Highly Thiolated Dendritic Mesoporous Silica Nanoparticles with High

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Highly-thiolated Dendritic Mesoporous Silica Nanoparticles with High-content Gold as Nanozyme: The Nano-Gold Size Matters Mohammad Kalantari, Trisha Ghosh, Yang Liu, Jun Zhang, Jin Zou, Chang Lei, and Chengzhong Yu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b01527 • Publication Date (Web): 19 Mar 2019 Downloaded from http://pubs.acs.org on March 19, 2019

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ACS Applied Materials & Interfaces

Highly-thiolated Dendritic Mesoporous Silica Nanoparticles

with

High-content

Gold

as

Nanozyme: The Nano-Gold Size Matters Mohammad Kalantari‡, Trisha Ghosh‡, Yang Liu‡, Jun Zhang‡, Jin Zou†, Chang Lei‡,*, Chengzhong Yu‡,*

‡Australian

Institute for Bioengineering and Nanotechnology, The University of

Queensland, Brisbane, QLD 4072, Australia.

†Materials

Engineering and Center for Microscopy and Microanalysis, The University

of Queensland, Brisbane, QLD 4072, Australia.

KEYWORDS: nanozyme, nano-gold, mesoporous silica, thiol, dendritic ABSTRACT: Thiolated dendritic mesoporous silica nanoparticles (T-DMSNs) with an ultrahigh density of thiol groups (284.6±9 μmol g-1) are synthesized and used to load gold nanoparticles with tunable sizes (1.2-2.7 nm) and a high content (34.0 wt%). It is demonstrated that the size of gold nanoparticles has a significant impact on their

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peroxidase-mimicking activity. At an optimized size of 1.9 nm, the T-DMSNs@Au exhibit the highest activity. Our contribution provides new insights into the rational design of nanozymes for future applications.

Introduction Nanozymes are nanomaterials with the capability of mimicking the catalytic function of natural enzymes.1 Nanozymes display several benefits over their natural counterparts including low cost, ease of mass production, long storage time, and more importantly, high stability in harsh environments.2 To date, many types of nanomaterials such as gold (Au) nanoparticles,3 ferromagnetic nanoparticles,4 graphene oxide,5 cerium oxide nanoparticles,6 metal-organic frameworks,7 and vanadium pentaoxide nanowires8 have been discovered as candidates for nanozymes. In particular, Au nanoparticles have attracted tremendous research interest due to their biocompatibility, stability and activity under mild conditions.9 The catalytic activity of Au nanoparticles is reported size-dependent in various applications.10-11 For example, Valden et al.12 prepared a range of Au clusters with diameters from 1 to 6 nm and discovered an optimal size of 3 nm with the maximum 2 ACS Paragon Plus Environment

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activity for carbon monoxide oxidation. In another case, Zhou and coworkers11 used Au nanoparticles with various sizes (2-15 nm) to catalyze the reduction of resazurin, showing that Au nanoparticles of 6 nm exhibited the highest activity. Nevertheless, the optimized size of Au nanoparticle (nano-Au) in nanozyme applications (e.g., peroxidase mimicking) is rarely reported. Although nano-Au has excellent catalytic functions, it has been documented that small Au nanoparticles tend to aggregate and in turn lose their activity.9 To alleviate this issue, Au nanoparticles are typically modified by capping agents.9 Nevertheless, the use of capping agents may limit the accessibility of Au active sites and decrease their activity.13 Anchoring Au nanoparticles on supporting materials such as carbon,14 graphene,15 silica16 and periodic mesoporous organosilica17 is another approach to improve the dispersity of bare nano-Au without sacrificing its efficiency. Silica nanoparticles, in particular, have drawn much attention compared to other supports owing to their tunable mesostructures, adjustable morphologies, and ease of surface functionalization.18-19 The abundant silanols (Si-OH) can be easily substituted by other functional groups (e.g., -NH2, -SH) to anchor nano-Au.9, 20-29 However, the Au contents in previous reports on peroxidase mimicking studies are typically low (≤10 wt%).23-24, 3 ACS Paragon Plus Environment


29

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Chen and co-workers30 recently deposited Au nanoparticles on silica to mimic

DNase-like activity by using Au nanoparticles with large sizes (16 nm) to increase the Au content (17.36%). Because ultra-small nano-Au is expected to show high peroxidase mimicking activity,9 it is still a challenge to load a high content of ultra-small Au nanoparticles with adjustable sizes on silica nanoparticles. Among various surface functionalization, thiol groups have a high affinity toward Au,3132

thus increasing the thiol density on silica surface is the key towards a high Au loading

content. Wang et al.33 recently prepared thiolated silica nanoparticles for protein immobilization using a grafting method on preformed silica nanoparticles, the thiol density is 127±12 μmol g-1, the highest value reported to date. It has also been reported that the co-condensation method typically leads to a higher content of surface functional groups than the grafting or post-synthesis modification method,34 suggesting that there is plenty of room to further increase the thiol density and hence the Au loading capacity. In addition, the performance of nanozymes is generally evaluated by their catalytic activity towards substrate conversion.23-24,

29

Thus, the

accessibility of substrate molecules to the active sites, which is mainly controlled by the structure of supporting materials, could significantly affect the nanozyme 4 ACS Paragon Plus Environment

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performance.35 Reported silica nanoparticles for Au anchoring typically exhibit small pore sizes and long pore channels,23-24 hence the limitation in diffusion and accessibility should be improved to achieve enhanced nanozyme activity.23-24 In this regard, dendritic mesoporous silica nanoparticles (DMSNs) with large and open mesopores

36-38

enable easy access to substrate and opportunities for nano-Au

loading with high contents. However, the highest loading of nano-Au in DMSNs is 5 wt% due to the choice of functionalities other than thiols (e.g., -NH2)29 or a low thiol content (1.9 wt%).39 The synthesis of DMSNs with an ultrahigh density of thiol moieties for the loading of high-content nano-Au with tunable sizes has not been reported. Herein, we report the synthesis of thiolated dendritic mesoporous silica nanoparticles (T-DMSNs) with a high thiol density of 284.6±9 μmol g-1 and a high loading of Au nanoparticles (34.0 wt%) with tunable sizes (1.2-2.7 nm). Both the thiol density and Au loading content are higher than the highest values reported in literature.30, 33 As illustrated in Figure 1, the nanostructure and the thiol density can be controlled by adjusting the delayed addition time of thiolated organosilica precursor (see details in Supporting Information). Moreover, for the first time, we demonstrate that the peroxidase-like activity of T-DMSNs-Au is nano-Au size dependent: the highest activity 5 ACS Paragon Plus Environment


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is achieved at the Au particle size of 1.9 nm (Figure 1B). Our contribution provides new understandings in the rational design of nanozymes for future diagnostic applications.

Figure 1 Schematic illustration of the effects of (A) delayed addition time on the structures of the final product and (B) nano-Au size on the peroxidase-like activity.

Experimental Section Materials All chemicals were used as received without further purification. Tetraethyl orthosilicate (TEOS), triethanolamine (TEA), (3-mercaptopropyl) trimethoxysilane (MPTMS), sodium salicylate (NaSal), cetyltrimethylammonium bromide (CTAB), ethanol, hydrochloric acid (37 %wt), sodium acetate, 5,5'-dithiobis(2-nitrobenzoic acid) 6 ACS Paragon Plus Environment

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(DTNB), Au (III) chloride hydrate (HAuCl4), sodium borohydride (NaBH4), horseradish peroxidase (HRP), 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2 were purchased from Sigma-Aldrich. Doubly distilled water achieved from a laboratory purification system was used throughout the experiments. To eliminate the possible oxidation of thiol groups into disulfide bond during experiments, water was deoxygenated in an ultrasonic bath and then purged with N2 gas for 15 min to remove dissolved oxygen.

Synthesis of T-DMSNs In a typical synthesis, TEA (68 mg) was dissolved in deoxygenated water (25 mL) at 80 °C. After vigorous stirring for 30 min, NaSal (84 mg) and CTAB (380 mg) were added to the solution and the mixture was kept stirring for 60 min, followed by the addition of TEOS (3.8 mL). 30 min later, MPTMS (0.16 mL) was introduced to the solution (the molar ratio of MPTMS to TEOS, r, was 0.05). The mixture was kept stirring for 5 h and the product was collected by centrifugation at 4700 rpm for 10 min, washed for three times with ethanol to remove the residual reactants. Subsequently, surfactant was extracted using acidic ethanol (3 mL of 37% HCl in 50 mL of absolute ethanol) for three times (6 h each time) at 70°C.



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To investigate the effect of delayed addition time (t, calculated from TEOS to MPTMS addition) on the final structure, t was adjusted to 0, 15, 45, and 60 min while all other synthesis parameters kept unchanged. To achieve a higher thiol content, T-DMSNs were prepared with increased r of 0.2 while MPTMS was added to the solution by four times (0.16 mL each time) at 30, 60, 90, 120 min after the addition of TEOS. The obtained T-DMSNs were denoted as S-r-t (see Table S1).

Materials Characterizations Field emission SEM (JEOL 7800 operated at 1 kV) was used to study the morphological characteristics of synthesized products. SEM specimens were prepared by dispersed in ethanol and then dropped to the aluminum foil pieces and attached to conductive carbon film on SEM mounts. TEM (HT7700EXALENS operated at 200 kV) was used to determine the structural and chemical characteristics of the synthesized products. EDS elemental mappings were obtained in the FEI Tecnai F20 TEM operated at 200 kV. The TEM specimens were prepared by dispersed synthesized products in ethanol by sonication and then deposited onto a holey carbon film supported by copper grids. Nitrogen adsorption-desorption isotherms were measured at 77 K on a nitrogen adsorption device (Micromeritics


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ASAP Tristar II 3020). The samples were degassed under vacuum for 18 h at 110 °C before analysis. The total pore volume was calculated from the amount adsorbed at a maximum relative pressure (P/P0) of 0.99. The Brunauer-Emmet-Teller (BET) method was used to estimate the specific surface area. The pore size distribution was derived from the adsorption branch of the isotherms using the Barret-Joyner-Halanda (BJH) method. X-ray photoelectron spectra (XPS) were collected on a Kratos Axis Ultra Xray photoelectron spectrometer (PerkinElmer). spinning (CP-MAS) and

13C

29Si

cross-polarization magic-angle

CP-MAS nuclear magnetic resonance (NMR) spectra

were recorded on a Bruker Avance III spectrometer with a 7T magnet, Zirconia rotor, 4 mm, rotated at 5 kHz. X-ray diffraction (XRD) patterns were collected using a German Bruker D8 X-ray Diffractometer with Ni-filtered Cu Ka radiation (λ=0.15406 nm). For XRD analysis, a 0.26° divergence slit and a 5.0 mm anti-scatter slit were used. The measurements were conducted in the 2 theta range of 10-90°. The step size was 0.02° and the scan speed was 1.2 s per step with a total scan time of 80 min. Ultraviolet-visible (UV–vis) absorption spectra were documented with a Shimadzu UV3600 in the range of 200-800 nm at room temperature.



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Fabrication of thiolated dendritic mesoporous silica nanoparticles using a post grafting method In a typical procedure, S-0.2-30 was calcined in air at 550 °C for 6 h. The calcined sample (100 mg) was dispersed in toluene (20 mL) under nitrogen. After stirring for 1 h at 110 °C, MPTMS (0.1 mL) was added and the mixture was refluxed for 18 h under nitrogen.40 The product was centrifuged, washed with toluene and ethanol three times, and then dried under vacuum at 50 °C. The sample prepared by the post grafting method was called S-0.2-30-G.

Ellman’s assay The surface density of thiol groups was quantified using Ellman’s assay.33 In a typical procedure, DTNB solution was made by dissolving sodium acetate (41 mg) and DTNB (7.96 mg) in water (10 mL). To prepare the blank sample, DTNB solution (50 μL) was added to Tris buffer solution (100 μL, 1 M, pH=8) and then diluted in water (840 μL). The nanoparticles solution (10 μL, 2.5 mg mL-1) was added via syringe to blank sample, mixed carefully using a pipette and then incubated at room temperature for 5 min. The mixture was centrifuged and the absorbance of the supernatant was measured at 412 nm. The quantity of the surface thiol groups was


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calculated by dividing absorbance by the extinction coefficient of the reagent (13600 M-1 cm-1).

Loading gold nanoparticles on particles Au nanoparticles were deposited on the pore walls of the particles according to a previous method reported by Tao and coworkers.24 Briefly, S-0.2-30 (25 mg) were dispersed in 10 mL distilled water by sonication of 5 min followed by magnetic stirring for 15 min. Then, HAuCl4 solution (0.75 mL; 100 mM) was added to the above aqueous solution and kept under stirring for 1 h. A freshly prepared cold NaBH4 solution (3.75 mL; 100 mM) was added to the above mixture under vigorous stirring. The resulting suspension was stirred for another 1 h. The obtained S-0.2-30@Au was collected by centrifugation and then washed with ethanol three times. For comparison, S-0.2-30@Au with different Au sizes were prepared by introducing different amounts of HAuCl4 and NaBH4 solutions (see Table S2 for more details), while keeping all other synthesis parameters constant. The final products were denoted as S-0.2-30@Au-s, where s stands for the size of of Au nanoparticles measured by TEM analysis (please see Table S2).



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Evaluation of peroxidase-like activity Peroxidase-like activity of synthesized S-0.230@Au-s was measured based on the steady-state kinetic study. The catalytic ability of S-0.2-30@Au-s was measured by UV-Vis spectrophotometric (UV-2450, Shimadzu Company).24 All assays were carried out at room temperature in quartz cuvettes (path length (l) = 1.0 cm) using sodium phosphate (100 mM, pH 4.0) as the reaction buffer. The absorbance of the produced blue color at 652 nm represents the released TMB diimine product.24 In a typical experiment, S-0.2-30@Au-s (300 μg) was dispersed in sodium phosphate (600 μL) and then placed in the cuvette. H2O2 (450 μL) and TMB (450 μL) were added to the cuvette and the increase of absorbance at 652 nm was immediately measured as a function of time (10 s intervals) for five minutes. The relative activity was defined as the absorbance of S-0.2-30@Au-s to that of [email protected].

Estimation of kinetic parameters To measure the kinetic parameters for [email protected], kinetic assays were conducted using S-0.2-30@Au-s (10 μg) dispersed in sodium phosphate (200 μL) and H2O2 (final concentration of 50 mM) as the reaction media at 35 °C. Various concentrations of TMB was added to the cuvette and the absorbance


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at 652 nm was instantly measured as a function of time with intervals of 6 s for three minutes. The obtained “Absorbance vs Time” plots were then used to calculate the slope at the initial point (SlopeInitial) of each reaction. The initial reaction velocity (v) was calculated by dividing SlopeInitial by (εTMB-652 nm × l), where εTMB-652 nm represents the molar extinction coefficient of TMB at 652 nm which equals to 3.9×104 M-1 cm-1.15 Nonlinear regression of the Michaelis−Menten equation (v = Vmax × [S]/(Km + [S])) was used to fit the plots of ν against TMB concentrations ([S]). Lineweaver-Burk plot was created from the Michaelis Menten equation to obtain the kinetic parameters, Km and

Vmax. Vmax and Km are the highest reaction rate and the Michaelis constant, respectively.39

Reusability assay The reusability of [email protected] was studied by repeating the use of nanozyme to catalyze the oxidation reaction of TMB. After each cycle (5 min), the absorbance was read and nanozyme was then separated from the reaction system by centrifugation, washed three times with buffer solution to remove all of the substrate and products from the sample and applied in the next activity measurement with fresh



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substrates. The relative activity was defined as the observed absorbance of each cycle to the absorbance of the first cycle.

Results and Discussion T-DMSNs were prepared in an aqueous system using TEA as the catalyst, NaSal and CTAB as structure directing agents, TEOS and MPTMS as silica and organosilica precursors, respectively (Figure S1). To fabricate dispersed T-DMSNs with controllable morphology and a high thiol density, the effects of the molar ratio of MPTMS to TEOS (r) and delayed addition time (t, calculated from TEOS to MPTMS addition) on the final structures have been investigated. The samples are named as S-r-t (see the experimental section and Table S1). Figure 2a is a SEM image taken from S-0.05-30, and shows the spherical morphology of particles with open pores on the surface. Figure 2b is a typical TEM image and shows the dispersity and dendritic structure. Figure 2c is a dark-field (DF) scanning TEM (STEM) image and Figure 2d-f are the corresponding EDS elemental mapping of Si, O and S, all demonstrating the homogenous elemental distribution.


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Figure 2 (a) SEM and (b) TEM images of S-0.05-30. (c) Dark-field STEM image and (d-f) corresponding EDS mapping of S-0.05-30. (g) Solid-state

29Si

and (h)

13C

CP-

MAS NMR spectra of S-0.05-30

The

29Si

NMR spectrum shows both Q and T resonances (Figure 2g) representing

inorganic and organic silica.34 The resonances at -96, -106, and -116 ppm are



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correspond to the Q2 (Si(OSi)2(OH)2), Q3 (Si(OSi)3(OH)), and Q4 (Si(OSi)4) sites, respectively, represents inorganic silica, while resonances at -64 and -74 ppm assigned to T2 (C-Si(OSi)(OH)2) and T3 (C-Si(OSi)2(OH)) sites, respectively, confirming the successful incorporation of organic moieties into the framework (Figure 2g).34 The 13C NMR spectrum shows three distinct resonances at 11, 27, and 29 ppm which are assigned to methylene C atom 1, 2, and 3 of Si-SH chain, respectively (Figure 2h).39 The absence of CTAB peaks authorizes the success of extraction process.34 The sulfur content on the external surface of sample measured by XPS was 5.02 wt% (Table 1), suggesting enriched thiol groups on the pore surface which could be easily utilized for further reaction. The density of thiol groups on the external surface was measured through Ellman’s test to be 138.8±9 μmol g-1 (Table 1), which is comparable to the highest value reported so far for silica nanoparticles (127 μmol g1).33

Nitrogen sorption analysis displays type IV isotherm, suggesting the mesopores

structure of the obtained T-DMSNs. The pore diameter, BET surface area and the total pore volume are 22.6 nm, 544 m2 g-1 and 1.92 cm3 g-1, respectively (Supporting Information, Figure S2 and Table 1). The large open pore is beneficial to the high loading of Au and enables the easy accessibility of substrate to Au. 16 ACS Paragon Plus Environment

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Table 1. Textural properties and chemical composition of samples.

a)

Sample

Dp

dp

S-0.05-0

NA

S-0.05-15

NA & 161

S-0.05-30

165

S-0.05-45

a)

b)

SBET

d)

SXPS e)

S f)

605

1.77

0.0

0.0

406

1.42

2.64

67.1±11

22.6

544

1.92

5.02

138.8±9

171

22.5

477

1.71

4.55

122.3±8

S-0.05-60

174

22.8

489

1.41

4.19

S-0.2-30

162

20.2

539

1.30

12.0

284.6±9

S-0.2-30-G

158

22.2

756

1.96

3.09

87.1±7

Average particle size (nm);

adsorption branch (nm);

c)

8.2

VP

c)

9.7 & 21.7

b)

117.5±1 2

Pore diameter calculated by BJH method from the

BET surface area (m2g-1);

Sulfur weight percentage estimated by XPS;

f)

d)

Total pore volume (cm3g-1);

e)

Thiol group density on the surface

measured by Ellman’s test (μmol g-1).

To study the effect of delayed addition time (t) on the self-assembled structure, t was adjusted in the range of 0-60 min. At t = 0 min, similar to the conventional cocondensation approach,41 organic and inorganic precursors were added at the same time. As shown in Figure S3a and e, the resultant nanoparticles (S-0.05-0) possessed



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aggregated disordered morphology. S-0.05-15 with a time gap of 15 min exposed a mixture of aggregated disordered particles and DMSNs with an average size of 161 nm (Figure S3b and f, Table 1). Increasing t to 45 and 60 min (S-0.05-45 and 60) led to spherical dendritic structures similar to S-0.05-30 with slightly increased particle sizes (171 and 174 nm, Figure S3). However, as summarized in Table 1, the sulfur content measured by XPS is the highest when t = 30 min. Either a lower (15 min) or higher t (45 or 60 min) caused smaller sulfur contents in the final structures. To understand the impact of the delayed addition time on the formation of particles, TEM analysis was conducted to monitor the structural evolution of S-0.05-30 and S0.05-0 as a function of time (Figure S4). For S-0.05-30, DMSNs with a well-defined structure and a particle size of around 100 nm already formed at the reaction time of 15 min (Figure S4 a1). After addition of MPTMS, the particle size progressively increased from 118 to 136 and 152 nm at 30, 45 and 60 min, respectively (Figure S4 a2, a3, and a4), suggesting that further co-condensation and assembly of MPTMS/TEOS occurred on pre-formed DMSNs. Further increasing the reaction time to 3-5 h (Figure S4 a5 and a6) resulted in little change in structure and slight increase in particle sizes (159 and 165 nm at 3 and 5 h, respectively). For S-0.05-0, 18 ACS Paragon Plus Environment

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disorganized and irregular structures were formed at 15 min (Figure S4 b1) without the delayed addition. No dendritic structures with uniform particle sizes were observed at prolonged reaction time (30 min – 5 h, Figure S4 b2, b3, and b4). The above observations suggest that when adding precursors at the same time (S-0.05-0), MPTMS (a hydrophobic organosilica precursor)42 could penetrate inside the CTAB micelles

43-44

and interrupt the self-assembly of DMSNs. On the other hand, a critical

delayed addition time window of 30 min provides adequate time for the formation of DMSNs, which act as the "template" and heterogeneous nucleation site for MPTMS and remaining TEOS/structure-directing agents to grow, eventually forming T-DMSNs with high thiol contents. The above mechanism can be used to explain the effect of t on the final structures of T-DMSNs. In the case of S-0.05-15, a mixture of disordered structures and DMSNs indicates that a sufficiently long t is essential to avoid the impurity phase. Compared to S-0.05-30, the lower sulfur contents of S-0.05-0 and S-0.05-15 (0 and 2.64% by XPS analysis, see Table 1) can be explained by the penetration of MPTMS inside CTAB micelles and lost during surfactant extraction process. The sulfur contents for S-0.05-t (t=45 and 60) were 4.55 and 4.19 wt% , also lower than that of S-0.05-30 19 ACS Paragon Plus Environment


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(5.02%). This is because with further prolonged t (i.e., 45 and 60 min), the DMSNs are already formed and most TEOS molecules are consumed before the addition of MPTMS. Consequently, the added MPTMS molecules have reduced chance to cocondense with TEOS to be incorporated into the silica framework, leading to reduced sulfur contents in the final products after solvent extraction. In line with XPS results, the densities of accessible thiol groups on the external surface of S-0.05-t samples (by Ellman’s test) also show that S-0.05-30 has the highest thiol density (Table 1). Nitrogen adsorption-desorption measurements revealed that all samples showed type IV isotherms, characteristic of mesoporous materials (Figure S2a). The structural parameters of all samples are also summarized in Table 1. Compared to other S-0.05-t samples, S-0.05-30 with the highest thiol density also exhibits the highest specific surface area (544 m2 g-1) and pore volume (1.92 cm3 g-1) as well as a large pore size of 22.6 nm. To further increase the thiol density, the molar ratio of MPTMS to TEOS, r, was increased to 0.2 while keeping t at 30 min (see the experimental part for details). The final product S-0.2-30 is typical DMSNs with large open pores and uniform particle sizes of about 162 nm (Figure 3a, 3b). For S-0.2-30, the sulfur content was 12.01% 20 ACS Paragon Plus Environment

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determined from XPS and the thiol density was 284.6±9 μmol g-1 by Ellman’s test, which is two times of the highest value reported so far (127±12 μmol g-1).33 S-0.2-30 owns typical type IV isotherms (Figure S5a). The pore size, BET surface area and total pore volume were measured to 20.2 nm, 539 m2 g-1 and 1.30 cm3 g-1 (Figure S5b and Table 1). By further increasing r to 0.4, organic impurities were shaped in the form of small particles (data are not shown), therefore, S-0.4-30 was not subjected to further characterization. It is suggested that in the high concentrations of MPTMS, the similar hydrolysis rates of TEOS and MPTMS under our synthesis conditions favours the polycondensation of organic moieties and formation of impurities.45 S-0.2-30 benefits from uniform particle size (162 nm), large pore size (20.2 nm), highly accessible large surface area (539 m2 g-1) and high pore volume (1.30 cm3 g-1), which make it an ideal platform for in situ loading of Au nanoparticles. In particular, the ultrahigh density of functional -SH groups (284.6±9 μmol g-1) on accessible dendritic pore walls allows for the formation of highly dispersed Au nanoparticles by firstly forming S-Au covalent bonds with auric chloride ions and then in-situ reduction by NaBH4. The formation of Au nanoparticle in S-0.2-30 is evidenced by the color change in the solution from white (bare silica) to brown (Figure S6a and b). Low-magnification 21 ACS Paragon Plus Environment


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TEM image shows no distinct change in the dendritic structure after the Au nanoparticle loading process (Figure 3c and S6c). The high-resolution TEM images (Figure 3d) clearly show the formation of well-dispersed Au nanoparticles with an average size of 1.9±0.3 nm (determined by measuring the size of 50 Au nanoparticles) and crystalline structure.46 The distances measured from the lattice planes on average is about  0.23 nm corresponding to the lattice spacing’s of Au (111) planes (insect of Figure 3d).46


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Figure 3 (a) SEM and (b) TEM images of S-0.2-30. (c) TEM and (d) high-resolution TEM images of [email protected]. Insect shows lattice spacing. (e-h) Corresponding EDS mapping, (i) XRD pattern, and (j) XPS spectra of [email protected].

The DF-STEM image and the EDS elemental mapping further demonstrate the uniform distribution of Si, S, and Au elements in the framework of [email protected] (Figure 3e-h). The EDS spectra also confirmed that [email protected] nanocomposite 23 ACS Paragon Plus Environment


Page 24 of 42

was composed of Si, O, S, C and Au elements (Figure S6d). The structure of [email protected] was further verified by XRD pattern and XPS. The XRD pattern displays four intense diffraction peaks at 2θ = 38°, 45°, 65° and 78°, which can be indexed as the (111), (200), (220), and (311) reflections of the face-centered-cubic structured Au0, respectively (Figure 3i).24 XPS spectra displays peaks from Au 4f (83 eV), C 1s (∼284 eV), O 1s (∼530 eV), Si 2s (∼153 eV), Si 2p (∼101 eV), and S 2p (∼163 eV).24, 47-48 The presence of Au 4f peak at XPS spectra clearly shows the successful loading of Au on S-0.2-30 (Figure 3j).39 The content of Au measured by XPS is 29.8 wt%, which is considerably higher than previously reported values,23-24,

39

highlighting the advantage of an

exceptionally high density of accessible thiol groups on the surface of silica nanoparticles to host Au nanoparticles. To further explore the contribution of thiol density on the loading of Au nanoparticles, S-0.05-30 with a lower content of thiol groups was also subjected to anchor Au nanoparticles. The amount of loaded Au nanoparticles measured by XPS is 13.9 wt%, lower than that for S-0.2-30, suggesting the direct connection between the density of thiol groups and the content of loaded Au nanoparticles. To demonstrate the superiority of the delayed addition strategy over conventional grafting or post24 ACS Paragon Plus Environment

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modification strategy, S-0.2-30-G was synthesized for comparison (Figure S7, Table 1, see experimental section for more details). The sulfur content, the thiol density and the content of Au nanoparticles of S-0.2-30-G was measured to be 3.09 wt%, 87.1±7 μmol g-1 and 7.2 wt%, respectively, much lower than those for S-0.2-30, suggesting that the delayed addition approach plays a key role to create an ultrahigh density of thiol groups to load a high-content of Au nanoparticles. The peroxidase-like activity of [email protected] was studied through monitoring the oxidation of TMB in the presence of H2O2 (see the experimental part).24 As the reaction progressed, the color of the reaction media turned into deep blue, indicating peroxidase-like activity of [email protected] (Figure S8).24 As a control, no color change was observed for S-0.2-30, suggesting the intrinsic catalytic property came from Au nanoparticles. To study the effect of pH value on the peroxidase-like activity of [email protected], the activity of [email protected] in pH values from 2 to 8 was studied. As illustrated in Figure S9, the activity of [email protected] was pH dependent with the maximum activity at pH 3.0. However, free HRP shows the maximum peroxidase activity at pH 4.24 In most literature reports of gold/silica composites, their



Page 26 of 42

peroxidase-like activities23-24, 29 were studied also at pH 4. To have a fair comparison, in this work pH 4 was selected to evaluate the catalytic performance of T-DMSNs@Au. In literatures, Au nanoparticles with sizes smaller than 5 nm exhibit higher enzymelike activity.23-24, 39 While the catalytic activity of Au nanoparticles is size-dependent in various applications,11-12,

49

this hypothesis has been rarely studied in nanozyme

applications (e.g., peroxidase mimicking). To study the contribution of Au nanoparticle size on enzyme-like activity, a series of S-0.2-30@Au-s (s=1.2±0.3, 1.4±0.4, 1.9±0.3, 2.1±0.3, and 2.9±0.3 nm) were synthesized (Figure S10, supporting information) by varying the initial amount of Au precursor. The Au content was measured to be 14.8, 24.1, 29.8, 31.0, and 34.0 wt% by XPS analysis. The peroxidase-like activity of S-0.2-30@Au-s was studied through in-situ UV-Vis spectroscopy by monitoring the increase of absorbance at 652 nm against that of a reference buffer with the same amount of TMB and H2O2. Figure 4a displays the absorbance of TMB oxidation product at 652 nm as a function of time for S-0.2-30@Au-s with the same Au contents (0.1 mg). The increased absorbance over the time and produced blue colour for S-0.2-30@Au-s suggest the intrinsic peroxidase-like activity of anchored Au nanoparticles (Figure 4a). The relative 26 ACS Paragon Plus Environment

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activities of [email protected], 1.4, 1.9, 2.1, and 2.7 were measured to be 0.07, 0.35, 1.00, 0.87, and 0.49, respectively (Figure 4b). As shown in Figure 4b, with the Au nanoparticle size decreasing from 2.7 to 1.9 nm, the relative activity increases from 0.49 to 1.00, suggesting that small-sized Au nanoparticles have more exposed catalytically active sites and thus higher activity.3 By further decreasing the Au nanoparticle size to 1.4 and 1.2 nm, the relative activity sharply drops to 0.35 and 0.07, respectively. As reported in literature, Au nanoparticles with very small sizes usually possess a less rounded shape which results in reduced amount of exposed active sites and catalytic activity,49-50, 50 similar to our observations (see Figure S11 for more details)” Collectively, the above findings demonstrate the size-dependent peroxidaselike activity of Au nanoparticles with an optimized size of 1.9 nm showing the highest activity.



Page 28 of 42

Figure 4 (a) The absorbance of TMB oxidation product at 652 nm as the function of time for S-0.2-30 and S-0.2-30@Au-s and (b) the corresponding relative activities. The relative activity was defined as the absorbance for S-0.2-30@Au-s to that for [email protected] after 5 min.

The supreme peroxidase-like activity of [email protected] was further confirmed by additional catalytic study. Michaelis-Menten plots were obtained and kinetic parameters such as Km (Michaelis constant) and Vmax (maximum velocity) were calculated through the Lineweaver-Burk equation (Figure S12). Km indicates the enzyme affinity to substrates and a high Km value signifies a lower affinity and vice versa.51 [email protected] showed a typical enzymatic Michaelis-Menten kinetics 28 ACS Paragon Plus Environment

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(Figure S12) with the Km and Vmax values of 0.0407 mM and 25.910-8 M s-1. The Km value of [email protected] was very close to that of the natural enzyme (horseradish peroxidase, 0.0411 mM),15 showing the advantages of the unique highly thiolated large-pore DMSNs as nanozyme supports to enable a high-content loading of Au nanoparticles, and to facilitate the catalytic performance due to easy diffusion of the substrates and products.29 The Vmax value of [email protected] was approximately 6 times higher than that of horseradish peroxidase (4.310-8 M s-1),15 indicating the excellent catalytic activity of nanozyme. The obtained Vmax values are superior compared to previously reported gold-silica materials,23-24,

29

ferromagnetic

nanoparticles and graphene oxide materials (Table S3),4-5 suggesting the benefit of Au nanoparticles with tailored size in mimicking enzyme-like activity. Furthermore, [email protected] showed excellent reusability and higher activities compared to free HRP over a broad temperature and pH range (Figure S13), suggesting its superiority over natural enzyme.

Conclusion



Page 30 of 42

In summary, T-DMSNs with an ultra-high density of accessible thiol moieties are fabricated using a delayed addition strategy. The ultra-high thiol content of T-DMSNs enables an extremely high loading of Au nanoparticles with superior peroxidase-like activity. It is found that the size of Au nanoparticles affects their peroxidase-like activity and the size of 1.9 nm exhibits the maximum catalytic activity. This study has provided new understandings in the rational design of nanozymes and a platform of functional materials with potential to enhance the sensitivity of diagnosis technologies, such as lateral flow assay and enzyme-linked immunosorbent assay.

ASSOCIATED CONTENT Further characterizations of materials and control samples. This supporting information is available free of charge via the internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author


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* Email: [email protected]; [email protected]; Fax: +61 7-334 63973; Tel: +61 7-334 63283

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources The authors acknowledge the financial support from the Australian Research Council. Dr Chang Lei acknowledges the support from Advance Queensland Fellowship.

Notes The authors declare no financial interest.

ACKNOWLEDGMENT



Page 32 of 42

We thank Australian National Fabrication Facility-Queensland Node (ANFFQ), the Australian Microscopy and Microanalysis Research Facility at the Centre for Microscopy and Microanalysis, The University of Queensland for technical assistance.

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Graphical Abstract


Highly Thiolated Dendritic Mesoporous Silica Nanoparticles with High

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