Multienzyme-nanoparticles amplification for sensitive virus genotyping in microfluidic microbeads array using Au...

Area of Science:

• Biotechnology
• Nanotechnology
• Molecular Diagnostics

Background:

• Human papillomavirus (HPV) is a common pathogen requiring sensitive detection methods.
• Existing genotyping techniques often lack the sensitivity and efficiency for early-stage disease detection.
• Microfluidic devices offer potential for miniaturized and high-throughput biological assays.

Purpose of the Study:

• To develop a novel microfluidic device for sensitive HPV genotyping.
• To utilize microbead arrays and gold nanoparticle bioconjugates for enhanced DNA detection.
• To demonstrate the capability of the on-chip system for discriminating and genotyping HPV DNA.

Main Methods:

• A microfluidic device with a microbead array was engineered, functionalized with capture probes and electron-rich proteins.
• Horseradish peroxidase (HRP)-functionalized gold nanoparticles served as labels with secondary DNA probes for sandwich hybridization.
• Signal amplification was achieved through biotin-tyramine oxidation, biotin deposition, and subsequent binding of streptavidin-labeled quantum dots.

Main Results:

• The on-chip genotyping method achieved a detection limit of 1 fmol/L for synthesized HPV oligonucleotides DNA.
• The chip-based assay demonstrated 1000 times higher sensitivity compared to off-chip methods.
• The system successfully discriminated and genotyped 10 copies/μL of HPV genomic DNA from PCR products.

Conclusions:

• The developed microfluidic device enables highly sensitive detection and genotyping of target DNA.
• The on-chip approach significantly enhances assay sensitivity through signal amplification strategies.
• This technology holds promise for early detection of disease-related biomolecules.