CRISPR/Cas12a-SERS biosensor based on sea urchin-like AuNPs for the detection of β-thalassemia mutant gene CD31

Nov 14, 2025Mikrochimica acta

CRISPR/Cas12a and gold nanoparticle sensor for detecting the β-thalassemia CD31 mutant gene

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Abstract

The biosensor achieves a detection limit of 0.1 fM for the β-thalassemia mutation gene CD31 within 40 minutes.

Approximately 1.5% of the global population carries mutations associated with β-thalassemia.
The biosensor utilizes CRISPR/Cas12a technology combined with surface-enhanced Raman spectroscopy for detection.
Magnetic separation technology is employed to improve specificity and reduce interference from serum matrix.
The method shows excellent linearity for target gene detection over a concentration range of 0.1 fM to 10 pM.
This technique is simple, rapid, and demonstrates high sensitivity and specificity compared to traditional methods.

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Beta-thalassemia is a single-gene recessive disorder caused by mutations in the HBB gene, and approximately 1.5% of the global population are carriers of β-thalassemia. It is therefore vital to establish a rapid and sensitive method to detect the mutant genes of β-thalassemia. In this study, a CRISPR/Cas12a-mediated amplification-free surface-enhanced Raman spectroscopy (SERS) biosensor was developed. This biosensor uses sea urchin-shaped gold nanoparticles (SUGNPs) as the SERS enhancement substrate and 4-mercaptobenzoic acid (4-MBA) as the Raman reporter. It couples the SUGNPs/4-MBA with magnetic beads through single-strand DNA (ssDNA) to form an SERS probe with magnetic responsiveness. The presence of the β-thalassemia target mutation gene CD31 activates the cleavage activity of Cas12a, leading to non-specific cleavage of single-stranded DNA (ssDNA) on the probe. This results in a significant reduction in SERS intensity. This signal change enables quantitative detection of the target gene, thereby significantly enhancing the sensitivity of nucleic acid testing. We employed magnetic separation technology to enrich target nucleic acids in serum while eliminating matrix interference, enabling specific recognition and quantitative detection of the mutated CD31 gene. This method exhibits excellent linearity over a concentration range 0.1 fM to 10 pM, with a detection limit of 0.1 fM and a detection time of only 40 min. Compared to traditional qPCR and other CRISPR methods, this approach is simple, rapid, and offers advantages such as high sensitivity, high specificity, and cost-effectiveness. By simply replacing the crRNA, it can detect multiple β-thalassemia and other disease genes, demonstrating broad clinical application potential.

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CRISPR/Cas12a-SERS biosensor based on sea urchin-like AuNPs for the detection of β-thalassemia mutant gene CD31

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