RESUMEN
Breast cancer, the most common malignant tumor in the world, seriously threatens human life and health. Early diagnosis of breast cancer may help enhance the survival rate. In this work, a colorimetric and fluorescent dual-mode biosensor based on the CRISPR-Cas12a system was constructed to detect the breast cancer biomarker BRCA1. The intact G4 DNA, with the assistance of K+ and hemin, catalyses the oxidation of o-phenylenediamine (OPD) with the assistance of hydrogen peroxide (H2O2), generating the oxidation product 2,3-diaminophenazine (DAP), which has distinct absorption and fluorescence peaks. The presence of the target BRCA1 activates the trans-cleavage activity of CRISPR-Cas12a, leading to the cleavage of G4 DNA and inhibiting the catalytic oxidation of OPD. Target BRCA1 was quantitatively determined by measuring both the absorbance and fluorescence intensity of DAP. The detection limits were calculated to be 0.615 nM for the colorimetric method and 0.289 nM for the fluorescence method. The dual-mode biosensor showed good selectivity and reliability for BRCA1 and can resist interference from complex substrates, and it has great potential in biomedical detection.
RESUMEN
Breast cancer is reported to be one of the most lethal cancers in women, and its multi-target detection can help improve the accuracy of diagnosis. In this work, a cluster regularly interspaced short palindromic repeats (CRISPR)-Cas13a/Cas12a-based system was established for the simultaneous fluorescence detection of breast cancer biomarkers circROBO1 and BRCA1. CRISPR-Cas13a and CRISPR-Cas12a were directly activated by their respective targets, resulting in the cleavage of short RNA and DNA reporters, respectively, thus the signals of 6-carboxyfluorescein (FAM) and 6-carboxy-xrhodamine (ROX) were restored. As the fluorescence intensities of FAM and ROX were dependent on the concentrations of circROBO1 and BRCA1, respectively, synchronous fluorescence scanning could achieve one-step detection of circROBO1 and BRCA1 with detection limits of 0.013 pM and 0.26 pM, respectively. The system was highly sensitive and specific, holding high diagnostic potential for the detection of clinical samples. Furthermore, the competing endogenous RNA mechanism between circROBO1 and BRCA1 was also explored, providing a reliable basis for the intrinsic regulatory mechanism of breast cancer.
