Solid State Biosensor for The Detection of Nucleic Acid Amplification

Abstract

Rapid detection of infectious pathogens has been a priority set by the World Health Organization in terms of their ASSURED agenda for developing biosensors. Developing an affordable, sensitive, selective, user-friendly, and reliable disease diagnostic tool is the need of the hour. In this context, nucleic acid amplification of genomic DNA or plate growth assay and immunocapture by aptamer/antibody are routine tests performed to identify causative pathogens. The readout of nucleic acid amplification is typically done using agarose gel electrophoresis and real-time fluorescence monitoring. However, these processes are usually lengthy and sophisticated involving high-end instrumentations (real-time fluorescence reader). Even with vast progress made in terms of reducing the size and cost of thermal cyclers, the means to get a rapid readout remains unmet. Approaching the diagnosis from an electrochemistry pathway is a relatively economical, decentralized, and yet highly sensitive route. This work combines the electrochemical sensing approach with isothermal nucleic acid amplification to achieve an efficacious and robust biosensing device with the aid of an electrochemically active redox probe. The work uniquely employs transition metal oxide (TMO) which is a relatively less explored class of material for nucleic acid amplification detection. TMO having a high degree of tunability in terms of electrical, morphological, and electrochemical properties proved to be an ideal candidate for applications. The TMO based electrochemical bi-electrode sensing devices (EBSD) were fabricated by a cost-effective method and further characterized using several techniques such as XRD, XPS, SEM, and electrical measurements. The optimized electrodes were utilized for sensing end-point nucleic acid amplification using voltammetry, electrochemical impedance spectroscopy (EIS), and amperometry techniques. The devices were tested for sensing various viral and bacterial nucleic acid sequences including dengue virus sequence DNA, Staphylococcus aureus genomic DNA, plasmid DNA and in vitro transcribed SARS-CoV-2 RdRp RNA. The performance was observed to be reliable and comparable to the standard techniques (standard quantitative real-time polymerase chain reaction (qPCR) fluorescence and electrochemical detection using screen-printed electrode). This work provided an economical, and straightforward approach for NAATs with the potential of scaling up via batch-processing.