Durable enzyme-based biosensor and process for drop deposition immobilization

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.

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.