Portable electroanalytical nucleic acid amplification tests using printed circuit boards and open-source electronics

Abstract

The realization of electrochemical nucleic acid amplification tests (NAATs) at the point of care (POC) is highly desirable, but it remains a challenge given their high cost and lack of true portability/miniaturization. Here we show that mass-produced, industrial standardized, printed circuit boards (PCBs) can be repurposed to act as near-zero cost electrodes for self-assembled monolayer-based DNA biosensing, and further integration with a custom-designed and low-cost portable potentiostat. To show the analytical capability of this system, we developed a NAAT using isothermal recombinase polymerase amplification, bypassing the need of thermal cyclers, followed by an electrochemical readout relying on a sandwich hybridization assay. We used our sensor and device for analytical detection of the toxic microalgae Ostreopsis cf. ovata as a proof of concept. This work shows the potential of PCBs and open-source electronics to be used as powerful POC DNA biosensors at a low-cost.

Fig. 1. Design and electrochemical characterization of the PCBs. (A) Each PCB has 4 gold electrodes. (B) Randles–Sevcik plots of bare gold electrodes and SAM-modified gold electrodes after conducting CVs in 5 mM ferricyanide at different scan rates (10 mV s⁻¹–300 mV s⁻¹) (n = 3). (C) Representative CV in 5 mM ferricyanide (scan rate of 30 mV s⁻¹) after gold electroplating: bare gold electrode and SAM-modified gold electrode.

Fig. 2. Schematics and pictures of the electroanalytical device. (A) Schematics of the potentiostat showing connection with the computer and the PCB for sample analysis. (B) Picture of the PCB with a card edge connector, fitting inside a 200 μL tube. The size of a PCB is 1.2 × 3.1 cm. (C) Picture of the main components of the potentiostat. The size of the potentiostat is 2.3 × 7 cm.

Fig. 3. Schematics and results of the NAAT. (A) Schematics of the RPA followed by a SHA with electrochemical detection: (1) RPA is performed in solution. The RPA product is a dsDNA sequence with specific ssDNA tails at each end (due to the use of tailed primers); (2) the RPA amplicon hybridizes with the capture probe. After a washing step, the HRP-labelled reporter probe hybridizes with the RPA product; and (3) once TMB/H₂O₂ enzymatic substrate is added, reduction of TMB is measured by chronoamperometry. (B) Representative gel electrophoresis confirming the successful RPA amplification of the tailed target (148 bp dsDNA + 35 bp ssDNA). (C) Chronoamperometric results using Autolab and the portable potentiostat for positive (1 pM target synthetic DNA) and blank (nuclease-free water) samples for gold working electrode 1 (WE1) and 2 (WE2). Chronoamperometry aggregated data showing a statistically significant between groups (n = 5) at p-value ≤0.05 (unpaired student's t test).