Abstract
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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References
Zhao Y, Chen F, Li Q, Wang L, Fan C (2015) Isothermal amplification of nucleic acids. Chem Rev 115(22):12491– 12545
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(8):4277–4284
Göransson J, Ke R, Nong RY, Howell WM, Karman A, Grawé J, Stenberg J, Granberg M, Elgh M, Herthnek D, Wikström P, Jarvius J, Nilsson M (2012) Rapid identification of bio-molecules applied for detection of biosecurity agents using rolling circle amplification. PLoS ONE 7:e31068
Clausson CM, Arngården L, Ishaq O, Klaesson A, Kühnemund M, Grannas K, Koos B, Qian X, Ranefall P, Krzywkowski T, Brismar H, Nilsson M, Wählby C, Söderberg O (2015) Compaction of rolling circle amplification products increases signal integrity and signal-to-noise ratio. Sci Rep 5(1):12317
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(14):e66
Jarvius J, Meln 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, Iván H N, 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
Strömberg M, Göransson J, Gunnarsson K, Nilsson M, Svedlindh P, Strømme M (2008) Sensitive molecular diagnostics using volume-amplified magnetic nanobeads. Nano Lett 8(3):816–821
Donolato M, Antunes P, de la Torre TZG, Hwu ET, 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. Biosensors Bioelectron 67:649–655
Mezger A, Fock J, Antunes P, FW Østerberg, Boisen A, Nilsson M, Hansen MF, Ahlford A, Donolato M (2015) Scalable DNA-based magnetic nanoparticle agglutination assay for bacterial detection in patient samples. ACS Nano 9:7374–7382
Li J, Deng T, Chu X, Yang R, Jiang J, Shen G, Yu R (2010) Rolling circle amplification combined with gold nanoparticle aggregates for highly sensitive identification of single-nucleotide polymorphisms. Anal Chem 82:2811–16
Peyman SA, Iles A, Pamme N (2009) Mobile magnetic particles as solid-supports for rapid surface-based bioanalysis in continuous flow. Lab Chip 9:3110–3117
Gijs MA, Lacharme F, Lehmann U (2010) Microfluidic applications of magnetic particles for biological analysis and catalysis. Chem Rev 110:1518–1563
Rödiger S, Liebsch C, Schmidt C, Lehmann W, Resch-Genger U, Schedler U, Schierack P (2014) Nucleic acid detection based on the use of microbeads: a review. Microchim Acta 181:1151–1168
Van Reenen A, De Jong AM, Den Toonder JM, Prins MW (2014) Integrated lab-on-chip biosensing systems based on magnetic particle actuation-a comprehensive review. Lab Chip 14(12):1966–1986
Pandit KR, Nanayakkara IA, Cao W, Raghavan SR, White IM (2015) Capture and direct amplification of DNA on chitosan microparticles in a single PCR-optimal solution. Anal Chem 87:11022–11029
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:e12
Palanisamy R, Connolly AR, Trau M (2010) Considerations of solid-phase DNA amplification. ACS Bioconjugate Chem 21:690–695
Schopf E, Fischer NO, Chen Y, Tok JB (2008) Sensitive and selective viral DNA detection assay via microbead-based rolling circle amplification. Bioorganic Med Chem Lett 18:5871–5874
Sato K, Ishii R, Sasaki N, Sato K, Nilsson M (2013) Bead-based padlock rolling circle amplification for single DNA molecule counting. Anal Biochem 437(1):43–45
Sato K, Tachihara A, Renberg B, Mawatari K, Sato K, Tanaka Y, Jarvius J, Nilsson M, Kitamori T (2010) Microbead-based rolling circle amplification in a microchip for sensitive DNA detection. Lab Chip 10:1262–1266
Ahlford A, Conde A, Sabourin D, Kutter J, Nilsson M, Dufva M, Brivio M (2011) A microfluidic platform for personalized cancer diagnostics by padlock probes ligation and circle-to-circle amplification. In: 15th International conference on miniaturized systems for chemistry and life sciences 2011 (MicroTAS 2011), pp 61–63
Kühnemund M, Witters D, Nilsson M, Lammertyn J (2014) Circle-to-circle amplification on a digital microfluidic chip for amplified single molecule detection. Lab Chip 14:2983
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. Biosensors Bioelectron 102:531–539
Schopf E, Liu Y, Deng JC, Yang S, Cheng G, Chen Y (2011) Mycobacterium tuberculosis detection via rolling circle amplification. Anal Methods 3:267–273
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
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
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. Biosensors Bioelectron 24:696–703
Strömberg M, Zardán Gómez De La Torre T, Göransson J, Gunnarsson K, Nilsson M, Svedlindh P, Strømme M (2009) Multiplex detection of DNA sequences using the volume-amplified magnetic nanobead detection assay. Anal Chem 81:3398–3406
Zardán Gómez De La Torre T, Strömberg M, Russell C, Göransson J, Nilsson M, Svedlindh P, Strømme M (2010) Investigation of immobilization of functionalized magnetic nanobeads in rolling circle amplified DNA coils. J Phys Chem B 114:3707–3713
Oishi M (2015) Enzyme-free and isothermal detection of microRNA based on click-chemical ligation-assisted hybridization coupled with hybridization chain reaction signal amplification. Anal Bioanal Chem 407(14):4165–72
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. Biosensors Bioelectron 24:3201–3207
Schopf E, Chen Y (2010) Attomole DNA detection assay via rolling circle amplification and single molecule detection. Anal Biochem 397:115–117
Yang X, Yang K, Zhao X, Lin Z, Liu Z, Luo S, Zhang Y, Wang Y, Fu W (2017) Terahertz spectroscopy for the isothermal detection of bacterial DNA by magnetic bead-based rolling circle amplification. Analyst 142(24):4661–4669
Acknowledgements
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.
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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
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DOI: https://doi.org/10.1007/s00604-019-3636-x