An ultrasensitive biosensor for H1N1 virus coupled with 3D spherical DNA nanostructure and CRISPR-Cas12a

Sep 8, 2025Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy

Highly sensitive H1N1 virus sensor using 3D DNA structures and CRISPR-Cas12a

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Abstract

A detection limit of 0.17 copies/μL was achieved for the H1N1 influenza virus using a novel biosensor.

The biosensor utilizes a 3D spherical DNA nanostructure combined with CRISPR/Cas12a for enhanced detection capabilities.
Binding of the H1N1 virus to specific nucleic acid aptamers triggers a change that activates the Cas12a enzyme, leading to signal amplification.
Spike recovery rates in various matrices, including chicken serum and milk, ranged from 91.89% to 104.03%, indicating reliable performance.
The total detection time is reduced to 40 minutes, which is three times faster than traditional qPCR methods.
The structural design of the 3D nanostructure contributes to its stability during storage, which was an unexpected finding.

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To achieve ultrasensitive and real-time detection of the H1N1 influenza virus, this study designed a nucleic acid-free fluorescent biosensor based on 3D spherical DNA nanostructure and CRISPR/Cas12a (3D-SDNC). The biosensor constructs a rigid 3D nano-framework via self-assembly of six oligonucleotide chains, with H1N1-specific nucleic acid aptamers and Cas12a activator strands strategically positioned at multi-spined vertices for precise spatial coupling between viral recognition and signal transduction. Upon aptamer-virus binding, the induced conformational change liberates the activator strand, thereby activating the trans-cleavage activity of the Cas12a/crRNA complex to efficiently cleave the HEX/BHQ1 double-labeled fluorescent probe and initiate cascade signal amplification. Experimental results demonstrate a detection limit of 0.17 copies/μL (S/N = 3), achieving qPCR-comparable sensitivity, with spike recovery rates of 91.89 % to 104.03 % (RSD < 5.12 %) in chicken serum, bovine serum, and milk matrices. The innovative nucleic acid-free extraction design reduces the total detection time to 40 min; its efficiency is three times higher than qPCR. Notably, we not only discovered the ultra-high sensitivity of the sensor to H1N1 but also unexpectedly found that the rigid structure of the 3D spherical DNA nanostructure conferred enhanced stability under storage conditions. This work establishes a groundbreaking molecular engineering paradigm for rapid pathogen diagnosis, combining ultrahigh sensitivity, fast response, and clinical utility.

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An ultrasensitive biosensor for H1N1 virus coupled with 3D spherical DNA nanostructure and CRISPR-Cas12a

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