Micro-scale thermofluidics enable autonomous and scalable CRISPR diagnostics for sexually transmitted infections screening

The development of clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection has recently been a center of interest for next-generation molecular diagnostics. Despite considerable advances, simple and effective strategies to harness the isothermal amplification reaction and CRISPR-based detection for maximal performance and minimal complexity are still desirable. Here, a thermofluidic approach leverages the micro-scale chemical and physical mechanism to perform autonomous and scalable CRISPR-based diagnostics (CRISPR-Dx) in a greatly simplified format, which was called “Thermofluidic CRISPR”. Originating from the concept of convective PCR, it utilizes looped microchannel reactors to perform approximatively undisturbed isothermal amplification reaction at balanced temperature by virtue of the restricted molecular diffusion across the microchannel, in which the reagents of two reactions are compartmentalized virtually; then it creates circulatory flow within the loop channel to mix the amplificons and CRISPR reagents via Rayleigh–Bénard thermal convection, by simply warming up one side of the loop channel. Due to the simplicity and scalability, a low-cost, battery-powered, portable diagnostic platform, incorporating with smartphone-enabled real-time fluorescence readout, to perform rapid (<30 min), highly sensitive (2 copies per reaction), quantitative and multiplexed CRISPR-Dx was constructed. Its diagnostic performance in rapid screening of multiple pathogens from 196 clinical samples for syndromic testing of sexually transmitted infections was evaluated, exhibiting 97.4 % sensitivity and 100 % specificity benchmarked against the laboratory-based testing. Leveraging the micro-scale chemical and physical mechanism to simplify workflows for CRISPR-Dx may enhance their versatility and facilitate their broader applicability at the point of care.