CA1162075A - Quantitative analysis of an analyte in a liquid using a test element having a fibrous spreading layer - Google Patents

Abstract

QUANTITATIVE ANALYSIS OF AN ANALYTE IN A LIQUID USING
A TEST ELEMENT HAVING A FIBROUS SPREADING LAYER
Abstract In a method for quantitative analysis wherein liquid is applied to a test element containing one or more reagents, and comprising a fibrous spreading layer and optionally a permeable layer, a liquid transport zone is provided at the point of introduction of the liquid to facilitate accurate and precise measurements.

Description

UANTITATIVE ANALYSIS OF AN ANALYTE IN A LIOUID USING
Q
A TEST ELEMENT HAVING A FIBROUS SPREADING LAYER
.
1) Field of the Invention The invention relates to a method for analysis of components of a liquid, for example, the clinical analysis of biological liquids.

2) Background of the Invention -In the field of clinical analysis, earlie~ attempts to determine the composition of biological liquid~ such as serum and urine required complicated, time-consuming liquid assays. To provide a more prompt ~creening of patient'~
serum and urine, a first improvement was the development o~
"dip-and-read" dry test monolayer elements designed to give qualitative indications of extreme conditions. For example, an above-normal amount of BUN was detectable by an element containing an amount of buffer that had to be overcome by NH3 generated by the BUN before an indicator dye would re-spond. Examples of such qualitative test element~ appear in U.S. Patent No. 3,145,086, issued August 18, 1964. Such test elements were in no way intended to generate precise readings of the amount of analyte present, and were constructed from materials that had gross irregularities, for example, fibrous materials such as filter paper.
A next step was to d~liver the analyte-containing liquid to a fibrous matrix in a more rapid manner, to aid in a more uniform color generation. The improved color in turn permitted operstors to roughly estimate a range of analytes by comparing the generated color against a color scale.
Examples of test elements designed for such use are included in, e.g., U.S. Patent No. 3,715,192, issued on February 6, 1973~ The test elements described therein have as f9 chief feature, a liquid path that pours through the open ends of the element to distribute ~he liquid to the fibrous rea8ent layer. However, as the matrix for the rea~ent layer was ~till a bare fibrous material with gross irregularities, the tests involved were still ~u~litative rather than preci~e determinations of the quantity of analyte present.
The restriction of fibrous materials to qualitative ~A

tests only is understandable in light of the nature of these materials. Such ma~erials have been found to be objection-able for quantitative assays, because the individual fiber~
tend to generate non-uniform reflectance or transmittance, and even non-uniform permeability. The non-uniform reflect-ance and trans~ittances are of course no problem to qualita-tive tests made by the human eye, because the eye integrates over the field in question. In contrast, a machine without expensive corrective devices will detece such variationfi aod create error and uncertainty in the reading, preven~ing the precision needed for quantitstive, automated as6ays.
The difficulties presented by fibrous matrices when used for quantitative assays are described in detail in U.S.
Patent No. 4,059,405, particularly in connection with Figs.
3A-3E. For reactions producing a relatively marked optical change, that is, a change in optical absorbance greater than about 0.2 (about 0.4 for reflection densities), fibrou~
matrices are described as producing unsatisfactory, highly non-uniform density profiles. The density values are large around the periphery of the area contacted with liquid and much less within the periphery. To avoid this problem, re-agent concentrations are described that are so low that the differential optical absorbance never exceeds about 0.20 Such a technique, while avoiding the problem created by a fibrous medium during quantit~tive assays, add~ its own problems. First, useful reagents are limited to only those having coefficients of extinction that are low enough to insure that high analyte values normally encountered will not produce differential densities that exceed the 0.2 absorb-ance. Second, detection of changes at such low levels i6difficult, necessitating the use of extremely 6ensitive ins~rumentation.
Therefore, for accurate quantitative a~says producing, for example, differentisl reflection densitie6 greater than sbout 0.4, there has not been available prior to this invention ~ completely ~atisfactory test element featuring fibrou~ matrices. Such exclusion of fibrou~ layer~
from quantitative test elements has meant that readily 16~07 available and inexpensive material~, such as filter paper, have had to be avoided. The purpose of ~his invention is to provide a quantitative assay of analytes using an element featuring such fibrous materials.

SUM~ARY OF THE INVENTION
This invention is directed to a method of obt~ining quantitative determinations of analyte concen~rations, u~ing improved test elements having fibrous spreadin~ layers.
More specifically, the method provides a quantitfl-tive determination of analyte in a liquid by placing a quantity of the liquid into a test element compri~ing a support; a porous, aqueous-wettable fibrous spreadin~ layer;
and optionally a layer that ia permeable to the liquid under analysis and is disposed between the support and the spreading layer. At least one of the spreading and the permeable layers includes a reagent that reacts with the analyte or with a precursor or a reaction product thereof to ~enerate a radiometrically detectable change that is predictably and precisely related to the amount of snalyte present. The reagent is present in an amount capable of generating an optical absorbance greater than about 0.2 for the expected amount of analyte.
In accord with one aspect of the invention, the liquid is transported over the surface of the spreading layer at a rate faster than it iB absorbed. A radiometric measurement is made of the change produced by the analyte-reagent reaction and the smount of change is correlated quantitatively with the concentration of the analyte in the liquid.

3 In accord with another aspect of the invention, the element further includes 8 cover sheet spaced ~way from the exterior surface of the 6preading layer a distsnce no greater than about 1 mm to create a zone of intended liquid trans-port. Access means are provided to permit introduction of liquid into the zone.
It is an advantage of this invention that the test elements of such quantita~ive assays sre prepared using inexpensive fibrous materials.
It is a further advantage of thi6 invention that a 1 ~620~l~

4-quantitative assay can be made with such ~est element6 regardless of the radiometric range likely to be encounter-ed. Specifically, the assay is ~till quantitative even for an optical absorbance greater than about 0.2.
Othe~ features and advantages will become apparent upon reference to the following Description of the Preferred Embodiments when read in light of the attached drawings.
BRIEF DESCRIPTION OF THE DRAWIN
Fi~. 1 is a plan view of a test element utilized in the quantitative assay of the invention; ~nd Fig. 2 is a section view taken along the line II-II
of Fi~. 1.
DESCRIPTION OF THE PREFERRED E~BODI~ENTS
-The method of this invention is described in con-nection with preferred embodiments featuring the assay of biological liquids and particularly blood serum. In addi-tion, the method is applicable to any liquid which has an analyte capable of being assayed. For example, industrial liquids can be ~o assayed.
This invention is based on the discovery that test elements featuring tibrous spreading layers are useful in ~
quantitative test element, and especially those producing a differential optical absorbance greater than about 0.2, if ~
liquid transport zone is disposed above the fibrous layer in the form of a selected spacing between portions of the tefit element. Although test elements containin~ capillary transport zones snd fibrous layer6 have been used prior to this invention for qualitative tests, it has not heretofore been recognized that they are useful for precise quantitaeive 3 testing over the entire radiometric range of use.
As used herein, "quantitative" is reserved for those measurements having fl high order of precision ~nd ~ccuracy.
Becsuse "quantitative" justifiably may be defined differently for non-fibrous tes~ elements, its use herein refers only to ; 35 test elements containing fibrous matrices, such as in the instant invention. SpecificalIy, it is the condition in which the concentration error, defined to mean all errors over the entire range of use, as determined by a comparison against ~ standard reference method, is not more than about 20%. By comparison, any test element measurement producin~
deviations or error greater than about ~ 20% is defined to be qualitative, whether it be due to the inherent constrUCtiOn of the elem~nt or the manner in which it is measured.
The Element A test element 10 for the assay comprises a ~ibrou6 spreadin~ layer 16, an optional liquid-permeable layer 14, and a support 12 for layers 16 and 14, in that order, to form a laminate 18, ~ig. 2. Support 12 is preferably A
transparent layer. Laminate 18 includes at least one reagent capable of reacting with the analyte in question, or ~
reaction product of the analyte, to produce a detectable change. Preferably the reagent~s) are located in layer 14.
Optionally, the spreading layer 16 also includes one or more of the reagents.
To minimize spreading irregularities that might oc-cur from the use of fibers in layer 16, a liquid transport zone is provided at the point of introduction of the liquid.
More specifically, the entire laminate 18 is preferably mounted in a frame 20 provided with a cover 6heet 22 spaced from exterior surface 24 of layer 1~ a distance "6" that i6 no greater than about 1 mm. This distance i6 effective to induce capillary flow of liquid placed between under-sur-face 23 of sheet 22 and ~urface 24. The spaced cover ~heet thus creates a zone 25 of intended liquid transport, that extends across the aurface 24 of layer 16. Preferably, distance s is maintained by sidewalls 26, 26' and 28, Fig. 1, &urrounding laminate 18 and to which cover sheet 22 i6 per-manently sealed by conven~ional mean~ 6uch as water-insoluble 3 adhesives or ultrasonic energy. That is, sidewall6 26, 26' and 28 have a height that exceeds the thickness of laminate 18 by distance ~. A bottom viewing layer 30 is similarly ~ealed to the surface 31 of sidewalls 26, 26' and 28 opposite to cover sheet 22. A view port 32 is formed in layer 30, Or alternatively portion 32 i~ rendered more transparent than the remainder of layer 30. As yet another alternative, layer 12 is formed as an inte~ral part of bottom layer 30, provid-ing a single support.
A~ noted, di~tance s, by being no greater than ~bout ~62~5 1 mm, provides capillary transport of a drop deposited between surface 24 of layer 16 and sheet 22. The actual value of s varies, depending upon factors such a9 the liquid being assayed and the material selected for layer 16.
Preferably, s is between about 0.002 cm and about 0.05 cm.
A possible variation for cover sheet 22 is ~he pro-vision of ener~y barriers such as grooves, not shown, in sur-face 23 to aid in the control of the shape of the liquid men-iscus or wave front that advances through zone 25o The bar-riers, if used, have any form and are preferably mutually parallel.
To permit introduction of the liquid into contact with laminate 18 and into zone 25, an access aperture 40 is provided in the element~ Preferably, it is located in sheet 22 at one end of zone 25, as shown, or above the center of the zone. Most preferably the aperture is placed in ~heet 22, but alternatively it can be in sidewall 26. Aperture 40 is sized to insure that, when a volume of liquid is deposited therein, that particular volume of liquid will contact both surface 23 of sheet 22 and surface 24 of layer 160 For example, if a liquid volume of 10 ~1 is desired for proper contact with the opposin~ surfaces, aperture 40 preferably has a diameter of between about 0.1 cm and about 0.5 cm.
As shown, aperture 40 i 8 circular in shape. In an alternative embodiment, not shown, the aperture has corners in its sidewall to form the shape of a regular polygon such as a hexagon, as described in U.S.Patent 4,254,U83, issued 3/3/81. ~Such corners aid in urging a quantity of liquid to enter the aperture, arrow 44, even thou~h the quantity might 3 not be exactly centered on the aperture.
To vent trapped air, a vent aperture 42 preferably is formed at the end of zone 25 opposite to the end with aperture 40, and preferably in sheet 22. Alternatively, it i6 located in sidewall 26' above layer 16.
Layer 16 is selected from any conventional fibrous material, such as cellulosic materials of any ty~e9 including filter paper; synthetic fibers such as glass fibers; fibrous mesh work, and the like. Preferably the fibers are aqueous-wettable, The void volume of such material is not critical, ~"

3 1~207~

although most prefer~bly such void volumes are between 50 and 80% of the total volume of layer 16. Layer 16 thufi serves pri~arily to distribute the liquid uniformly to the rest of the test element, for example, the reagent(s) th~t are present ~or reaction with the 3nalyte. 1~ layer 14 is omitted, then layer 16 is ~upported directly by support 12 rather than indirectly and contains all the reagents necessary for the assay.
Preferably, element 10 includes an opaque reflective material, most pre~erably in layer 16, to allow radiometric changes in the reagent(s) to be detected reflectively. For example, a white pigment ~uch as TiO2 is useful when incor-porated into the fibers of layer 16 to create ~ reflective back~round~ Alternatively, i~ the fibers are themselves white, for example, dyed white, the pigments can be omitted.
In yet another alternate embodiment, surface 23 of sheet 22 is given a reflective coating, particularly if it i6 desired that layer 16 be free of any pigments or dye other than the product of the reaction between the reagent (6) and the analyte.
As noted, the reagents are primarily in layer 14 but can also be in layer 16. As used herein "reagent" i~ any màterial capable of chemical or physical interaction with an analyte, a precursor of an analyte, a decomposition product of an analyte, or An intermediate, to ultimately result in a detectable product. Thus, reagents include enzymes, chromo-genic indicators, buffers etc. For certain analyses, ~
plurality of reagents are preferred, and in some cafie~ they are distributed between the two layers 14 and 16. Because 3 limited air volume is present during testing, as described hereinafter, it i~ preferred that the reagents be selected to be oxygen independent.
If present, layer 14 also preferably comprises a radiation-transmissive binder permeable to the liquid to be analyzed. As used herein, a radiation-tran~missive binder iB
one that is effective to permit psssage of electromagnetic radiation used for the radiometric detection of the change produced by the reagent(s). "~adiometric detection" i used in its conventional 6ense, that is, one that includes both 1~2075 colorimetric and fluorimetric detection. Useful examples of binders for layer 14 include poly(vinyl alcohol), acrylamide polymers, agarose, gum srabic, etcO, and particularly gelatin.
Other layers, not shown, are optionally added be-tween layers 14 and 16, or between layer6 12 ~nd 14, for example, barrier layers to exclude interferants, filter layers, registration layers, mordant layers and the like.
These are all conventional, requiring no further explana~ion, and are useful depending on the assay in question.
Examples of representative reagents and binders, if any, for the layers are found in U.S. Pa~ent Nos. 4,066,403, issued January 3, 1978; 4,132,528, issued January 2, 1979;
and 4,069,016 or '017, issued January 17, 1978; for testing for BUN, total protein, and bilirubin, respectively. For example, a total protein test element constructed as ~hown for Fig. 2 comprises a filter paper spreading layer, and under that, a reagent layer comprising acrylamide-N-vinyl-2-pyrrolidone, CuS04, tartaric acid, LiOH and a nonylphenoxy-polyglycerol surfactant obtainable from Olin Mathieson under the trademark "lOG". Or alternatively, the separate reagent layer is omitted and all of the aforementioned rea~ents, including the polymer, are imbibed into the filter paper.
The polymer is useful even in the paper matrix as it provides protection to reagents against the degradative effect~ of the high pH of the lithium hydroxide.
The amount of reagents to be used is such that, preferably, for maximum amounts of analyte to be tested, a radiometric change is produced that exceeds that correspond-ing to ~ 0.2 optical absorbance. For example, ~t high concentrations of analyte within the expected dynamic range, differential reflection densities are often encountered that exceed 0.4. However, it will be readily appreciated that the invention is ~till useful for detecting a low amount of analyte present in 8 certain liquid ~ample. Such ~ lo~
amount, for a given example, produces a radiometric chan~e that corresponds to less than a differential 0.2 optical absorbance. The amount of reagent is not changed for such a contingency but rather the amount i~ selected to accommodate both ends of the analyte range.

l 1~20 In use, layers 14 and 16 are in liquid con~act, that is, liquid reaching the bottom portion of layer 16 then con-t&cts the upper portion of layer 14 and permeate~ into it.
Most preferably, layers 14 and 16 are conti~uous and fl~t to provide such liquid contact, but ~hey al60 function if ~paced slightly by a distance that still insures ~uch liquid contact.
The layers of lamina~e 18 can be preformed ~s separate members. In that event layer 16 is coated from ~ol-ution or disper~ion on a surface from which layer 16 i~ then 10 physically stripped when dried. However, a convenient pro-cedure which avoids problems of multiple ~tripping and lsmi-nation steps when contiguous layers are desired, i8 to coat layer 14 on a stripping surface or support layer 12, a~ de-sired, and thereafter to form layer 16 directly on lsyer 14 coated previously. Such coatings are conveniently accomplished by various well-known coatin~ technique~, such as those described in U.S. Patent 3,992,158. Any interlayer adhesion problems can be overcome without harmful e~fect by means of surface treatmentE, including extremely thin Pppli-cation of subbing materials such as are used in photoRraphicfilms.
For coatable layers, a coating solution or di~per-6ion is used, and the layer is coated and dried to form ~
dimensionally stable layer. The thickness of optional layer 14 and its degree of permeability are widely variable ~nd depend on actual usage. 9ry thicknesses of from about 10 microns to about 100 microns are useful, althou~h a greater range of thicknesses may be preferable in certain cir-cumstances. Useful reagent containing fibrous layers 16 ~re formed by impregnation of the fibrous matrix, in accordance with well-known techniques. Preferably layer 16 has thickness of between about 25 and about 375 micron~.
Useful support materials for layer 12 include a vsr-iety of polymeric materials such as cellulose ~cetate, poly-(ethylene terephthalate), polycarbonates and polyvinyl com-pounds ~uch as polystyrenes, etc.
Method In accordance with one aspect of the inveneion~ the quantitative assay proceeds by the placement of ~ predeter-.. . , .. . . _ .. . _ . _ . . .. _ . . . .. .

l 162~7~

mined quantity of liquid, such a~ blood serum, into aperture 40. The quantity is deposited either in drop form or ~6 a quantity that is injected into the apereure. In the latter case, the applied pressure is 6elected to be effective only in introducing the liquid and to cause a minimum amount of transport ~low. Because of the spacing 6 of the transport zone, the liquid will flow under capillary attraction and the pressure need not be effective to fill zone 25.
The spacing insures that horizontal transport ln direction 50 take~ place more rapidly than it does without the presence of the cover sheet. Further, this spacing provides a rate of transport in the horizontal direction (arrow 50) that exceeds the rate of absorption into layer 16. It is believed that this mechanism provides the improved uniformity of the detectable change notwi~hstanding the irregularities introduced by the fibers of layer 16. That is, it appears that chromatographing through and along the fibers of layer 16, such as could cause separation of liquid components and irregular density production, is reduced if transport occurs throughout zone 25 before absorption occurs into layer 16. Spot quality defects, ~uch as a darker ring of density around a lighter central spot, described in prior systems as "ringing", are minimized. The quality of the radiometric change to be detected permits quantitative as~ays to be run, even for changes corresponding to differential optical absorbances greater than about 0.2.
The filling of zone 25 with liquid, prior to absorp-tion in layer 16, tends to form a meniscus trap at apertures 40 and 42, thereby cutting off oxygen flow into zone 25. The trap tends to remain even after sbsorption. It is for this reason that the reagents are preferably selected to be oxygen-independent.
After the liquid is transported through zone 25 and absorbed into the layers, the change produced by the reagent~
i~ measured by a radiometer, e.g., a photometer or a fluorimeter. A variety of Guch instruments is useful, ~or example, the radiometer di~clo~ed in German OLS 2,755,334, published June 29, 1978, or the photometer described in ~ 1620~5 U.S. Patent No. 4,119,381, i~sued on October 10, 1978. One preferred detection mode is the reflection mode, wherein incident light is reflected by the spreading lsyer~ ~rrow 6 in inverse proportion to the amount of density developed by the reagent(~).
Correlation of the detected change and the concen~
tration of the analyte comprises, first, measuring the changes produced-when the analyte in question is present in several known concentrations, i.e., in calibrator~ or standards at several different concentration levels. One of these levels frequently i~ selected to produce ~ differential optical absorbance greater than 0.2. These changes are then expressed as a mathematical relationship between the ccn-centration of the analyte and the detected change. There-after, any test element change measured for an unknown liquidis checked against this relationship to determine the concentration of the analyte. Preferably, this correlation is handled by conventional data proces~ors, as is well known. The correlation for such processors is periodically readjusted as a result of retesting the elements by means of the calibrators.
The invention has been described in detail with par-ticular reference to preferred embodiments thereof, but it will be understood that variations and modifications can be effected within the ~pirit and ~cope of the invention.

3o ... . . .

Claims (6)

WHAT IS CLAIMED IS:

1. A method for quantitatively determining an analyte in a liquid, comprising placing a quantity of the liquid into a test element that comprises (a) a support; (b) a porous, aqueous-wettable fibrous spreading layer; and, optionally, (c) a layer permeable to said liquid and disposed between said support and said spreading layer and covered by said spreading layer, at least one of said spreading and said permeable layer including a reagent that reacts with said analyte or with precursor or reaction product thereof to generate a radiometrically detectable change that is predictably and precisely related to the amount of analyte present, said re-agent being present in an amount capable of generating a differential optical absorbance greater than about 0.2 for said analyte amount; said element further including (d) a cover sheet spaced away from the exterior surface of said spreading layer a distance no greater than about 1 mm to create a zone of intended liquid transport, and (e) access means to permit introduction of liquid into said zone;
radiometrically measuring the detectable change pro-duced by the analyte; and quantitatively correlating the amount of change with the concentration of the analyte in the liquid.

2. A method as defined in claim 1, wherein said access means comprises an aperture in said cover sheet, said aperture being configured to promote contact of the liquid with the spreading layer and the cover sheet, and wherein said placing step comprises the step of depositing the quant-ity of liquid into said aperture, whereby the liquid spreads more rapidly over the spreading layer than it spreads without said cover sheet and said transport zone.

3. A method as defined in claim 1, wherein said measuring step comprises the step of detecting photo-metrically the amount of incident light that is reflected from said element.

4. A method as defined in claim 1, wherein said correlating step comprises the steps of a) measuring the amounts of detectable change pro-duced by standard containing known amounts of said analyte, b) establishing from said amounts a relationship of measured change vs . concentration, and c) determining from this relationship the concen-tration of analyte in aid liquid that corresponds to said detectable change measured for said liquid.

5. A method for quantitatively determining an amount of an analyte in a liquid, comprising placing a quantity of the liquid into a test element that comprises (a) a support; (b) a porous, aqueous-wettable fibrous spreading layer; and, optionally, (c) a layer permeable to said liquid and disposed between said support and said spreading layer and covered by said spreading layer, at least one of said spreading and said permeable layer including a reagent that reacts with said analyte or with precursor or reaction product thereof to generate a radiometrically detectable change that is predictably and precisely related to the amount of analyte present, said re-agent being present in an amount capable of generating a differential optical absorbance greater than about 0.2 for said analyte amount; said element further including (d) a cover sheet spaced away from the exterior surface of said spreading layer a distance no greater than about 1 mm to create a zone of intended liquid transport, and (e) access means to permit introduction of liquid into said zone;
transporting the liquid over the surface of said spreading layer st a rate that exceeds the rate of absorption of the liquid into said spreading layer;
radiometrically measuring the detectable change pro-duced by the analyte; ant quantitatively correlating the amount of change with the concentration of the analyte in the liquid.

6. A method as defined in claim 5, wherein the liquid is transported over said surface by capillary action.