Facile synthesis of gold nanoparticles on propylamine

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Facile synthesis of gold nanoparticles on propylamine functionalized SBA-15 and effect of surface functionality of its enhanced bactericidal activity against gram positive bacteria

This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 Mater. Res. Express 2 075402 (http://iopscience.iop.org/2053-1591/2/7/075402) View the table of contents for this issue, or go to the journal homepage for more

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Mater. Res. Express 2 (2015) 075402

doi:10.1088/2053-1591/2/7/075402

PAPER

RECEIVED

19 March 2015 REVISED

14 June 2015

Facile synthesis of gold nanoparticles on propylamine functionalized SBA-15 and effect of surface functionality of its enhanced bactericidal activity against gram positive bacteria

ACCEPTED FOR PUBLICATION

26 June 2015 PUBLISHED

29 July 2015

Diganta Bhuyan1, Animesh Gogoi2, Mrinal Saikia1, Ratul Saikia2 and Lakshi Saikia1 1 2

Materials Science Division, CSIR-North East Institute of Science and Technology Jorhat—785006, Assam, India Biotechnology Division, CSIR-North East Institute of Science and Technology Jorhat—785006, Assam, India

E-mail: [email protected] Keywords: SBA-15, Au nanohybrid, bactericidal activity, Gram positive bacteria Supplementary material for this article is available online

Abstract The facile synthesis of an SBA-15-pr-+NH3.Au0 nano-hybrid material by spontaneous autoreduction of aqueous chloroaurate anions on propylamine functionalized SBA-15 was successfully demonstrated. The as-synthesized SBA-15-pr-+NH3.Au0 nano-hybrid material was well characterized using low and wide angle x-ray diffraction (XRD), N2 adsorption–desorption isotherms, Fourier transform infrared (FTIR), transmission electron microscopy (TEM), scanning electron microscopy-energy dispersive x-ray spectroscopy (SEM-EDX), x-ray photoelectron spectroscopy (XPS), UV-Visible spectroscopy and atomic absorption spectroscopy (AAS). The activity of the nano-hybrid material as a potent bactericidal agent was successfully tested against Gram positive/negative bacteria viz. Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. The colony killing percentage of Gram positive bacteria was found to be higher than Gram negative bacteria due to the stronger electrostatic interaction between the positively-charged amine functionality of SBA-15 and the negatively charged functionality of the bacterial cell wall.

1. Introduction Since the discovery of ordered mesoporous materials (OMMs), they have attracted much attention from researchers owing to their outstanding properties, such as their mesoporosity, tunable pore sizes, large pore volumes, high surface areas, etc [1–9]. SBA-15 is one of the most well studied materials because of its unique properties like high surface area, 2D hexagonal array of pores, large and uniform pore channels and good thermal stability [2]. Since the discovery of SBA-15, it has undergone tremendous synthetic modification to improve its material properties [3, 10–12, 21, 22]. The surface functionalization strategy is an emerging field of research in SBA-15 materials for various applications like catalysis [10–12], drug delivery [13, 14], adsorption and separation [15, 16], chemical sensing [17, 18], biomedical applications, etc [19, 20]. For the stabilization of nanoparticles, SBA-15 is often functionalized using a variety of organic functional moieties such as NH2, SH, and SO3H [21, 22]. Nowadays, there is tremendous interest in synthesis of advanced hybrid materials by incorporating metal nanoparticles in silica materials. Many research groups have reported the synthesis of nanohybrid materials by generating different metal nanoparticles in SBA-15 supports like gold, silver, palladium, platinum, etc [23, 24]. M Sastry et al reported the spontaneous autoreduction of chloroaurate anions into gold nanoparticles [25, 26]. In this report, we have functionalized mesoporous SBA-15 using 3-APTMS and after that, aqueous AuCl 4− was adsorbed in the SBA-15 through an acid–base neutralization reaction. A spontaneous autoreduction reaction took place that converted adsorbed AuCl 4− into Au0 nanoparticles. The synthesized SBA-15-pr-+NH3.Au0 nano-hybrid material was well characterized using low angle and wide angle XRD, N2 adsorption–desorption isotherms, FTIR, TEM, SEM-EDX, XPS, UV-Visible and AAS. A tentative mechanism for the formation of Au0 nanoparticles was also proposed. © 2015 IOP Publishing Ltd


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Bacterial infection is currently a serious medical issue which is associated with significant mortality and health care costs [27]. Many commercially available antibiotic drugs are used to combat different Gram positive/ negative bacteria. However, the broad and prolonged use of these antibiotics has caused increased bacterial resistance against multiple antibiotics [28, 29]. Recently, multidrug resistant (MDR) superbugs have created significant health hazards. Some typical examples include Gram positive vancomycin-resistant Enterococcus faecium (VRE), methicillin-resistant Staphylococcus aureus (MRSA), and Gram negative MDR Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa, and Enterobacter species, abbreviated as ‘ESKAPE’ [28, 29]. Metal nanoparticles, especially silver nanoparticles, have a very good bactericidal activity and silver is generally the metal of choice for bactericidal activity studies against MDR bacteria, however, instability and toxicity have caused concern about its practical usability [30]. In contrast, gold nanoparticles are chemically stable, biocompatible, nontoxic toward human cells and their easy surface modification either using small molecules or by forming bioconjugates makes them a very useful bactericidal agent [31, 32]. Reportedly, surface modified gold nanoparticles exhibited pronounced efficacy against MDR bacteria [33, 34]. The surface modification and surface charge of gold nanoparticles are also important factors for their activity against Gram positive/negative bacteria [32, 33]. SBA-15 has also been reported as a support for silver metal which has exhibited effective bactericidal activity against S. aureus and E. coli [34, 35]. In this report, we have tested the bactericidal activity of SBA-15-pr-+NH3.Au0 nano-hybrid material against Gram positive/negative bacteria viz. B. subtilis, S. aureus, E. coli and P. aeruginosa. The probable mechanistic explanation for the selective bactericidal activity of the assynthesized nano-hybrid material against Gram positive bacteria was also explained.

2. Experimental section 2.1. Materials Tetraethyl orthosilicate (TEOS), Pluronic P123 triblock copolymer (EO20PO70EO20, Mn ̴ 5800), and 3-amino propyltrimethoxysilane (3-APTMS) were purchased from Aldrich. HCl (Merck), HF (Merck), hydrogen tetrachloroaurate (III) trihydrate (Acros Organics), nutrient broth (NB) (g L−1 peptone 5 g, beef extract 3 g, sodium chloride 5 g; pH 7; HiMedia, Mumbai, India) were purchased from the companies mentioned. All chemicals were analytical grade and used as received without further purification. 2.2. Synthesis of SBA-15 material SBA-15 was synthesized according to the literature2(a) using tetraethyl orthosilicate (TEOS) as the silica source, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly (ethylene glycol) as the template and HCl as the pH controlling agent. 2.3. Surface functionalization of SBA-15 material Surface functionalization of SBA-15 material was accomplished by condensation of 3-amino propyltrimethoxysilane (3-APTMS) with its surface silanol groups according to the reported procedure [21, 22]. In a typical synthetic approach, 3 g of calcined SBA-15 was first activated under vacuum at 423 K for 4 h and dispersed in 100 ml of dry toluene taken in a 250 ml double-necked round-bottom flask fitted with a watercooled condenser. To it, 3.10 g of 3-APTMS was added slowly over 10 min The contents of the flask were refluxed for 24 h in nitrogen. The solid was filtered off, dried and Soxhlet extracted, initially with toluene (12 h) and then with dichloromethane (for 12 h). The propylamine functionalized SBA-15 (hereafter referred to as SBA-15-pr-NH2 material) thus-obtained was dried at 353 K for 6 h and used for further preparations. 2.4. Synthesis of SBA-15-pr-+NH3.Au0 nano-hybrid material The spontaneous autoreduction of adsorbed AuCl 4− ions to Au0 nanoparticles in the SBA-15-pr-NH2 material proceeded through the following experimental procedure. In a typical experiment, 0.5 g of SBA-15-pr-NH2 and 100 ml of 1 mM HAuCl4 aqueous solution were mixed by sonicating for 30 min and then kept under continuous stirring for 48 h at room temperature. After the specified time, the material was washed with distilled water and acetone several times. The synthesized material (hereafter referred to as SBA-15-pr-+NH3.Au0 nano-hybrid material) which acquired a pink color was then dried under vacuum. 2.5. Characterization Low angle XRD was performed using a Philips X’Pert Pro diffractometer in the 2θ range from 0.5–5° using Cu Kα radiation and a proportional counter as a detector. Wide angle XRD patterns were recorded on a Rigaku, Ultima IV x-ray diffractometer in the 2θ range of 10–90° using a Cu Kα source (λ = 1.54 Å). Specific surface area, pore volume, and average pore diameter were measured using an Autosorb-1 (Quantachrome, USA). Specific surface areas of the samples were measured by the adsorption of nitrogen gas at 77 K and applying Brunauer– 2


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Figure 1. Low angle XRD patterns of SBA-15, SBA-15-pr-NH2 and SBA-15-pr-+NH3.Au0 nano-hybrid materials.

Emmett–Teller (BET) calculations. Prior to adsorption, the samples were degassed at 250 °C for 3 h. Pore size distributions were derived from desorption isotherms using the Barrett–Joyner–Halenda (BJH) method. FTIR spectra (4000–400 cm−1) were recorded using KBr pellets in a Shimadzu IR Affinity-1 spectrophotometer. TEM images were taken using a JEOL (Model JEM-2011) and Scanning electron microscopy (SEM) images and energy dispersive x-ray spectroscopy (EDX) patterns were obtained from a JSM-6360, Jeol operated at 20 kV. XPS were recorded on a VG Microtech Multilab ESCA 3000 instrument with non-monochromatized Mg Kα radiation (hν = 1253.6 eV). The absorbance spectra were recorded at room temperature using a UV-visible spectrophotometer (Model Shimadzu 1601 pc) on an aqueous dispersion. AAS measurements were carried out using a PerkinElmer (Model-AAnalyst-700) spectrometer. Prior to analysis, the sample was digested in aqua regia. The zeta potential was measured using a Malvern Zetasizer, 3000 HSA. Prior to measurements, a definite amount of sample was dispersed in double distilled water. 2.6. Bactericidal activity of SBA-15-pr-+NH3.Au0 nano-hybrid material For bactericidal activity studies, 10 mg of SBA-15-pr-NH2 /SBA-15-pr-+NH3.Au0 nano-hybrid material was dispersed in 5 ml of water by ultrasonication and then this aqueous suspension at different concentrations was used against two Gram positive bacterial strains viz. B. subtilis (MTCC 441) and S. aureus (MTCC 96) and two Gram negative bacterial strains viz. E. coli (MTCC 739) and P. aeruginosa (MTCC 2453) obtained from the Microbial Type Culture Collection, IMTECH, Chandigarh, India. The bacterial strains were grown in NB at 30 ± 2 °C with continuous agitation at 180 rpm for 24 h. The concentration the gold nanoparticles is 32.8 ng μl−1. The batch cultures were prepared at five different concentrations of SBA-15-pr-NH2/SBA-15-pr-+NH3.Au0 nano-hybrid material as aqueous suspensions (viz. 25, 75, 150, 250 and 300 μl), keeping the bacterial strain inoculated with SBA-15 as negative control as it does not show any bactericidal activity. The corresponding concentrations of gold nanoparticles in the nano-hybrid material as aqueous suspensions were 0.82, 2.46, 4.92, 8.2 and 9.84 μg mL−1, respectively. The aqueous suspensions of SBA-15-pr-NH2/SBA-15-pr-+NH3.Au0 nanohybrid material at different concentrations were added to 100 mL of sterile NB in an Erlenmeyer flask (250 mL) and was fully sonicated to prevent aggregation of the SBA-15-pr-+NH3.Au0 nano-hybrid material under sterile conditions [36].

3. Results and discussion 3.1. Characterization The low angle XRD pattern of SBA-15 (figure 1) consists of (100), (110), and (200) reflections corresponding to a two dimensional hexagonal p6 mm symmetry and long-range mesopore ordering for SBA-15 appeared at 2θ values of 0.979, 1.649, and 1.879°, respectively [3, 10–12, 21, 22]. On propylamine functionalization of SBA-15, these peaks shifted to higher 2θ values of 0.989, 1.659 and 1.889 (figure 1) and the intensity of the respective peaks also decreased compared to unmodified SBA-15. In the case of the SBA-15-pr-+NH3.Au0 nano-hybrid material the 2θ values shifted to lower values of 0.959, 1.629 and 1.869 compared to unmodified SBA-15 (figure 1). It was previously reported that on organic functionalization or incorporation of metal nanoparticles, the 2θ values of the XRD peaks can shift to higher or lower values and the intensity of the characteristic peaks of SBA-15 also decreases [10–12, 21, 22]. The wide angle XRD patterns of the SBA-15 materials are depicted in 3


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Figure 2. Wide angle XRD patterns of SBA-15, SBA-15-pr-NH2 and SBA-15-pr-+NH3.Au0 nano-hybrid materials.

Table 1. Chemical compositions and structural properties of SBA-15 materials. Elemental analysis (wt%)

System SBA-15 SBA-15-pr-NH2 SBA-15-pr-NH2. Au0

N2 adsorption–desorption isotherm

C

H

N

Average pore diameter (nm)

Specific surface area (m2 g−1)

Total pore volume (cm3 g−1)

0.3 8.75 8.82

0.8 2.0 2.45

0 1.29 1.27

7.4 7.0 6.5

559 453 301

1.03 0.80 0.59

figure 2. SBA-15 and SBA-15-pr-NH2 have only one broad peak between 2θ values of 20–30° which is characteristic for mesoporous SBA-15. In the case of the SBA-15-pr-+NH3.Au0 nano-hybrid material this broad peak is retained [37]. The XRD pattern of the SBA-15-pr-+NH3.Au0 exhibited another five extra diffraction peaks which can be indexed to diffraction from the (111), (200), (220), (311) and (222) planes of the face centered cubic (fcc) structure of metallic gold (JCPDS card no. 04-0784) [38]. The N2 adsorption– desorption isotherm of SBA-15 shows a typical type IV curve (figure S1 (a), ESI) with a clear H1 hysteresis loop characteristic of a mesoporous material with uniform pores and long-range cylindrical channels [3, 10– 12, 21, 22]. The sharp inflection in the relative pressure range (P/P0 = 0.6–0.8) corresponds to capillary condensation of the uniform mesopores. The BET surface area of SBA-15 was found to be 559 m2 g−1 with an average BJH pore size of 7.4 nm (figure S1 (a) inset, ESI) and the total pore volume is 1.03 cm3 g−1 (table 1). Upon propylamine functionalization, SBA-15 did not alter its characteristic type IV curve (figure S1 (b), ESI). The BET surface area, BJH average pore size (figure S1 (b) inset, ESI) and total pore volume were found to be 453 m2 g−1, 7.0 nm and 0.80 cm3 g−1, respectively (table 1) The observed decrease is due to propylamine functionalization of surface silanol groups [10–12, 21, 22]. The inflection in the relative pressure range (P/ P0 = 0.4–0.8) corresponds to capillary condensation of the uniform mesopores which is not as sharp as for SBA15 due to the functionalization of the pores. In the case of the SBA-15-pr-+NH3.Au0 nano-hybrid material, the typical type IV curve of mesoporous SBA-15 is retained (figure S1 (c), ESI) wherein, the BET surface area, BJH average pore size (figure S1 (c) inset, ESI), and total pore volume were found to be 301 m2 g−1, 6.5 nm and 0.59 cm3 g−1, respectively. The decrease in the textural parameters compared to SBA-15 and SBA-15-pr-NH2 is due to the partly blocking of the pores of the mesoporous channels by gold nanoparticles. The typical inflection in the relative pressure range (P/P0 = 0.6–0.8) corresponds to capillary condensation of the uniform mesopores. FTIR spectra of all three synthesized SBA-15 materials are illustrated in figure S2, ESI. The FTIR peaks around 3800–3000 and 1645 cm−1 are due to O–H stretching and bending vibrations of surface silanol and adsorbed water molecules, the peaks at 1260–1000, 957, and 798 cm−1 are attributed to Si–O–Si stretching vibrations and the peak at 461 cm−1 is assigned to the bending mode vibration of the Si–O–Si group of SBA-15. For propylamine functionalized SBA-15, the N–H stretching vibration appeared at 1547 cm−1 and the N–H bending vibration peak appeared around 687–680 cm−1, confirming the presence of amino groups. The C–H stretching vibrations appeared at 2938 and 2882 cm−1. The C–N stretching vibration generally appears around 4


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Figure 3. TEM images of (a) SBA-15, (b) SBA-15-pr-NH2, (c) SBA-15-pr-+NH3.Au0 nano-hybrid material with SAED pattern (inset), and (d) SBA-15-pr-NH2. Au0 nano-hybrid material and one individual Au0 nanoparticles (inset).

1200–1000 cm−1 but this peak cannot be resolved because of its overlap with Si–O–Si vibrations. All of these data confirm the propylamine functionalization of mesoporous SBA-15 and all of the peaks due to functionalization were retained in the case of the SBA-15-pr-+NH3.Au0 nano-hybrid material [10–12]. The TEM images of the SBA-15 materials revealed the presence of a highly uniform and ordered honeycomb-like hexagonal array of pores, as depicted in figure 3(a) and it is retained after propylamine functionalization, as shown in figure 3(b). Observing figures 3(c) and (d), it can be commented that the mesoporous arrangement was preserved after synthesis of gold nanoparticles. The gold nanoparticles which are within the mesoporous channels are 5–6 nm in size and those which remain on the surface of SBA-15 are 10–25 nm. The SAED pattern is shown in figure 3(c) inset which confirms that the synthesized gold nanoparticles are single crystalline in nature and one individual gold nanoparticle is shown in figure 3(d) inset. SEM images reveal that the synthesized SBA-15 materials (figure 4(a)) aggregated into wheat-like macrostructures [3, 10–12, 21, 22]. No change in morphology was observed after propylamine functionalization of SBA-15 (figure 4(b)). The gold nanoparticles are attached to the surface of the wheat-like macrostructures in the case of the SBA-15-pr-+NH3.Au0 nano-hybrid material (figure 4(c)). The EDX pattern in figure 4(d) shows that gold is the only detectable metal in the SBA-15-pr-+NH3.Au0 nano-hybrid material. The UV-Visible absorbance spectra of the aqueous suspension of the SBA-15-pr-+NH3.Au0 nano-hybrid material (figure 5) contains the typical SPR band of gold nanoparticles observed around 532 nm, whereas for the SBA-15-pr-NH2 material there is no absorbance peak [39–41]. The oxidation state of gold in the SBA-15-pr-+NH3. Au0 nano-hybrid material was investigated by XPS. The results reported in figure 6 show a binding energy of 87.7 eV for 4 f5/2 electrons and 84.2 eV for 4 f7/2 electrons [40–42]. The binding energy indicates exclusively Au0 nanoparticles without traces of Au(III) or Au(I) species. The amount of metallic gold in the SBA-15-pr-+NH3.Au0 nano-hybrid material was found to be 3.28 wt% from AAS. From elemental analysis, the amount of C, H and N was obtained for all the as-synthesized SBA-15 materials (table 1).The amount of C, H and N was found to be 5


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Figure 4. SEM images of (a) SBA-15, (b) SBA-15-pr-NH2, (c) SBA-15-pr-+NH3.Au0 and (d) EDX analysis of SBA-15-pr-+NH3.Au0 nano-hybrid material. (Area taken for analysis is whole area in figure 2(c)).

Figure 5. UV-visible spectra of SBA-15-pr-NH2 and SBA-15-pr-NH2.Au0 nano-hybrid material and photographs of the aqueous suspension of (a) SBA-15-pr-NH2 and (b) SBA-15-pr-NH2.Au0 nano-hybrid material (inset).

almost the same for SBA-15-pr-NH2 and the SBA-15-pr-+NH3.Au0 nano-hybrid material. The zeta potential measurements (figure S3, ESI.) of the SBA-15-pr-NH2 and SBA-15-pr-+NH3.Au0 nano-hybrid materials showed average potentials of +4.9 mV and +40.2 mV, respectively, in aqueous medium. This may be due to the protonation of some of the amine moieties of SBA-15-pr-NH2 in water and due to the acid–base neutralization reaction, the amine moieties of SBA-15-pr-+NH3.Au0 are most likely to stay protonated. 3.2. Tentative mechanistic explanation for autoreduction of aqueous AuCl4− ions to Au0 nanoparticles It has been previously reported that HAuCl4 can be immobilized on a silica support modified by propylamine through an acid–base neutralization reaction [43, 44]. Both amine and hydroxyl groups can reportedly reduce 6


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Figure 6. Au 4f XPS spectra of SBA-15-pr-+NH3.Au0 nano-hybrid material.

Scheme 1. Synthesis of Au0 nanoparticles by spontaneous autoreduction of chloroaurate ions with propylamine functionalized mesoporous SBA-15 material.

gold salts to metallic gold nanoparticles [25, 26, 42, 45], and reports are also available for the reduction of gold salt to metallic gold nanoparticles by biopolymer-like chitosan which has both amine and hydroxyl groups [46]. As described in scheme 1, the formation of gold nanoparticles in SBA-15 is a three step process. In the first step, mesoporous SBA-15 was functionalized with 3-APTMS. The second and third step were a continuous process. The aqueous chloroaurate anions (AuCl 4−) were adsorbed onto the SBA-15 surface and channels through an acid–base neutralization reaction of the amine moieties of SBA-15-pr-NH2 and aqueous HAuCl4, and the spontaneous autoreduction of the adsorbed AuCl 4− resulted in the formation of Au0 nanoparticles on SBA-15pr-NH2. It is believed that spontaneous autoreduction of adsorbed AuCl 4− to Au0 took place through the 7


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Figure 7. Images of colonies formed by the Gram positive and Gram negative bacteria for (a) SBA-15 (control), (b) 25 μl, (c) 75 μl, (d) 150 μl, (e) 250 μl and (f) 300 μl of SBA-15-pr-+NH3.Au0 nano-hybrid material as aqueous suspension.

reduction of AuCl 4− by both free silanol and amine groups present in the pores and at the surface of the SBA-15pr-NH2 material. The bigger size of Au0 nanoparticles on the surface may be due to the fact that the preformed small Au0 nanoparticles catalyze the formation of other very small gold nanoparticles from the unadsorbed aqueous AuCl 4− ions and these deposited nanoparticles lead to bigger sized Au0 nanoparticles on the surface. However, in the case of the channels, the sizes of the Au0 nanoparticles cannot be bigger than the pore diameter of channels. 3.3. Bactericidal activity of SBA-15-pr-+NH3.Au0 nano-hybrid material The bactericidal activity of the SBA-15-pr-+NH3.Au0 nano-hybrid material was studied against two Gram positive bacteria viz. B. subtilis and S. aureus and two Gram negative bacteria viz. E. coli and P. aeruginosa on nutrient agar (NA) plates using variable concentrations of the SBA-15-pr-+NH3.Au0 nano-hybrid material. The number of bacterial colonies grown on the NA plates as a function of the concentration of SBA-15-pr-+NH3.Au0 nano-hybrid material when approximately 108 CFU mL−1 was applied to the NA plates is shown in figure 7. The correlation of the percentage of killed bacterial colonies with the variable concentration of the SBA-15-pr-+NH3. Au0 nano-hybrid material as an aqueous suspension is depicted in figure 8. It was observed that 100% killing of the bacterial colonies was found using 300 μl of SBA-15-pr-+NH3.Au0 nano-hybrid material in the case of B. subtilis while the same volume shows 99.44% colony killing in the case of S. aureus. However, in the case of both Gram negative bacteria almost the same colony killing percentages were found which are about 75.05% and 74.43% for E. coli and P. aeruginosa, respectively, using 300 μl of the SBA-15-pr-+NH3.Au0 nano-hybrid material. The bacterial colony killing percentage of SBA-15-pr-NH2 was determined for all bacterial strains using SBA-15 as negative control (table S1, ESI). The colony killing percentage of SBA-15-pr-NH2 for all bacterial strains viz. B. subtilis, S. aureus, E. coli and P. aeruginosa was found to be 28.97, 30.55, 12.33 and 13.55%, respectively. The observed data were very low as compared to the SBA-15-pr-+NH3.Au0 nano-hybrid material, indicating the main role of gold nanoparticles in the bactericidal activity. The colony killing percentage of SBA15-pr-NH2 was higher against Gram positive bacteria than Gram negative bacteria which proves that SBA-15pr-NH2 interacts well with the cell wall of Gram positive bacteria. 8


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Figure 8. Correlation of killing percentage for bacterial colonies with different concentrations of SBA-15-pr-+NH3.Au0 nano-hybrid material.

3.4. Tentative explanation for the higher bactericidal activity of SBA-15-pr-+NH3.Au0 nano-hybrid material against Gram positive bacteria The positively-charged SBA-15-pr-+NH3.Au0 nanohybrid material with a zeta potential of +40.2 mV has strong electrostatic interactions with the negatively charged components of the bacterial surface such as teichoic acid (Gram positive bacteria) and lipopolysaccharides (Gram negative bacteria). As we have found almost the same bactericidal activity for the SBA-15-pr-+NH3.Au0 nano-hybrid material against two Gram positive bacteria (approx.100%) and two Gram negative bacteria (approx. 75%), the electrostatic interaction between gold nanoparticles and the bacterial cell wall is the most probable reason for the selective bactericidal activity against Gram positive bacteria. Since the SBA-15-pr-+NH3.Au0 nanohybrid material is hydrophilic in nature, we used an aqueous suspension of this nanohybrid material in the bactericidal activity test. The higher activity of the positively-charged nano-hybrid toward Gram positive bacteria could be due to the higher negative charge imparted by teichoic acids compared to Gram negative bacteria.

4. Conclusion In this report, we have been able to demonstrate the spontaneous autoreduction of AuCl 4− ions to Au0 nanoparticles with SBA-15-pr-NH2 material without adding any external reducing agent. The gold nanoparticles are synthesized through an eco-friendly green methodology and exhibited excellent bactericidal activity. Using the same concentration of SBA-15-pr-+NH3.Au0 nano-hybrid material as an aqueous suspension we have found that the material exhibited better bactericidal activity against Gram positive bacteria (B. subtilis, S. aureus) than Gram negative bacteria (E. coli, P. aeruginosa). The leakage of sugars and proteins was also enhanced in the case of this nano-hybrid material compared to the control experiment. The effective bactericidal activities of the as-synthesized SBA-15-pr-+NH3.Au0 nano-hybrid material are encouraging for research of applications of this type of material as a drug carrier that can selective attack MDR bacteria in the foreseeable future. Moreover, the higher surface area and aqueous dispersibility of this nano-hybrid material can have potential applications in different areas such as catalysis, drug delivery and biosensing.

5. Acknowledgements The authors are grateful to the Director, CSIR-North East Institute of Science and Technology, Jorhat, Assam, India, for his kind permission to publish the work. Thanks also go to DST, New Delhi (DST Fast Track project GPP-0267 and DST-RFBR project GPP-0287) and non-network project MLP-6000(WP-1) for financial support. The authors are thankful to Gitashree Darabdhara and Dr M R Das for providing facilities for zeta potential measurements.

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