Optomagnetic Detection of Rolling Circle Amplification Products.

Rolling circle amplification (RCA) of a synthetic nucleic acid target is detected using magnetic nanoparticles (MNPs) combined with an optomagnetic (OM) readout. Two RCA assays are developed with on-chip detection of rolling circle products (RCPs) either at end-point where MNPs are mixed with the sample after completion of RCA or in real time where MNPs are mixed with the sample during RCA. The plastic chip acts as a cuvette, which is positioned in a setup integrated with temperature control and simultaneous detection of four parallel DNA hybridization reactions between functionalized MNPs and products of DNA amplification. The OM technique probes the small-angle rotation of MNPs bearing oligonucleotide probes complementary to the repeated nucleotide sequence of the RCPs. This rotation is restricted when MNPs bind to RCPs, which can be observed as a turn-off of the signal from MNPs that are free to rotate. The amount of MNPs bound to RCPs is found to increase in response to the amplification time as well as in response to the synthetic DNA target concentration (2-40 pM dynamic range). We report OM real-time results obtained with MNPs present during RCA and compare to relevant end-point OM results for RCPs generated for different RCA times. The real-time approach avoids opening of tubes post-RCA and thus reduces risk of lab contamination with amplification products without compromising the sensitivity and dynamic range of the assay.

Integration of microbead DNA handling with optomagnetic detection in rolling circle amplification assays.

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. Graphical Abstract Schematic illustration of magnetic microbeads transporting a DNA target through the steps in a rolling circle amplification assay. Optomagnetic measurements detect the binding of magnetic nanoparticles to amplicons released from microbeads (top) or the pH-induced release of magnetic nanoparticles trapped in amplicons (bottom).