Open-channel microfluidic device for TiO2NTs@Fe3O4NPs-assisted viral RNA extraction and amplification-free RNA fluorescence status evaluation

• Published in: Microchemical journal 2024-11, Vol.206, p.111554, Article 111554
• Main Authors: Matejkova, Nikola, Smela, Denisa, Beranek, Martin, Capek, Jan, Michalcova, Lucie, Michalkova, Lenka, Svoboda, Jakub, Skeren, Marek, Svobodova, Zuzana, Sopha, Hanna, Macak, Jan M., Korecka, Lucie, Pacinkova, Anna, Gancarcikova, Marketa, Bolehovska, Radka, Koblizek, Vladimir, Bilkova, Zuzana
• Format: Article
• Language: English
• Subjects: Fluorescence detection; Immunocapturing; Open-channel microfluidic device; RNA extraction; RNA virus; TiO2 nanotube
• ISSN: 0026-265X
• DOI: 10.1016/j.microc.2024.111554

[Display omitted] •TiO2NTs@Fe3O4NPs-assisted open-channel device for viral RNA extraction.•Direct viral RNA detection via simple fluorescence status evaluation.•Method validated by 145 SARS-CoV-2 samples against PCR-based method.•Nanomaterial-assisted device meets the criteria of point-of-care testing. Most routine viral RNA detection assays available today rely on RNA amplification steps. The sensitivity of such methods comes at the expense of time, cost, and equipment availability. Given the constraints during the COVID-19 pandemic, such as the limited capacity of RNA amplification instruments and the global shortage of isolation kits, there has been a demand for amplification-free assays which should meet POCT criteria: affordable, sensitive, specific, user-friendly, rapid, and equipment-free. This study aims to design and validate a microanalytical system for direct viral RNA detection which meets these criteria. The presented RNA detection assay consists of four consecutive steps: immunocapturing viral particles, viral particles lysis, TiO2NTs@Fe3O4NPs-assisted RNA extraction, and finally viral RNA detection. The last three steps occur within a microfluidic device with open channels (OC-MFD). The OC-MFD is manufactured from polycarbonate film by unique mechanical replication techniques in a roll-to-roll setup. This method has been experimentally validated to offer the advantages of low-cost and extremely rapid production (1 mil. pieces of OC-MFDs per hour). Two permanent magnets situated under the application/extraction zones enabled to fix magnetically active particles/nanotubes. At first, viral particles are quantitatively captured from the entire sample volume using immunomagnetic particles. The lysed viral particles are transferred to the application zone of OC-MFD, followed by RNA extraction using magnetic TiO2 nanotubes decorated with Fe3O4 nanoparticles (TiO2NTs@Fe3O4NPs). The effectivity of the nanomaterial to attract viral RNA molecules was evaluated against a standard RT-qPCR-based laboratory test. Equivalence tests were performed with 143 positive and negative SARS-CoV-2 samples. Spearman rank correlation coefficients demonstrated high agreement for both measured biomarker genes (the E gene equaled 0.76, the RdRp gene equaled 0.73, the p-value