Controlling Dynamic DNA Reactions at the Surface of Single-Walled Carbon Nanotube Electrodes to Design Hybridization Platforms with a Specific Amperometric Readout

Abstract

Carbon nanotube (CNT)-based electrodes are cheap, highly performing, and robust platforms for the fabrication of electrochemical sensors. Engineering programmable DNA nanotechnologies on the CNT surface can support the construction of new electrochemical DNA sensors providing an amperometric output in response to biomolecular recognition. This is a significant challenge, since it requires gaining control of specific hybridization processes and functional DNA systems at the interface, while limiting DNA physisorption on the electrode surface, which contributes to nonspecific signal. In this study, we provide design rules to program dynamic DNA structures at the surface of single-walled carbon nanotubes electrodes, showing that specific DNA interactions can be monitored through measurement of the current signal provided by redox-tagged DNA strands. We propose the use of pyrene as a backfilling agent to reduce nonspecific adsorption of reporter DNA strands and demonstrate the controlled formation of DNA duplexes on the electrode surface, which we then apply in the design and conduction of programmable DNA strand displacement reactions. Expanding on this aspect, we report the development of novel amperometric hybridization platforms based on artificial DNA structures templated by the small molecule melamine. These platforms enable dynamic strand exchange reactions orthogonal to conventional toehold-mediated strand displacement and may support new strategies in electrochemical sensing of biomolecular targets, combining the physicochemical properties of nanostructured carbon-based materials with programmable nucleic acid hybridization.

A Folding-Based Electrochemical Aptasensor for the Single-Step Detection of the SARS-CoV-2 Spike Protein

“…A free amine group was introduced instead at the 5′ terminus of the aptamer sequence to allow covalent anchoring to the electrode surface through the formation of an amide bond with the carboxylic groups present on the SWCNTs, using EDC/NHS as a coupling mixture. This coupling reaction had been already used in several works − and was further optimized by diluting the aptamer in carbonate buffer (pH = 9) with 0.1% SDS to increase the wettability of the CNT hydrophobic surface. The concentration of the aptamer in the carbonate buffer solution used for covalent immobilization on the electrode surface was 500 nM, based on a previous work in which we investigated different concentrations of biotinylated amine-modified DNA probes to maximize surface functionalization, using an enzyme-based reaction to generate an electrochemical signal proportional to the amount of DNA probes attached to the electrode surface (Figure S1).…”

“…The concentration of the aptamer in the carbonate buffer solution used for covalent immobilization on the electrode surface was 500 nM, based on a previous work in which we investigated different concentrations of biotinylated amine-modified DNA probes to maximize surface functionalization, using an enzyme-based reaction to generate an electrochemical signal proportional to the amount of DNA probes attached to the electrode surface (Figure S1). The surface density of the aptamer was estimated by means of fluorescence spectroscopy following the emission of the AttoMB2 tag at λ = 680 nm before and after the immobilization of the aptamer onto the surface (Figure S2). A value of (1.7 ± 0.4) · 10 13 aptamer molecules per mm 2 was obtained, which is higher than the average values generally found in the literature for E-DNAs based on gold electrode substrates. , The electrode surface was eventually treated with a solution of pyrene in DMSO as a backfilling agent to minimize non-specific adsorption of the biomolecules contained in the analyzed samples.…”

“…A value of (1.7 ± 0.4) · 10 13 aptamer molecules per mm 2 was obtained, which is higher than the average values generally found in the literature for E-DNAs based on gold electrode substrates. , The electrode surface was eventually treated with a solution of pyrene in DMSO as a backfilling agent to minimize non-specific adsorption of the biomolecules contained in the analyzed samples. This pyrene-based backfilling strategy was developed and applied in previous works and proved particularly efficient in reducing non-specific adsorption of non-target biological molecules when compared to other common blocking strategies such as BSA. , …”

“…This pyrene-based backfilling strategy was developed and applied in previous works and proved particularly efficient in reducing non-specific adsorption of non-target biological molecules when compared to other common blocking strategies such as BSA. 43,44 3.2. S1 Protein-Aptamer Interaction Analysis.…”

Point-of-care biochemical assays using electrochemical technologies: approaches, applications, and opportunities

“…Electrochemical enzyme sensors can make use of organic molecules (e.g., polyacrylamide), materials with porous frameworks such as metal organic frameworks and stabilizers to enhance the immobilization of the enzyme [72]. Similar approaches apply to electrochemical immunosensors, electrochemical DNA sensors, and other types of devices as well [73][74][75]. Furthermore, in order to miniaturize and produce electrochemical biosensors on a large scale, microfluidic techniques could be ingeniously utilized in sweat sampling.…”