Denatured proteins as a novel template for the synthesis of well-defined, ultra-stable and water-soluble metal nanostructures for catalytic applications

The templated synthesis of noble metal nanoparticles using biomass, such as proteins and polysaccharides, has generated great interest in recent years. In this work, we report on denatured proteins as a novel template for the preparation of water-soluble metal nanoparticles with excellent stability even after high speed centrifugation or storage at room temperature for one year. Different noble metal nanoparticles including spherical gold and platinum nanoparticles as well as gold nanoflowers are obtained using sodium borohydride or ascorbic acid as the reducing agent. The particle size can be controlled by the concentration of the template. These metal nanoparticles are further used as catalysts for the hydrogenation reaction of p-nitrophenol to p-aminophenol. Especially, spherical gold nanoparticles with an average size of 2 nm show remarkable catalytic performance with a rate constant of 1.026 × 10− 2 L s− 1 mg− 1. These metal nanoparticles with tunable size and shape have great potential for various applications such as catalysis, energy, sensing, and biomedicine.


Introduction
Due to their high surface-to-volume ratios and quantum size effects, noble metal nanoparticles exhibit distinct optical, thermal, and chemical properties from their bulk counterparts [1,2]. These nanoparticles with controlled sizes and shapes have received tremendous attention in a myriad of areas including catalysis, energy, optics, sensing, biomedicine, and leather industry [3][4][5][6][7]. For example, gold nanoparticles (AuNPs) have shown remarkable catalytic activities for many organic transformations such as addition to carbon-carbon bonds, oxidation of carbon monoxide and alcohols, selective hydrogenations, and carbon-carbon coupling reactions [8][9][10]. However, metal nanoparticles aggregate easily which greatly hinders their usage in catalysis, biomedicine, and many other fields. To solve this problem, a variety of substances including thiolcontaining surfactants and polymers have been developed for surface functionalization of metal nanoparticles with long-term stability and dispersity [11][12][13][14]. It should be noted that this strategy may involve tedious synthetic and purification steps, harsh reaction conditions, and the use of toxic organic solvents [15,16].
Recently, the environmentally friendly synthesis and stabilization of metal nanoparticles using biopolymers have become a trend [15][16][17][18][19]. For instance, chitosan [20][21][22], cellulose [23], gelatin [24], bovine serum albumin [25,26], and collagen that can be extracted from leather products [27], have been reported as templates for the synthesis of gold nanoparticles at room temperature. In the past decade, our group has developed a facile synthesis route to unfold native proteins including human serum albumin (HSA) and lysozyme to well-defined linear biopolymers [28,29]. Such protein-derived polymers offer many fascinating characteristics such as biocompatibility, biodegradability, predetermined length, narrow size distribution, and a defined number of functional groups at distinct positions along the polypeptide backbone [30]. In addition, the conjugated PEG chains can reduce protein binding and prolong circulation time in the blood stream. Therefore, these proteinderived polymers are particularly attractive for biomedical applications and as templates for preparation of precision nanomaterials. For example, multifunctional nanoparticles have been constructed by coating these protein-derived biopolymers to the surface of quantum dots, gold nanostructures, and nanodiamonds [28,[31][32][33]. The resulting nanohybrids which display excellent stability and biocompatibility, have been successfully used in gene transfection, cell imaging, and cancer therapy [31,32,34].
In this work, we report on unfolded protein backbones that possess a large number of amino groups able to bind metal ions and using them to act as novel templates for the preparation of noble metal nanoparticles with controlled sizes and shapes. Two reducing agents, ascorbic acid and sodium borohydride, are used to control the shape, and the template concentration is discovered to control the size of nanoparticles. Anisotropic gold nanoflowers (AuNFs) as well as ultrasmall spherical AuNPs and platinum nanoparticles (PtNPs) with high water solubility, excellent stability and good biocompatibility have been obtained. The template is derived from natural polymers, and the whole process is conducted in aqueous solution at room temperature. Therefore, the route can be regarded as an environmentally friendly procedure. More significantly, the well-defined nanoparticles demonstrate excellent catalytic performances for the hydrogenation reaction of p-nitrophenol to p-aminophenol. Collectively, these metal nanoparticles prepared using the novel protein-derived template may find great potential in surface-enhanced Raman spectroscopy, photothermal therapy, catalysis, biomedicine, textiles, and functional coatings for leather products.
2.2 Synthesis of cationic HSA (cHSA) [32,34] HSA (150 mg, 2.26 μmol) was dissolved in 15 mL of degassed ethylenediamine solution (2.5 M), and the pH was tuned to 4.75 by using HCl. After adding EDC·HCl (4 mmol, 766 mg) and stirring for two hours at room temperature, acetate buffer (1 mL, 4 M, pH 4.75) was added to terminate the reaction. The obtained reaction solution was purified twice with acetate buffer (100 mM, pH 4.75) and thrice with deionized water by ultracentrifugation using a Vivaspin 20 concentrator (MWCO 30 kDa). The resulting solution was lyophilized to afford the product as a white fluffy solid (160 mg, yield: 98%, MALDI-ToF MS: 72.3 kDa). [32,34] cHSA (101 mg, 1.40 μmol) was firstly dissolved in degassed phosphate buffer (30 mL, 50 mM, pH 8.0). NHS-PEG (159.6 mg, 79.8 μmol) was dissolved in 0.6 mL of DMSO and then added into the cHSA solution. After stirring at room temperature for four hours, the reaction solution was purified five times with deionized water by ultracentrifugation using a Vivaspin 20 concentrator (MWCO 30 kDa). The resulting solution was lyophilized to obtain the product as a white fluffy solid (206 mg, yield: 90%). The MALDI-ToF MS indicates a molecular weight of 164.3 kDa which means on average 46 PEG chains were conjugated into each cHSA backbone.

Synthesis of AuNPs using NaBH 4 as the reducing agent
The molar ratio of HAuCl 4 to amino groups in PEG-dcHSA for the synthesis of AuNPs was tuned from 3:0 to 3:3. Take the molar ratio of 3:3 as an example, 1.5 mg of PEG-dcHSA was dissolved in 4.4 mL of Milli-Q water and then 0.5 mL of HAuCl 4 aqueous solution (2 mM) was added and stirred for one hour. Afterwards, 0.1 mL of freshly prepared NaBH 4 solution (100 mM) was added dropwise under vigorous stirring. The color of the solution changed immediately to yellow. After stirring for one more hour, the solution was measured by UV-vis spectrometry. The product was purified five times with deionized water by ultracentrifugation using a Vivaspin 6 concentrator (MWCO 30 kDa).

Synthesis of AuNFs using ascorbic acid as the reducing agent
For the preparation of AuNFs, the reducing agent solution (100 mM) was obtained by dissolving L-ascorbic acid (176.1 mg) in 10 mL of degassed Milli-Q water. Most procedures and conditions are the same with those used for the synthesis of AuNPs in section 2.5, and the only difference is that L-ascorbic acid was used to replace NaBH 4 as the reducing agent.

Synthesis of PtNPs using NaBH 4 as the reducing agent
The procedures for the synthesis of PtNPs are similar to those used for preparing AuNPs. H 2 PtCl 6 solution (2 mM) was obtained by dissolving 10 mg H 2 PtCl 6 ·xH 2 O (39% Pt) in 10 mL Milli-Q water. The molar ratios of H 2 PtCl 6 to amino groups in PEG-dcHSA were 3:2 and 3: 0. The solutions were stirred overnight before use.

Catalytic reduction of p-nitrophenol to p-aminophenol
To a 20 mL glass bottle, 3 mL of freshly prepare p-nitrophenol aqueous solution (1 mM, pH = 12) and 15 mL of NaBH 4 aqueous solution (50 mM dissolved in Milli-Q water) were firstly added and gently shaken. Followed 1.2 mL of the mixed solution of NaBH 4 and p-nitrophenol was added into a vial and determined amounts of metal nanocatalysts were added and the reaction was monitored by UV-vis spectrometer. The absorbance at 400 nm is subtracted from that at 500 nm to correct for background absorption and then used to calculate the conversion and apparent rate constant (k app ). The conversion (C) is calculated using the following equation: where A 0 and A t are the absorbance at time 0 and t. The apparent rate constant (k app ) is determined as the slope of ln(A 0 /A t ) at 400 nm against the reaction time.

Characterization
UV-vis absorbance spectra were collected using a TECAN (Spark 20 M) microplate reader. The samples were added in a Greiner 96 flat transparent plate. The wavelength range was set from 250 to 800 nm. Matrixassisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry was performed on Bruker rapifleX spectrometer. Saturated solution of sinapinic acid dissolved in a 1:1 water/acetonitrile with 0.2% trifluoroacetic acid was used as the matrix solution and different concentrations of samples were measured. Transmission electron microscopy (TEM) samples were prepared by adding 4 μL of the metal nanoparticle solution onto a carbon-coated copper grid. After drying in air for 10 min, the remaining solution was removed by blotting with a filter paper. The measurement was conducted on a JEOL JEM-1400 TEM operating at an accelerating voltage of 120 kV. ImageJ software was used for the analysis of particle size.

Unfolding native proteins to well-defined biopolymers
The synthesis of protein-derived biopolymer PEG-dcHSA and its application as a novel template for the preparation of metal nanoparticles are schematically illustrated in Fig. 1a. Firstly, the native protein HSA was cationized by converting carboxyl groups on the surface to primary amino groups. In order to prevent protein aggregation during the following denaturation step, short polyethylene glycol (PEG) chains with an average molecular weight of 2000 g mol − 1 were introduced. These biocompatible PEG chains also afford PEG-cHSA with improved solubility and stability due to the steric effect between different chains. Subsequently, PEG-cHSA was unfolded in urea-phosphate buffer in the presence of TCEP as a reducing agent. In this step, urea could break the hydrogen bonds and other supramolecular forces. The 17 disulfide bridges in the 3D structure of HSA were also destroyed by TCEP, and the generated thiol groups were capped by MI-NH 2 . Therefore, the number of primary amino groups was further increased, which resulted in better solubility and metal ion binding ability of PEG-dcHSA. MALDI-ToF mass spectra in Fig. 1b show the increase of molecular mass in each step, confirming the successful synthesis of the PEGylated linear polypeptide PEG-dcHSA.

Ultrasmall AuNPs prepared using NaBH 4 as the reducing agent
The protein-derived linear biopolymer PEG-dcHSA which possesses abundant primary amino groups that can bind various metal-containing anions was used as a template for the in situ synthesis of different metal nanoparticles. First, we investigated the templated synthesis of AuNPs using NaBH 4 as the reducing agent. In a typical procedure, PEG-dcHSA was dissolved in Milli-Q water and then mixed with HAuCl 4 aqueous solution for one hour. Subsequently, freshly prepared NaBH 4 solution was added dropwise under vigorous stirring. The reaction solution changed immediately to yellow, pink or red depending on the added amount of PEG-dcHSA. After stirring for one more hour, the product was purified by five ultracentrifugation cycles with deionized water using a Vivaspin 6 concentrator (MWCO 30 kDa). As PEG-dcHSA is a precision polymer derived from HSA, the average number of primary amino groups in PEG-dcHSA was calculated as 147 (see supplementary information for more details). The molar ratio of chloroauric acid (HAuCl 4 ) to amino groups in PEG-dcHSA was tuned from 3:0 to 3:3 by fixing the amount of HAuCl 4 and gradually increasing the amount of the denatured proteins. The details for the synthesis of AuNPs with different HAuCl 4 /−NH 2 molar ratios are summarized in Table 1.
The reaction was very fast as indicated by the swift change of the solution color after adding the reducing agent. As displayed on the top panel of Fig. 2a, the color gradually shifts from red to light brown with the increased amount of denatured proteins. The UV-vis absorbance spectrum in Fig. 2b shows a characteristic peak at 512 nm for the sample without adding the template (HAuCl 4 /−NH 2 molar ratio = 3:0). In contrast, the peaks of the other three samples are weaker and a blue shift is observed when the amount of PEG-dcHSA is increased, indicating reduction of size for these AuNPs [35,36]. TEM was further used to directly observe the morphology of AuNPs and the results are displayed in Fig. 2ck. We can see that AuNPs prepared under four different HAuCl 4 /−NH 2 molar ratios all display good dispersity, narrow distributions, and spherical shapes. The AuNPs prepared with a HAuCl 4 /−NH 2 molar ratio of 3:0 has an average diameter of 6.4 ± 2.2 nm. Ultrasmall AuNPs with the size of 2~5 nm were obtained when the denatured protein template was added to the reaction solutions. Increasing the added amount of PEG-dcHSA made the particle size smaller, which is consistent with the UV-vis spectra. Therefore, we can conclude that the proteinderived biopolymer can be used as an ideal template for the preparation of ultrasmall AuNPs together with the use of NaBH 4 as the reducing agent.  Crucially, the samples prepared with denatured proteins demonstrated significantly enhanced stability compared to AuNPs prepared without the template (HAuCl 4 /−NH 2 molar ratio 3:0). After the formation of AuNPs, the reaction solutions were purified five times by centrifugation at a high speed of 5000 rpm. As shown in the bottom panel of Fig. 2a, colors of AuNPs prepared in the presence of the biopolymer template did not change even though they were repeatedly concentrated and recovered. The solutions stayed homogeneous and there was no obvious change for the UV spectra of these solutions ( Figure S1). In contrast, the solution of the AuNPs prepared in the absence of the template changed to colorless after the purification step and black precipitates were observed at the bottom (see Figure S2). As displayed in the TEM image, severe aggregation occurred for the AuNPs without protection by denatured proteins (Fig. 2l). For AuNPs prepared in the presence of the biopolymer, all of them remained well-dispersed and no size change was observed (Fig. 2m-o). In addition, the AuNPs prepared by templated synthesis show excellent stability and dispersity even after storage at room temperature for one year ( Figure S3). Therefore, the protein-derived biopolymer is indispensable for good water-solubility and excellent stability of AuNPs.

AuNFs prepared using ascorbic acid as the reducing agent
The choice of reducing agent has a great impact on the size and shape of AuNPs formed by templated synthesis. We further investigated the influence on particle morphology and size the when using L-ascorbic acid as the reducing agent and PEG-dcHSA as the template. By replacing NaBH 4 with L-ascorbic acid, four Au nanostructures were synthesized with similar conditions as in Table 1. As shown in Fig. 3a, the reaction solutions showed different colors from red to purple and to blue when the amount of denatured proteins was increased. UV-vis spectra in Fig. 3b show characteristic peaks of gold nanoparticles and a red shift is observed when the molar ratio of chloroauric anions to amine groups in denatured protein was changed from 3:0 to 3:3. Both the colors and UV-vis spectra indicate an increase of particle size after adding more biopolymers. Furthermore, we used TEM to directly characterize the size and morphology of these Au nanostructures (Fig. 3c-k). Very interestingly, AuNFs with multiple extrusions were obtained, indicating a different particle formation mechanism when ascorbic acid was used as the reducing agent. More specifically, some very small gold dots were firstly formed, which served as seeds for the following reduction of HAuCl 4 . Because L-ascorbic acid is a mild reducing agent, the reactions were slower than those using NaBH 4 . Under slow reaction kinetics, gold atoms and small dots had enough time to diffuse and meet each other. Because the added biopolymer template may favor to bind certain crystalline phases [37], the nanoparticles thus grew along specific facets and formed branched morphologies. The diameter of AuNFs was tuned in 30 70 nm by varying the amount of the denatured protein based on analysis of more than 100 nanoparticles in TEM images for each sample. The increase of biopolymer concentration leads to larger-sized AuNFs, which is a different trend in comparison to the synthesis of ultrasmall AuNPs using NaBH 4 as the reducing agent. The reason for this interesting phenomenon is not clear. One possible reason is the larger area covered by the presence of more PEG-dcHSA. This facilitated the anisotropic growth of flower branches and the size increased. For the sample prepared in absence of the biopolymer template, spherical AuNPs with an average size of 35.4 ± 10.0 nm were obtained. In addition, the TEM image in Fig. 3d even shows aggregation before purification for the sample prepared without the denatured protein template, indicating the poor stability of naked AuNPs. Surprisingly, all three AuNFs prepared in the presence of different amounts of denatured proteins demonstrated excellent stability despite their larger sizes compared to those ultrasmall AuNPs prepared using NaBH 4 as the reducing agent (Fig. 3m-o and Figure S4).

Catalytical reduction of p-nitrophenol by Au nanostructures
Due to their high surface-to-volume ratios, gold nanomaterials are ideal catalysts for many organic reactions [8][9][10]. For example, the hydrogenation reaction of p-nitrophenol to p-aminophenol by NaBH 4 , which can be easily monitored by UV-vis spectroscopy, is a model reaction to evaluate the catalytic performance of noble metal catalysts [38]. Here, both ultrasmall AuNPs and AuNFs prepared under different conditions were used for the catalytical reduction of p-nitrophenol. As displayed in Fig. 4a-d, the characteristic peaks at 400 nm decreased for all spherical AuNPs prepared using NaBH 4 as the reducing agent, indicating their catalytic activity for the hydrogenation reaction. More importantly, there is a clear trend that the reaction became faster when the AuNPs were prepared in the presence of more PEG-dcHSA. The time to convert 90% catalyzed by AuNPs prepared without the biopolymer template (HAuCl 4 / −NH 2 molar ratio = 3:0) is more than 40 min. In contrast, it took only 24 min for the AuNPs prepared with a HAuCl 4 /−NH 2 molar ratio of 3:3 (Fig. 4e). By plotting ln(A 0 /A t ) at 400 nm against the reaction time, the apparent rate constant (k app ) is determined as the slope of fitted lines. As shown in Fig. 4f, the AuNPs prepared with a HAuCl 4 /−NH 2 molar ratio of 3:3 show the highest k app of 1.673 × 10 − 3 s − 1 . The apparent rate constants for AuNPs prepared with HAuCl 4 /−NH 2 molar ratios of 3:0, 3:1, 3:2 are 9.273 × 10 − 4 s − 1 , 1.032 × 10 − 3 s − 1 , 1.488 × 10 − 3 s − 1 , respectively. The higher catalytic activity of AuNPs synthesized with more templates is attributed to their smaller size and therefore higher surface-to-volume ratio to provide more catalytically active sites. Although the biopolymer coating may have a negative influence on the catalytic performance, it seems that the size effect is more significant. As the Au concentration (C Au ) in this catalytic reaction is only 0.163 mg L − 1 , the rate constant (k app /C Au ) of the AuNPs prepared with a HAuCl 4 /−NH 2 molar ratio of 3:3 is as high as 1.026 × 10 − 2 L s − 1 mg − 1 .
UV-vis spectra showing the catalytic properties of AuNPs and AuNFs prepared with ascorbic acid for the hydrogenation of p-nitrophenol are depicted in Fig. 5ad. By plotting the conversion against the time, we can see that the reaction catalyzed by spherical AuNPs showed a relatively high speed (Fig. 5e). However, AuNFs prepared in the presence of PEG-dcHSA demonstrate slower reaction kinetics especially in the beginning stages. This phenomenon probably is due to the biopolymer coating of AuNFs, which took some time for small molecules to diffuse. Fig. 5f shows the plot of ln(A 0 /A t ) at 400 nm against the reaction time, the k app of spherical AuNPs (HAuCl 4 /−NH 2 molar ratios of 3:0) was determined as 1.518 × 10 − 3 s − 1 . For AuNFs, we also calculated the k app for the period after diffusion. As the Au concentration (C Au ) here is 2.627 mg L − 1 , the rate constants (k app /C Au ) catalyzed by AuNFs prepared with HAuCl 4 / −NH 2 molar ratios of 3:1, 3:2, 3:3 are 3.64 × 10 − 4 L s − 1 mg − 1 , 4.21 × 10 − 4 L s − 1 mg − 1 , and 4.69 × 10 − 4 L s − 1 mg − 1 , respectively. Although the catalytic activity is not as high as ultrasmall AuNPs, they are still among the very effective Au catalysts in the literature [39][40][41][42].

Synthesis of PtNPs and their catalytic performance
In addition to well-defined AuNPs and AuNFs, other noble metal nanoparticles can also be prepared using the protein-derived biopolymer as template. For example, PtNPs have also attracted broad attention in catalysis and nanomedicine [43][44][45][46]. We further explored the preparation of PtNPs in the presence of PEG-dcHSA by reducing H 2 PtCl 6 with NaBH 4 . The molar ratio of PtCl 6 2− anions to amine groups of PEG-dcHSA in the solution was set as 3:2. As shown in Fig. 6a, water-soluble PtNPs  with good dispersity and an average size of~3 nm were obtained. In a control experiment, very severe aggregation was observed for PtNPs prepared without adding the denatured protein (Fig. 6b). In addition, the size and dispersity of PtNPs by templated synthesis did not change even after purification several times by ultracentrifugation at a high speed of 5000 rpm, indicating their excellent stability (Fig. 6c). We further evaluated the catalytic performance of PtNPs using the same hydrogenation reaction. As shown in Fig. 6d, the characteristic peak at 400 nm of p-nitrophenol gradually decreased in two hours. The conversion reaches 90% after reacting for 100 min (Fig. 6e). By plotting ln(A 0 /A t ) at 400 nm against the reaction time, the k app was obtained as 3.913 × 10 − 4 s − 1 (Fig. 6f). Therefore, PtNPs synthesized and stabilized with PEG-dcHSA can also serve as an effective catalyst for the hydrogenation reaction. In comparison, a catalytic study for PtNPs prepared without the biopolymer template was not possible because the particles were difficult to purify and the amount could not be determined.

Conclusion
In summary, denatured proteins have been reported as a novel template for the synthesis of water-soluble, ultrastable, and well-defined noble metal nanoparticles (AuNPs, AuNFs, PtNPs). Au nanostructures of different shapes (spherical and flower-like) have been prepared using NaBH 4 or ascorbic acid as the reducing agent. The sizes of Au nanostructures have been controlled by tuning the molar ratio of metal ions to amino groups of the template. Compared to nanoparticles prepared without the template, these metal nanoparticles prepared by templated synthesis demonstrated much better stability even after high speed centrifugation or storage at room temperature for one year. Moreover, these nanoparticles