WO2005003774A1

WO2005003774A1 - Electrochemicalbiosensor test strip and reagent for analyzing physiologicsl sample including blood corpuscles

Info

Publication number: WO2005003774A1
Application number: PCT/KR2004/001627
Authority: WO
Inventors: Yon-Chan Ahn; Im-Ok Kim; Mi-Suk Park; Seung-Joo Kang
Original assignee: Allmedicus Co., Ltd
Priority date: 2003-07-03
Filing date: 2004-07-02
Publication date: 2005-01-13
Also published as: KR20050003653A; KR100611510B1

Abstract

Disclosed is an electrochemical biosensor test strip that is capable of effectively compensating for the interference effect of the hematocrit. The electrochemical biosensor test strip does not use conventionally widely used erythrocyte blocking methods but use a system of compensating for a reduction in a signal, shown in conventional methods, using an electrical signal obtained from a substance in erythrocytes. The electrochemical biosensor test strip includes a first insulating substrate, a plurality of electrodes formed on the first insulating substrate, and a reagent, which is immobilized on the plurality of electrodes formed on the first insulating substrate, to react with an analyte of interest in a physiological sample to generate a charge corresponding to the concentration of the analyte, wherein the reagent includes a corpuscle interference correcting agent to react with corpuscles in the physiological sample to generate the charge corresponding to the concentration of the analyte.

Description

ELECTROCHEMICAL BIOSENSOR TEST STRIP AND REAGENT FOR ANALYZING PHYSIOLOGICAL SAMPLE INCLUDING BLOOD CORPUSCLES

Technical Field

The present invention, in general, relates to a test strip and a reagent for use in electrochemical biosensors, and, more particularly, to an electrochemical biosensor test strip and a reagent for quantitative analysis of an analyte of interest in a physiological sample containing blood corpuscles.

Background Art

Blood consists of about 55% fluid (so called plasma) and about 45% formed elements including several kinds of corpuscles. The blood plasma is composed of 92% water, 6.5-7% plasma proteins and 1-1.5% trace elements including inorganic salts, enzymes, hormones, vitamins, lipids and sugars. The formed elements, corpuscles comprise three main types: red blood cells (RBC) or erythrocytes; white blood cells (WBC) or leukocytes; and platelets. Erythrocytes are the most numerous cells in the blood. When blood samples are analyzed by electrochemical biosensors, the greatest problem is the interference of the formed elements, especially erythrocytes, present in the samples . The volume occupied by erythrocytes in blood is represented by hematocrit (Hct) , which is expressed as a percentage. Normal hematocrit values vary between adults, pregnant women, newborns, and the like. Hematocrit values differ between individuals, but, in general, normal hematocrit ranges are 35-50% in adults, lower percentages in pregnant women, and higher percentages in newborns . This wide range of hematocrit values can cause errors in other measurements made by electrochemical biosensor systems. For example, in the case of measuring blood sugar, hematocrit values have clinically important meaning in adults, pregnant women, newborns, and the like. Typically, high hematocrit blood samples display lower blood sugar than the true value, while low hematocrit blood samples exhibit higher blood sugar than the true value. Such distortion of the measured values is caused by the following factors: 1) decrease of the diffusion rate of a reagent into a plasma, according to the increased volume of erythrocytes in a blood sample; 2) adherence of erythrocytes and proteins to the surfaces of electrodes, leading to a reduction in the surface area of the electrodes; 3) change of sample viscosity; 4) formation of micro-aggregates; and 5) hemolysis. In case of analyzing blood samples using electrochemical systems, the irregular adherence of blood cells and proteins to electrode surfaces is a major cause. The factors distorting measured values make accurate measurement for analytes difficult. Efforts were made to reduce the adherence of erythrocytes, proteins and other blood solid particles to electrodes in order to achieve accurate measurement . As a result, a membrane-type electrochemical enzyme electrode as shown in FIG. 1 was developed, and already marketed as "YSI 2300 STAT PLUS (Yellow Spring Instrument, Inc.)". As shown in FIG. 1, this analyzer comprises three membrane layers: an enzyme layer 101; an outer layer 103; and an inner layer 105, and a platinum electrode 107. This system has the following feature: an enzyme to react with an analyte is prepared in a thin film, and functional polymer membrane layers composed of cellulose acetate and polycarbonate are formed respectively in the front and the back of the enzyme layer, thereby effectively blocking absorption of erythrocytes and proteins onto the surface of the electrode. However, this enzyme electrode system is expensive and is not disposable but repeatedly used in hospitals or research centers, and is not portable and thus unsuitable for use at the point of care. Also, the enzyme electrode system is disadvantageous in terms of requiring a lot of blood and having relatively slow response. U.S. Pat. Nos. 5,708,247 and 5,951,836 (each titled "Disposable Glucose Test Strips And Methods And Compositions For Making Same") and U.S. Pat. No. 6,241,862 (titled "Disposable Test Strips With Integrated Reagent/Blood Separation Layer") disclose methods of preparing carbon paste electrode systems using screen printing techniques and a reagent/blood separation layer immobilized on the surface of an electrode, respectively. In the U.S. patents, a non-conductive silica filler is introduced into a carbon electrode in combination with hydroxyethyl cellulose to reduce the interference effect of the hematocrit . The non-conductive silica is dried along with a reagent by a balance of hydrophobicity and hydrophilicity to form a two-dimensional network on the electrode, thereby inhibiting absorption of erythrocytes, proteins and other blood elements onto the electrode. However, the carbon paste electrode, prepared by a screen printing method, is relatively unstable compared to metal electrodes because of its uneven surface and because of degradation and leakage of the electrode material with the passage of time. Also, when the non-conductive silica has an unbalance in its hydrophobicity and hydrophilicity, the two-dimensional network becomes unstable and thus affects the diffusion rate of a sample into an electrode, resulting in distortion of measured values . As described above, reduction of the interference effect of the hematocrit is conventionally achieved by introducing outer and inner membrane layers into a membrane-type enzyme electrode or forming a two-dimensional network on a carbon paste electrode using a non-conductive substance such as silica. The membrane-type enzyme electrode, so called a membrane technique", is very effective in removing the hematocrit interference effect. However, because of requiring a complex manufacturing process and high cost to introduce the three layers, that is, the enzyme layer 101, the outer layer 103 and the inner layer 105, into an electrode system, the membrane-type enzyme electrode is difficult to mass produce as a disposable self-diagnostic sensor at competitive cost. Also, the membrane-type enzyme electrode requires a large amount of blood, and has a long response time because a sample reacts with the electrode after passing through three layers. Therefore, in spite of having an excellent effect on reducing the hematocrit interference, the membrane-type enzyme electrode is problematic in use as a component of disposable self-diagnostic sensors . Another method of preventing the hematocrit interference, that is, the absorption of erythrocytes, proteins and other elements in blood samples onto an electrode, is to introduce a variety of polymers individually or in a mixed form in combination with silica onto the surface of the electrode. However, this electrode has severe problems as follows. Since generally used polymers are hydrophilic, when a sample enters the electrode, they are dissolved along with the sample ^' so that a network cannot serve as an effective blocker. In the case that the polymers are hydrophobic, surface repulsion occurs between the polymers and a hydrophilic reagent layer, thereby making establishment of a physically stable structure difficult.

Disclosure of the Invention

Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and an object of the present invention is to provide a disposable self-diagnostic sensor capable of effectively compensating for the hematocrit interference. Another object of the present invention is to provide an electrochemical biosensor capable of being manufactured at low cost as well as effectively compensating for the hematocrit interference . A further object of the present invention is to provide an electrochemical biosensor capable of being mass produced by a simple process as well as effectively compensating for the hematocrit interference. The present invention for accomplishing the above objects differs from conventional techniques in terms of not using conventionally widely used erythrocyte blocking methods but providing a system ^' of compensating for a reduction in a signal, shown in conventional methods, using an electrical signal obtained from a substance in erythrocytes . The present invention provides an electrochemical biosensor test strip for quantitative analysis of an analyte of interest in a physiological sample containing blood corpuscles, comprising a first insulating substrate, a plurality of electrodes formed on the first insulating substrate, and a reagent, which is immobilized on the plurality of electrodes formed on the first insulating substrate, to react with an analyte of interest in a physiological sample to generate a charge corresponding to the concentration of the analyte, wherein the reagent includes a corpuscle interference correcting agent to react with corpuscles in the physiological sample to generate the charge corresponding to the concentration of the analyte. The electrochemical biosensor test strip may further comprise a second insulating substrate, which is placed on the first insulating substrate, to form a passage to introduce the physiological sample into the reagent. Preferably, the corpuscle interference correcting agent includes a lysis agent to destroy erythrocytes in the physiological sample to release hemoglobin from the erythrocytes to the exterior, and a redox mediator to transfer the charge generated by a reaction with the hemoglobin to the electrode. The lysis agent is selected from the group consisting of saponin, sodium deoxycholate, EDTA, lysis buffers, detergents and combinations thereof. In addition, the present invention provides an electrochemical biosensor reagent for quantitative analysis of an analyte of interest in a physiological sample containing blood corpuscles, comprising a biochemical substance to specifically react with the analyte in the physiological sample to generate a charge corresponding to the concentration of the analyte, a lysis agent to destroy erythrocytes in the physiological sample to release hemoglobin from the erythrocytes to the exterior, and a redox mediator to transfer the charge generated by a reaction of the biochemical substance with the hemoglobin to a predetermined electrode. The present invention has advantages of not using the conventional membrane technique, and effectively blocking absorption of erythrocytes present in samples onto the surface of an electrode and reducing the hematocrit interference effect without introduction of hydrophilic and hydrophobic polymers to form a specific network structure. The advantages further include that the present invention provides a disposable electrochemical test strip capable of being mass produced at low cost by a simple process.

Brief Description of the Drawings FIG. 1 is an exploded perspective view of a conventional membrane-type electrochemical enzyme electrode; FIG. 2 is an exploded perspective view of an electrochemical biosensor test strip according to the present invention; FIG. 3 is a view showing the concept of a glucose sensor using an enzyme-redox mediator reaction according to the present invention; FIG. 4 is a view showing the concept of a hemoglobin sensor using a hemoglobin-redox mediator reaction according to the present invention; FIG. 5 is a view for describing hematocrit compensation by the present invention; FIG. 6 is a graph showing changes in measured values against hematocrits; FIG. 7 is a graph showing the interference effect of hematocrit in the case of using a conventional reagent not containing an erythrocyte lysis agent; and FIG. 8 is a graph showing the interference effect of hematocrit in the case of using a reagent containing an erythrocyte lysis agent according to the present invention.

Best Mode for Carrying Out the Invention Hereinafter, an embodiment of the present invention will be described in detail with reference to the accompanying drawings. In the embodiment, the present invention is applied to glucose detection, but the embodiment is provided only to illustrate the present invention. Thus, it is to be understood that the present invention can be used for quantitative analysis of any analyte of interest. FIG. 2 is an exploded perspective view of an electrochemical biosensor test strip according to an embodiment of the present invention. As shown in FIG. 2, an electrochemical biosensor test strip 200 includes an insulating substrate 201, electrodes 203 and 205 formed on the insulating substrate 201, and a reagent 207, which is immobilized on the electrodes 203 and 205 formed on the insulating substrate 201, to react with an analyte of interest in a physiological sample to generate a charge corresponding to the concentration of the analyte. The reagent 207 includes a corpuscle interference correcting agent to react with corpuscles in the physiological sample to generate the charge corresponding to the concentration of the analyte. Referring to FIG. 2, a reference electrode 203 and a working electrode 205 are provided on the insulating substrate 201, and the insulating substrate 201 is attached to another insulating substrate 211, wherein the insulating substrates are separated by spacers 209. A two electrode system is illustrated in FIG. 2, but it is to be understood that a three electrode system can be employed. As shown in FIG. 2, the reagent 207 is transversely immobilized on the reference electrode 203 and the working electrode 205, and may be coated on an electrode region on the insulating substrate 201 corresponding to a gap 210 with the aid of an automatic dispenser or by use of a screen printing, a roll coating, or a spin coating technique. When a physiological sample (or specimen) is applied to the test strip, the reagent 207 reacts with the sample to generate a charge. When a proper electric potential is applied to the two electrodes 203 and 205, a current flows through the electrodes 203 and 205, which corresponds to the concentration of an analyte of interest in the sample. The reagent 207 may include a biochemical substance (eg., enzymes, antibodies and proteins) capable of reacting with an analyte of interest and being detected by intermolecular recognition, a redox mediator capable of effectively transfering a charge generated by a biochemical reaction to the surface of an electrode, a hydrophilic polymer to be used as a support between the electron surface and the biochemical substance, and a surfactant to be used a dispersing agent. Available enzymes vary depending on substances to be detected. For example, when glucose is to be detected or analyzed, glucose oxidase may be used. The hydrophilic polymer is required to be easily able to immobilize the reagent on the electrodes, and is exemplified by cellulose and hydroxyethyl cellulose. The surfactant facilitates introduction of a sample to be analyzed into the sample introduction passage formed by the gap 210, and is exemplified by Triton X-100. A detailed preparation method of such reagents, and available reagents and redox mediators can be referred to U.S. Pat. No. 5,762,770, which is incorporated herein by reference. As describe above, the reagent 207 further includes a corpuscle interference correcting agent (not shown) . The corpuscle interference correcting agent may include a lysis agent to destroy erythrocytes in a physiological sample to release hemoglobin from the erythrocytes to the exterior, and a redox mediator to transfer a charge generated by a reaction with the released hemoglobin to the electrode 205. In the case that a redox mediator is already contained in the reagent, the corpuscle interference correcting agent may not include the redox mediator. The lysis agent is selected from the group consisting of saponin, sodium deoxycholate, EDTA, lysis buffers, detergents and combinations thereof. FIG. 3 shows the concept of a glucose sensor using an enzyme-redox mediator reaction according to the present invention. As shown in FIG. 3, first, glucose oxidase (GOD; 301) reacts with glucose to generate gluconic acid. The reduced glucose oxidase (GOD_recι) transfers electrons (e^") to a redox mediator and returns to the original glucose oxidase (GOD_OX) , and the redox mediator 303 transfers the electrons (e^~) to an electrode 305 to which a predetermined electric potential has been applied. By this mechanism, the enzyme-redox mediator complex shown in FIG. 3 allows a current corresponding to the concentration of glucose contained in a physiological sample to flow through the electrode when the physiological sample does not contain corpuscles including erythrocytes. However, when the physiological sample contains corpuscles including erythrocytes, the current flowing through the electrode is inversely proportional to the amount of erythrocytes due to the interference effect of the hematocrit as noted above. This relationship is illustrated at A in FIG. 5. In FIG. 5, the horizontal axis represents the hematocrit of the sample, and the vertical axis represents the size of a current flowing through an electrode of a test strip. FIG. 4 shows the concept of a hemoglobin sensor using a hemoglobin-redox mediator reaction according to the present invention. First, when red blood cells (RBCs) are lysed with a lysis agent, hemoglobin is released from RBCs to the exterior. A redox mediator 401 reacts with the released hemoglobin to receive electrons (e^~) and transfers the electrons (e^~) to the electrode 305. By this mechanism, the hemoglobin-redox mediator reaction allows a current to flow through the electrode in proportion to the amount of red blood cells in a physiological sample. This relationship is illustrated at B in FIG. 5. In addition to an enzyme and a redox mediator, the electrochemical biosensor reagent according to the present invention includes a lysis agent. Thus, a current practically flowing through an electrode, that is, output current, is a sum of a current (the A of FIG. 5) flowing through an electrode by a glucose sensor using an enzyme- redox mediator reaction and another current (the B of FIG. 5) flowing through an electrode by a hemoglobin sensor using a hemoglobin-redox mediator reaction. Thus, a current flowing through an electrode of a test strip, illustrated at C in FIG. 5, is constant regardless of the hematocrit. These results indicate that a reduction in the current A by the enzyme-redox mediator reaction, caused by an increase in red blood cells, is compensated for by the current B by the hemoglobin-redox mediator reaction. The reagent used in the embodiment of the present invention is prepared as follows. First, 0.75 g of polyvinyl alcohol and 1 g of Triton X-100 are dissolved in 100 ml of potassium phosphate buffer (100 itiM, pH 7.4). After mixing for one hour at room temperature, this solution is supplemented with 6.59 g of potassium ferricianide and 3 g of a redox enzyme, glucose oxidase, and mixed for one hour. To the resulting solution, erythrocyte lysis agents listed in Table 1, below are added, and the solution is mixed for 40 hrs at 8°C.

TABLE 1

Using the resulting enzyme solution, a glucose sensor is prepared. As shown in FIG. 2, gold (Au, 99.9%) electrodes 203 and 205 of 100 nm or less are formed on the substrate 201 by a sputtering process, and a PET film 209 is then attached to the substrate 201 using a laminator. The electrodes 203 and 205 are coated with the enzyme solution and dried for one hour. Another PET film 211 is attached to the substrate 201. The resulting product is sectioned into a size of 30 mm long x 7.5 mm wide to provide a glucose strip. Using a sensor prepared as described above, blood sugar levels are measured according to the changes in the hematocrit. First, whole blood is centrifuged to separate plasma and corpuscles. The separated plasma and corpuscles are mixed at proper ratios to generate blood samples with various hematocrit values of 20%, 30%, 40%, 50% and 60%. All samples are adjusted to have a blood sugar level of 180 mg/dl when blood sugar is measured using an YSI analyzer. The results are given in FIG. 6. FIG. 6a is a graph showing the relation of the hematocrit and the blood sugar. In FIG. 6a, the horizontal axis represents the hematocrit expressed as %, and the vertical axis represents the output current expressed as μA. In FIG. 6a, "control" is the case of using a conventional reagent not containing a lysis agent, and "agenti" are the cases of using a reagent prepared by adding a lysis agent to the conventional reagent according to the present invention. "Agentl", "agent2" and "agent3" indicate the cases of using different types and concentrations of the lysis agent. Blood sugar levels are converted to percentages based on the blood hematocrit value (44%) . The results are given in FIG. 6b. As shown in FIGS. 6a and 6b, the cases (agent i) of using the lysis agent have lower dependence on the hematocrit than the case (control) of not using the lysis agent. These results indicate that an electric signal from hemoglobin released from lysed erythrocytes reduces the dependence of conventional sensors on the hematocrit. Also, the lysis of erythrocytes reduces the absorption of erythrocytes onto the electrode surface and eliminates the increased viscosity of samples due to the hematocrit. Blood sugar is measured in blood taken from a vein of each of fifty diabetic patients with various concentrations of blood sugar and a wide range of hematocrit. The measured blood sugar is compared to YSI glucose measured using an YSI blood glucose analyzer to investigate its relationship with the YSI glucose. An error range of the measured YSI glucose is plotted against a measured hematocrit range. The results are given in FIGS. 7 and 8. FIG. 7 shows the results of using a conventional reagent, and FIG. 8 displays the results of using a reagent containing a lysis agent according to the present invention. Compared to FIG. 7, in FIG. 8, a large increase in the relationship was found, and the error rate was greatly reduced in a hematocrit range of 30-50% . The preferred embodiments of the present invention have been disclosed for illustrative purposes to facilitate those skilled in the art to understand and practice the present invention, but are not to be constructed to limit the present invention. Therefore, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the appended claims.

Industrial Applicability

As described hereinbefore, because of comprising a reagent layer prepared by mixing an enzyme, a redox mediator, a hydrophilic polymer, a surfactant and an erythrocyte lysis agent, the electrochemical biosensor test strip of the present invention can provide effective, accurate measurement for an analyte of interest in a manner insensitive to the hematocrit of a sample. Therefore, the biosensor test strip of the present invention can be easily applied to patients with different hematocrit levels, for example, adults, newborns and pregnant women. The present biosensor test strip is advantageous in terms of being capable of being manufactured at low cost as well as effectively compensating for the interference effect of the hematocrit. Also, the present biosensor test strip is advantageous in terms of being capable of being mass produced by a simple process as well as effectively compensating for the interference effect of the hematocrit .

Claims

1. An electrochemical biosensor test strip for quantitative analysis of an analyte of interest in a physiological sample containing blood corpuscles, comprising: a first insulating substrate; a plurality of electrodes formed on the first insulating substrate; and a reagent, which is immobilized on the plurality of electrodes formed on the first insulating substrate, to react with the analyte of interest in the physiological sample to generate a charge corresponding to concentration of the analyte, wherein the reagent includes a corpuscle interference correcting agent to react with the blood corpuscles in the physiological sample to generate the charge corresponding to the concentration of the analyte.

2. The electrochemical biosensor test strip according to claim 1, further comprising a second insulating substrate, which is placed on the first insulating substrate, to form a passage to introduce the physiological sample into the reagent .

3. The electrochemical biosensor test strip according to claim 1, wherein the corpuscle interference correcting agent comprises a lysis agent to destroy erythrocytes in the physiological sample to release hemoglobin from the erythrocytes to the exterior; and a redox mediator to transfer the charge generated by a reaction with the hemoglobin to the electrode.

4. The electrochemical biosensor test strip according to claim 3, wherein the lysis agent is selected from the group consisting of saponin, sodium deoxycholate, EDTA, lysis buffers, detergents and combinations thereof.

5. An electrochemical biosensor test strip for quantitative analysis of an analyte of interest in a physiological sample containing erythrocytes, comprising: a biochemical substance to specifically react with the analyte in the physiological sample to generate a charge corresponding to the concentration of the analyte; a lysis agent to destroy the erythrocytes in the physiological sample to release hemoglobin from the erythrocytes to the exterior; and a redox mediator to transfer the charge generated by a reaction of the biochemical substance with the hemoglobin to a predetermined electrode.

6. An electrochemical biosensor test strip for quantitative analysis of an analyte of interest in a physiological sample containing blood corpuscles, comprising: a first insulating substrate; a plurality of electrodes formed on the first insulating substrate; a reagent, which is immobilized on the plurality of electrodes formed on the first insulating substrate, to react with the analyte of interest in the physiological sample to generate a charge corresponding to concentration of the analyte; and a second insulating substrate, which is placed on the first insulating substrate, to form a passage to introduce the physiological sample into the reagent, wherein the reagent comprises: a biochemical substance to specifically react with the analyte in the physiological sample to generate a charge corresponding to the concentration of the analyte; a lysis agent to destroy erythrocytes in the physiological sample to release hemoglobin from the erythrocytes to the exterior; and a redox mediator to transfer the charge generated by a reaction of the biochemical substance with the hemoglobin to a predetermined electrode .