WO1994018542A1

WO1994018542A1 - Non-instrumented measurement device with control

Info

Publication number: WO1994018542A1
Application number: PCT/US1994/001400
Authority: WO
Inventors: Prithipal Singh
Original assignee: Chemtrak, Inc.
Priority date: 1993-02-09
Filing date: 1994-02-08
Publication date: 1994-08-18

Abstract

A self-contained non-instrumented measurement device with control is provided, where an analyte may be measured by measuring the distance of a detectable signal from a predetermined location. A measurement control is simultaneously provided which verifies the operation of the device and desirably the accuracy of the measurement. The device has sample measurement and control pathways. The sample measurement pathway provides two bibulous strips (62 and 64) separated by a gap (80), where the sample flow path is initiated by moving a sample into the gap (80). The control pathway also provides two bibulous strips (68 and 70) separated by a gap (90), wherein the control is based on the signal produced from a known amount of a compound present in the control pathway. Means for releasing an eluent (100) are also provided, which eluent is transported through the sample strip (62) and control strip (68) into measurement regions (74 and 84) on each strip (62 and 68) for the determination of the amount of analyte. Various chemistries may be used for providing the detectable signal and control.

Description

NON-INSTRUMENTED MEASUREMENT DEVICE WITH CONTROL

INTRODUCTION Technical Field

The field of the subject invention concerns devices that provide for analyte measurement with a control measurement for operational accuracy.

Background

Devices and methods for quantitatively measuring a wide variety of compounds, both naturally occurring and synthetic, have become increasingly important; particularly for those compounds which are physiologically active.

Historically, quantitative analyses or assays (including immunoassays) of physiological fluids, non- physiological fluids, drugs, organic chemicals, inorganic chemicals, and so on, have required analytical laboratory or clinical laboratory determinations or measurements. However, with the need to reduce medical costs, diminish travel to the doctor's office or chemical laboratory, monitor various drugs and physiological fluids on an ongoing basis, as well as because of the rapidly increasing costs of laboratory or clinical evaluation, there is an increasing awareness of the importance of being able to conduct an assay, either quantitative or qualitative, at home, at work, in a doctors's office, or at some site remote from analytical or clinical laboratory facilities. Such devices as these are applicable to virtually any analyte. The determination of blood glucose level to monitor diabetics, the determination of blood cholesterol levels, pregnancy determination, the determination of the presence of environmental contaminants, and application to field wildlife management situations, are just common examples of the applicability of such a device.

Assays performed outside the realm of traditional laboratories require that the assays have a simple protocol and be relatively free from sensitivity to small changes in the environmental or procedural conditions under which the assay is carried out. Importantly, there should be minimal handling and measurement of reagents by the user. Devices having some of these attributes are known such as those described in U.S. Patent Numbers 4,999,287; and 5,132,086; and are herein incorporated by reference. Furthermore, as these devices proliferate and they become an acknowledged replacement rather than a supplement to the more traditional measurement protocols and methodologies, the accuracy, reliability, and reproducability of quantitative determinations from these devices must be assured. Therefore, there is a need for an integral control or standard by which the assay determination may be verified. As with the measurement, this control should have a simple protocol and vary in response to small changes in the environmental or procedural conditions under which it is carried out, in the same manner as the analyte measurement. Measurements of any required reagents and sample should be avoided or minimized. Ideally, the control will provide a response that is substantially identical to that of the measurement for an analogous amount of analyte, and provide a manner of indication that is not confusing to the individual using the device. It is also desirable to avoid differences due to matrix effects from the background constituents of the sample. Such control will assure that the device was operated as intended, that there has been no spoilage or contamination of the device such as through improper storage or handling, and that the results may be relied upon.

Alternatively it may be desirable to measure multiple (2, 3, or more) analytes at one time from a single sample. This would allow the measurement of appropriate groups of tests such as: a lipid panel including two or more of the following, total cholesterol, HDL cholesterol, LDL cholesterol, triglyceride, LP(a), apo B or apo A, a diabetic panel including glucose, hemoglobulin Ale and fructosamine; a blood bank panel including hemogloblin and ALA. Such a device is applicable to both direct assays, where the analyte gives a measurable product, and to indirect assays, where the analyte reacts with an intermediate entity which subsequently reacts to give a signal. Exemplary of a need for a non-instrumented home or doctor's office device is the need today to be able to determine cholesterol levels, or low or high density lipoprotein levels in blood. There is a relationship between total blood cholesterol and coronary artery disease. Guidelines have been established for adults over 20 years of age to identify risk groups associated with blood cholesterol level. Since cholesterol levels can be controlled by both diet and cholesterol lowering drugs, the key is to be able to identify those at risk and then to monitor the individual's cholesterol level. Being able to monitor one's own cholesterol level at home for those individuals at risk will provide a significant tool in monitoring cholesterol levels and reducing the potential for heart disease. The measuring of other naturally occurring compounds of physiologic importance and synthetic drugs is also of great interest. For example, therapeutic dosage monitoring, drugs of abuse, iodothyronines, alcohol, cytokines, as well as numerous other haptens and antigens are monitored. Microorganisms, β-HCG for ectopic births, antibodies associated with disease, and the like are also of interest, for home or doctor's office determination.

Relevant Literature

Zuk et al., "Enzyme immunochromatography — A quantitative immunoasεay requiring no instrument", Clin . Chem . 31:1144 (1985) . Liu and Green, "A monoclonal antibody enzyme immunoassay for the detection of hepatitis B surface antigen utilizing a biotin avidin system", Clin . Chem . 31:202 (1985). Tobias et al. , "A monoclonal antibody radioimmunoassay for carcinoembryonic antigen with improved specificity", Clin . Chem . 31:191 (1985) .

SUMMARY OF THE INVENTION Methods and devices are provided for the measurement of an analyte in a sample, and simultaneous verification of the operability of the device using a control. The device employs continuous sample and control flow paths; each flow path having a fluid transport region, and a measurement region, the sample path including a sample region. Other regions may also be provided. Depending on the assay protocol, the flow paths may differ as to organization and compositions present in the flow path or the elements of the flow path.

The sample flow path provides a measurement of the analyte in the sample. The control flow path verifies the functioning of the device and desirably the accuracy of the measurement. A preferred embodiment has the operation of the control pathway substantially the same as the sample pathway. In this manner, each of the pathways will be similarly affected. The sample assay may be performed in accordance with the protocols described in U.S. Patent Nos. 4,959,324 and 5,132,086, for example. The control pathway uses various reagents for generating a controlled amount of control product. For example, a controlled amount of hydrogen peroxide (H₂0₂) is generated in a device for measuring cholesterol. The control product, such as H₂0₂, is then transported in the measurement region where the distance of the color border indicates whether the device is operating properly.

BRIEF DESCRIPTION OF THE FIGURES The accompanying drawings, which are incorporated in and form a part of the specification, illustrate embodiments of the present invention and, together with the description, serve to explain the principles of the invention. In the drawings:

Fig. 1 is an illustration of one embodiment of the present invention;

Fig. 2 is an illustration of another embodiment of a device according to the subject invention with;

Figs. 2a, 2b, and 2c, being a diagrammatic plan view of the external surface of the cover, base, and slide; Fig. 3 is an illustration of another embodiment of a device with;

Fig. 3a, 3b, and 3c, being diagrammatic plan views of the external surface of the cover, base, and slide; Fig. 4 is an illustration of another embodiment of the invention with diagrammatic plan views as indicated for Fig. 3; and

Fig. 5 is an illustration of another embodiment of the invention with diagrammatic plan views as indicated for Fig. 3.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS A device according to the present invention comprises sample and control flow paths, a signal producing system, and an eluent, for producing detectable signals in measurement regions of the pathways. Members of the signal producing system are selected to provide a detectable signal for a specific analyte or class of analytes. The amount of analyte present is indicated by the distance traversed by a color front along the measurement region of the sample path. The control path verifies device function and desirably the accuracy of the measurement in the sample path. Verification is provided by the appearance of a color front at a predetermined location along the measurement region of the control path. The location corresponds to the distance that should be traversed by a known amount of analyte in the sample path. The control indication afforded by the control methods and devices of the present invention instills user confidence that the assay value is correct and may be relied upon. Control is achieved using a simple and reliable protocol, a protocol that is related to the sample path assay protocol. Substantially analogous path protocols for the sample and control are desirable, because the measurement and control paths will be similarly affected by the same factors, such as test conditions, e.g. temperature, humidity, etc., prior device storage history, and so on. An adverse control indication invalidates an assay measurement in spite of the fact that the assay measurement value may be within a typical range.

Conversely, some aspects of the control path will be different from the sample path in order to provide a known standard for comparison and verification. The differences derive from three principal aspects: potential differences in the compound to be measured in the respective paths and the amount of a compound present in each path. In the sample path, an assay sample comprising an unknown quantity of analyte is provided from a source external to the device. However, in the control path, a known amount of compound is provided. This compound may be the same compound as the analyte, or a different compound that can be related to a known amount of the analyte. The desirability for a substantial degree of similarity between the paths establishes a relationship between them that is reflected in the path elements and organization, the number and identity of members of the signal producing system, and so on. It is therefore appropriate to consider a description of a device control path in light of the description of the sample path which provides the desired analyte measurement. The general design for non-instrumented assay devices having a sample path, but without the control or other aspects of the present invention, are set forth in U.S. Patent Nos. 4,973,549; 4,959,324; and 5,132,086; and U.S. Application Serial Nos. 07/789,059; and 07/724,919; which are incorporated herein by reference in their entirety.

The sample flow path comprises a minimum number of regions: a transport region, a sample region, and a measurement region. Other optional intermediate regions such as capture and conversion regions may also be provided. There may also be an optional test complete indicator region, generally present at a portion of the sample flow path downstream from the measurement region. The sample flow path may be of any suitable configuration, usually a bibulous elongated strip which has the various regions aligned in the direction of eluent flow. The material of the flow path comprises a substrate which is capable of and directs liquid flow.

The sample path elements or regions are now set forth, the description proceeding generally in the direction of eluent flow (downstream) through the device. An eluent is provided which serves as a transport fluid that moves via capillary action down the path. In simple devices, the eluent may be provided in a container into which elements of the device are dipped to initiate the wicking of the eluent. In more sophisticated devices, the eluent is provided in a well or reservoir contained within the device.

The sample path transport region receives the eluent from the well or reservoir and initiates the flow of eluent into the sample path. Where the eluent is contained in the device, means for releasing eluent may be provided such as by an apparatus having an arm to score or tear a seal covering the well or pouch containing the eluent. Methods for increasing the efficiency of release include providing a bibulous strip with a tongue extending from the scoring arm to the well adjacent the transport region, where upon movement of the scoring arm, the bibulous strip moves into the chamber and initiates flow. Alternatively, the scoring arm may undergo a rapid drop after having scored a substantial portion of the length of the seal so that the sudden shock encourages eluent flow. Generally, the transport region will be a short bibulous element, such as filter paper or membrane strip, which serves by capillary action to wick or transport the eluent to the next region, normally the sample region.

The sample region serves a plurality of functions. First, it serves to receive the sample applied to the device and may optionally have one or more members of the signal producing system present. Generally, the members will be non-diffusively bound, so as to be retained in the sample region. In other situations, members may be diffusively bound, thereby permitting them to move downstream with the eluent. Whether other members are present, and how they are bound, generally depends on the analyte to be measured and the protocol used.

The sample region also serves as a bridge for transferring the eluent to the next region. U.S. Patent No. 4,959,324 describes a device in which regions adjacent to the sample region are not in fluid transporting relationship with the sample region prior to the start of the assay, but are placed in fluid transport relation when the assay begins. In that device, means for moving the sample region between two positions are provided by a slide. After receiving the sample, the sample receiving region is moved on a slide to fill a gap and to permit the sample region to be a bridging element for eluent flow between adjacent regions. Isolating the sample region from adjacent regions prevents the sample from spreading prior to the start of the assay, and allows the sample to be confined after application. The confinement allows optional processing of the sample within the device, including being measured or metered or the like.

Providing a measured amount of sample is important because a device according to the present invention measures the total amount of analyte present, and not the concentration per unit of applied sample. Various means for providing a measured amount of sample to the sample region may be employed. Measuring means, such as a micropipet, capillary or the like, may be used to measure an amount of sample and then releasing the sample to the sample region. Alternatively, an automatic mechanism may be provided as part of the device, which receives the sample on a sample region in the form of a pad having a predetermined liquid absorbing capacity. The pad is moved from a site where the sample is received, past a wiping mechanism that meters the sample volume to provide the desired volume, to the site where the pad serves as a bridge between adjacent regions in the flow path. In such cases it is only necessary to apply an excess of sample directly to the sample region, such as by a hanging drop of blood.

When a sample contains cells, e.g. RBC, or other particulate matter, that may interfere with the assay, or with the detection of the measurement signal, filtration means may be provided. These filtration means is interposed between a sample application site and the sample region to provide a filtered sample. The use of various means for filtering blood cells from a sample are described in U.S. Patent No. 5,132,086, for a Non- Instrumented Cholesterol Assay.

The measurement region is generally an extended region, which allows for the flow of a reagent solution through the measurement region, by means of capillary action. The measurement region will have one or more members of the signal producing system present. Generally at least one member of the signal producing system will be non-diffusively bound in the measurement region. A color front or boundary appears at a distance where the reaction between members of the signal producing system ends. The distance along the measurement region where the color front occurs is related to the amount of analyte in the sample. A variety of assay protocols may be devised and implemented, based on a limited amount of material migrating along the path to produce a boundary at a distance proportional to the amount of analyte. The particular protocol is implemented in part by organization of the flow path, when appropriate, including the optional regions, and suitable selection of the members of the signal producing system. In general, any technique that allows for reaction or binding of a detectable entity in proportion to an analyte of interest may be employed for the measurement. General and specific examples of protocols suitable for measuring an analyte are described in U.S. Patent No. 4,959,324 and the other incorporated references.

Sample path intermediate regions, between the sample region and the measurement region, may be provided for some protocols. One or more members of the signal producing system may be present in such region or the region may simply be provided for mixing or as a delay so that a reaction may be completed to a predetermined degree prior to entering the measurement region. The reagents in this region may variously serve to control the dynamic range of the signal by capturing an amount of analyte or related entity, or to provide a time delay for mixing or reaction before moving into the measurement region. Where the analyte is a reactant which reacts with a member of the signal producing system, a conversion regent is provided which reacts with the analyte to produce an intermediate product. If the conversation reagent is at other than the sample region, upon movement of the eluent through the path, the conversion reagent is transported into contact with the sample or the sample is transported into contact with the conversion reagent, depending upon whether the conversion reagent is placed upstream or downstream of the sample region. In the former case, a separate conversion region may be provided for this purpose. The conversion region contains at least one member of the signal producing system, which serves to convert the analyte into an intermediate product. The intermediate product then undergoes a subsequent reaction with other members of the signal system to produce a detectable signal.

For example, in a cholesterol assay, a conversion region may be provided downstream from the sample region, which conversion region contains cholesterol esterase and cholesterol oxidase diffusively or non-diffusively bound to convert serum or plasma cholesterol and cholesterol ester to hydrogen peroxide. Alternatively, cholesterol esterase may be provided on the sample pad while the cholesterol oxidase may be present either upstream or downstream from the sample pad. Furthermore, the enzymes may be in the eluent as the eluent traverses the path. The hydrogen peroxide subsequently reacts with an intermediate which reacts with a leuco dye in the measurement region to produce a color change. The amount of analyte present is translated into a value by the conversion reaction stoichiometry.

An optional capture region may also be employed in the path. The capture reagent is placed downstream from the sample region, and may be either upstream or downstream from the conversion region, for assays where a conversion region is present. The capture reagent essentially prevents a predetermined amount of substance (such as analyte or analyte intermediate product that results from the reaction with the conversion reagent) from affecting the measurement region. This allows for shorter measurement regions and reduced wicking time, while still expanding the distance traversed for an incremental value of the analyte.

The capture region may employ the same reagent as the measurement region, but at a higher density. Generally, the density will be at least 20% greater, usually at least 1.5 fold greater and may be 5 fold or more greater. Usually the capture region will be less than half the area of the measurement region, usually less than about one-quarter. The capture region should be large enough to ensure substantially complete capture of the amount of reagent desired.

The reaction in the capture region may involve specific binding pair member complex formation, chemical reactions involving transformation of reactants into a product, or the like. For example, one may provide for antibodies to cholesterol, which prevents the cholesterol from reacting with the conversion reagents. Thus, one can withdraw a predetermined amount of cholesterol from the eluent or sample prior to reaction, so that in order to have a signal in the measurement region, the amount of cholesterol must be greater than the amount that reacts within the capture region. Alternatively, one can provide for a relatively high density of a component of the signal producing system in a relatively small area, so that there will be a strong band of signal produced in this narrow area, which may be disregarded. Rather than using a member of the signal producing system, one may use a different compound which reacts with a signal producing system component which is present in a limited amount. For example, in the case of hydrogen peroxide as a member of the signal producing system, various reactants may be present which will react with hydrogen peroxide to produce unreactive products. Illustrative reagents include metal ions or ions such as iodide. Examples of how capture regions may be implemented in an assay are described in U.S. Patent No. 5,132,086.

The measuring region will usually be an extended member, which allows for the flow of the eluent through the measuring region. The measuring region will have one or more members of the signal producing system non-diffusively bound. Members of the signal producing system may be uniformly distributed or spread in the form of alternating equal or unequal height bands. These bands effectively extend the signal height for a given sample amount. In an assay for cholesterol, the bound member reacts, either directly or indirectly, with the product of the cholesterol and conversion reagent to produce a detectable signal, e.g., a colored region with a discernible border. The height or distance of the observable border as a result of a detectable signal of the detectable signal reagent system, e.g., distance from the sample receiving element to the signal front, will be related to the amount of cholesterol in the sample.

A viewing window is provided in some embodiments that enclose the measurement strip in a housing to permit the user to visually observe the location of the color front. The window may also provide reference or calibration marks including numerals to indicate the amount of analyte that corresponds to the location of the color front.

For the sample path, the proportion of the path length allocated to operate as transport region, sample region, conversion region, capture region, and measurement region will generally vary depending on the analyte to be measured and the protocol. In general, the entire flow path may have a length of about 25 to 200 mm, more usually from about 50 to 150 mm, preferably about 100 mm. In general, about 25% to 90% of the length of the sample flow path will be the measurement region. The conversion region and/or capture regions will generally be about 5 to 50% of the flow path. The bibulous strips of the transport region will generally be from about 5 to 25 mm, more usually about 10 to 20%, of the length of the flow path. The sample region will generally be from about 1 to 10% of the length, more usually from about 2 to 8% of the length of the flow path; the longer the flow path, the larger the sample region may normally be. The width of the strips may be varied, usually being at least about 2 mm and not more than about 10 mm, preferably about 3 to 7 mm. Any convenient material may be used for the various bibulous parts. Usually, the thickness of the bibulous components will be in the range of about 0.05 to 2.0 mm, more usually 0.15 to 0.75 mm. A wide variety of bibulous supports may be employed, particularly cellulosic supports, such as chromatography paper, silica on a support, alumina on a support, and polymeric membranes such as nitrocellulose and nylon. The characteristics of the bibulous material employed for the measurement region include the need in many instances to covalently or irreversibly bind an indicator molecule to the support, to develop a clear and sharp color, and to ensure that the fluid is capable of flowing at a convenient rate through the bibulous member. The signal producing system comprises members appropriate to the detection of an analyte. Considering the assay for cholesterol as exemplary of such systems where analyte is a reactant, the system comprises a conversion reagent which reacts with the cholesterol to produce a stoichiometric amount of an intermediate product which in turn reacts, directly or indirectly, with another member of the signal producing system bound to the flow path to produce a detectable, e.g., colored, signal on the path or strip. The signal producing system may also comprise additional reagents, such as other intermediate components or catalysts needed to produce a detectable signal. The conversion reagent preferably includes reagents which react with cholesterol esters and cholesterol to form hydrogen peroxide. Such reagents are most preferably cholesterol esterase (EC:3.1.1.13) and cholesterol oxidase (EC: 1.1.3.6) . The serum cholesterol ester is hydrolyzed by the cholesterol esterase and subsequent oxidation of the cholesterol is accomplished by the cholesterol oxidase to produce a stoichiometric amount of hydrogen peroxide. The hydrogen peroxide can in turn react with a peroxidase substrate on the continuous flow path in the presence of a catalytic agent such as horseradish peroxidase or other peroxidase to react with a coupling compound to form a colored region on the path. The size of the colored region relates to the amount of cholesterol in the sample. The cholesterol esterase can be immobilized at 5 to 50 units/ml most preferably at about 18 units/ml. The cholesterol oxidase can be immobilized at 10 to 100 units/ml, most preferably at about 50 units/ml. The horseradish peroxidase may be immobilized along the flow path of the assay strip at immobilization concentrations 0.05 to 2.5 mg/ml, usually at 0.5 mg/ml. Preferably, peroxidase is included in the eluent at 0.0005 to 0.050 mg/ml.

By appropriate choice of members of the signal producing system, visually observable color fronts, fluorescent signals, or the like may be obtained for a quantitative assay.

The conversion reagent of the signal producing system can be placed at any position prior to the measurement region; e.g. in the eluent; in the eluent well to be dissolved by the eluent; in the transport region; the sample region; or the region, e.g. conversion region, between the sample region and measurement region. Upon movement of the transport medium through the path, the conversion reagent is transported into contact with the sample or vice versa depending upon whether the conversion reagent is placed upstream or downstream of the sample receiving region. Any additional reagents of the signal producing system, other than the bound reagent in the measurement region, may be similarly placed as the conversion reagent. In an embodiment of a device for a cholesterol assay a signal producing system comprising the following reagents has been effective. The eluent comprises buffered horseradish peroxidase. The region between the sample and measurement regions comprises cholesterol esterase and cholesterol oxidase, while the sample region comprises cholesterol esterase or is blank. The measurement region comprises N-methylbenzthiazole hydrazone (MBTH) and immobilized N,N-dimethylaniline (DMA) . An alternative device that produces good color fronts is provided by a modified arrangement. The eluent comprises buffered horseradish peroxidase, and the transport region comprises cholesterol oxidase. Cholesterol esterase is present in the sample region and/or the transport region. The conversion region is used only as a mixing/reaction region since the typical conversion reagents, cholesterol esterase and cholesterol oxidase are present upstream. The measurement region comprises MBTH and DMA. The cholesterol oxidase and/or horseradish peroxidase may be provided in tablet form in the eluent well or may be incorporated in the transport region.

Instead of having analyte as a substrate, as in the case of cholesterol, glucose, uric acid, etc., immunoassays may also be performed. For the most part the format will be the same, except that a conjugate will be employed of an enzyme and a member of a specific binding pair, consisting of ligand and receptor. The ligand or receptor of the conjugate will have the same binding specificity as the analyte. Instead of a dye forming compound in the measurement region, the complementary member of the conjugate will be non-diffusibly bound. The assay protocol provides for competition between conjugate and analyte for the complementary member bound to the bibulous support or competition between analyte and the complementary bound member for the conjugate. Also, substrate is provided to the conjugate after migration of the conjugate is complete. Instead of an enzyme label, a fluorescent label may be employed.

The sample flow path having been described generically, and in the exemplary context of a cholesterol assay; attention is now directed to the characteristics of the control flow path. The control flow path has most features analogous to those of the sample path, but also important differences.

The primary differences between the control path and the sample path concerns the sample region. There may also be differences with the optional regions including conversion, capture, mixing, and/or reaction regions. Also, the blood filtering device may be different. The control path characteristics for a particular embodiment of the invention generally depend on the type of analyte and protocol. They particularly depend on whether the compound measured in the control path is provided by a compound other than the analyte or a compound the same as the analyte. The control path is first described as to similarities followed by differences.

The control flow path for a device will comprise at least a transport region, a control region and a measurement region. Also, there will be a signal producing system and an eluent. The means for releasing the eluent, and one or more members of the signal producing system and the eluent may be shared with the sample assay system. Other regions may also be provided.

The eluent that serves as a transport fluid and flows through the control path will usually be identical to that in the sample path. Identity is not necessarily maintained however as the eluent flows through the different paths. Different eluents may be employed, but are not generally preferred because of the added complexity to the device. The control path transport region is generally similar to that of the sample path. It comprises the same type of bibulous element, and may even share all or part of the bibulous element with the sample path. The bibulous regions of the two paths may comprise separate segments or may overlap, particularly as to a single transport region. Different segments may have members of the signal producing system thereupon.

Desirably, the signal producing system reagents will be distributed analogously to their distribution in the sample pathway. One can provide a reactive compound (may be one or more compounds, e.g. cholesterol, cholesterol ester or glucose, etc.) to be present in a control region which is analogously situated as the sample region. By removing analyte from a portion of the sample to provide a matrix and using the analyte free portion ("matrix") to mix with the reactive compound, the properties of the sample may be substantially reproduced. The control path measurement region is substantially the same as the sample path measurement region. Generally, it is desirable to establish a known amount of reactive compound in a region upstream from the control path measurement region, and then to provide substantially the same protocols in each of the sample and control path measurement regions so that like factors will have like effects. For example, the control path measurement region of an assay for cholesterol may comprise substantially the same members of the signal producing system as comprise the sample path measurement region. These members have been described above.

The signal producing system includes means for producing a product so that a detectable signal is produced for a predetermined value of the reactive compound. Members of the signal producing system are present in at least one of the eluent or the control pathway prior to the measurement region, and in the measurement region, so that a product is produced by reaction of the reactive compound and the signal producing system. This reaction results in a colored boundary in the control measurement region at a predetermined site.

The means for producing a product may be located in the control pathway at a control product site. The particular characteristics of the control product site are addressed below in conjunction with the description of use of a compound which is the same as or different from the analyte.

The control product site will be situated between the transport region and the measurement region. In an embodiment that comprises a control region for receiving a portion of the sample, the control product site, if present, will generally be situated between the control region and the measurement region. In order to approximate the assay protocol, various compounds may be used which undergo enzymatic reaction to produce hydrogen peroxide: with cholesterol, cholesterol oxidase; with cholesterol ester, cholesterol esterase and cholesterol oxidase; with glucose, glucose oxidase; etc. In each instance a peroxidase is employed with the appropriate dye intermediate.

The amount of reactive compound present in the control path is indicated by the height (distance) of a visual signal, which height is related to a known amount of analyte. If the control signal agrees within some acceptable tolerance to the anticipated border site for the control, then the assay has functioned properly and the sample path measurement is verified. Desirably the control path will include the sample matrix without analyte. The presence of the matrix may affect the sample path measurement, and one would wish to have a like effect for the control. To the extent that the control path protocol is similar, the control will be similarly affected by the matrix. The matrix may be applied to the control region, control product site or other convenient location. In some instances a prepared matrix may be provided upstream for the measurement region. In order to use the sample as a source of matrix, the matrix will be treated to remove analyte. This may be accomplished by removing or otherwise deactivating the naturally occurring analyte so that it will not react to produce a signal. The means may comprise an antibody to the analyte, or an analyte deactivating enzyme, or both. Other means for particular analytes may also be provided. In an assay for cholesterol, the means for preventing signal production may comprise antibody to cholesterol or a cholesterol deactivating enzyme e.g. a kinase or other esterifying enzyme. For other analytes, similar reagents may be used for preventing signal production.

The control path comprises a control region, where the reactive compound and optionally, the sample matrix sample is received, and a control product site, where means for producing a product of the signal producing system with the control reactive compound is provided. The product results in a detectable signal, a defined border, related to the predetermined amount of reactive compound present.

The device may advantageously include a control region (equivalent to the sample region) moving from a first position out of contact with the transport and measurement regions of the control pathway, to a second position where it serves as a bridge between the transport and measurement regions. The structure and function of this movable region is analogous to the movable sample receiving region in the sample path. The device comprises means for filtering a blood sample. To provide analyte-free sample, the filter may include a pathway feeding the control pathway comprising antibodies to the analyte to remove analyte from the sample matrix. When the sample matrix is used for the control, there may be provided a means for distributing an aliquot of a single sample to the sample and control paths. The device user applies a single volume of sample to an application site such as an orifice leading from the external surface of the device to the interior which contains the two regions. Interposed between the orifice and each of the sample region and the control region is means for distributing the sample to the two regions. The means for distributing may comprise: two capillary tubes or bibulous members extending between the orifice and the sample and control regions; the sample may be applied to an area that is divided between a sample region and a control region. This later structure may be provided, for example, by a pad of material that has been effectively divided into two halves by impregnating it with a boundary wall or region of hydrophobic material, such as a wax or other means for preventing fluid transport between the two halves. The structure effectively allows a sample to distribute between the two areas when initially applied, but precludes any significant migration across the boundary region. The post-application isolation is important. Providing a matrix that is from the same source to both paths enhances the reliability of the measurement. It is also advantageous because it reduces potential sources of operating error.

If solubility of the reactive compounds is a problem, additional reagents may be provided to enhance solubility. For example, proteins may be provided to bind the analyte. Alternatively, solubilizing agents, such as detergents, may be provided. When using a dry agent might have undesirable affects on the control, it may be possible to provide a wet agent. In such embodiments, the agent could be provided in a hermetically sealed device, or a wet agent within the device may be separately sealed, then unsealed just prior to the start of the measurement. The devices may advantageously comprise a second control product site and measurement region in the control pathway to provide a second control value. The second product site and measurement region may simply provide a redundant control indication, or different amounts of reactive compound may be provided to give control indications over different ranges.

One or more control path measurement region viewing windows may be provided in embodiments of the device that enclose the measurement strip in a housing. As for the sample path, these windows permit the user to visually observe the location of the color front. The window may also provide a control verification mark or marks which indicate the distance that should be traversed by the color front for the known amount of sample in the path. For assays other than when the analyte is a substrate, signal developer, e.g. enzyme substrate, may be desirable or necessary for sample path and control path protocols to make a signal visible at the completion of signal front migration. Such procedure comprises flooding the strip with a suitable reagent to develop the color. In some embodiments of the invention, this additional procedure is implemented by providing a second sealed pouch or a containment well with a peelable poly-foil cover containing the developer reagent. At least one developer fluid transport channel leading from the development containment well is provided so that when the developer reagent is released into the channel, it floods the strip, initiating a reaction that makes the color front visible. Means for controlling flow of a developer fluid from the developer containment well to the developer fluid transport channel may also be provided. This means for controlling the flow of developer may be provided by pulling a second slide having protruding barbs at the appropriate time in the measurement and control protocols, so as to cut or otherwise open the pouch or poly-foil seal.

The subject invention is now considered in light of the drawings which depict several embodiments of the present invention. The embodiments are examples and not limitations of the subject invention.

In the embodiment illustrated in Fig. 1, a device 20 is provided for measurement of an analyte with simultaneous control. Device 20 includes a strip 22 having a notch to bifurcate the strip into two connected portions or strips, sample strip 30, and control strip 40. Notched strip 22 has scored areas 24 and 25 which includes sample pad 26 as the sample region for receiving the sample, and control pad 27 as the control region for receiving a different aliquot of analyte-free sample.

Pads 26, 27 may be removed from the scored areas 24, 25 and a known amount of sample applied to sample pads 26. An aliquot of the sample freed of analyte may be added to the control pad 27 where a reactive compound may be present and is dissolved in the sample matrix. The aliquot will be the same volume as the sample volume. The pads 26, 27 are then returned to the scored areas and fitted so as to be part of device 20 and be in fluid transport relation to notched strip 22. The notched strip is then dipped into eluent 23 which will be then be wicked through transport region 28 into sample region 26 and control region 27 and thereby transport the sample and reactive compound into downstream regions of device 20.

The sample strip 30 and the control strip 40 usually have similar organizations. The embodiment illustrated in Fig. 1 has substantially similar organization of the regions in both the sample strip 30 and in the control strip 40. If the analyte is not removed from the sample aliquot for the control, pad 27 may have an antibody to analyte, so as to deactivate any analyte in the applied sample. Downstream from the control region 27 is a control product region 29. Depending on the protocol, the reactive compound may be on the control pad 27 or in the control produced region 28.

Downstream from the sample region 26 and control product region 29 are conversion regions 32, 42. The conversion regions 32, 42 may be reguired where the analyte is measured by formation of an intermediate compound. For example, in an assay for cholesterol, one would have cholesterol esterase and cholesterol oxidase as conversion reagents in regions 32, 42 for reacting with the cholesterol in the sample and control to produce the intermediate product hydrogen peroxide.

The hydrogen peroxide reacts with a leuco dye intermediate downstream in measurement regions 36, 46 in the presence of horseradish peroxidase (HRP) to produce a compound which reacts with a coupling agent bound to the support. A border results which determines the cholesterol level in the sample. Rockets or color fronts as depicted by broken lines 38, 48 are obtained, where one can delineate the top of the rocket from the region immediately upstream from lines 38, 48. Desirably, the side edges of sample and control strip regions 30, 40 are perforated or serrated (not shown) , since this appears to provide for a sharper delineation of the color border. See U.S. Patent No. 4,757,004 which is herein incorporated by reference.

A more sophisticated device is shown in the embodiment illustrated in Figs. 2a, 2b, and 2c. The housing of this embodiment of the invention may be fabricated from three injection molded parts, or by any other convenient process. The parts comprise a base plate 50, a slide 52, and a cover plate 54, as shown in Figs. 2a, 2b, and 2c. The base plate 50 consists of a cutout 56 to accept the slide 52, a first slot 58 with locating pins 60 into which a sample path measurement strip 62 and transport strip 64 are precisely positioned, a second slot 66 with locating pins 61 into which a control path measurement strip 68 and transport strip 70 are precisely positioned. The base plate 50 also has a well 72 designed to capture eluent upon release. If two different elements are used in the sample and control paths, the base plate 50 would comprise two separate wells for capturing the two different released transport solutions.

The sample strip 62 comprises a measurement region 74, and may optionally comprise a conversion region 76, and capture region 78. A gap 80 of about 3 mm is maintained between the sample strip 62 and the transport strip 64. The gap 80 may be wider or narrower depending on the dimensions of the sample region 94, which may relate to the quantity of sample used in the measurement. The control strip 68 comprises a measurement region 84, and may optionally comprise a conversion region 86, and may contain capture region 88 for symmetry to the sample region. A gap 90 of about 3 mm is maintained between the control path measurement strip 68 and the transport strip 70. The gap may be wider or narrower depending on the dimensions of the control region 96. The slide 52 consists of a vented receptor site 92 into which the sample pad 94 is inserted, a control region 96 having a control reactive compound 98 present thereupon. Means for releasing the eluent, such as an arm 100 with shearing action, such as by barbs or protrusions 102 attached to the arm 100, and designed to facilitate the release of the transport solution from a shearable vessel is provided. The vessel is housed in well 104 of 5 cover plate 54. A snap 106 to lock the slide 52 in place, once pulled to initiate the measurement and control is also present.

The cover plate 54 consists of a well 100, which houses a sealed foil pouch containing the eluent or the

10 well 100 may be filled with eluent and covered with a peelable seal, such as a peelable poly-foil seal. The cover plate 54 has an orifice 108 for the introduction of the sample into the device. Underneath orifice 108 are filters 110, for separating particulates, such as cells , 15 from whole blood samples, from the fluid portion of the sample. For example, in a measurement for cholesterol, the filtration system may comprise dual glass fiber disks and a final filtration membrane in order to deliver blood cell free plasma to the sample region 94 from a sample of

20 whole blood.

The cover plate 54 also comprises the squeegee metering bar (not shown) , which serves to control the volume of sample absorbed by the pads 94 and 96. The squeegees are situated to squeeze the sample pad 94 and

25 control region 96 during movement toward the pathways.

Also present is a sample path measurement region viewing slot 112, and a control path measurement region viewing slot 114. At the top of the viewing slot 112 is a test complete indicator viewing hole 116, to view a color

30 change when the test is complete. The external surface of cover plate 54 may also comprise measurement scales 120, 122 adjacent the measurement region viewing slots 112, 114.

An assembled device is obtained by introducing the

35 slide 52 into base plate 50, positioning transport strips 64, 70, sample strip 62, and control strip 68, at their appropriate sites, introducing the eluent pouch into well 104 or as indicated above, filling well 100 with eluent and sealing with a poly-foil seal, assembling the cover plate and base plate and then sealing, conveniently by sonic welding, the base plate 50 and the cover plate 54. This procedure locates the sample receiving region 94 of the slide 52 directly beneath the filtration media 110 of the cover plate 54, as well as locating the shearing points of arm 100 of the slide 52 beneath the foiled sealed pouch located in the cover plate 54.

In order to carry out a measurement with control, the user applies an amount of sample to the application site 124. The application site may have characteristics which facilitate the application of an approximate amount of sample such as a central sample well 126 with a contrasting color border. The user then waits a sufficient period of time to allow adequate filtration and recovery of the liquid aliquot component of the sample onto the sample pad 94 and an analyte-free sample, aliquot is directed to the control region 96, where the analyte is removed, before pulling slide 52 to initiate the measurement. The slide is locked into the second position by snap 106.

At this point the sample region 94 containing the liquid component of the sample has been metered by the squeegee metering bar and is brought into fluid transferring relationship with the transport strip 64 and the sample strip 62. Similarly, control region 96 has been brought into contact and fluid transferring relationship with the control path transport strip 70 and the control strip 68. The shearing points 102 of the slide 52 have also pierced the foil seal of the pouch. Upon contact of the transport strip 64 with the eluent, the strip 64 begins wicking up the eluent which carries any catalytic agents or enzymes which transports the sample in sample pad 94 to sample strip. The eluent further carries any reaction product that results from the reaction of the reagents and the sample into the measurement region 74. The operation is analogous for the control flow path. The reaction results in a colored region with a defined boundary, the location of the boundary providing the user a precise reading of the amount of analyte. This reading is made when the color indicator site above the viewing slot shows the test is complete. In general, the user must wait a period of time for the measurement in the sample and control paths to be completed.

In an embodiment illustrated in Figure 3, a modification of the device shown in Figure 2 is shown, where an additional liquid reagent is supplied for flooding the sample and control strips 62, 68 to provide post-signal production color development. It is noted that the relative locations of the sample strip 62 and the control strip 68 have been interchanged in the embodiment of Figure 3. The development reagent located in developer well 140, flows through channels 142, 144 so as to flood the sample and control strips 62, 68. The locations of measurement viewing window 112, control viewing window 114, and test completion indicator 116, have similarly been interchanged.

When the eluent transport has been completed, the development slide 148, positioned analogously to slide 52 under eluent holding well 152 is pulled and tangs 150 on the arm of the slide 148 cause the contents of a poly-foil sealed container or pouch (not shown) in eluent holding well 152 to be released into developer well 140 in base 50. The developer reagent will then be channeled into slot 58 through channel 142, and through channel 144 to flood the sample and control strips 62, 68. Two or more channels are advantageous since they promote more rapid and uniform flooding of the strips.

Another embodiment is illustrated in Fig. 4. The embodiment in Figure 4 also contemplates the provision of a single sample applied at orifice 108 which is distributed to sample region 182, and control region 184, which are in close spatial proximity and located substantially under orifice 108 when the slide is in its first position. Regions 182 and 184 are separated in a line extending throughout their thickness impregnated by a material 186 that substantially prevents the transport or migration of material between the two regions. A hydrophobic material, such as a wax or polymer, may provide a suitable barrier for an aqueous eluent. The separation is important because one wishes to avoid mixing between the sample and the analyte-free matrix of the control, as well as communication during the assay. The geometrical structure of transport strip 160, sample strip 62, and control strip 68, in the spatial vicinity of gaps 80, 90 is modified over that shown in the earlier embodiments to interface with the closer spacing of sample regions 182, 184 when the slide 52 is in its second position. Figure 5 illustrates an embodiment of the invention with two features: a fixed binding element for transfer of eluent to the flow path; two different concentrations of a substance which serves as an analyte member in the assay. In this embodiment, the slide 52 in Fig. 5C has only the sample pad 200.. The slide 52 moves from an original position where the sample pad receives the sample to a final position where the sample pad 200 serves as a bridge to complete the flow of eluent in the sample pathway. During the movement, the slide passes under the bridging element 186, so as not to disturb its location. A known amount of reactive compound is placed at position 188 and 192, where the amounts are selected to provide for a low and high indication. Two measurement regions are provided, lower region 192 and upper region 194, where the amounts of reactive compound and the amounts of reagent in the measurement regions will provide for borders which relate to a low value of analyte in the sample and a high value of analyte in the sample. Therefore, if the borders do not appear in approximately the appropriate position, the determination may be rejected. The upper housing 54 has a lower window 196 and an upper window 198. The side(s) of the windows may be indexed indicating the region where the borders should be for a reliable determination.

It is evident from the above description that the subject device provides for greatly improved capability in carrying out quantitation determinations where little or no technical competence or instrumentation is required. The subject devices allow for greatly increased security that an observed result is accurate. The subject device allows for monitoring of operability of devices without requiring the use of samples of known concentration and the reproducibility of devices against a standardized measurement. By using the control measurements, device lots may be monitored and readily compared.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. An assay measurement and verification device for measuring one or more analytes, comprising substantially contiguous sample and control flow pathways, wherein there is a separate sample pathway for each analyte, and a signal producing system including an eluent solution for producing a detectable signal in measurement regions of said flow pathways, said device comprising: having at least two pathways comprising substrates capable of liquid transport; said pathways comprising a transport region for transporting eluent to separate measurement regions for said sample and control pathways; a sample receiving region for moving from a first position out of contact with said sample transport and measurement regions to a second position serving as a bridge between said sample transport and measurement regions; means for producing a product of said signal producing system resulting in a detectable signal for a predetermined value in said measurement region of said control pathway; and members of said signal producing system in at least one of said eluent or in said pathways prior to said measurement region, and in said measurement region, whereby a product is produced by said signal producing system in said sample pathway which results in a colored boundary in said sample measurement region at a distance related to the amount of an analyte in said sample, and a colored boundary is produced in said control measurement region at a predetermined site in said measurement region.

2. A device according to Claim 1, wherein said device further comprises: eluent in a sealed container; a member of said signal producing system on said substrates; means for releasing said eluent to be transported by said transport region after said sample has been applied to said sample region, whereby said eluent travels through said transport region, said sample receiving region and said measurement regions.

3. A device according to Claim 2, wherein said producing product means is located at least in part in said control pathway at a control product site, and further comprising: a sample intermediate region between said sample site and measurement region in said sample pathway, and a control intermediate region between said control product site and said measurement region in said control pathway.

4. A device according to Claim 3, wherein: said means for producing product comprises a known amount of a compound which produces said product upon enzymatic reaction; and said sample and control intermediate regions comprise enzymatic members of said signal producing system which react with said analyte and/or said compound to produce said product.

5. An assay measurement and verification device for measuring an analyte, comprising substantially contiguous sample and control flow pathways, an eluent solution, and a signal producing system for producing a detectable signal in measurement regions of said pathways, said device comprising: two pathways comprising bibulous solid substrates, said pathways comprising a transport region for transporting said eluent and separate measurement regions for said sample and control pathways; a sample receiving region for moving from a first position out of contact with said transport and measurement region to a second position serving as a bridge between said transport and measurement region; means for producing a product of said signal producing system at a control product site resulting in a detectable signal for a predetermined value in said measurement region of said control pathway; means for adding analyte-free sample to said control pathway prior to said measurement region; and members of said signal producing system in at least one of said eluent or in said pathways prior to said measurement region, and in said measurement region, whereby a product is produced by reaction of said analyte and at least one member of said signal producing system in said sample pathway which results in a colored boundary in said sample measurement region, and a colored boundary is produced in said control measurement region at a predetermined site in said measurement region.

6. A device according to Claim 5, wherein said device further comprises: eluent in a sealed container; means for releasing said eluent to be transported by said transport region after said sample has been applied to said sample region, whereby said eluent travels through said transport region, said sample region and said measurement regions.

7. A device according to Claim 6, wherein said analyte-free sample is produced by reaction of said analyte with at least one of antibody to said analyte or analyte deactivating enzyme.

8. A device according to Claim 5 comprising means for removing red blood cells from a blood sample and applying aliquots of said red blood cell free sample to said sample region and a control region.

9. An assay measurement and verification device for measuring cholesterol, comprising substantially contiguous sample and control pathways, an eluent solution, and a signal producing system for producing a detectable signal in measurement regions of said pathways, said device comprising: two pathways comprising bibulous solid substrates, said pathways comprising a transport region for transporting said eluent and separate measurement regions for said sample and control pathways; a sample receiving region and a control region moving from a first position out of contact with said transport and measurement regions to a second position serving as a bridge between said transport and measurement regions; means for producing a product of said signal producing system at a control product site resulting in a detectable signal for a predetermined value in said measurement region of said control pathway, said control product site being situated between said control region and said measurement region and comprising at least one of cholesterol and cholesterol ester or glucose; means for adding analyte-free sample analyte in said control pathway prior to said measurement region; and members of said signal producing system in at least one of said eluent or in said pathways prior to said measurement region, and in said measurement region, whereby a product is produced by reaction of said analyte and at least one member of said signal producing system in said sample pathway which results in a colored boundary in said sample measurement region, and a colored boundary is produced in said control measurement region at a predetermined site in said measurement region, said signal producing system members for producing hydrogen peroxide from cholesterol and cholesterol ester comprising cholesterol oxidase -and cholesterol esterase, and from glucose comprising glucose oxidase.

10. A device according to Claim 9, wherein said device further comprises: eluent in a sealed container; means for releasing said eluent to be transported by said transport region after said sample has been applied to said sample region, whereby said eluent travels through said transport region, said sample region and said measurement regions.

11. A device according to Claim 9, comprising means for removing red blood cells from a blood sample and applying aliquots of said red blood cell free sample to said sample region and said control region.

12. A device according to Claim 11, wherein said red blood cell removing means further comprises means for removing analyte from said aliquot applied to said control region.

13. A device according to Claim 12, further comprising a second control product site and measurement region in said control pathway to provide a second control value.

14. An assay measurement and verification device for measuring an analyte, comprising substantially contiguous sample and control flow pathways, for use with a signal producing system for producing a detectable signal by means of an enzymatic reaction, said detectable signal produced in said pathways in measurement regions as discernible boundaries; said device comprising: two pathways comprising bibulous substrates; said pathways comprising a transport region for transporting eluent to separate measurement regions for said sample and control pathways; a sample receiving region and a control region between said transport region and said sample and control measurement regions, respectively, said control region comprising a reactive compound capable of producing a product resulting in a detectable signal; and means for adding red-blood-cell-free sample to said sample receiving region and analyte-free red-blood-cell- free sample to said control region.