A CRISPR/Cas12a-assisted on-fibre immunosensor for ultrasensitive small protein detection in complex biological samples

Tracking trace amounts of analytes directly from low volumes of complex biological samples remains an ongoing challenge in precision diagnostics, as the commonly used immunosorbent assays have limited sensitivity. Herein, a CRISPR/Cas12a assisted on-fibre immunosensor (CAFI) was developed based on an antibody-analyte-aptamer sandwich structure, in which a single strand DNA aptamer was applied to detect the analyte while triggering the CRISPR/Cas12a fluorescent detection system to amplify the analyte signal. This novel CAFI biosensing system was fabricated on a glass fibre surface with an antifouling PEG polymer brush modified for the detection of a spectrum of small molecules from complex media. In comparison with a conventional ELISA system, CAFI has a 1,000-fold higher sensitivity with the limit of detection for IFN-γ down to 1 fg mL-1 (58.8 aM). It also has a tuneable linear detection range that can be easily adjusted within the range 1 fg mL-1 to 100 pg mL-1 (5 orders of magnitude), meeting the requirements of the demanding diagnostic scenarios. CAFI has successfully been demonstrated by detecting IFN-γ from a diverse complex biological sample type, including human serum, whole blood, perspiration, and saliva. Moreover, CAFI is applicable for the detection of other analytes by simply modifying the capture antibody and detection aptamer, demonstrated here with insulin. All these superior capabilities of CAFI make it a suitable technology to measure proteins in low (100 μL) volume complex biological samples.