Amplification-free CRISPR/Cas12a biosensor integrating AuNPs-mediated surface plasmon resonance for human papillomavirus detection and genotyping

Sep 6, 2025Biosensors & bioelectronics

CRISPR/Cas12a biosensor using gold nanoparticles and surface plasmon resonance for detecting and identifying human papillomavirus

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Abstract

The platform achieves a detection limit of 300 aM for HPV DNA.

An innovative platform combines CRISPR/Cas12a with alkaline phosphatase to detect high-risk HPV.
Sensitivity is enhanced by 10,000-fold compared to previous detection methods using p-nitrophenyl phosphate.
The system can specifically identify nine HPV subtypes targeted by the nine-valent HPV vaccine.
Results are generated within 2.5 hours and validated with cervical swab samples.
The method is designed to be simple, portable, and cost-effective, suitable for resource-limited settings.

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Screening for high-risk human papillomavirus (hrHPV) infection is essential for cervical cancer prevention. However, developing a simple, portable, and low-cost hrHPV genotyping method remains challenging, particularly in resource-limited settings. Herein, we present an innovative amplification-free, point-of-care hrHPV genotyping platform integrating CRISPR/Cas12a with alkaline phosphatase (ALP)-mediated surface plasmon effect. The platform detects HPV DNA through the Cas12a-crRNA complex recognition, activating the cleavage of ALP-labeled oligonucleotides within microwells and releasing ALP. The output signal is generated by changes in surface plasmon resonance of gold nanoparticles (AuNPs) induced by the ALP-mediated reaction of AuNPs with p-aminophenyl phosphate. This ALP-integrated CRISPR/Cas12a biosensing strategy enhances sensitivity by 10,000-fold compared to Cas12a-based detection integrating ALP-mediated p-nitrophenyl phosphate (p-NPP) hydrolysis. This approach allows the sensitive detection of HPV DNA with a detection limit of 300 aM. Moreover, integration with microplate separation allows specifically screen for the nine HPV subtypes targeted by the nine-valent HPV vaccine within 2.5 h. The platform's performance is validated using cervical swab samples, confirming its accuracy for HPV genotyping. Overall, this strategy provides a simple, portable, and cost-effective solution for multiplex nucleic acid targets detection without preamplification or instrumentation.

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Amplification-free CRISPR/Cas12a biosensor integrating AuNPs-mediated surface plasmon resonance for human papillomavirus detection and genotyping

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