Development of a PNA-DiSc2 based portable absorbance platform for the detection of pathogen nucleic acids.

We report the development of a portable absorption (PortAbs)-based pathogen nucleic acid detection system using peptide nucleic acid (PNA) and a cyanine dye, DiSc2(5). When the dye binds to the PNA-DNA hybrid, it results in a characteristic ∼110 nm shift in the dye absorbance, which we measure using PortAbs. The protocol involves amplification of the target DNA, PNA-DNA hybridization and dye complexing steps followed by absorption measurement. The system is built using a broad-spectrum photodiode whose output is amplified and then measured by a high resolution (24 or 32 bit) analog-to-digital converter. The excitation pulses of light are delivered by a color-changing LED. The sequence of excitation, measurement and display of results are all controlled by an embedded Raspberry-Pi board (or alternatively a laptop). At higher concentrations of the target amplicon (∼200 ng), the color change can be detected visually. At lower concentrations, PortAbs outperforms a plate reader and can detect target DNA as low as 30 ng or approximately 10 nM which is at least 10 fold better than previously reported studies. We validate the methodology using SARS-CoV-2 clinical samples containing about 1000 copies of the viral RNA and show that the entire workflow takes about 90 min. The cost of the complete standalone system is less than INR 40 000 (approx. 500 USD).

Electrochemical sensing of SARS-CoV-2 amplicons with PCB electrodes

We present a low-cost electrochemical DNA biosensor based on printed circuit board (PCB) electrodes for wastewater monitoring using portable PCR instruments, such as miniPCR®, without the requirement for qPCR reagents. PCB electrodes are attractive candidates for low-cost and sensitive DNA biosensors of relevance in a pandemic such as COVID-19, and facilitate the opportunity to map disease spread in Low-Middle Income Countries (LMICs) through monitoring of environmental samples such as wastewater. The biosensor reported in this work is capable of detecting PCR amplicons through the intercalation of methylene blue (MB) with DNA, which increases the voltammogram peak current at the redox potential of MB. We describe how these changes are likely to result from the adsorption of MB-DNA complex on the electrode surface. The electrodes are reusable, easy to clean, do not undergo any surface modification and represent a cost-effective solution with long shelf-life. We also explore the impact that MB concentration and DNA length have upon our biosensor performance and provide insights useful to other investigators in the field. The sensor reported here is capable of detecting SARS-CoV-2 nucleocapsid gene amplicons at concentrations as low as 10pg/μ l (approximately 1.7fM) and can detect nucleotides amplified after 10 PCR cycles. Furthermore, using the PCB electrode and approaches described here, SARS-CoV-2 amplicons were detected in simulated wastewater sample, by spiking wastewater collected from a sewage treatment plant in Mumbai, India with SARS-CoV-2 RNA.