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Oct 8, 2016 - State Key Laboratory of Chemical Engineering, Tianjin Key Laboratory of Membrane Science and Desalination Technology, School of Chemical...
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Superior Catalytic Performance of Gold Nanoparticles Within Small Cross-Linked Lysozyme Crystals Mingyue Liu, Libing Wang, Renliang Huang, Yanjun Yu, Rongxin Su, Wei Qi, and Zhimin He Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b02544 • Publication Date (Web): 08 Oct 2016 Downloaded from http://pubs.acs.org on October 9, 2016

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Superior Catalytic Performance of Gold Nanoparticles Within Small Cross-Linked Lysozyme Crystals Mingyue Liu,†, ǁ Libing Wang,†, ǁ Renliang Huang, § Yanjun Yu,† Rongxin Su,*, †, ‡ Wei Qi,†, ‡ and Zhimin He † †

State Key Laboratory of Chemical Engineering, Tianjin Key Laboratory of Membrane Science

and Desalination Technology, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, P. R. China ‡

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin

300072, P. R. China §

School of Environmental Science and Engineering, Tianjin University, Tianjin 300072, P. R. China

KEYWORDS: gold nanoparticle, cross-linked lysozyme crystal, catalytic reduction, 4-nitrophenol

ǁ

M. Liu and L. Wang contributed equally to this work.

* Correspondence concerning this article should be addressed to R. Su at [email protected]

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ABSTRACT. Bionanomaterials synthesized by bio-inspired templating methods have emerged as a novel class of composite materials with varied applications in catalysis, detection, drug delivery, and biomedicine. In this study, two kinds of cross-linked lysozyme crystals (CLLCs) of different sizes were applied for the in situ growth of Au nanoparticles (AuNPs). The resulting composite materials were characterized by light microscopy, scanning electron microscopy, transmission electron microscopy, thermogravimetric analysis, and X-ray photoelectron spectroscopy. The catalytic properties of the prepared materials were examined in the catalytic reduction of 4-nitrophenol (4-NP) to 4-aminophenol (4-AP). It was found that the size of the AuNPs increased with an increase in Au loading for both small and large crystals. In addition, small crystals favored homogeneous adsorption and distribution of the metal precursors. And the size of the AuNPs within small crystals could be maintained below 2.5 nm by managing the HAuCl4/lysozyme molar ratio. Furthermore, the lysozyme functional groups blocked the AuNP activity sites, therefore reducing their catalytic activity. This effect was more pronounced for small AuNPs. Moreover, the mass transfer of reactants (4-NP) from solution to AuNPs within the crystals restricted their catalytic reduction, leading to superior catalytic performance of the AuNPs within small cross-linked lysozyme crystals (Au@S-CLLCs) compared to those within large cross-linked lysozyme crystals (Au@L-CLLCs) at similar Au loadings. Finally, an increase in Au loading clogged the crystal channels with increased quantities of larger aggregated AuNPs, thus impeding the catalytic performance of Au@S-CLLCs.

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INTRODUCTION Nanomaterials, and in particular noble metal nanoparticles (NPs), have attracted increasing attention for a range of potential applications, including catalysis,1–2 nanodevices,3 biosensors,4 photonics,5 and biomedicine,6 because of their intriguing physicochemical properties when compared with their bulk materials. Indeed, the catalytic properties of noble metal NPs are particularly attractive because of their excellent reactivities and selectivities,7 which result from their large surface area to volume ratios and their unusual electronic structures.8 However, small nanocrystals tend to aggregate due to van der Waals attractions9 and high surface energies,10 resulting in a reduction in catalytic activity and limited catalyst recyclability. Moreover, the removal of nanoscopic catalysts from reaction media can be challenging. Therefore, the design and preparation of noble metal NP-based catalysts with long-term dispersion stability and facile separation is desirable. To date, various strategies have been attempted to address these issues, such as the use of dendrimers to stabilize noble metal NPs,11 the assembly of encapsulated noble metal NPs using core-shell structures,12 and the immobilization of noble metal NPs on or into a solid matrix such as TiO2,13 carbon nanotubes,14 or eggshell membrane.15 Recently, macroscopic biomaterials exhibiting three dimensional porous structures have emerged as a novel class of template for the synthesis of bionanohybrid materials. Protein crystals, which present highly ordered three dimensional structures with high porosities (0.5–0.8), large surface areas (800–2000 m2 g−1), and interpenetrating nanoporous and mesoporous solvent channels of 0.5–10 nm diameter, were traditionally used for the determination of protein structures,16 until their inherent fragility and solubility were addressed 3 ACS Paragon Plus Environment

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by cross-linking technology. These cross-linked protein crystals then emerged as a novel class of nanoporous materials,17 with a broad range of applications in biocatalysis,18–19 separation,20 and adsorption.21 In addition, cross-linked protein crystals have been widely used for the fabrication of bionanohybrid materials since Colvin et al. reported that their nanoporous structures can be applied in the immobilization of NPs.22 For example, Mann et al. adopted cross-linked lysozyme crystals (CLLCs) as a template in the synthesis of nanoplasmonic arrays,23 while Lu et al. employed native lysozyme crystals to study the mechanism of biomolecule-directed AuNP formation.24 They also confirmed that AuNPs grown in situ within lysozyme crystals could be used for the catalytic reduction of 4-NP to 4-AP by NaBH4,25 and reported that the size of AuNPs grown within the crystals could be controlled by varying the growth time. However, since these crystals were not cross-linked, the utilization of such composite materials for practical applications was restricted. Thus, to take advantage of the reducing ability of amino acid residues,26–28 our group recently reported a facile, reductant-free, and green synthetic route to prepare AuNPs within CLLCs (Au@CLLCs) for the catalytic reduction of 4-NP to 4-AP.29 Cross-linked lysozyme crystal (CLLC) is an ideal academic model system because of its highly ordered three dimensional porous structure and uniform size of solvent channels. Furthermore, it could be also used to fabricate nanoparticles at large scale for catalysis due to the facile crystallization and relative low price of lysozyme. To successfully form AuNPs within CLLCs, the metal precursors must diffuse into the CLLC solvent channels from the bulk solution. In CLLCs of varying sizes, the distance from the surface to the center differs, and as such, the metal precursors follow different diffusion paths, thus altering their distribution within the 4 ACS Paragon Plus Environment

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crystals and potentially affecting the size and spatial distribution of the AuNPs. However, to date, such an effect has not been reported, and so we herein prepared two kinds of CLLCS with widths30 (i.e., the distance between two parallel (110) crystal surfaces) of ~25 μm and 400 μm. Four different HAuCl4 to lysozyme molar ratios were employed to grow a range of Au@CLLCs, and the catalytic properties of the prepared Au@CLLCs were examined in the catalytic reduction of 4-nitrophenol (4-NP) to 4-aminophenol (4-AP). Furthermore, we investigated the effect of CLLC size on the size distribution of AuNPs, the correlation between Au loading and AuNP size, and the effect of CLLC and AuNP sizes on catalytic performance (Scheme 1). Scheme 1. Schematic illustration of the effect of lysozyme crystal size and Au loading on the size distribution of AuNPs and on AuNP activity in the catalytic reduction of 4-NP to 4-AP in the presence of excess NaBH4.

EXPERIMENTAL SECTION

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Materials. Ultrapure grade lysozyme (egg white) was purchased from Amresco (Solon, OH, USA) and was used without further purification. Chloroauric acid (HAuCl4·3H2O, Au≥48%), disodium hydrogen phosphate (Na2HPO4, ≥99%), sodium dihydrogen phosphate (NaH2PO4, ≥99%), sodium acetate (CH3COONa, ≥99%), acetic acid (CH3COOH, ≥99.9%), 4-nitrophenol (C6H5NO3, 4-NP, ≥98%), sodium borohydride (NaBH4, ≥98%), polyethylene glycol (PEG, MW = 10000), and glutaraldehyde (50 wt%) were purchased from Aladdin Reagent Co. Sodium chloride (NaCl, ≥99.9%) was purchased from Alfa Aesar and sodium hydroxide (NaOH, 98%) was purchased from Solarbio Science and Technology. All water was purified by a Sartorius arium® pro VF water purification system (18.2 MΩ resistivity). Lysozyme crystal growth and cross-linking. A batch method based on previous literature reports30–31 was employed for lysozyme crystallization. Protein buffer was prepared by dissolving lysozyme powder in the buffer, and precipitant buffer was prepared by dissolving NaCl and PEG in the buffer. Small lysozyme crystals were grown in a 10 mL polypropylene centrifuge tube containing the crystallization solution (8 mL), which was prepared by the addition of protein buffer (2 mL) to precipitant buffer (6 mL). The final crystallization solution contained 9 mg/mL lysozyme, 15 wt% NaCl, and 6 wt% PEG in 0.5 M sodium acetate buffer at pH 4. Large lysozyme crystals were grown in a 10 mL vial containing the crystallization solution (2 mL), which was prepared by the addition of protein buffer (1 mL) to precipitant buffer (1 mL). The final crystallization solution contained 30 mg/mL lysozyme, 3 wt% NaCl, and 6 wt% PEG in 0.1 M sodium phosphate buffer at pH 6. All protein buffers and precipitant buffers were passed through 0.22 μm sterile filters before 6 ACS Paragon Plus Environment

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use in preparation of the crystallization solution. The samples were then sealed and stored at 4 °C. After 24 h, small crystals were obtained, and each solution was centrifuged (2 min, 2600 g, 4 °C). The supernatant was carefully removed and the pellet was resuspended in the cross-linking solution (8 mL), which contained 2.5 wt% glutaraldehyde, 15 wt% NaCl, and 6 wt% PEG in 0.5 M sodium acetate buffer at pH 4. In contrast, the large crystals were obtained following 48 h growth, after which time, the supernatant was carefully removed. In this case, the pellet was resuspended in a cross-linking solution (8 mL), which contained 2.5 wt% glutaraldehyde, 3 wt% NaCl, and 6 wt% PEG in 0.1 M sodium phosphate buffer at pH 6. All of the above samples were maintained at 20 °C for 48 h, after which time, the solution was removed and the crystals were washed six times with ultrapure water. To acquire the mass of crystals in each sample, the lysozyme concentration in the crystallization solution both before and after crystallization was determined by UV-Vis absorbance at 280 nm.32 The lysozyme concentrations of all crystallization solutions were then calculated from the standard curve shown in Figure S1. Preparation of Au@CLLCS with varying Au loadings. To obtain a quantified Au loading of the Au@CLLCs composite materials, specific volumes of HAuCl4 solution (10 mM) were added to the crystals according to their mass, giving HAuCl4 to lysozyme molar ratios of 3:1, 5:1, 7:1, and 9:1. Immediately following injection of the HAuCl4 solutions, the small cross-linked lysozyme crystals (S-CLLCs) and large cross-linked lysozyme crystals (L-CLLCs) samples were heated at 30 °C for 30 min and 120 min, respectively. After the above times, the colorless supernatant was removed, both the small and large 7 ACS Paragon Plus Environment

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crystals were washed to remove unbound metal precursors, and each sample was redispersed in water (4 mL). Subsequently, NaOH (4 M, 80 μL) was added, and the samples were placed in a shaking water bath (Julabo, Germany) at 37 °C for 36 h. Finally, the resulting composite materials were washed 6 times with ultrapure water. The Au@L-CLLCs samples (AuNPs grown within L-CLLCs) were dried at 60 °C for 24 h prior to further use, and the Au@S-CLLCs samples (AuNPs grown within S-CLLCs) were freeze-dried to prevent the agglomeration of small crystals in the subsequent heating operation (i.e., drying at 60 °C for 24 h). Characterization. A polarized-light microscope (ShunYu XP, China) with an attached charge-coupled device video camera was used for optical microscopy imaging. Scanning electron microscopy (SEM, Hitachi S-4800) was used to observe S-CLLCs morphology. To analyze the morphology and size of the Au@CLLCs, high-resolution transmission electron microscopy (HRTEM) (JEM-2100F) was performed. The HRTEM samples were prepared as outlined in our previous report.29 X-ray photoelectron spectroscopy (XPS, PHI-5000 Versa Probe) of Au@S-CLLCs9:1 was carried out using an Al Kα source to determine the valence state of Au within the CLLCs. Thermogravimetric experiments were conducted on a Mettler-Toledo TGA/DSC1 1100 system under an O2 atmosphere. Catalytic reduction of 4-NP. The catalytic activity of Au@CLLCs was investigated for the catalytic reduction of 4-NP in the presence of NaBH4. In a typical catalytic experiment, 4-NP (0.75 mL, 3 mM) and Au@CLLCs (4 mg Au@L-CLLCs or 2 mg Au@S-CLLCs) were added to ultrapure water (7 mL), then fresh NaBH4 solution (1 mL, 0.3 M) was added to initiate the reduction at 37 °C with continuous stirring. Immediately after NaBH4 addition and at specific 8 ACS Paragon Plus Environment

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points throughout the reaction, samples of the reaction solution (~0.4 mL) were transferred into a quartz cuvette using a syringe, and the UV-Vis absorption spectra were recorded from 240 nm to 550 nm to monitor the progress of the catalytic reaction. Following each measurement, the transferred reaction solution was reintroduced into the reaction mixture. The reaction vessels, magnetons, and stirring rates employed were maintained constant for all measurements. RESULTS AND DISCUSSION Preparation of Au@CLLCs composite materials. Figure 1 shows the optical micrographs of the tetragonal lysozyme crystals with average widths of ~25 and 400 μm. Following AuNP growth within the CLLCs, little change in crystal size was observed, although the crystal surfaces were slightly eroded, and the edges of the crystals were not distinct. In addition, preparation of Au@S-CLLCs and Au@L-CLLCs using different HAuCl4 to lysozyme molar ratios gave no significant difference in morphology or size (Figs. S2 and S3).

Figure 1. Optical micrograph images of (a) S-CLLCs, (b) Au@S-CLLCs, (c) L-CLLCs, and (d) Au@L-CLLCs. 9 ACS Paragon Plus Environment

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As S-CLLCs have a specific surface area ~16 times larger than L-CLLCs (treat a single crystal as cube), the absorption rate of equal amounts of AuCl4− ions (relative to CLLC mass) is slower for L-CLLCs. This was confirmed by monitoring the change in solution color following injection of the HAuCl4 solution to L-CLLCs and S-CLLCs samples in equal HAuCl4 to lysozyme molar ratios. Due to the yellow color of the HAuCl4 solution, the adsorption rate could be assessed based on color fading. This color change was observed for the S-CLLCs samples ~10 min following HAuCl4 injection (Figure S4), while the fading was observed only after ~30 min for the L-CLLCs samples (Figure S5). Interestingly, during the adsorption of metal precursors, S-CLLCs were suspended in the solution, while the L-CLLCs accumulated at the bottom of the vessel, leading to inhomogeneous adsorption by the L-CLLCs in the initial stages. This resulted not only in the inhomogeneous distribution of AuCl4− ions in different sections of each crystal,33 but also caused varying adsorption between crystals due to the accumulation of L-CLLCs. Finally, although distribution of the AuCl4− ions in each crystal may reach an equilibrium through the diffusion of ions from high concentration to low concentration, such an equilibrium is not achieved between different crystals. Therefore, considering that the S-CLLCs remained suspended in solution during diffusion, equal adsorption conditions were present on all crystal faces, resulting in comparable adsorption of AuCl4− ions, and a more homogeneous adsorption pattern than the L-CLLCs samples. TGA measurements were then performed to analyze the Au loading on the samples. As shown in Fig. 2, the CLLCs sample decomposed completely, indicating that the cross-linked lysozyme of the Au@CLLCs behaved similarly, allowing the remaining mass to be used to 10 ACS Paragon Plus Environment

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calculate the Au loading by excluding the mass of water for each sample. Figure 2 shows the weight loss of both the Au@S-CLLCs and the Au@L-CLLCs at 25–700 °C under an O2 atmosphere. As previously reported, weight loss below 150 °C could be attributed to the evaporation of absorbed and bound water in the crystals.34 Interestingly, the decomposition of the sample containing the higher Au loading reached completion at a lower temperature, showing that Au@L-CLLC3:1 was completely decomposed at 592 °C, in comparison to 565 °C for Au@L-CLLC9:1. This suggests that Au may act as a catalyst for decomposition of the cross-linked lysozyme at high temperatures.35 Thus, a higher Au loading likely results in a lower total decomposition temperature for these species.

Figure 2. TGA of (a) Au@S-CLLCs and (b) Au@L-CLLCs (b). The insets show an amplification of the temperature range where sample decomposition reached completion.

Table 1 shows the calculated Au loadings of all samples, which correlate well with the theoretical values calculated from the HAuCl4 to lysozyme molar ratios. Interestingly, the Au loadings of the Au@L-CLLCs samples and of Au@S-CLLC9:1 were slightly lower than the

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expected values, likely due to insufficient absorption of the metal precursors. Furthermore, the calculated values of the remainder of the Au@S-CLLCs samples were slightly higher than the expected values due to crystal loss (i.e., of small CLLCs) during the washing stages that followed cross-linking. Table 1. Expected and calculated Au loading capacities, mean AuNP diameters, estimated delay time, apparent rate constant k, and normalized apparent rate constants (kcat, and kAu) for the eight samples Au@S-CLLCs

Au@L-CLLCs

HAuCl4 to lysozyme molar ratio

3:1

5:1

7:1

9:1

3:1

5:1

7:1

9:1

Expected Au loading capacity (wt%)

3.96

6.44

8.79

11.03

3.96

6.44

8.79

11.03

Calculated Au loading capacity (wt%)

4.06

6.47

8.81

10.79

3.94

6.37

8.34

9.86

Mean AuNP diameter (nm)

1.28

1.82

2.43

2.52

1.19

2.05

2.24

2.33

2

2

2

2

4

4

4

4

1.12

2.39

3.62

4.60

0.04

1.28

3.34

5.39

0.56

1.20

1.81

2.30

0.01

0.32

0.84

1.35

13.79

18.47

20.54

21.32

0.26

5.02

10.01

13.67

650

340

220

150

900

500

350

200

Catalyst dosage (mg) −3 −1

k (10 s ) kcat (10−3 mg−1 s−1) −3

−1 −1

kAu (10 mg s ) Delay time (s)

XPS analysis of the Au@S-CLLC9:1 sample (i.e., the sample with the highest Au loading) was conducted to verify whether all AuCl4− ions were reduced to Au0. In the wide scan XPS spectra shown in Fig. 3a, the Cl 2p peak at a binding energy of ~197 eV was absent (see inset of Fig. 3a), which suggests that all Cl− ions had been removed, and that no AuCl4− ions remained.36 In addition, Fig. 3b shows the binding energies of Au4f7/2 and Au4f5/2 to be 83.5 and 87.4 eV, respectively, which was consistent with the Au0 binding energy.37 Furthermore, no band corresponding to Au(I) was observed at 84.9 eV, further confirming this conclusion.38 Thus, as the Au@S-CLLC9:1 samples possess the highest Au loading, we can conclude that Au was

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present largely in the form of Au0 for all samples.

Figure 3. XPS spectra of Au@S-CLLC9:1. (a) Wide scan spectrum (inset: Cl 2p core-level spectrum). (b) Au4f core-level spectrum. Examination of morphology using TEM. The morphology of the AuNPs grown in L-CLLCs and S-CLLCs using different Au loadings was characterized by TEM. As shown in Figure 4, the resulting AuNPs were relatively spherical, and a few aggregates were also formed.

Figure 4. Representative TEM images of AuNPs within (a) Au@S-CLL3:1, (b) Au@S-CLLC5:1, (c) Au@S-CLLC7:1, (d) Au@S-CLLC9:1, (e) Au@L-CLLC3:1, (f) Au@L-CLLC5:1, (g) Au@L-CLLC7:1, and (h) Au@L-CLLC9:1. 13 ACS Paragon Plus Environment

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Figure 5 shows the size distribution of the 8 AuNP samples calculated from the TEM images. The mean AuNP diameters (dA) were based on a count of more than 150 individual nanoparticles. For AuNPs grown within S-CLLCs, the mean diameters increased from 1.28, 1.82, and 2.43 nm to 2.52 nm upon increasing the Au loading, while the mean diameters of the Au@L-CLLCs increased from 1.19, 2.05, and 2.24 nm to 2.33 nm. Interestingly, the sample with the lowest Au loading was occupied by the smallest AuNPs, while the sample with the highest Au loading was occupied by the largest AuNPs for both L-CLLCs and S-CLLCs. Indeed, such a dependence of the AuNPs size on the ratio of oxidizing to reducing agent has previously been reported.36, 39–40. As an increased Au loading corresponds to an increased ratio of oxidizing agent (HAuCl4) to reducing agent (lysozyme), this results in fewer nuclei for the further growth of particles. In addition, higher Au loadings lead to an increase in crystal solvent channels taking up Au atoms, thus providing more probability for the aggregation of Au atoms, and the formation of larger particles. However, the interpenetrating solvent channels prevent further particle aggregation, and as such, the growth of AuNPs with increased Au loading was restricted. Therefore, upon increasing the Au loading from 8.81% (Au@S-CLLC7:1) to 10.79% (Au@S-CLLC9:1), the mean diameter increased by only 0.1 nm, which is significantly lower than the increase of 0.6 nm from 6.47% (Au@S-CLLC5:1) to 8.81% (Au@S-CLLC7:1).

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Figure 5. Size distribution of AuNPs within (a) Au@S-CLLCs and (b) Au@L-CLLCs calculated from the TEM images.

As the Au loading was increased, the AuNP size distribution became wider in all S-CLLCs and L-CLLCs supports with the exception of Au@L-CLLC9:1. Indeed, for equal HAuCl4 to lysozyme ratios, the AuNPs were slightly larger in Au@S-CLLCs than in Au@L-CLLCs. In addition to the insufficient absorption of AuCl4− ions, which reduces the amount of Au atoms available for AuNP formation, the inhomogeneous distribution of AuCl4− ions in L-CLLCs may generate crystals with higher Au loadings than expected, yielding an unexpected AuNP size distribution. Nevertheless, due to the homogeneous distribution of metal precursors in the crystals of each sample, the size of the AuNPs (i.e., 2.5 nm) within CLLCs using additional reductant and investigate the correlation between their size and catalytic performance are currently in progress. ASSOCIATED CONTENT This material is available free of charge via the Internet at http://pubs.acs.org. Standard curve of lysozyme in buffer solutions, SEM images of Au@S-CLLCs, optical micrographs of Au@L-CLLCs, additional experiments of HAuCl4 diffusion to S-CLLCs and L-CLLCs, lattice fringe image of synthesized AuNPs, and time-dependent evolution of UV-Vis spectra of the reactions catalyzed by Au@S-CLLCs and Au@L-CLLCs.

AUTHOR INFORMATION

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Corresponding Author *E-mail: [email protected]. Phone: +86 22 27407799. Fax: +86 22 27407599.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the Natural Science Foundation of China (51473115 and 21276192), the Ministry of Science and Technology of China (No. 2012YQ090194), and the Natural Science Foundation of Tianjin (No. 16JCZDJC37900). ABBREVIATIONS 4-AP, 4-aminophenol; 4-NP, 4-nitrophenol; Au@CLLCs, AuNPs within CLLCs; Au@L-CLLCs, AuNPs within large cross-linked lysozyme crystals; AuNP, Au nanoparticle; Au@S-CLLCs, AuNPs within small cross-linked lysozyme crystals; L-CLLCs, large cross-linked lysozyme crystals; CLLC, cross-linked lysozyme crystal; NP, nanoparticle; PB, sodium phosphate buffer; S-CLLCs, small cross-linked lysozyme crystals.

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