Ionic Strength and Hybridization Position near Gold Electrodes Can Significantly Improve Kinetics in DNA-Based Electrochemical Sensors

Abstract

A variety of electrochemical (EC) biosensors play critical roles in disease diagnostics. More recently, DNA-based EC sensors have been established as promising for detecting a wide range of analyte classes. Since most of these sensors rely on the high specificity of DNA hybridization for analyte binding or structural control, it is crucial to understand the kinetics of hybridization at the electrode surface. In this work, we have used methylene blue-labeled DNA strands to monitor the kinetics of DNA hybridization at the electrode surface with square-wave voltammetry. By varying the position of the double-stranded DNA segment relative to the electrode surface as well as the bulk solution’s ionic strength (0.125–1.00 M), we observed significant interferences with DNA hybridization closer to the surface, with more substantial interference at lower ionic strength. As a demonstration of the effect, toehold-mediated strand displacement reactions were slowed and diminished close to the surface, while strategic placement of the DNA binding site improved reaction rates and yields. This work manifests that both the salt concentration and DNA hybridization site relative to the electrode are important factors to consider when designing DNA-based EC sensors that measure hybridization directly at the electrode surface.

Building a Nucleic Acid Nanostructure with DNA–Epitope Conjugates for a Versatile Approach to Electrochemical Protein Detection

“…The nanostructure is somewhat flexible and subjected to tethered diffusion and Brownian motion at the surface 30 with a short oligonucleotide spacer to avoid detrimental double-layer effects on the double-stranded DNA formation. 31 When a large enough target molecule is bound to the nanostructure, this movement slows down, likely due to the combination of increased molecular weight and steric hindrance effects. Hence, the diffusional motion of methylene blue near the surface slows down and provides an EC-based quantification method to analyze the target molecule's presence.…”

“…In our recent work, we introduced the electrochemical DNA nanostructure for the versatile detection of clinically relevant analytes. , As it follows the enzymatic ligation of three different short single-stranded DNA on the electrode surface for immobilization, recognition, and signaling, each nanostructure becomes a stable, single molecular unit on the electrode surface (Figure ). The nanostructure is somewhat flexible and subjected to tethered diffusion and Brownian motion at the surface with a short oligonucleotide spacer to avoid detrimental double-layer effects on the double-stranded DNA formation . When a large enough target molecule is bound to the nanostructure, this movement slows down, likely due to the combination of increased molecular weight and steric hindrance effects.…”

Toward Continuous Molecular Testing Using Gold-Coated Threads as Multi-Target Electrochemical Biosensors

“…We performed a SHA [ 46 , 47 , 48 ] using the capture probes immobilized on the Au thread and oligonucleotide-tagged HRP reporter probes ( Figure 2 A). In this assay, HRP catalyzes a reaction in the presence of 3,3′,5,5′ tetramethylbenzidine (TMB) and hydrogen peroxide in which TMB is oxidized, allowing an electrochemical analysis with chronoamperometry at −0.2 V. This voltage was chosen based on initial investigation with circular voltammetry on Au threads in TMB and H 2 O 2 ( Figure S5 ).…”

Building a nucleic acid nanostructure with DNA-epitope conjugates for a versatile electrochemical protein detection platform

“…30 , with a short oligonucleotide spacer to avoid detrimental double-layer effects on the double stranded DNA formation. 31 When a large enough target molecule is bound to the nanostructure, this movement slows, likely due to a combination of increased molecular weight and steric hindrance effects. Hence, diffusional motion of methylene blue near the surface slows and provides an EC-based quantification method to analyze the target molecule's presence.…”