Nicking enzyme-free strand displacement amplification-assisted CRISPR-Cas-based colorimetric detection of prostate-specific antigen in serum samples.

Immunosorbent assay is the gold standard diagnostic technique for the detection of protein biomarkers. However, this technique tends to have low sensitivity and requires laborious manipulation. Although advanced CRISPR-Cas-based biosensors offer advantages of simplicity, low cost and high accuracy, the synergy of using CRISPR-Cas-assisted dual signal amplification system for rapid diagnosis of protein biomarkers remains scarce. In this work, we report a synergetic signal amplification system comprising CRISPR-Cas12a and nicking enzyme-free strand displacement amplification (SDA) technique for accurate detection of prostate-specific antigen (PSA). The presence of PSA will initiate the nicking enzyme-free SDA process, generating amplicons that can be recognized by the CRISPR-Cas12a system. The activated CRISPR-Cas system will then mediate trans-ssDNA cleavage of neighboring linker DNA, which unlocks the gold nanoparticles (AuNPs) signal probes and gives a distance-dependent colorimetric readout. This assay could detect PSA in aqueous buffer sensitively and selectively with a limit of detection (LOD) down to 0.030 ng mL. Importantly, this assay was successfully applied for discriminating four blood samples from prostate cancer patients among thirteen blood samples from normal individuals/cancer patients accurately. This work will open an avenue for the development of SDA-CRISPR-AuNPs hybrid sensing systems, offering great potential for the development of non-invasive point-of-care diagnostic tools for prostate cancer.