Spectroscopic investigations of the changes in ligand conformation during the synthesis of soy protein-templated fluorescent gold nanoclusters
Graphical abstract
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.
Section snippets
Materials
Soybean seeds were provided by Shandong Yuwang Industrial Co. Ltd. (Shandong, China). Defatted soybean protein was laboratory-made from soybean seed according to a well-established literature procedure [23] and its purity was measured using the Kjeldahl method (N × 6.25). Chloroauric acid (HAuCl4·3H2O, ≥99%) was purchased from Shaen Chem. Tech. Co. Ltd. (Shanghai, China). Heavy water (D2O, ≥99.9%) was purchased from Sigma-Aldrich (St Louis, MO). All other reagents were of analytical grade and
FL data analysis
Fig. 1 shows the fluorescence emission spectra recorded for the SP-AuNCs formed at the different reaction times studied. Before starting the reaction (0 min), the reaction system did not present any fluorescence signal within the wavelength range of 500–750 nm. After 15 min of reaction, a weak fluorescence peak began to appear at ~600 nm. The fluorescence peak gradually increased upon prolonging the reaction time until reaching a maximum at 360 min (the fluorescence peak intensity showed a
Conclusions
The conformational changes in the fluorescence and protein ligands during the synthesis of SP-AuNCs were determined and analyzed using FL, CD and FT-IR spectroscopy. According to the FL analysis, the intensity of the fluorescence peak before the synthesis reaction before 60 min was dominated by the Au nuclei, which was then determined by the Au nucleus and the protein ligand after 60 min. 2D correlation analysis of the FL spectra showed that the fluorescence peak of the SP-AuNCs observed at
CRediT authorship contribution statement
Yuliang Cheng: Conceptualization, Methodology, Writing - original draft, preparation, Funding acquisition. Jiannan Chen: Investigation, Data curation, Formal analysis, Visualization. Bin Hu: Resources. Fuwei Pi: Software. Hang Yu: Software. Yahui Guo: Resources. Yunfei Xie: Validation. Weirong Yao: Writing - review & editing. He Qian: Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The work reported in this article is supported by the National Key Research and Development Program of China (Project No.2019YFC1606000).
References (34)
- et al.
A label-free fluorescent assay for free chlorine in drinking water based on protein-stabilized gold nanoclusters
Talanta
(2015) - et al.
Ultrasensitive fluorescence detection of glutaraldehyde in water samples with bovine serum albumin-Au nanoclusters
Microchem. J.
(2011) - et al.
Au-nanocluster emission based glucose sensing
Biosens. Bioelectron.
(2011) - et al.
pH-Induced conformational changes of BSA in fluorescent AuNCs@BSA and its effects on NCs emission
Vib. Spectrosc.
(2013) - et al.
Temperature-induced optical property and conformational change of BSA-protected gold nanoclusters
J. Mol. Struct.
(2014) - et al.
Preparation and in vitro evaluation of calcium-induced soy protein isolate nanoparticles and their formation mechanism study
Food Chem.
(2012) - et al.
Thermally induced structural changes in glycinin, the 11S globulin of soya bean (Glycine max) - an in situ spectroscopic study
Biochim. Et Biophys. Acta-Proteins Proteomics
(2003) - et al.
Molecular-receptor-specific, non-toxic, near-infrared-emitting Au cluster-protein nanoconjugates for targeted cancer imaging
Nanotechnology
(2010) - et al.
Ultrasmall near-infrared gold nanoclusters for tumor fluorescence imaging in vivo
Nanoscale
(2010) - et al.
Time-dependent, protein-directed growth of gold nanoparticles within a single crystal of lysozyme
Nat. Nanotechnol.
(2011)
Synthesis and characterization of human transferrin-stabilized gold nanoclusters
Nanotechnology
Luminescent quantum clusters of gold in transferrin family protein, lactoferrin exhibiting FRET
Nanoscale
Luminescent quantum clusters of gold in bulk by albumin-induced core etching of nanoparticles: metal ion sensing, metal-enhanced luminescence, and biolabeling
Chem. -A Eur. J.
Bio-directed synthesis and assembly of nanomaterials
Chem. Soc. Rev.
Protein-assisted synthesis route of metal nanoparticles: exploration of key chemistry of the biomolecule
J. Nanopart. Res.
Controlling assembly of paired gold clusters within apoferritin nanoreactor for in vivo kidney targeting and biomedical imaging
J. Am. Chem. Soc.
Facile one-pot synthesis of near-infrared luminescent gold nanoparticles for sensing copper (II)
Nanotechnology
Cited by (4)
Natural plant compounds in synthesis and luminescence modulation of metal nanoclusters: Toward sustainable nanoprobes for sensing and bioimaging
2022, Materials Today AdvancesCitation Excerpt :The SP-AuNCs prepared under the optimized conditions can be used for sensitive detection of Cu2+. Qian's group also studied the correlation between SP conformation and fluorescence emission of the SP-AuNCs [53]. The investigation showed that the secondary structure of the SP ligand was affected by both Au–S coordination and growth of the Au nuclei during the preparation of SP-AuNCs, experiencing a transformation from the ordered to disordered and then to ordered structures (Fig. 4A).
One facile fluorescence strategy for sensitive determination of baicalein using trypsin-templated copper nanoclusters
2022, Spectrochimica Acta - Part A: Molecular and Biomolecular SpectroscopyCitation Excerpt :Meanwhile, MNCs have been employed in many fields of drug detection [27–29], catalysis [30,31], and cell imaging [32–34]. Specifically, Cu NCs display stronger fluorescence and lower cost than Au NCs, Ag NCs and Pt NCs [35–37]. The Cu NCs have great application prospects and attracted intensive interest in recent years [38,39].
Advances in the synthesis of gold nanoclusters (AuNCs) of proteins extracted from nature
2024, Nanotechnology ReviewsSoy Protein Gold Nanocluster as an “Off-On” Fluorescent Probe for the Detection of Bacillus Anthracis Biomarkers DPA
2023, Guang Pu Xue Yu Guang Pu Fen Xi/Spectroscopy and Spectral Analysis
- 1
These authors contributed equally to this work and should be considered co-first authors.