Aptamer-based electrochemical biosensors for protein detection: Integrating immobilization strategies, plasma surface engineering, and enzymatic amplification

Abstract

Biosensors have been an integral part of medical diagnosis and biochemical monitoring. The capture probe of biosensors has undergone modifications from isolated enzymes and antibodies to artificial biorecognition elements. Aptamers have emerged as a novel artificial capture probe with a similar affinity detection mechanism to naturally occurring antibodies. This thesis investigates aptamer-based sensor (aptasensor) fabrication to detect two protein targets: SARS-CoV-2 spike protein and, at a greater depth, a coagulation cascade protein, thrombin.

The high demand for accessible tools for coronavirus disease 2019 diagnostics was the first project’s motif as demonstrated in chapter 2, in which an aptasensor based on self-assembled monolayer formation (SAM) of thiol-modified single-stranded DNA on a gold screen-printed electrode (SPE) was developed. The aptamer binding to the spike protein was observed by Electrochemical Impedance Spectroscopy (EIS). However, due to the rapid mutations of SARS-CoV-2 and the complexity of the aptamer redesign, this project was discontinued.

In chapter 3, we introduced a regeneration strategy for recycling gold SPE wastes by using an air plasma cleaning method, resulting in enhanced electrochemical properties and immobilization efficiency of a thrombin-binding aptamer. Surface chemistry studies confirmed that the air plasma created a hydrophilic surface, improved the electron transfer kinetics, and increased surface roughness.

In chapter 4, we utilized a plasma-enhanced chemical vapor deposition method, known as plasma-activated coating (PAC), to covalently tether the thrombin-binding aptamer to a printed carbon electrode. X-ray photoelectron spectroscopy of surface phosphorus signals increased by 6-fold, and surface nitrogen increased by 5-fold after aptamer immobilization on a PAC-modified surface compared to the unmodified electrodes.

In chapter 5, we showed that EIS can be used for binding analysis of the thrombin aptamer, but with limited sensitivity. Therefore, thrombin’s enzymatic cleavage was measured electrochemically by an electrogenic substrate that was cleaved by thrombin to liberate para-nitroaniline. Square-wave voltammetry enabled real-time monitoring of the liberated para-nitroaniline at 0.12 V, producing a 90-fold greater signal than EIS under identical conditions.

Our work provides the experimental knowledge for the development of sensitive biosensors for protein detection, with applications in biomedical devices for point-of-care coagulation health monitoring.