Using Cu 2+ ions as a detection material to verify the synthesis mechanism of Au nanoclusters mediated by wool keratin and silk fibroin resilience network

Abstract

Nanoclusters, with their ultrasmall sizes, have emerged as an indispensable tool in designing structural materials with a wide range of applications, but predicting the synthesis mechanism and structures remains challenging. This work delineates a synthesis mechanism of gold nanoclusters (AuNCs), which is realized by functionalizing a wool keratin (WK) and silk fibroin (SF) resilience network structure via self-assembly with controllable microstructure transformation. We synthesized such AuNCs by reducing the thiol groups of WK into WK@AuNCs and then entering the WF&SF resilience framework during the reconstruction, achieving WK@AuNCs/WK&SF with high fluorescence intensity for selective quenching of heavy metal Cu²⁺ ions. Further investigation indicated that α-helix and β-crystallites resulted in a soft–hard molecular segment denoted as WK&SF resilience network, which held and separated the WK@AuNCs into the nanocages. Owing to the synergism of these features, WK@AuNCs/WK&SF displayed superior fluorescence performances compared with WK@AuNCs.

Keywords

Gold nanoclusters soft–hard segment structure synthesis mechanism fluorescence quenching

In recent years single or multiple metallic nanoclusters have undergone rapid development due to their discrete energy levels and strong fluorescence.¹ Especially, noble metal nanoclusters (i.e. Au, Ag, Pt, and Pd) have attracted considerable attention because they have a high surface-to-volume ratio and intense intrinsic fluorescence.^2–5 Currently, gold or silver nanoclusters are synthesized by the use of environmentally benign templates, such as insulin, protein, enzyme, and peptide.^6–9 The fluorescence intensity of nanoclusters is strongly influenced by the templates, nanoclusters sizes, and the heavy metal ions (i.e. Hg²⁺, Cu²⁺, Zn²⁺, and Cr³⁺).^9–12 Cu²⁺ ions, as one of the most important heavy ions, affect human health, in which the increased level of Cu²⁺ ions will result in neurodegenerative diseases such as Wilson disease,^13–15 Menkes disease,¹⁶ Alzheimer’s disease,^17–19 and prion diseases.²⁰,²¹ Various sensors have been widely used to detect heavy metal ions.²²,²³ Interestingly, the quenching mechanism can reflect the microstructural transformation of materials. To overcome these issues, a controllable microstructure of gold nanoclusters (AuNCs), which is designed by functionalizing AuNCs into a soft–hard segment resilient network but hindering the aggregation of AuNCs to obtain high fluorescence, is needed.

A promising material regarding the required resilient network is a wool keratin (WK) and silk fibroin (SF) network, with a network structure of alternating α-helix and β-sheets, due to their low expense and biocompatibility, respectively.²⁴,²⁵ Pioneering work was done using bovine serum albumin or WK to reduce Au³⁺ into AuNCs which were then incorporated and assembled into SF networks.²⁶,²⁷ However, these methods do not enable the construction of soft–hard resilience networks, but rather just SF networks. Also, WK might provide more α-helix networks rather than mercapto (-SH). When using one-step synthesis from WK and SF, the WK&SF@AuNCs can grow dynamically and involve the variation of Au nanocrystals with a bimodal distribution.⁷ Although the synthesis mechanism of single metal clusters has been extensively studied, there is still some research space in the mechanism between AuNCs and self-assembled templates. Therefore, understanding the AuNCs’ microstructure transformation and preventing the aggregation process is of great importance for the preparation of strong fluorescence materials.

Herein, on the basis of the aforementioned design, we report a strategy to functionalize the WK and SF resilience network structure via self-assembly with controllable microstructure transformation. We synthesized AuNCs by reducing the thiol groups of WK into WK@AuNCs and then entering WF&SF resilience framework during the reconstruction. The strategy gives rise to high fluorescence intensity for selective quenching of heavy metal Cu²⁺ ions.

Experimental

Materials and apparatus

Merino 70s wool was supplied by Nanshan group Co., Ltd (Shandong, China). Silkworm pupa was purchased from Mengjin Silk Co., Ltd (Hangzhou, China). All chemicals are of analytical grade and used as received without further purification. Acetone, anhydrous ethanol, urea, sodium sulfide nonahydrate (Na₂S·9H2O), chloroauric acid tetrahydrate (HAuCl₄·4H₂O), sodium hydroxide (NaOH), and other salts (including various ions) were obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Sodium dodecyl sulfate (SDS) and Lithium bromide (LiBr) were purchased from Lingfeng Chemical Reagent Co., Ltd (Shanghai, China). Ultrapure water (resistivity > 18.2 MΩ·cm⁻¹) from the Millipore Milli-Q system was used in all experiments.

Fluorescence measurements were performed using a QM/TM fluorescence spectrometer (PTI, USA) with a 1.0 cm quartz cell. Fourier transform infrared spectroscopy (FTIR) was recorded on a FTIR spectrometer (NicoletTM 5700, USA) to examine the molecular conformation of the WK@AuNCs/WK&SF solution. For each measurement, 128 scans were performed with a resolution of 4 cm⁻¹ and a scope of 400–4000 cm⁻¹. A UV-VIS-NIR Spectrophotometer (Hitachi U-400, Japan) was used to record in the region between 200 and 800 nm. Transmission electron microscope (TEM) images of the nanoclusters were obtained using a JEM-2100 (JEOL Ltd, Tokyo, Japan) at an accelerating voltage of 200 kV. Measurement of the stability of the samples was performed using the Zeta-potential analysis and particle size analyzer (NANO ZS, British Malvern Instrument Co., Ltd., UK).

Preparation of regenerated WK and SF Solutions

Pretreated wool fibers (5 g) were treated in a mixed solution containing Na₂S (0.26M), SDS (0.13M), and urea (9M) with a bath ratio of 1:10 in a water bath at 60°C for 12 h. After the dissolution was completed, the WK mixed solution was added to a centrifuge tube, centrifuged at 5000 r/min for 30 min, and the supernatant liquid was taken to remove the undissolved wool fiber to obtain a pale yellow wool keratin solution. After filtering and dialysis for 72 h, the pure WK solution was obtained. The obtained WK solution was stored at 4°C before further use.

Degummed silk fibers (5 g) were dissolved in 9.3 mol/L LiBr solution at 60°C for 4 h to obtain a pale yellow transparent SF solution. Then the SF macromolecule was collected by dialysis bag with molecular weight of 3500 Da, in which the SF solution was placed in deionized water for 72 h. The obtained SF aqueous solution was stored at 4°C before further use.

Preparation of WK@AuNCs/WK&SF solution

A WK solution (2.5%wt) was first blended with a SF solution (2.5%wt) at 37°C for 10 min, and then a HAuCl₄ solution (5 mL, 10 mmol/L) was introduced to carry out a chelation reaction. After the chelation reaction, a NaOH aqueous solution (0.5 mL, 1 mol/L) was added to adjust the pH of the solution. The mixture was reacted in an incubator at a temperature of 37°C and a relative humidity of 50% for 12 h to obtain a WK@AuNCs/WK&SF solution. The WK@AuNCs/WK&SF solution percentage of WK: SF was AuNCs1(100:0), AuNCs2(80:20), AuNCs3(60:40), AuNCs4(40:60), AuNCs5(20:80), and AuNCs6(0:100), respectively. For one-step synthesis, 5 mL of 10 mM HAuCl4 aqueous solution was added to 5 mL of keratin and fibroin solutions with vigorous stirring at 37°C.

Sensing Cu²⁺ ions in probe solution

The WK@AuNCs/WK&SF solution of 2.5wt% was diluted to 0.1wt% using phosphate buffered saline to obtain the AuNCs probe solution. To determine the feasibility of using a Cu²⁺ ions solution as a detection material, a certain concentration (10⁻² M) of a Cu²⁺ standard solution was added to the WK@AuNCs/WK&SF solution. At the same time, different kinds of ions solutions, such as Na⁺, Mg²⁺, Sr²⁺, etc., were added to the WK@AuNCs/WK&SF solution at the same concentration (10⁻² M) to detect the specific selectivity of the WK@AuNCs/WK&SF solution.

Results and discussion

Design and synthesis mechanism of WK@AuNCs/WK&SF system

The synthesis mechanism of the WK@AuNCs/WK&SF system is different from one-step⁷ or two-step methods,²⁷ and its design and synthesis mechanism is illustrated in Figure 1. The microstructure of wool and silk fibers main consists of α-helix and β-sheets, respectively. The two right-handed α-helix chains of polypeptides in wool fibers are stabilized by the hydrogen bonds, in which the right-handed α-helix chains form the dimers by disulfide bonds between the ends and the sides of dimers. Our aim is to break the disulfide bonds crosslinking and form thiol groups as the reducing agents. These unstable sulfhydryl groups will reduce Au³⁺ to gold nanoclusters with 25 gold atoms, which will emit red fluorescence under UV light (λ = 365 nm).²⁸ However, for the silk fibers, the antiparallel β-sheets are held together by intermolecular hydrogen bonds but lack disulfide bond crosslinking. The α-helix, β-turns and random coils are unstable and soluble in aqueous solutions.²⁶ Here, we employed the WK solution with 40–60 kDa molecular mass composed of more thiol groups due to the breakage between disulfide bonds. Accordingly, the SF solution has larger molecular weight and is more prone to crosslinking by hydrogen bonds compared with the WK solution. If WK and SF solutions are first mixed, the β-sheets in SF can stack the α-helix and random coil in WK along the pre-existing β-sheets in SF to assemble new mixed β-sheets, as shown in Figure 1.⁷ Meanwhile, the α-helix in WK solution can crosslink with new mixed β-sheets to assemble the new resilient network structure. The α-helix, like a spring coil, has a high elongation and recovery elasticity, while the β-sheets, with a relatively stable and orderly arrangement, constitute the β-crystallites. The thiol groups are distributed inside the resilient network structure. Next, when chloroauric acid (HAuCl₄) solution is introduced, there is a significant synergistic effect between the WK&SF resilient network and the HAuCl₄. The order of addition of the materials has a great influence on the synthesis mechanism. On the one hand, the α-helix, β-sheets, β-turns, and random coils in SF can quickly form a crosslinked network structure with WK by self-assembly in the initial stage.²⁹ On the other hand, the thiol groups in WK are reduced by HAuCl₄ and form WK@AuNCs. Moreover, the WK&SF resilient network can separate the WK@AuNCs into a cage network structure to decrease the WK@AuNCs aggregation, resulting in higher fluorescence intensity. Due to the presence of α-helix in the SF&WK network structure, the nanocages can hold the WK@AuNCs tightly and do not easily aggregate. The synthesis mechanism of the WK@AuNCs/WK&SF system is difficult to characterize by direct experiments. Therefore, in this paper, we indirectly speculate the assembly and synthesis mechanism of WK@AuNCs/WK&SF by introducing Cu²⁺ ions into the system and observing the luminescence and quenching of the materials.

Figure 1.

Structure design and synthesis mechanism of the WK@AuNCs/WK&SF system.

Different characterization methods, including UV spectroscopy and photoluminescence spectroscopy, were carried out on the as-prepared WK@AuNCs/WK&SF system. Different ratios (R_WK:SF) (AuNCs1(100:0), AuNCs2(80:20), AuNCs3(60:40), AuNCs4(40:60), AuNCs5(20:80), and AuNCs6(0:100)) were used in our synthesis, and the most optimal ratio for the WK&SF@AuNCs system was AuNCs4 (Figure 2). It should be noted that when the SF to WK ratio increased, the red fluorescence and the fluorescence intensity gradually increased within a certain range except for AuNCs5 and AuNCs6. The pure WK@AuNCs (AuNCs1) gave the fluorescence intensity of 1.63 × 10⁵, demonstrating the fluorescence effect. Introducing the WK&SF resilience network into the WK@AuNCs remarkably enhanced the fluorescence intensity values, giving the value of 4.12 × 10⁵ (AuNCs4), and confirming the synergistic effect of SF. In addition, with the introduction of SF, the characteristic peaks were transferred from 670 nm to 657 nm for AuNCs1 and AuNCs4, respectively. The results showed that the synergistic effect between WK@AuNCs and WK&SF network gave rise to the increase of the fluorescence intensity. Under UV light (λ = 365 nm), the AuNCs1–5 emitted red fluorescence and AuNCs6 did not emit light (Figure 2(a)). The color of the solution changed from light brown, brown, and finally red-brown (Figure 2(b), inset). When the SF to WK ratio was 80:20, AuNCs5 emitted bright but shallow red fluorescence and the fluorescence intensity was close to AuNCs1 (pure WK), indicating the formation of WK@AuNCs. However, because the amount of the WK content in AuNCs5 was only 20%, part of the WK was first used to construct the elastic network, and then the remainder was used for the formation of WK@AuNCs. Therefore, the fluorescence intensity decreased drastically. When it was mediated by pure SF, the solution appeared reddish brown and the fluorescence intensity was almost zero with no fluorescence effect. This was because pure SF lacked sulfhydryl groups and had no reducing property, so it was impossible to synthesize WK@AuNCs. Figure 2(c, d) displays the UV absorption spectra of WK@AuNCs at various WK and SF concentrations. The resulting spectrum (Figure 2(c)) showed that the pure SF and SF@AuNCs had only one UV absorbance peak, at 275 and 287 nm, respectively, while for the pure WK and WK@AuNCs, the positions of the peaks were at 293 and 350 nm, related to amino acids tyrosine, tryptophan, and phenylalanine in the region.³⁰ In addition, we noticed that as the SF content increased, the integral area gradually decreased, excluding SF ratios of 80% and 100%, suggesting that the interactions were reconstructed between SF and WK during the formation of WK@AuNCs.

Figure 2.

(a) Fluorescence spectra and photographs under UV light (λ = 365 nm) of AuNCs1–6. (b) The maximum intensity value and photographs under visible light of AuNCs1–6. (c) UV absorption spectra of AuNCs1–6. (d) Corresponding peak area of UV absorption spectrum of AuNCs1–6. (e) FTIR infrared spectra of AuNCs1–6 in the ranges of 1720–1573 cm⁻¹. (f) Secondary structure percentage of AuNCs1–6.

To further quantitatively investigate the synthesis mechanism of the WK@AuNCs/WK&SF system, FTIR spectroscopy was performed to verify the microstructure transformation of WK@AuNCs/WK&SF system. The bands in amide I region were analyzed in accordance with procedures in the literature.³¹ A quantitative analysis on the amide I band was performed using the area of each component calculated as a percentage of the sum of the areas of all amide I component presented in the 1573–1720 cm⁻¹ spectral region, as shown in Figure 2(e). The amide I band can be fitted into 11 peaks, in which 1605–1615 cm⁻¹ is attributed to aggregated strands, 1616–1637 cm⁻¹ is associated to β-sheets, 1638–1655 cm⁻¹ is attributed to random coil, and 1656–1662 cm⁻¹ and 1663–1695 cm⁻¹ are assigned to α-helix and β-turns, respectively, as shown in Figure 2(e). By comparing the results of AuNCs1-6 as indicated in Figure 2(e, f), it is worth noting that as the SF content increased, the AuNCs4 with the highest fluorescence intensity had the lowest percentage of α-helix (15.17%) and the greatest percentage of β-sheets (25.52%). The results demonstrated that α-helix structures and β-sheets structures played a decisive role in the fluorescence performance of the WK@AuNCs/WK&SF system. Even more, with the increase of SF content, the side chains and random coils percentage of WK@AuNCs decreased, while the percentage of β-turns structure showed an upward trend. This showed that in the process of forming WK@AuNCs, the presence of SF might promote the transformation of irregular structures into more regular structures.

Selectivity of WK@AuNCs/WK&SF system toward detection of different metal ions

In preliminary experiments, a variety of different types of heavy metal ions including Li⁺, Na⁺, Mg²⁺, Al³⁺, K⁺, Ca²⁺, Cr⁶⁺, Fe³⁺, Cu²⁺, Zn²⁺, Sr²⁺, Ag⁺, and Pb²⁺ were tested to investigate their effects on the fluorescence intensity of AuNCs4, and the results are shown in Figure 3. As is quite obvious from Figure 3, only when Cu²⁺ ions were added to AuNCs4 did the fluorescence intensity at 665 nm significantly reduce to almost zero, and the fluorescence of AuNCs4 was quenched. In addition, the other metal ions possessed negligible effects on the fluorescence intensity. To better reflect the specific quenchability of AuNCs4 to Cu²⁺ ions, the relative fluorescence intensity of AuNCs4 with different kinds of metal ions added at 665 nm is shown in Figure 3(b). We can observe more clearly that only the relative fluorescence intensity of the AuNCs4 with Cu²⁺ ions was below 0.2, which more accurately indicated the specificity of the WK @AuNCs/WK&SF system. Figure 3(c) is photographs showing the luminescence of AuNCs4 before and after the action of Cu²⁺ ions in visible light and UV light (λ = 365 nm). It can be seen that under visible light, the addition of Cu²⁺ ions made AuNCs4 clear, and the pale yellow color became transparent. Under UV light irradiation, we found that the addition of Cu²⁺ ions caused the red fluorescence of AuNCs4 to disappear. Figure 3(d) illustrates the UV absorption spectra of AuNCs4 and AuNCs4 after the addition of Cu²⁺ ions. For AuNCs4, there was a strong peak at 270 nm and at 336 nm, which was attributed to the ligand protein in the system and its own absorption, respectively. It is worth noting that when a sufficiently large concentration of Cu²⁺ ions was added to AuNCs4, the characteristic peak of the UV absorption spectrum disappeared, demonstrating that the structure of AuNCs4 had been destroyed.

Figure 3.

(a) Fluorescence spectrum of AuNCs4 added to different kinds of metal ions. (b) Relative fluorescence intensity of AuNCs4 added to different kinds of metal ions. (c) Photographs of AuNCs4 solution before and after addition of Cu²⁺ under visible light and UV light (λ = 365 nm). (d) Ultraviolet absorption spectra of AuNCs4 and AuNCs4 solution after addition of Cu²⁺.

Assembly mechanism of WK&SF@AuNCs system verified by the fluorescence changes

Based on the above experimental results, Cu²⁺ ions were selected as the detection material for verifying the assembly mechanism of the WK@AuNCs/WK&SF system. The effect of Cu²⁺ ions with different concentrations (1.0 × 10⁻⁸–5.0 × 10⁻²M) added to the AuNCs4 and AuNCs1 is illustrated in Figure 4 (a–f). When the relative fluorescent intensity was lower than 0.3, the AuNCs4 and AuNCs1 system was quenched.³² It is worth noting that the fluorescence intensity of AuNCs4 and AuNCs1 gradually decreased with the increasing concentration of Cu²⁺ ions. The values of I and I₀ seemed to be divided into three parts in the presence of various concentrations of Cu²⁺, as illustrated in Figure 4(e, f). For AuNCs4 and AuNCs1, the first part of the concentration of Cu²⁺ ions was less than 5.0 × 10⁻⁵M and 1.0 × 10⁻⁵ M, the second part of the concentration of Cu²⁺ ions was between 5.0 × 10⁻⁵ M to 1.0 × 10⁻³ M and 1.0 × 10⁻⁵ M to 5.0 × 10⁻⁴M, and the third portion of the concentration of Cu²⁺ was greater than 1.0 ×10⁻³ M and 5.0 × 10⁻⁴ M, respectively. According to the analytical results, at the same concentration of Cu²⁺ ions, AuNCs4 was difficult to quench. Meanwhile, the Stern–Volmer equation was established to describe the quenching process. The equation was $\frac{I_{0}}{I} = 1 + K_{s v} [I]$ , where I₀ and I represent the fluorescence intensity of AuNCs4 or AuNCs1 in the absence and the presence of Cu²⁺ ions, respectively. K_sv is the Stern–Volmer quenching constant and [I] is the concentration of Cu²⁺ ions.³² As is clearly seen, all three parts showed a good linear relationship for both probes, which was consistent with the Stern–Volmer plot, as shown in Figure 4(e, f). From the nature of the Stern–Volmer graphs as depicted in Figure 4e and f, the magnitudes of K_sv increased and then decreased with the rise of concentration of Cu²⁺ ions. For AuNCs4 and AuNCs1, at the first and second stage, the values of K_sv (0.0303 and 0.2252) for AuNCs1 was bigger than that of K_sv (0.0145 and 0.1929) for AuNCs4, respectively, suggesting that the occurrence of quenching of AuNCs4 required a larger amount of Cu²⁺ than that of AuNCs1. Moreover, at the third stage, we notice that Ksv (0.0398) for the AuNCs4 was larger than that of AuNCs1 (0.0230), demonstrating that the quenching rate for AuNCs4 was faster, but the fluorescence intensity was still greater than AuNCs1. By comparing the three region fitting curves of the two probe solutions, this further indicated that more Cu²⁺ ions were indeed needed during the quenching of AuNCs4.

Figure 4.

Fluorescence spectra of (a) AuNCs4 and (b) AuNCs1; Relative fluorescent intensity (c) AuNCs4 and (d) AuNCs1; Fitting results of relative fluorescent intensity of (e) AuNCs4 (f) AuNCs1 at different concentrations of Cu²⁺ ions.

Synthesis mechanism of WK@AuNCs/WK&SF system as Cu²⁺ ions probe

The TEM images of AuNCs4 with the introduction of Cu²⁺ ions were also measured, as shown in Figure 5(a–c). The increasing concentration of Cu²⁺ ions to AuNCs4 had greater effect on the size of nanoclusters. As shown in Figure 5(b), we can clearly observe that the surface crystal lattice (0.236 nm) of AuNCs4 faded. With the increase of Cu²⁺ ions concentration, the size of AuNCs4 with the Cu²⁺ ions concentration (5.0 × 10⁻⁴M) increased to 3.593 nm. When the Cu²⁺ ions concentration (5.0 × 10⁻²M) was sufficient to completely quench AuNCs4, the average diameter reached about 3.782 nm of more than 100 individual particles, and there was almost no individual with a diameter less than 2 nm in the system, as shown in Figure 5(c). Furthermore, the surface lattice of the WK@AuNCs/WK&SF had all disappeared. The zeta potential before and after the addition of Cu²⁺ to the AuNCs1-5 is given in Table 1. The zeta potentials were, in essentially all cases, negative. The absolute value of the zeta potential (–48.33±4.96 mV) was typically maximized at AuNCs4 without the addition of Cu²⁺. With the increase of SF, the zeta value reduced. The results indicated that SF imparts stability to the gold nanoparticles, avoiding aggregation between particles by both electrostatic forces and steric interactions.³³,³⁴ Especially, the self-assembling network of WK and SF prevented neighboring gold nanoparticles from aggregation, as illustrated in Figure 6. It can be found that the addition of Cu²⁺ ions significantly reduced the absolute value of the zeta potential of the system, indicating the stability of the WK@AuNCs/WK&SF system.³⁵ Based on the above results, we speculate that when Cu²⁺ enters the WK@AuNCs/WK&SF system, they are divided into two parts. A portion of the Cu²⁺ ions first reacts with the SF&WK wrapped in the outer layer to form a complex, while another portion of the Cu²⁺ ions continues to advance through the SF to form Au–WK–Cu with the WK@AuNCs.³⁶ Therefore, the WK@AuNCs/WK&SF system required more Cu²⁺ ions to complete fluorescence quenching (relative fluorescence intensity less than 0.1), as shown in Table 1. When the ratio of WK: SF was 20:80, the <20% WK and 80% SF firstly constructed a thicker cage, in which most of the WK was involved in the construction of the network. With the introduction of HAuCl₄, the amount of WK was not enough to build the WK@AuNCs. Therefore, the fluorescence intensity was significantly reduced, as illustrated in Figure 2(a), and it is difficult to quench owing to the thicker cage.

Figure 5.

(a–c) TEM image and size distribution map of AuNCs4 treated with Cu²⁺ ions at different concentration (a) 0M, (b) 5 × 10 ⁻⁴ M.

Table 1.

Zeta potential of the system before and after the addition of Cu²⁺ ions and Cu²⁺ ions concentration completely quenched of AuNCs1–5.

Samples	AuNCs1	AuNCs2	AuNCs3	AuNCs4	AuNCs5
Zeta potential (mV) before	–27.57 ± 1.07	–30.67 ± 1.34	–33.70 ± 2.86	–48.33 ± 4.96	–28.20 ± 3.13
Zeta potential (mV) after	–20.6 ± 3.04	–23.4 ± 0.69	–21.0 ± 1.65	–22.6 ± 0.65	–7.92 ± 2.72
concentration (M)	0.0005	0.001	0.005	0.01	0.05

Figure 6.

Schematic illustrations for the quenching mechanism of Cu²⁺ ions on the WK@AuNCs/WK&SF system and WK&SF@AuNCs of one-step synthesis.

As shown in Figure 6, Cu²⁺ ions were introduced into AuNCs1 and AuNCs4, which may have led to the “two-phase” and “three-phase” structure in the mixed solution, respectively. Generally, AuNCs1 was composed of the WK@AuNCs phase in which the individual WK@AuNCs were crosslinked on WK macromolecular chains,³² as illustrated in Figure 6(a). The WK@AuNCs and Cu²⁺ ions mixed solution was considered as two phases, including the uniform WK@AuNCs phase and the aggregated WK@AuNCs phase. The three-phase structure model contained the above two-phase structure consisting of the Au–WK–Cu structure and network phase made up of WK&SF structure. Figure 6(b) demonstrates the reaction between WK@AuNCs/WK&SF system and Cu²⁺ ions. With the increase of Cu²⁺ ions, it is obvious that the lattice fringe faded away from a distinct lattice spacing of 0.236 nm³⁷ to none, as shown in Figure 5, which was consistent with the crystal lattice of the gold crystal face. The results indicated that the Cu²⁺ ions led to the aggregated WK@AuNCs phase, thereby giving rise to the fluorescence quenching. The results showed that the complex (WK–Cu–WK) at the 1:2 ratio was produced between copper (II) ions and WK@AuNCs, the copper (II) ions through carboxyl groups and amine groups. Due to the interaction between copper (II) ions and WK molecules, WK@AuNCs systems form AuNCs–WK–Cu–WK–AuNCs complex, which has the ability to destroy the stable structure of WK@AuNCs systems. Therefore, the fluorescence of WK@AuNCs is quenched. The WK&SF structure, which contained the molecular network reconstruction of WK and SF, had formed the α-helix and β-sheets network structures. The β-sheets in WK and SF network fabricated the form of β-crystallites due to the interactions between the WK and SF molecules, and the α-helix chains of WK were located among the crystallites chains via the hydrogen-bond interactions.³⁸ The WK&SF molecular network protected WK@AuNCs from aggregation and led to fluorescence-quenching difficulties via Cu²⁺ ions. In addition, we notice that WK@AuNCs was uniformly distributed in WK&SF molecular network by hydrogen bonds and Van der Waals forces, as illustrated in Figure 6(b). The mechanism of WK@AuNCs/WK&SF system was in good agreement with the relative fluorescence of AuNCs1 and AuNCs4 with various concentrations of Cu²⁺ ions, which indicated high quenching rate at low concentration and relative low reaction rate at high concentration of Cu²⁺ ions. To speculate about the separation role of the WK&SF resilience network, the one-step synthesis of WK&SF@AuNCs is illustrated in Figure 6 (c). We notice that new WK@AuNCs continued to emerge and were aggregated under the electron beam irradiation.⁷ We calculated the distance between the centers of two WK@AuNCs in Figure 6(b) and (c), 2.161 nm for AuNCs4 and 6.229 nm for one-step synthesis, respectively, demonstrating the separation role of the WK&SF resilience network. The α-helix chains provided an elastic effect, and the β-crystallites played a key role in the network stability of the analyzed WK&SF resilience network. Furthermore, once the WK@AuNCs were generated, this elastic network can separate and wrap them into the WK&SF system. Therefore, the results confirmed the superior separate effect of the WK&SF resilience network, maximizing the synergistic effect between WK@AuNCs and the WK&SF resilience network.

Stability and application of WK@AuNCs/WK&SF system

Figure 7(a) shows the fluorescence performance stability of the WK@AuNCs/WK&SF system placed at various days. It can be seen that the fluorescence intensity of AuNCs4 was lower at the beginning. With the increase of time, it was obvious that the maximum peak at 670 nm shifted to 657 nm when treated at 3, 7, and 14 days. Meanwhile, the fluorescence intensity increased, indicating that more thiol groups formed Au–S covalent bonds with gold and required more reaction time. The results of the secondary structural analyses for AuNCs4 are given in Figure 7(b). As indicated, when the time was changed from 1 day to 14 days, the α-helix percentage decreased from 18.13% to 14.58% and the β-sheets percentage increased from 24.21% to 26.29%. The reason for this situation was that the thiol groups formed more Au–S bonds and led to the decrease of α-helix percentage in AuNCs4. In addition, due to the crosslinking of hydrogen bonds, the β-sheets accordingly increased. Therefore, the lower component of α-helix was responsible for the higher fluorescence intensity. Figure 7(c, d) reflects the fluorescence stability of WK@AuNCs/WK&SF in buffer systems at various pH values. It is evident that the fluorescence intensity increased with increasing pH values. Moreover, the relative fluorescence intensity of AuNCs4 did not show a large difference with a pH value from 5.5 to 8.0, indicating that WK@AuNCs/WK&SF had higher stability in a certain range of acidity and alkalinity. The appropriate concentration of WK@AuNCs/WK&SF ink was prepared by using phosphate buffer solution and placed in an inkjet printer cartridge, in which the Donghua University emblem was printed by PC, and the image is shown in Figure7(d). As can be seen from Figure7(d), the pattern printed with AuNCs4 as ink was clearly visible. The printing paper was lavender under UV light and the Donghua University emblem emitted red fluorescence. This result indicated that the WK@AuNCs/WK&SF solution was used as a printing ink to achieve fluorescence imaging. This application not only indicated that the WK@AuNCs/WK&SF had good adaptability to common printer nozzles, but also different fluorescent printing papers for security can be easily prepared. Therefore, the method provides new ideas for the application of WK@AuNCs/WK&SF, for example, detection of heavy metal ions, inkjet LED materials, and functional scaffolds.

Figure 7.

(a) Fluorescence spectra of AuNCs4 placed at different days. (b) Secondary structure percentage of AuNCs4 at different days. (c) Fluorescence spectra of AuNCs4 in phosphate buffer with different pH values at room temperature. (d) Printing image using AuNCs4 ink.

Conclusion

In summary, we reported a synthesis mechanism of a WK@AuNCs/WK&SF system, which was realized by fixation of WK@AuNCs into a WK&SF resilience network with controllable microstructure transformation. Such materials combined the functions of heavy metal selectivity by fluorescence quenching. The order in which WK, SF, and HAuCl₄ were added to determine the macromolecular network. In addition, WK and SF percentages were critical in determining the fluorescent performance. In this design, the key success was to reconstruct a WK&SF soft–hard segment molecular network to separate the WK@AuNCs, which maximized the synergy between the resilient network and WK@AuNCs, exhibiting superior fluorescence performances. The synthesis mechanism of WK@AuNCs/WK&SF may open an avenue for microstructure construction for developing highly efficient nanoclusters for heavy metal detection and self-fluorescent emission devices.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Hongling Liu

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