# US 12,213,784 B2 · Durable enzyme-based biosensor and process for drop deposition immobilization > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,213,784 B2 - Document ID: US12213784B2 - Status: Granted - Kind: B2 - Application number: 16/468,146 - Filed: 2017-12-11 - Published: 2020-12-31 - Granted: 2025-02-04 - Earliest claimed: 2016-12-09 - Assignees: Northeastern University - Applicants: Northeastern University - Inventors: Yunqing Du; Ming L. Wang - Web page: https://patentgrep.com/patent/US12213784B2 ## CPC - A61B 5/1486: using enzyme electrodes, e.g. with immobilised oxidase - A61B 5/14532: {for measuring glucose, e.g. by tissue impedance measurement} - G01N 27/3271: {Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood (amperometry per se G01N27/49; aspects concerning the enzyme reagent C12Q1/001)} - A61B 2562/0285: Nanoscale sensors - G01N 27/3278: {involving nanosized elements, e.g. nanogaps or nanoparticles (nanopores G01N33/48721; magnetic beads G01N27/745)} - A61B: DIAGNOSIS; SURGERY; IDENTIFICATION (usefulness limited to only animals A61D) - 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) ## Abstract A sensor having improved shelf life is provided for determining concentration of a biomarker in a liquid sample. The sensor functions by electrochemical detection and requires use of a biomolecule, e.g., an enzyme that catalyzes an electron transfer reaction specific for the biomarker. The sensor requires use of a quaternary ammonium compound as a bio-linker. It further requires crosslinking of the biomolecule for improved stability. ## Claims ### Claim 1 A sensor for determining a concentration of a biomarker in a liquid sample, the sensor comprising: an insulating or semiconducting substrate; at least one working electrode, a counter electrode, and a reference electrode, and a sample placement area on a surface of the substrate for containing the liquid sample during a determination of the biomarker concentration in the liquid sample; wherein each of the working electrode(s), counter electrode, and reference electrode comprises a conductive metal layer deposited on the substrate in the sample placement area; wherein the working electrode is coated in the sample placement area with a plurality of sensor elements; wherein the sensor elements are functionalized with a functionalization coating comprising a quaternary ammonium compound, a plurality of metal nanoparticles, a crosslinked enzyme that catalyzes an electron transfer reaction specific for the biomarker; wherein the working electrode, counter electrode, and reference electrode are connected to an amperometry circuit; wherein the sensor measures electron transfer into the working electrode; wherein an output voltage of the amperometry circuit correlates with concentration of the biomarker in the liquid sample deposited in the sample placement area; and wherein the sensor has been fabricated by a method comprising drop deposition of the functionalization coating from a single suspension comprising the quaternary ammonium compound, the metal nanoparticles, and the enzyme. ### Claim 2 (depends on 1) The sensor of claim 1, wherein the sensor elements comprise a material selected from the group consisting of single-walled carbon nanotubes (SWNT), double-walled carbon nanotubes, multi-walled carbon nanotubes, graphite, graphene, carbon nanofibers, carbon nanowires, carbon nanorods, and combinations thereof. ### Claim 3 (depends on 1) The sensor of claim 1, wherein the functionalization coating does not contain a polycationic polymer. ### Claim 4 (depends on 1) The sensor of claim 1, wherein the quaternary ammonium compound comprises alkyl chains having a chain length from about C2 to about C16. ### Claim 5 (depends on 1) The sensor of claim 1, wherein the quaternary ammonium compound is a chloride or bromide salt. ### Claim 6 (depends on 1) The sensor of claim 1, wherein the quaternary ammonium compound is selected from the group consisting of tetraethylammonium bromide, tetrapropylammonium bromide, tetrabutylammonium bromide, hexadecyltrimethylammonium bromide, benzalkonium chloride, alkyldimethylbenzylammonium chloride having alkyl chain lengths from C12 to C16, and dialkyldimethylammonium chloride having alkyl chain lengths from C8 to C10. ### Claim 7 (depends on 1) The sensor of claim 1, wherein the enzyme is crosslinked using a homobifunctional or heterobifunctional crosslinking reagent. ### Claim 8 (depends on 1) The sensor of claim 1, wherein the enzyme is crosslinked by a reagent selected from the group consisting of glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, N-hydroxysulfosuccinimide, dicyclohexylcarbodiimide, dimethyl adipimidate, dimethyl suberimidate, and combinations thereof. ### Claim 9 (depends on 1) The sensor of claim 1, further comprising a protective membrane covering the functionalized sensor elements. ### Claim 10 (depends on 1) The sensor of claim 1 having a shelf life of at least nine months. ### Claim 11 (depends on 1) The sensor of claim 1, wherein the enzyme is glucose oxidase and the biomarker is glucose. ### Claim 12 (depends on 11) The sensor of claim 11 that is capable of detecting glucose at concentrations down to 5 μM. ### Claim 13 (depends on 11) The sensor of claim 11 that is configured for determination of glucose concentration in saliva. ### Claim 14 (depends on 1) A biomarker analysis system comprising: the sensor of claim 1; a signal conditioning and/or analysis device that processes an electrical signal from the sensor. ### Claim 15 (depends on 1) A biomarker sensor array comprising a plurality of sensors according to claim 1 sharing a common substrate. ### Claim 16 (depends on 1) A kit comprising the sensor of claim 1 in a vacuum storage container. ### Claim 17 A method of determining a concentration of a biomarker in a liquid sample, the method comprising the steps of: (a) providing a sensor comprising: an insulating or semiconducting substrate; at least one working electrode, a counter electrode, and a reference electrode, and a sample placement area on a surface of the substrate for containing the liquid sample during a determination of the biomarker concentration in the liquid sample; wherein each of the working electrode(s), counter electrode, and reference electrode comprises a conductive metal layer deposited on the substrate in the sample placement area; wherein the working electrode is coated in the sample placement area with a plurality of sensor elements; wherein the sensor elements are functionalized with a functionalization coating comprising a quaternary ammonium compound, a plurality of metal nanoparticles, a crosslinked enzyme that catalyzes an electron transfer reaction specific for the biomarker; wherein the working electrode, counter electrode, and reference electrode are connected to an amperometry circuit; wherein the sensor measures electron transfer into the working electrode; and wherein an output voltage of the amperometry circuit correlates with concentration of the biomarker in the liquid sample deposited in the sample placement area; wherein the sensor has been fabricated by a method comprising drop deposition of the functionalization coating from a single suspension comprising the quaternary ammonium compound, the metal nanoparticles, and the enzyme; (b) introducing a liquid sample into the sample placement area of the sensor; and (c) determining the concentration in the liquid sample from an electrical output of the sensor. ### Claim 18 (depends on 17) The method of claim 17 further comprising the steps of: (d) removing the liquid sample introduced in step (b); (e) introducing a new liquid sample into the sample placement area of the sensor; and (f) determining a new biomarker concentration in the new liquid sample from an electrical output of the sensor. ### Claim 19 (depends on 17) The method of claim 17, wherein the liquid sample is saliva. ### Claim 20 A method of fabricating a biomarker sensor, comprising the steps of: (a) microfabricating one or more working electrodes, a reference electrode, and a counter electrode on the surface of an insulating substrate, wherein each of said electrodes contacts a sample placement area on the substrate; (b) depositing a plurality of sensor elements onto the working electrode; (c) depositing a functionalization coating onto the sensor elements, the functionalization coating comprising one or more functionalization layers; wherein each functionalization layer comprises a quaternary ammonium compound, a plurality of metal nanoparticles, an enzyme that catalyzes an electron transfer reaction specific for the biomarker, wherein the one or more functionalization layers are deposited through drop deposition of a single suspension comprising the quaternary ammonium compound, the metal nanoparticles, and the enzyme; and (d) crosslinking the enzyme by applying a crosslinking reagent to the functionalization coating. ### Claim 21 (depends on 20) The method of claim 20, wherein the crosslinking reagent is applied to the top of the functionalization coating, or wherein the crosslinking reagent is applying by its inclusion in one or more of the one or more functionalization layers. ### Claim 22 (depends on 20) The method of claim 20, wherein the single suspension further comprises a homobifunctional or heterobifunctional crosslinking reagent. ### Claim 23 (depends on 20) The method of claim 20, wherein the sensor elements are deposited onto the working electrode by a self-assembly process comprising depositing a liquid suspension of sensor elements onto the electrode.