Role of the Functionalization of the Gold Nanoparticle Surface on the Formation of Bioconjugates with Human Serum Albumin

Abstract

The protein−gold nanoparticle bioconjugates are playing an important role in the studies of biological systems. The nature of the interaction and the magnitude of the binding affinity together with the conformational changes in the protein upon binding are the most addressed topics in relation to the uses of the bioconjugates in different organisms. In this work, we study the human serum albumin (HSA) protein−gold nanoparticle (AuNP) interactions focusing on the nature of the gold nanoparticle surface modification. We have found that the interactions of the HSA with the AuNPs are mainly electrostatic and that the concentration of protein necessary to stabilize the conjugates decreases when the overall negative charge on the nanoparticle surface increases. The changes in the localized surface plasmon resonance (LSPR) signals of the gold nanoparticles (13 nm diameter) are used to determine the number of protein molecules necessary to stabilize the conjugates in a high ionic strength medium. Fluorescence spectroscopy (stationary and time-resolved) is used to characterize the different bioconjugates and determine the binding constants under different experimental conditions. Moreover, the use of an extrinsic fluorescence probe (1-anilino-8-naphthalenesulfonic acid, ANS) gives us some information about the existence of partial unfolding of the protein upon binding to the nanoparticle.

Direct Detection of α-1 Antitrypsin in Serum Samples using Surface Plasmon Resonance with a New Aptamer–Antibody Sandwich Assay

“…SPR sensing of target proteins in crude serum via specific via direct affinity interactions has been demonstrated, [16][17][18][19][20] as well as achieving greater signal enhancement and 4 selectivity via the use of a secondary probe conjugated to particles. 16,[21][22][23][24][25][26] In addition, localized SPR measurements has also been explored for the sensing of antibiotin 27 and human epididymis secretory protein 28 in serum samples. Thus, after characterising the interactions between AAT with antiAAT and the AAT-sepcific aptamer in buffer, we also demonstrate a methodology for the analysis of AAT in serum samples at concentrations as low as 10 fM with a tuneable dynamic range depending on the antiAAT concentration.…”

1.3 μm emitting SrF2:Nd3+ nanoparticles for high contrast in vivo imaging in the second biological window

“…This is the case, for instance, of semiconductor quantum dots (QDs) or rare earth doped nanoparticles (RENPs) in which the spectral working range can be controlled by an adequate selection of synthesis conditions or of the doping fluorescent ions for QDs and RENPs, respectively. In addition, chemical methods have evolved such that a fine tuning of the LNPs surface coating is now possible [19][20][21]. This has been vital for the production of colloidal solutions of LNPs dispersed in biocompatible liquids, thus allowing for their direct injection in the Bloodstream for in vivo bioimaging experiments.…”

A Generalized Model on the Effects of Nanoparticles on Fluorophore Fluorescence in Solution

“…This is because the NP concentration in eq 9 and in the Stern−Volmer equation is the equilibrium concentration of ligand-free NPs, but not the total NP concentration used in much existing literature. 4,8,[11][12][13][14][15][16][17]42 Unfortunately, it is currently impossible to determine the equilibrium concentration of free NP (without bound ligands) in NP binding experiments.…”

“…As an example, the AuNP concentration is usually more than 2 orders of magnitude lower than that of protein in the fluorescence studies of protein/AuNP binding. 11,12,[14][15][16][17]42 It is, therefore, unreliable to use the total NP concentration to determine the NP/protein binding equilibrium constants.…”

“…Consequently, the IFE-corrected fluorescence intensity in BSA/AuNP samples can be calculated with eq 12 by replacing η AuNP in eq 11 with η pAuNP calculated with eq 4. (12) The data shown in Figure 1 indicate both BSA/p 2k AuNP and BSA/p 30k AuNP can serve as the external reference for correcting the fluorescence IFE effect in the BSA/AuNP samples because no BSA adsorption was observed in these two samples. In this work, we use BSA/p 2k AuNP as the external reference for correcting the fluorescence IFE effect in the BSA/ AuNP solutions, and the BSA/p 30k AuNP samples were used as independent validation samples for evaluation of the performance of the external reference method and two mathematical methods employed in recent literature.…”