CA1316087C - Volume independent diagnostic device - Google Patents
Volume independent diagnostic deviceInfo
- Publication number
- CA1316087C CA1316087C CA000566513A CA566513A CA1316087C CA 1316087 C CA1316087 C CA 1316087C CA 000566513 A CA000566513 A CA 000566513A CA 566513 A CA566513 A CA 566513A CA 1316087 C CA1316087 C CA 1316087C
- Authority
- CA
- Canada
- Prior art keywords
- liquid
- bibulous
- bibulous matrix
- matrix
- test
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
- G01N33/525—Multi-layer analytical elements
Abstract
VOLUME INDEPENDENT DIAGNOSTIC DEVICE
Abstract of the Disclosure 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, provid-ing more uniform and accurate quantitative analyte determinations.
Abstract of the Disclosure 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, provid-ing more uniform and accurate quantitative analyte determinations.
Description
1 3 ~ 7 VOLU~5E INDEPENDENT DIAGNOSTIC DEVICE
.. .. _ _ , . . _ _ _ _ .
Field of the Invention The present invention relates to a process and device for quantitatively determining analyte concentrations in liquids. More particu-larly, the present invention relates to an im-proved 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 ellmi-nated. Overall, more accurate and more repro-ducible analyte determinations result.
Background of the Invention Presently, th~re are numerous test devices available to simply and rapidly test liquids for the presence or absence of a particu-lar analyte. For example, in regards to body fluids, tests are available to detect glucose, uric acid or protein in urine and to detect glu-cose, potassium ion or cholesterol in blood.
~istorically, most of the available diagnostic test devices have been sensi~ive to the volume o~ the test sample, as well as to ~he concentra-tion 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 es-~3~
pecially desirable in small or private medical offices, where fast and accurate results are required, but the volume of assay samples i5 low enough to preclude the investment in expensive diagnostic devices. In addition, the medical profession requires simple ~nd essentially fool-proof 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 l'dip-and-read" type devices. These devices are widely used in the chemical analysis of biological fluids. For examplej 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 re-flected 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 semiquantita-tive measur~ements. Thus, by measuring the re-sponse 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 physi-cian with a facile diagnostic tool as well as the ability to gauge the extent of disease or of bodily malfunctio~.
1316~87 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 detect-able response, e.g., a color change, in the pres-ence of a specific test sample analyte. Depending on the test reagent syste~ incorporated into a particular bibulous matrix, the test devices can detect the presence of glucose, ketone bodies~
cholesterol, triglycerides, bilirubin, uro-bilinogen, occult blood, nitrite, protein~ urea, potassium, and other substances. A speci~ic change in the intensity of color observed within a specific time range after contacting the test device with a sample, is indicative of the pre-sence of a particular analyte and of the concen-tration 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 se~ond 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 ~L31~7 --4~
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 i5 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 separat-ing 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 incon-- sistent 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.
Ot~her considerations also arise in developing a process and device for testing li-quids for a specific analyté. One important consideration i5 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"
.
1 31 ~087 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 importan~ 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 concen- -trations 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 users are demanding more accurate test methods, for more analytes, on smaller liquid test samples~
~Diagnostic device users are especially eager to reduce the possibi}ity of test inaccuracy, usually by making the test method simpler and less opera-MS~1487 .
1 3 ~ 7 tor 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 diag-nostic device. It is to the lattex objective thatthe method and device of the present invention is directed.
Indicative of the work conducted in this field is U.S. Patent No. 3,798~004 to Zerachia et al, disclosing a semiquantitative method for determining analyte concentrations with a laminated device including a reagent-impregnated ~atrix placed between a pair of liquid impervious members. The analyte-containing sample contacts the reagent-impregnated matrix along the matrix-exposed edye of the test device. As the a~alyte-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. Patent 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, ~316~87 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 an~lyte concentrationO The method disclosed in the Morison patent is volume dependent, as the amount of analyte, and therefore the degree of the chro-mogenic reaction, increases with sample volume.
Nussbaum in U.S. Patent 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 throuyh the translu--cent plastic used to encase the reagent-impreg-nated paper. The method and device of the Nussbaum patent are directed to qualitatively determining the presence or absence of a particu-lar 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 tempera-tures. As a result, the volume or weight of test sample is an unimportant variable in the method disclosed in the Nussbaum patent.
U.S. Patent 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~ re- -agent-impregnated matrix. Although the device does reduce voIume dependencet the configuration of the matrices is specifically designed to pro-vide a uniform bilirubin distribution to the reagent-impregnated matrix for uniform binding and chromogenic reaction.
~3~ 6~87 ~ondo et al UOS. Patent 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 distribu-tion. Accordingly, the spreading layer does not and could not act as a barrier layer.
U.K. Patent 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 wi~h a re agent.
.S. Patent 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 brie, the present invention is directed to a process and device for quantita-tively determinLng analyte concentrations in liquids, whereby assay sensitivity tv sample volume is~essentially eliminated. It has been found that the method and device of the present invention unexpectedly give accurate and reprodu-cible 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.
13~87 g According to the method of the present invention, an excess sample amount o 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 imperme-able coating or film serves to assist in metering the li~uid sample into the first and second bibu-lous matrices and to ac~ 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 ~uantitative 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 o the device. The liquid test sample is directed to~the test reagent-treated assay area of the bibulous matrix by the wicking ac~ion of the bibulous matrix from the area of sample application. The reagent-treated .
~ 3 ~ 7 assay area of the matrices absorbs liquid test sample only up to the point o matrix saturation, thereby providing a specific volume of liquid test sample for analyte determinalion from an initially variable volume of applied liquid test sample.
~ ore particularly, in accvrdance with the present invention, the device includes one or more hydrophilic bibulous matrices securely adhered to a hydrophobic substrateO 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-txeated bibulous matrix, or tes~-reagent treated~portion of the bibulous ma~rix, is laminated with a liquid-impermeable coating or film to act as a barrier and prohibit the test sample from directly con-tacting 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 ~iquid-impermeable coating or film. After the test sample saturates the bibulous matrices, no urther liquid sample enters the matrices, thereby making the method . and device essentially volume independent. If the technician inadvertently applies some of the ~-1487 13~ ~0~7 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 o~E the coating or film before quantitative analyte determination.
In accordance ~ith an important feature of the present invention, the test sample passes chromatographically to an assay area of the same, 10 or an adjacent, bibulous matrix, containing a suitable te~t 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 indi-cate 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 cQncen-tration of a particular analyte~ independent of the test sample volume applied to the device.
More~particularly~ in accordance with another impor~tant 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 li~uid test sample is metered to the test-reagent treated bibulous matrix for~qualitative or quantitative determina-tion of a particular analyte.
Accordin~ to the method of the present invention, an excess amount of test sample is deposited on the area of the bibulous matrix 3S that is not covered by the liquid-impervious , ~L3~6~
coating or film The test sample, metered into the bibulous matrices by the liquld-impervious coating or film, passes chromatographically through the bibulous matrix to th~e assay area of the same bibulous matrix t or to ~he 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 amvunt 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 bîbulous 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 cont~minating, 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-imper~eable film or coating ~ 31 6~87 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 ancl 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 determina-tions that are conducted primarily on bibulous matrices such as paper. These analyte determina-tions include assays for tri~lycerides, galactose, `
glucose, potassium ions, AST, cholesterol, creati-nine, ALT, phenobarbital, bilirubin, theophylline, urea, dye samples and other immunochemîcal assays.
Therefore, it is an object of the pre-sent invention to provide a method and device for determining analyte concentrations in liquid samples quickly, effectlvely~and accurately.
It is also an ob~ect of the present invention to provide a method and device for the rapid, convenient and effective analysis of ana-lyte 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 inde-pendent of sample volume applied to the device.
~3~ 6~7 Another object of the present invention is to provide a method and devlce that delivers a constant and reproducible amount of analyte-containing liquid to an assay area of the device r independent of the amount of exce~s sample applied to the deYice.
Another object of the present invention is to provide a method and device to determine analyte concentrations in small l;;quid samples with a mlnimum of manipulative steps~
Another object of the present invention is to provide a method and device to test ~mall 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.
BRIEF DESCRIPTION OF THE_DRAWING
The above and other objects and advan-tages of the present invention will become appar- :
ent from the following detailed description of - the present invention taken in conjunction with the drawings, wherein:~
FIGS. 1 through 20 are graphs showing : the volume dependence of several different diagnos-: 25 tic test device configurations by plotting the Kubelka-Munk function (K/S) versus the volume of test solutions added to the diagnostic device;
FIG. 21 is a perspective view of a : volu~e independent diagnostic device constructed in accordance with one embodiment of the present invention for determining analyte concentrations in liguid samples having a liquid impervious ; barrier extending from a portion of the firstbibulous matrix and completely covering tha second testing matrix and secured to the top of contact-~S-14~7 13~6~
ing adjacent bibulous matrices to precisely meter test sample from a first matrix to a second test-ing matrix, and to prevent test sample spillover rom the first matrix onto the second testing matrix;
FIG~ 22 is a view similar to FIG. 21 showing the first matri~ an~ the second testiny matrix separated and connected by a thin, absorb-ent tissue bridge;
FIGSo 23 and 24 are views similar to FIGS. 21 and 22 showing alternate configurations of the device of the present invention; and : ~ FIG. 25 is a view similar to FIGS. 21-24 showing another construction of the device of the present invention including only one bibulous matrix.
DETAILED DESCRIPTION OF THE INVENTION
. _ In accordance with the present inven-tion, ac~urate and reproducible quantitative ~ 20 analyte determinations can be performed on liquid :~ samples,~ uch that the assay results are essenti-: : ; ally independent of the test sample volume applied : : to the diagnostic device~ According to the method and device o the:present invention, a small : ; 25 liquid sample~, such as a~pinprick amount, is : sufficient for:quantitative anal~yte determina-- tions, as opposed to the relatively large volume test samples~required in the typical dip-and- :
: test method of analyte~determination. Unexpected-~
30 ~ ly, the device of the present invention, after the introduc~tion of an excess amount of the test:~
:sample, meters a precise and constant amount of : test sample to a te~t reagent-treated bibulous : ; : : matrix. The test sample then is assaye~ for a ~: , :
: MS-1487 1~6~87 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 wi~hout having-to add a predetermined and precise amount of test sample to the diagnostic deviceO Overall, the process and device oE 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 chro-mogenic or other visual responses to test for the presence, absence or concentration of various analytes in liquid test samples. For quan~itative analyte determinations, it i5 of primary import-ance 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 sensi-tive to the concentration of the analyte of in-terest, but are also sensitive to the volume of test sample applied to the diagnostic device.
Table I illustrates the affect o sample volume size upon various q~antitative analyte determina-tions made on whole blood. For example, for each 1% change in sample voIumeD the quantitative assay of AST will change by 0.3%.
~3~ 6~8~
TABLE
VOLU~5E DEPENDENCY OF ANALYTE DETERMINATIONS
. .
Volume Dependence ~ Change in Assay for a 5 Analyte 1~ Change in Sample_Volume) AST
Creatinine 0 to 0.5 Cholesterol 1.3 Triglycerides 1.0 Potassium 0.1 Dye Sa~ples 1.0 In accordance with the present inven tion, it has been found that sample volume inde-pendence 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 inven-tion 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, absorb-ent matrix that is amenable to treatment with a test reagent. The bibulous matrix also should 0 ~ 7 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 con-taminate 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 appre!ciably altering the test sample in a way to make the subsequen~
analyte assays inconclusive, inaccurate or doubt-ful.
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 ~atrix.
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 hydro-philic materiaI 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 a~ filter paper or chromato-graphic paper, synthetic or modified naturally-occurring polymers, such as nitrocellulose, cel-lulose acetate, polyvinyl chloride, polyacryl-amide, polyacrylates, polyurethanes, crosslinked dextran, agarose, and other such crosslinked and ::
13~8~
noncrosslinked water-insoluble hydrophilic poly-mers. Hydrophobic substances, such as a hard~
porou~ 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 charac~eristics and can be of dif~erent chemical compositions or a mixture of chemical compositions. The matrices can also vary in regards to smoothness and roughness combined with hardness and softness. ~owever, in every instance, the bibulous matrix mus~ include a hydrophilic material. Regardless of the éxact 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, bibu-lous matrix includes a cellulosic material, such - as paper, and preferably a fiber-containing paper, such as filter paper. Fil~er paper possesses all of the qualities required of a bibulous matrix Oe 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 re-agent, 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 8 ~
thicknesses and porosities. Since the method of the present invention requires the test sample ~o be metered into the bibulous matrix and subse-quently chromatographed to the assay area of the bibulous matrix, it is well within the experiment-al 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 fcrm of a padj 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 o~ 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 inven-tion, the first bibulous matrix, after appropriate sizing, e.g., 0O5 cm x 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.
MS-14~7 ~ 3 ~ 7 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 imper- :
vious film or coating prohibits excess liquid test sample from entering the bibulous matrices, and thereby making the method and device volume indepe~dent. It has been found that by applying an excess amount of liquid test sample to a bibu-: lous matrix of a diagnostic device having two contact~ng, 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 matri~
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 :
: 20 discussed more fully hereinafter, the liquid ~ : ~
impermeable film:or coating coveri~ng the two ~ ;
bibulous~matrices pr~events any excess liquid~
test sample from entering the second bibulous matrix :: 25 : ~ ; At the present time, reagent-strip~ :~
: test formats utilize:a test-reagent treated bibu~
: lous:matr~ix, whereby a fixed volume of test sample is~applied to the reagent test strip, and the - ~ refléctance is measured at a fixed time or series 30 ~ ~of:times.~ In using this~forma~t~ iE~ the~:volume ; of~ test:sample var~ies,: the quantitative determina-tion~ of analy~te 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 .
: ' :~3:L~087 -~2-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, utllizing a single test reagent-treated bibulous matrix, with the test sample applied directly to the test area.
1. ~he film cover format, wherein an un-treated bibulous matrix i5 placed adja-cent to and in con~act 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 ample is applied to the uncovered and untreated bibulous matrix and chromato-graphs through the untreated bibulous matrix to the reagent-treated bibulous matrix. Afte saturation, no further sample enters the bibulous matrices and any excess sample then is removed.
.. .. _ _ , . . _ _ _ _ .
Field of the Invention The present invention relates to a process and device for quantitatively determining analyte concentrations in liquids. More particu-larly, the present invention relates to an im-proved 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 ellmi-nated. Overall, more accurate and more repro-ducible analyte determinations result.
Background of the Invention Presently, th~re are numerous test devices available to simply and rapidly test liquids for the presence or absence of a particu-lar analyte. For example, in regards to body fluids, tests are available to detect glucose, uric acid or protein in urine and to detect glu-cose, potassium ion or cholesterol in blood.
~istorically, most of the available diagnostic test devices have been sensi~ive to the volume o~ the test sample, as well as to ~he concentra-tion 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 es-~3~
pecially desirable in small or private medical offices, where fast and accurate results are required, but the volume of assay samples i5 low enough to preclude the investment in expensive diagnostic devices. In addition, the medical profession requires simple ~nd essentially fool-proof 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 l'dip-and-read" type devices. These devices are widely used in the chemical analysis of biological fluids. For examplej 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 re-flected 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 semiquantita-tive measur~ements. Thus, by measuring the re-sponse 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 physi-cian with a facile diagnostic tool as well as the ability to gauge the extent of disease or of bodily malfunctio~.
1316~87 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 detect-able response, e.g., a color change, in the pres-ence of a specific test sample analyte. Depending on the test reagent syste~ incorporated into a particular bibulous matrix, the test devices can detect the presence of glucose, ketone bodies~
cholesterol, triglycerides, bilirubin, uro-bilinogen, occult blood, nitrite, protein~ urea, potassium, and other substances. A speci~ic change in the intensity of color observed within a specific time range after contacting the test device with a sample, is indicative of the pre-sence of a particular analyte and of the concen-tration 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 se~ond 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 ~L31~7 --4~
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 i5 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 separat-ing 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 incon-- sistent 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.
Ot~her considerations also arise in developing a process and device for testing li-quids for a specific analyté. One important consideration i5 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"
.
1 31 ~087 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 importan~ 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 concen- -trations 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 users are demanding more accurate test methods, for more analytes, on smaller liquid test samples~
~Diagnostic device users are especially eager to reduce the possibi}ity of test inaccuracy, usually by making the test method simpler and less opera-MS~1487 .
1 3 ~ 7 tor 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 diag-nostic device. It is to the lattex objective thatthe method and device of the present invention is directed.
Indicative of the work conducted in this field is U.S. Patent No. 3,798~004 to Zerachia et al, disclosing a semiquantitative method for determining analyte concentrations with a laminated device including a reagent-impregnated ~atrix placed between a pair of liquid impervious members. The analyte-containing sample contacts the reagent-impregnated matrix along the matrix-exposed edye of the test device. As the a~alyte-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. Patent 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, ~316~87 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 an~lyte concentrationO The method disclosed in the Morison patent is volume dependent, as the amount of analyte, and therefore the degree of the chro-mogenic reaction, increases with sample volume.
Nussbaum in U.S. Patent 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 throuyh the translu--cent plastic used to encase the reagent-impreg-nated paper. The method and device of the Nussbaum patent are directed to qualitatively determining the presence or absence of a particu-lar 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 tempera-tures. As a result, the volume or weight of test sample is an unimportant variable in the method disclosed in the Nussbaum patent.
U.S. Patent 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~ re- -agent-impregnated matrix. Although the device does reduce voIume dependencet the configuration of the matrices is specifically designed to pro-vide a uniform bilirubin distribution to the reagent-impregnated matrix for uniform binding and chromogenic reaction.
~3~ 6~87 ~ondo et al UOS. Patent 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 distribu-tion. Accordingly, the spreading layer does not and could not act as a barrier layer.
U.K. Patent 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 wi~h a re agent.
.S. Patent 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 brie, the present invention is directed to a process and device for quantita-tively determinLng analyte concentrations in liquids, whereby assay sensitivity tv sample volume is~essentially eliminated. It has been found that the method and device of the present invention unexpectedly give accurate and reprodu-cible 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.
13~87 g According to the method of the present invention, an excess sample amount o 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 imperme-able coating or film serves to assist in metering the li~uid sample into the first and second bibu-lous matrices and to ac~ 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 ~uantitative 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 o the device. The liquid test sample is directed to~the test reagent-treated assay area of the bibulous matrix by the wicking ac~ion of the bibulous matrix from the area of sample application. The reagent-treated .
~ 3 ~ 7 assay area of the matrices absorbs liquid test sample only up to the point o matrix saturation, thereby providing a specific volume of liquid test sample for analyte determinalion from an initially variable volume of applied liquid test sample.
~ ore particularly, in accvrdance with the present invention, the device includes one or more hydrophilic bibulous matrices securely adhered to a hydrophobic substrateO 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-txeated bibulous matrix, or tes~-reagent treated~portion of the bibulous ma~rix, is laminated with a liquid-impermeable coating or film to act as a barrier and prohibit the test sample from directly con-tacting 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 ~iquid-impermeable coating or film. After the test sample saturates the bibulous matrices, no urther liquid sample enters the matrices, thereby making the method . and device essentially volume independent. If the technician inadvertently applies some of the ~-1487 13~ ~0~7 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 o~E the coating or film before quantitative analyte determination.
In accordance ~ith an important feature of the present invention, the test sample passes chromatographically to an assay area of the same, 10 or an adjacent, bibulous matrix, containing a suitable te~t 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 indi-cate 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 cQncen-tration of a particular analyte~ independent of the test sample volume applied to the device.
More~particularly~ in accordance with another impor~tant 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 li~uid test sample is metered to the test-reagent treated bibulous matrix for~qualitative or quantitative determina-tion of a particular analyte.
Accordin~ to the method of the present invention, an excess amount of test sample is deposited on the area of the bibulous matrix 3S that is not covered by the liquid-impervious , ~L3~6~
coating or film The test sample, metered into the bibulous matrices by the liquld-impervious coating or film, passes chromatographically through the bibulous matrix to th~e assay area of the same bibulous matrix t or to ~he 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 amvunt 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 bîbulous 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 cont~minating, 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-imper~eable film or coating ~ 31 6~87 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 ancl 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 determina-tions that are conducted primarily on bibulous matrices such as paper. These analyte determina-tions include assays for tri~lycerides, galactose, `
glucose, potassium ions, AST, cholesterol, creati-nine, ALT, phenobarbital, bilirubin, theophylline, urea, dye samples and other immunochemîcal assays.
Therefore, it is an object of the pre-sent invention to provide a method and device for determining analyte concentrations in liquid samples quickly, effectlvely~and accurately.
It is also an ob~ect of the present invention to provide a method and device for the rapid, convenient and effective analysis of ana-lyte 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 inde-pendent of sample volume applied to the device.
~3~ 6~7 Another object of the present invention is to provide a method and devlce that delivers a constant and reproducible amount of analyte-containing liquid to an assay area of the device r independent of the amount of exce~s sample applied to the deYice.
Another object of the present invention is to provide a method and device to determine analyte concentrations in small l;;quid samples with a mlnimum of manipulative steps~
Another object of the present invention is to provide a method and device to test ~mall 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.
BRIEF DESCRIPTION OF THE_DRAWING
The above and other objects and advan-tages of the present invention will become appar- :
ent from the following detailed description of - the present invention taken in conjunction with the drawings, wherein:~
FIGS. 1 through 20 are graphs showing : the volume dependence of several different diagnos-: 25 tic test device configurations by plotting the Kubelka-Munk function (K/S) versus the volume of test solutions added to the diagnostic device;
FIG. 21 is a perspective view of a : volu~e independent diagnostic device constructed in accordance with one embodiment of the present invention for determining analyte concentrations in liguid samples having a liquid impervious ; barrier extending from a portion of the firstbibulous matrix and completely covering tha second testing matrix and secured to the top of contact-~S-14~7 13~6~
ing adjacent bibulous matrices to precisely meter test sample from a first matrix to a second test-ing matrix, and to prevent test sample spillover rom the first matrix onto the second testing matrix;
FIG~ 22 is a view similar to FIG. 21 showing the first matri~ an~ the second testiny matrix separated and connected by a thin, absorb-ent tissue bridge;
FIGSo 23 and 24 are views similar to FIGS. 21 and 22 showing alternate configurations of the device of the present invention; and : ~ FIG. 25 is a view similar to FIGS. 21-24 showing another construction of the device of the present invention including only one bibulous matrix.
DETAILED DESCRIPTION OF THE INVENTION
. _ In accordance with the present inven-tion, ac~urate and reproducible quantitative ~ 20 analyte determinations can be performed on liquid :~ samples,~ uch that the assay results are essenti-: : ; ally independent of the test sample volume applied : : to the diagnostic device~ According to the method and device o the:present invention, a small : ; 25 liquid sample~, such as a~pinprick amount, is : sufficient for:quantitative anal~yte determina-- tions, as opposed to the relatively large volume test samples~required in the typical dip-and- :
: test method of analyte~determination. Unexpected-~
30 ~ ly, the device of the present invention, after the introduc~tion of an excess amount of the test:~
:sample, meters a precise and constant amount of : test sample to a te~t reagent-treated bibulous : ; : : matrix. The test sample then is assaye~ for a ~: , :
: MS-1487 1~6~87 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 wi~hout having-to add a predetermined and precise amount of test sample to the diagnostic deviceO Overall, the process and device oE 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 chro-mogenic or other visual responses to test for the presence, absence or concentration of various analytes in liquid test samples. For quan~itative analyte determinations, it i5 of primary import-ance 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 sensi-tive to the concentration of the analyte of in-terest, but are also sensitive to the volume of test sample applied to the diagnostic device.
Table I illustrates the affect o sample volume size upon various q~antitative analyte determina-tions made on whole blood. For example, for each 1% change in sample voIumeD the quantitative assay of AST will change by 0.3%.
~3~ 6~8~
TABLE
VOLU~5E DEPENDENCY OF ANALYTE DETERMINATIONS
. .
Volume Dependence ~ Change in Assay for a 5 Analyte 1~ Change in Sample_Volume) AST
Creatinine 0 to 0.5 Cholesterol 1.3 Triglycerides 1.0 Potassium 0.1 Dye Sa~ples 1.0 In accordance with the present inven tion, it has been found that sample volume inde-pendence 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 inven-tion 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, absorb-ent matrix that is amenable to treatment with a test reagent. The bibulous matrix also should 0 ~ 7 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 con-taminate 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 appre!ciably altering the test sample in a way to make the subsequen~
analyte assays inconclusive, inaccurate or doubt-ful.
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 ~atrix.
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 hydro-philic materiaI 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 a~ filter paper or chromato-graphic paper, synthetic or modified naturally-occurring polymers, such as nitrocellulose, cel-lulose acetate, polyvinyl chloride, polyacryl-amide, polyacrylates, polyurethanes, crosslinked dextran, agarose, and other such crosslinked and ::
13~8~
noncrosslinked water-insoluble hydrophilic poly-mers. Hydrophobic substances, such as a hard~
porou~ 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 charac~eristics and can be of dif~erent chemical compositions or a mixture of chemical compositions. The matrices can also vary in regards to smoothness and roughness combined with hardness and softness. ~owever, in every instance, the bibulous matrix mus~ include a hydrophilic material. Regardless of the éxact 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, bibu-lous matrix includes a cellulosic material, such - as paper, and preferably a fiber-containing paper, such as filter paper. Fil~er paper possesses all of the qualities required of a bibulous matrix Oe 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 re-agent, 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 8 ~
thicknesses and porosities. Since the method of the present invention requires the test sample ~o be metered into the bibulous matrix and subse-quently chromatographed to the assay area of the bibulous matrix, it is well within the experiment-al 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 fcrm of a padj 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 o~ 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 inven-tion, the first bibulous matrix, after appropriate sizing, e.g., 0O5 cm x 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.
MS-14~7 ~ 3 ~ 7 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 imper- :
vious film or coating prohibits excess liquid test sample from entering the bibulous matrices, and thereby making the method and device volume indepe~dent. It has been found that by applying an excess amount of liquid test sample to a bibu-: lous matrix of a diagnostic device having two contact~ng, 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 matri~
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 :
: 20 discussed more fully hereinafter, the liquid ~ : ~
impermeable film:or coating coveri~ng the two ~ ;
bibulous~matrices pr~events any excess liquid~
test sample from entering the second bibulous matrix :: 25 : ~ ; At the present time, reagent-strip~ :~
: test formats utilize:a test-reagent treated bibu~
: lous:matr~ix, whereby a fixed volume of test sample is~applied to the reagent test strip, and the - ~ refléctance is measured at a fixed time or series 30 ~ ~of:times.~ In using this~forma~t~ iE~ the~:volume ; of~ test:sample var~ies,: the quantitative determina-tion~ of analy~te 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 .
: ' :~3:L~087 -~2-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, utllizing a single test reagent-treated bibulous matrix, with the test sample applied directly to the test area.
1. ~he film cover format, wherein an un-treated bibulous matrix i5 placed adja-cent to and in con~act 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 ample is applied to the uncovered and untreated bibulous matrix and chromato-graphs through the untreated bibulous matrix to the reagent-treated bibulous matrix. Afte 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 cQntrol format d. 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.
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 131~7 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 bibuIous 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.
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 bridye 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 ~ mm. A thin bridge of tissue paper connects the two bibulous ma- ~
trices. The test sample is applied t`o the test reagent-treated bibulous ma-trix, and excess~sample can wick to ; ~ the untreated bibulous matrix~through the tissue;paper bridge.
~ 7r Apply~to ad~jacent 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 :
.
. .
. ' ~
~3~87 -2~-is applied to the untreated bibulous matrix.
8. Apply to adjacent pad with bridge for-mat, wherein the test deYice is prepared as in format 6, however, the test sample i5 applied to the untreated bibulous matrix and wicks through the tissue paper bridge to the test reagent-treated bibulous matrix.
9. ~ip reagent format, utilizing the stand-ard 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 solu-tions 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 appro-priate test reagent for that particular analyte solution. Individual test results were determined by taking a reflectance measurement with a re-flectance photometer at a suitable time and wave-length for that particular analyte determination.
The reflectance, as taken from the reflectance scale of zero to one, was incorporated into the Kubelka-Munk function:
K/S - (l-R)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 ~ 316~87 that as reflectance decreases, the K~S value increases.
For example, FIG. 1 show~ 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 x 10-5 M, 2 x 10-5 M and 3 x 10-5 ~, were used.
; ~he 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 testst with simiIarly tabulated test results.
:: : :
:: .
: ~
~: .
: :
:
.
~3~L60~7 . Example 1 Dye Solution Applied to Untreated Fil~er Pa~r - Standard Control Foxmat ~0) FIG~ 1: Tests performed in duplic:ate, using two instruments, two 0.5 x 0.5 cm:ilt:er paper ma-trices, wavelength - 630 nm.
VOLUME OF
DYE SOLUTION
APPLIED TO
: ~ 10 TEST DEVICE CONCENTRATION OF DYE SOLUTION
1 x 10-5 M 2 x 10-5 M 3 x 10-5 M
STD. STD. STD.
DEVIA- DEVIA- DEVIA-K/S TION K/S TION K/S TION
20 u~ 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 ~.012 1.633 0.09~ 2.896 ~ 0 201 Exam~le 2 : `Cholesterol Solution Applied to Treated Filter Paper - Standard Control Format (0) FIG. 2: Data obtained as in Example 1, using ~:: 25 appropriate commercial test reagent:and wavelength 600~nm.
: ~ :
:
VOLUME OF
CHOLESTEROL
SOLUTION
APPLIED TO
TEST DEVICE CONCENTRATION OF CHOLESTEROL SOI,UTION
_ LOW ME~IUM 3IGH
STD. STD. STD.
DEVIA- DEVIA- DEVIA-K/S TION K/S TION K/S TION
20 uL 0.450 0.006 0.663 0.003 0.910 0.U25 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 Exam~le 3 Triglyceride ~olution Applied to Treated Filter Pa~er - Standard Control Format ~0) FIG. 3: Data obtained as in Example 1, using appropriate commercial test reagent and wavelength - 580 nm.
VOLUME OF
TRIG~YCERIDE
:SOLUTION
APPLIED TO
TEST DEVICE CONCENTRATION OF TRIGLYCERIDE_SOLUTION
LOW MEDIUM HIGH
_ _ _ : STD. STD. STD.
DEVIA- DEVIA- DEVIA-K/S TION R/S TION K/S TION
-- _ 20 uL 0.301 0.007 0.723 0.013 10146 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 PaE~r - Standard Control Format (0) FIG. 4: Data obtained as in ~xample 1, using __ appropriate commercial test reagent and wavelength - 640 nm.
VOLUME OF
POTASSIUM ION
SOLUTI ON
APPLIED TO
l'EST DEVICE CONCENTRATION OF POTASSIUM ION SOLUTION
LOW MEDIUM HIGH
STD. STD. STD.
DEVIA- DEVIA- DEVIA-K/S TION K/S TION K/S TION
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 o.ooa 0.693 0.017 ExamPle 5 Glucose Solution Applied to Treated Filter Paper - Standard Control Forma__(0) FIG. 5: Data obtained as in Example 1, using : 20 appropriate commercial test reagent and wavelength - 620 nm.
VOLUME OF
CLUCOSE~
SOLUTION
APPLIED TO
TEST DEVICE CONCENTRATION OF GLUCOSE SOLUTION
LOW MEDIUM_ HIGH _ STD. STDo STD.
: : DEVIA- DEVIA- DEVIA-K/S ION ~ TION K/S TION
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 ~3~ 6~7 As shown in FIGS. l through 5, the dependence upon sample volume for test samples applied direct-ly to devices having the standard control format ~0) is quite large. The volume dependence is seen in the large slope of the graphed unctions in FIGS. l 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 necessitat-ing the application of a precisely-measured test sample volume to test devices having the configura-tion of format (0).
However, by applying varying volumes of a dye solution to a device of the present invention, the film cover format (l), 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. l through 5 and, even more surprisingly the slopes of the graphs in : 25 FIG. 6 approach zero, thereby approaching a volume dependence of zero for a device having the film cover format (l).
Exam~le 6 Dye~Solution Applied to Untreated : 30 Filter Pa~er - F_lm Cover F_rmat (l) FIG. 6: Tests per~ormed in duplicate, using two instruments, two 0.5 x 1.0 cm. filter paper ma : ~ trices, wavelenyth - 630 nm.
.
~ 3 ~
VOLUME OF
DYE SOLUTION
APPLIED TO
TEST DEVICE ~=~=_~
5 1 x 10-5 M 2 x 11~-5 M 3 x 10-5 M
STD. STD. STD.
DEVIA- DEVIA- DEVIA-K/S TION K/S TION K/S TION
40 uL 0.270 0.004 0.473 9.015 0.639 :0.022 0.260 ~.018 ~.473 0.018 0.626 0.044 60 uL 0.266 0.012 0.476 0.023 0.617 0.024 0.26g 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 a.013 0.472 0.013 0.6~0 0.034 Example 7 Cholesterol Solution Applied to Treated Filter ~Gover Format (1) Data for Examples 7 and 8 was obtained as in Example 6, using the film cover format (1) ; ; 20 and thé appropriate test reagent 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.
CHOLESTEROL
SOLUTION
: APPLIED TO
TEST DEVICE CONCENTRATION OF CHOL~5T3Ro~ .OLil5~O;
::~ 30 : LOW~ _ MEDIUM _ HIGH
STD. STD. STD.
:: DEVIA- DEVIA- DEVIA-K/S TION K/S TION K/S TION
:~ Not Quanti- 0.442 0.012 0.629 0.018 :0.795 0.011 35 tatively 0.454 0.022 0.69g 0.030 0.855 0.042 Measured MS-14~7 ~L31~87 Example 8 Triglyceride Solution Applied to Treated ~ C~er rormat (1) VOLUME OF
TRIGLYCERIDE
SOLUTION
APPLI.ED TO
TEST DEVICE CONCENTRATION ~F TRIGLYCERIDE SOLUTION
.._ . . . . . _ ~.
LOW MEDIUM EIG~
~ . , _ . . . ___ STD. STD. STD.
DEVIA- DEVIA- DEVIA- .
K/S TION K/S TION K/S TION
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 i5 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 con~rol format (0) and : having a precise volume of test sample applied : 25 : to the test device (Ex. 1-5).
Exam~e_9 Cholesterol Solution Applied to Treated Filter Pa~er ~ ~o~chorl ~rma~ (2~:
:
FIG. 7: Data obtained as in Example 2.
.
:
~3~6(~8~
VOLUME OF
CHOLESTEROL
SOLUTION
APPLIED TO
TEST DEVICECONCENTRATION OE CElOLESTEROL SOLUTION
LOW MEDIUM HIGH
STD. STD. STD.
DEVIA- DEVIA- DEVIA-K/S TION R/S TION K/S TION
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 Tri~lyceride SoIution Applied to Treated Filter Paper - Touchoff Format (2) FIG. 8: Data obtained as in Example 3.
VOLUME OF
TRIGLYCERIDE
SOLUTION
APPLIED TO
20 : TEST DEVICE CONCENTRATION OF TRIGLYCERIDE SOLUTION
LOW _ MEDIUM HIGH _ STD.: STD. STD.
DEVIA- DEVIA- DEVIA-: KtS TION R/S TION K/S TION
: :25 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 ~ormat ~23 ~ - :30 FIG. 9 Data obtained as in Fxample 4-::::
13~L6~87 VOLUME OF
POTASSIUM ION
SOLUTION
APPLIED TO
TEST DEVICE CONCENTRATION OF POTASSIUM ION SOLUTION _ oW MEDIUM _ HIGH
STD. STD~ STD.
DEVIA- DEVIA- DFVIA- -~ TION K/S TION K/S TION
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 Exam~le 12 Glucose Solution Applied to Treated Filter Paper -_Touchoff Format (2) 15 FIG. 10: Data obtained as in Example 5.
VOLUME OF
GLUCOSE
SOLUTION
APPLIED TO
TEST DEVICE CONCENTRATION OF GLUCOSE S _UTION
LOW MEDIUM_ HIGH
STD. STD. STD.
DEVIA- DEVIA- DEVIA- .
K/S TION K/S TION K/S TION
30 uL 0.136 d.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 tes:t sample for removal, therefore 20-microliter test sample volumes were not tested.
As shown by the slopes of the graphed function in FIGS. 8 through 10, a diagnostic device utiliz-~1 3~6~7 ing the tvuchoff format is relatively independent of applied test sample volume. However, this format has the disadvantages of being significant-ly dependent upon user technique alnd of requiring an additional manipulative step within the test.
Example 13 Cholesterol Solution Applied to Treated Filter Paper - lot Li~htly Format (3 _IG. ll: Data obtained as in Æxample 2.
VOLUME OF
CHOLESTEROL
SOLUTION
APPLIED TO
TEST DEVICE CONCENTRATION OF CHOLESTEROL SOLUTION
LOW MEDIUM HIGH _ STD7 STD o STD
DEVIA- DEVIA- DEVIA-K/S TION K/S TION K/S TION
: 30 uL ~D333 0.~38 0.464 0.009 0.639 0.020 40 uL 0.335 0.031 0.473 0.028 0.617: ~ 0.036 Example 14 Trig:lyceride Solution Applied to Treated Filter P per - Blot_Li~ht1y Format (3) FIG. 12: Data obtained as in Example 3.
~ :
:
:: ~ : .
:131~7 VOLUME OF
TRIGLYCERIDE
SOLUTION
APPLTED TO
TEST DEVICECONCENTRATION OF TRIGLYCERIDE SOLUTION
LOW MEDIUM ~IGE
STD. STD. STD.
DEVIA- DEVIA- DEVIA-K/S TION K/S TION K/S TION
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 - ~lot Li ~tly_Format (3) FIG. i3: Data obtained as in Example 4.
VOLUME OF
POTASSIUM ION
SOLUTION
APPLIED TO
TEST DEVICE _ CQNCENTRATION OF POTASSIUM ION SOLUTION
_ LOW MEDIUM HIGH
~STD. STD. STD .
DEVIA- DEVIA- DEVIA-K/S TION ~ TION R/S TION
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 Li~htly Format _3) FIG. 14: Data obtained as in Example 5.
1 3 ~ 7 VOLUME OF
GLUCOSE
SOLUTI ON
APPL I ED TO
S TEST DEVICE CONCENTRATION OP GLUCOSE SOLUTION
LOW MEDIUM HIGH
. _ . . . . _ STDo STD. STD.
DEVIA- DEVIA- DEVIA--K/S TION ~ TION K/S TION
30 uL 0.111 0~002 0.232 0.016 0.385 0.012 40 uL 0.108 0.~)07 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 ~o the standard control format (0), however, the blotting lightly format (3) is highly dependent upon user technique. The volume independence oE 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 : heavi~ly format (4). ~ -- ~ Ex~ple 17 Cholesterol Solution Applied to Treated Filter Paper - Blot V ay~ g~ 4) 25 FIG._15: :Data obtained as in Example 2.
VOLUME OF
CHOLESTERO L
SOLU T I ON
APPLIED TO:
TEST DEVICE CONCENTRATïO~ (:)F CHOLESTEROL SOLUTION
_ LOW MEDIUM HIGH
, : STD .: STD . STD .
DEVIA-- DEVIA- DEVIA-K/S TION K/S TION K/S TION
30 UL 0~142 0.015 0.236 0.040 0.228 0.027 13~087 40 uL 0.122 0.010 0.180 0.016 0.217 0.031 Exam~le 18 Triglyceride Solution Applied to Treated Filter Paper - Blot Heavily ]?ormat ~4) 5 FIG. 16: Data obtained as in Example 3.
VOLUME OE
TRIGLYCERIDE
SOLUTION
APPLIED TO
TEST DEVICE CONCENTRATION OF TRIGLYCERIOE SOLUTTON
LOW MEDIUM HIG~
STD. STD. STD.
DEVIA- DEVIA- DEVIA-K/S TION K/S TION K/S TION
30 uL 0.110 0.008 0.253 0.032 0.420 0.025 40 uL 0.112 0.015 00223 0.018 0.335 0.073 Example_l9 Potassium Ion Solution Applied to Treated ilter Paper -_Blot Heavily Format (4) : 20 FIG. 17 Data obtained as in Example 4.
::
VOLUME OF
POTASSIUM ION
: SOLUTION
APPLIED TO
: 25 TEST DEVICE _ CONCENTR~TION OF POTASSIUM ION SOLUTION _ LOW MEDIUM HIG~I _ : STD. STD. : STD.
DEVIA- DEVIA- DEVIA-K/S : TION K/S TIOM K/S TION
~, _.
: ~ 30 30 uL 0~352 0.0I3 0.461 0.015 0.608 0.014 40 uL 0.351 Q.010 0.515 0.002 0.629 0.010 .
~3~87 ~38-Exam~le 20 Gluco~e Solution Applied to Treated Filter Paper - Blot Heavily Format ~4 FIG. 18: Data obtained as in Example 5.
VOLUME OF
GLUCOSE
: SOLUTION
APPLIED TO
TEST DEVICE _ CONCENTRATION C)F GLUCOSE SOLUTION
LOW MED IUM H I GH
STD. STD. STD.
DEVIA- DEVIA- DEVIA-K/S TION K/S TION K/S TION
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 extreme-ly dependent upon the actual pressure used in : blottin~. As can be seen by comparing the da~a and yraphs of Examples 17 through 20 to the data and graphs of the blot lightly techFIique~ Examples :25 13 through 16, the negative slopes oP several of the ~raphed functions in FIGS. 15 through 18 : : .shows the effect of blotting too heavily and thereby removing too much test sample from the : b:ibulous matrix.
:Dye Solution Applied to Untreated Filter Paper - Adjacent Dr~ Pad Format _5 :
FIG. l9s Data obtained as in Example 1.
--VOLUME OF
DYE SOLUTION
APPLIED TO
TEST DEVICECONCENTRATIC)N OF DYB SOLUTION
l x 10-5 M 2 x 10-5 M 3 x lG-5 M
STD. STD. STD.
DEVIA- `DEVIA- DEVIA-20 uL 0~129 0.017 0~312 0.121 0.427 0.060 0~183 0.017 0.337 0.12~ 0.480 0.184 30 uL 0.187 0.016 0.368 0.00~ 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. l9, ~he use of an additional bibulous matrix does reduce the dependence upon sample volume~ How-ever, the format does not exhibit sample volume independent to a sufficient degree to give accur-ate and reproducible analytical results on vari~
able test sample volumes. For instance, the RjS
values using 40 uL of a 2 x 10 ~5 M dye solution are larger than the K/S values found using 20 uL
of 3 x 10-5 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 .
, ~S-14~7 VOLUME OF
DYE SOLUTION
APPLIED TO
TEST DEVICE CONCENTRATION OF DYE SOLUTION
3 x 10-5 M
STD .
I:~EVI A-K/S Tl:ON
lQ 20 uL 0.563 0.033 0 . 658 0 . 0~3 30 uI. O . 709 0 . 144 0.772 1~.0D~) 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.
Dye Solution Applied to Untreated Filter Pa~er - 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 bibu-lous substrate. Variable amounts of excess test sample were visually present on the testing bibu-lous substrate. This format essen~ially did not reduce test sample volume dependence.
:
~3~6~87 Example 24 Dye Solution Applied to Untreated Fil~er Paper - Apply to Adjacent .
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 matrixO Variable amounts of excess test sample were visually present on t~e testing bibulous matrix. This format essenti-ally did not reduce the test sample volume dep~nd-ence.
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
: Law MEDIUM HIGH ~
. _ STD. ~ STD. STD
DEVIA- DEVIA- DEVIA-K/S TION K/S TION K/S TION
; ~ Not Quanti- 0.449 0.024 0.696 0.025 0.952 0.072 tativel~
Measured Example 26 :
Triglyceride Solution Applied to Treated Filter Paper - DiP Reaqent Format (9~
n Data obtained~ as in Example 3. Volume of applied test solution varies.
~:
:
~ 3 ~ 8 7 -4~-CONCENTRATION OF TRIGLYCERIDE SOLUTION
LOW MEDIUM HIGH
STD. STD. STD.
DEVIA- DEVIA- DEVIA-K/S TION ~ TION K/S TION
Not Quanti- 0.310 0.009 0.746 0.021 1.212 0.031 tatively Measured Example 2?
Potassium Ion Solution Applied to Treated Filter Pa~er - Dip Rea~ent Format (9) Data obtained as in Example 4. Volume of applied test solution varies.
CONCENTRATION OF POTASSIUM ION SOLUTION
LOW MEDIUM HIGH
. _ .
STD. STD. STD.
DEVIA- DEVIA- DEVIA-~ TION K/S TION K/S TION
Not ~uanti- 0.334 0.002 0.424 0.005 0.554 0.024 tatively Measured Exampl-e 28 Glucose Solut:ion:Applied to Treated : ilter ~ 9) : 25 Data obtained as in Example 5. Volume of ;
applied test solution varies.
CONCENTRATION OF GLUCOSE SOLUTION
- - - -- -- _ : LOW MEDIUM HIGH
--- ~
: ~ : 30 - STD. STD. STD.
DEVIA- DEVIA- DEVIA-K/S TION K/S TION ~ TION
- - Not Quanti- 0.135 0.007 0.293 0.014 0.445 0.022 tatively Measured:
1 3 ~
Examples 25 through 28, using the dip reagent format (9), demonstrate an intrinsically volume independent test method and devic:e because the bibulous matrix in this format can only absorb the volume of liquid test sample ~o required saturate the matrix. However, for several olini-cal tests, and in particular for blood ser~m tests,-the dip reagent format (g~ is impractical because it requires a large test sample to provide sufficient volume for completely~dipping the test device~ The dip reagent format also intro-duces the risk of contamination of the test sample by the diagnostic device, thereby leading ~o erroneous results o~ causing interference with any subsequent tests to be run on the same test sample.
~he summary of results from the data tabulat-ed in Examples 1 through 28 and the graphs in FIGS. 1 through 20 are included in Table II.
:~ 20 From the Examples utilizing these various methods ; and devices~ it is seen ~hat several of the de-:~ vices have formats demonstrating sample volume : independence. ~owever, these devices, including the touchoff~ blot;lightlyl blot heavily and dip :
~: : 25 reagent formats, also have She 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 : 30 6 through 8 and in FIG. 6, shows essentially : complète volume independence and is free of any technique or manipulative disadvantages.
In Table II,~ the volume dependence is ex-pressed~re:lative to the volume dependency of the :: 35 skandard control format (0). For a basis of :
:~ MS-1487 ~ 31 ~8~
comparison, the volume dependency of the standard con-trol 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
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 bibu-lous matrices covered by a liquid impervious coating or film. The matrices are so treated X
~3~ ~87 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 o a bibulous matrix such that the test sample is metered into the bibulous matrix chromatographically. By wicking actiont 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.
5pecifically, the positioning of the bibulous matrices and the testing-reagent, and the metering of the sample, may be better under-stood by reference to FIGS. 21 through 25. FIG.
21 shows a perspective view of a volume independ-ent diagnostic device 10 including a first bibu-lous matrix 14; and a second bibulous matrix 16 : impregnated with a suitable testing reagent, : both bibulous matrices securely adhered to a support str;ip or handle~12. As will become more : apparent hereinafter, in order to facilitate the quanti~ative determination of analytes, it i5 preferred that the support strip or handle be manufactured from a hydrophobic material. Prefer-ably, the hydrophobic material is translucent, and can be formed from materials such as cellulose : 30 acetate, polyethylene, terephthala~e, polycarbon-~ : ate and polystyreneO 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 : : 35 14 and to prevent sample spillover onto the second ~31~7 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 with the second bibulous matrix 16. The barrier 18 extends to comple~ely 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 place-ment 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 chromo- -genic test within the assay area. If barrier 18 : 25 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 ~est sample cannot penetrate through the barrier : 35 18 to directly contact the second bibulous sub-~31~0~7 strate 16. The barrier 18 is preferably a tran~
parent or translucent material. However, if the substrate 12 i9 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 smoo~h, water repellent and nontoxic. Types of waxes that can be utilized in the method and devi.ce of the pre-sent invention include natural wa~es, 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, cere-sin, montan; and petroleum waxes, such as paraffin, microcrystalline, petrolatum; as well as synthetic waxes such as ethylenic polymers and polyolether-esters, sorbitol and chlorinated napthalenes and other hydrocarbon waxes.
Another coniguration 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 ; 25 by a bibulous thin-tissue bridge 219 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 30 : 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 configura-: tions 30 (40) wherein the amount of the test sample:reguired may be increased or decreased ~3~87 -48~
per dose by varying the size of the second bibu lous matrix 36 (46) relative to the size of the first bibulous matrix 34 (44). Such configura-tions attached to substrates 32 (42) and contain-ing a barrier 38 (48) allows flexibility in ana-lyte determinativns by making chromogenic reac-tions more responsive to quantitative determina-tion.
FIG. 25 illustrates a confiyuration 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 w~ith~an important feature of the present invention, an excess amount of test sample, usually in excess of approximat~ely 30 microliters, is applied to the diagnostic device in the area of the bibulous~matrix that i5 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 matri~.
The liquid-impervious barrier helps meter the test sample into the bibulous matrices. The liquid chromatographs through the bibulous ma-krices by wicking action up to the point of matrix saturation. After matrix saturation by the test .
. , . . , ~
~ 3 1~
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 bibu-lous matrix. Therefore, a constant volume oftest sample, in relation to the size of the bibu lous matrix, is directed to the assay area of the device, resulting in accurate and reproducible analyte concentration determinations.
rhe particular 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 ~ 20 amount required to saturate the test reagent-; - treated pad. When the test reagent-treated pad becomes saturated further flow of test sample stops, and the remainder of the test sample re-;~ mains separated ~rom the reagent pad by the coat-ing or ilm layer and does not thereby affect the chromogenic reaction~
In accordance with an important eature of the process and device of the present inven-tion, in addition to a constant and reproducible amount of test sample reaching the assay a~ea of ~the device, the test sample reaches the assay area o the device with an essentially unaltered composition. Tests performed on blood samples showed no increase in potassium ion or loss of ch~lesterolj as the test sample chromatographed~
;
~ 3~ 7 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 axcess test sample to assure test sample satura-tion 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 ren-ders more accurate and reproducible analyte deter-minations. 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 de~ermined by those skilled in the art.
~ n addition to the fast and efficient analyte determinations of liquid test samples, and the essentially complete migration o un-altered test sample to the assay area, the methodand device of the present invention permit quan-titative analyte determinations without dilutio~
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 ~est sample. The extent of the chromogenic reaction, and therefore the quan-MS~lA87 1 3 ~ 7 titative amount of the analytet then can be deter-mined by the chromogenic detection techniques that are well-known in the art.
Obviously, many modifications and vari ations o the invention as hereinbefore set forth can be made without departing from the spirit and scope thereof and therefore only such limita-tions should be imposed as are indicated by the appended claims.
-, . :
trices. The test sample is applied t`o the test reagent-treated bibulous ma-trix, and excess~sample can wick to ; ~ the untreated bibulous matrix~through the tissue;paper bridge.
~ 7r Apply~to ad~jacent 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 :
.
. .
. ' ~
~3~87 -2~-is applied to the untreated bibulous matrix.
8. Apply to adjacent pad with bridge for-mat, wherein the test deYice is prepared as in format 6, however, the test sample i5 applied to the untreated bibulous matrix and wicks through the tissue paper bridge to the test reagent-treated bibulous matrix.
9. ~ip reagent format, utilizing the stand-ard 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 solu-tions 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 appro-priate test reagent for that particular analyte solution. Individual test results were determined by taking a reflectance measurement with a re-flectance photometer at a suitable time and wave-length for that particular analyte determination.
The reflectance, as taken from the reflectance scale of zero to one, was incorporated into the Kubelka-Munk function:
K/S - (l-R)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 ~ 316~87 that as reflectance decreases, the K~S value increases.
For example, FIG. 1 show~ 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 x 10-5 M, 2 x 10-5 M and 3 x 10-5 ~, were used.
; ~he 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 testst with simiIarly tabulated test results.
:: : :
:: .
: ~
~: .
: :
:
.
~3~L60~7 . Example 1 Dye Solution Applied to Untreated Fil~er Pa~r - Standard Control Foxmat ~0) FIG~ 1: Tests performed in duplic:ate, using two instruments, two 0.5 x 0.5 cm:ilt:er paper ma-trices, wavelength - 630 nm.
VOLUME OF
DYE SOLUTION
APPLIED TO
: ~ 10 TEST DEVICE CONCENTRATION OF DYE SOLUTION
1 x 10-5 M 2 x 10-5 M 3 x 10-5 M
STD. STD. STD.
DEVIA- DEVIA- DEVIA-K/S TION K/S TION K/S TION
20 u~ 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 ~.012 1.633 0.09~ 2.896 ~ 0 201 Exam~le 2 : `Cholesterol Solution Applied to Treated Filter Paper - Standard Control Format (0) FIG. 2: Data obtained as in Example 1, using ~:: 25 appropriate commercial test reagent:and wavelength 600~nm.
: ~ :
:
VOLUME OF
CHOLESTEROL
SOLUTION
APPLIED TO
TEST DEVICE CONCENTRATION OF CHOLESTEROL SOI,UTION
_ LOW ME~IUM 3IGH
STD. STD. STD.
DEVIA- DEVIA- DEVIA-K/S TION K/S TION K/S TION
20 uL 0.450 0.006 0.663 0.003 0.910 0.U25 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 Exam~le 3 Triglyceride ~olution Applied to Treated Filter Pa~er - Standard Control Format ~0) FIG. 3: Data obtained as in Example 1, using appropriate commercial test reagent and wavelength - 580 nm.
VOLUME OF
TRIG~YCERIDE
:SOLUTION
APPLIED TO
TEST DEVICE CONCENTRATION OF TRIGLYCERIDE_SOLUTION
LOW MEDIUM HIGH
_ _ _ : STD. STD. STD.
DEVIA- DEVIA- DEVIA-K/S TION R/S TION K/S TION
-- _ 20 uL 0.301 0.007 0.723 0.013 10146 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 PaE~r - Standard Control Format (0) FIG. 4: Data obtained as in ~xample 1, using __ appropriate commercial test reagent and wavelength - 640 nm.
VOLUME OF
POTASSIUM ION
SOLUTI ON
APPLIED TO
l'EST DEVICE CONCENTRATION OF POTASSIUM ION SOLUTION
LOW MEDIUM HIGH
STD. STD. STD.
DEVIA- DEVIA- DEVIA-K/S TION K/S TION K/S TION
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 o.ooa 0.693 0.017 ExamPle 5 Glucose Solution Applied to Treated Filter Paper - Standard Control Forma__(0) FIG. 5: Data obtained as in Example 1, using : 20 appropriate commercial test reagent and wavelength - 620 nm.
VOLUME OF
CLUCOSE~
SOLUTION
APPLIED TO
TEST DEVICE CONCENTRATION OF GLUCOSE SOLUTION
LOW MEDIUM_ HIGH _ STD. STDo STD.
: : DEVIA- DEVIA- DEVIA-K/S ION ~ TION K/S TION
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 ~3~ 6~7 As shown in FIGS. l through 5, the dependence upon sample volume for test samples applied direct-ly to devices having the standard control format ~0) is quite large. The volume dependence is seen in the large slope of the graphed unctions in FIGS. l 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 necessitat-ing the application of a precisely-measured test sample volume to test devices having the configura-tion of format (0).
However, by applying varying volumes of a dye solution to a device of the present invention, the film cover format (l), 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. l through 5 and, even more surprisingly the slopes of the graphs in : 25 FIG. 6 approach zero, thereby approaching a volume dependence of zero for a device having the film cover format (l).
Exam~le 6 Dye~Solution Applied to Untreated : 30 Filter Pa~er - F_lm Cover F_rmat (l) FIG. 6: Tests per~ormed in duplicate, using two instruments, two 0.5 x 1.0 cm. filter paper ma : ~ trices, wavelenyth - 630 nm.
.
~ 3 ~
VOLUME OF
DYE SOLUTION
APPLIED TO
TEST DEVICE ~=~=_~
5 1 x 10-5 M 2 x 11~-5 M 3 x 10-5 M
STD. STD. STD.
DEVIA- DEVIA- DEVIA-K/S TION K/S TION K/S TION
40 uL 0.270 0.004 0.473 9.015 0.639 :0.022 0.260 ~.018 ~.473 0.018 0.626 0.044 60 uL 0.266 0.012 0.476 0.023 0.617 0.024 0.26g 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 a.013 0.472 0.013 0.6~0 0.034 Example 7 Cholesterol Solution Applied to Treated Filter ~Gover Format (1) Data for Examples 7 and 8 was obtained as in Example 6, using the film cover format (1) ; ; 20 and thé appropriate test reagent 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.
CHOLESTEROL
SOLUTION
: APPLIED TO
TEST DEVICE CONCENTRATION OF CHOL~5T3Ro~ .OLil5~O;
::~ 30 : LOW~ _ MEDIUM _ HIGH
STD. STD. STD.
:: DEVIA- DEVIA- DEVIA-K/S TION K/S TION K/S TION
:~ Not Quanti- 0.442 0.012 0.629 0.018 :0.795 0.011 35 tatively 0.454 0.022 0.69g 0.030 0.855 0.042 Measured MS-14~7 ~L31~87 Example 8 Triglyceride Solution Applied to Treated ~ C~er rormat (1) VOLUME OF
TRIGLYCERIDE
SOLUTION
APPLI.ED TO
TEST DEVICE CONCENTRATION ~F TRIGLYCERIDE SOLUTION
.._ . . . . . _ ~.
LOW MEDIUM EIG~
~ . , _ . . . ___ STD. STD. STD.
DEVIA- DEVIA- DEVIA- .
K/S TION K/S TION K/S TION
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 i5 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 con~rol format (0) and : having a precise volume of test sample applied : 25 : to the test device (Ex. 1-5).
Exam~e_9 Cholesterol Solution Applied to Treated Filter Pa~er ~ ~o~chorl ~rma~ (2~:
:
FIG. 7: Data obtained as in Example 2.
.
:
~3~6(~8~
VOLUME OF
CHOLESTEROL
SOLUTION
APPLIED TO
TEST DEVICECONCENTRATION OE CElOLESTEROL SOLUTION
LOW MEDIUM HIGH
STD. STD. STD.
DEVIA- DEVIA- DEVIA-K/S TION R/S TION K/S TION
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 Tri~lyceride SoIution Applied to Treated Filter Paper - Touchoff Format (2) FIG. 8: Data obtained as in Example 3.
VOLUME OF
TRIGLYCERIDE
SOLUTION
APPLIED TO
20 : TEST DEVICE CONCENTRATION OF TRIGLYCERIDE SOLUTION
LOW _ MEDIUM HIGH _ STD.: STD. STD.
DEVIA- DEVIA- DEVIA-: KtS TION R/S TION K/S TION
: :25 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 ~ormat ~23 ~ - :30 FIG. 9 Data obtained as in Fxample 4-::::
13~L6~87 VOLUME OF
POTASSIUM ION
SOLUTION
APPLIED TO
TEST DEVICE CONCENTRATION OF POTASSIUM ION SOLUTION _ oW MEDIUM _ HIGH
STD. STD~ STD.
DEVIA- DEVIA- DFVIA- -~ TION K/S TION K/S TION
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 Exam~le 12 Glucose Solution Applied to Treated Filter Paper -_Touchoff Format (2) 15 FIG. 10: Data obtained as in Example 5.
VOLUME OF
GLUCOSE
SOLUTION
APPLIED TO
TEST DEVICE CONCENTRATION OF GLUCOSE S _UTION
LOW MEDIUM_ HIGH
STD. STD. STD.
DEVIA- DEVIA- DEVIA- .
K/S TION K/S TION K/S TION
30 uL 0.136 d.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 tes:t sample for removal, therefore 20-microliter test sample volumes were not tested.
As shown by the slopes of the graphed function in FIGS. 8 through 10, a diagnostic device utiliz-~1 3~6~7 ing the tvuchoff format is relatively independent of applied test sample volume. However, this format has the disadvantages of being significant-ly dependent upon user technique alnd of requiring an additional manipulative step within the test.
Example 13 Cholesterol Solution Applied to Treated Filter Paper - lot Li~htly Format (3 _IG. ll: Data obtained as in Æxample 2.
VOLUME OF
CHOLESTEROL
SOLUTION
APPLIED TO
TEST DEVICE CONCENTRATION OF CHOLESTEROL SOLUTION
LOW MEDIUM HIGH _ STD7 STD o STD
DEVIA- DEVIA- DEVIA-K/S TION K/S TION K/S TION
: 30 uL ~D333 0.~38 0.464 0.009 0.639 0.020 40 uL 0.335 0.031 0.473 0.028 0.617: ~ 0.036 Example 14 Trig:lyceride Solution Applied to Treated Filter P per - Blot_Li~ht1y Format (3) FIG. 12: Data obtained as in Example 3.
~ :
:
:: ~ : .
:131~7 VOLUME OF
TRIGLYCERIDE
SOLUTION
APPLTED TO
TEST DEVICECONCENTRATION OF TRIGLYCERIDE SOLUTION
LOW MEDIUM ~IGE
STD. STD. STD.
DEVIA- DEVIA- DEVIA-K/S TION K/S TION K/S TION
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 - ~lot Li ~tly_Format (3) FIG. i3: Data obtained as in Example 4.
VOLUME OF
POTASSIUM ION
SOLUTION
APPLIED TO
TEST DEVICE _ CQNCENTRATION OF POTASSIUM ION SOLUTION
_ LOW MEDIUM HIGH
~STD. STD. STD .
DEVIA- DEVIA- DEVIA-K/S TION ~ TION R/S TION
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 Li~htly Format _3) FIG. 14: Data obtained as in Example 5.
1 3 ~ 7 VOLUME OF
GLUCOSE
SOLUTI ON
APPL I ED TO
S TEST DEVICE CONCENTRATION OP GLUCOSE SOLUTION
LOW MEDIUM HIGH
. _ . . . . _ STDo STD. STD.
DEVIA- DEVIA- DEVIA--K/S TION ~ TION K/S TION
30 uL 0.111 0~002 0.232 0.016 0.385 0.012 40 uL 0.108 0.~)07 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 ~o the standard control format (0), however, the blotting lightly format (3) is highly dependent upon user technique. The volume independence oE 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 : heavi~ly format (4). ~ -- ~ Ex~ple 17 Cholesterol Solution Applied to Treated Filter Paper - Blot V ay~ g~ 4) 25 FIG._15: :Data obtained as in Example 2.
VOLUME OF
CHOLESTERO L
SOLU T I ON
APPLIED TO:
TEST DEVICE CONCENTRATïO~ (:)F CHOLESTEROL SOLUTION
_ LOW MEDIUM HIGH
, : STD .: STD . STD .
DEVIA-- DEVIA- DEVIA-K/S TION K/S TION K/S TION
30 UL 0~142 0.015 0.236 0.040 0.228 0.027 13~087 40 uL 0.122 0.010 0.180 0.016 0.217 0.031 Exam~le 18 Triglyceride Solution Applied to Treated Filter Paper - Blot Heavily ]?ormat ~4) 5 FIG. 16: Data obtained as in Example 3.
VOLUME OE
TRIGLYCERIDE
SOLUTION
APPLIED TO
TEST DEVICE CONCENTRATION OF TRIGLYCERIOE SOLUTTON
LOW MEDIUM HIG~
STD. STD. STD.
DEVIA- DEVIA- DEVIA-K/S TION K/S TION K/S TION
30 uL 0.110 0.008 0.253 0.032 0.420 0.025 40 uL 0.112 0.015 00223 0.018 0.335 0.073 Example_l9 Potassium Ion Solution Applied to Treated ilter Paper -_Blot Heavily Format (4) : 20 FIG. 17 Data obtained as in Example 4.
::
VOLUME OF
POTASSIUM ION
: SOLUTION
APPLIED TO
: 25 TEST DEVICE _ CONCENTR~TION OF POTASSIUM ION SOLUTION _ LOW MEDIUM HIG~I _ : STD. STD. : STD.
DEVIA- DEVIA- DEVIA-K/S : TION K/S TIOM K/S TION
~, _.
: ~ 30 30 uL 0~352 0.0I3 0.461 0.015 0.608 0.014 40 uL 0.351 Q.010 0.515 0.002 0.629 0.010 .
~3~87 ~38-Exam~le 20 Gluco~e Solution Applied to Treated Filter Paper - Blot Heavily Format ~4 FIG. 18: Data obtained as in Example 5.
VOLUME OF
GLUCOSE
: SOLUTION
APPLIED TO
TEST DEVICE _ CONCENTRATION C)F GLUCOSE SOLUTION
LOW MED IUM H I GH
STD. STD. STD.
DEVIA- DEVIA- DEVIA-K/S TION K/S TION K/S TION
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 extreme-ly dependent upon the actual pressure used in : blottin~. As can be seen by comparing the da~a and yraphs of Examples 17 through 20 to the data and graphs of the blot lightly techFIique~ Examples :25 13 through 16, the negative slopes oP several of the ~raphed functions in FIGS. 15 through 18 : : .shows the effect of blotting too heavily and thereby removing too much test sample from the : b:ibulous matrix.
:Dye Solution Applied to Untreated Filter Paper - Adjacent Dr~ Pad Format _5 :
FIG. l9s Data obtained as in Example 1.
--VOLUME OF
DYE SOLUTION
APPLIED TO
TEST DEVICECONCENTRATIC)N OF DYB SOLUTION
l x 10-5 M 2 x 10-5 M 3 x lG-5 M
STD. STD. STD.
DEVIA- `DEVIA- DEVIA-20 uL 0~129 0.017 0~312 0.121 0.427 0.060 0~183 0.017 0.337 0.12~ 0.480 0.184 30 uL 0.187 0.016 0.368 0.00~ 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. l9, ~he use of an additional bibulous matrix does reduce the dependence upon sample volume~ How-ever, the format does not exhibit sample volume independent to a sufficient degree to give accur-ate and reproducible analytical results on vari~
able test sample volumes. For instance, the RjS
values using 40 uL of a 2 x 10 ~5 M dye solution are larger than the K/S values found using 20 uL
of 3 x 10-5 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 .
, ~S-14~7 VOLUME OF
DYE SOLUTION
APPLIED TO
TEST DEVICE CONCENTRATION OF DYE SOLUTION
3 x 10-5 M
STD .
I:~EVI A-K/S Tl:ON
lQ 20 uL 0.563 0.033 0 . 658 0 . 0~3 30 uI. O . 709 0 . 144 0.772 1~.0D~) 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.
Dye Solution Applied to Untreated Filter Pa~er - 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 bibu-lous substrate. Variable amounts of excess test sample were visually present on the testing bibu-lous substrate. This format essen~ially did not reduce test sample volume dependence.
:
~3~6~87 Example 24 Dye Solution Applied to Untreated Fil~er Paper - Apply to Adjacent .
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 matrixO Variable amounts of excess test sample were visually present on t~e testing bibulous matrix. This format essenti-ally did not reduce the test sample volume dep~nd-ence.
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
: Law MEDIUM HIGH ~
. _ STD. ~ STD. STD
DEVIA- DEVIA- DEVIA-K/S TION K/S TION K/S TION
; ~ Not Quanti- 0.449 0.024 0.696 0.025 0.952 0.072 tativel~
Measured Example 26 :
Triglyceride Solution Applied to Treated Filter Paper - DiP Reaqent Format (9~
n Data obtained~ as in Example 3. Volume of applied test solution varies.
~:
:
~ 3 ~ 8 7 -4~-CONCENTRATION OF TRIGLYCERIDE SOLUTION
LOW MEDIUM HIGH
STD. STD. STD.
DEVIA- DEVIA- DEVIA-K/S TION ~ TION K/S TION
Not Quanti- 0.310 0.009 0.746 0.021 1.212 0.031 tatively Measured Example 2?
Potassium Ion Solution Applied to Treated Filter Pa~er - Dip Rea~ent Format (9) Data obtained as in Example 4. Volume of applied test solution varies.
CONCENTRATION OF POTASSIUM ION SOLUTION
LOW MEDIUM HIGH
. _ .
STD. STD. STD.
DEVIA- DEVIA- DEVIA-~ TION K/S TION K/S TION
Not ~uanti- 0.334 0.002 0.424 0.005 0.554 0.024 tatively Measured Exampl-e 28 Glucose Solut:ion:Applied to Treated : ilter ~ 9) : 25 Data obtained as in Example 5. Volume of ;
applied test solution varies.
CONCENTRATION OF GLUCOSE SOLUTION
- - - -- -- _ : LOW MEDIUM HIGH
--- ~
: ~ : 30 - STD. STD. STD.
DEVIA- DEVIA- DEVIA-K/S TION K/S TION ~ TION
- - Not Quanti- 0.135 0.007 0.293 0.014 0.445 0.022 tatively Measured:
1 3 ~
Examples 25 through 28, using the dip reagent format (9), demonstrate an intrinsically volume independent test method and devic:e because the bibulous matrix in this format can only absorb the volume of liquid test sample ~o required saturate the matrix. However, for several olini-cal tests, and in particular for blood ser~m tests,-the dip reagent format (g~ is impractical because it requires a large test sample to provide sufficient volume for completely~dipping the test device~ The dip reagent format also intro-duces the risk of contamination of the test sample by the diagnostic device, thereby leading ~o erroneous results o~ causing interference with any subsequent tests to be run on the same test sample.
~he summary of results from the data tabulat-ed in Examples 1 through 28 and the graphs in FIGS. 1 through 20 are included in Table II.
:~ 20 From the Examples utilizing these various methods ; and devices~ it is seen ~hat several of the de-:~ vices have formats demonstrating sample volume : independence. ~owever, these devices, including the touchoff~ blot;lightlyl blot heavily and dip :
~: : 25 reagent formats, also have She 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 : 30 6 through 8 and in FIG. 6, shows essentially : complète volume independence and is free of any technique or manipulative disadvantages.
In Table II,~ the volume dependence is ex-pressed~re:lative to the volume dependency of the :: 35 skandard control format (0). For a basis of :
:~ MS-1487 ~ 31 ~8~
comparison, the volume dependency of the standard con-trol 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
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 bibu-lous matrices covered by a liquid impervious coating or film. The matrices are so treated X
~3~ ~87 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 o a bibulous matrix such that the test sample is metered into the bibulous matrix chromatographically. By wicking actiont 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.
5pecifically, the positioning of the bibulous matrices and the testing-reagent, and the metering of the sample, may be better under-stood by reference to FIGS. 21 through 25. FIG.
21 shows a perspective view of a volume independ-ent diagnostic device 10 including a first bibu-lous matrix 14; and a second bibulous matrix 16 : impregnated with a suitable testing reagent, : both bibulous matrices securely adhered to a support str;ip or handle~12. As will become more : apparent hereinafter, in order to facilitate the quanti~ative determination of analytes, it i5 preferred that the support strip or handle be manufactured from a hydrophobic material. Prefer-ably, the hydrophobic material is translucent, and can be formed from materials such as cellulose : 30 acetate, polyethylene, terephthala~e, polycarbon-~ : ate and polystyreneO 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 : : 35 14 and to prevent sample spillover onto the second ~31~7 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 with the second bibulous matrix 16. The barrier 18 extends to comple~ely 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 place-ment 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 chromo- -genic test within the assay area. If barrier 18 : 25 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 ~est sample cannot penetrate through the barrier : 35 18 to directly contact the second bibulous sub-~31~0~7 strate 16. The barrier 18 is preferably a tran~
parent or translucent material. However, if the substrate 12 i9 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 smoo~h, water repellent and nontoxic. Types of waxes that can be utilized in the method and devi.ce of the pre-sent invention include natural wa~es, 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, cere-sin, montan; and petroleum waxes, such as paraffin, microcrystalline, petrolatum; as well as synthetic waxes such as ethylenic polymers and polyolether-esters, sorbitol and chlorinated napthalenes and other hydrocarbon waxes.
Another coniguration 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 ; 25 by a bibulous thin-tissue bridge 219 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 30 : 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 configura-: tions 30 (40) wherein the amount of the test sample:reguired may be increased or decreased ~3~87 -48~
per dose by varying the size of the second bibu lous matrix 36 (46) relative to the size of the first bibulous matrix 34 (44). Such configura-tions attached to substrates 32 (42) and contain-ing a barrier 38 (48) allows flexibility in ana-lyte determinativns by making chromogenic reac-tions more responsive to quantitative determina-tion.
FIG. 25 illustrates a confiyuration 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 w~ith~an important feature of the present invention, an excess amount of test sample, usually in excess of approximat~ely 30 microliters, is applied to the diagnostic device in the area of the bibulous~matrix that i5 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 matri~.
The liquid-impervious barrier helps meter the test sample into the bibulous matrices. The liquid chromatographs through the bibulous ma-krices by wicking action up to the point of matrix saturation. After matrix saturation by the test .
. , . . , ~
~ 3 1~
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 bibu-lous matrix. Therefore, a constant volume oftest sample, in relation to the size of the bibu lous matrix, is directed to the assay area of the device, resulting in accurate and reproducible analyte concentration determinations.
rhe particular 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 ~ 20 amount required to saturate the test reagent-; - treated pad. When the test reagent-treated pad becomes saturated further flow of test sample stops, and the remainder of the test sample re-;~ mains separated ~rom the reagent pad by the coat-ing or ilm layer and does not thereby affect the chromogenic reaction~
In accordance with an important eature of the process and device of the present inven-tion, in addition to a constant and reproducible amount of test sample reaching the assay a~ea of ~the device, the test sample reaches the assay area o the device with an essentially unaltered composition. Tests performed on blood samples showed no increase in potassium ion or loss of ch~lesterolj as the test sample chromatographed~
;
~ 3~ 7 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 axcess test sample to assure test sample satura-tion 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 ren-ders more accurate and reproducible analyte deter-minations. 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 de~ermined by those skilled in the art.
~ n addition to the fast and efficient analyte determinations of liquid test samples, and the essentially complete migration o un-altered test sample to the assay area, the methodand device of the present invention permit quan-titative analyte determinations without dilutio~
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 ~est sample. The extent of the chromogenic reaction, and therefore the quan-MS~lA87 1 3 ~ 7 titative amount of the analytet then can be deter-mined by the chromogenic detection techniques that are well-known in the art.
Obviously, many modifications and vari ations o the invention as hereinbefore set forth can be made without departing from the spirit and scope thereof and therefore only such limita-tions should be imposed as are indicated by the appended claims.
-, . :
Claims (11)
1. A device for metering a constant volume of a liquid to an analyte assay area com-prising a bibulous matrix having upper and lower major surfaces, the lower major surface disposed against a hydrophobic substrate, and the upper major surface partially covered by a liquid-impermeable barrier such that liquid applied to an area of the upper major bibulous matrix surface spaced from the liquid-impermeable barrier is metered into the bibulous matrix, wherein the liquid flows through the bibulous matrix to satur-ate the bibulous matrix in the analyte assay area with a constant volume of liquid per unit volume of bibulous matrix.
2. A device for metering a constant volume of a liquid to an analyte assay area and for determining analyte concentrations in liquid samples comprising a first bibulous matrix, a second bibulous matrix containing a suitable test reagent in a sufficient quantity for inter-action with a particular analyte constituent of the liquid to produce a detectable change in the second bibulous matrix upon contact with the liquid, said second bibulous matrix being disposed sufficiently close to the first bibulous matrix and so positioned and sized that the interaction between the analyte constituent of the liquid and the testing reagent is quantitatively de-tected; and a liquid impermeable barrier disposed over a portion of the first bibulous matrix and at least a portion of the second bibulous matrix such that liquid applied to the first bibulous matrix at an area spaced from the second bibulous matrix will be metered into the second bibulous matrix only to the point of liquid saturation of the second bibulous matrix such that the quanti-tative determination of the analyte constituent of the liquid is independent of the volume of liquid sample applied to the first bibulous ma-trix.
3. The device of claim 2 wherein the first bibulous matrix and the second bibulous matrix are the same or different hydrophilic materials selected from the group consisting of inorganic powders, sponge materials, argillaceous materials, cloth, hydrophilic naturally-occurring polymers, hydrophilic naturally-occurring modified polymers, hydrophilic synthetic polymers, silica gel, alumina, diatomaceous earth, cellulosic materials, nitrocellulose, cellulose acetate, polyvinyl chloride, polyacrylamide, polyurethanes, polyacrylates, crosslinked dextran, agarose, and mixtures thereof.
4. The device of claim 3 wherein the first and second bibulous matrices are filter paper.
5. A volume independent diagnostic device comprising at least one bibulous matrix, said bibulous matrix treated with a test reagent in a reagent-treated test area, the test area of the bibulous matrix being covered by a liquid impermeable barrier, said barrier so positioned to meter a liquid from a point spaced from the test area to the test area of the bibulous matrix, wherein the bibulous matrix permits the liquid to pass through the bibulous matrix to introduce a constant, saturating amount of liquid into the test area per unit volume of bibulous matrix, said liquid-impermeable barrier covering all of the reagent treated test area of the bibulous matrix and a portion of the untreated bibulous matrix.
6. The device of claim 5 wherein the test reagent treated portion of the bibulous matrix and the untreated portion of the bibulous matrix are the same or different hydrophilic materials selected from the group consisting of inorganic powders, sponge materials, argillaceous materials, cloth, hydrophilic naturally-occurring polymers, hydrophilic naturally-occurring modi-fied polymers, hydrophilic synthetic polymers, silica gel, alumina, diatomaceous earth, cellulo-sic materials, nitrocellulose, cellulose acetate, polyvinyl chloride, polyacrylamide, polyurethanes, polyacrylates, crosslinked dextran, agarose, and mixtures thereof.
7. The device of claim 6 wherein the bibulous matrix portions are filter paper.
8. A method of introducing a constant volume of a liquid from a liquid application area to an assay area of a diagnostic device comprising contacting a bibulous matrix with a quantity of liquid at least sufficient to saturate the assay area of the bibulous matrix, said matrix having a liquid-impervious covering disposed between the liquid-application area of the bibu-lous matrix and the assay area of the bibulous matrix to prevent the liquid from initially con-tacting the assay area of said bibulous matrix and to meter the liquid from the liquid applica-tion area to the assay area of said bibulous matrix, allowing a quantity of the liquid to flow through said bibulous matrix and under the liquid-impervious covering, to saturate the assay area of the bibulous matrix.
9. A method of determining analyte concentrations in a liquid comprising contacting a first area of a bibulous matrix with an excess quantity of the liquid, said first area of said bibulous matrix disposed in contact with a second area of said bibulous matrix, said second area containing a sufficient quantity of a suitable testing reagent for a particular analyte for interaction with the liquid to produce a detect-able change in the second area of the bibulous matrix; said first area of the bibulous matrix and the second area of the bibulous matrix covered by a liquid-impermeable covering disposed to prevent the liquid from initially contacting an upper surface of the second area of the bibulous matrix, said covering metering the liquid from the first area of the bibulous matrix, under the liquid-impermeable covering to saturate the second area of the bibulous matrix to cause the liquid to interact with the testing reagent resulting in a detectable change in the second area of the bibulous matrix, and detecting the detectable change in said second bibulous matrix in the second area of the bibulous matrix.
10. The method of claim 9 wherein the first bibulous matrix and the second bibulous matrix are the same or different hydrophilic materials selected from the group consisting of inorganic powders, sponge materials, argillaceous materials, cloth, hydrophilic naturally-occurring polymers, hydrophilic naturally-occurring modified polymers, hydrophilic synthetic polymers, silica gel, alumina, diatomaceous earth, cellulosic materials, nitrocellulose, cellulose acetate, polyvinyl chloride, polyacrylamide, polyurethanes, polyacrylates, crosslinked dextran, agarose, and mixtures thereof.
11. The method of claim 10 wherein the first and second bibulous matrices are filter paper.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US63,925 | 1979-08-06 | ||
| US07/063,925 US4810470A (en) | 1987-06-19 | 1987-06-19 | Volume independent diagnostic device |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1316087C true CA1316087C (en) | 1993-04-13 |
Family
ID=22052401
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000566513A Expired - Fee Related CA1316087C (en) | 1987-06-19 | 1988-05-11 | Volume independent diagnostic device |
Country Status (10)
| Country | Link |
|---|---|
| US (1) | US4810470A (en) |
| EP (1) | EP0303784B2 (en) |
| JP (1) | JPS6421361A (en) |
| AU (1) | AU591599B2 (en) |
| CA (1) | CA1316087C (en) |
| DE (1) | DE3870356D1 (en) |
| DK (1) | DK332588A (en) |
| ES (1) | ES2030788T5 (en) |
| IE (1) | IE60139B1 (en) |
| ZA (1) | ZA883910B (en) |
Families Citing this family (128)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5622871A (en) | 1987-04-27 | 1997-04-22 | Unilever Patent Holdings B.V. | Capillary immunoassay and device therefor comprising mobilizable particulate labelled reagents |
| US4935346A (en) * | 1986-08-13 | 1990-06-19 | Lifescan, Inc. | Minimum procedure system for the determination of analytes |
| DE3856421T2 (en) | 1987-04-27 | 2000-12-14 | Unilever Nv | Specific binding test procedures |
| EP0654659B1 (en) * | 1988-06-09 | 1997-02-12 | Boehringer Mannheim Corporation | Defined volume test device |
| US5082626A (en) * | 1988-08-08 | 1992-01-21 | Boehringer Mannheim Gmbh | Wedge shaped test strip system useful in analyzing test samples, such as whole blood |
| US6352862B1 (en) | 1989-02-17 | 2002-03-05 | Unilever Patent Holdings B.V. | Analytical test device for imuno assays and methods of using same |
| US5234813A (en) * | 1989-05-17 | 1993-08-10 | Actimed Laboratories, Inc. | Method and device for metering of fluid samples and detection of analytes therein |
| US6395227B1 (en) * | 1989-08-28 | 2002-05-28 | Lifescan, Inc. | Test strip for measuring analyte concentration over a broad range of sample volume |
| US5036861A (en) * | 1990-01-11 | 1991-08-06 | Sembrowich Walter L | Method and apparatus for non-invasively monitoring plasma glucose levels |
| GB9014903D0 (en) * | 1990-07-05 | 1990-08-22 | Unilever Plc | Assays |
| US5169599A (en) * | 1990-08-30 | 1992-12-08 | Teknekron Sensor Development Corporation | Method and apparatus for optically detecting presence of immunological components |
| US5869345A (en) * | 1991-05-29 | 1999-02-09 | Smithkline Diagnostics, Inc. | Opposable-element assay device employing conductive barrier |
| US5607863A (en) * | 1991-05-29 | 1997-03-04 | Smithkline Diagnostics, Inc. | Barrier-controlled assay device |
| US5998220A (en) | 1991-05-29 | 1999-12-07 | Beckman Coulter, Inc. | Opposable-element assay devices, kits, and methods employing them |
| US6168956B1 (en) | 1991-05-29 | 2001-01-02 | Beckman Coulter, Inc. | Multiple component chromatographic assay device |
| US5468648A (en) * | 1991-05-29 | 1995-11-21 | Smithkline Diagnostics, Inc. | Interrupted-flow assay device |
| US5877028A (en) | 1991-05-29 | 1999-03-02 | Smithkline Diagnostics, Inc. | Immunochromatographic assay device |
| GB9304452D0 (en) * | 1993-03-04 | 1993-04-21 | Bunce Roger A | Analytical devices |
| US5843691A (en) * | 1993-05-15 | 1998-12-01 | Lifescan, Inc. | Visually-readable reagent test strip |
| EP0772484B1 (en) * | 1994-07-28 | 2008-02-27 | Pall Corporation | Fibrous web and process of preparing same |
| US5582907A (en) * | 1994-07-28 | 1996-12-10 | Pall Corporation | Melt-blown fibrous web |
| US6638415B1 (en) | 1995-11-16 | 2003-10-28 | Lifescan, Inc. | Antioxidant sensor |
| US5962215A (en) | 1996-04-05 | 1999-10-05 | Mercury Diagnostics, Inc. | Methods for testing the concentration of an analyte in a body fluid |
| US5879951A (en) | 1997-01-29 | 1999-03-09 | Smithkline Diagnostics, Inc. | Opposable-element assay device employing unidirectional flow |
| US20050033132A1 (en) | 1997-03-04 | 2005-02-10 | Shults Mark C. | Analyte measuring device |
| US6741877B1 (en) * | 1997-03-04 | 2004-05-25 | Dexcom, Inc. | Device and method for determining analyte levels |
| US6001067A (en) * | 1997-03-04 | 1999-12-14 | Shults; Mark C. | Device and method for determining analyte levels |
| US7192450B2 (en) | 2003-05-21 | 2007-03-20 | Dexcom, Inc. | Porous membranes for use with implantable devices |
| US5939252A (en) | 1997-05-09 | 1999-08-17 | Lennon; Donald J. | Detachable-element assay device |
| US9066695B2 (en) | 1998-04-30 | 2015-06-30 | Abbott Diabetes Care Inc. | Analyte monitoring device and methods of use |
| US6949816B2 (en) | 2003-04-21 | 2005-09-27 | Motorola, Inc. | Semiconductor component having first surface area for electrically coupling to a semiconductor chip and second surface area for electrically coupling to a substrate, and method of manufacturing same |
| US8688188B2 (en) | 1998-04-30 | 2014-04-01 | Abbott Diabetes Care Inc. | Analyte monitoring device and methods of use |
| US8346337B2 (en) | 1998-04-30 | 2013-01-01 | Abbott Diabetes Care Inc. | Analyte monitoring device and methods of use |
| US6175752B1 (en) | 1998-04-30 | 2001-01-16 | Therasense, Inc. | Analyte monitoring device and methods of use |
| US8480580B2 (en) | 1998-04-30 | 2013-07-09 | Abbott Diabetes Care Inc. | Analyte monitoring device and methods of use |
| US8974386B2 (en) | 1998-04-30 | 2015-03-10 | Abbott Diabetes Care Inc. | Analyte monitoring device and methods of use |
| US8465425B2 (en) | 1998-04-30 | 2013-06-18 | Abbott Diabetes Care Inc. | Analyte monitoring device and methods of use |
| US6312888B1 (en) | 1998-06-10 | 2001-11-06 | Abbott Laboratories | Diagnostic assay for a sample of biological fluid |
| US6656741B1 (en) * | 1999-03-03 | 2003-12-02 | Diabetes Diagnostics, Inc. | Air gap for controlling sample absorption and hematocrit dependence |
| US6458326B1 (en) | 1999-11-24 | 2002-10-01 | Home Diagnostics, Inc. | Protective test strip platform |
| US6627057B1 (en) | 1999-12-23 | 2003-09-30 | Roche Diagnostic Corporation | Microsphere containing sensor |
| US6612111B1 (en) | 2000-03-27 | 2003-09-02 | Lifescan, Inc. | Method and device for sampling and analyzing interstitial fluid and whole blood samples |
| US6571651B1 (en) * | 2000-03-27 | 2003-06-03 | Lifescan, Inc. | Method of preventing short sampling of a capillary or wicking fill device |
| US6603403B2 (en) | 2000-12-12 | 2003-08-05 | Kimberly-Clark Worldwide, Inc. | Remote, wetness signaling system |
| US6583722B2 (en) | 2000-12-12 | 2003-06-24 | Kimberly-Clark Worldwide, Inc. | Wetness signaling device |
| US6560471B1 (en) | 2001-01-02 | 2003-05-06 | Therasense, Inc. | Analyte monitoring device and methods of use |
| US6525330B2 (en) | 2001-02-28 | 2003-02-25 | Home Diagnostics, Inc. | Method of strip insertion detection |
| US6562625B2 (en) | 2001-02-28 | 2003-05-13 | Home Diagnostics, Inc. | Distinguishing test types through spectral analysis |
| US6541266B2 (en) | 2001-02-28 | 2003-04-01 | Home Diagnostics, Inc. | Method for determining concentration of an analyte in a test strip |
| WO2002078512A2 (en) | 2001-04-02 | 2002-10-10 | Therasense, Inc. | Blood glucose tracking apparatus and methods |
| US6702857B2 (en) | 2001-07-27 | 2004-03-09 | Dexcom, Inc. | Membrane for use with implantable devices |
| US20030032874A1 (en) | 2001-07-27 | 2003-02-13 | Dexcom, Inc. | Sensor head for use with implantable devices |
| US7613491B2 (en) | 2002-05-22 | 2009-11-03 | Dexcom, Inc. | Silicone based membranes for use in implantable glucose sensors |
| US8010174B2 (en) | 2003-08-22 | 2011-08-30 | Dexcom, Inc. | Systems and methods for replacing signal artifacts in a glucose sensor data stream |
| US8364229B2 (en) | 2003-07-25 | 2013-01-29 | Dexcom, Inc. | Analyte sensors having a signal-to-noise ratio substantially unaffected by non-constant noise |
| US9247901B2 (en) | 2003-08-22 | 2016-02-02 | Dexcom, Inc. | Systems and methods for replacing signal artifacts in a glucose sensor data stream |
| US8260393B2 (en) | 2003-07-25 | 2012-09-04 | Dexcom, Inc. | Systems and methods for replacing signal data artifacts in a glucose sensor data stream |
| US7226978B2 (en) * | 2002-05-22 | 2007-06-05 | Dexcom, Inc. | Techniques to improve polyurethane membranes for implantable glucose sensors |
| US20060258761A1 (en) * | 2002-05-22 | 2006-11-16 | Robert Boock | Silicone based membranes for use in implantable glucose sensors |
| US7134999B2 (en) | 2003-04-04 | 2006-11-14 | Dexcom, Inc. | Optimized sensor geometry for an implantable glucose sensor |
| US7875293B2 (en) * | 2003-05-21 | 2011-01-25 | Dexcom, Inc. | Biointerface membranes incorporating bioactive agents |
| US7467003B2 (en) * | 2003-12-05 | 2008-12-16 | Dexcom, Inc. | Dual electrode system for a continuous analyte sensor |
| US7366556B2 (en) | 2003-12-05 | 2008-04-29 | Dexcom, Inc. | Dual electrode system for a continuous analyte sensor |
| WO2005012871A2 (en) * | 2003-07-25 | 2005-02-10 | Dexcom, Inc. | Increasing bias for oxygen production in an electrode system |
| US7424318B2 (en) | 2003-12-05 | 2008-09-09 | Dexcom, Inc. | Dual electrode system for a continuous analyte sensor |
| US9763609B2 (en) | 2003-07-25 | 2017-09-19 | Dexcom, Inc. | Analyte sensors having a signal-to-noise ratio substantially unaffected by non-constant noise |
| US7651596B2 (en) | 2005-04-08 | 2010-01-26 | Dexcom, Inc. | Cellulosic-based interference domain for an analyte sensor |
| US20070173709A1 (en) * | 2005-04-08 | 2007-07-26 | Petisce James R | Membranes for an analyte sensor |
| US8423113B2 (en) | 2003-07-25 | 2013-04-16 | Dexcom, Inc. | Systems and methods for processing sensor data |
| US7460898B2 (en) * | 2003-12-05 | 2008-12-02 | Dexcom, Inc. | Dual electrode system for a continuous analyte sensor |
| WO2007120442A2 (en) * | 2003-07-25 | 2007-10-25 | Dexcom, Inc. | Dual electrode system for a continuous analyte sensor |
| US20050176136A1 (en) * | 2003-11-19 | 2005-08-11 | Dexcom, Inc. | Afinity domain for analyte sensor |
| US7591801B2 (en) | 2004-02-26 | 2009-09-22 | Dexcom, Inc. | Integrated delivery device for continuous glucose sensor |
| US20190357827A1 (en) | 2003-08-01 | 2019-11-28 | Dexcom, Inc. | Analyte sensor |
| US7774145B2 (en) | 2003-08-01 | 2010-08-10 | Dexcom, Inc. | Transcutaneous analyte sensor |
| US7519408B2 (en) | 2003-11-19 | 2009-04-14 | Dexcom, Inc. | Integrated receiver for continuous analyte sensor |
| US8622905B2 (en) | 2003-08-01 | 2014-01-07 | Dexcom, Inc. | System and methods for processing analyte sensor data |
| US8060173B2 (en) | 2003-08-01 | 2011-11-15 | Dexcom, Inc. | System and methods for processing analyte sensor data |
| US20080119703A1 (en) | 2006-10-04 | 2008-05-22 | Mark Brister | Analyte sensor |
| US8160669B2 (en) | 2003-08-01 | 2012-04-17 | Dexcom, Inc. | Transcutaneous analyte sensor |
| US8845536B2 (en) | 2003-08-01 | 2014-09-30 | Dexcom, Inc. | Transcutaneous analyte sensor |
| US8275437B2 (en) | 2003-08-01 | 2012-09-25 | Dexcom, Inc. | Transcutaneous analyte sensor |
| US9135402B2 (en) | 2007-12-17 | 2015-09-15 | Dexcom, Inc. | Systems and methods for processing sensor data |
| US8233959B2 (en) | 2003-08-22 | 2012-07-31 | Dexcom, Inc. | Systems and methods for processing analyte sensor data |
| US7920906B2 (en) | 2005-03-10 | 2011-04-05 | Dexcom, Inc. | System and methods for processing analyte sensor data for sensor calibration |
| US20140121989A1 (en) | 2003-08-22 | 2014-05-01 | Dexcom, Inc. | Systems and methods for processing analyte sensor data |
| US20050090607A1 (en) * | 2003-10-28 | 2005-04-28 | Dexcom, Inc. | Silicone composition for biocompatible membrane |
| US8615282B2 (en) | 2004-07-13 | 2013-12-24 | Dexcom, Inc. | Analyte sensor |
| US9247900B2 (en) | 2004-07-13 | 2016-02-02 | Dexcom, Inc. | Analyte sensor |
| US8364231B2 (en) | 2006-10-04 | 2013-01-29 | Dexcom, Inc. | Analyte sensor |
| EP1711790B1 (en) | 2003-12-05 | 2010-09-08 | DexCom, Inc. | Calibration techniques for a continuous analyte sensor |
| US11633133B2 (en) | 2003-12-05 | 2023-04-25 | Dexcom, Inc. | Dual electrode system for a continuous analyte sensor |
| EP2228642B1 (en) * | 2003-12-08 | 2017-07-19 | DexCom, Inc. | Systems and methods for improving electrochemical analyte sensors |
| EP2316331B1 (en) * | 2003-12-09 | 2016-06-29 | Dexcom, Inc. | Signal processing for continuous analyte sensor |
| US8808228B2 (en) | 2004-02-26 | 2014-08-19 | Dexcom, Inc. | Integrated medicament delivery device for use with continuous analyte sensor |
| US8277713B2 (en) * | 2004-05-03 | 2012-10-02 | Dexcom, Inc. | Implantable analyte sensor |
| US8792955B2 (en) | 2004-05-03 | 2014-07-29 | Dexcom, Inc. | Transcutaneous analyte sensor |
| WO2006127694A2 (en) | 2004-07-13 | 2006-11-30 | Dexcom, Inc. | Analyte sensor |
| US7654956B2 (en) | 2004-07-13 | 2010-02-02 | Dexcom, Inc. | Transcutaneous analyte sensor |
| US20060016700A1 (en) * | 2004-07-13 | 2006-01-26 | Dexcom, Inc. | Transcutaneous analyte sensor |
| US8565848B2 (en) | 2004-07-13 | 2013-10-22 | Dexcom, Inc. | Transcutaneous analyte sensor |
| US7783333B2 (en) | 2004-07-13 | 2010-08-24 | Dexcom, Inc. | Transcutaneous medical device with variable stiffness |
| US8452368B2 (en) | 2004-07-13 | 2013-05-28 | Dexcom, Inc. | Transcutaneous analyte sensor |
| US8744546B2 (en) | 2005-05-05 | 2014-06-03 | Dexcom, Inc. | Cellulosic-based resistance domain for an analyte sensor |
| US20060249381A1 (en) * | 2005-05-05 | 2006-11-09 | Petisce James R | Cellulosic-based resistance domain for an analyte sensor |
| US7803319B2 (en) * | 2005-04-29 | 2010-09-28 | Kimberly-Clark Worldwide, Inc. | Metering technique for lateral flow assay devices |
| US7279136B2 (en) | 2005-12-13 | 2007-10-09 | Takeuchi James M | Metering technique for lateral flow assay devices |
| US7618810B2 (en) * | 2005-12-14 | 2009-11-17 | Kimberly-Clark Worldwide, Inc. | Metering strip and method for lateral flow assay devices |
| JP4876646B2 (en) * | 2006-03-13 | 2012-02-15 | 富士レビオ株式会社 | Immunoassay strip and immunoassay device |
| US7920907B2 (en) | 2006-06-07 | 2011-04-05 | Abbott Diabetes Care Inc. | Analyte monitoring system and method |
| KR100770440B1 (en) * | 2006-08-29 | 2007-10-26 | 삼성전기주식회사 | Nitride semiconductor light emitting device |
| US7831287B2 (en) * | 2006-10-04 | 2010-11-09 | Dexcom, Inc. | Dual electrode system for a continuous analyte sensor |
| US20200037874A1 (en) | 2007-05-18 | 2020-02-06 | Dexcom, Inc. | Analyte sensors having a signal-to-noise ratio substantially unaffected by non-constant noise |
| US20080306434A1 (en) | 2007-06-08 | 2008-12-11 | Dexcom, Inc. | Integrated medicament delivery device for use with continuous analyte sensor |
| US9452258B2 (en) | 2007-10-09 | 2016-09-27 | Dexcom, Inc. | Integrated insulin delivery system with continuous glucose sensor |
| US8417312B2 (en) | 2007-10-25 | 2013-04-09 | Dexcom, Inc. | Systems and methods for processing sensor data |
| US8535617B2 (en) * | 2007-11-30 | 2013-09-17 | Kimberly-Clark Worldwide, Inc. | Blood cell barrier for a lateral flow device |
| US8290559B2 (en) | 2007-12-17 | 2012-10-16 | Dexcom, Inc. | Systems and methods for processing sensor data |
| CA2715628A1 (en) | 2008-02-21 | 2009-08-27 | Dexcom, Inc. | Systems and methods for processing, transmitting and displaying sensor data |
| EP2106784B1 (en) * | 2008-04-03 | 2015-04-22 | Rohm and Haas Company | Hair styling composition |
| US8956859B1 (en) | 2010-08-13 | 2015-02-17 | Aviex Technologies Llc | Compositions and methods for determining successful immunization by one or more vaccines |
| ES2847578T3 (en) | 2011-04-15 | 2021-08-03 | Dexcom Inc | Advanced analyte sensor calibration and error detection |
| US9528987B2 (en) | 2011-06-23 | 2016-12-27 | University Of Washington | Reagent patterning in capillarity-based analyzers and associated systems and methods |
| CN105050720A (en) | 2013-01-22 | 2015-11-11 | 华盛顿大学商业化中心 | Sequential delivery of fluid volumes and associated devices, systems and methods |
| MX2017002795A (en) * | 2014-09-02 | 2018-01-23 | Clinical Genomics Pty Ltd | Test device and method. |
| US10293340B2 (en) | 2017-10-11 | 2019-05-21 | Fitbit, Inc. | Microfluidic metering and delivery system |
| US11331022B2 (en) | 2017-10-24 | 2022-05-17 | Dexcom, Inc. | Pre-connected analyte sensors |
| CN111246797A (en) | 2017-10-24 | 2020-06-05 | 德克斯康公司 | Pre-attached analyte sensors |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NL286850A (en) * | 1961-12-18 | 1900-01-01 | ||
| US3798004A (en) * | 1972-04-18 | 1974-03-19 | Ames Yissum Ltd | Test device |
| US3917453A (en) * | 1974-08-16 | 1975-11-04 | Polaroid Corp | Method and device for determining the concentration of a substance in a fluid |
| US4069017A (en) * | 1977-01-14 | 1978-01-17 | Eastman Kodak Company | Colorimetric assay for bilirubin |
| JPS55164356A (en) * | 1979-06-08 | 1980-12-22 | Fuji Photo Film Co Ltd | Multi-layer analysis sheet for liquid sample analysis |
| JPS5697872A (en) * | 1980-01-07 | 1981-08-06 | Fuji Photo Film Co Ltd | Measuring material for concentration of hemoglobin in blood |
| US4647430A (en) * | 1985-06-20 | 1987-03-03 | Miles Laboratories, Inc. | Volume independent test device |
-
1987
- 1987-06-19 US US07/063,925 patent/US4810470A/en not_active Expired - Fee Related
-
1988
- 1988-05-11 CA CA000566513A patent/CA1316087C/en not_active Expired - Fee Related
- 1988-06-02 ZA ZA883910A patent/ZA883910B/en unknown
- 1988-06-07 DE DE8888109038T patent/DE3870356D1/en not_active Expired - Fee Related
- 1988-06-07 ES ES88109038T patent/ES2030788T5/en not_active Expired - Lifetime
- 1988-06-07 EP EP88109038A patent/EP0303784B2/en not_active Expired - Lifetime
- 1988-06-17 IE IE185488A patent/IE60139B1/en not_active IP Right Cessation
- 1988-06-17 JP JP63148452A patent/JPS6421361A/en active Pending
- 1988-06-17 AU AU18130/88A patent/AU591599B2/en not_active Ceased
- 1988-06-17 DK DK332588A patent/DK332588A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| DK332588D0 (en) | 1988-06-17 |
| US4810470A (en) | 1989-03-07 |
| IE881854L (en) | 1988-12-19 |
| AU1813088A (en) | 1988-12-22 |
| EP0303784B2 (en) | 1995-09-06 |
| EP0303784A1 (en) | 1989-02-22 |
| JPS6421361A (en) | 1989-01-24 |
| DE3870356D1 (en) | 1992-05-27 |
| DK332588A (en) | 1988-12-20 |
| IE60139B1 (en) | 1994-06-01 |
| AU591599B2 (en) | 1989-12-07 |
| ES2030788T3 (en) | 1992-11-16 |
| EP0303784B1 (en) | 1992-04-22 |
| ZA883910B (en) | 1988-12-19 |
| ES2030788T5 (en) | 1996-01-16 |
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| Date | Code | Title | Description |
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| MKLA | Lapsed | ||
| MKLA | Lapsed |
Effective date: 19951015 |