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

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

Research output: Contribution to journalJournal articleResearchpeer-review

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.
Original languageEnglish
Article number528
JournalMicrochimica Acta
Volume186
Issue number8
Number of pages10
ISSN0026-3672
DOIs
Publication statusPublished - 2019

Keywords

  • Assay integration
  • Biosensor
  • DNA capture
  • DNA melting
  • Magnetic microbead

Cite this

Minero, Gabriel Antonio S. ; Cangiano, Valentina ; Garbarino, Francesca ; Fock, Jeppe ; Hansen, Mikkel Fougt. / Integration of microbead DNA handling with optomagnetic detection in rolling circle amplification assays. In: Microchimica Acta. 2019 ; Vol. 186, No. 8.
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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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Integration of microbead DNA handling with optomagnetic detection in rolling circle amplification assays. / Minero, Gabriel Antonio S.; Cangiano, Valentina; Garbarino, Francesca; Fock, Jeppe; Hansen, Mikkel Fougt.

In: Microchimica Acta, Vol. 186, No. 8, 528, 2019.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Integration of microbead DNA handling with optomagnetic detection in rolling circle amplification assays

AU - Minero, Gabriel Antonio S.

AU - Cangiano, Valentina

AU - Garbarino, Francesca

AU - Fock, Jeppe

AU - Hansen, Mikkel Fougt

PY - 2019

Y1 - 2019

N2 - 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.

AB - 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.

KW - Assay integration

KW - Biosensor

KW - DNA capture

KW - DNA melting

KW - Magnetic microbead

U2 - 10.1007/s00604-019-3636-x

DO - 10.1007/s00604-019-3636-x

M3 - Journal article

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VL - 186

JO - Microchimica Acta

JF - Microchimica Acta

SN - 0026-3672

IS - 8

M1 - 528

ER -