Single-Layer Radially Compartmentalized Paper Chip (RCP-Chip) for Rapid Isothermal Multiplex Detection of SARS-CoV-2 Gene Targets

Abstract

The study presents the development of a radially compartmentalized paper chip (RCP-Chip) designed for the rapid detection of multiple gene targets of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay. The RCP-Chip features reaction chambers for multiplexing, fluidic resistors to prevent backflow, fluidic ports for excess fluid venting, and an outer ring to contain overflowing fluids, all within a single-layer cellulose paper platform. Optimization results successfully achieve a color readout sensitivity of 10 copies µL⁻¹ RNA spiked in water (singleplex device) and 2040 copies µL⁻¹ RNA spiked saliva samples (multiplex device). The chip demonstrates the capability to simultaneously detect the envelope (E) and nucleocapsid (N) genes of SARS-CoV-2 in a single run. Remarkably, the RCP-Chip technology enables rapid qualitative amplification of SARS-CoV-2 RNA in as little as 4 min using pH-based assay. It further enhances this visual detection by implementing in situ synthesis of gold nanoparticles (AuNPs) within the single-layer paper platform, achieving a total assay time of 9 min. The adaptability of RCP-Chip can extend beyond SARS-CoV-2 detection: It can be modified and optimized for detecting gene targets of other microbial pathogens across diverse environments pertaining to cause diseases.