# US 11,079,351 B2 · Active enzyme protection system for an electrochemical biosensor > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,079,351 B2 - Document ID: US11079351B2 - Status: Granted - Kind: B2 - Application number: 16/194,489 - Filed: 2018-11-19 - Published: 2019-10-17 - Granted: 2021-08-03 - Earliest claimed: 2017-11-22 - Applicants: Jim Connolly - Inventors: Jim Connolly - Web page: https://patentgrep.com/patent/US11079351B2 ## CPC - G01N 27/3272: {Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels (optical biosensors G01N33/52)} - G01N 33/54393: {Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding} - C12Q 1/001: {Enzyme electrodes} - G01N 33/54366: {Apparatus specially adapted for solid-phase testing} - G01N 33/5438: {Electrodes} - 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) - 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 ## Abstract Immobilization zones through which a sample flows before reaching the proteolytic agents and/or active enzyme and associated electrode/s of an electrochemical test sensor may be used to trap or chemically deactivate active enzyme and/or proteolytic agent deactivating agents. Through either trapping or chemical deactivation, the immobilization zones substantially prevent the enzyme deactivating agents from reaching, and thus reducing the activity of the active enzyme or enzymes. Similarly, trapping or chemical deactivation may be used to prevent or substantially reduce the incidence of the proteolytic deactivating agent or agents from reaching the proteolytic agent. ## Claims ### Claim 1 A method of electrochemically analyzing an undiluted whole blood sample in a test sensor, the method comprising: introducing an undiluted whole blood sample to an inlet of a test sensor, the test sensor having at least one isolated flow path; contacting the undiluted whole blood sample with a lysing agent at a first chemical reaction zone in the at least one isolated flow path to release red blood cell constituents, then contacting the undiluted whole blood sample with an interference reduction matrix that reduces lysate interference, then contacting the undiluted whole blood sample with a proteolytic agent at a second chemical reaction zone in the at least one isolated flow path; then transferring the sample to a protease inactivation zone in the at least one isolated flow path; reducing the activity of the proteolytic agent by altering the chemistry of the sample at the protease inactivation zone; then transferring the sample from the protease inactivation zone to an electrochemical reaction zone in the at least one isolated flow path; then contacting the sample with an active enzyme that selectively undergoes a redox reaction with an analyte in the sample to produce a measurable species; and electrically altering the oxidation state of the measurable species with an electrode pair in response to an applied potential to generate an output signal component responsive to the analyte concentration of the sample. ### Claim 2 (depends on 1) The method of claim 1, where the reducing the activity of the proteolytic agent by altering the chemistry of the sample at the protease inactivation zone reduces the activity of the proteolytic agent by at least 30%. ### Claim 3 (depends on 1) The method of claim 1, where the reducing the activity of the proteolytic agent by altering the chemistry of the sample at the protease inactivation zone reduces the activity of the proteolytic agent by at least 70%. ### Claim 4 (depends on 1) The method of claim 1, where contacting the sample with the lysing agent and contacting the sample with the interference reduction matrix occur sequentially at the first chemical reaction zone. ### Claim 5 (depends on 1) The method of claim 1, where the first chemical reaction zone is separated from the second chemical reaction zone by 4 to 30 millimeters. ### Claim 6 (depends on 1) The method of claim 1, where the second chemical reaction zone is separated from the protease inactivation zone by 2 to 20 millimeters. ### Claim 7 (depends on 4) The method of claim 4, where the interference reduction matrix irreversibly chemically alters the released red blood cell constituents. ### Claim 8 (depends on 1) The method of claim 1, where the sample is present at the first chemical reaction zone from 0.5 to 2.5 seconds before transferring to the second chemical reaction zone. ### Claim 9 (depends on 1) The method of claim 1, where the sample is present at the second chemical reaction zone from 1 to 4 seconds before transferring to the protease inactivation zone. ### Claim 10 (depends on 1) The method of claim 1, where the sample is present at the protease inactivation zone from 0.5 to 1.5 seconds before transferring to the electrochemical reaction zone. ### Claim 11 (depends on 1) The method of claim 1, further comprising reducing the temperature of the electrochemical reaction zone by at least 10 degrees C. in relation to the temperature of the second chemical reaction zone. ### Claim 12 (depends on 1) The method of claim 1, further comprising reducing the temperature of the electrochemical reaction zone by at least 15 degrees C. in relation to the temperature of the second chemical reaction zone. ### Claim 13 (depends on 1) The method of claim 1, where the pH of the second chemical reaction zone is approximately 8. ### Claim 14 (depends on 1) The method of claim 1, where the protease inactivation zone is a pH adjustment zone. ### Claim 15 (depends on 14) The method of claim 14, where the pH of the pH adjustment zone is approximately 6. ### Claim 16 (depends on 1) The method of claim 1, where the protease inactivation zone is a chemical inactivation zone. ### Claim 17 (depends on 16) The method of claim 16, where the chemical inactivation zone comprises a chemical inactivator selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), ferric chloride, polyoxyethylene (23) lauryl ether (Brij 35), polyethylene glycol sorbitan monolaurate (TWEEN 20), and combinations thereof. ### Claim 18 (depends on 16) The method of claim 16, where the chemical inactivation zone comprises ethylenediaminetetraacetic acid.