Click chemical ligation-enabled digital particle counting for multiplexed microRNA analysis

Area of Science:

• Biomedical Engineering
• Nanotechnology
• Molecular Biology

Background:

• Digital counting assays offer sensitive biomarker analysis at the single-molecule level.
• Traditional digital counting biosensors often require complex amplification techniques and specialized instruments.
• MicroRNA (miRNA) detection is crucial for biological research and diagnostics.

Purpose of the Study:

• To develop a simple, sensitive, and versatile digital counting platform for microRNA (miRNA) analysis.
• To overcome limitations of traditional methods by employing click chemistry for nanoparticle immobilization.
• To enable multiplexed and enzyme-free detection of miRNA targets.

Main Methods:

• Utilized miRNA-templated click chemical ligation to anchor quantum dot-doped nanoparticles (QDNPs) to microplate wells.
• Employed a common wide-field fluorescence microscope for direct counting of QDNPs.
• Implemented an enzyme-free cycling click ligation strategy for enhanced sensitivity.
• Leveraged the tunable emission spectra of QDNPs for multiplex detection.

Main Results:

• Achieved enhanced stability and higher sensitivity compared to traditional sandwich hybridization methods.
• Reached a low detection limit of 8 fM for miRNA.
• Demonstrated simultaneous detection of multiple miRNA targets without cross-interference.
• Successfully analyzed miRNA in complex biological samples.

Conclusions:

• The developed platform offers a simple, sensitive, and generalizable approach for digital miRNA counting.
• This method pioneers a new route for digital counting assays with potential in miRNA-related research.
• The enzyme-free, click chemistry-based approach provides a robust alternative for biomarker quantification.