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Development and application of a label-free fluorescence method for determining the composition of gold nanoparticle-protein conjugates.

Sotnikov DV, Zherdev AV, Dzantiev BB - Int J Mol Sci (2014)

Bottom Line: 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 Article: PubMed Central - PubMed

Affiliation: A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky Prospect 33, Moscow 119071, Russia. sotnikov-d-i@mail.ru.

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.

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Characteristics of the gold nanoparticles. (A) Histogram of particle size distribution; (B) An image of gold nanoparticles from the electron micrograph.
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ijms-16-00907-f001: Characteristics of the gold nanoparticles. (A) Histogram of particle size distribution; (B) An image of gold nanoparticles from the electron micrograph.

Mentions: Electron microscopy of the gold nanoparticles showed a high degree of size uniformity. The 57 tested particles had an average diameter of 23.9 ± 2.7 nm, and an axial ratio of 1.20 ± 0.09 (Figure 1). The hydrodynamic diameter of the particles according to data from dynamic light scattering was 25.8 ± 1.5 nm.


Development and application of a label-free fluorescence method for determining the composition of gold nanoparticle-protein conjugates.

Sotnikov DV, Zherdev AV, Dzantiev BB - Int J Mol Sci (2014)

Characteristics of the gold nanoparticles. (A) Histogram of particle size distribution; (B) An image of gold nanoparticles from the electron micrograph.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4307282&req=5

ijms-16-00907-f001: Characteristics of the gold nanoparticles. (A) Histogram of particle size distribution; (B) An image of gold nanoparticles from the electron micrograph.
Mentions: Electron microscopy of the gold nanoparticles showed a high degree of size uniformity. The 57 tested particles had an average diameter of 23.9 ± 2.7 nm, and an axial ratio of 1.20 ± 0.09 (Figure 1). The hydrodynamic diameter of the particles according to data from dynamic light scattering was 25.8 ± 1.5 nm.

Bottom Line: 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 Article: PubMed Central - PubMed

Affiliation: A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky Prospect 33, Moscow 119071, Russia. sotnikov-d-i@mail.ru.

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.

Show MeSH
Related in: MedlinePlus