An amplification-free CRISPR-SERS biosensor for specific, sensitive and rapid detection of Salmonella Typhimurium in poultry

Abstract

CRISPR (Clustered regularly interspaced short palindromic repeats) has been a cutting-edge biotechnology in both gene editing and biosensing due to its desirable features, such as high base resolution and set-and-forget operation. However, as a nucleic acid detection method, an DNA/RNA amplification procedure is still inevitable in CRISPR biosensors. In this study, a highly-ordered nanopore array SERS substrate, coated with silver nanoparticles, was fabricated and utilized as a platform for the specific detection of Salmonella Typhimurium (S. Typhimurium) without the time-consuming DNA amplification step. The smart sensing strategy was established by introducing an ssDNA labeled with a Raman active molecule as a signal report probe to show the changes during target identification and detection. The CRISPR-SERS biosensor was developed by combining the ultra-sensitive sensing mechanism of CRISPR technology with the SERS analytical method, aiming at detecting the invasin A gene (invA) of S. Typhimurium. The sensing materials and operation conditions were optimized to maximize the output signal and minimize the cost. The results showed that the proposed biosensor could specifically detect S. Typhimurium with a limit of detection (LOD) of 110 CFU/mL in poultry samples within 2 h. This CRISPR-SERS sensing strategy could provide an alternative way for pathogen detection in food safety monitoring.