Spectroscopic investigations of the changes in ligand conformation during the synthesis of soy protein-templated fluorescent gold nanoclusters

Highlights

• Ligand’s conformational changes during the synthesis of SP-AuNCs were investigated.

• The fluorescence intensity was dominated by the Au nucleus in early phases.

• An ordered-disordered-ordered structure transition happened to the ligand.

• The growth of Au core and LMCT caused the structural changes of protein ligand.

Abstract

In this paper, the potential relationship between fluorescence and changes in the ligand conformation observed during the synthesis of soy protein-templated fluorescent gold nanoclusters (SP-AuNCs) was studied using a series of spectroscopic techniques. The results show that the determinants of the fluorescence effect in SP-AuNCs changed with the reaction time during the synthesis process. In the early stage of the reaction (within 60 min), the fluorescence intensity was dominated by the Au nucleus, followed by the combination of the Au nucleus and protein ligand. The structure of the protein ligand also underwent a transition from ordered to disordered to ordered. At the same time, its role in the reaction also changed from providing the reducing power to protecting the Au nucleus and contributing to the transition of the fluorescence effect in the AuNCs via ligand-to-metal charge transfer (LMCT). Using two-dimensional (2D) photon spectra correlation analysis, the formation and growth of the Au nuclei and the LMCT effect observed during the synthesis of the SP-AuNCs were found to be the major causes for the changes in the conformation of the protein ligand. Our results are an important discovery and can be used to explain the mechanism of protein ligands in the synthesis of gold nanoclusters.

Introduction

Protein-templated gold nanoclusters (AuNCs) have excellent fluorescence performance and favorable biocompatibility [1], [2]. The super-large spatial molecular structure of protein ligands and the large number of reactive groups (–COOH, –NH₂, –OH and –SH, etc.) contained therein provide high stability to AuNCs, as well as infinite possibilities for further functional surface modification. In recent years, research on AuNCs capped by proteins (P-AuNCs) has mainly focused on the exploration of new protein ligands [3], [4], [5], [6], novel synthesis strategies and mechanisms [7], [8], [9] and the expansion of their possible application fields [10], [11], [12], [13]. However, nearly all of these studies are based on the fluorescence properties of AuNCs, such as how to enhance their fluorescence intensity or an investigation of some new application.

In order to carry out the above work effectively, it is necessary to have a sufficient understanding of the fluorescence mechanism of P-AuNCs. At present, the authoritative theory explaining the fluorescence emission of AuNCs protected by macromolecular ligands is the ligand to metal nanoparticle-core charge transfer (LMNCT) theory proposed by Wu et al. [14] in 2010, which is based on the LMCT (ligand-metal charge transfer) effect discovered by Forward et al. [15]. According to LCMT theory, if the ligand molecules, located on the outer shell layer to protect the gold nanoclusters, contain electron-rich atoms (such as O, N, etc.) or groups (such as –COOH, NH₂, etc.), the electrons will transfer from the ligands to the Au nuclei via LMNCT, thus enhancing the overall fluorescence intensity of the AuNCs. Subsequently, LMNCT theory has been used in many studies to explain the fluorescence mechanism of gold nanoclusters protected using various ligands such as PEG [16], DNA [17], PAMAM [18] and PEI [19]. Protein molecules contain a large number of electron-rich atoms and groups. Therefore, LNMCT theory must also be applied to elucidate and analyze the fluorescence mechanism of P-AuNCs. Shang et al. [20] have reported the most representative example, which involves the adsorption of human serum albumin (HSA) to the surface of DHLA-AuNCs to greatly increase its fluorescence intensity (6 fold). However, the changes in the protein ligands themselves during the synthesis of the AuNCs have been neglected because most attention has been focused on the ligand effect on the fluorescence emission. According to LMNCT theory, the protein ligands directly contribute to the fluorescence performance of AuNCs, and at the same time, as the outer shell they are directly in contact with the external environment, medium and target. It can be concluded that the protein ligands directly determine the fluorescence emission and fluorescence application of P-AuNCs. We believe that it is of great importance and very necessary to carry out an in-depth study on any possible changes in the protein ligands during the synthesis of P-AuNCs. In order to expand the applications of P-AuNCs in the construction of fluorescence sensors, researchers have carried out some work focused on the relationship between the fluorescence intensity and the changes in the conformation of the protein ligands caused by the external experimental parameters, such as pH [21] and temperature [22]. However, to the best of our knowledge, how the protein ligands influence the fluorescence emission during the synthesis of P-AuNCs, whether the protein ligands themselves experience any changes, how they change and how the changes are related to the fluorescence or whether there is a link between them, have not been reported to date.

In this study, the evolution of the fluorescence emission of AuNCs and the conformational changes in the soy protein (SP) ligand were investigated and analyzed systematically using a variety spectroscopic techniques including fluorescence spectroscopy, circular dichroism and Fourier transform infrared spectroscopy. The dynamic relationship between the fluorescence emission and protein ligand structure (especially the role it plays) during the synthesis of SP-AuNCs was fully explored using the research idea of “Phenomenon + Inference + Verification”, combining with two-dimensional (2D) correlation analysis technology. According to our spectroscopic analysis results, the fluorescence evolution of AuNCs and the conformational changes in protein ligands, as well as its roles in the synthesis process were mutually verified. The mutual advancement and dynamic structural changes observed between the conformation of the protein ligands and the fluorescence of the AuNCs observed during the SP-AuNCs synthesis have been systematically elucidated for the first time.