Enzyme-based biosensor system for monitoring the freshness of fish

Claim 3

The method of claim 1, wherein said extract is produced by obtaining a preselected quantity of fish muscle, mixing said preselected quantity of fish muscle with a solution comprising an acid suitable to break the cell membrane of said preselected quantity of said fish muscle and homogenizing said mixture to produce an extract.

Claim 4

A method according to claim 2, wherein said solution is a 10% trichloroacetic acid solution.

Claim 5

The method of claim 1, wherein said enzyme nucleotidase is immobilized by a crosslinking agent on a polymeric tube.

Claim 6

A method according to claim 1, wherein said amperometric probe consists of a platinum anode and a silver/silver chloride cathode, wherein said anode is polarized at +0.5 V to +0.7 V with respect to said cathode.

Claim 7

A method according to claim 1, wherein said enzymes xanthine oxidase and nucleoside phosphorylase are co-immobilized on a polymeric membrane, said polymeric membrane being on said probe to form an enzyme electrode.

Claim 8

A method according to claim 6, wherein said enzymes are co-immobilized through glutaraldehyde cross-linking with bovine serum albumin and deposited on a nylon 66 membrane having a pore size of about 3 μm.

Claim 9

A method according to claim 1, wherein said enzyme nucleotidase is immobilized on a polymeric support.

Claim 10

A method according to claim 8, wherein said nucleotidase is immobilized through a glutaraldehyde activation on the wall of a polymeric tube precoated with a thin layer of polyethyleneimine.

Claim 11

An enzyme biosensor system for use in the determination of fish freshness from a fish extract obtained by breaking the cell membrane of said fish, the freshness of said fish being determined by monitoring the autolytic degradation of adenosine triphosphate in fish muscles to inosine monophosphate, inosine and hypoxanthine, said system including a biosensor comprising in combination an amperometric probe having thereon a porous membrane having the enzymes xanthine oxidase and nucleoside phosphorylase co-immobilized thereon to form an enzyme electrode and a polymeric support having the enzyme nucleotidase immobilized thereon, a first portion of said extract being contacted with said enzyme electrode to electrochemically measure a value d 1 =[HxR]+[Hx] from the simultaneous determination of the amount of hydrogen peroxide and uric acid resulting from the degradation hypoxanthine and inosine in said first extract portion by said enzyme electrode enzymes wherein [HxR] is the concentration of inosine and [Hx] is the concentration of hypoxanthine, a second portion of said extract being contacted with said enzyme electrode and said polymeric support to electrochemically measure a value d 2 =[IMP]+[HxR]+[Hx] from the simultaneous determination of the amount of hydrogen peroxide and uric acid resulting from the degradation of inosine monophosphate, inosine and hypoxanthine in said second extract portion by said enzyme electrode enzymes and said polymeric support enzyme, wherein [HxR] is the concentration of inosine, [Hx] is the concentration of hypoxanthine and [IMP] is the concentration of inosine monophosphate, whereby the index of freshness is determined from the formula K=d 1 /d 2 , wherein K represents the index of freshness.

Claim 12

An enzyme biosensor system for use in the determination of fish freshness from a fish extract obtained by breaking the cell membrane of said fish, the freshness of said fish being determined by monitoring the autolytic degradation of adenosine triphosphate in fish muscles to inosine monophosphate, inosine and hypoxanthine, said system including a biosensor comprising in combination an amperometric probe having thereon a porous membrane having the enzymes xanthine oxidase and nucleoside phosphorylase co-immobilized thereon to form an enzyme electrode and a polymeric support having the enzyme nucleotidase immobilized thereon, a first portion of said extract being contacted with said enzyme electrode to electrochemically measure a value d 1 =[HxR]+[Hx] from the simultaneous determination of the amount of hydrogen peroxide and uric acid resulting from the degradation hypoxanthine and inosine in said first extract portion by said enzyme electrode enzymes wherein [HxR] is the concentration of inosine and [Hx] is the concentration of hypoxanthine, a second portion of said extract being contacted with said enzyme electrode and said polymeric support to electrochemically measure a value d 2 =[IMP]+[HxR]+[Hx] from the simultaneous determination of the amount of hydrogen peroxide and uric acid resulting from the degradation of inosine monophosphate, inosine and hypoxanthine in said second extract portion by said enzyme electrode enzymes and said polymeric support enzyme, wherein [HxR] is the concentration of inosine, [Hx] is the concentration of hypoxanthine and [IMP] is the concentration of inosine monophosphate, whereby the index of freshness is determined from the formula K=d 1 /d 2 , wherein K represents the index of freshness.

Claim 13

An enzyme biosensor according to claim 10, wherein said porous membrane is a nylon 66 membrane having a pore size of about 3 μm.

Claim 14

An enzyme biosensor according to claim 10, wherein said xanthine oxidase and nucleoside phosphorylase enzymes are co-immobilized through glutaraldehyde cross-linking with bovine serum albumin.

Claim 15

An enzyme biosensor system according to claim 10, wherein nucleotidase is immobilized through glutaraldehyde activation on the wall of a polystyrene tube precoated with a thin layer of polyethyleneimine.

Claim 16

An enzyme biosensor according to claim 10, wherein said amperometric probe consists of a platinum anode and a silver/silver chloride cathode wherein said anode is polarized at 0.5 V to 0.7 V with respect to said cathode.