Primary and Secondary Mesoscopic Hybrid Materials of Au

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Functional Nanostructured Materials (including low-D carbon)

Primary and Secondary Mesoscopic Hybrid Materials of Au Nano Particles@Silk Fibroin and Applications Chenyang Shi, Yao Xing, Aniruddha Patil, Zhaohui Meng, Rui Yu, Naibo Lin, Wu Qiu, Fan Hu, and Xiang Yang Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b07846 • Publication Date (Web): 01 Aug 2019 Downloaded from pubs.acs.org on August 2, 2019

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ACS Applied Materials & Interfaces

Primary and Secondary Mesoscopic Hybrid Materials of Au Nano Particles@Silk Fibroin and Applications

Chenyang Shi,a Yao Xing,a Aniruddha Patil, a,b Zhaohui Meng, a Rui Yu, a,b Naibo Lin, a

Wu Qiu, a,b Fan Hu, a,c Xiang Yang Liu a,b,d*

a Research

Institute for Biomimetics and Soft Matter of Xiamen University, College of

Materials, Xiamen University, Xiamen 361005, China. b

Fujian Provincial Key Laboratory for Soft Functional Materials Research, College of

Physical Science and Engineering, Jiujiang Research Institute, Xiamen University, Xiamen 361005, China. c Advanced

Soft Matter Group, Department of Chemical Engineering, Delft University

of Technology, Vander Maasweg 9, 2629 HZ, Delft, The Netherlands. d

Department of Physics, National University of Singapore, 2 Science Drive 3,

Singapore, 117542.

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ABSTRACT In this work, we demonstrate the principle of mesoscopic construction of silk fibroin (SF) hybrid materials that endows the materials with new performance. In implementing this strategy, mediating molecules, wool keratin (WK) molecules, were adopted to in-line synthesize Au nano particles (WK@AuNPs), which further create the stable linkage of Au nano-particles with SF nanofibrils networks via templated crystallization. It follows from the combined techniques of Fourier transform infrared spectroscopy (FTIR)- X-ray diffraction (XRD) and directly imaged by Atomic Force Microscopy (AFM) that the mesoscopic hybrid network structure of the hybrid materials is different from neat SF materials, which gives rise to various new performance ie. long-stable fluorescent emission. As the fluorescence emission can be characteristically annealed by Cu ions, therefore be adopted as the highly selective ions probes. Moreover, as WK@AuNPs homogeneously are connected to SF nanofibrils networks, the carbonization of the materials leads to secondary hybrid materials of carbon-Au, where nano sized Au particles are well distributed in carbonized mesoscopic conductive carbon networks. Such hybrid materials of carbon-Au can be further fabricated into electrochemical (ie. dopamine) sensors, which demonstrate to have excellent sensing performance.

KEYWORDS: silk fibroin, wool keratin, molecular nano cage, nano bridge, sensor


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1. INTRODUCTION Recently, many studies have shown that the mesoscopic structure of many functional soft materials plays an important role in their behaviors, which provides a detailed guidance to acquire functional performance of soft matters.1-3 It has been shown that the relationship between the properties and mesoscopic structure of soft materials is related to the following five elements: (1) topology, (2) correlation length, (3) ordering/system, (4) strength of linkages or interactions, and (5) hierarchy of structure.45

Due to the correlation mentioned above, a systematic approach for functionalizing

these mesoscopic materials has been adopted, which focuses on the reconstruction or modification of the mesoscopic structure of soft materials, such as cocoon silk, spider silk and wool materials. As typical class of soft materials, Bombyx mori silk fibroin (SF) has shown excellent behaviors in biomedical/electrical applications because of its promising

biocompatibility,

strong

mechanical

properties,

controllable

biodegradability.6-10 Acquire other additional properties of SF materials provides us with numerous opportunities to fabricate the next generation of SF devices.11 We note that quantum dots and functional molecules have been incorporated into silk materials;12-13 However, the applications of functionalized SF are restricted by the weak singular hydrogen bond interactions of functional molecules SF networks. The synthesis of novel functional molecules or particles to functionalize soft materials at the mesoscale in a controllable manner is urgently needed. Subnanometer noble metal particles have attracted the attention of many researchers in recent years. As their size approaches the Fermi wavelength (ca. 0.7 nm) of electrons, gold nanoparticles display molecule-like emission.14 The size-dependent optical



properties, coupled with potentially high photostability, make them promising candidates for optoelectronic applications.14-16 Meanwhile, the high surface area of subnanometer nanoparticles makes their synthesis particularly challenging. Unless protected by proper passivating ligands, small metal particles tend to agglomerate into larger particles.16 In the literature, these ultrasmall metal particles have been obtained by templates such as phosphine, thiol, dendrimers, DNA, peptides and proteins.16-21 In the various ligands mentioned above, proteins with mercapto groups (-SH) as a template exhibit highly bioactive and stable biocompatibility and provide bioactive functionalities throughout the nanocrystal surface for further biological interactions or couplings.22-23 However, more novel templating materials for metal nanoparticles synthesis are needed to broaden applications. To synthesize novel, highly efficient gold nanoparticles, wool keratin (WK) molecules were introduced due to the high content of the mercapto group (-SH) and unique macromolecular network structure.24-26 This work is to explore a new and generic approach of mesoscopic-hybrid materials for soft materials (ie. SF), which endows the materials with acquired new and extraordinary performance. In this context, wool keratin (WK) molecules are employed as Au nanoparticles synthesizers (WK@AuNPs) and hybrid facilitators to in-line synthesize Au nanoparticles (Au NPs), and to deliver and fix them into the mesoscopic structure of SF materials. It shows that WK@AuNPs are bounded to silk fibroin networks via linking β-crystallization between SF and WK molecules, endowing the materials with some particular properties (ie. fluorescent), the meso-hybrid materials SF materials can be applied as a highly sensitive and selective probe for rapid detection of Cu2+ in


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drinking water via a visual and nontoxic inspection. In addition, we harvested the mesohybrid carbon-Au materials after carbonizing the mesoscopic hybrid materials. The obtained meso-hybrid carbon-Au materials turn out to be high performance electrode materials for dopamine-sensing. 2. EXPERIMENTAL SECTION Materials. Wool fibers and cocoons of Bombyx mori silkworm silk were kindly supplied by Tongxiang Dushi Woolen Material Co., Ltd., and Guangxi Sericulture Technology Co., Ltd., respectively. Reagents and solvents were used as received without further purification unless otherwise mentioned. Chloroauric acid tetrahydrate (HAuCl4∙4H2O), sodium carbonate anhydrous and ethyl alcohol were purchased from Sinopharm Chemical Reagent. Sodium dodecylsulfate (SDS), sodium sulfide nonahydrate (Na2S∙9H2O), urea, acetone and sodium hydroxide (NaOH) were purchased from Xilong Chemical Industry. Lithium bromide was purchased from Aladdin Industrial Corporation. Solutions of metal ions, including Na+, Pb2+, Mg2+, K+, Ca2+, Cu2+, Fe2+, Al3+, Mn2+, Zn2+, Co2+, and Ni2+, were prepared from NaCl, Pb(NO3)2, MgCl4.6H2O, KCl, CaCl2, Cu(NO3)2, Fe(NO3)3, Al(NO3)3, MnCl2, Zn(NO3)2, CoCl2 and Ni(NO3)2, respectively. Water (18 MΩ cm-1) used for all the experiments was purified by the Milli-Q System. Dopamine was purchased from Sigma-Aldrich Ltd. Preparation of regenerated WK solution. The WK aqueous solution was prepared from pretreated wool fiber following a well-established procedure.8 Briefly, pretreated wool fibers were dissolved in 0.1 L of an aqueous solution containing 4 M urea, 0.1 M Na2S and 0.02 M SDS and heated to 50 ºC for 12 h. The mixed solution was then dialyzed



against distilled water using dialysis cassettes (Solarbio, molecular cut-off approximately 3500 Da) for 3 days, resulting in a 50 mg mL-1 aqueous solution of WK. Preparation of regenerated Bombyx mori SF solution. Regenerated aqueous SF solution was produced following previously reported protocols.5 In brief, to remove the sericin proteins from the fiber, the cocoons were boiled in a solution of NaHCO3 (0.02 M) (30 min, twice) and then rinsed thoroughly with distilled water to remove sericin and NaHCO3. Then, 1 g of degummed silk was dissolved in 7 mL of a 9.3 M lithium bromide solution (60 ºC, 4 h). The dissolved solution was then poured into dialysis cassettes (Solarbio, molecular cut-off approximately 3500 Da) and dialyzed against distilled water for 2 days with a change of water every 2 h, resulting in a 60 mg mL-1 aqueous solution of SF. Preparation of WK@AuNPs. The wool keratin-templated AuNPs were synthesized through a simple and convenient method. In a typical experiment, an aqueous HAuCl4 solution (5 mL, 10 mM) was added to the wool keratin solution (5 mL, 25 mg/mL) under vigorous stirring in order to bind Au3+ to the WK molecules sufficiently. Two minutes later, an NaOH solution (0.5 mL, 1 M) was introduced with continuous stirring for 8 min, followed by placing at 37 ºC for another 12 h. Finally, the obtained WK@AuNPs were stored at 4 ºC for later usage. Preparation of WK@AuNP-SF composite sponges: The sponges were fabricated by the freeze-drying method.27 Before the solution was frozen, a specified volume of WK@AuNPs was added into the silk solution (40 mg/mL) to reach six different final weight ratios: 0, 10%, 20%, 30%, 40% and 50%. Then, the hybrid solution was firstly


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transferred into a mold and frozen to -50 °C for 24 h, followed by freeze-drying (at -108 °C, 24 h) to obtain 3D porous functional sponges. The final samples SF, SF/Au10, SF/Au20, SF/Au30, SF/Au40, SF/Au50 refer to 0 wt%, 10 wt%, 20 wt%, 30 wt%, 40 wt% and 50wt%of WK@AuNPs in the sponges, respectively. Preparation of carbon materials: The WK@AuNP powders and SF/Au20 sponge which contain Au NPs with the same mass, followed by annealing at 700 ℃ under a nitrogen atmosphere for 2 h to obtain the Au@WK-C and Au@WK/SF-C, respectively. WK was added into the silk solution (40 mg/mL) to reach final weight ratios (20%), then, the hybrid solution was transferred into a mold and frozen at -50 °C for 24 h, followed by being placed in a vacuum freeze drier for 24 h. After that, SF/WK-C would be obtained from same carbonization methods. Characterization of WK@AuNPs and WK@AuNP-SF materials: Photographs of WK@AuNPs were captured under nature light and UV light (365 nm, UV-vis absorption spectra were recorded on a UV-vis spectrophotometer (Lambda 750, PerkinElmer). The fluorescence emission spectra were measured by a fluorescence spectrometer at room temperature with an excitation wavelength of 390 nm (FluoroMax-4, HORIBA). The absolute quantum yields were measured through an integrating sphere assessor (Quanta-φ, Horiba F-3029), which was connected to the spectroscopy fluorometer by two optical fiber bundles. Light from the sample compartment was directed into the sphere via a fiber-optic cable and an F-3000 fiber optic adapter and returned to the sample compartment via a second fiber optic cable and the F-3000 fiber. Transmission electron microscopy (TEM) was performed



througha JEM-2100F (JEOL, Japan), and X-ray photoelectron spectroscopy (Quantum 2000, PHI, USA) was employed to characterize the inner element chemical state. FTIR spectra were obtained with a Thermo Scientific Nicolet iZ10 spectrometer (Thermo Fisher, USA) to analyze the secondary structures of composite sponges. The crystallite structures of the composite sponges were characterized by X-ray diffraction (XRD, Bruker D8 AVANCE) with a beam size of 0.5 mm in the range of 0 to 60°. The morphology of the composite sponges was recorded with a field emission gun scanning electron microscope (SEM, Hitachi SU70) with an accelerating voltage of 5 kV. The compressive properties of the sponges were obtained by an Instron Microtester 5525X at room temperature. The samples were pretreated in a 0.01 M PBS solution for 24 h. The height and diameter of each cylinder-shaped sponge was measured by a ruler. Compressive modulus was calculated from the gradient of the linear-elastic region of the stress-strain curve. Detection of Cu2+: Six mg of the as-prepared SF/Au20 sponge was redissolved in a 2 mL aqueous solution containing different concentrations of Cu ions (0, 1 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, and 100 μM); then, the mixed solution was gently vibrated and incubated for 10 min at 25 °C before the luminescence measurement. To obtain the specificity of Au-hybrid sponge for sensing Cu2+, we carried out interference tests with Na+, Pb2+, Mg2+, K+, Ca2+, Cu2+, Fe2+, Al3+, Mn2+, Zn2+, Co2+, and Ni2+ under the same conditions. Detection of dopamine: Electrochemical performance of prepared carbonized composite material were examined by using a three-electrode system (CHI660D,


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Chenhua Instruments, China). That is an elongated polyethylene terephthalate (PET) strip having three electrode screen printed electrode (SPE) consisting Ag/AgCl reference electrode, carbon counter and working electrode. The working electrode is prepared by deposition of 10 µL of working ink (i.e. Au@WK/SF-C (5 mg/mL)) on a layer of a non-reactive electrically conductive material. Subsequent to the drying process, the electrode was cured for 1h at 70 ℃. The cyclic voltammetry (CV) and differential pulse voltammetry (DPV) measurements were carryout by use of electrochemical workstation (CHI 760E, Shanghai Chenhua, China).

3. RESULTS AND DISCUSSION 3.1. Hierarchical Network Structures of SF and WK Bombyx mori silk fibroin materials are composed of a light (L)-chain polypeptide and a heavy (H)-chain polypeptide with a molecular weight of ~ 390 kDa.6,

28

Regenerated SF materials have hierarchical nanofibril network structures (Figure 1a). The β-sheet structure forms through a self-assembled peptide, which is cross-linked by abundant hydrogen bonding and can be further stacked into β-nanocrystallites.2, 4-5, 7, 2930

The β-crystallites connected by SF protein molecules give rise to β-crystal networks

(cf. Figure 1a).4, 31 Such β-crystallite networks appear as silk nanofibrils with a diameter of 20-30 nm.2, 5 WK materials consist of keratin intermediate filaments (60 wt%) and matrix proteins (40 wt%), which have average molecular masses of 40-60 kDa and 1126 kDa, respectively.24 Intermediate filaments can be organized into a coiled structure (7 nm in diameter) because of their hierarchical structures (Figure 1b). To stabilize the structure, disulfide crosslinks are of great importance in wool fibers. The intermediate



filaments have a low sulfur content, and the matrix proteins have a high sulfur content.25 In regenerating WK materials, some of the disulfide bonds are broken. Each broken disulfide bond is divided into two mercapto groups (-SH), which are unstable and can be used as reducing agents to acquire Au nano-particles from Au3+ ions. In addition, the main structure of wool keratin molecules is α-keratin, which consists of an α-helix structure. The helical structure is stabilized by hydrogen bonds inside the helical chain, causing the chain to twist and exhibit an α-helical shape.

3.2. Mesoscopic hybrid The key steps of meso-hybrid materials construction include enclosing and binding functional molecules/nanoparticles into the mesoscopic network structure of silk fibroin materials.1,

4

This endows the materials with additional functions without

jeopardizing their original performance. Concerning the active molecular nanocage effect, the mercapto groups (-SH) in WK molecules serve as the chelating and reducing centers to reduce Au3+ to AuNPs inside the WK molecules. Then, the AuNPs are further encapsulated and stabilized by the WK molecules, which preserve the high fluorescence efficiency of the AuNPs (Figure 2a).23 The active molecular nanocages of WK are shown in Figure 2a. The active molecular nanocage effect allows WK@AuNPs to accumulate at a high density without jeopardizing their stability. This can be seen from the fact that WK@AuNPs acquire a high fluorescent intensity without suffering from fluorescence quenching.32 WK molecules were employed as active molecular nanocages to synthesize AuNPs in-line and as mediating molecules to incorporate them into the mesoscopic structure of SF materials. Mediated by WK molecules, the SF


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nanofibrils networks were subsequently reconstructed into SF-WK@AuNPs mesohybrid networks (Figure 2b). Concurrently, the WK@AuNPs built into SF nanofibrils network appear to be fluorescent emission centers and electrochemical reaction centers. In the process of preparing the AuNP-WK solution, a series of parameters in the reaction were observed by photographs and fluorescence spectroscopy, such as pH, temperature and time, as shown in Figure S1. In the experiments, the single-factor method was employed for qualitative analysis of the influence of synthesis. The results show that an alkaline condition (pH = 12), reaction temperature of 37 ℃ and solution mixing time of 12 hours are optimum conditions for the preparation of a AuNPs-WK solution. 3.2.1. Fluorescent properties of meso-hybrid materials Here, the fluorescent emission of Au NPs was utilized to identify the distribution in the ambient phase and to characterize the effect of SF meso-hybrid materials. While a neat WK solution appears pale yellow in daylight, it emits a weak blue light under UV light (λ= 365 nm) (Figure 3a) due to the aromatic side groups containing tryptophan, tyrosine (Tyr) and phenylalanine.33-34 On the other hand, the WK@AuNPs solution appears deep brown in daylight and emits a bright red fluorescence under UV light (λ = 365 nm). Furthermore, a sharp peak in the UV-vis absorption spectrum of WK solutions was observed at 280 nm, which was caused by aromatic acid groups (Figure 3b (left)). In contrast, WK@AuNPs display a broader peak at 280 nm due to the alteration of the microenvironment by Au atoms. To distinguish the fluorescence emission of WK@AuNPs from localized surface plasmon resonance (LSPR), we note that the fluorescence emission associated with the localized surface plasmon resonance



of Au nanoparticles normally occurs at approximately 520 nm.35 In our case, no peak was observed at this wavelength. In the fluorescence emission spectra of WK and WK@ AuNPs given in Figure 3b (right), a characteristic fluorescence emission peak at 690 nm was observed instead. On the other hand, neat WK solutions have no similar fluorescent peak at this wavelength (550~850 nm). X-ray photoelectron spectroscopy (XPS) determines the oxidation state of the AuNPs. In Figure 3c, WK@AuNPs exhibit six major elements: C, O, N, Au, Na, and Cl. The deconvolution of the Au spectrum gives rise to two distinct components centered at 83.95 and 87.3eV on the binding energies plot, which can be assigned to Au(0) and Au(I), respectively (Figure 3c & Figure S3).36 As shown in the observations of the fluorescence of composite sponges in Figure 3e, SF, SF/Au10~SF/Au50 refers to sponges with a content of WK@AuNPs from 0 wt% to 50 wt%, which appear from weak blue to bright red, respectively, under the 365 nm UV light. The fluorescence intensity increases with the weight ratio of WK@AuNPs. The uniform dispersion and stable fluorescence emission of AuNPs in the SF materials are subjected to the molecular cage effect of WK molecules. Concerning the fluorescence emission of WK@AuNP-SF solutions under different chemical microenvironments, the quantum yields of 20 wt% WK@AuNP-SF solutions were measured at different pH values. With increasing pH from 5, 7, 9, 11, to 13, the quantum yield (QY) of 20 wt% WK@AuNPs-SF increased from 6.73% to 15.31% (Figure 3g). The more effective fluorescent radiation transition of the QY of WK@AuNP-SF at high pH may be attributed to the change in conformation associated with the increment of


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organic-metal bonding and stabilization under the alkaline microenvironment.14 Based on the unique hierarchical nanofibrils network structures, the regenerated silk fibroin solution was further processed easily into different forms of materials to suit a range of potential applications.6 Consequently, processing WK@AuNP-SF materials into different forms is crucial (cf. Figure S5). For instance, WK@AuNPs-SF hybrid sponges can be applied for bioengineering, bioimaging and fluorescence probing, and the WK@AuNP-SF solutions or gels can be applied as “inks” to fabricate various sensors and flexible photonic/electronic devices by inkjet printing (work in progress). WK@AuNPs- SF hybrid fibers and films can be applied to fabricate flexible electronics and photonics components (work in progress). 3.2.2. Structural Synergy of meso-hybrid materials To study the mechanism of WK facilitated meso-hybrid materials construction, the structural and interaction synergies between WK@AuNPs and silk fibroin molecules were further examined fourier transform infrared spectroscopy (FTIR). The absorption frequency of the amide I peak (C=O stretching) in FTIR is quite sensitive to the secondary structure of SF materials.37-38 Therefore, the secondary structural changes of SF materials caused by hybridized WK@AuNPs were measured by analyzing the amide I bands in the FTIR spectra (cf. Figure S6). On the other hand, the β-crystalline phases of SF materials can be investigated by X-ray diffraction (XRD) technology.27, 39 In this regard, the change of β-crystalline of SF materials caused by hybridized WK@AuNPs was examined by XRD (cf. Figure S6). As shown in Figure S6, the actual percentage of total β-conformations (β-sheets + β-crystalline) in SF-WK@AuNPs hybrid materials is much higher than the estimated percentage of WK@AuNPs + SF



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based on simple and ideal mixing. The increments of the β-sheet and β-crystallite at different Au NPs fraction in SF materials are illustrated in Figure 4A-c. This result implies that the incorporation of WK@AuNPs into the SF materials causes the occurrence of mixed β-conformation and β-crystallites. This can be attributed to the synergetic interaction between SF and WK@AuNPs. As shown in Figure 4A-a, the lattice spacing of AuNPs are consistent with those of metallic Au crystal lattice spacing of the (111) plane that is 0.235 nm. Notice that the ordered structure of the surfaces of Au NPs will provide ideal templates for the nucleation of β-crystallization, which will trigger SF nanofibrils formation.4-5 This gives rise to the shortening of the gelation time of a SF solution (50 mg/ml) once WK@AuNPs (5 mg/ml) was added (see Figure 4Ab), and the extra mixed β-crystallites formation (cf. Figure 4A-c). Due to the fact that the formation of SF materials is controlled by the nucleation of β-crystallites,2,

5

WK@AuNPs may serve as nucleation seeds to promote the formation of mixed βcrystallites, which lead to the formation of SF nanofibrils (β-crystallite networks) then the gelation of SF solutions. Therefore, the fact that the promotion of WK@AuNPs for the formation of β-crystallites can be substantiated by the significantly shortening of the gelation time of SF solutions, which has been verified by our experiments (cf. Figure 4A-b). The mixed β-crystallites actually function as “crystallite bridges” to bind WK@AuNPs with the SF nanofibril networks (Figure 2b), so that the WK@AuNPs are incorporated uniformly and linked firmly into SF nanofibrils networks of the materials. This leads to a high intensity and stable fluorescent emission. In meso-hybrid process, WK@AuNPs serve as the centers to initiate the formation


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of SF nanofibrils (Figure 2b) so that they are incorporated into SF nanofibrils networks. The incorporation of WK@AuNPs into the bulk of SF films can be examined by transmission electron microscopy (TEM). The white circles of ~70 nm in Figure 4B-c indicate WK@AuNPs complexes. There are multiple dots in a circle, indicating that WK@AuNPs are the multiple encapsulation of AuNPs by WK molecules (cf. Figure 2a). The surface morphology of SF films hybridized by WK@AuNPs was investigated by atomic force microscopy (AFM). As shown by Figure 4B-a, the surface morphology of a neat SF film displays as matrices of SF nanofibrils while in the WK@AuNPs-SF hybrid films, the WK@AuNPs spheres are submerged in the SF nanofibril matrices (Figure 4B-b). One can see that some of SF fibrils are connected to the WK@AuNPs spheres as displayed by Figure 2b.

3.3. Highly Sensitive/Selective Ion Probing Since the as-synthesized fluorescent SF sponge has relatively high quantum yield and good water solubility (Table S3 & Figure S7), and the hybrid AuNPs appear to be giant fluorescent emission centers, which are protected by WK molecules. WK@AuNPs induced high fluorescence emission can be annealed once WK and SF molecular protection were vanished. This will happen exclusively when a trace amount Cu2+ was introduced. This is subject to the fact that Au-S-in WK mediating molecules can be substituted by Cu-S- once Cu2+ was added, this leads to the aggregation of the Au nanoparticles, further quenching the fluorescence (Figure 5a).40-41 On the other hand, the exclusive annealing effect of WK@AuNPs-SF materials can be applied to the



sensing a trace amount of Cu2+. In this regard, the fluorescent WK@AuNPs-SF hybrid sponges can be further adopted as the detector for copper ions. In recent years, Cu ion (Cu2+) pollution has received increasing attention throughout the world due to adverse effects on the environment and, more importantly, human health.42 As reported, if people ingest excessive Cu2+ from drinking water or other materials, Cu ions can accumulate in the human body for a long time, leading to the disruption of the central nervous system, this can also cause irreversible kidney damage.42-43 Therefore, the detection of Cu2+ is of great importance in our daily life. Traditional liquid fluorescence Cu2+ probes are difficult to store and inconvenient to use, and currently available materials deteriorate easily in water, leading to a reduction in fluorescence efficiency. In our approach, it is necessity for the fluorescent agents (WK@AuNPs) in the SF networks to transform from the fluid state into the solid state. This protects the fluorescence emission in an aqueous environment and allows convenient detection. As shown in Figure S8-9, after freeze-drying, the gold particles solution is transformed into a solid powder or into the porous sponge once SF molecules are added. As mentioned before, when adding the silk fibroin network into the WK@AuNPs solution, the WK@AuNPs are uniformly dispersed in the silk fibroin matrix (Figure 3e), giving rise to a slight increase in fluorescence intensity (Figure S10). Meanwhile, no quenching in the fluorescence of the WK@AuNPs-SF sponge was observed during this process (Figure 5b, c). These results imply that the SF molecules can effectively protect Au nanoparticles from aggregation, which leads to stable fluorescence via molecule-mediated mesoscopic material assembly. Another


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remarkable feature of the meso-hybrid fluorescent SF sponges is their long-term fluorescent stability, which is lacking for WK@AuNPs solutions. As shown in Figure 5d, the fluorescence intensity of a WK@AuNPs solution decreases sharply with time at room temperature, while a solution from the corresponding powder-redissolution displayed almost no fluorescence. However, WK@AuNPs-SF hybrid materials show incredible fluorescence stability: a solution from the re-dissolution of a SF/Au20 sponge (SF sponge with 20 wt% WK@AuNPs hybridization) remains almost unchanged fluorescent emission for a long period (> 60d) at room temperature (cf. Figure 5d). This is due to the fact that once WK@AuNPs are incorporated uniformly and linked firmly into SF nanofibrils networks of the materials in any state, they will be very stable in the structure (Figure 2B), leading to high intensity and stable fluorescent emission. To quantitatively describe the response sensitivity of this sensing system, 6 mg SF/Au20 sponge was added to a series of solutions (2 mL) containing different concentrations of Cu2+ (1 μM-1 mM). As shown in Figure 5e, the fluorescence of the redissolved SF/Au20 was reduced with increasing Cu2+ concentration. This decrease in fluorescence intensity with Cu2+ concentration is nearly linear within the concentration range of 0-100 μM (Figure 5f), and the corresponding linear function was F0/F =0.008Ccu + 0.976 (R2 =0.987). The detection limit was calculated to be 0.15 μM (3σ/slope), which is noticeably lower than the safe concentration (20 μM). To investigate the detection selectivity of the fluorescence SF sponge for metal ions, several metal ions were tested (Na+, Pb2+, Mg2+, K+, Ca2+, Cu2+, Fe2+, Al3+, Mn2+, Zn2+,



Co2+, and Ni2+) under the same experimental conditions. The results show a significant decrease in fluorescence intensity in the presence of Cu2+, while the fluorescence remained unaltered in the presence of the other metal ions (Figure S11). Hence, the fluorescent SF sponge can be used as a highly sensitive and selective luminescence sensor for Cu2+.

3.4. Carbonization of meso-hybrid materials for bio-sensing In carbonization under high temperature heat treatment (700 ℃), the nanofibril networks in the hybrid materials can be further transformed to aligned polyaromatic carbon structures due to intermolecular dehydration (Figure 2c).9-10, 44-45 Whereas, the meso-connections between SF nanofibrils and Au particles lead to well distribution of nano sized Au particles in carbonized conductive carbon networks. The meso-hybrid carbon-Au (Au@WK/SF-C) materials can be obtained by further calcination (see XRD in Figure S12), which exhibit potential as electrochemical sensors. In comparison to Au@WK-C (derived by WK@AuNPs), Au nanoparticles found well distributed in Au@WK/SF-C (Figure 6c-d & Figure S13-14), which provide more electrochemical active sites to promote the electron transfer and to enhance the adsorption strength of biomolecules.46 This prevents the aggregation of Au particles due to that the tightly anchored of WK@AuNPs and SF nanofibrils networks in carbonization. To further verify the ultrahigh superiority of the meso-hybrid carbon materials, we adopted Au@WK/SF-C to fabricate electrochemical sensors to examine the detection capability of dopamine (DA). DA is an indispensable neurotransmitter in the central nervous


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system, the levels of which relate to various neurological diseases such as Parkinson’s disease, schizophrenia and HIV.47 The electrochemical performances of DA on the asprepared electrodes were firstly investigated by cyclic voltammetry (CV). The performance of as prepared materials to DA analysis was investigated using the three electrode screen printed electrode (SPE) configuration with Ag/AgCl reference electrode, carbon counter and working electrode was employed. The electrochemical performance of Au@WK/SF-C, Au@WK-C, SF/WK-C, and bare C were investigated by cyclic voltammetry (CV) in 1mM DA solution at a scan rate of 50 mV s-1. Figure 6e shows cyclic voltammograms (CVs) of SF/WK-C, Au@WK-C, Au@WK/SF-C, and bare carbon in 0.01 M PBS (pH 7.0) containing the mixture of 1mM DA. There are no obvious peaks on the bare carbon electrode, while the well-defined oxidation peaks can be observed at 0.09 V (DA) at the Au@WK/SF-C, which was much more prominent than the Au@WK-C, SF/WK-C, indicating an enhanced response sensitivity of the Au@WK/SF-C samples. In addition, as shown in Figure 6f, remarkable current peaks can be observed for the electrolytes with DA at different scan rates (20-200 mV S-1), and both the intensities of oxidation peaks (Ipa) and reduction peaks (Ipc) are linearly dependent on the scan rates (Figure 6g), indicating that the DA molecule on the Au@WK/SF-C undergoes a surface-controlled process. Moreover, the CV fluctuated in a small range after 100 cycles, suggesting that a long-term stable redox reaction (Figure 6h). Such remarkable response peaks on Au@WK/SF-C could be owed to good electronic performance. Additionally, the differential pulse voltammetry (DPV) performances of simultaneous determination for DA (0.488 μM-125 μM) in PBS (pH



7.0) solution were studied on those carbon samples as well, recorded in Figure 6i and Figure S15. For Au@WK/SF-C, as shown in Figure 6j, it can be seen that the intensity of the peak gradually increases with the concentration of DA during the test process. The oxidation peak currents were linearly dependent on the concentrations of DA in the ranges of 0.488 μM-125 μM with detection limits of 0.1 µM (S/N = 3), which demonstrates that the as-developed biosensor have excellent electrochemical performance. By comparing the electrochemical performance of meso-hybrid carbon (Au@WK/SF-C) with reported work, our Au@WK/SF-C has remarkable detection properties with a relatively simple preparation process (Table 1). Wherein, the peak potential separations, linear detection ranges obtained in meso-hybrid carbon were better with the results from other carbonized sample (Figure S15). The simultaneous detection of ascorbic acid, DA and uric acid on Au@WK/SF-C were carried out by DPV measurement to exhibit the fabricated sensor had a good selectivity and was an appropriate tool for detecting dopamine (Figure S16). Indicating the meso-carbonized strategy endow a carbon electrode with extraordinary electrochemical functions, and will open a facile route to prepare carbon nanomaterial with high electrochemical performance which derived by soft material and boost its electrochemical applications.

4. CONCLUSIONS In conclusion, the concentration of this work is mainly focused on the rational design and new materials strategy of mesoscopic re-construction for SF hybrid materials. The functionalizing reconstruction of silk materials was implemented by introducing


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WK@AuNPs at the mesoscale. The in-line synthesized AuNPs are adopted as functional nano-dopants or seeds for mesoscopic network reconstruction. The functional components WK@AuNPs are built into SF nanofibrils networks through βcrystallite bridging between WK@AuNPs and SF nanofibrils. This endows SF materials with various added functionalities with stable and ultra performance. More specifically, the performance of WK@AuNPs-SF hybrid fluorescent sponge overrides the neat WK@AuNPs or SF in all aspects, such as exhibit long-term fluorescent stability, high intense fluorescence emission (QYs of >15%, 50% higher than other AuNCs complexes) and display highly sensitive and selective responses towards metal ions. The in-line synthesized AuNPs are adopted as functional nano-dopants or seeds for mesoscopic network reconstruction and the structure has been directly imaged by AFM (cf. Figure 4). In this regard, the meso-hybrid of WK@AuNPs with a stable reconstructed mesoscopic structure subject to templated β-crystallization. In addition, we put forward a new concept of primary and secondary functional hybrid materials. The secondary result from the carbonization of the primary mesoscopic hybrid materials, producing the carbon-Au hybrid materials, and offer a new type of hybrid materials for electronic sensing. Although the secondary inherit to some extent the structure (meso-network structures) and components (ie. AuNPs) of the primary, the functionalities and the applications of the both are very much different. In other words, we develop two different types of hybrid functional meso-materials from the same initial materials. Moreover, the primary and the secondary hybrid materials are further applied to fabricate various types of chemical sensors to acquire the much higher



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performance and stability based on the particular mesoscopic structures. Specifically, the hybrid materials provide a visual and nontoxic observation and high selectivity for rapid detection of Cu2+ in drinking water without elaborate equipment. Notice that the WK@AuNPs silk meso-hybrid materials, the nano sized Au particles are well distributed SF nanofibril network structures. After carbonization, Au particles will be highly distributed in the carbonized mesoscopic conductive carbon networks which have good electrical conductivity. This becomes excellent Au sensing materials for working electrodes in biochemical sensing. By fabricating the chemical sensors using the meso-hybrid Au-carbon materials, the sensors demonstrate to have much more enhancement in dopamine sensing than those without gold other carbon derived from neat WK@AuNPs, neat SF/WK, respectively. We found that the stable bound of WK@AuNPs with SF nanofibril network derived carbon is a critical factor for achieving good electrochemical performances. This meso-hybrid materials fabricating strategy

will

inspire

more

functionalized

soft

materials

with

additional

physical/chemical/biological properties (such as electrical, mechanical, and fluorescent) in a convenient and controlled way.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: Fluorescence spectra, XPS spectra analysis of AuNP, TEM image of WK@AuNPs, FTIR spectra and XRD pattern of protein, SEM images of sponges, data of Cu2+ detection, XRD pattern of carbon materials, electrochemical data, comparison of quantum yields of Au fluorescent materials.


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AUTHOR INFORMATION Corresponding Author All the correspondence should be addressed to [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. C.Y., Y.X., and A.P. contributed equally. ORCID Chenyang Shi: 0000-0002-3029-4770 Aniruddha Patil: 0000-0002-8423-2815 Fan Hu: 0000-0001-5251-6795 Xiang Yang Liu: 0000-0002-5280-5578 ACKNOWLEDGMENTS One of the authors, X.-Y. Liu’s primary affiliation is Department of Physics, National University of Singapore.” This work was financially supported by NUS AcRF Tier 1 (R-144-000-416-114), 111 project (B16029), National Nature Science Foundation (Nos. U1405226), Doctoral Fund of the Ministry of Education (20130121110018), Science and Technology Project of Xiamen City (3502Z20183012), Science and Technology Planning Project of Guangdong Province (2018B030331001) and the 1000 Talents Program funding from the Xiamen University. We are also grateful to the National Natural Science Foundation of China (Grant Nos. 51773171), the Fujian Provincial Department of Science and Technology (Grant No. 2017J06019). References (1) Lin, N.; Cao, L.; Huang, Q.; Wang, C.; Wang, Y.; Zhou, J.; Liu, X.-Y. Functionalization of Silk Fibroin Materials at Mesoscale. Adv. Funct. Mater. 2016, 26, 8885-8902. (2) Liu, R.; Deng, Q.; Yang, Z.; Yang, D.; Han, M.-Y.; Liu, X. Y. “Nano-Fishnet” Structure Making Silk Fibers Tougher. Adv. Funct. Mater. 2016, 26, 5534-5541. (3) Ling, S.; Kaplan, D. L.; Buehler, M. J. Nanofibrils in Nature and Materials Engineering. Nat.



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Platform for Dopamine Detection. Sensors Actuators B: Chem. 2017, 238, 1043-1051.

Figures and Table



Figure 1. Hierarchical structures of SF and WK materials and schema of the fabrication of SFWK@AuNPs hybrid protein materials. (a) The hierarchical network structures of SF materials. (b) The schematic illustration of WK materials. The α-helix shows the location of the hydrogen bonds (yellow ellipse) and the 0.51 nm pitch of the helix. (c) Schematic of fabricating SF-WK@AuNPs hybrid protein materials. WKs are applied to acquire WK@AuNPs that are further blended into SF to obtain the mesoscopic functional materials to acquire fluorescent emission and then serve as the carbon precursor to obtain meso-hybrid carbon-Au under 700 ℃. Protein under carbonization in inert atmosphere may form highly conductive N-doped sp2 hybridized graphitic structures through a simple heat treatment without sophisticated chemical procedures.9 Base on meso-hybrid materials strategy, the functionalized SF materials may cause the Multiple effects (primary meso hybrid effect to secondary meso hybrid effect, cf. (c)) which can obtain various applications ie. probing, biosensing. “bio-imaging”,11 “Supercapacitors”,48 “organocatalysis”.10 Reprinted with permission from ref 11, Copyright 2017, Wiley-VCH. Reprinted with permission from ref 48, Copyright 2013, Wiley-VCH. Reprinted with permission from ref 10, Copyright © 2018, Springer Nature under Creative Commons license BY 4.0 (https://creativecommons.org/licenses/by/4.0/).


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Figure 2. Schematic illustrations mesoscopic hybrid materials. (a) Mesoscopic WK complex Au NPs dopants. Self-active WK molecules reduce in-line Au3+ to Au NPs and entrap them inside. (b) In SF mesoscopic hybrid materials construction, the WK molecules serve as mediating molecules to deliver and bind AuNPs onto the nanofibril networks of SF materials. (c) Schematic illustration showing the silk nanofibrils can transform into an aligned polyaromatic carbon structure under heat treatment.



Figure 3. Fluorescence emission of WK@AuNPs. (a) The photographs of a neat WK solution (1) and a WK@AuNPs solution (2) under natural light (top) and UV light (bottom, =365 nm). (b) Optical absorption (left) and optical emission (right, =365 nm) of a neat WK solution (black) and a WK@AuNPs solution (red). (c) X-ray photoelectron spectroscopy (XPS) spectra of WK@AuNPs. (d) Simple optical transitions involved for WK@AuNPs. (e) Photographs of sponges SF,SF/Au10 ~ SF/Au50 (gradually from left to right) under daylight (i) and UV light (ii, =365 nm). Under daylight, sponge Au0 appears pure white, while the SF materials exhibit a deeper brown with rising percentage of WK@AuNPs in the SF sponges. Similarly, WK@AuNP-SF sponges show an intense red fluorescence emission except for sponge SF, which emits a weak blue light under UV light (=365 nm). (f) The fluorescence spectra of sponges SF/Au10~SF/Au50 caused by the energy transition of WK@AuNPs. The spectra of Au0 samples display a strong peak at 468 nm (excited at 390 nm), while no peak is detectable at approximately 690 nm. For sponges SF/Au10~SF/Au50, there is a broad emission peak ranging from 550-850 nm with the maximum emission at approximately 670 nm (excited at 390 nm), and a slight blue shift of the fluorescent peaks is identified when compared to the characteristic peak of WK@AuNP solutions (690 nm). (g) Quantum yields of sponge SF/Au20 at different pH values measured by a calibrated integrating sphere (ISF-513). With increasing pH, the QYs of WK@AuNP sponges rise dramatically from 6% to 15%.


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Figure 4. Structural analysis to explain the mesoscopic hybrid SF networks by WK@AuNPs and the synergetic interconnections between WK@AuNPs and SF networks. (A) (a) HRTEM image of WK@AuNPs in SF solution. (b) Normalized optical density (OD) vs time for a neat SF solution (50 mg/ml) and a SF solution (50 mg/ml) with WK@AuNPs (5 mg/ml). T = 25℃. Gelation time (tg) was defined as the time when sharply increases.4 WK@AuNPs promote the β-crystallization can be seen from the shortening of gelation time (tg =108 h => tg =82 h). (c) The increment of βconformation (β-sheet + β-crystalline) with different WK@AuNPs contents. This indicates that the interaction between WK@AuNPs and SF molecules promotes the formation of β-conformation. (B) (a) AFM height image of neat SF material. (b) and (c) AFM height and TEM images of WK@AuNP-SF composite material. The proportion of WK@AuNPs to SF was 20:80 (w/w). The inset in (b) is the schematic illustration of the interaction and linkage between silk nanofibril networks and WK@AuNPs complexes. The formation of mixed β-crystallite provides linkages (bridges) between silk nanofibril networks and WK@AuNPs.



Figure 5. Fluorescence media for SF annealing system. (a) The schematic illustrations of fluorescent Au NPs and its application for highly selective and sensitive detection of Cu2+. Due to the breaking-bond action of Cu2+ ions between WK molecules and AuNPs, the AuNPs become close without the stabilizer and the fluorescence is quenched. (b) Photographs of the WK@AuNP powders and SF/Au20 sponge under daylight (i) and UV light (ii), the photographs of corresponding redissolutions under daylight (iii) and UV light (iv). These solid samples (powder or sponge) contain Au NPs with the same mass. The WK@AuNP powders were difficult to redissolve. (c) Fluorescent spectra (λex = 390 nm) of different solutions (black line: initial WK@AuNP solution, blue line: WK@AuNP powder redissolution, red line: SF/Au20 sponge redissolution). (d) Fluorescence matter are placed at room temperature (25 °C) for various time spans to probe their fluorescentstability. F0690 is the fluorescent intensity of WK@AuNPs solutions or WK@AuNPs powder redissolution or SF/Au20 sponge redissolution located at 690 nm for initial period, F690 is the fluorescent intensity of WK@AuNPs solutions or WK@AuNPs powder redissolution or SF/Au20


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sponge at 690 nm for different periods (10 days-60 days) (25 °C). The fluorescence intensity of WK@AuNPs decrease obviously once storage at room temperature, while SF/Au20 change only slightly preserve ~98% of the initial intensity even after 60 days placement at room temperature. (e) Fluorescent spectra of sponge SF/Au20 after redissolving in the presence of different Cu2+ concentrations (from top to bottom: 0-1 mM). (f) The correlation between quenching efficiency and Cu2+ concentration, showing the linear detection range of 1-100 μM of Cu2+.



Figure 6. Meso-hybrid SF for electrochemical sensors. (a) The schematic illustrations and picture of electrochemical sensors. (b) The schematic illustrations of redox reaction. Working principle of Au@WK/SF-C composite toward dopamine and other bio-molecule sensing applications. (c) TEM image of Au@WK-C. (d) TEM image of Au@WK/SF-C. Owing to the fact that WK@AuNPs are incorporated uniformly and linked firmly into SF nanofibrils networks of the materials in any state, they will be very stable in the structure even if carbonized, leading to highly dispersed AuNPs in carbonized network, while high electrical conductivity and effective electrochemical performance. (e) CV curves of carbon samples recorded in 1 mM DA solution at scan rate 50 mV s-1. (f) The CV responses of Au@WK/SF-C in 0.01 M PBS (pH 7.0) at different scan rates. (g) The plots of CV peak current versus the scan rates. (h) The CV responses of Au@WK/SF-C after 100 cycles. (i) DPV curves obtained in 0.1 M PBS solution in the presence of various concentrations of DA (0.448 μM-125 μM). (j) Linear relationship between the intensity of current peaks and the concentration of DA.


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Table 1. The specific electrochemical biosensors for detecting dopamine. Sensor

Method

Detection Limit/μM

Linear Range/μM

Reference

Meso-hybrid Au/Carbon

DPV

0.1

0.488-125

This work

GF

DPV

1.2

0.125-70

47

Au/RGO/GCE

DPV

1.4

6.8-41

49

Pristine graphene

i-t

2

5-710

50

PEDOT modified LSG

DPV

0.33

1-150

51

Au-Cu2O/rGO

DPV

0.0039

10-90

52

Graphene ink

DPV

2.21

3-140

53

N-MWCNTs

DPV

1.4

12-322

54

Chitosan/graphene

DPV

1

1-24

55

Graphene

DPV

2.64

4-100

56

PyC

DPV

2.3

18-270

57

Co3O4/rGO/GCE

i-t

0.277

1-30

58



Table of Contents (TOC) Functionalization of silk fibroin materials is implemented via meso-hybrid strategy using fluorescent Au nanoparticles mediated by wool keratin (WK). Meso-hybrid WK@AuNPs-silk fibroin materials can be applied to bioimaging, biosensing, biooptics and bioelectronics, etc.


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Primary and Secondary Mesoscopic Hybrid Materials of Au

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