Optomagnetic Detection of Rolling Circle Amplification Products

Gabriel Antonio S. Minero, Valentina Cangiano, Jeppe Fock, Francesca Garbarino, Mikkel Fougt Hansen

Research output: Chapter in Book/Report/Conference proceedingBook chapterEducationpeer-review

Abstract

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.
Original languageEnglish
Title of host publicationNucleic Acid Detection and Structural Investigations : Methods and Protocols
Volume2063
PublisherSpringer
Publication date2020
Pages3-15
Chapter1
ISBN (Print)978-1-0716-0137-2
ISBN (Electronic)978-1-0716-0138-9
DOIs
Publication statusPublished - 2020
SeriesMethods in Molecular Biology
ISSN1064-3745

Cite this

Minero, G. A. S., Cangiano, V., Fock, J., Garbarino, F., & Hansen, M. F. (2020). Optomagnetic Detection of Rolling Circle Amplification Products. In Nucleic Acid Detection and Structural Investigations: Methods and Protocols (Vol. 2063, pp. 3-15). Springer. Methods in Molecular Biology https://doi.org/10.1007/978-1-0716-0138-9_1