filter Paper--Touchoff Format (2) 
FIG. 9: Data obtained as in Example 4. 
__________________________________________________________________________ 
VOLUME OF POTASSIUM 
CONCENTRATION OF POTASSIUM ION SOLUTION 
ION SOLUTION APPLIED 
LOW MEDIUM HIGH 
TO TEST DEVICE K/S 
STD. DEVIATION 
K/S 
STD. DEVIATION 
K/S 
STD. DEVIATION 
__________________________________________________________________________ 
30 uL 0.330 
0.002 0.418 
0.005 0.546 
0.024 
40 uL 0.417 
0.004 0.535 
0.006 0.691 
0.008 
__________________________________________________________________________ 
EXAMPLE 12 
Glucose Solution Applied to Treated Filter Paper--Touchoff Format (2) 
FIG. 10: Data obtained as in Example 5. 
__________________________________________________________________________ 
VOLUME OF GLUCOSE 
CONCENTRATION OF GLUCOSE SOLUTION 
SOLUTION APPLIED 
LOW MEDIUM HIGH 
TO TEST DEVICE 
K/S 
STD. DEVIATION 
K/S 
STD. DEVIATION 
K/S 
STD. DEVIATION 
__________________________________________________________________________ 
30 uL 0.136 
0.010 0.321 
0.020 0.551 
0.013 
40 uL 0.137 
0.003 0.329 
0.019 0.576 
0.028 
__________________________________________________________________________ 
In Examples 9 through 12, a test sample volume of 20 microliters is 
approximately the saturation volume for the reagent-treated bibulous 
matrix. This volume of test sample does not provide excess test sample for 
removal, therefore 2-microliter test sample volumes were not tested. As 
shown by the slopes of the graphed function in FIGS. 8 through 10, a 
diagnostic device utilizing the touchoff format is relatively independent 
of applied test sample volume. However, this format has the disadvantages 
of being significantly dependent upon user technique and of requiring an 
additional manipulative step within the test. 
EXAMPLE 13 
Cholesterol Solution Applied to Treated Filter Paper--Blot Lightly Format 
(3) 
FIG. 11: Data obtained as in Example 2. 
__________________________________________________________________________ 
VOLUME OF CHOLESTEROL 
CONCENTRATION OF CHOLESTEROL SOLUTION 
SOLUTION APPLIED LOW MEDIUM HIGH 
TO TEST DEVICE K/S STD. DEVIATION 
K/S STD. DEVIATION 
K/S STD. 
__________________________________________________________________________ 
DEVIATION 
30 uL 0.333 
0.038 0.464 
0.009 0.639 
0.020 
40 uL 0.335 
0.031 0.473 
0.028 0.617 
0.036 
__________________________________________________________________________ 
EXAMPLE 14 
Triglyceride Solution Applied to Treated Filter Paper--Blot Lightly Format 
(3) 
FIG. 12: Data obtained as in Example 3. 
__________________________________________________________________________ 
VOLUME OF TRIGLYCERIDE 
CONCENTRATION OF TRIGLYCERIDE SOLUTION 
SOLUTION APPLIED TO 
LOW MEDIUM HIGH 
TEST DEVICE K/S STD. DEVIATION 
K/S STD. DEVIATION 
K/S STD. 
__________________________________________________________________________ 
DEVIATION 
30 uL 0.205 
0.009 0.476 
0.018 0.717 
0.077 
40 uL 0.156 
0.017 0.478 
0.044 0.791 
0.032 
__________________________________________________________________________ 
EXAMPLE 15 
Potassium Ion Solution Applied to Treated Filter Paper--Blot Lightly Format 
(3) 
FIG. 13: Data obtained as in Example 4. 
__________________________________________________________________________ 
VOLUME OF POTASSIUM 
CONCENTRATION OF POTASSIUM ION SOLUTION 
ION SOLUTION APPLIED 
LOW MEDIUM HIGH 
TO TEST DEVICE K/S 
STD. DEVIATION 
K/S 
STD. DEVIATION 
K/S 
STD. DEVIATION 
__________________________________________________________________________ 
30 uL 0.346 
0.005 0.472 
0.019 0.656 
0.017 
40 uL 0.395 
0.004 0.512 
0.004 0.711 
0.013 
__________________________________________________________________________ 
EXAMPLE 16 
Glucose Solution Applied to Treated Filter Paper--Blot Lightly Format (3) 
FIG. 14: Data obtained as in Example 5. 
__________________________________________________________________________ 
VOLUME OF GLUCOSE 
CONCENTRATION OF GLUCOSE SOLUTION 
SOLUTION APPLIED TO 
LOW MEDIUM HIGH 
TEST DEVICE K/S 
STD. DEVIATION 
K/S 
STD. DEVIATION 
K/S 
STD. DEVIATION 
__________________________________________________________________________ 
30 uL 0.111 
0.002 0.232 
0.016 0.385 
0.012 
40 uL 0.108 
0.007 0.251 
0.017 0.393 
0.023 
__________________________________________________________________________ 
Examples 13 through 16 and FIGS. 11 through 14 show that the lightly 
blotting format does reduce the volume dependence compared to the standard 
control format (0), however, the blotting lightly formast (3) is highly 
dependent upon user technique. The volume independence of this format is 
highly dependent on the pressure of blotting, as can be seen by comparing 
Examples 13-16 to the following data obtained from the blotting heavily 
format (4). 
EXAMPLE 17 
Cholesterol Solution Applied to Treated Filter Paper--Blot Heavily Format 
(4) 
FIG. 15: Data obtained as in Example 2. 
__________________________________________________________________________ 
VOLUME OF CHOLESTEROL 
CONCENTRATION OF CHOLESTEROL SOLUTION 
SOLUTION APPLIED TO 
LOW MEDIUM HIGH 
TEST DEVICE K/S STD. DEVIATION 
K/S STD. DEVIATION 
K/S STD. 
__________________________________________________________________________ 
DEVIATION 
30 uL 0.142 
0.015 0.236 
0.040 0.228 
0.027 
40 uL 0.122 
0.010 0.180 
0.016 0.217 
0.031 
__________________________________________________________________________ 
EXAMPLE 18 
Triglyceride Solution Applied to Treated Filter Paper--Blot Heavily Format 
(4) 
FIG. 16: Data obtained as in Example 3. 
__________________________________________________________________________ 
VOLUME OF TRIGLYCERIDE 
CONCENTRATION OF TRIGLYCERIDE SOLUTION 
SOLUTION APPLIED TO 
LOW MEDIUM HIGH 
TEST DEVICE K/S STD. DEVIATION 
K/S STD. DEVIATION 
K/S STD. 
__________________________________________________________________________ 
DEVIATION 
30 uL 0.110 
0.008 0.253 
0.032 0.420 
0.025 
40 uL 0.112 
0.015 0.223 
0.018 0.335 
0.073 
__________________________________________________________________________ 
EXAMPLE 19 
Potassium Ion Solution Applied to Treated Filter Paper--Blot Heavily Format 
(4) 
FIG. 17: Data obtained as in Example 4. 
__________________________________________________________________________ 
VOLUME OF POTASSIUM 
CONCENTRATION OF POTASSIUM ION SOLUTION 
ION SOLUTION APPLIED 
LOW MEDIUM HIGH 
TO TEST DEVICE K/S 
STD. DEVIATION 
K/S 
STD. DEVIATION 
K/S 
STD. DEVIATION 
__________________________________________________________________________ 
30 uL 0.352 
0.013 0.461 
0.015 0.608 
0.014 
40 uL 0.351 
0.010 0.515 
0.002 0.629 
0.010 
__________________________________________________________________________ 
EXAMPLE 20 
Glucose Solution Applied to Treated Filter Paper--Blot Heavily Format (4) 
FIG. 18: Data obtained as in Example 5. 
__________________________________________________________________________ 
VOLUME OF GLUCOSE 
CONCENTRATION OF GLUCOSE SOLUTION 
SOLUTION APPLIED TO 
LOW MEDIUM HIGH 
TEST DEVICE K/S 
STD. DEVIATION 
K/S 
STD. DEVIATION 
K/S 
STD. DEVIATION 
__________________________________________________________________________ 
30 uL 0.080 
0.002 0.178 
0.032 0.248 
0.022 
40 uL 0.082 
0.002 0.170 
0.032 0.230 
0.035 
__________________________________________________________________________ 
As shown in Examples 17 through 20 and in FIGS. 15 through 18, the blot 
heavily technique (4) does reduce the dependence on sample volume compared 
to the standard control format (0), however, the blotting heavily 
technique is extremely dependent upon the actual pressure used in 
blotting. As can be seen by comparing the data and graphs of Examples 17 
through 20 to the data and graphs of the blot lightly technique, Examples 
13 through 16, the negative slopes of several of the graphed functions in 
FIGS. 15 through 18 shows the effect of blotting too heavily and thereby 
removing too much test sample from the bibulous matrix. 
EXAMPLE 21 
Dye Solution Applied to Untreated Filter Paper--Adjacent Dry Pad Format (5) 
FIG. 19: Data obtained as in Example 1. 
__________________________________________________________________________ 
VOLUME OF DYE CONCENTRATION OF DYE SOLUTION 
SOLUTION APPLIED TO 
1 .times. 10.sup.-5 M 
2 .times. 10.sup.-5 M 
3 .times. 10.sup.-5 M 
TEST DEVICE K/S 
STD. DEVIATION 
K/S 
STD. DEVIATION 
K/S 
STD. DEVIATION 
__________________________________________________________________________ 
20 uL 0.129 
0.017 0.312 
0.121 0.427 
0.060 
0.183 
0.017 0.337 
0.128 0.480 
0.184 
30 uL 0.187 
0.016 0.368 
0.008 0.535 
0.026 
0.161 
0.011 0.363 
0.010 0.525 
0.004 
40 uL 0.215 
0.004 0.494 
0.011 0.743 
0.028 
0.206 
0.002 0.471 
0.005 0.720 
0.009 
__________________________________________________________________________ 
As seen from the above data and FIG. 19, the use of an additional bibulous 
matrix does reduce the dependence upon sample volume. However, the format 
does not exhibit sample volume independent to a sufficient degree to give 
accurate and reproducible analytical results on variable test sample 
volumes. For instance, the K/S values using 40 .mu.L of a 
2.times.10.sup.-5 M dye solution are larger than the K/S values found 
using 20 .mu.L of 3.times.10.sup.-5 M solution. 
EXAMPLE 22 
Dye Solution Applied to Untreated Filter Paper--Adjacent Dry Pad with 
Bridge Format (6) 
FIG. 20: Data obtained as in Example 1 
______________________________________ 
VOLUME OF CONCENTRATION OF 
DYE SOLUTION DYE SOLUTION 
APPLIED TO 3 .times. 10.sup.-5 M 
TEST DEVICE K/S STD. DEVIATION 
______________________________________ 
20 uL 0.563 0.033 
0.658 0.083 
30 uL 0.709 0.144 
0.772 0.040 
40 uL 0.797 0.014 
0.848 0.033 
______________________________________ 
The above data and FIG. 20 shows improved volume independence compared to 
Example 21 and FIG. 19, however a device based on this format still is not 
sufficiently volume independent to provide a method and device for giving 
accurate and reproducible analyte determinations. 
EXAMPLE 23 
Dye Solution Applied to Untreated Filter Paper--Apply to Adjacent Pad 
Format (7) 
The tests utilizing this format resulted in test sample runover of the test 
sample from the saturated bibulous substrate to the testing bibulous 
substrate. Variable amounts of excess test sample were visually present on 
the testing bibulous substrate. This format essentially did not reduce 
test sample volume dependence. 
EXAMPLE 24 
Dye Solution Applied to Untreated Filter Paper--Apply to Adjacent Pad with 
Bridge Format (8) 
The tests utilizing this format resulted in a syphoning of the liquid test 
sample from the saturated bibulous matrix, across the bridge, to the 
testing bibulous matrix. Variable amounts of excess test sample were 
visually present on the testing bibulous matrix. This format essentially 
did not reduce the test sample volume dependence. 
EXAMPLE 25 
Cholesterol Solution Applied to Treated Filter Paper--Dip Reagent Format 
(9) 
Data obtained as in Example 2. Volume of applied test solution varies. 
__________________________________________________________________________ 
CONCENTRATION OF CHOLESTEROL SOLUTION 
LOW MEDIUM HIGH 
K/S STD. DEVIATION 
K/S 
STD. DEVIATION 
K/S 
STD. DEVIATION 
__________________________________________________________________________ 
Not Quanti- 
0.449 
0.024 0.696 
0.025 0.952 
0.072 
tatively 
Measured 
__________________________________________________________________________ 
EXAMPLE 26 
Triglyceride Solution Applied to Treated Filter Paper--Dip Reagent Format 
(9) 
Data obtained as in Example 3. Volume of applied test solution varies. 
__________________________________________________________________________ 
CONCENTRATION OF TRIGLYCERIDE SOLUTION 
LOW MEDIUM HIGH 
K/S STD. DEVIATION 
K/S 
STD. DEVIATION 
K/S 
STD. DEVIATION 
__________________________________________________________________________ 
Not Quanti- 
0.310 
0.009 0.746 
0.021 1.212 
0.031 
tatively 
Measured 
__________________________________________________________________________ 
EXAMPLE 27 
Potassium Ion Solution Applied to Treated Filter Paper--Dip Reagent Format 
(9) 
Data obtained as in Example 4. Volume of applied test solution varies. 
__________________________________________________________________________ 
CONCENTRATION OF POTASSIUM ION SOLUTION 
LOW MEDIUM HIGH 
K/S STD. DEVIATION 
K/S 
STD. DEVIATION 
K/S 
STD. DEVIATION 
__________________________________________________________________________ 
Not Quanti- 
0.334 
0.002 0.424 
0.005 0.554 
0.024 
tatively 
Measured 
__________________________________________________________________________ 
EXAMPLE 28 
Glucose Solution Applied to Treated Filter paper--Dip Reagent Fromat (9) 
Data obtained as in Example 5. Volume of applied test solution varies. 
__________________________________________________________________________ 
CONCENTRATION OF GLUCOSE SOLUTION 
LOW MEDIUM HIGH 
K/S STD. DEVIATION 
K/S 
STD. DEVIATION 
K/S 
STD. DEVIATION 
__________________________________________________________________________ 
Not Quanti- 
0.135 
0.007 0.293 
0.014 0.445 
0.022 
tatively 
Measured 
__________________________________________________________________________ 
Examples 25 through 28, using the dip reagent format (9), demonstrate an 
intrinsically volume indepeddent test method and device because the 
bibulous matrix in this format can only absorb the volume of liquid test 
sample to required saturate the matrix. However, for several clinical 
tests, and in particular for blood serum tests, the dip reagent format (9) 
is impractical because it requires a large test sample to provide 
sufficient volume for completely dipping the test device. The dip reagent 
format also introduces the risk of contamination of the test sample by the 
diagnostic device, thereby leading to erroneous results or causing 
interference with any subsequent tests to be run on the same test sample. 
The summary of results from the data tabulated in Examples 1 through 28 and 
the graphs in FIGS. 1 through 20 are included in Table II. From the 
Examples utilizing these various methods and devices, it is seen that 
several of the devices have formats demonstrating sample volume 
independence. However, these devices, including the touchoff, blot 
lightly, blot heavily and dip reagent formats, also have the disadvantages 
and drawbacks of technique sensitivity, laboriousness or the need of a 
large sample volume. However, the method of the present invention, using 
the film cover format, and as illustrated in Examples 6 through 8 and in 
FIG. 6, shows essentially complete volume independence and is free of any 
technique or manipulative disadvantages. 
In Table II, the volume dependence is expressed relative to the volume 
dependency of the standard control format (0). For a basis of comparison, 
the volume dependency of the standard control format was arbitrarily 
assigned a value of unity. The term "CV" is the coefficient of variation 
and is determined by multiplying the standard deviation by 100 then 
dividing by the average K/S value. 
TABLE II 
______________________________________ 
Format 
Example Volume 
No. Nos. Method Dependence 
Comment 
______________________________________ 
0 1-5 Standard 1 Control 
1 6-8 Film cover 0 Best, CV low 
2 9-12 Touch off 0 Good, but 
laborious 
3 13-16 Blot 0 Technique 
lightly sensitive 
4 17-20 Blot 0 Very technique 
heavily sensitive, high 
CV 
5 21 Adjacent 0.5 Some improve- 
dry pad ment 
6 22 Adjacent 0.25 More improve- 
dry pad ment 
with bridge 
7 23 Apply to 1 No improvement 
adjacent pad 
8 24 Apply to 1 No improvement 
adjacent pad 
with bridge 
9 25-28 Dip reagent 
0 Requires large 
sample volume 
______________________________________ 
As previously described, the device of the present invention includes one 
or more bibulous matrices covered by a liquid impervious coating or film. 
The matrices are so treated and arranged to quantitatively determine 
analyte concentrations in a liquid test sample independent of test sample 
size. The liquid test sample is deposited on a portion of a bibulous 
matrix such that the test sample is metered into the bibulous matrix 
chromatographically. By wicking action, the liquid test sample travels to 
an assay region of the device that has been previously treated with a 
suitable test reagent for a particular analyte. In accordance with an 
important feature of the present invention, the test sample is metered 
into the bibulous matrices only to the point of liquid saturation of the 
matrices. 
Specifically, the positioning of the bibulous matrices and the 
testing-reagent, and the metering of the sample, may be better understood 
by reference to FIGS. 21 through 25. FIG. 21 shows a perspective view of a 
volume independent diagnostic device 10 including a first bibulous matrix 
14; and a second bibulous matrix 16 impregnated with a suitable testing 
reagent, both bibulous matrices securely adhered to a support strip or 
handle 12. As will become more apparent hereinafter, in order to 
facilitate the quantitative determination of analytes, it is preferred 
that the support strip or handle be manufactured from a hydrophobic 
material. Preferably, the hydrophobic material is translucent, and can be 
formed from materials such as cellulose acetate, polyethylene, 
terephthalate, polycarbonate and polystyrene. A hydrophobic barrier 18 is 
disposed above the two bibulous matrices 14 and 16 attached to substrate 
12 to help meter the test sample into the first bibulous matrix 14 and to 
prevent sample spillover onto the second bibulous matrix 16. In this 
embodiment, the hydrophobic barrier 18 is positioned above the bibulous 
matrices 14 and 16, near the end of the first bibulous matrix 14 that is 
in contact wth the second bibulous matrix 16. The barrier 18 extends to 
completely cover the second bibulous substrate 16. 
To achieve the full advantage of the present invention, the test sample 
should be introduced in the area of the arrow. For the arrangement 
illustrated in FIG. 21, such a placement allows barrier 18 to help meter 
the sample into the first bibulous matrix 14. The wicking action of 
bibulous matrix 14 allows the test sample to chromatograph through matrix 
14 to the second bibulous matrix 16. Upon saturation of the second 
bibulous substrate 16 with test sample, no further test sample can be 
metered into the first bibulous substrate 14. The hydrophobic barrier 18 
also prevents spillover of excess test sample onto the second bibulous 
matrix to prohibit excess sample addition into bibulous matrix 16 and 
therefore interfere with the chromogenic test within the assay area. If 
barrier 18 is absent, test sample may run onto and flood bibulous matrix 
16 as opposed to chromatographing through the bibulous matrix 14. This 
results in excess test sample entering the assay area of bibulous matrix 
16 yielding inaccurate analyte determinations. 
In accordance with an important feature of the present invention, the 
barrier 18 comprises a liquid impermeable material, such that the test 
sample cannot penetrate through the barrier 18 to directly contact the 
second bibulous substrate 16. The barrier 18 is preferably a transparent 
or translucent material. However, if the substrate 12 is transparent, and 
readings are taken through substrate 12, then the barrier 18 can be 
opaque. Suitable materials include tape, silicones, rubber, plastics, and 
waxes. Waxes that are especially useful are smooth, water repellent and 
nontoxic. Types of waxes that can be utilized in the method and device of 
the present invention include natural waxes, such as animal wax, beeswax, 
spermaceti, lanolin, shellac wax; vegetable waxes, such as carnauba, 
candelilla, bayberry, sugar cane; mineral waxes, such as fossil or earth 
waxes, including ozocerite, ceresin, montan; and petroleum waxes, such as 
paraffin, microcrystalline, petrolatum; as well as synthetic waxes such as 
ethylenic polymers and polyoletheresters, sorbitol and chlorinated 
napthalenes and other hydrocarbon waxes. 
Another configuration of the device 20 of the present invention is 
illustrated in FIG. 22, wherein the two bibulous matrices 24 and 26 
attached to a substrate 22 are not in intimate contact, but physically 
separated and connected by a bibulous thin-tissue bridge 21, whereby the 
test sample can travel from the first bibulous matrix 24 to the second 
bibulous matrix 26 for assay. A hydrophobic barrier 28 is disposed to 
cover the thin tissue bridge 21 to help meter the sample to the bibulous 
matrices 24 and 26 and avoid contamination of the assay area by the test 
sample. 
FIGS. 23 and 24 are alternate configurations 30 (40) wherein the amont of 
the test sample required may be increased or decreased per dose by varying 
the size of the second bibulous matrix 36 (46) relative to the size of the 
first bibulous matrix 34 (44). Such configurations attached to substrates 
32 (42) and containing a barrier 38 (48) allows flexibility in analyte 
determinations by making chromogenic reactions more responsive to 
quantitative determination. 
FIG. 25 illustrates a configuration 50 utilizing a single bibulous matrix 
generally designated 55 attached to a substrate 52. The testing reagent is 
impregnated in one portion 57 of matrix 55 covered by barrier or coating 
53, and the test sample is applied in the area of the arrow. Although a 
single matrix test device can be constructed as indicated, to achieve the 
full advantage of the present invention, the device is fabricated such 
that the test reagent is introduced in an assay area of the bibulous 
matrix spaced from the area of the bibulous matrix where the test sample 
is applied. 
In accordance with an important feature of the present invention, an excess 
amount of test sample, usually in excess of approximately 30 microliters, 
is applied to the diagnostic device in the area of the bibulous matrix 
that is not covered by the liquid-impervious coating or film. This sample 
volume is sufficient to provide an excess amount of test sample, thereby 
assuring saturation of each bibulous matrix. The liquid-impervious barrier 
helps meter the test sample into the bibulous matrices. The liquid 
chromatographs through the bibulous matrices by wicking action up to the 
point of matrix saturation. After matrix saturation by the test sample, 
the metering and wicking action stops such that no free liquid test sample 
enters the assay area of the diagnostic device to fill the voids between 
the materials comprising the bibulous matrix. Therefore, a constant volume 
of test sample, in relation to the size of the bibulous matrix, is 
directed to the assay area of the device, resulting in accurate and 
reproducible analyte concentration determinations. 
The pariicular test reagent composition contained in the bibulous matrix 
depends on the particular analyte to be measured and is within the skill 
of those in the art. Normally the test reagents are impregnated into the 
bibulous matrix prior to the attachment of the bibulous matrix to a 
suitable hydrophobic substrate. 
Thus, in accordance with the present invention, the amount of test sample 
placed onto the diagnostic device is in excess of the sample amount 
required to saturate the test reagenttreated pad. When the test 
reagent-treated pad becomes saturated further flow of test sample stops, 
and the remainder of the test sample remains separated from the reagent 
pad by the coating or film layer and does not thereby affect the 
chromogenic reaction. 
In accordance with an important feature of the process and device of the 
present invention, in addition to a constant and reproducible amount of 
test sample reaching the assay area of the device, the test sample reaches 
the assay area of the device with an essentially unaltered composition. 
Tests performed on blood samples showed no increase in potassium ion or 
loss of cholesterol, as the test sample chromatographed through the 
bibulous matrices. Generally, the proper amount of test sample has reached 
the assay area when the assay area is saturated with test sample. This is 
accomplished by using an excess test sample to assure test sample 
saturation of the assay area. The size of the test sample can be increased 
or decreased by adjusting the relative sizes of the first bibulous 
substrate and the second test reagent-containing bibulous matrix such that 
the assay area will be saturated with test sample. The sizes of bibulous 
matrices will depend upon a predetermined test sample size and the testing 
reagent and method utilized. This process assures an essentially fixed 
amount of test sample to reach the assay area, and renders more accurate 
and reproducible analyte determinations. The variables of minimum test 
sample size, bibulous matrix size, and the amount of test reagent to 
incorporate into the assay area bibulous matrix easily can be determined 
by those skilled in the art. 
In addition to the fast and efficient analyte determinations of liquid test 
samples, and the essentially complete migration of unaltered test sample 
to the assay area, the method and device of the present invention permit 
quantitative analyte determinations without dilution of the liquid sample. 
Testing the undiluted serum or plasma both omits a manipulative step and, 
more importantly, eliminates the possibility of technician error. The 
proper amount of a suitable chromogenic reagent can be incorporated into 
the assay area for immediate reaction with the undiluted liquid test 
sample. The extent of the chromogenic reaction, and therefore the 
quantitative amount of the analyte, then can be determined by the 
chromogenic detection techniques that are well-known in the art. 
Obviously, many modifications and variations of the invention as 
hereinbefore set forth can be made without departing from the spirit and 
scope thereof and therefore only such limitations should be imposed as are 
indicated by the appended claims.