# US 11,898,191 B2 · Enzymatic electrochemical method for the quantification of analytes in biological fluid samples > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,898,191 B2 - Document ID: US11898191B2 - Status: Granted - Kind: B2 - Application number: 17/126,971 - Filed: 2020-12-18 - Published: 2021-04-08 - Granted: 2024-02-13 - Earliest claimed: 2018-06-21 - Assignees: Biolan Health, S.L. - Applicants: Biolan Health, S.L. - Inventors: Laura González-Macia; Maitane Urien Berrio; Roberto González Rioja; Arrate Jaureguibeitia Cayrols; Israel Sánchez Moreno; Carmen Hermida Díaz - Web page: https://patentgrep.com/patent/US11898191B2 ## CPC - C12Q 1/005: {involving specific analytes or enzymes (including groups of enzymes, e.g. oxydases; C12Q1/004 takes precedence)} - G01N 27/3274: {Corrective measures, e.g. error detection, compensation for temperature or hematocrit, calibration (coding of calibration information G01N33/48771)} - C12N 9/0016: {with NAD or NADP as acceptor (1.4.1)} - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract An enzymatic electrochemical method for the quantification of analytes in isolated samples of biological fluids based on a dual biosensor strip electrochemical system. The sample is analyzed in parallel using first and second different biosensor test strips (blank and complete) and the results are obtained by subtracting a response obtained in the electrodes of the first biosensor test strip from a response obtained in the electrodes of the second biosensor test strip, thus eliminating the effect of interferences and isolating the signal corresponding only to the analyte determination. ## Claims ### Claim 1 An enzymatic electrochemical method for quantification of an analyte in a biological fluid sample, the method comprising: introducing the biological fluid sample in an electrochemical system formed by a first biosensor strip and a second biosensor strip, the first biosensor strip including a first working electrode modified with an enzyme and an electrochemical mediator, a first reference electrode and a first auxiliary electrode, the second biosensor strip including a second working electrode, a second reference electrode and a second auxiliary electrode, the second working electrode being modified with the enzyme, the electrochemical mediator and a cofactor; applying a constant potential to the first and second working electrodes, the first and second reference electrodes and the first and second auxiliary electrodes during a set period of time; obtaining current intensity data by measuring a current intensity produced by a re-oxidation of the cofactor on the second working electrode after the cofactor being initially reduced due to a reaction between the enzyme and the analyte on each of the first and second biosensor strips, and subtracting a response obtained in each of the first working electrode, first reference electrode and first auxiliary electrode from a response respectively obtained in each of the second working electrode, second reference electrode and second auxiliary electrode, the re-oxidation of the cofactor being facilitated by the electrochemical mediator; translating the current intensity data by use of a calibration curve obtained from samples of a synthetic biological fluid comprising different concentrations of the analyte to be determined. ### Claim 2 (depends on 1) The method according to claim 1, wherein the enzyme present on the first and second working electrodes is an oxidoreductase. ### Claim 3 (depends on 2) The method according to claim 2, wherein the enzyme is a dehydrogenase enzyme. ### Claim 4 (depends on 3) The method according to claim 3, where the dehydrogenase enzyme is selected from the group consisting of lactate dehydrogenase, glucose dehydrogenase, methanol dehydrogenase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, glycerol-3-phosphate dehydrogenase, glycerol dehydrogenase, alcohol dehydrogenase, and D-xylose-1-dehydrogenase. ### Claim 5 (depends on 1) The method according to claim 1, wherein the electrochemical mediator present on each of the first and second working electrodes is selected from the group consisting of tetramethylbenzidine (TMB), potassium ferricyanide, toluidine blue, hydroquinone, tetrathiafulvalene (TTF), and ferrocene. ### Claim 6 (depends on 1) The method according to claim 1, wherein the cofactor present on the second working electrode is selected from the group consisting of nicotinamide adenine dinucleotide (NAD+), nicotinamide adenine dinucleotide phosphate (NADP+), flavin adenine dinucleotide (FAD), and flavin mononucleotide (FMN). ### Claim 7 (depends on 6) The method according to claim 6, wherein the cofactor does not form part of a structure of the enzyme. ### Claim 8 (depends on 1) The method according to claim 1, wherein the potential applied is between −0.2 V and +0.2 V vs. Ag/AgCI. ### Claim 9 (depends on 8) The method according to claim 8, wherein the potential applied is between −0.05 V and +0.15 V vs. Ag/AgCI. ### Claim 10 (depends on 1) The method according to claim 1, wherein the set period of time is between 20 seconds and 120 seconds. ### Claim 11 (depends on 1) The method according to claim 1, wherein the set period of time is between 30 seconds and 90 seconds. ### Claim 12 (depends on 1) The method according to claim 1, wherein the biological fluid sample is an isolated biological sample selected from the group consisting of urine, blood, serum, plasma, pleural fluid and saliva. ### Claim 13 (depends on 1) The method according to claim 1, wherein the first and second biosensor strips reside on a same substrate. ### Claim 14 (depends on 13) The method according to claim 13, wherein the first and second reference electrodes are one and the same. ### Claim 15 (depends on 13) The method according to claim 13, wherein the first and second auxiliary electrodes are one and the same. ### Claim 16 (depends on 14) The method according to claim 14, wherein the first and second auxiliary electrodes are one and the same.