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

Caneira CRF, Rosa RR, Chu V, Nilsson M, Madaboosi N, Soares RRG, Conde JP

Anal. Chim. Acta 1351 (-) 343834 [2025-05-15; online 2025-02-23]

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. 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. 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."

PubMed 40187866

DOI 10.1016/j.aca.2025.343834

Crossref 10.1016/j.aca.2025.343834

pii: S0003-2670(25)00228-4