Temperature-programmed microfluidic CRISPR diagnostics enable rapid and automatous point-of-care testing for syphilis

Abstract

The rising incidence of syphilis highlights the urgent need to develop molecular tests of Treponema pallidum at the point-of-care (POC). Traditional molecular approaches based on polymerase chain reaction (PCR) often involve expensive equipment, expert technicians, and lengthy turnaround time. Recently emerged approaches coupling CRISPR detection with isothermal amplification techniques hold great promise to advance the POC diagnostics, but commonly require multiple manual operations for postamplification reaction transfer or suffer from compromised performance in terms of sensitivity or assay time. To address these issues, we develop a thermometer-inspired microfluidic biosensing platform that seamlessly integrates CRISPR detection and isothermal amplification through temperature-programmed air expansion for intended fluidic manipulation. This platform not only allows for automated running of the canonical two-step recombinase polymerase amplification-CRISPR/Cas12a assay via a simple two-step temperature profile, but also provides a smartphone-enabled fluorescence readout with a limit of detection of a few DNA copies in 30 min. We detail the rational design of a thermally-responsive microfluidic cartridge and showcase how it harnesses the temperature difference in preamplification and CRISPR detection to implement the assay workflow without the need for an external driving mechanism or human intervention. Furthermore, we validate its applicability for rapid and sensitive detection of Treponema pallidum DNA in clinical samples (n = 30), exhibiting 100 % sensitivity and 100 % specificity compared to the quantitative PCR technique. The simplicity and effectiveness may lead this platform to be a powerful upgrade kit compatible with the existing CRISPR approaches, thus facilitating their widespread point-of-care diagnostic applications.