CRISPR-initiated exponential amplification on fluorescently-barcoded microspheres for deep learning-assisted...

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostics

Background:

• Multiplexed nucleic acid testing is crucial for human health but faces challenges in speed and cost.
• CRISPR-Cas systems offer high specificity but integration into near-patient multiplexed platforms is limited.

Purpose of the Study:

• To develop a novel biosensing platform for multiplexed detection of human papillomavirus (HPV) DNA.
• To integrate CRISPR-initiated amplification with quantum-dot encoded microbeads and deep learning for automated analysis.

Main Methods:

• Utilized CRISPR/Cas9 for specific initiation of exponential nucleic acid amplification.
• Employed quantum-dot encoded microbeads for localized fluorescent readout of amplified products.
• Implemented a custom deep-learning algorithm for automated quantification of bead fluorescence.

Main Results:

• Achieved simultaneous detection of HPV16, HPV18, and HPV33 DNA.
• Demonstrated low detection limits as low as 0.2 pM for HPV DNA.
• Significantly simplified the workflow through recognition-triggered amplification and deep-learning assisted readout.

Conclusions:

• The developed platform offers a universal and practical strategy for molecular diagnostics.
• The integrated approach shows strong potential for enabling near-patient testing applications.
• This method enhances the accessibility and efficiency of multiplexed nucleic acid detection.