A CRISPR-Cas12a System Integrated with Metal-Enhanced Light-Up Aptamer-Fluorophore Nanoreporter for Ultrasensitive Detection of Prostate-Specific Antigen.

In this study, a CRISPR-Cas12a-based biosensing system integrated with metal-enhanced light-up aptamer fluorescence (MELAF) nanoreporters was developed for ultrasensitive detection of prostate-specific antigen (PSA). Here, the MELAF nanoreporters are constructed with a core-shell architecture consisting of a gold nanorod core, a silver inner shell, a mesoporous silica spacer, and surface linked light-up DNA aptamer-fluorogen complexes, enabling cascade fluorescence enhancement. This cascade fluorescence enhancement is accomplished through a two-stage process: (i) aptamer-fluorogen binding restricts intramolecular rotation, thereby activating fluorogen emission; and (ii) spectrally and spatially optimized Au@Ag core-shell structure provides plasmonic amplification, further boosting the fluorogen signal. In the presence of PSA, the PSA-specific aptamers preferentially bind the antigen, thereby blocking activation of the CRISPR-Cas12a system and preserving the "On" fluorescence state of the nanoreporter. In the absence of PSA, unbound PSA-specific aptamers activate the CRISPR-Cas12a system, inducing trans-cleavage of the MELAF nanoreporter and simultaneously abolishing both the light-up effect and plasmonic enhancement, which leads to a pronounced reduction in fluorescence. As a proof of concept, the platform enables rapid (approximately 75 min) and highly sensitive detection of PSA with a limit of detection of 0.36 pg/mL. The assay exhibits excellent specificity and robustness in complex biological matrices, and measurements in clinical specimens demonstrate high accuracy and diagnostic utility.