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

Menglu Gao1, Chen Yang2,3, Wu Si1

  • 1Department of Laboratory Medicine, Center for Single-Cell Omics and Tumor Liquid Biopsy, Zhongnan Hospital of Wuhan University, Wuhan 430071, China.

ACS Applied Materials & Interfaces
|March 28, 2025
PubMed
Summary
This summary is machine-generated.

This study enhances fluorescence signal intensity for Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based assays by incorporating microsphere arrays. This innovation improves pathogen detection sensitivity and enables rapid, cost-effective screening for diseases like COVID-19 and Shigella.

Keywords:
CRISPRfluorescence enhancementmicrospheresnucleic acid detectionpathogen detection

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Area of Science:

  • Biotechnology and Biosensing
  • Molecular Diagnostics
  • Food Safety

Background:

  • Public health relies on swift and sensitive detection of food contamination and pathogens.
  • CRISPR-based nucleic acid detection assays offer high specificity but require sensitivity enhancement.
  • Existing research focuses on Cas protein refinement and crRNA optimization for improved CRISPR assay sensitivity.

Purpose of the Study:

  • To introduce a novel strategy for enhancing fluorescence signal output in CRISPR-based assays from a physical perspective.
  • To develop a practical and cost-effective method for lowering detection thresholds in CRISPR assays.
  • To create a versatile platform for rapid pathogen detection applicable in various settings.

Main Methods:

  • Integrated microsphere arrays into a microfluidic chip to amplify fluorescence signals via self-assembly.
  • Employed Recombinase Polymerase Amplification (RPA) for target sequence amplification.
  • Utilized CRISPR-Cas enzyme activity on FAM-labeled reporters triggered by crRNA binding for signal generation, detected using a smartphone.

Main Results:

  • Successfully identified SARS-CoV-2 and *Shigella* samples, including clinical and food contamination sources.
  • Achieved low limits of detection (LoD): 75 fM for SARS-CoV-2 and 100 fM for *Shigella* (further improved with POCT USB camera).
  • Demonstrated 100% sensitivity and specificity in small-sample validation, with combined portable and automated detection.

Conclusions:

  • The developed method provides an efficient and cost-effective strategy to enhance fluorescence signaling in CRISPR-based assays.
  • The platform is suitable for pathogen detection in cold-chain food, community hospitals, and resource-limited areas.
  • This approach offers a paradigm for advancing CRISPR fluorescence sensor commercialization and practical application across diverse settings.