Immunomagnetic Separation Combined with RCA-CRISPR/Cas12a for the Detection of <i>Salmonella typhimurium</i> on a Figure-Actuated Microfluidic Biosensor

Abstract

A figure-actuated microfluidic biosensor was developed for the rapid and sensitive detection of <i>Salmonella typhimurium</i> using immunomagnetic separation to separate target bacteria and rolling circle amplification (RCA) combined with CRISPR/Cas12a to amplify the detection signal. The magnetic nanoparticles (MNPs) modified with the capture antibodies (MNPs@Ab₁) and RCA primer linked with recognized antibodies (primer@Ab₂) were first used to react with <i>S. typhimurium</i>, resulting in the formation of MNPs@Ab₁-<i>S. typhimurium</i>-primer@Ab₂ complexes. Then, the RCA and CRISPR/Cas12a reagents were successively pumped into the chamber and incubated at the appropriate conditions. With the help of a 3D-printed signal detector, the fluorescence signal was collected and analyzed using the smartphone APP for the determination of bacterial concentration. This biosensor exhibited a wide linear range for the detection of <i>S. typhimurium</i> with a low limit of detection of 1.93 × 10² CFU/mL and a mean recovery of about 106% in the spiked milk sample.