Method of using enzyme electrode

Claim 1

A method of assaying the concentration of a substance in an undiluted whole blood or undiluted plasma sample, which includes catalase, comprising providing a polarographic cell that includes an electrode assembly comprising a reference electrode and a hydrogen peroxide sensor electrode having a laminated membrane covering a sample-contacting face of said sensor electrode, said laminated membrane comprising an outer membrane permeable to said substance and oxygen while preventing the passage of proteins and red blood cells, an inner membrane permeable to hydrogen peroxide and located adjacent the face of said sensor electrode, and an enzyme layer between said inner membrane and said outer membrane, said enzyme layer comprising an enzyme that can oxidize said substance to generate hydrogen peroxide, said outer membrane having pores that allow passage of said substance and oxygen from said undiluted sample to said enzyme layer, the thickness of said outer membrane and the size of said pores being selected so that said passage of said substrate is sufficiently hindered relative to said passage of said oxygen that a steady state response proportional to the substance concentration with said electrode assembly can be obtained at substance concentrations in an undiluted whole blood sample of at least 300 mg/dl where the oxygen concentration is equivalent to the oxygen concentration in a plasma sample derived from a normal human subject, and said outer membrane having a thickness of between 20 and 100μ to impair the passage of hydrogen peroxide from said enzyme layer to said undiluted sample to an extent great enough to permit said steady state to be reached; contacting said outer membrane with said undiluted sample comprising said substrate to permit said substance and oxygen in said undiluted sample to pass through said outer membrane to said enzyme layer so that said enzyme oxidizes said substance to generate hydrogen peroxide, said generated hydrogen peroxide passing through said inner layer to contact said face of said sensor electrode; maintaining said contact between said outer membrane and said undiluted sample for a sufficient period of time to generate a steady state responses proportional to the substrate concentration; and measuring said steady state response to determine said substrate concentration.

Claim 2

A method of assaying the concentration of a substance in an undiluted whole blood or undiluted plasma sample, which includes catalase, comprising providing a polarographic cell that includes an electrode assembly comprising a reference electrode and a hydrogen peroxide sensor electrode having a laminated membrane covering a sample-contacting face of said sensor electrode, said laminated membrane comprising an outer membrane permeable to said substance and oxygen while preventing the passage of proteins and red blood cells, an inner membrane permeable to hydrogen peroxide and located adjacent the face of said sensor electrode, and an enzyme layer between said inner membrane and said outer membrane, said enzyme layer comprising an enzyme that can oxidize said substance to generate hydrogen peroxide, said outer membrane having pores that allow passage of said substance and oxygen from said undiluted sample to said enzyme layer, the thickness of said outer membrane and the size of said pores being selected so that said passage of said substrate is sufficiently hindered relative to said passage of said oxygen that a steady state response proportional to the substance concentration with said electrode assembly can be obtained at substance concentrations in an undiluted whole blood sample of at least 300 mg/dl where the oxygen concentration is equivalent to the oxygen concentration in a plasma sample derived from a normal human subject, and said outer membrane having a thickness of between 20 and 100μ to impair the passage of hydrogen peroxide from said enzyme layer to said undiluted sample to an extent great enough to permit said steady state to be reached; contacting said outer membrane with said undiluted sample comprising said substrate to permit said substance and oxygen in said undiluted sample to pass through said outer membrane to said enzyme layer so that said enzyme oxidizes said substance to generate hydrogen peroxide, said generated hydrogen peroxide passing through said inner layer to contact said face of said sensor electrode; maintaining said contact between said outer membrane and said undiluted sample for a sufficient period of time to generate a steady state responses proportional to the substrate concentration; and measuring said steady state response to determine said substrate concentration.