Next-generation biosensing tools based on CRISPR/Cas
have revolutionized
the molecular detection. A number of CRISPR/Cas-based biosensors have
been reported for the detection of nucleic acid targets. The establishment
of efficient methods for non-nucleic acid target detection would further
broaden the scope of this technique, but up to now, the concerning
research is limited. In the current study, we reported a versatile
biosensing platform for non-nucleic acid small-molecule detection
called SMART-Cas12a (small-molecule aptamer regulated test using CRISPR/Cas12a).
Simply, hybridization chain reaction cascade signal amplification
was first trigged by functional nucleic acid (aptamer) through target
binding. Then, the CRISPR/Cas system was integrated to recognize the
amplified products followed by activation of the trans-cleavage. As such, the target can be ingeniously converted to nucleic
acid signals and then fluorescent signals that can be readily visualized
and analyzed by a customized 3D-printed visualizer with the help of
a home-made App-enabled smartphone. Adenosine triphosphate was selected
as a model target, and under the optimized conditions, we achieved
fine analytical performance with a linear range from 0.1 to 750 μM
and a detection limit of 1.0 nM. The satisfactory selectivity and
recoveries that we have obtained further demonstrated this method
to be suitable for a complex sample environment. The sample-to-answer
time was less than 100 min. Our work not only expanded the reach of
the CRISPR-Cas system in biosensing but also provided a prototype
method that can be generalized for detecting a wider range of analytes
with desirable adaptability, sensitivity, specificity, and on-site
capability.
It is imperative to develop practicable pathogenic bacteria detection methods. We devised a biosensor for the ultrasensitive detection of Salmonella typhimurium (S. typhi), termed as SCOUT‐dCas9 (ultrasensitive, cross‐validating, on‐site, and dUal‐mode test using CRISPR/dCas9). Simply, the species‐specific invA gene of S. typhi was amplified using loop‐mediated isothermal amplification with a biotinylated primer, which can be specifically “pulled down” by “antibody‐like” dCas9‐single guide RNA to form a ternary complex. SYBR Green I and streptavidin‐modified alkaline phosphatase were used to functionalize them to generate fluorescent and colorimetric signals, respectively. With this strategy, the target could be dexterously converted into bimodal signals that were cross‐validated to afford more reliable results. For both modes, SCOUT‐dCas9 was able to detect as low as 1 CFU/mL with a dynamic range from 1 to 109 CFU/mL. Lastly, SCOUT‐dCas9 had satisfactory selectivity and was capable of detecting S. typhi‐contaminated real food samples. SCOUT‐dCas9 provides a robust platform for bacterial detection.
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