Analytical and Bioanalytical Chemistry

, Volume 406, Issue 24, pp 5967–5976 | Cite as

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

  • Maria-Nefeli TsaloglouEmail author
  • Adrian Jacobs
  • Hywel Morgan
Research Paper


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.


Bioassays Immunoassays/ELISA Digital microfluidics Electrowetting 



Bovine serum albumin


Cardiac muscle troponin I


Digital microfluidics




Glycolic acid ethoxylate 4-nonylphenyl ether


Immunoglobulin E


Immunoglobulin G


Limit of detection



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.

Supplementary material

216_2014_7997_MOESM1_ESM.pdf (68 kb)
ESM 1 (PDF 62 kb)

(WMV 1531 kb)


  1. 1.
    Anderson NL, Anderson NG (2002) The human plasma proteome. Mol Cell Proteomics 1(11):845–867. doi: 10.1074/mcp.R200007-MCP200 CrossRefGoogle Scholar
  2. 2.
    Takeda S, Yamashita A, Maeda K, Maeda Y (2003) Structure of the core domain of human cardiac troponin in the Ca2+-saturated form. Nature 424(6944):35–41. doi: 10.1038/nature01780 CrossRefGoogle Scholar
  3. 3.
    Ritchie RF, Ledue TB (2001) The immunoassay handbook. In: Wild D (ed) Clin Chem, 2nd edn. Washington, DC 47:1876–1885Google Scholar
  4. 4.
    Voller A, Bartlett A, Bidwell DE (1978) Enzyme immunoassays with special reference to ELISA techniques. J Clin Pathol 31(6):507–520. doi: 10.1136/jcp.31.6.507 CrossRefGoogle Scholar
  5. 5.
    Stratis-Cullum DN, Griffin GD, Mobley J, Vass AA, Vo-Dinh T (2002) A miniature biochip system for detection of aerosolized Bacillus globigii spores. Anal Chem 75(2):275–280. doi: 10.1021/ac026068+
  6. 6.
    Leira F, Vieites JM, Vieytes MR, Botana LM (2000) Characterization of 9H-(1,3-dichlor-9,9-dimethylacridin-2-ona-7-yl)-phosphate (DDAO) as substrate of PP-2A in a fluorimetric microplate assay for diarrhetic shellfish toxins (DSP). Toxicon 38(12):1833–1844. doi: 10.1016/s0041-0101(00)00111-2 CrossRefGoogle Scholar
  7. 7.
    Zeng Y, Wang T (2013) Quantitative microfluidic biomolecular analysis for systems biology and medicine. Anal Bioanal Chem 405(17):5743–5758. doi: 10.1007/s00216-013-6930-1 CrossRefGoogle Scholar
  8. 8.
    Lippmann G (1875) Relations entre les phénomènes électriques et capillaries. Ann Chim Phys 5:494–549Google Scholar
  9. 9.
    Froumkine A (1936) Couche double, électrocapillarité, surtension. Actualités scientifiques et industrielles 373(1):5–36Google Scholar
  10. 10.
    Berge B, Peseux J (2000) Variable focal lens controlled by an external voltage: an application of electrowetting. Eur Phys J E 3(2):159–163. doi: 10.1007/s101890070029 CrossRefGoogle Scholar
  11. 11.
    Pamula VK, Srinivasan V, Chakrapani H, Fair RB, Toone EJ (2005) A droplet-based lab-on-a-chip for colorimetric detection of nitroaromatic explosives. In: Micro Electro Mechanical Systems, 2005. MEMS 2005. 18th IEEE International Conference on, 30 Jan–3 Feb 2005. pp 722–725. doi: 10.1109/memsys.2005.1454031
  12. 12.
    Sista R, Hua Z, Thwar P, Sudarsan A, Srinivasan V, Eckhardt A, Pollack M, Pamula V (2008) Development of a digital microfluidic platform for point of care testing. Lab Chip 8(12):2091–2104. doi: 10.1039/B814922D CrossRefGoogle Scholar
  13. 13.
    Shah GJ, Veale JL, Korin Y, Reed EF, Gritsch HA, Kim CJ (2010) Specific binding and magnetic concentration of CD8+T-lymphocytes on electrowetting-on-dielectric platform. Biomicrofluidics 4(4):44106. doi: 10.1063/1.3509457 CrossRefGoogle Scholar
  14. 14.
    Shah GJ, Ding H, Sadeghi S, Chen S, Kim C-J, Dam RM (2013) On-demand droplet loading for automated organic chemistry on digital microfluidics. Lab Chip 13:2785–2795. doi: 10.1039/C3LC41363B CrossRefGoogle Scholar
  15. 15.
    Eydelnant IA, Uddayasankar U, Li BB, Liao MW, Wheeler AR (2012) Virtual microwells for digital microfluidic reagent dispensing and cell culture. Lab Chip 12(4):750–757. doi: 10.1039/C2LC21004E CrossRefGoogle Scholar
  16. 16.
    Hadwen B, Broder GR, Morganti D, Jacobs A, Brown C, Hector JR, Kubota Y, Morgan H (2012) Programmable large area digital microfluidic array with integrated droplet sensing for bioassays. Lab Chip 12(18):3305–3313. doi: 10.1039/c2lc40273d CrossRefGoogle Scholar
  17. 17.
    Delattre C, Allier CP, Fouillet Y, Jary D, Bottausci F, Bouvier D, Delapierre G, Quinaud M, Rival A, Davoust L, Peponnet C (2012) Macro to microfluidics system for biological environmental monitoring. Biosens Bioelectron 36(1):230–235. doi: 10.1016/j.bios.2012.04.024 CrossRefGoogle Scholar
  18. 18.
    Choi K, Ng AHC, Fobel R, Wheeler AR (2012) Digital microfluidics. Annu Rev Anal Chem 5(1):413–440. doi: 10.1146/annurev-anchem-062011-143028 CrossRefGoogle Scholar
  19. 19.
    Fair RB (2007) Digital microfluidics: is a true lab-on-a-chip possible? Microfluid Nanofluid 3(3):245–281. doi: 10.1007/s10404-007-0161-8 CrossRefGoogle Scholar
  20. 20.
    Jebrail MJ, Bartsch MS, Patel KD (2012) Digital microfluidics: a versatile tool for applications in chemistry, biology and medicine. Lab Chip 12(14):2452–2463. doi: 10.1039/c2lc40318h CrossRefGoogle Scholar
  21. 21.
    Vergauwe N, Witters D, Ceyssens F, Vermeir S, Verbruggen B, Puers R, Lammertyn J (2011) A versatile electrowetting-based digital microfluidic platform for quantitative homogeneous and heterogeneous bio-assays. J Micromech Microeng 21(5):054026. doi: 10.1088/0960-1317/21/5/054026 CrossRefGoogle Scholar
  22. 22.
    Sista RS, Eckhardt AE, Srinivasan V, Pollack MG, Palanki S, Pamula VK (2008) Heterogeneous immunoassays using magnetic beads on a digital microfluidic platform. Lab Chip 8(12):2188–2196. doi: 10.1039/B807855F CrossRefGoogle Scholar
  23. 23.
    Ng AHC, Choi K, Luoma RP, Robinson JM, Wheeler AR (2012) Digital microfluidic magnetic separation for particle-based immunoassays. Anal Chem 84(20):8805–8812. doi: 10.1021/ac3020627 CrossRefGoogle Scholar
  24. 24.
    Vermeir S, Witters D, Vergauwe N, Knez K, Gijs M, Puers R, Lammertyn J (2012) Ferromagnetic particles for an improved heterogeneous bioassay performance on a digital lab-on-chip. In: 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Okinawa, Japan, October 28–November 1. pp 1042–1044Google Scholar
  25. 25.
    Miller E, Ng A, Uddayasankar U, Wheeler A (2011) A digital microfluidic approach to heterogeneous immunoassays. Anal Bioanal Chem 399(1):337–345. doi: 10.1007/s00216-010-4368-2 CrossRefGoogle Scholar
  26. 26.
    Ash J, Baxevanakis G, Bilandzic L, Shin H, Kadijevic L (2000) Development of an automated quantitative latex immunoassay for cardiac troponin I in serum. Clin Chem 46(9):1521–1522Google Scholar
  27. 27.
    Rotman B, Zderic JA, Edelstein M (1963) Fluorogenic substrates for f3-D-galactosidases and phosphatases derived from fluorescein (3, 6-dihydroxyfluoran) and its monomethyl ether. Proc Natl Acad Sci U S A 50(1):1–6CrossRefGoogle Scholar
  28. 28.
    Lewkowich IP, Campbell JD, HayGlass KT (2001) Comparison of chemiluminescent assays and colorimetric ELISAs for quantification of murine IL-12, human IL-4 and murine IL-4: chemiluminescent substrates provide markedly enhanced sensitivity. J Immunol Methods 247(1–2):111–118. doi: 10.1016/S0022-1759(00)00306-9 CrossRefGoogle Scholar
  29. 29.
    Dodeigne C, Thunus L, Lejeune R (2000) Chemiluminescence as diagnostic tool. A review. Talanta 51(3):415–439. doi: 10.1016/S0039-9140(99)00294-5 CrossRefGoogle Scholar
  30. 30.
    Mahajan VS, Jarolim P (2011) How to interpret elevated cardiac troponin levels. Circulation 124(21):2350–2354. doi: 10.1161/circulationaha.111.023697 CrossRefGoogle Scholar
  31. 31.
    Jenkins WT, D’Ari L (1966) The kinetics of alkaline phosphatase. J Biol Chem 241(2):295–296Google Scholar
  32. 32.
    Berry SM, Maccoux LJ, Beebe DJ (2012) Streamlining immunoassays with immiscible filtrations assisted by surface tension. Anal Chem 84(13):5518–5523. doi: 10.1021/ac300085m CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Maria-Nefeli Tsaloglou
    • 1
    Email author
  • Adrian Jacobs
    • 2
  • Hywel Morgan
    • 1
  1. 1.Electronics and Computer Science and Institute for Life SciencesUniversity of SouthamptonSouthamptonUK
  2. 2.Sharp Labs of EuropeOxfordUK

Personalised recommendations