Novel Multimode\nAssay Based on Asymmetrically Competitive\nCRISPR and Raman Barcode Spectra for Multiple Hepatocellular Carcinoma\nBiomarkers Detection

Commercial pregnancy test strips (PTS) possess the advantages\nof\nlower price, higher stability, and better repeatability and have been\npopularized to integrate with novel sensing strategies to detect other\ndisease biomarkers, which accelerates the commercialization process\nof those novel sensing strategies. However, the current integration\nof novel sensing strategies into commercial PTS still faced the problems\nof insufficient quantification, low sensitivity, and lack of multiple\ndetection capabilities. Hence, we proposed the concept of “visual\nclassification recognition, spectral signal subdivision” for\nmultiple hepatocellular carcinoma biomarkers (miRNA122 and miRNA233)\ndetection with dual signals based on asymmetric competitive CRISPR\n(acCRISPR) and surface-enhanced Raman spectroscopy coupling with PTS,\nnamed the acCRISPR-PTS-SERS assay. In this assay, acCRISPR was used\nas a nonamplified cascaded signal amplification method to improve\nthe sensitivity of detection. Two AuNPs-based core–shell Raman\ntags, each corresponding to different miRNA biomarkers, were used\nto achieve both visual recognition and spectral segmentation to enhance\nthe quantification of PTS detection and the capability for multiple\ndetection. Under the optimal conditions, the LOD for miRNA122 and\nmiRNA223 were 10.36 and 4.65 fM, respectively. The sensitivity was\nenhanced by nearly 2 orders of magnitude. In the future, simultaneous\nhand-held detection for fingerprint barcodes of different cancers\ncan be achieved with the assistance of a microfluidic chip and smartphone.