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Int. J. Mol. Sci. 2015, 16(1), 907-923; doi:10.3390/ijms16010907

Development and Application of a Label-Free Fluorescence Method for Determining the Composition of Gold Nanoparticle–Protein Conjugates

A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky Prospect 33, Moscow 119071, Russia
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Academic Editor: Bing Yan
Received: 2 December 2014 / Accepted: 18 December 2014 / Published: 31 December 2014
(This article belongs to the Special Issue Bioactive Nanoparticles 2014)
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Abstract

A method was developed for determining the composition of the conjugates between gold nanoparticles and proteins based on the intrinsic fluorescence of unbound protein molecules. The fluorescence was evaluated after separation of the conjugates from the reaction mixture by centrifugation. Gold nanoparticles obtained using the citrate technique (average diameter 24 nm) were conjugated at pH 5.4 with the following four proteins: human immunoglobulin G (IgG), bovine serum albumin (BSA), recombinant streptococcal protein G (protein G), and Kunitz-type soybean trypsin inhibitor (STI). The compositions of these conjugates were determined using the developed method. The conjugate compositions were dependent on the concentration of the added protein, and in all cases reached saturation. The equilibrium dissociation constants of the gold nanoparticle conjugates with IgG, BSA, protein G, STI in the initial section of the concentration dependence curve were 4, 6, 10, and 15 nM, respectively. Close to saturation, the corresponding values were 25, 76, 175, and 100 nM, respectively. The maximal binding capacities of a single gold nanoparticle for IgG, BSA, Protein G, and STI were 52, 90, 500, and 550, respectively, which agrees well with the hypothesis of monolayer immobilization. View Full-Text
Keywords: gold nanoparticles; protein conjugates; tryptophan fluorescence gold nanoparticles; protein conjugates; tryptophan fluorescence
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Sotnikov, D.V.; Zherdev, A.V.; Dzantiev, B.B. Development and Application of a Label-Free Fluorescence Method for Determining the Composition of Gold Nanoparticle–Protein Conjugates. Int. J. Mol. Sci. 2015, 16, 907-923.

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