Understanding Signal and Background in a Thermally Resolved, Single-Branched DNA Assay Using Square Wave Voltammetry

Abstract

Electrochemical bioanalytical sensors with oligonucleotide transducer molecules have been recently extended for quantifying a wide range of biomolecules, from small drugs to large proteins. Short DNA or RNA strands have gained attention recently due to the existence of circulating oligonucleotides in human blood, yet challenges remain for adequately sensing these targets at electrode surfaces. In this work, we have developed a quantitative electrochemical method which uses target-induced proximity of a single-branched DNA structure to drive hybridization at an electrode surface, with readout by square-wave voltammetry (SWV). Using custom instrumentation, we first show that precise control of temperature can provide both electrochemical signal amplification and background signal depreciation in SWV readout of small oligonucleotides. Next, we thoroughly compared 25 different combinations of binding energies by their signal-to-background ratios and differences. These data served as a guide to select the optimal parameters of binding energy, SWV frequency, and assay temperature. Finally, the influence of experimental workflow on the sensitivity and limit of detection (LOD) of the sensor is demonstrated. This study highlights the importance of precisely controlling temperature and SWV frequency in DNA-driven assays on electrode surfaces while also presenting a novel instrumental design for fine-tuning of such systems.

A review of microfabricated electrochemical biosensors for DNA detection

“…However, there are reports which look at development of assays for DNA sequences, establish the optimum parameters for the SWV measurement and involve the use of a redox tagged DNA probe in order to generate the Faradaic signal (Cao et al, 2014). More specifically, binding energies, hybridization temperature and SWV parameters were assessed to find the optimum 535 conditions for detecting short oligonucleotides (Somasundaram et al, 2018). Also, the signal gain of microfabricated gold DNA sensor systems in square wave mode was assessed with the optimal square-wave frequency depending on the structure of the probe, the nature of the redox reporter, and other features of the sensor (Dauphin-Ducharme and Plaxco, 2016).…”

Non-Faradaic Current Suppression in DNA Based Electrochemical Assays with a Differential Potentiostat

“…Photolithographically patterned gold on glass electrodes (GoG) and PDMS electrochemical cells were used by following similar fabrication steps presented in our previous work. 2 Briefly, GoG slides were placed in an oven at 200 °C for a minimum of 15 min then cooled to room temperature (RT). A layer of ~20 µm thickness of AZ40XT photoresist was spin-coated onto the GoG then baked at 126 o C for 6 min.…”

“…PDMS Chambers. 3D-printed templates 2,3 were designed in Sketchup (Trimble, Inc.; Sunnyvale, CA) to align with GoG electrodes, and models were sliced in Makerbot Desktop software (New York, NY) then printed on a Makerbot 2 3D printer at high resolution (100 µm layer) with 75% infill. PDMS precursors were mixed (10:1, prepolymer:initiator), degassed, and poured over a silanized 100-mm polished silicon wafer.…”

“…We followed a similar immobilization protocol explained in our previous work. 2 Briefly, 200 µM thiolated DNA was combined with 10 mM TCEP (reducing agent) at a 1:3 ratio and incubated 1 h at RT in darkness. The reduced thiolated DNA was then diluted to 1.25 µM and introduced into the electrochemical cell.…”

Ligand-Based Stability Changes in Duplex DNA Measured with a Microscale Electrochemical Platform

“…Having a heat source that is compatible with microdevice architectures opens the opportunity of arrays with locally heated sites. Prior examples of coupling electrochemical detection with controlled temperature capability include: thermoelectric modules [21,22], heating wells of solutions [23,24] and heated wire electrodes [25,26]. The incorporation of copper resistive heaters on the backside of fiberglass epoxy sheets has been used for quantitative polymerase chain reaction (PCR) biosensors [27].…”