DNA triangular prism nanostructure and CRISPR/Cas12a empowered electrochemical biosensor for dual detection of alpha-fetoprotein and microRNA 122

Jun 15, 2025Biosensors & bioelectronics

Electrochemical sensor using DNA nanostructures and CRISPR technology for detecting alpha-fetoprotein and microRNA 122

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Abstract

An electrochemical biosensor can sensitively detect HCC biomarkers miRNA 122 and alpha-fetoprotein (AFP) in real serum samples.

The biosensor utilizes a DNA triangular prism nanostructure integrated with a signal amplification strategy involving DNAzyme, hybridization chain reaction, and CRISPR/Cas12a.
Initial electrochemical signals are generated through the formation of G-quadruplex/hemin complexes facilitated by target recognition.
The activation of CRISPR/Cas12a leads to a significant decrease in current signal, indicating successful detection.
The DTP nano-framework improves nucleic acid hybridization and reduces nonspecific adsorption, enhancing detection sensitivity.
The platform shows versatility, allowing for sensitive detection through minimal sequence modifications while maintaining the same amplification system.

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Developing sensitive multi-target-combined detection method is of great significance for the early and accurate diagnosis and prognosis of hepatocellular carcinoma (HCC). Herein, an electrochemical biosensor was proposed for detecting HCC biomarkers miRNA 122 and alpha-fetoprotein (AFP), employing a DNA triangular prism (DTP) nanostructure as an efficient supporting platform integrated with a DNAzyme-hybridization chain reaction (HCR)-CRISPR/Cas12a triple signal amplification strategy. The ssDNA portion at the top of the DTP hybridized with ssDNA (S6) rich in guanine, thereby capturing hemin and forming G-quadruplex/hemin complexes, generating a strong initial electrochemical signal. Target recognition process released Mg-dependent DNAzyme, which cleaved the substrate hairpin DNA and produced trigger for HCR. The product of HCR activated CRISPR/Cas12a, causing non-specific cleavage of S6 and the ssDNA portion of DTP, which hindered the formation of G-quadruplex/hemin and led to a significant decrease in current signal. The rigid, stable, and size-controllable DTP nano-framework enhanced nucleic acid hybridization and signal molecule binding while minimizing nonspecific adsorption, eliminating masking agent requirements, with its ordered assembly facilitating CRISPR/Cas12a access to ssDNA for improved cleavage efficiency and detection sensitivity. Additionally, the programmable biosensing platform enabled sensitive detection of miRNA 122 and AFP through minimal sequence modifications in target recognition, while maintaining the identical cascade amplification system and DTP framework, demonstrating excellent versatility. Meaningfully, the biosensor sensitively detected miRNA 122 and AFP in real serum samples, and effectively distinguished healthy individuals from HCC patients, indicating its enormous potential for clinical diagnosis. 2+

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DNA triangular prism nanostructure and CRISPR/Cas12a empowered electrochemical biosensor for dual detection of alpha-fetoprotein and microRNA 122

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