Sensitive and accurate detection of protein biomarkers is crucial for disease diagnosis, especially for early diagnosis. Here, we describe surface-enhanced Raman scattering (SERS)-based CRISPR/Cas12a assays (S-CRISPR) for protein biomarker detection. Firstly, an S-CRISPR-driven enzyme-linked immunosorbent assay (S-CasLISA) was developed utilizing a capture antibody coated on a microplate to recognize the target and a detection antibody labeled with active DNA to trigger the activity of CRISPR/Cas12a. With this assay, we achieved detection of prostate-specific antigen (PSA) as models at the picogram level. The limit of detection (LoD) of S-CasLISA was 0.17 pg mL-1 and in the range of 0.1 pg mL-1 to 10 ng mL-1. Further, we applied aptamer to S-CRISPR (S-Apt-CRISPR), combining the high sensitivity of SERS with the high selectivity of aptamers, while simplifying the operation process of CRISPR detection of protein biomarkers. The proposed S-Apt-CRISPR also could detect picogram-level PSA and without repeated washing steps. The LoD of S-Apt-CRISPR was 0.35 pg mL-1 and in the range of 0.1 pg mL-1 to 10 ng mL-1. Both SERS-based CRISPR/Cas12a assays were validated with clinical samples and demonstrated accuracy consistent with the chemiluminescence immunoassay. The introduction of the CRISPR/Cas12a system with SERS has the effect of improving the analytical capabilities of the system, thereby broadening and facilitating its application in the analysis of sensitive and accurate protein biomarkers.
Keywords: Aptamer; CRISPR/Cas12a; Enzyme-linked immunosorbent assay; Protein biomarker detection.
© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.