WO2001025760A1

WO2001025760A1 - Timing independent method for determining a proper time for measurement of a reaction between a sample fluid and a reagent

Info

Publication number: WO2001025760A1
Application number: PCT/US2000/027370
Authority: WO
Inventors: Richard Riedel
Original assignee: Umm Electronics, Inc.
Priority date: 1999-10-07
Filing date: 2000-10-04
Publication date: 2001-04-12
Also published as: AU7855000A

Abstract

The present invention relates to a timing independent method for determining a proper time for measurement of a reaction between a sample fluid and a reagent on an analyte strip. Sample fluid, such as whole blood, is applied to a reagent matrix and a characteristic of matrix (such as reflected light, current, etc.) is periodically measured both before and after application of the sample fluid. A transformation is then made of this measurement data into a function that is independent in time or at most varies linearly in time. The second derivative of the transformed function is then analyzed to determine when the second derivative falls below a predetermined threshold. At this time, the transformed function will yield the analyte concentration in the sample fluid (such as glucose concentration in a whole blood sample).

Description

TIMING INDEPENDENT METHOD FOR DETERMINING A

PROPER TIME FOR MEASUREMENT OF A REACTION BETWEEN A SAMPLE FLUID AND A REAGENT

TECHNICAL FIELD OF THE INVENTION

The present invention generally relates to a method for measuring chemical and biochemical components (analytes) in aqueous fluids on an analyte test strip and, more particularly, a timing independent method for determining a proper time for measurement of a reaction between a sample fluid and a reagent.

BACKGROUND OF THE INVENTION The quantification of chemical and biochemical components in colored aqueous fluids, in particular colored biological fluids such as whole blood and urine and biological fluid derivatives such as serum and plasma, is of ever- increasing importance. Important applications exist in medical diagnosis and treatment and in the quantification of exposure to therapeutic drugs, intoxicants, hazardous chemicals and the like. In some instances, the amounts of materials being determined are either so miniscule - in the range of a microgram or less per deciliter - or so difficult to precisely determine that the apparatus employed is complicated and useful only to skilled laboratory personnel. In this case, the results are generally not available for some hours or days after sampling. In other instances, there is often an emphasis on the ability of lay operators to perform the test routinely, quickly and reproducibly outside a laboratory setting with rapid or immediate information display. One common medical test is the measurement of blood glucose levels by diabetics. Current teaching counsels diabetic patients to measure their blood glucose level from two to seven times a day depending on the nature and severity of their individual cases. Based on the observed pattern in the measured glucose levels, the patient and physician together make adjustments in diet, exercise and insulin intake to better manage the disease. Clearly, this information should be available to the patient immediately.

Previously, a method widely used in the United States employs a test article of the type described in U.S. Patent No. 3,298,789 issued January 17, 1967 to Mast. In this method, a sample of fresh, whole blood (typically 20-40 μl) is placed on an ethylcellulose-coated reagent pad containing an enzyme system having glucose oxidase and peroxidase activity. The enzyme system reacts with glucose and releases hydrogen peroxide. The pad also contains an indicator which reacts with the hydrogen peroxide in the presence of peroxidase to give a color proportional in intensity to the sample's glucose level.

Another previous blood glucose test method employs similar chemistry but in place of the ethylcellulose-coated pad employs a water-resistant film through which the enzymes and indicator are dispersed. This type of system is disclosed in U.S. patent No. 3,630,957 issued December 28, 1971 to Rey et al.

In both cases the sample is allowed to remain in contact with the reagent pad for a specified time (typically one minute). Then in the first case the blood sample is washed off with a stream of water while in the second case it is wiped off the film. The reagent pad or film is then blotted dry and evaluated. The evaluation is made either by comparing color generated with a color chart or by placing the pad or film in a diffuse reflectance instrument to read a color intensity value.

While the above methods have been used in glucose monitoring for years, they do have certain limitations. The sample size required is rather large for a finger stick test and is difficult to achieve for some people whose capillary blood does not express readily.

In addition, these methods share a limitation with other simple lay-operator colorimetric determinations in that their result is based on an absolute color reading which is in turn related to the absolute extent of reaction between the sample and the test reagents. The fact that the sample must be washed or wiped off the reagent pad after the timed interval requires that the user be ready at the end of the timed interval and wipe or apply a wash stream at the required time. The fact that the reaction is stopped by removing the sample leads to some uncertainty in the result, especially in the hands of the home user. Overwashing can give low results and underw ashing can give high results. Another problem that often exists in simple lay-operator colorimetric determinations is the necessity for initiating a timing sequence when blood is applied to a reagent pad. A user will typically have conducted a finger stick to obtain a blood sample and will then be required to simultaneously apply the blood from the finger to a reagent pad while initiating a timing circuit with his or her other hand, thereby requiring the use of both hands simultaneously. This is particularly difficult since it is often necessary to insure that the timing circuit is started only when blood is applied to the reagent pad.

In order to eliminate the need for the user to initiate a timing sequence upon application of the blood sample to the reagent pad, U.S. Patent No. 5,049,487 issued September 17, 1991 to Phillips et al. teaches the use of a reagent pad and reflectance measurement system as illustrated schematically in FIG. 1. The Phillips et al. patent teaches an apparatus for determining the presence of an analyte in a fluid as well as a test strip for use with the apparatus. The fluid to be analyzed is applied to the test strip and the test strip is analyzed by the apparatus. In a preferred embodiment, the test strip comprises a single layer hydrophilic porous matrix 10 to which the chemical reagents are bound. The chemical reagents react with the analyte in the sample applied to the test strip in order to produce a dye that is characteristically absorptive at a wavelength other than the wavelength that the assay medium substantially absorbs. In other works, reaction of the chemical reagent with the analyte produces a color change in the sample. The reagent matrix 10 is coupled to the underside of an inert test strip carrier 12 containing an orifice 14 therethrough. The analyte sample is applied to the orifice 14 and the apparatus analyzes the opposite side of the test strip by reflecting light from an LED 16 off of the bottom surface of the reagent matrix 10 and sensing the amount of reflected light with a photodiode 18. It is therefore necessary for the sample to diffuse through the test strip prior to being analyzed. In such systems, the amount of time that the analyte is allowed to react with the reagent prior to measurement of a color change is critical to the accuracy of the measurement. The beginning of this "incubation period" must be measured as precisely as possible. In the Phillips et al. patent, as the analyte sample penetrates the reagent matrix 10 and wets the bottom surface, an initial change in reflectance of this measurement surface occurs. The apparatus detects this change in reflectance by sensing a decrease in the amount of light reflected to the photodetector 18. The apparatus then begins the timing of the incubation period upon detection of this change in reflectance. After a predetermined incubation time period, during which the sample containing the analyte reacts with the reagent chemicals in the matrix 10, a second reflectance measurement is made in order to determine the color change in the sample. By accurately measuring the beginning of the incubation period and the time delay before measurement, the accuracy of the apparatus is greatly improved over prior methods.

Because the start of the incubation period in the Phillips et al. method begins with a determination that surface wetting of the underside of the reagent matrix 10 has occurred (in the embodiment of FIG.1), the processing circuitry coupled to the photodetector 18 must have some method for determining when the reflectance measurements indicate surface wetting. Referring to FIG. 2, there is shown a graph of remission (percent reflection) v. the apparatus system time (in which one unit of system time equals .25 seconds of actual time). As can be seen from the graph, the reflectivity of the reagent matrix 10 prior to sample application is a constant value (approximately 88%). After sample application, the reflectivity of the underside of the reagent matrix 10 steadily drops as the sample fluid migrates to the underside of the reagent matrix 10. The remission also drops due to a color change in the reagent caused by reaction with the analyte sample. At some point, the analyte fluid has reached the undersurface of the reagent matrix 10 and further drops in remission are caused only by color change of the reagent. The prior art method analyzes this data in order to make a determination of when surface wetting has occurred on the underside of the reagent matrix 10. This determination is made by sensing when the remission value has dropped by a predetermined, fixed amount from its steady state value prior to sample application. For example, in one commercial version of this prior art system, surface wetting is assumed to have occurred when the remission value drops by approximately 38% (i.e. when a remission value of 50% is observed). When this change in remission (or ΔR) is observed, the prior art device starts the timing of the incubation period, after which the sample measurement will be made. The prior art method described in Phillips et al. suffers from the problem that the start of the incubation period, by using a fixed reflectance drop, must be tailored to a specific chemistry. A changing in the base reflectance, such as may occur for different enzymes or indicators, requires a predetermination of the fixed reflectance drop. In this sense, a given fixed drop is not generally applicable to different systems.

There is therefore a need for a system and method for determining the application of a sample fluid on an analyte strip which is effective for use with any analyte strip, regardless of the specific chemistry or strip-to-strip variations therein. The present invention is directed toward meeting this need.

SUMMARY OF THE INVENTION The present invention relates to a timing independent method for determining a proper time for measurement of a reaction between a sample fluid and a reagent on an analyte strip. Sample fluid, such as whole blood, is applied to a reagent matrix and a characteristic of matrix (such as reflected light, current, etc.) is periodically measured both before and after application of the sample fluid. A transformation is then made of this measurement data into a function that is independent in time or at most varies linearly in time. The second derivative of the transformed function is then analyzed to determine when the second derivative falls below a predetermined threshold. At this time, the transformed function will yield the analyte concentration in the sample fluid (such as glucose concentration in a whole blood sample).

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic perspective view of a prior art optical reflectance analyte measurement system.

FIG. 2 is a graph of remission v. system time, illustrating a prior art method for determining a start of the reaction incubation period in an optical reflectance meter.

FIG. 3 is a first graph of remission v. system time for several different analyte samples.

FIG. 4 is a second graph of remission v. system time for several different analyte samples.

FIG. 5 is a graph of a transformation of the data of FIG. 4, plotted v. system time.

FIG. 6 is a graph of the first derivative of the data of FIG. 5, plotted v. system time. FIG. 7 is a graph of K/S v. system time for several different analyte samples.

FIG. 8 is graph of the first derivative of the data of FIG. 7, plotted v. system time.

FIG. 9 is a graph of current v. system time for several different analyte samples.

FIG. 10 is a graph of a transformation of the data of FIG. 9, plotted v. system time.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS For the purposes of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, and alterations and modifications in the illustrated device, and further applications of the principles of the invention as illustrated therein are herein contemplated as would normally occur to one skilled in the art to which the invention relates. FIG. 3 illustrates the problem with establishing the start of the incubation period with a predetermined drop in remission. It is characteristic of remission v. time data for a whole blood sample to exhibit the general shape of the curve of FIG. 3. However, FIG. 3 plots the remission v. time data for several different whole blood samples, illustrating that the minimum remission value and the speed with which the graph transitions to the minimum remission value is highly variable and dependent upon several factors, including the glucose concentration within the sample. Therefore, while picking a predetermined drop in remission value in order to start the incubation period may work well when the remission v. time graph has the expected form (as in FIG. 2), this method may not work well when the remission v. time graph for different glucose samples varies widely, as shown in FIG. 3. The 50% remission threshold of FIG. 2 is not effective for the varying curves of FIG. 3, as plainly evident from an examination of this figure. In fact, one of the remission v. time curves never reaches the 50% remission value. Utilizing a predetermined drop in remission as the start of the incubation period is therefore undesirable in many real-world test scenarios. For the chemistry of FIG. 3 it is obvious that reducing the magnitude of the predetermined drop will cause a start time to be triggered for all three curves. However, it is by no means certain that the same magnitude will be valid for a chemistry where the background material has a different density, or the enzyme-indicator mix has a different base color. In theory, it is possible that n different strip lots could require n different "predetermined" drops. The present invention utilizes a method for determining the concentration of an analyte which requires no timing whatsoever. In general, when determining the concentration (c) of an analyte, the variable being measured (i.e. reflectance, c rent, fluorescent signal, etc.) can be described by a function f(t,c) which depends upon both time and concentration of the analyte. Because of this dependence upon time, most prior art methods require c to be inferred from f(t,c) where t is a fixed time after sample application to the test strip. For example, in an electrochemical measurement, the current I = f(t,c) may be measured 30 seconds after sample application. In this case, c is given by the function c = G(30,I), where G is an inverse function related to f via G(a,I) = f(a,c), where a is the fixed time after sample application at which the measurement is taken.

The explicit form of f(t,c) may not be known from a theoretical standpoint; however, this function can always be experimentally (empirically) determined. Once f(t,c) is known, one can transform f(t,c) into a function s(t,c) which is independent in time or at most varies linearly in time (i.e. s(t,c) = a(c) + b(c)t). By using the transformed function, the analyte concentration (c) may be determined using a timing independent determination.

By calculating the second derivative of the transformed function s, one can ascertain whether the reaction system has "settled" into the linear state. When the magnitude of the second derivative of the transformed function is below a predetermined threshold, the transformed function can be considered to be at most linear in time. At this point, the value of the analyte concentration c may be determined from the transformed function. Typically, only the slope of the transformed function will depend upon the analyte concentration c (s(t,c) = a + b(c)t); however, in general, both coefficients can depend upon the analyte concentration c (s(t,c) = a(c) + b(c)t).

Consider, for example, the colormetric measurement of an analyte applied to a test strip. FIG. 4 illustrates a graph of remission (or reflectance) R(t) vs. time for test samples having a variety of different glucose values. By using the transformation G(t) = t(R(t)), we obtain the curves shown in FIG. 5. The first derivatives of the functions G(t) of FIG. 5 are illustrated in FIG. 6. When the second derivative of G(t) is less than a predetermined threshold value, we may measure the smoothed slope of G(t), which is related to the glucose concentration c of the sample. This method relies on the function R(t) being well modeled as R(t) = A + ^B/ ₁, where the coefficients A, B both depend upon glucose concentration. By using the slope of the function tR(t) as a measure of glucose, we are in effect using the coefficient A. The point at which the second derivation of the data in FIG. 6 is less than the predetermined threshold value is shown by the regions spanned by the arrows. It will be appreciated that the threshold value is chosen such that the slopes of the first derivative data are approximately constant for times greater than the ranges marked by the double arrows.

In another embodiment of the present invention, a suitable transformation for some test strip architectures is a transformation of the reflectance data R to K/S (K/S = ((1-R2)/2R)). This transformation is shown in FIG. 7 for test samples having a variety of glucose concentrations. The slopes of the K/S(t) curves can be seen to be almost constant after the test strip hydration phase. The first derivative of the K/S(t) data of FIG. 7 is illustrated in FIG. 8. When the second derivative of this data is below a predetermined threshold (as shown by the double arrows in the graph), the smoothed derivative may be calculated in order to determine the glucose concentration. The slopes of the curves in FIG. 8 can be seen to be approximately constant for times greater than the ranges delineated by double anows.

A further embodiment of the present invention is applicable for use with an electrochemical sensor. For example, FIG. 9 is a graph of current v. time (I(t)) for various sample glucose concentrations. FIG. 10 illustrates the function I'(t) = max (I(t), I'(t - δ)) v. time (where δ is a small time increment). The nature of the transformation is to produce a function which is constant after the peak in the value of I(t). The second derivative will therefore be zero for times after this peak. Possible appropriate times to determine peak cunent (and hence glucose concentration) are indicated by the double arrow regions in FIG. 10. Typically, in an ampeometric system, the cunent can be written as I(t) = h(t) + g(t) where h(t) is not a function of the analyte (glucose) present, while g(t) is. Their sum gives the cunent I(t). g(t) generally develops a value which reaches a peak upon full hydration and then decays as a boundary layer is formed. Because the same enzymatic chemistry occurs during this transition (mixing) phase, g(t) will be a good indicator of the amount of glucose present.

While the invention has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the prefened embodiment has been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected.

Claims

What is claimed is:

1. A method for determining a proper time for measurement of a reaction between a sample fluid and a reagent in order to determine an analyte concentration in the sample fluid, comprising the steps of: a) measuring the reagent at intervals prior to application of the sample fluid to the reagent; b) applying the sample fluid to the reagent; c) measuring the reagent/sample fluid combination at intervals after performing step (b); d) Transforming the measurement data taken at steps (a) and (c) into a function that at most varies linearly in time; e) calculating a second derivative data of the function; f) identifying a time conesponding to a point where the second derivative data falls below a predetermined threshold; and g) determining the analyte concentration using the function determined in step (d) and the time identified in step (f).

2. The method of claim 1, wherein step (a) comprises measuring an amount of light reflected off of the reagent and step (c) comprises measuring an amount of light reflected off of the reagent/sample fluid combination.

3. The method of claim 2, wherein the measurement data taken at steps (a) and (c) is of the form R(t) and the function comprises t(R(t)).

4. The method of claim 2, wherein the measurement data taken at steps (a) and (c) is of the form R(t) and the function comprises k s = ((1- 2R(t))/2R(t)).

5. The method of claim 1, wherein step (a) comprises measuring an amount of cunent flowing through the reagent and step (c) comprises measuring an amount of cunent flowing through the reagent/sample fluid combination.

6. The method of claim 5, wherein the measurement data taken at steps (a) and (c) is of the form I(t) and the function comprises I'(t) = max (I(t), I'(t- δ)), wherein δ is a small increment of time.

7. The method of claim 1, wherein the sample fluid is whole blood.

8. The method of claim 7, wherein the reagent reacts with the whole blood to produce a color change in proportion to an amount of glucose in the whole blood.

9. The method of claim 7, wherein the reagent reacts with the whole blood to produce a conductivity change in proportion to an amount of glucose in the whole blood.

10. The method of claim 1, wherein the intervals are fixed intervals.