A systematic implementation of padlock probing-based rolling circle amplification in an integrated microfluidic device for quantitative biomolecular analyses

Abstract

Background

Pathogen detection in primary care is crucial not only to identify viruses like SARS-CoV-2 but also for antibiotic-resistant bacteria. While microfluidic devices enable point-of-care diagnostics, they often lack sufficient sensitivity. On-chip isothermal amplification techniques, such as padlock probing-based rolling circle amplification (PLP-RCA), can enhance specificity and sensitivity while keeping device complexity low. However, integrating PLP-RCA on-chip requires precise optimization of enzyme concentrations, flow conditions, and target capture to achieve its full potential.

Results

This study demonstrates a microfluidic RCA assay using porous agarose microbeads as a solid-phase capture, packed inside a microfluidic device. Various target capture strategies were systematically compared and quantitatively investigated, progressing from single-stranded synthetic DNA oligonucleotides to double-stranded Staphylococcus aureus genomic DNA. The best strategy for double-stranded Staphylococcus aureus genomic DNA used a primer bound to the beads that capture the PLP and the target genomic DNA. The system integrates an amorphous-hydrogenated silicon (a-Si:H) thin film p-i-n photodiode and a high-pass interference filter, enabling on-chip fluorescence signal acquisition of amplicons. This integration allows for a fully functional PLP-RCA assay on-chip, along with the added merits of device portability and compatibility with clinical demands.

Significance and Novelty

This study systematically evaluates single- and double-stranded target capture for on-chip PLP-RCA assays. It demonstrates the successful integration of microfluidics with a solid-phase capture medium and fluorescence detection system. The findings highlight the potential of this platform for developing sensitive, portable pathogen detection devices suited for clinical applications.”