Rolling circle amplification (RCA) is a linear isothermal amplification technique that is widely applied in biomolecular assays due to its high specificity. Handling of a target sample using magnetic microbeads (MMBs) in a multi-step assay is appealing as the MMBs enable separation and transportation using an external magnet. Detection of amplicons using optomagnetic measurements of the rotational diffusion properties of magnetic nanoparticles (MNPs) is also appealing as it can be performed on any transparent sample container. Two strategies are described for integration of MMB sample handling in an RCA assay with on-chip optomagnetic detection of the amplification products. The first strategy relies on selective and irreversible release of the amplicons from the MMBs so that the binding of functionalized MNPs to the amplicons can be detected optomagnetically. The second strategy relies on the incorporation of MNPs into RCA products during RCA, followed by their separation on MMBs and subsequent optomagnetic detection upon release from the RCA products. Using MMB handling of RCA steps, the limits of detection (LODs) for a synthetic DNA target representative of Victoria Influenza type B were found to be between 4 and 20 pM with total assay times between 2 and 2.5 h. Without magnetic microbead sample handling, the LOD was 200 fM. The findings provide deeper insight into the use of magnetic microbeads as solid substrates to handle a DNA target for integration of RCA as well as other DNA-based assays.
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We thank Mats Nilsson (SciLifeLab, Stockholm) for design of Influenza target and probes as well as for the routine protocols of MMB-free circularization and rolling circle amplification.
The author(s) declare that they have no competing interests.
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The work was supported by DFF project (#4184-00121B). VC thanks Erasmus+ Program (Key action 1 A.Y. 2017-18) and Roberto Raiteri for sponsoring of the Erasmus program. JF acknowledges MUDP for support (MST-141-01415).
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Minero, G.A.S., Cangiano, V., Garbarino, F. et al. Integration of microbead DNA handling with optomagnetic detection in rolling circle amplification assays. Microchim Acta 186, 528 (2019). https://doi.org/10.1007/s00604-019-3636-x
- Assay integration
- DNA capture
- DNA melting
- Magnetic microbead