Integration of rolling circle amplification and optomagnetic detection on a polymer chip

Francesca Garbarino, Gabriel Antonio S. Minero, Giovanni Rizzi, Jeppe Fock, Mikkel Fougt Hansen

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Rolling circle amplification (RCA) combined with padlock probe recognition of a DNA target is attractive for on-chip nucleic acid testing due to its high specificity and isothermal reaction conditions. However, the integration of RCA on an automated chip platform is challenging due to the different reagents needed for the reaction steps and the temperature sensitivity of the phi29 polymerase. Here, we describe the integration of an RCA assay on a single-use polymer chip platform where magnetic microbeads are used as solid support to transport the DNA target between three connected reaction chambers for (i) padlock probe annealing and ligation, (ii) RCA, and (iii) optomagnetic detection of RCA products. The three chambers were loaded with reagents by sequential filling combined with passive microfluidic structures. After loading, the on-chip assay steps were automated. For an assay in which all steps but the padlock probe annealing on the target were performed on-chip, we found a limit of detection (LOD) for a synthetic influenza target of 2 pM after 45 min of RCA, which is comparable to the corresponding laboratory assay. The entire assay, including padlock probe annealing, could be performed on-chip with an LOD of 20 pM after 45 min of RCA. This LOD can likely be reduced by further optimizing the microbead mixing. The results present important steps towards the integration and automation of RCA and potentially also other complex multi-step assays on a single-use polymer chip for molecular analysis.
Original languageEnglish
Article number111485
JournalBiosensors and Bioelectronics
Volume142
Number of pages7
ISSN0956-5663
DOIs
Publication statusPublished - 2019

Keywords

  • Lab-on-a-chip
  • Magnetic microparticles
  • Magnetic nanoparticles
  • Microfluidics
  • Nucleic acid analysis

Cite this

Garbarino, Francesca ; Minero, Gabriel Antonio S. ; Rizzi, Giovanni ; Fock, Jeppe ; Hansen, Mikkel Fougt. / Integration of rolling circle amplification and optomagnetic detection on a polymer chip. In: Biosensors and Bioelectronics. 2019 ; Vol. 142.
@article{a586424d0b0c46feac277322968e23b0,
title = "Integration of rolling circle amplification and optomagnetic detection on a polymer chip",
abstract = "Rolling circle amplification (RCA) combined with padlock probe recognition of a DNA target is attractive for on-chip nucleic acid testing due to its high specificity and isothermal reaction conditions. However, the integration of RCA on an automated chip platform is challenging due to the different reagents needed for the reaction steps and the temperature sensitivity of the phi29 polymerase. Here, we describe the integration of an RCA assay on a single-use polymer chip platform where magnetic microbeads are used as solid support to transport the DNA target between three connected reaction chambers for (i) padlock probe annealing and ligation, (ii) RCA, and (iii) optomagnetic detection of RCA products. The three chambers were loaded with reagents by sequential filling combined with passive microfluidic structures. After loading, the on-chip assay steps were automated. For an assay in which all steps but the padlock probe annealing on the target were performed on-chip, we found a limit of detection (LOD) for a synthetic influenza target of 2 pM after 45 min of RCA, which is comparable to the corresponding laboratory assay. The entire assay, including padlock probe annealing, could be performed on-chip with an LOD of 20 pM after 45 min of RCA. This LOD can likely be reduced by further optimizing the microbead mixing. The results present important steps towards the integration and automation of RCA and potentially also other complex multi-step assays on a single-use polymer chip for molecular analysis.",
keywords = "Lab-on-a-chip, Magnetic microparticles, Magnetic nanoparticles, Microfluidics, Nucleic acid analysis",
author = "Francesca Garbarino and Minero, {Gabriel Antonio S.} and Giovanni Rizzi and Jeppe Fock and Hansen, {Mikkel Fougt}",
year = "2019",
doi = "10.1016/j.bios.2019.111485",
language = "English",
volume = "142",
journal = "Biosensors and Bioelectronics",
issn = "0956-5663",
publisher = "Elsevier",

}

Integration of rolling circle amplification and optomagnetic detection on a polymer chip. / Garbarino, Francesca; Minero, Gabriel Antonio S.; Rizzi, Giovanni; Fock, Jeppe; Hansen, Mikkel Fougt.

In: Biosensors and Bioelectronics, Vol. 142, 111485, 2019.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Integration of rolling circle amplification and optomagnetic detection on a polymer chip

AU - Garbarino, Francesca

AU - Minero, Gabriel Antonio S.

AU - Rizzi, Giovanni

AU - Fock, Jeppe

AU - Hansen, Mikkel Fougt

PY - 2019

Y1 - 2019

N2 - Rolling circle amplification (RCA) combined with padlock probe recognition of a DNA target is attractive for on-chip nucleic acid testing due to its high specificity and isothermal reaction conditions. However, the integration of RCA on an automated chip platform is challenging due to the different reagents needed for the reaction steps and the temperature sensitivity of the phi29 polymerase. Here, we describe the integration of an RCA assay on a single-use polymer chip platform where magnetic microbeads are used as solid support to transport the DNA target between three connected reaction chambers for (i) padlock probe annealing and ligation, (ii) RCA, and (iii) optomagnetic detection of RCA products. The three chambers were loaded with reagents by sequential filling combined with passive microfluidic structures. After loading, the on-chip assay steps were automated. For an assay in which all steps but the padlock probe annealing on the target were performed on-chip, we found a limit of detection (LOD) for a synthetic influenza target of 2 pM after 45 min of RCA, which is comparable to the corresponding laboratory assay. The entire assay, including padlock probe annealing, could be performed on-chip with an LOD of 20 pM after 45 min of RCA. This LOD can likely be reduced by further optimizing the microbead mixing. The results present important steps towards the integration and automation of RCA and potentially also other complex multi-step assays on a single-use polymer chip for molecular analysis.

AB - Rolling circle amplification (RCA) combined with padlock probe recognition of a DNA target is attractive for on-chip nucleic acid testing due to its high specificity and isothermal reaction conditions. However, the integration of RCA on an automated chip platform is challenging due to the different reagents needed for the reaction steps and the temperature sensitivity of the phi29 polymerase. Here, we describe the integration of an RCA assay on a single-use polymer chip platform where magnetic microbeads are used as solid support to transport the DNA target between three connected reaction chambers for (i) padlock probe annealing and ligation, (ii) RCA, and (iii) optomagnetic detection of RCA products. The three chambers were loaded with reagents by sequential filling combined with passive microfluidic structures. After loading, the on-chip assay steps were automated. For an assay in which all steps but the padlock probe annealing on the target were performed on-chip, we found a limit of detection (LOD) for a synthetic influenza target of 2 pM after 45 min of RCA, which is comparable to the corresponding laboratory assay. The entire assay, including padlock probe annealing, could be performed on-chip with an LOD of 20 pM after 45 min of RCA. This LOD can likely be reduced by further optimizing the microbead mixing. The results present important steps towards the integration and automation of RCA and potentially also other complex multi-step assays on a single-use polymer chip for molecular analysis.

KW - Lab-on-a-chip

KW - Magnetic microparticles

KW - Magnetic nanoparticles

KW - Microfluidics

KW - Nucleic acid analysis

U2 - 10.1016/j.bios.2019.111485

DO - 10.1016/j.bios.2019.111485

M3 - Journal article

VL - 142

JO - Biosensors and Bioelectronics

JF - Biosensors and Bioelectronics

SN - 0956-5663

M1 - 111485

ER -