Nucleic acid detection is crucial for early disease diagnosis and related clinical applications. Exploring nucleic acid amplification-free yet highly sensitive detection strategies is of significance for the development of accurate and user-friendly point-of-care testing devices. Herein, a modularized universal electrochemical sensing strategy was proposed based on the synergistic effect of CRISPR/Cas12 system and nanoporous materials. The trans -cleavage activity of Cas12a, which was activated by HPV-16 target, was utilized to cut the gatekeepers of mesoporous silica nanoparticle nanocontainers. The released methylene blue was entrapped by a mercaptobenzoic acid functionalized nanoporous gold (NPG) sensing platform. By virtue of the excellent electrocatalytic ability of NPG, ultrasensitive detection of HPV-16 was achieved over a broad linear range of 7 orders of magnitude with a detection limit as low as 0.41 fM. Moreover, determination of HPV-18 was easily achieved by only altering crRNA, revealing the remarkable universality of this strategy and its superiority of modularized design. The practical applicability of the proposed sensing strategy was verified in terms of specificity, stability, and complex biological sample analysis, demonstrating its promising prospects in the development of reliable, universal, robust, and user-friendly point-of-care diagnostic devices.
Yinghao He,Yinhuan Liu,Lingjun Cheng,Yuanyuan Yang,Bin Qiu,Longhua Guo,Yan Wang,Zhenyu Lin,Guolin Hong
Most DNA-based electrochemiluminescence (ECL) biosensors are established through the self-assembly of thiolated single-stranded DNA (ssDNA) probes on the Au electrode surface. Because of this random assembly process, a significant discrepancy exists in the distribution of a modified DNA film on different electrodes, which greatly affects the reproducibility of a biosensor. In this study, a porous bovine serum albumin (BSA) layer was first modified on the electrode surface, which can improve the position distribution and spatial orientation of the self-assembly ssDNA probe. It was then coupled with hyperbranched rolling circle amplification to develop the high-reproducibility-and-sensitivity ECL biosensor for human papillomavirus 16 E6 and E7 oncogene detection. In the presence of the target DNA, the surface of the electrode accumulates abundant amplified products through reaction, which contain double-stranded DNA (dsDNA) fragments of different lengths, followed by plentiful dichlorotris (1,10-phenanthroline) ruthenium(II) hydrate (Ru(phen)32+, acting as an ECL indicator) insertion into grooves of dsDNA fragments, and a strong signal can be detected. There is a linear relationship between the signal and the target concentration range from 10 fM to 15 pM, and the detection limit is 7.6 fM (S/N = 3). After the BSA modification step, the relative standard deviation was reduced from 9.20 to 3.96%, thereby achieving good reproducibility. The proposed ECL strategy provides a new method for constructing high-reproducibility-and-sensitivity ECL biosensors.
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