A simple and versatile CRISPR/Cas12a-based immunosensing platform: Towards attomolar level sensitivity for small protein diagnostics

Recent advances in CRISPR/Cas biosensing have led to impressive performance in sensitivity, specificity, and speed for nucleic acid detection. However, the remarkable advantages (such as universality, ultralow, attomolar detection limits) of CRISPR/Cas biosensing systems are limited in testing non-nucleic acid targets. Herein, by synthesizing a functional hybrid conjugate of antibody and single strand DNA oligonucleotide, we had successfully demonstrated the capability to integrate CRISPR/Cas12a-based signal amplification into different types of immunoassay schemes without the need for any additional recognition molecule or molecular synthesis during the detection process, thus providing a simple but generally applicable approach to improve the conventional immunoassays with attomolar sensitivity for small protein detections, referred as the CRISPR-based Universal Immunoassay Signal Enhancer (CRUISE). CRUISE is capable of being integrated into various immunoassays either through the primary antibody or the secondary antibody, with sensitivity down to 1 fg mL(-1) (~50 aM) and 6 logs of linear range for detecting cytokines, such as IFN-gamma and EGFR, under 3-4 h. It has a 103 times higher sensitivity compared to a commercial IFN-gamma ELISA kit, but uses the same experimental scheme. The same 1 fg mL(-1) sensitivity along with 6 logs of linear range was realized for IFN-gamma detection in human plasma samples. We are expecting that our CRUISE provides an alternative but simple, user-friendly and effective strategy for those who rely on the use of immunoassays, while struggling with the limits of their sensitivity or detection ranges.

Automated summary

The study demonstrates the successful integration of CRISPR/Cas12a-based signal amplification into immunoassays using a functional hybrid conjugate of antibody and DNA oligonucleotide. This approach improves the sensitivity of conventional immunoassays, allowing for the detection of small proteins with attomolar sensitivity. The CRISPR-based Universal Immunoassay Signal Enhancer (CRUISE) can be applied to various immunoassays and has the potential to enhance sensitivity and expand the detection range.