A Laminated Paper chip for Field-Deployable One-Pot CRISPR Diagnostics

Abstract

Point-of-care molecular diagnostics are needed to provide rapid and sensitive testing outside centralized laboratories. Although lateral flow immunoassays are simple, widely deployable, and suitable for mass production, their analytical sensitivity is generally lower than that of nucleic acid amplification based assays. Molecular diagnostics offer high sensitivity and specificity, but their implementation in broadly distributable formats remains challenging because of complex handling, contamination risk, evaporation, and limited manufacturing scalability. This study presents a laminated paper chip for one-pot RPA–CRISPR based molecular detection, designed to combine the deployability of paper based platforms with the sensitivity of nucleic acid detection and the mass producibility of lamination based manufacturing. The chip incorporated a paper membrane sealed with laminating film, together with an inlet, outlet, PTFE membrane, and observation window for passive fluid delivery, air venting, and fluorescence readout. Evaluation of membrane materials, laminating films, and primer sets identified glass fiber membrane, polypropylene laminating film, and an appropriate primer/crRNA set for the paper chip format. Using the selected configuration, the laminated paper chip produced target-dependent fluorescence and distinguished 10 copies/µL of SARS-CoV-2 RNA from the no template control. Further analysis to guide performance improvement indicated that the lower performance than tube-based reactions was mainly associated with reduced RPA amplification in the paper chip. The laminating film adhesive layer was considered a potential contributor, while BSA showed the greatest potential among the tested blocking reagents. These results demonstrate the feasibility of the laminated paper chip for sensitive molecular detection. With further optimization, the platform could serve as a mass-producible and widely deployable point-of-care molecular diagnostic system.