Integration of CRISPR/Cas12a and a Fiber Optic Particle Plasmon Resonance Sensor for Single Nucleotide Polymorphism Detection in an Aldehyde Dehydrogenase 2 Gene

Jul 9, 2025ACS sensors

Using CRISPR and a Fiber Optic Sensor to Detect a Single DNA Change in the Aldehyde Dehydrogenase 2 Gene

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Abstract

The CRISPR-FONLISA system can detect the ALDH2 rs671 SNP with a limit of detection of 71 aM.

A combination of CRISPR/Cas12a technology and fiber optic nanogold-linked sorbent assay (FONLISA) is proposed for SNP identification.
Cas12a targets double-stranded DNA from the ALDH2 SNP and activates trans-cleavage, which amplifies the detection signal.
The method detects SNPs through a signal generated by the release of a gold nanoparticle-labeled DNA probe.
The system shows potential for accurate detection of the ALDH2 rs671 SNP using both cell lines and unamplified DNA from blood samples.

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The highly prevalent single nucleotide polymorphism (SNP, rs671) of the aldehyde dehydrogenase (ALDH2) gene in Asian populations instigates various human pathologies and thus accentuates the urgent need for effective diagnostic tools. In this study, we present an ultrasensitive biosensing method by a combination of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a with the fiber optic nanogold-linked sorbent assay (FONLISA) for precise SNP identification. This method leverages the sequence-specific recognition capability of the CRISPR/Cas system and the ultrahigh sensitivity via the dual signal enhancement mechanisms by integrating the trans-cleavage mechanism of Cas12a to amplify the signal from an activity reporter and the subsequent waveguide-enhanced nanoplasmonic absorption by a signaling reporter. In this method, Cas12a targets a double-stranded DNA from the ALDH2 SNP and then activates the degradation of the activity reporter, a free biotin-labeled single-stranded DNA probe (ssDNA), by trans-cleavage. An unhybridized complementary single-stranded DNA probe (ssDNA) labeled with a gold nanoparticle (AuNP) as the signaling reporter (AuNP@ssDNA) is subsequently released and captured by the immobilized ssDNAon the fiber core surface, resulting in a detectable nanoplasmonic absorption signal. The method also utilized an indispensable nanoplasmonic signal generator, carboxymethyl dextran-coated AuNP, to improve the preparation and bioconjugation processes. The CRISPR-FONLISA system demonstrates the ability to analyze the ALDH2 rs671 SNP from double-stranded DNA with a limit of detection of 71 aM. Furthermore, both cell lines and unamplified DNA extracted from blood samples were conducted to verify the system accuracy for ALDH2 rs671 SNP detection. b c c b

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Integration of CRISPR/Cas12a and a Fiber Optic Particle Plasmon Resonance Sensor for Single Nucleotide Polymorphism Detection in an Aldehyde Dehydrogenase 2 Gene

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