1. Field of the Invention
Chemical analysis of liquids such as water, foodstuffs like milk, and biological liquids is often desirable or necessary. Various elements to facilitate liquid analyses are known. Such elements have often included a reagent for a substance under analysis, termed analyte herein, which reagent, upon contacting a liquid sample containing the analyte, effects formation of a colored material or another detectable change in response to the presence of the analyte. Such elements include, for example, pH test strips and similar indicators wherein a paper or other highly absorbent carrier is impregnated with a material, chemically reactive or otherwise, that responds to contact with liquid containing hydrogen ion or other analyte and either generates color or changes color. Depending on the selection of responsive material, the change is usually qualitative or, at best, semiquantitative. In certain fields, it is often required that analytical techniques yield rapid, quantitative results. Much recent development work has attempted to provide elements useful in diagnostic chemical analysis, where testing of biological liquids including body fluids such as blood, serum, urine and the like must produce highly quantitative results, rapidly and conveniently.
Solution chemical techniques have enjoyed broad acceptance in the clinical laboratory environment, particularly in automated analysis. Such techniques, however, require analyzer equipment having intricate solution handling and transport capabilities. Analytical equipment of the "wet chemistry" variety, illustrated for example in U.S. Pat. No. 2,797,149, is often expensive and may require skilled personnel, both for operation and to maintain the high level of cleanliness that is needed to avoid sample to sample contamination.
As an alternative to solution chemistry, various multi-layer integral elements for non-solution, essentially dry chemical analysis have been proposed. The term "integral", as used herein to describe analytical elements, refers to elements containing two or more desirably discrete layers that under conditions of use are superposed and in substantially continuous intimate contact with adjacent layers in the element. Although essentially dry analysis offers substantial storage, handling and other conveniences as compared to wet chemistry, variations of the "dry" approach have enjoyed only limited success and have been used primarily for qualitative and semi-quantitative test purposes.
2. Description of Related Art
A basic variety of integral analytical element is described in U.S. Pat. No. 3,092,465. Such multi-layer elements use an absorbent fibrous carrier impregnated with one or more reagents, typically including a color former, over which is coated a semi-permeable membrane. Upon contact with a test liquid, analyte passes through the membrane and into the fibrous carrier to generate color in an amount related to the concentration of analyte. The membrane prevents passage and absorption of certain interfering components, such as red blood cells, that could impair accurate reading of the color provided as a test result.
Analytical elements that rely on absorbent filter papers or other fibrous media to receive and distribute a liquid sample have not been popular, compared to wet chemical procedures, in applications such as clinical laboratory testing, presumably due to their inability to produce high accurate, quantitative results. It is described in the literature that diagnostic elements using impregnated bibulous materials, such as fibrous filter papers, can produce non-uniform test results. In U.S. Pat. No. 3,050,373, it is mentioned that precipitation can occur in the impregnating solutions, thereby impairing uniform distribution of reagent within the bibulous carrier or matrix. Also, elements using fibrous, bibulous materials are susceptible to the occurrence of a non-uniformity in test result that is termed "banding." "Banding" is exemplified by a test result occurring to a greater extent in one portion of the region of the element experiencing a test result, such as at the periphery of the region penetrated by an applied sample. It is apparently the result of extensive and extremely non-uniform migration of sample components or reagent chemicals within the bibulous material, possibly due to chromatographing, to provide high local concentrations of such chemicals. Gelatin and gelatin-like materials are described in U.S. Pat. Nos. 3,061,523 and 3,104,209 as useful constituents of the impregnating solution, due apparently to their ability to restrain the high rate of such migration and consequently to encourage improvements in test result uniformity. However, gelatin and gelatin-like materials in the fibrous, reagent containing bibulous matrix decrease the rate of sample uptake as compared to the more highly absorbent gelatin-free matrix. Such decreased absorption can leave surface liquid on the element and necessitate washing the element to remove the excess prior to making a test determination. As a result, an upper limit on the amount of gelatin to be impregnated into a bibulous matrix is typically specified. Such properties also characterize analytical elements using, without more, layers solely of gelatin or similar materials, as discussed in U.S. Pat. No. 3,526,480.
Integral analytical elements adapted for automated test procedures have also been described, such as in U.S. Pat. Nos. 3,368,872 and 3,526,480. Such descriptions refer to means for avoiding chromatographic effects (often called ringing, targeting, doughnuting or banding) in the element by immobilizing reagent or including a means to decrease the tendency of an applied sample to exert a washing effect on incorporated reagent, as by use of simple porous members over an absorbent, reagent containing material, such as fibrous filter paper. However, there is no suggestion in such descriptions of using within an element a means that not only takes up a liquid sample but also provides a uniform apparent concentration of a sample component such as analyte to substantially the entire portion of a reagent layer surface that is contacted by an applied sample. Such uniform apparency of concentration is extremely important in obtaining test results appropriate for interpretation by automated readout, whether densitometric, colorimetric, fluorimetric, or otherwise. This is true even in the absence of gross non-uniformities such as those introduced by chromatrographic effects.
A means to provide somewhat uniform concentration of analyte to the reagent areas of an element for dry analysis has been by a technique that can be termed sample confinement. Usually, as is described in U.S. Pat. No. 3,368,872, a barrier is included on the element to confine an applied sample in a predetermined region of the element's surface, with the result that excess liquid is usually present on the element after sample application. This can create inconveniences, as in the handling and cleanup of excess sample remaining on the element and, more seriously, can require extremely precise sample volume delivery when applying sample to the element.
There has been some recognition of the need to promote or avoid, as desired, the migration of material between layers of integral analytical elements, as is discussed in U.S. Pat. Nos. 2,761,813; 2,672,431; 2,672,432; 2,677,647; 2,923,669; 3,814,670 and 3,843,452. However, this has been in the context of elements for determining the presence of micro-organisms, and the elements described for such purposes typically include at least one layer comprising a fibrous matrix and require non-discrete layers, the interface of which is a blend of the adjacent layers.
Until very recently, there was no suggestion in art relating to analytical elements of a layer or other means to receive sample constituents and to encourage them to distribute within that layer to achieve therein an apparent concentrational uniformity of analyte, analyte products or other substances to be provided, in such uniform apparent concentration, to an associated layer for analytical reactions or similar activity. In fact, as was apparently well recognized, the structural and chemical characteristics of bibulous and other fibrous materials used in most known analytical elements (such as absorbent cellulosic filter papers, glass fiber papers, wood, etc.) might impair such a result for reasons of physical restraint, non-uniform permeation of sample constituents or undesirable chemical binding. Additionally, the choice of fibrous materials can frustrate highly accurate measurement due to severe non-uniformity in properties such as structure and texture. It is known, for example, that in the preparation of papers, starting fibers are often processed to form smaller constituent fibers, called tendrils, that increase the strength of the resultant paper. The term "fibrous," as used herein to describe materials such as papers and the like, refers to materials prepared using preformed fibers or strands that are present in the finished material. Exemplary fibers used in preparing fibrous materials are described in U.S. Pat. No. 3,867,258.
Non-uniformity in the detectable color response or other test result obtained when using integral analytical elements incorporating fibrous materials has been recognized as a problem associated with the use of such elements. Improved devices using such materials to provide absorbent layers have sought to overcome the gross effect of such non-uniformity, but they have not succeeded in avoiding the problem. As an example, U.S. Pat. No. 3,723,064 describes an analytical element that includes regions of different effective permeability to an analyte or reaction product of an analyte and produces a plurality of spaced-apart, threshold color indications as an analytical result. Although the desirability of a smoothly continuous response is manifest, an element made as described in the U.S. Pat. No. 3,723,064 patent can only yield an approximate analytical result, the accuracy of which varies indirectly with increased spacing between thresholds. If the difference in permeability between regions were decreased, to narrow the interval between thresholds in the interest of increased response precision over the intended dynamic range, the complexity of elements made in accordance with the U.S. Pat. No. 3,723,064 patent would increase dramatically. No suggestion is made as to how one might improve the uniformity and precision of a continuously varying test result and, however optimized, elements of the U.S. Pat. No. 3,723,064 patent would produce a discontinuous response that would apparently be non-uniform within each region of permeability due to non-uniformities associated with the use of filter papers and other fibrous materials present in that element.
U.S. Pat. No. 3,791,933 describes a multi-component device for the assay of enzyme substrates and metabolites, such as in body fluids. It describes a clamped array adapted to receive a test sample, filter out or otherwise remove large sample constituents (such as proteins) and effect a test reaction to produce a detectable result, such as the generation of a color. Although glass fiber paper is disclosed as assisting in distributing a reaction mixture across a plastic viewing window, such material apparently merely assists the outward diffusion of liquid sample within the glass fiber layer to enlarge the region of the element exhibiting a test result and thereby render the result more easily visible. There is no suggestion of any means to form within the region of diffusion a concentrational uniformity of analyte, which, of course, is extremely important for the production of an analytical result that is of a uniform nature and, as such, precisely detectable. Further, the glass fiber is apparently observable to a means of detection.
Improved multilayer integral analytical elements are described in French Patent Application 7,323,599, filed June 28, 1973, now French Pat. No. 2,191,734, and in U.S. Patent Application Ser. No. 538,072, filed Jan. 2, 1975 now U.S. Pat. No. 3,992,158. Such elements can receive a liquid sample and spread the sample within a spreading layer of the element to obtain in the element a uniform apparent concentration of analyte, other appropriate sample constituent or analyte product and produce in the presence of analyte an analytical result that, by virtue of its uniformity, can be measured quantitatively by automated devices, using techniques such as spectrophotometry, fluorimetry, etc. Elements disclosed in French Pat. No. 2,191,734, include spreading layers and reagent layers that contain a reactive or otherwise interactive material that, by virtue of its activity, promotes in the element a radiometrically detectable change, such as a color change.
In providing an analytical result, whether in a bibulous test strip, other predominantly fibrous element or in an element of the type described in French Pat. No. 2,191,734 and in the above-mentioned U.S. Application Ser. No. 538,072, difficulties in obtaining or measuring a detectable change within the element may be experienced. For example, the fluid under analysis or by-products of an analytical reaction can provide, within the reagent matrix, constituents that interfere with detection of the test result. Also, popular reagent matrix materials such as filter papers can be opaque, allowing detection of an analytical result only at the surface of the matrix material and not throughout its thickness. This can diminish the observable magnitude and range of an analytical result, resulting in an inability to measure effectively both low concentrations of analyte and subtle changes in analyte concentration. It is considered desirable, therefore, to have elements for analysis of liquids in which the materials characterizing the test result can be detected without hindrance from chemical or optical interferants, opaque matrix materials, or the like.
Accordingly, although various test strips and improved analytical elements, e.g., those of French Pat. No. 2,191,734 can be desirable for qualitative and quantitative dry analysis of liquids, comparable, more interference-free elements would be desirable.