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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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Number of molecules of protein adsorbed on a single nanoparticle. The dependence of the number of molecules of protein adsorbed on a single nanoparticle (RL) on the protein concentration used in the synthesis ([L0]). Graph (A) is for bovine serum albumin (BSA); (B) is for Kunitz-type soybean trypsin inhibitor (STI); (C) is for recombinant streptococcal protein G (protein G); and (D) is for human immunoglobulin G (IgG).
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ijms-16-00907-f004: Number of molecules of protein adsorbed on a single nanoparticle. The dependence of the number of molecules of protein adsorbed on a single nanoparticle (RL) on the protein concentration used in the synthesis ([L0]). Graph (A) is for bovine serum albumin (BSA); (B) is for Kunitz-type soybean trypsin inhibitor (STI); (C) is for recombinant streptococcal protein G (protein G); and (D) is for human immunoglobulin G (IgG).

Mentions: The obtained values of protein adsorbed on a single nanoparticle for different added concentrations of four studied proteins are given as curves at Figure 4. As can be seen, the saturation of adsorption capacity is reached for all cases. The concentrations necessary for the saturation lie in the range from 40 to 60 μg/mL.


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)

Number of molecules of protein adsorbed on a single nanoparticle. The dependence of the number of molecules of protein adsorbed on a single nanoparticle (RL) on the protein concentration used in the synthesis ([L0]). Graph (A) is for bovine serum albumin (BSA); (B) is for Kunitz-type soybean trypsin inhibitor (STI); (C) is for recombinant streptococcal protein G (protein G); and (D) is for human immunoglobulin G (IgG).
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-00907-f004: Number of molecules of protein adsorbed on a single nanoparticle. The dependence of the number of molecules of protein adsorbed on a single nanoparticle (RL) on the protein concentration used in the synthesis ([L0]). Graph (A) is for bovine serum albumin (BSA); (B) is for Kunitz-type soybean trypsin inhibitor (STI); (C) is for recombinant streptococcal protein G (protein G); and (D) is for human immunoglobulin G (IgG).
Mentions: The obtained values of protein adsorbed on a single nanoparticle for different added concentrations of four studied proteins are given as curves at Figure 4. As can be seen, the saturation of adsorption capacity is reached for all cases. The concentrations necessary for the saturation lie in the range from 40 to 60 μg/mL.

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