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A fluorogenic heterogeneous immunoassay for cardiac muscle troponin cTnI on a digital microfluidic device

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Abstract

We describe a fluorogenic two-site noncompetitive heterogeneous immunoassay with magnetic beads on a low-voltage digital microfluidic platform using closed electrowetting-on-dielectric (EWOD). All the steps of an enzyme-linked immunosorbent assay (ELISA) were performed on the device using 9H-(1, 3-dichloro-9, 9-dimethylacridin-2-one-7-yl) phosphate as the fluorogenic substrate for the enzyme alkaline phosphatase. The performance of the system was demonstrated with cardiac marker Troponin I (cTnI) as a model analyte in phosphate-buffered saline samples. cTnI was detected within the diagnostically relevant range with a limit of detection of 2.0 ng/mL (CV = 6.47 %). Washing of magnetic beads was achieved by movement through a narrow region of buffer bridging one drop to another with minimal fluid transfer. More than 90 % of the unbound reagents were removed after five washes. Further experiments testing human blood serum on the same platform demonstrated a sample-to-answer time at ∼18.5 min detecting 6.79 ng/mL cTnI.

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Abbreviations

BSA:

Bovine serum albumin

cTnI:

Cardiac muscle troponin I

DMF:

Digital microfluidics

EWOD:

Electrowetting-on-dielectric

GAENE:

Glycolic acid ethoxylate 4-nonylphenyl ether

IgE:

Immunoglobulin E

IgG:

Immunoglobulin G

LOD:

Limit of detection

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Acknowledgments

This work would not have been possible without the assistance from Campbell Brown, Ben Hadwen and Jason Hector from Sharp Labs Europe. The authors would like to thank Prof. Peter Roach and Martyn Hiscox for use of their fluorescence plate reader. Mike Reeve kindly advised on DDAO-P and Cathy Rushworth assisted with the serum experiments. Funding from Sharp Labs Europe is gratefully acknowledged. This work was partly undertaken as an independent research by the National Institute for Health Research (Invention for Innovation (i4i), Rapid detection of infectious agents at point of triage (PoT), II-ES-0511-21002). The views expressed in this publication are those of the author(s) and not necessarily those of the NHS, the National Institute for Health Research or the Department of Health.

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Authors and Affiliations

  1. Electronics and Computer Science and Institute for Life Sciences, University of Southampton, Southampton, SO17 1BJ, UK

    Maria-Nefeli Tsaloglou & Hywel Morgan

  2. Sharp Labs of Europe, Oxford Science Park, Edmund Halley Rd, Oxford, OX4 4GB, UK

    Adrian Jacobs

Authors
  1. Maria-Nefeli Tsaloglou
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  2. Adrian Jacobs
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  3. Hywel Morgan
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Correspondence to Maria-Nefeli Tsaloglou.

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Tsaloglou, MN., Jacobs, A. & Morgan, H. A fluorogenic heterogeneous immunoassay for cardiac muscle troponin cTnI on a digital microfluidic device. Anal Bioanal Chem 406, 5967–5976 (2014). https://doi.org/10.1007/s00216-014-7997-z

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