Dual-mode biosensors for ultrasensitive detection of Legionella pneumophila using CRISPR/Cas12a integrated recombinase polymerase amplification

Legionella pneumophila (L. pneumophila) can cause respiratory disease in humans when it infects water sources, posing a major challenge to public health and the environment. Herein, we further explore the potential application of CRISPR/Cas12a and develop CRISPR/Cas12a-drived fluorescent and colorimetry dual-mode biosensors for detection L. pneumophila. In this design, Amplified part not only realizes fast and convenient operation, but also avoids aerosol pollution. Bi2Fe4O9 NSs have excellent colorimetric performance and Bi2Fe4O9 NSs-coated DNA hydrogel can serve as independent unit. Experimental results demonstrate that the limit of detection (LOD) for the fluorescent biosensor is 7.606 × 10−6 ng/μL (3.8 copies/μL) with 60 min, which is 10,000 times better than the industry standard (SN/T 2528 - 2010). While the colorimetry achieves a LOD is 7.606 × 10−3 ng/μL (3800 copies/μL) with 100 min, which is 10 times better than the industry standard. In the actual sample test, the selectivity of the detection scheme is 100%, and the positive detection rate is higher than the industry standard 10.71%. We take the lead in integrating RPA and CRISPR/Cas12a for detecting L. pneumophila, which has been further broadened the detection strategy by incorporating colorimetric sensing. Moreover, this biosensor demonstrates excellent repeatability and strong universality, along with practical applicability for various water environment monitoring purposes.