Pathogenic bacteria infection severely threatens human health and causes substantial medical and financial concern. Rapid, sensitive, specific, and reliable detection of pathogenic bacteria is crucial. In the current study, a CRISPR-Cas12a-powered dual-mode biosensor was developed for ultrasensitive and cross-validating detection of pathogenic bacteria. Simply, the amplicons of Salmonella (used as a model)-specific invA sequence triggered CRISPR-Cas12a-based indiscriminate degradation of single-stranded DNAs that were supposed to link two gold nanoparticle (AuNP) probe pairs, inducing an aggregation-to-dispersion change. This generated observable color changes that became even more apparent after centrifugation. The color changes can be discerned by naked eyes and recorded using a portable colorimeter. Meanwhile, the photothermal effect of CRISPR-Cas12-powered AuNPs was explored for the first time through 808 nm near-infrared irradiation such that quantitative measurement can be carried out by recording temperatures using a thermal camera. For both modes, a limit of detection of 1 CFU/mL and a dynamic range of detection from 100 to 108 CFU/mL were obtained, which were comparable with or better than previously reported methods. Our proposed biosensor demonstrated satisfactory selectivity for Salmonella against other interfering cells. Furthermore, this biosensor proved to be capable of Salmonella detection in food samples. Regarding the real applications, the result from each mode can be used for cross-validation. Only the case having doubly confirmed positive or negative results can be assured, which rendered a more dependable biosensing conclusion. This technology not only expands the reach of the CRISPR-Cas system in biosensing but also provides a general method for bacteria sensing with desirable sensitivity, specificity, and cross-validating capacity.

CRISPR-Cas12a-Powered Dual-Mode Biosensor for Ultrasensitive and Cross-validating Detection of Pathogenic Bacteria

A CRISPR-based and post-amplification coupled SARS-CoV-2 detection with a portable evanescent wave biosensor.

Novel non-nucleic acid targets detection strategies based on CRISPR/Cas toolboxes: A review.

A smartphone-based visual biosensor for CRISPR-Cas powered SARS-CoV-2 diagnostics

Signal amplification and output of CRISPR/Cas-based biosensing systems: A review.

CRISPR/Cas system is now playing an important role in biosensing applications, especially when the trans cleavage activity of several Cas effectors is discovered. Recently, CRISPR/Cas system combining with multiple detection techniques has been developed for various targets detection, which broadens the application scope of CRISPR/Cas system and makes the system more mature. In this review, the characteristics of four commonly used Cas effectors are first analyzed. Detection strategies based on the binding activity and cleavage activity of CRISPR/Cas system are fully discussed. Various targets including nucleic acids, protein, bacteria, metal ions and small molecules can be detected by CRSIPR/Cas system. However, there are still some challenges need to be addressed in the future, like off-target effect, target sequence limitation, multiplex and quantitative detection limitation, etc. As the CRISPR technology continues to develop, the CRSIPR/Cas system with its unique glamour would provide infinite possibilities for various targets detection in the future.

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Versatile detection with CRISPR/Cas system from applications to challenges