Abstract
In this work, we used a model assay system (polyclonal human IgG–goat antihuman IgG) to elucidate some of the key factors that influence the analytical performance of bioassays that employ metal-enhanced fluorescence (MEF) using silver nanoparticles (NPs). Cy5 dye was used as the fluorescent label, and results were compared with a standard assay performed in the absence of NPs. Two sizes of silver NPs were prepared with respective diameters of 60 ± 10 and 149 ± 16 nm. The absorption spectra of the NPs in solution were fitted accurately using Mie theory, and the dipole resonance of the 149-nm NPs in solution was found to match well with the absorption spectrum of Cy5. Such spectral matching is a key factor in optimizing MEF. NPs were deposited uniformly and reproducibly on polyelectrolyte-coated polystyrene substrates. Compared to the standard assay performed without the aid of NPs, significant improvements in sensitivity and in limit of detection (LOD) were obtained for the assay with the 149-nm NPs. An important observation was that the relative enhancement of fluorescence increased as the concentration of antigen increased. The metal-assisted assay data were analyzed using standard statistical methods and yielded a LOD of 0.086 ng/mL for the spectrally matched NPs compared to a value of 5.67 ng/mL obtained for the same assay in the absence of NPs. This improvement of ∼66× in LOD demonstrates the potential of metal-enhanced fluorescence for improving the analytical performance of bioassays when care is taken to optimize the key determining parameters.
Similar content being viewed by others
References
Chowdhury MH, Ray K, Aslan K, Lakowicz JR, Geddes CD (2007) J Phys Chem C 111:18856–18863
Zhang J, Lakowicz JR (2007) Opt Express 15:2598–2606
Matveeva EG, Gryczynski I, Barnett A, Leonenko Z, Lakowicz JR, Gryczynski Z (2007) Anal Biochem 363:239–245
Matveeva EG, Gryczynski Z, Lakowicz JR (2005) J Immunol Methods 302:26–35
Lochner N, Lobmaier C, Wirth M, Leitner A, Pittner F, Gabor F (2003) Eur J Pharm Biopharm 56:469–477
Matveeva E, Gryczynski Z, Malicka J, Gryczynski I, Lakowicz JR (2004) Anal Biochem 334:303–311
Mie G (1908) Ann Phys 25:377–445
Ihara M, Tanaka K, Sakaki K, Honma I, Yamada K (1997) J Phys Chem B 101:5153–5157
Lakowicz JR, Shen B, Gryczynski Z, D’Auria S, Gryczynski I (2001) Biochem Biophys Res Commun 286:875–879
Chen Y, Munichuka K, Ginger DS (2007) Nano Lett 7:690–696
Szmacinski H, Smith DS, Hanson MA, Kostov Y, Lakowicz JR, Rao G (2008) Biotech Bioeng 100:448–457
Yamaguchi T, Kaya T, Takei H (2007) Anal Biochem 364:171–179
Nooney RI, Stranik O, McDonagh C, MacCraith BD (2008) Langmuir 24:11261–11267
Amersham CyDye™ momo-reactive NHS Esters, Product Booklet, GE Healthcare, UK Limited
Tan YW, Li YF, Zhu DB (2003) J Coll Int Sci 258:244–251
Eirich FR (1977) J Coll Int Sci 58:423–436
Horisberger M (1981) In: Johari O (ed) Scanning electron microscopy, vol. 2. SEM Inc. AMF O'Hare, Chicago
Horisberger M, Vauthey M (1984) Histochemistry 80:13–18
Martinez A (2005) J Braz Chem Soc 16:337–344
Diamandis EP, Christopoulos TK (1996) Immunoassay. Academic, New York
Finlay JNA, Smith WC, Lee JW, Nordblom GD, Das I, DeSilver BS, Khan MN, Bousher RR (2000) J Pharm Biomed Anal 21:1249–1273
West W, Pearce S (1965) J Phys Chem 69:1894–1903
Malynych S, Chumanov G (2007) J Quant Spectrosc Radiat Transfer 106:297–303
Spillman SD, McEvoy HM, MacCraith BD (2009) Langmuir 25:1403–1411
Grabar KC, Smith PC, Musick MD, Davis JA, Walter DG, Jackson MA, Guthrie AP, Natan MJ (1996) J Am Chem Soc 118:1148–1153
Malicka J, Gryczynski I, Lakowicz JR (2003) Biochem Biophys Res Commun 306:213–218
Lakowicz JR, Malicka J, D’Auria S, Gryczynski I (2003) Anal Biochem 320:13–20
Lakowicz JR (2006) Principles of fluorescence spectroscopy, 3rd edn. Springer, New York
Lakowicz JR, Ray K, Chowdhury M, Szmacinski H, Fu Y, Zhang J, Nowaczyk K (2008) Analyst 133:1308–1346
Xu H, Zhao X, Grant C, Lu JR, Williams D, Penfold J (2006) Langmuir 22:6313–6320
Xu H, Zhao X, Lu JR, Williams D (2007) Biomacromolecules 8:2422–2428
Acknowledgment
This material is based upon a work supported by the Science Foundation Ireland under grant no. 05/CE3/B754.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Nooney, R., Clifford, A., LeGuevel, X. et al. Enhancing the analytical performance of immunoassays that employ metal-enhanced fluorescence. Anal Bioanal Chem 396, 1127–1134 (2010). https://doi.org/10.1007/s00216-009-3357-9
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00216-009-3357-9