Apparatus for amperometric diagnostic anagnostic analysis

Abstract

The present invention relates to a novel method and apparatus for the amperometric determination of an analyte, and in particular, to an apparatus for amperometric analysis utilizing a novel disposable electroanalytical cell for the quantitative determination of biologically important compounds from body fluids.

We claim:  
     
         1 . A sample cell for determining the concentration of a selected compound in a sample aqueous fluid, comprising 
 a first electrode which acts as a working electrode.    a second electrode of substantially the same size as said first electrode and being made of the same electrically conducting material as said first electrode, said second electrode being operatively associated with said first electrode, and    at least one non-conducting layer member having an opening therethrough, said layer member being disposed in contact with at least one of said electrodes and said layer member being sealed against at least one of said first and second electrodes to form a known electrode area within said opening such that said opening forms a well to receive said sample aqueous fluid and to place said fluid in said known electrode area in contact with said first electrode and second electrode, whereby substantially the entire contents of said well is capable of being substantially simultaneously subjected to a predetermined reaction.    
     
     
         2 . The sample cell of  claim 1  wherein said first and second electrodes comprise palladium.  
     
     
         3 . The sample cell of  claim 1  wherein said second electrode is a reference electrode.  
     
     
         4 . An apparatus for measuring compounds in a sample fluid, comprising 
 a) a housing having an access opening therethrough.    b) a sample cell receivable into said access opening of said housing, said sample cell being composed of 
 a first electrode which acts as a working electrode,  
 a second electrode which acts to fix the system potential and provide opposing current flow with respect to said first electrode, said second electrode being of substantially the same size as said first electrode and being made of the same electrically conducting material as said first electrode, said second electrode being operatively associated with said first electrode,  
 at lease one non-conducting layer member having an opening therethrough, said layer member being and said layer member being sealed against at least one of said first and second electrode to form a known electrode area within said opening such that said opening forms a well to receive said sample fluid and to place said fluid in said known electrode area in contact with said first electrode and said second electrode,  
 (c) means for applying an electrical potential to said first electrode and said second electrode,  
 (d) means for creating an electrical circuit between said first electrode and said second electrode through said sample,  
 (e) means for measuring Cottrell current through said sample and  
 (f) means for visually displaying results of said measurement.  
   
     
     
         5 . The apparatus of  claim 4  further including means for obtaining a plurality of readings of current in said sample over a plurality of measurement times, after said sample fluid has ben placed in said well.  
     
     
         6 . The cell of  claim 1 , said cell also including a reagent layer positioned within said well created by said opening.  
     
     
         7 . The cell of  claim 6  wherein said reagent layer contains an oxidant, a buffer and a binding agent.  
     
     
         8 . The cell of  claim 7  wherein said reagent layer is a layer of said oxidant, buffer and binding agent coated onto a porous matrix and said matrix is positioned within said cell.  
     
     
         9 . The cell of  claim 8  wherein said reagent layer is a mixture of said oxidant, buffer and binding agent deposited directly into said cell.  
     
     
         10 . The cell of  claim 7  wherein said oxidant is selected from the group consisting of benzoquinone, ferricyanide, ferricinium, Cobalt (III) orthophenanthroline, and Cobalt (III) dipyridyl.  
     
     
         11 . The cell of  claim 7  wherein said reagent layer also includes an enzyme and said enzyme is an oxidoreductase.  
     
     
         12 . The cell of  claim 1  wherein said first electrode and said second electrode comprises a nonconducting substrate to which said electrically conducting material has been applied, wherein said electrically conducting material is the same for each electrode and selected from the group consisting of platinum, gold, palladium, silver and carbon.  
     
     
         13 . The cell of  claim 1  wherein a first non-conducting layer member has an opening therethrough and is positioned on said first electrode, and said first electrode is positioned on said second electrode, and said second electrode being positioned on a second non-conducting layer member.  
     
     
         14 . The cell of  claim 1  wherein said first layer member includes a plurality of notches therein exposing and defining an electrical contact area on said first electrode, and said first electrode has a notch therein to expose and define an electrical contact area on said second electrode.  
     
     
         15 . The cell of  claim 1  wherein said first and second electrodes are co-planarly positioned on a single substrate.  
     
     
         16 . The cell of  claim 1  wherein said second electrode has circular opening therein and said opening of said layer member is concentric with said opening of said layer member is concentric with said openings of said second electrode, and said opening in said second electrode is of smaller diameter than said opening of said layer member whereby a circular functional electrode area is defined on said second electrode, and said first electrode is positioned beneath said second electrode such that said opening in said second electrode exposes and defines a functional electrode area on said first electrode.  
     
     
         17 . The apparatus of  claim 13  also comprising means for initiating an electrical potential upon insertion of said sample aqueous fluid to detect the presence of said sample aqueous fluid, and said initiating means also having means for signaling microprocessor means to commence a reaction timing sequence when the presence of said sample aqueous fluid is detected, and means for removing said potential during said reaction timing sequence.  
     
     
         18 . A method of measuring the amount of a selected compound in body fluids comprising: 
 a) providing a measuring cell having at least a first and second electrode of substantially the same size and made of the same electrically conductive material, said cell further containing an oxidant and a buffer,    b) placing a sample of fluid to be tested in said cell,    c) reconstituting said oxidant and buffer with said sample fluid to generate a predetermined reaction,    d) allowing said reaction to proceed substantially to completion,    e) applying a potential across said electrodes and sample, and    f) measuring the resulting Cottrell current to determine the concentration of said selected compound present in said sample.    
     
     
         19 . The method as set forth in  claim 18  including providing as said first electrode a working electrode and as said second electrode a reference electrode.  
     
     
         20 . The method of  claim 18  including also providing in said cell and enzyme as a catalyst and said enzyme is an oxidoreductase.  
     
     
         21 . A method for measuring the amount of glucose in blood, comprising 
 a) providing a measuring cell having at least a f irst and second electrode of substantially the same size and made of the same electrically conductive material, said cell further containing an oxidant, a buffer and an enzyme,    b) placing a blood sample to be tested in said cell,    c) reconstituting said oxidant, buffer and enzyme with said blood sample to generate a predetermined reaction,    d) essentially immediately applying a potential across said electrodes and blood sample, and    e) measuring the resultant Cottrell current when the reaction has proceeded to completion to determine the concentration of said glucose present in said blood sample.    
     
     
         22 . The method of  claim 21  including selecting said oxidant from the group consisting of benzoquinone, ferricyanide, ferricinium, Cobalt (III) orthophenanthroline, and Cobalt (III) dipyridyl.  
     
     
         23 . The method of  claim 21  including selecting said oxidant from the group consisting of benzoquinone, ferricyanide, ferricinium, Cobalt (III) orthophenanthroline, and Cobalt (III) dipyridyl.  
     
     
         24 . The method of  claim 21  including providing as said first electrode a working electrode and said second electrode a reference electrode.  
     
     
         25 . The method of  claim 21  including adding as said enzyme, glucose oxydase.  
     
     
         26 . The method of  claim 1  wherein in step b) said placing of the blood sample to be tested in the cell generates a current and initiates a timing sequence, and wherein the reaction of step d) is allowed to proceed with an open circuit between said first and second electrode.  
     
     
         27 . The method of  claim 18  wherein the measuring of said Cottrell current includes obtaining a plurality of readings of current in said cell over a plurality of measurement times after said potential has been applied across said electrodes.  
     
     
         28 . A method of measuring the amount of an analyte in a fluid sample, comprising: 
 a. adding the fluid sample to an electrochemical cell having at least a first and second electrode of substantially the same size and comprising the same electrically conductive material, said electrochemical cell including an electron transfer agent that will react in a reaction involving the analyte, thereby forming a detectable species;    b. incubating the reaction involving analyte and electron transfer agent in an open circuit for a specified period of time;    c. applying a sufficient potential difference between the electrodes of the electrochemical cell, after the incubation step, to readily transfer at least one electron between the detectable species and one of the electrodes, thereby resulting in a Cottrell current;    d. measuring the Cottrell current; and    e. correlating the measured Cottrell current to the amount of analyte in the fluid sample.    
     
     
         29 . The method of  claim 28 , wherein adding the fluid sample to the electrochemical cell causes a sudden charging current, which automatically initiates incubation step b) performed under open circuit.  
     
     
         30 . The method of  claim 29 , wherein the Cottrell current is measured at a preset time following the incubation step.  
     
     
         31 . The method of  claim 28 , wherein the electrochemical cell further includes a catalyst in sufficient amount to catalyze the reaction involving the analyte and the electron transfer agent.  
     
     
         32 . The method of  claim 31 , wherein the catalyst is an enzyme.  
     
     
         33 . The method of  claim 32 , wherein the analyte is glucose and the enzyme is glucose oxidase.  
     
     
         34 . The method of  claim 28 , wherein the electron transfer agent is included in a reagent layer that is coated directly onto the electrochemical cell or is incorporated into a supporting matrix that is placed into the electrochemical cell.  
     
     
         35 . The method of  claim 34 , wherein the supporting matrix is filter paper, membrane, filter, woven fabric, or nonwoven fabric.  
     
     
         36 . The method of  claim 34 , wherein the reagent layer further includes a binder.  
     
     
         37 . The method of  claim 36 , wherein the binder is gelatin, carrageenan, methylcellulose, polyvinyl alcohol, or polyvinylpyrrolidone.  
     
     
         38 . The method of  claim 37 , wherein a dispersing, spreading, or wicking layer overlays the reagent layer.  
     
     
         39 . The method of  claim 34 , wherein adding the fluid sample to the electrochemical cell causes a sudden charging current, which automatically initiates incubation step b) performed under open circuit.  
     
     
         40 . The method of  claim 39 , wherein the Cottrell current is measured at a preset time following the incubation step.  
     
     
         41 . The method of  claim 40 , wherein the reagent layer further includes an enzyme catalyst in sufficient amount to catalyze the reaction involving the analyte and the electron transfer agent.  
     
     
         42 . The method of  claim 41 , wherein the analyte is glucose in a concentration from about 1 milligram glucose per deciliter of fluid sample to about 1000 milligrams glucose per deciliter of fluid sample, and the fluid sample is blood.  
     
     
         43 . The method of  claim 42 , wherein the electron transfer agent is ferricyanide, ferricinium, cobalt (III) orthophenanthroline, cobalt (III) dipyridyl, or benzoquinone.  
     
     
         44 . The method of  claim 28 , wherein the analyte is glucose, TSH, T 4 , a hormone, a cardiac glycoside, an antiarrhythmic, an antiepileptic, an antibiotic, cholesterol, or a non-therapeutic drug.  
     
     
         45 . The method of  claim 28 , wherein the measuring of said Cottrell current includes obtaining a plurality of readings of current in said cell over a plurality of measurement times after said potential has been applied between said electrodes.  
     
     
         46 . A method of measuring the amount of an analyte in a fluid sample, comprising: 
 a. adding the fluid sample to an electrochemical cell that includes 
 at least a first and second electrode of substantially the same size and comprising the same electrically conductive material, wherein the conductive material is selected from the group consisting of palladium, platinum, gold, silver, and carbon,  
 an electron transfer agent,  
 a first catalyst in sufficient amount to catalyze a first reaction involving the analyte, and  
 a second catalyst in sufficient amount to catalyze a second reaction involving a product of the first reaction and the electron transfer agent, thereby forming a detectable species;  
   b. incubating the first and second reactions in an open circuit for a specified period of time;    c. applying a sufficient potential difference between electrodes of the electrochemical cell, after the incubation step, to readily transfer at least one electron between the detectable species and one of the electrodes, thereby resulting in a Cottrell current;    d. measuring the Cottrell current; and    e. correlating the measured Cottrell current to the amount of analyte in the fluid sample.    
     
     
         47 . The method of  claim 46 , wherein adding the fluid sample to the electrochemical cell causes a sudden charging current, which automatically initiates incubation step b) performed under open circuit.  
     
     
         48 . The method of  claim 47 , wherein the Cottrell current is measured at a preset time following the incubation step.  
     
     
         49 . A method of measuring the amount of an analyte in a fluid sample, comprising: 
 a. adding the fluid sample to an electrochemical cell that includes 
 first and second electron transfer agents,  
 a first catalyst in sufficient amount to catalyze a first reaction involving the analyte,  
 a second catalyst in sufficient amount to catalyze a second reaction involving a product of the first reaction and the first electron transfer agent, thereby forming an intermediate species that reacts with the second electron transfer agent, thereby forming a detectable species;  
   b. incubating the reactions of step a) in an open circuit for a specified period of time;    c. applying a sufficient potential difference between electrodes of the electrochemical cell having substantially the same surface area and comprising the same material, after the incubation step, to readily transfer at least one electron between the detectable species and one of the electrodes, thereby resulting in a Cottrell current;    d. measuring the Cottrell current; and    e. correlating the measured Cottrell current to the amount of analyte in the fluid sample.    
     
     
         50 . The method of  claim 49 , wherein the measuring of said Cottrell current includes obtaining a plurality of readings of current in said cell over a plurality of measurement times after said potential has been applied between said electrodes.  
     
     
         51 . A method for measuring the amount of a selected compound in a fluid sample, comprising: 
 providing a measuring cell having at least first and second electrodes of substantially the same size and comprising the same electrically conductive material, for contact with the fluid sample introduced into the cell,    applying a potential to the electrodes to detect the presence of the fluid sample in the cell,    placing the fluid sample into the cell,    removing the potential to the electrode after the fluid sample is detected in the cell,    selectively oxidizing the compound in the fluid sample with an oxidized electron acceptor to produce an oxidized form of the selected compound and a reduced electron acceptor, and    re-applying a potential across the cell electrodes and measuring the resulting Cottrell current, said current being proportional to the concentration of the reduced electron acceptor and the selected compound in the fluid sample.    
     
     
         52 . A method for measuring the amount of glucose in blood, comprising: 
 providing a measuring cell consisting of first and second electrodes for contact with blood introduced into the cell, said electrodes being of substantially the same size and comprising the same electrically conductive material,    applying a potential across the electrodes,    placing a volume of blood into the cell,    removing the potential across the electrodes after the volume of blood is placed into the measuring cell,    oxidizing the glucose in the blood with an oxidized electron acceptor in the presence of glucose oxidase to produce gluconic acid and a reduced electron acceptor,    re-applying a potential across the measuring cell electrodes, and    measuring the Cottrell current through the cell, the Cottrell current being proportional to the glucose concentration in the blood.    
     
     
         53 . The method of  claim 52 , wherein placing the fluid sample into the measuring cell causes a sudden charging current, which automatically initiates removal of the potential from the electrodes and performance of the selective oxidation of the selected compound under open circuit.  
     
     
         54 . The method of  claim 53 , wherein the Cottrell current is measured at the preset time after re-application of a potential across the measuring cell electrodes.  
     
     
         55 . The method of  claim 52 , wherein placing the volume of blood into the measuring cell causes a sudden charging current, which automatically initiates removal of the potential across the electrodes and performance of the oxidation of glucose in the blood under open circuit.  
     
     
         56 . The method of  claim 55 , wherein the Cottrell current is measured at a preset time after re-application of a potential across the measuring cell electrodes.  
     
     
         57 . The method of  claim 56 , wherein the measuring of Cottrell current includes obtaining a plurality of readings of current in said cell after a plurality of pre-set measurement times after said potential has been applied across said measuring cell electrodes.  
     
     
         58 . A device for analyzing an analyte, comprising: 
 a. a first electrical insulator;    b. a pair of electrodes consisting of working and second electrodes of substantially the same size, the electrodes being made of the same electrically conducting materials and being supported on the first electrical insulator;    c. a second electrical insulator, overlaying the first electrical insulator and the electrodes and including a cutout portion that exposes substantially equal surface areas of the working and second electrodes; and    d. a reagent substantially covering the exposed electrode surfaces in the cutout portion and comprising the oxidized form of a redox mediator, an enzyme, and a buffer, 
 the oxidized form of the redox mediator being of sufficient type to receive at least one electron from a reaction involving enzyme, analyte, and oxidized form of the redox mediator and being in sufficient excess to insure that the diffusion limited electrooxidation of the redox mediator at the working electrode surface is the principle limiter of current flow through the device and to resist a shift in potential between the electrodes,  
 the enzyme being of sufficient type and in sufficient amount to catalyze the reaction involving enzyme, analyte, and oxidized form of the redox mediator, and  
 the buffer being unreactive with respect to the reduced and oxidized form of the redox mediator and being of sufficient type and in sufficient amount to provide and maintain a pH at which the enzyme catalyzes the reaction involving enzyme, analyte, and oxidized form of the redox mediator.  
   
     
     
         59 . A reagent incorporated into a sample receiving portion of an electrochemical device that measures an analyte and that has a pair of electrodes consisting of working and second electrodes of substantially the same size, the electrodes being made of the same electrically conducting materials and having substantially equal surface areas in the sample receiving portion, comprising: 
 the oxidized form of a redox mediator, an enzyme, and a buffer,    the oxidized form of the redox mediator being of sufficient type to receive at least one electron from a reaction involving enzyme, analyte, and oxidized form of the redox mediator and being in sufficient amount to insure that current produced by diffusion limited electrooxidation is limited by the oxidation of the reduced form of the redox mediator at the working electrode surface,    the enzyme being of sufficient type and in sufficient amount to catalyze the reaction involving enzyme, analyte, and oxidized form of the redox mediator, and    the buffer having a higher oxidation potential than the reduced form of the redox mediator and being of sufficient type and in sufficient amount to provide and maintain a pH at which the enzyme, analyte, and oxidized form of the redox mediator.    
     
     
         60 . A reagent incorporated into a sample receiving portion of an electrochemical device that measures an analyte and that has a pair of electrodes consisting of working and second electrodes of substantially the same size, the electrodes being made of the same electrically conducting materials and having substantially equal surface areas in the sample receiving portion, comprising: 
 the reduced form of a redox mediator, an enzyme, and buffer,    the reduced form of a redox mediator being of sufficient type to donate at least one electron from a reaction involving enzyme, analyte, and reduced form of the redox mediator and being in sufficient amount to insure that current produced by diffusion limited electroreduction is limited by the reduction of the oxidized form of the redox mediator at the working electrode surface,    the enzyme being of sufficient type and in sufficient amount to catalyze the reaction involving enzyme, analyte, and the reduced form of the redox mediator, and    the buffer having a lower reduction potential than the oxidized form of the redox mediator and being of sufficient type and in sufficient amount to provide and maintain a pH at which the enzyme catalyzes the reaction involving enzyme, analyte, and the reduced form of the redox mediator.    
     
     
         61 . A method of determining the concentration of an analyte in a fluid, comprising the steps of: 
 a. contacting the fluid with a reagent that covers substantially equal surface areas of first and second electrodes and includes the oxidized form of a redox mediator, an enzyme, and a buffer, 
 the oxidized form of the redox mediator being of sufficient type to receive at least one electron from a reaction involving enzyme, analyte, and oxidized form of the redox mediator and being in sufficient amount to insure that current produced by diffusion limited electrooxidation is limited by the oxidation of the reduced form of the redox mediator at the working electrode surface,  
 the enzyme being of sufficient type and in sufficient amount to catalyze the reaction involving enzyme, analyte, and the oxidized form of the redox mediator, and  
 the buffer having a higher oxidation potential than the reduced form of the redox mediator and being of sufficient type and in sufficient amount to provide and maintain a pH at which the enzyme catalyzes the reaction involving enzyme, analyte, and the oxidized form of the redox mediator;  
   b. allowing the reaction involving the enzyme, analyte, and the oxidized form of the redox mediator to go to completion;    c. subsequently applying a potential difference between the electrodes sufficient to cause diffusion limited electrooxidation of the reduced form of the redox mediator at the surface of the first electrode;    d. thereafter measuring the resulting diffusion limited current; and    e. correlating the current measurement to the concentration of the analyte in the fluid.    
     
     
         62 . The method of  claim 61 , wherein the reagent further includes a supporting matrix material of sufficient type and in sufficient amount to disperse the redox mediator in the reagent.  
     
     
         63 . The method of  claim 62 , wherein the reagent further includes a surfactant of sufficient type and in sufficient amount to wet the fluid upon contact with the reagent.  
     
     
         64 . The method of  claim 63 , wherein the analyte is glucose, the oxidized form of the redox mediator is ferricyanide, the buffer is phosphate, and the supporting matrix delays dissolution of the reagent until said reagent has adsorbed said fluid.  
     
     
         65 . The apparatus of  claim 8 , further including a wicking layer positioned over said reagent layer and held in place with an overlay tape.  
     
     
         66 . The apparatus of  claim 1 , wherein the first and second electrodes are spaced apart by a distance of at least about O.lmm and at most about lcm.