Abstract:
A new electrochemical sensor for determining hydrogen peroxide concentration having a mixed-valence metal oxide of M x O y  deposited on an electrode surface thereof is disclosed, wherein M is a transition metal and has two or more than two valences. M x O y , for example, is M 3 O 4  where M is Mn, Fe, Co or Pb, Tb 4 O 7  or Pr 6 O 11 . Further, this invention also discloses an electrochemical sensor for determining a concentration of a hydrogen peroxide precursor, wherein a catalyst is immobilized in the matrix or on the surface of the mixed-valence metal oxide on the electrode. In a typical biochemical system, the catalyst can be a glucose oxidase and blood glucose is catalyzed to form hydrogen peroxide, so that the concentration of blood glucose is determined.

Description:
FIELD OF THE INVENTION  
       [0001]     This invention is related to an electrochemical sensor for determining hydrogen peroxide concentration in a solution. Further, this invention is related to an electrochemical sensor for determining the concentration of a hydrogen peroxide precursor in a solution, which will form hydrogen peroxide under appropriate reaction conditions. In particular, this invention uses a mixed-valence metal oxide as a working electrode and a fixed potential ranging from +0.2 V to −0.3V between the working electrode and a reference electrode of 3 M KCl Ag/AgCl to catalyze the reduction of hydrogen peroxide, so that the concentration of hydrogen peroxide is measured.  
       BACKGROUND OF THE INVENTION  
       [0002]     In U.S. Pat. No. 6,042,714 one of the inventors of the present invention and his co-workers disclose a new chemical sensor to monitor H 2 O 2  concentration in a liquid. The H 2 O 2  chemical sensor includes a transducer which is able to conduct an electric current and a mixed-valence compound deposited on a surface of the transducer. The mixed-valence compound has a formula as follows: 
 
M y   Z+ [Fe(II)(CN) 6 ]
 
 where M can be Co, Ni, Cr, Sc, V, Cu, Mn, Ag, Eu, Cd, Zn, Ru, or Rh; z is the valence of M; and y=4/z. This prior art invention also reveals a chemical sensor to monitor a concentration of a H 2 O 2  precursor. The H 2 O 2  precursor is defined as a compound that can produce H 2 O 2  in said liquid under appropriate reaction conditions. The H 2 O 2  precursor chemical sensor contains the transducer, and a composition deposited on a surface of the transducer. The composition comprises the mixed-valence compound and a catalyst capable of catalyzing the reaction. This prior art invention uses a fixed potential ranging from +0.1 V to −0.2V between the working electrode and a reference electrode of 3 M KCl Ag/AgCl to catalyze the reduction of hydrogen peroxide, so that the concentration of hydrogen peroxide is measured. As a result, the transducer modified by the mixed-valence compound is able to monitor the H 2 O 2  concentration at a potential which will not be interfered by other undesirable biochemical compounds in blood (such as ascorbic acid, uric acid, dopamine, cysteine and acetaminophen, etc.). Furthermore, by adding proper electrolyte and pH buffer the interference from oxygen (a strong reducible compound) is also prevented. The disclosure of U.S. Pat. No. 6,042,714 is incorporated herein by reference. 
 
       SUMMARY OF THE INVENTION  
       [0003]     The invention of the present application uses a mixed-valence metal oxide to develop chemical sensors for measuring concentrations of H 2 O 2  and H 2 O 2  precursor in environmental detection and medical tests.  
         [0004]     A chemical sensor constructed according to the present invention comprises a transducer which is able to conduct an electric current and a mixed-valence metal oxide deposited on a surface of the transducer, wherein said mixed-valence metal oxide has a formula as follows: 
 
M x O y   (I) 
 
 wherein M is a transition metal and has two or more than two different valences; x and y represent moles of said transition metal, M, and oxygen, respectively, provided that 2y=(x 1 )(z 1 )+(x 2 )(z 2 ) . . . +(x n )(z n ), and x 1 +x 2 + . . . +x n =x, wherein z 1 , z 2 , . . . z n  represent the valences of M; x 1 , x 2 , . . . x n  represent moles of M having valences of z 1 , z 2 , . . . z n , respectively, and n is a positive integer. 
 
         [0005]     Preferably, M is selected from the group consisting of Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Ga, Nb, Mo, Tc, Ru, Rh, Pd, Ag, In, Sn, W, Re, Ir, Pt, Au, Tl, Pb, Pr and Tb. For examples, said mixed-valence metal oxides are Mn 3 O 4 , Fe 3 O 4 , CO 3 O 4  or Pb 3 O 4 , where M is Mn, Fe, Co or Pb, and has valences of +2 and +3; Tb 4 O 7  and Pr 6 O 11 , where M is Tb or Pr, and has valences of +3 and +4.  
         [0006]     The mixed-valence metal oxide (I) per se is very stable chemically, and not easy to be affected by humidity, temperature, and light irradiation, etc. Further, the mixed-valence metal oxide has advantages such as a good electron transfer capability, lower price, and easy availability, which make the chemical sensors of the present invention more feasible to be commercialized. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0007]      FIG. 1  shows a calibration curve of a working electrode upon successive injections of H 2 O 2  solution to provide an increment in H 2 O 2  concentration of 0.1 mM for each addition in an amperometry analysis, where the x-axis is the concentration of H 2 O 2  (mM), and the y-axis is current (μA). The working electrode is a chemical sensor containing Mn 3 O 4  prepared according to Example 1 of the present invention.  
         [0008]      FIG. 2  shows a calibration curve of a working electrode upon successive injections of H 2 O 2  solution to provide an increment in H 2 O 2  concentration of 0.1 mM for each addition in an amperometry analysis, where the x-axis is the concentration of H 2 O 2  (mM), and the y-axis is current (μA). The working electrode is a chemical sensor containing Fe 3 O 4  prepared according to Example 2 of the present invention.  
         [0009]      FIG. 3  shows a calibration curve of a working electrode upon successive injections of H 2 O 2  solution to provide an increment in H 2 O 2  concentration of 0.1 mM for each addition in an amperometry analysis, where the x-axis is the concentration of H 2 O 2  (mM), and the y-axis is current (μA). The working electrode is a chemical sensor containing CO 3 O 4  prepared according to Example 3 of the present invention.  
         [0010]      FIG. 4   a  is a plot of current (μA) versus concentration of H 2 O 2  (mM) upon successive injections of H 2 O 2  solution to provide an increment in H 2 O 2  concentration for each measurement in a chronoamperometry analysis, where the x-axis is the concentration of H 2 O 2  (mM), and the y-axis is current (μA). The working electrode is a chemical sensor containing Pb 3 O 4  prepared according to Example 4 of the present invention.  
         [0011]      FIG. 4   b  shows the partial calibration curve of the working electrode within a H 2 O 2  concentration range of 0.1 mM to 9 mM in  FIG. 4   a.    
         [0012]      FIG. 5  shows a calibration curve of a working electrode upon successive injections of glucose solution to provide an increment in glucose concentration of 1 mM for each addition in an amperometry analysis, where the x-axis is the concentration of glucose (mM), and the y-axis is current (μA). The working electrode is a glucose biochemical sensor containing Fe 3 O 4  prepared according to Example 5 of the present invention. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0013]     Novel chemical sensors designed to measure concentrations of H 2 O 2  and H 2 O 2  precursors are provided in the present invention. The novel chemical sensors comprise the mixed-valence metal oxide (I) deposited on a surface of a transducer, for example, an electrochemical electrode. The mixed-valence metal oxide provides the chemical sensors with electrode assisted catalysis in an amperometric measurement of H 2 O 2  concentration in a given solution, wherein the chemical sensor is used as a working electrode.  
         [0014]     The mixed-valence metal oxide (I) deposited on the transducer having a catalysis characteristic of reducing H 2 O 2  is a metal oxide formed by two metallic nuclei connected by oxygen atom acting as a coordinating ligand. The two metallic nuclei have their own valences, and electrons within the tow metallic nuclei are in the delocalization state. As a result, the mixed-valence metal oxide can be used as an electron transferring route through the inter-valence charge transfer characteristic of the coordinating ligand. In the amperometric measurement of H 2 O 2 , the mixed-valence metal oxide can accomplish charge transfer and catalysis of H 2 O 2 , and even enhance the conductivity of the chemical sensors.  
         [0015]     When an electrode modified with the mixed-valence metal oxide (I) is used as a working electrode in an amperometric measurement of H 2 O 2 , the mixed-valence metal oxide (I) is oxidized from a reduction state to an oxidation state by H 2 O 2 , and creates an electronic hole therein. The electronic hole is then transferred to the transducer via the inter-valence charge transfer characteristic of the mixed-valence metal oxide (I), so that a current loop is formed, and a signal in response to H 2 O 2  concentration is obtained with a smaller reduction potential being applied. It is apparent that an electrode modified with this mixed-valence metal oxide (I) is also feasible for use in amperometric measurement of oxygen concentration in a given solution. But, in the measurement of hydrogen proxide, the lower applied potential can avoid the reduction of oxygen and the oxidation of easily oxidizable compounds simultaneously.  
         [0016]     The H 2 O 2  chemical sensor of the present invention has a fast response time (t 90% ), a broad linear range of concentration vs. current, and a high sensitivity, when it is used as a working electrode in an amperometric measurement of H 2 O 2  concentration in a given solution and when the reduction potential of the chemical sensor is at 0.2 to −0.3V (vs. 3 M KCl Ag/AgCl reference electrode).  
         [0017]     When a catalyst is immobilized in the matrix or on a surface of the mixed-valence metal oxide (I) deposited on the transducer, and the catalyst can catalyze a compound in a given solution to produce H 2 O 2 , it is apparent that this modified transducer is able to be used to determine the compound concentration. The catalyst is called “identifier” and the compound is call “a H 2 O 2  precursor” in this invention.  
         [0018]     By blending the mixed-valence metal oxide (I) with various oxidases (such as glucose oxidase (EC 1.1.3.4), uricase (EC 1.7.3.3), cholesterol oxidase (EC 1.1.3.6), glycerophosphate oxidase (EC 1.1.3.21), sarcosine oxidase (EC 1.5.3.1), polyamine oxidase (EC 1.4.3.10)) a series of new biochemical sensors for measuring blood glucose, urea, high and low density cholesterols, triglyceride, creatinine and polyamine in blood can be prepared and applied to medical, biomedical research, including diagnostic applications. The new biochemical sensors derived from this invention have excellent specificity originated from the specificity of the oxidases. In addition, the transducer modified by the mixed-valence metal oxide is able to monitor the H 2 O 2  concentration at a potential which will not be interfered by other undesirable biochemical compounds in blood (such as ascorbic acid, uric acid, dopamine, cystein and acetaminophen, etc.) and dissolved oxygen.  
         [0019]     The mixed-valence metal oxide (I) in this invention has low solubility in water and high chemical stability, and thus it can be applied to interfacial chemistry of electrochemical analysis. The preparation of the H 2 O 2  chemical sensor of the present invention is simple. For example, the mixed-valence metal oxide can mixed with an electrical conductive ink, and depositing the resulting mixture on a surface of an electrode by coating, chemical modification, sputtering or chemical vapor deposition to form a thick film electrode, which is ready for use when the ink is dry.  
         [0020]     When the identifier is to be incorporated to the transducer together with the mixed-valence metal oxide (I) for measuring the concentration of the H 2 O 2  precursor, several approaches can be taken such as polymer coating and trapping to cover the identifier under a polymer membrane, or surface adsorption and covalent bond crosslinking, thereby the identifier is immobilized to avoid the undesired variations of signals due to losing or distribution change of the identifier caused by agitation in the course of the measurement.  
         [0021]     The invention will be further illustrated by the following examples. The following examples are only meant to illustrate the invention, but not to limit it.  
       EXAMPLE 1  
     Chemical Sensor Based on Mn 3 O 4    
       [0000]     (1). Pretreatment of Electrode  
         [0022]     A rotating disk glassy carbon electrode (RDE 0032, Princeton Applied Research, 6 mm outer diameter) was polished using 3 μm and 1 μm diamond suspension, and sonicated for five minutes in deionized water. The electrode surface was then polished with 0.1 μm Al 2 O 3  powder, sonicated for 5 minutes in a deionized water twice followed by rinsing with deionized water twice. Subsequently, the electrode surface was checked by a cyclic voltammetry (BAS 100W, Bioanalytical Systems) to ensure free of contamination.  
         [0000]     (2). Preparation of a Working Electrode  
         [0023]     A mixture having 15% of Mn 3 O 4  was prepared by well mixing Mn 3 O 4  and electrically conductive ink, which was then diluted with cyclohexanone to obtain a viscosity suitable for coating (the amount of cyclohexanone added was about one half of the mixture). The pretreated rotating disk glassy carbon electrode was coated with the resulting Mn 3 O 4  mixture, and dried at 40° C. for 30 minutes.  
         [0000]     (3). Measurement Conditions  
         [0024]     The working electrode prepared above, a homemade 3M KCl Ag/AgCl reference electrode, and a platinum wire counter electrode were immersed in a 0.05 M, pH=10, glycine buffer, with 0.1 M NaCl solution to improve conductivity in an electrochemical cell. A bi-potentiostat (model PAR, 366A, Princeton Applied Research) was used to control the applied voltage at −50 mV (vs. Ag/AgCl). The detection temperature of the electrochemical cell was kept at 25° C. with a circulator (Model B402, Firstek Scientific). The glycine buffer in the cell was stirred constantly at 900 rpm with a motor controlled rotor (Model 636, Princeton Applied Research).  
         [0025]     H 2 O 2  solution was added to the glycine buffer in the cell at a constant time interval to provide an increment in H 2 O 2  concentration of 0.1 mM so that steady-state amperometric measurements of hydrogen peroxide concentration were conducted.  
         [0000]     (4). Results  
         [0026]     After the successive injections of H 2 O 2  solution to the cell, the electric current response versus time is used to establish a correlation curve of the chemical sensor prepared.  
         [0027]     At H 2 O 2  concentration of 0.1 mM, the response time that between 10% and 90% of the maximum signal (t 90% ) was 10.2 seconds (not shown in the drawing). By plotting H 2 O 2  concentration vs. current (μA), it was found that there was a linear relationship within a range from 0.1 mM to 3 mM (correlation coefficient=0.999). A slope of 2.737 μA/mM-mm 2  was obtained using the least square method, as shown in  FIG. 1 .  
         [0028]     The measurement was repeated for 20 times using 0.1 mM H 2 O 2 , and a relative standard deviation of 3.5% was observed. Based on the signal-to-noise characteristics (S/N=3), it was found that the detection limit of H 2 O 2  was 50 μM.  
         [0029]     Further interference experiments indicated there was no substantial interference when measuring 0.1 mM H 2 O 2  solution in the presence of 0.2 mM of ascorbic acid, uric acid, dopamine, cysteine or acetaminophen.  
       EXAMPLE 2  
     Chemical Sensor Based on Fe 3 O 4    
       [0000]     (1). Pretreatment of Electrode  
         [0030]     A rotating disk graphite electrode was polished using 0.1 μm Al 2 O 3  suspension, and sonicated for three minutes in deionized water. The procedures were repeated once. The electrode surface was then rinsed with deionized water twice. Finally, the electrode surface was checked by a cyclic voltammetry (BAS 100W, Bioanalytical Systems) to ensure free of contamination.  
         [0000]     (2). Preparation of a Working Electrode  
         [0031]     A mixture having 50% of Fe 3 O 4  was prepared by well mixing Fe 3 O 4  and electrically conductive ink, which was then diluted with cyclohexanone to obtain a viscosity suitable for coating (the amount of cyclohexanone added was four times of the mixture). The pretreated rotating disk graphite electrode was coated with the resulting Fe 3 O 4  mixture, and dried at room temperature (25° C.) for 30 minutes.  
         [0000]     (3). Measurement Conditions  
         [0032]     The working electrode prepared above, a homemade 3M KCl Ag/AgCl reference electrode, and a platinum wire counter electrode were immersed in a 0.05 M, pH=3, citrate buffer, with 0.1 M KCl solution to improve conductivity in an electrochemical cell. A bi-potentiostat (model PAR, 366A, Princeton Applied Research) was used to control the applied voltage at −200 mV (vs. Ag/AgCl). The detection temperature of the electrochemical cell was kept at 25° C. with a circulator (Model B402, Firstek Scientific). The citrate buffer in the cell was stirred constantly at 900 rpm with a motor controlled rotor (Model 636, Princeton Applied Research).  
         [0033]     H 2 O 2  solution was added to the citrate buffer in the cell at a constant time interval to provide an increment in H 2 O 2  concentration of 0.1 mM so that steady-state amperometric measurements of hydrogen peroxide concentration were conducted.  
         [0000]     (4). Results  
         [0034]     After the successive injections of H 2 O 2  solution to the cell, the electric current response versus time is used to establish a correlation curve of the chemical sensor prepared.  
         [0035]     At H 2 O 2  concentration of 0.1 mM, the response time that between 10% and 90% of the maximum signal (t 90% ) was 5.2 seconds (not shown in the drawing). By plotting H 2 O 2  concentration vs. current (μA), it was found that there was a linear relationship within a range from 0.05 mM to 1.5 mM (correlation coefficient=0.9993). A slope of 0.89 μA/mM-mm 2  was obtained using the least square method, as shown in  FIG. 2 .  
         [0036]     The measurement was repeated for 20 times using 0.1 mM H 2 O 2 , and a relative standard deviation of 2.18% was observed. Based on the signal-to-noise characteristics (S/N=3), it was found that the detection limit of H 2 O 2  was 81 μM.  
         [0037]     Further interference experiments indicated there was no substantial interference when measuring 0.1 mM H 2 O 2  solution in the presence of 0.2 mM of ascorbic acid, uric acid, dopamine, cysteine or acetaminophen.  
       EXAMPLE 3  
     Chemical Sensor Based on CO 3 O 4    
       [0000]     (1). Pretreatment of Electrode  
         [0038]     A rotating disk graphite electrode was polished using 0.1 μm Al 2 O 3  suspension, and sonicated for three minutes in deionized water. The procedures were repeated once. The electrode surface was then rinsed with deionized water twice. Finally, the electrode surface was checked by a cyclic voltammetry (BAS 100W, Bioanalytical Systems) to ensure free of contamination.  
         [0000]     (2). Preparation of a Working Electrode  
         [0039]     A mixture having 10% of CO 3 O 4  was prepared by well mixing CO 3 O 4  and electrically conductive ink, which was then diluted with cyclohexanone to obtain a viscosity suitable for coating (the amount of cyclohexanone added was the same as the mixture). The pretreated rotating disk graphite electrode was coated with the resulting CO 3 O 4  mixture, and dried at room temperature (25° C.) for 30 minutes.  
         [0000]     (3). Measurement Conditions  
         [0040]     The working electrode prepared above, a homemade 3M KCl Ag/AgCl reference electrode, and a platinum wire counter electrode were immersed in a 0.05 M, pH=9, tris-(hydroxymethyl)aminomethane buffer, with 0.1 M NaCl solution to improve conductivity in an electrochemical cell. A bi-potentiostat (model PAR, 366A, Princeton Applied Research) was used to control the applied voltage at −150 mV (vs. Ag/AgCl). The detection temperature of the electrochemical cell was kept at 25° C. with a circulator (Model B402, Firstek Scientific). The tris-(hydroxymethyl)aminomethane buffer in the cell was stirred constantly at 625 rpm with a motor controlled rotor (Model 636, Princeton Applied Research).  
         [0041]     H 2 O 2  solution was added to the tris-(hydroxymethyl)aminomethane buffer in the cell at a constant time interval to provide an increment in H 2 O 2  concentration of 0.1 mM so that steady-state amperometric measurements of hydrogen peroxide concentration were conducted.  
         [0000]     (4). Results  
         [0042]     After the successive injections of H 2 O 2  solution to the cell, the electric current response versus time is used to establish a correlation curve of the chemical sensor prepared.  
         [0043]     At H 2 O 2  concentration of 0.1 mM, the response time that between 10% and 90% of the maximum signal (t 90% ) was 12.3 seconds (not shown in the drawing). By plotting H 2 O 2  concentration vs. current (μA), it was found that there was a linear relationship within a range from 0.1 mM to 14 mM (correlation coefficient=0.999). A slope of 0488 μA/mM-mm 2  was obtained using the least square method, as shown in  FIG. 3 .  
         [0044]     The measurement was repeated for 20 times using 0.1 mM H 2 O 2 , and a relative standard deviation of 3.5% was observed. Based on the signal-to-noise characteristics (S/N=3), it was found that the detection limit of H 2 O 2  was 3.3 μM.  
         [0045]     Further interference experiments indicated there was no substantial interference when measuring 0.1 mM H 2 O 2  solution in the presence of 0.2 mM of ascorbic acid, uric acid, dopamine, cysteine or acetaminophen.  
       EXAMPLE 4  
     Chemical Sensor Based on Pb 3 O 4    
       [0000]     (1). Pretreatment of Electrode  
         [0046]     A rotating disk glassy carbon electrode (RDE 0032, Princeton Applied Research, 6 mm outer diameter) was polished using 3 μm and 1 μm diamond suspension, and sonicated for five minutes in deionized water. The electrode surface was then polished with 0.1 μm Al 2 O 3  powder, sonicated for 5 minutes in a deionized water twice followed by rinsing with deionized water twice. Subsequently, the electrode surface was checked by a cyclic voltammetry (BAS 100W, Bioanalytical Systems) to ensure free of contamination.  
         [0000]     (2). Preparation of a Working Electrode  
         [0047]     A mixture having 50% of Pb 3 O 4  was prepared by well mixing Pb 3 O 4  and electrically conductive ink, which was then diluted with cyclohexanone to obtain a viscosity suitable for coating (the amount of cyclohexanone added was about the same as the mixture). The pretreated rotating disk glassy carbon electrode was coated with the resulting Pb 3 O 4  mixture, and dried at 40° C. for 30 minutes.  
         [0000]     (3). Measurement Conditions  
         [0048]     The working electrode prepared above, a homemade 3M KCl Ag/AgCl reference electrode, and a platinum wire counter electrode were immersed in a 0.2 M, pH=6, acetate buffer in an electrochemical cell. Chronoamperometry was conducted to measure H 2 O 2  concentrations, wherein a working voltage at −200 mV (vs. Ag/AgCl) jumped from an initial voltage of 500 mV was used, and the sampling time was three seconds. The detection temperature of the electrochemical cell was kept at 25° C. with a circulator (Model B402, Firstek Scientific). The acetate buffer in the cell was stirred homogenously.  
         [0049]     H 2 O 2  solution was added to the acetate buffer in the cell at a constant time interval to provide an increment in H 2 O 2  concentration of 0.1 mM so that stable amperometric measurements of hydrogen peroxide concentration were conducted.  
         [0000]     (4). Results  
         [0050]     After the successive injections of H 2 O 2  solution to the cell, the electric current response versus H 2 O 2  concentration was plotted. Two linear sectors were observed, which ranges from 0.1 mM to 9 mM and form 9 mM to 46 mM with correlation coefficients of 0.9995 and 0.9997, respectively, as shown  FIG. 4   a . A slope of 0.11 μA/mM-mm 2  was obtained in the sector of 0.1 mM to 9 mM using the least square method, as shown in  FIG. 4   b.    
         [0051]     The measurement was repeated for 20 times using 0.1 mM H 2 O 2 , and a relative standard deviation of 4.657% was observed. Based on the signal-to-noise characteristics (S/N=3), it was found that the detection limit of H 2 O 2  was 20 nM.  
         [0052]     Further interference experiments indicated there was no substantial interference when measuring 0.1 mM H 2 O 2  solution in the presence of 0.2 mM of ascorbic acid, uric acid, dopamine, cysteine or acetaminophen.  
       EXAMPLE 5  
     Glucose Biochemical Sensor Based on Fe 3 O 4    
       [0000]     (1). Pretreatment of Electrode  
         [0053]     A rotating disk graphite electrode was polished using 0.1 μm Al 2 O 3  suspension, and sonicated for three minutes in deionized water. The procedures were repeated once. The electrode surface was then rinsed with deionized water twice. Finally, the electrode surface was checked by a cyclic voltammetry (BAS 100W, Bioanalytical Systems) to ensure free of contamination.  
         [0000]     (2). Preparation of a Working Electrode  
         [0054]     A mixture having 50% of Fe 3 O 4  was prepared by well mixing Fe 3 O 4  and electrically conductive ink, which was then diluted with cyclohexanone to obtain a viscosity suitable for coating (the amount of cyclohexanone added was four times of the mixture). The pretreated rotating disk graphite electrode was coated with the resulting Fe 3 O 4  mixture, and dried at room temperature (25° C.) for 30 minutes. An aqueous solution containing glucose oxidase and cross-linking reagent was added to the surface of the resulting electrode drop by drop until reached 5 units of addition of glucose oxidase, dried at room temperature, and an ethanol solution of Nafion® was dropped on the dried surface of the electrode and dried to immobilize the glucose oxidase.  
         [0000]     (3). Measurement Conditions  
         [0055]     The working electrode prepared above, a homemade 3M KCl Ag/AgCl reference electrode, and a platinum wire counter electrode were immersed in a 0.05 M, pH=7, phosphate buffer, with 0.1 M NaCl solution to improve conductivity in an electrochemical cell. A bi-potentiostat (model PAR, 366A, Princeton Applied Research) was used to control the applied voltage at −200 mV (vs. Ag/AgCl). The detection temperature of the electrochemical cell was kept at 25° C. with a circulator (Model B402, Firstek Scientific). The phosphate buffer in the cell was stirred constantly at 900 rpm with a motor controlled rotor (Model 636, Princeton Applied Research).  
         [0056]     Glucose solution was added to the phosphate buffer in the cell at a constant time interval to provide an increment in glucose concentration of 1 mM so that steady-state amperometric measurements of glucose concentration were conducted.  
         [0000]     (4). Results  
         [0057]     After the successive injections of glucose solution to the cell, the electric current response versus time is used to establish a correlation curve of the biochemical sensor prepared.  
         [0058]     At glucose concentration of 1 mM, the response time that between 10% and 90% of the maximum signal (t 90% ) was 8.4 seconds (not shown in the drawing). By plotting glucose concentration vs. current (μA), it was found that there was a linear relationship within a range from 1 mM to 8 mM (correlation coefficient=0.999). A slope of 0.89 μA/mM-mm 2  was obtained using the least square method, as shown in  FIG. 5 .  
         [0059]     The measurement was repeated for 20 times using 1 mM glucose, and a relative standard deviation of 2.18% was observed. Based on the signal-to-noise characteristics (S/N=3), it was found that the detection limit of glucose was 81 μM.  
         [0060]     Further interference experiments indicated there was no substantial interference when measuring 1 mM glucose solution in the presence of 0.2 mM of ascorbic acid, uric acid, dopamine, cysteine or acetaminophen.  
         [0061]     In the Examples, the working electrodes (6 mm diameter) are relatively small in size in comparison with the volume of the buffer solutions in the cell, so that a steady-state amperometric measurement is possible only when the buffer solutions are in a homogenous phase under sufficient stirring. However, a minute working electrode can be prepared by screen printing a small amount of the mixed-valence metal oxide/electrical conductive ink mixture on an insulation plate. In this case, a small amount of solution which is able to cover the minute working electrode, the reference electrode and the counter electrode, and an instant current detected are sufficient to determine the H 2 O 2  concentration or the H 2 O 2  precursor concentration. When the volume of the solution is very small such that no substantial potential drop caused by the solution, the reference electrode can be omitted. Therefore, this invention also discloses a technique of preparing a minute chemical sensor and determining H 2 O 2  concentration including the following steps: 
        a) forming a working electrode, a reference electrode and a counter electrode on an insulation plate by screen printing, wherein the working electrode contains the mixed-valence metal oxide, and electrical conductive ink, the reference electrode contains a Ag/AgCl ink, and the counter electrode contains an electrical conductive ink;     b) contacting an unknown solution with the working electrode, the reference electrode and the counter electrode on the plate at the same time;     c) conducting a chronoamperometric measurement so that an instant current is detected from the H 2 O 2  chemical sensor; and     d) determining H 2 O 2  concentration of the unknown solution by comparing a magnitude of the instant current obtained from step c) with a H 2 O 2  calibration curve previously established from known H 2 O 2  concentration solutions by the chronoamperometric measurement under same conditions.        
 
         [0066]     This invention further discloses a new technique of preparing a minute biochemical sensor and of determining H 2 O 2  precursor concentration including the following steps: 
        a) forming a working electrode, a reference electrode, a counter electrode and an identifier on an insulation plate by screen printing, wherein the working electrode contains the mixed-valence metal oxide and electrical conductive ink, the reference electrode contains a Ag/AgCl ink, the counter electrode contains an electrical conductive ink, and the identifier contains an enzyme, usually an oxidase;     b) contacting an unknown solution with the working electrode, the reference electrode, the counter electrode and the identified on the plate at the same time, wherein H 2 O 2  will be generated from the reaction of the H 2 O 2  precursor and the identified;     c) conducting a chronoamperometric measurement so that an instant current is detected from the biochemical sensor; and     d) determining H 2 O 2  precursor concentration of the unknown solution by comparing a magnitude of the instant current obtained from step c) with a H 2 O 2  precursor calibration curve previously established from solutions of known H 2 O 2  precursor concentrations by the chronoamperometric measurement under same conditions.        
 
         [0071]     Although particular embodiments of the invention have been described, various alternations, modifications, and improvements will readily occur to those skilled in the art. Accordingly, the forgoing description is by way of example only and is not intended as limiting. This invention is limited only as defined in the following claims and the equivalents thereto.