Coupling CRISPR/Cas12a and Recombinase Polymerase Amplification on a Stand-Alone Microfluidics Platform for Fast and Parallel Nucleic Acid Detection

Abstract

Timely identification of human papillomavirus (HPV) infection is crucial for the prevention of cervical cancer. Current HPV detection methods mainly rely on polymerase chain reaction (PCR), which often requires bulky equipment and a long assay time. In this work, we report a heating-membrane-assisted multiplexed microfluidics platform that couples recombinase polymerase amplification (RPA) and CRISPR technology (termed M3-CRISPR) for fast and low-cost detection of multiple HPV subtypes. The heating membrane can provide convenient temperature control for the on-chip RPA and CRISPR assays. This stand-alone system allows simultaneous detection of HPV16 and HPV18 with high specificity and detection sensitivity (0.5 nM and 1 × 10–18 M for unamplified and amplified plasmids, respectively) in 30 min with a fluorescence-based readout. Furthermore, we introduced an optimized lateral flow dipstick (LFD) into the portable system to allow visualized detection of HPV DNA. The LFD-based readout also reached a detection sensitivity of 1 × 10–18 M for amplified plasmids and realized successful detection of HPV subtypes in the clinical samples. Finally, we established an automatic microfluidic system that enables the sample-in-answer-out detection of HPV subtypes. We believe that this fast, convenient, and affordable molecular diagnostic platform can serve as a useful tool in point-of-care testing of HPV or other pathogens.

Recent progress on the CRISPR/Cas system in optical biosensors

“…Designing an integrated, multi-channel digital platform emerges as a solution to mitigate environmental influences on RNA, enabling the simultaneous detection of multiple targets and improving biosensor practicality. By designing an integrated, multi-channel digital platform, 97,128 the influence of the environment on RNA can be solved to achieve the purpose of detecting multiple targets at one time and improve the practicality of biosensors.…”

“…Moreover, they achieved the detection of three transgenes via the multiplexed microfluidic device embedded in LFD. Similar work based on RPA and Cas12a has also been used to detect HPV, 97,98 Staphylococcus aureus, 99 and drug-resistant genes. 100,101…”

Intelligent point of care testing for medicine diagnosis

“…In 2023, Zhou et al reported a heated membrane-assisted multiplex microfluidic platform that combines RPA and CRISPR technologies for rapid and cost-effective detection of multiple HPV subtypes (Figure 3A). 31 The heated membrane facilitates temperature control for RPA and CRISPR detection on the chip. This system allows simultaneous detection of HPV16 and HPV18 within 30 min, demonstrating high specificity and detection sensitivity, with fluorescence-based readout capability.…”

“…Reproduced with permission. 31 Copyright 2023, American Chemical Society. (B) Workflow for the detection of SARS-CoV-2 using the portable POC device.…”

Recent Advances in Molecular and Immunological Diagnostic Platform for Virus Detection: A Review

“…The signal was collected by fluorescence measurements or a lateral flow strip [ 119 ]. Recently, Zhou et al reported a heating-membrane-assisted multiplexed microfluidics for the fast and low-cost detection of HPV subtypes using a combination of RPA and CRISPR assays [ 120 ]. This device could simultaneously detect HPV16 and HPV18 within 30 min, and the LOD was approximately 1 × 10 –18 M using plasmids.…”