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.
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References
Cheung-Hoi Yu A, Vatcher G, Yue X, Dong Y, Hua Li M, K Tam PH, Tsang PY, Wong AK, Hui MH, Yang B, Tang H, Lau L-T (2012) Nucleic acid-based diagnostics for infectious diseases in public health affairs. Front Med 6:173–176
Ali MM, Li F, Zhang Z, Zhang K, Kang DK, Ankrum JA, Le XC, Zhao W (2014) Rolling circle amplification: a versatile tool for chemical biology, materials science and medicine. Chem Soc Rev 43:3324–3341
Pavankumar AR, Engström A, Liu J, Herthnek D, Nilsson M (2016) Proficient detection of multi-drug-resistant Mycobacterium tuberculosis by padlock probes and lateral flow nucleic acid biosensors. Anal Chem 88:4277–4284
Nilsson M, Gullberg M, Dahl F, Szuhai K, Raap AK (2002) Real-time monitoring of rolling-circle amplification using a modified molecular beacon design. Nucleic Acids Res 30:e66
Zhang S, Wu Z, Shen G, Yu R (2009) A label-free strategy for SNP detection with high fidelity and sensitivity based on ligation-rolling circle amplification and intercalating of methylene blue. Biosens Bioelectron 24:3201–3207
Strömberg M, Zardán Gómez de la Torre T, Göransson J, Gunnarsson K, Nilsson M, Strømme M, Svedlindh P (2008) Microscopic mechanisms influencing the volume amplified magnetic nanobead detection assay. Biosens Bioelectron 24:696–703
Yasui T, Ogawa K, Kaji N, Nilsson M, Ajiri T, Tokeshi M, Horiike Y, Baba Y (2016) Label-free detection of real-time DNA amplification using a nanofluidic diffraction grating. Sci Rep 6:31642
Hernández-Neuta I, Pereiro I, Ahlford A, Ferraro D, Zhang Q, Viovy JL, Descroix S, Nilsson M (2018) Microfluidic magnetic fluidized bed for DNA analysis in continuous flow mode. Biosens Bioelectron 102:531–539
Sun J, de Hoog S (2012) Hyperbranching rolling circle amplification, an improved protocol for discriminating between closely related fungal species. In: Fungal diagnostics. Humana, Totowa, NJ, pp 167–175
Neumann F, Hernández-Neuta I, Grabbe M, Madaboosi N, Albert J, Nilsson M (2018) Padlock probe assay for detection and subtyping of seasonal influenza. Clin Chem 64:1–9
Minero GAS, Nogueira C, Rizzi G, Tian B, Fock J, Donolato M, Strömberg M, Hansen MF (2017) Sequence-specific validation of LAMP amplicons in real-time optomagnetic detection of Dengue serotype 2 synthetic DNA. Analyst 142:3441–3450
Maier T, Kainz K, Barišić I, Hainberger R (2015) An impedimetric sensor for real-time detection of antibiotic resistance genes employing rolling circle amplification. Int J Electrochem Sci 10:2026–2034
Seichi A, Kokuka N, Kashima Y, Tabata M, Goda T, Matsumoto A, Iwasawa N, Citterio D, Miyahara Y, Suzuki K (2016) Real-time monitoring and detection of primer generation-rolling circle amplification of DNA using an ethidium ion-selective electrode. Anal Sci 32:505–510
Yang L, Fung CW, Eun Jeong Cho A, Ellington AD (2007) Real-time rolling circle amplification for protein detection. Anal Chem 79:3320–3329
Jarvius J, Melin J, Göransson J, Stenberg J, Fredriksson S, Gonzalez-Rey C, Bertilsson S, Nilsson M (2006) Digital quantification using amplified single-molecule detection. Nat Methods 3:725–727
Kühnemund M, Wei Q, Darai E, Wang Y, Hernández-Neuta I, Yang Z, Tseng D, Ahlford A, Mathot L, Sjöblom T, Ozcan A, Nilsson M (2017) Targeted DNA sequencing and in situ mutation analysis using mobile phone microscopy. Nat Commun 8:13913
Nallur G, Luo C, Fang L, Cooley S, Dave V, Lambert J, Kukanskis K, Kingsmore S, Lasken R, Schweitzer B (2001) Signal amplification by rolling circle amplification on DNA microarrays. Nucleic Acids Res 29:e118
Fock J, Jonasson C, Johansson C, Hansen MF (2017) Characterization of fine particles using optomagnetic measurements. Phys Chem Chem Phys 19:8802–8814
Fock J, Balceris C, Costo R, Zeng L, Ludwig F, Hansen MF (2017) Field-dependent dynamic responses from dilute magnetic nanoparticle dispersions. Nanoscale 10:2052–2066
Bejhed RS, de la Torre TZG, Donolato M, Hansen MF, Svedlindh P, Strömberg M (2015) Turn-on optomagnetic bacterial DNA sequence detection using volume-amplified magnetic nanobeads. Biosens Bioelectron 66:405–411
Donolato M, Antunes P, de la Torre TZG, Te HE, Chen CH, Burger R, Rizzi G, Bosco FG, Strømme M, Boisen A, Hansen MF (2015) Quantification of rolling circle amplified DNA using magnetic nanobeads and a Blu-ray optical pick-up unit. Biosens Bioelectron 67:649–655
Minero GAS, Fock J, McCaskill JS, Hansen MF (2017) Optomagnetic detection of DNA triplex nanoswitches. Analyst 142:582–585
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Minero, G.A.S., Cangiano, V., Fock, J., Garbarino, F., Hansen, M.F. (2020). Optomagnetic Detection of Rolling Circle Amplification Products. In: Astakhova, K., Bukhari, S. (eds) Nucleic Acid Detection and Structural Investigations. Methods in Molecular Biology, vol 2063. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0138-9_1
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DOI: https://doi.org/10.1007/978-1-0716-0138-9_1
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