AJAC  Vol.2 No.4 , August 2011
An Indirect Immunoassay for Detecting Antigen Based on Fluorescence Resonance Energy Transfer
Abstract: An indirect immunoassay for detecting antigen was developed. It was based on fluorescence resonance energy transfer (FRET) and quenching of gold nanoparticles. Bovine serum albumin (BSA) was chosen as model antigen. Fluorescein isothiocyanate (FITC) was attached to anti-BSA antibody (anti-BSA–FITC) as FRET donor, while BSA was conjugated to gold nanoparticles (GNPs–BSA) as FRET acceptor. The formation of anti-BSA–BSA immunocomplex resulted in the FRET between anti-BSA–FITC and GNPs–BSA. Thus, the fluorescence of FRET donor was quenched, and the decreasing fluorescence intensity responded linearly to the concentration of acceptor within the linear range. The concentration of BSA we obtained according to the stoichiometric ratio between BSA and GNPs. Following this approach, we were able to specifically detect BSA. The detection limit for BSA was 0.5 nM and the linear range of the assay was 2.9 - 43.5 nM. It had been successfully applied to specific detection of BSA in serum samples.
Cite this paper: nullP. Yang, S. Yao, W. Wei and J. Cai, "An Indirect Immunoassay for Detecting Antigen Based on Fluorescence Resonance Energy Transfer," American Journal of Analytical Chemistry, Vol. 2 No. 4, 2011, pp. 484-490. doi: 10.4236/ajac.2011.24058.

[1]   S. I. Stoeva, F. W. Huo, J. S. Lee and C. A. Mirkin, “Three-Layer Composite Magnetic Nanoparticle Probes for DNA,” Journal of the American Chemical Society, Vol. 127, No. 44, 2005, pp. 15362-15363. doi:10.1021/ja055056d

[2]   S. Lee, S. Kim and J. Choo, “Biological Imaging of HEK293 Cells Expressing PLCγ1 Using Surface-En- hanced Raman Microscopy,” Analytical Chemistry, Vol. 79, No. 3, 2007, pp. 916-922. doi:10.1021/ac061246a

[3]   K. Yum, H. N. Cho and J. Hu, “Individual Nano-tube-Based Needle Nanoprobes for Electrochemical Stu-dies in Picoliter Microenvironments,” ACS Nano, Vol. 1, No. 5, 2007, pp. 440-448. doi:10.1021/nn700171x

[4]   R. Freeman, T. Finder and R. Gill, “Probing Protein Ki-nase (CK2) and Alkaline Phosphatase with CdSe/ZnS Quantum Dots,” Nano Letters, Vol. 10, No. 6, 2010, pp. 2192-2196. doi:10.1021/nl101052f

[5]   Q. D. Wei, M. Lee and X. B. Yu, “Development of an Open Sandwich Fluoroimmuno-Assay Based on Fluores-cence Resonance Energy Transfer,” Analytical Biochemi-stry, Vol. 358, No. 1, 2006, pp. 31-37. doi:10.1016/j.ab.2006.08.019

[6]   K. Lymperopoulos, R. Crawford and J. Torella, “Sin-gle-Molecule DNA Biosensors for Protein and Ligand Detection,” Angewandte Chemie International Edition, Vol. 49, No. 7, 2010, pp. 1316-1320. doi:10.1002/anie.200904597

[7]   S. Ram, P. Vajpayee and R. Shanker, “Rapid Cul-ture-Independent Quantitative Detection of Enterotoxigenic Escherichia Coli in Surface Waters by Real-Time PCR with Molecular Beacon,” Environmental Science & Technology, Vol. 42, No. 12, 2008, pp. 4577-4582.

[8]   K. K. Haldar, T. Sen and A. Patra, “Metal Conjugated Semiconductor Hybrid Nanoparticle-Based Fluorescence Resonance Energy Transfer,” The Journal of Physical Chemistry C, Vol. 114, No. 11, 2010, pp. 4869-4874. doi:10.1021/jp911348n

[9]   J. Zhang, L. H. Wang and H. Zhang, “Aptamer-Based Multicolor Fluorescent Gold Nanoprobes for Multiplex Detection in Homogeneous Solution,” Samll, Vol. 6, No. 2, 2010, pp. 201-204. S. Pihlasalo, J. Kirjavainen and P. Hanninen, “Ultrasensi-tive Protein Concentration Measurement Based on Particle Adsorption and Fluorescence Quenching,” Analytical Chemistry, Vol. 81, No. 12, 2009, pp. 4995-5000. doi:10.1021/ac9001657

[10]   S. Mayilo, M. A.Kloster and M. Wunderlich, “Long- -Range Fluorescence Quenching by Gold Nanoparticles in a Sandwich Immunoassay for Cardiac Troponin T,” Nano Letters, Vol. 9, No. 12, 2009, pp. 4558-4563. doi:10.1021/nl903178n

[11]   N. Kato and F. Caruso, “Homogeneous, Competitive Fluorescence Quenching Immunoassay Based on Gold Nanoparticle/Polyelectrolyte Coated Latex Particles,” The Journal of Physical Chemistry B, Vol. 109, No. 42, 2005, pp. 19604-19612. doi:10.1021/jp052748f

[12]   D. Z. Yang, S. K. Xua and Q. F. Chen, “One System with Two Fluorescence Resonance Energy Transfer (FRET) Assembles among Quantum Dots, Gold Nanoparticles and Enzyme,” Colloids and Surfaces A: Physicochemical and Engineering Aspects, Vol. 329, No. 1-2, 2008, pp. 38-43. doi:10.1016/j.colsurfa.2008.06.048

[13]   J. M. Kürner, O. S.Wolfbeis and I. Klimant, “Homoge-neous Luminescence Decay Time-Based Assay Using Energy Transfer from Nanospheres,” Analytical Chemistry, Vol. 74, No. 9, 2002, pp. 2151-2156. doi:10.1021/ac0111098

[14]   G. Frens, “Controlled Nucleation for the Regulation of the Particle Size in Monodisperse Gold Suspensions,” Nature Physical Science, Vol. 241, No. 105, 1973, pp. 20-22.

[15]   R. C. Jin, G. S. Wu and Z. Li, “What Controls the Melting Properties of DNA-Linked Gold Nanoparticle As-semblies?” Journal of the American Chemical Society, Vol. 125, No. 6, 2003, pp. 1643-1654. doi:10.1021/ja021096v

[16]   B. Nikoobakht and M. A. El-Sayed, “Preparation and Growth Mechanism of Gold Nanorods (NRs) Using Seed-Mediated Growth Method,” Chemistry of Materials, Vol. 15, No. 10, 2003, pp. 1957-1962.

[17]   P. Xu. “Application of Fluorescence and Immunofluo-rescence Staining Technique,” People’s Medical Pub-lishing House, Beijing, 2000.

[18]   S. H. Brewer, W. R. Glomm and M. C. Johnson, “Probing BSA Binding to Citrate-Coated Gold Nanoparticles and Surfaces,” Langmuir, Vol. 21, No. 20, 2005, pp. 9303-9307. doi:10.1021/la050588t

[19]   X. Liu, Q. Dai and L. Austin, “A One-Step Homogeneous Immunoassay for Cancer Biomarker Detection Using Gold Nanoparticle Probes Coupled with Dynamic Light Scattering,” Journal of the American Chemical Society, Vol. 130, No. 9, 2008, pp. 2780-2782. doi:10.1021/ja711298b

[20]   B. F. Pan, D. X. Cui and P. Xu, “Study on Interaction between Gold Nanorod and Bovine Serum Albumin,” Colloids and Surfaces A: Physicochemical and Engi-neering Aspects, Vol. 295, No. 1-3, 2007, pp. 217-222. doi:10.1016/j.colsurfa.2006.09.002