Combining CRISPR-Cas12a with Microsphere Array-Enhanced Fluorescence for Portable Pathogen Nucleic Acid Detection

Abstract

The detection of food contamination in a swift and sensitive manner is essential for safeguarding public health. Clustered regularly interspaced short palindromic repeats (CRISPR)-based assays for nucleic acid detection are renowned for their high specificity and convenient, related studies have focused on refining the Cas protein and optimizing the CRISPR (cr)RNAs design within CRISPR-based assays for enhancing the sensitivity of nucleic acid detection. Our research offers innovative insights into enhancing the fluorescence signal output intensity from a physical standpoint, thereby presenting a practical and cost-effective strategy to lower the detection thresholds in CRISPR-based assays. By a layer of microsphere arrays was spread onto the bottom of the microfluidic chip to enhance the fluorescence signal of the sample via self-assembly of the microspheres. Recombinase polymerase amplification (RPA) was used to amplify target sequences, followed by crRNA binding to activate Cas enzyme, cleaving fluorescein amidite (FAM)-labeled reporters and emitting a fluorescent signal. The method successfully identified SARS-CoV-2 positive samples (10 clinical samples and 8 environmental contamination samples) and distinguished them from negative samples. Meanwhile, it successfully detected 4 food contamination Shigella samples and 5 clinical Shigella samples. In this study, the developed method exhibited a detection limit (LoD) of 75 fM for SARS-CoV-2 (POCT with USB camera: 50 fM) and 100 fM for Shigella (POCT with USB camera: 75 fM). It also demonstrated promising sensitivity (100%) and specificity (100%) in a small-sample validation. Combined portable and automated detection was achieved using a smartphone to receive and process the fluorescent signals obtained from the samples. The detection platform developed in this study is not only applicable for the detection of pathogens in cold-chain food products, but also extends to pathogen detection in community hospitals and resource-limited areas, providing an efficient solution for rapid pathogen screening in different settings. Moreover, different nucleic acid samples can be detected by changing the RPA primer and CRISPR crRNA. This method provides a paradigm for studying enhanced fluorescence signaling and holds significant potential to advance the commercialization and practical use of CRISPR fluorescence sensors.