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Using the M13 Phage as a Biotemplate to Create Mesoporous Structures Decorated with Gold and Platinum Nanoparticles L. Irais Vera-Robles,*,† Jaqueline González-Gracida,† Armin Hernández-Gordillo,† and Antonio Campero‡ Departamento de Química, Á rea de Biofisicoquímica, Universidad Autónoma Metropolitana-Iztapalapa, San Rafael Atlixco No. 186, Col. Vicentina, 09340 México D.F., México ‡ Departamento de Química, Á rea de Inorgánica, Universidad Autónoma Metropolitana-Iztapalapa, San Rafael Atlixco No. 186, Col. Vicentina, 09340 México D.F., México †

Downloaded by UNIV OF PRINCE EDWARD ISLAND on August 23, 2015 | http://pubs.acs.org Publication Date (Web): August 14, 2015 | doi: 10.1021/acs.langmuir.5b01741

S Supporting Information *

ABSTRACT: By taking advantage of the physical and chemical properties of the M13 bacteriophage, we have used this virus to synthesize mesoporous silica structures. Major coat protein p8 was chemically modified by attaching thiol groups. As we show, the resulting thiolated phage can be used as a biotemplate able to direct the formation of mesoporous silica materials. Simultaneously, this thiol functionality acts as an anchor for binding metal ions, such as Au3+ and Pt4+, forming reactive M13−metal ionic complexes which evolve into metal nanoparticles (NPs) trapped in the mesoporous network. Interestingly, Au3+ ions are reduced to Au0 NPs by the protein residues without requiring an external reducing agent. Likewise, silica mesostructures decorated with Au and Pt NPs are prepared in a one-pot synthesis and characterized using different techniques. The obtained results allow us to propose a mechanism of formation. In addition, gold-containing mesoporous structures are tested for the reduction of 4nitrophenol (4-NP) and methylene blue (MB) in the presence of NaBH4. Although all of the gold-containing catalysts exhibit catalytic activity, those obtained with thiolated phages present a better performance than that obtained with M13 alone. This behavior is ascribed to the position of the Au NPs, which are partially embedded in the wall of the final mesostructures. disordered carbon frameworks.5 Liang et al. obtained ordered mesostructures of PtRu NPs using two types of structuredirecting molecules, namely, Brij56 and heptane.6 Interestingly, other types of biomolecules have emerged as new templates and scaffolds for creating mesoporous materials. In particular, viral particles represent great promise when they are used as templates due to their properties such as precise shapes and dimensions, chemical nature (which can be genetically controlled), and size monodispersity. Moreover, they also can be modified by chemical bioconjugation to incorporate new functionalities, either those found in nature or entirely synthetic. In addition, viruses are able of self-assembly and self-organize, thus becoming promising templates for creating new materials. Mann’s group exploited all of these viral properties as they pioneered the use of the rod-shaped tobacco mosaic virus (TMV) as a biotemplate to synthesize mesoporous silica structures.7 Another widely employed viral particle is bacteriophage (phage) M13. This is a filamentous virus, which is ∼6.5 nm in diameter and 880 nm in length. There are five types of proteins in the protein cage, with p8 being the major coating protein

1. INTRODUCTION Since the discovery of mesoporous (silica) systems, intensive research effort on this subject has taken place due to the abundance of possible applications centered in these materials, ranging from the field of adsorbents to catalysts and drugdelivery systems, just to mention a few examples. The size, shape, and morphology of the pores are a function of the template molecules, which are eliminated primarily by calcination at the end of the synthesis. In contrast, the chemical nature and surface properties of the mesoporous materials depend on the type of silicon precursor. Several strategies have been used in introducing new functionalities inside the mesopores; for instance, nanoparticles1 (NPs) and enzymes2 have been grafted to silica walls. In particular, many attempts have been made to introduce metal NPs inside the porous structure in view of their potential applications in catalysis; moreover, using the surface plasmon resonance (SPR) of the NPs it is possible to trigger different processes such as drug delivery3 or to mimic enzymatic reactions.4 Nevertheless, many of these functionalizations have been made following mesostructure synthesis, but in only a few cases functionalization has proceeded simultaneously with the metal−oxide framework progress. Baumann et al. attempted to introduce metal salts during the synthesis; however, the self-assembly of the surfactant molecules was disturbed by the salts, yielding © XXXX American Chemical Society

Received: May 13, 2015 Revised: August 3, 2015

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DOI: 10.1021/acs.langmuir.5b01741 Langmuir XXXX, XXX, XXX−XXX

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ratio) was added to 320 μL of the M13 phage, and then deionized water was incorporated into the suspension. Such a suspension was kept for 4 days at room temperature and held for 2 more days at ∼45 °C. The sample was centrifuged, washed three times with water, and dried at room temperature. Finally, the obtained powder was annealed at 600 °C for 4 h. 2.2. Synthesis of Mesoporous SHM13 Decorated with Metal NPs. This synthesis was the same as that used for M13 mesoporous network formation; however, thiolated phage was used instead (SHAM13 or SHPM13). The M13 phage−silicon precursor mixture started to react, and after ∼30 min a gel was clearly visible; at this point a solution of either gold or platinum precursor was incorporated (77 μL, 69.4 mM). Samples were designated as XY@SiO2 and XY@ SiO2C before and after calcination, respectively, and X means Au or Pt NPs and Y means the type of phage (M13, SHAM13, or SHPM13) that was employed in the synthesis. 2.3. Characterization. The phage purity and concentration were measured by UV−vis spectroscopy according to ref 15 by using a UV1800 Shimadzu spectrometer or a Nanodrop 2000c (Thermo Scientific) spectrophotometer. A JEOL 2010 transmission electron microscope operated at 200 kV was employed to investigate the particle size and the morphology of the mesoporous materials. X-ray diffraction (XRD) analyses at a normal angle were performed with Cu Kα (1.5406 Å) radiation using a Bruker D-8 Advance diffractometer. Small-angle X-ray scattering (SAXS) characterization was performed on a laboratory SAXS/WAXS beamline (Xeuss, Xenocs) equipped with a Cu Kα X-ray source (GeniX 3D Cu ULD, Xenocs) and a hybrid pixel detector (Pilatus 300 K, Dectris). The sample-to-detector distance was 1.28 m. Infrared spectra (IR) of the mesoporous structures were recorded on a PerkinElmer Spectrum GX 2000 FT-IR spectrometer. SPR measurements of the solid samples were recorded in a Cary 5 UV−vis−NIR spectrometer (Varian) with the help of a Praying Mantis diffuse reflectance accessory. 2.4. Catalytic Test. Before any catalytic experiments were run, samples were heated to 80 °C in vacuum for 2 h to eliminate any substance adsorbed within the pores or on the surface during storage (aging). The gold-containing catalyst was redissolved by sonication in water to a concentration of 1 mg/mL. Two compounds were selected for catalytic testing: 4-NP and MB. For the reduction of 4-NP, 0.5 mL of catalyst solution was added to a quartz cuvette previously filled with 1.5 mL of water and 30 μL of 4-NP (10 mM). Then, 0.5 mL of a fresh solution of NaBH4 (0.25 M) was added to the cuvette. For MB reduction, 0.1 mL of catalyst solution was added to 1.199 mL of water and 0.231 mL of a MB solution (0.13 mM), followed by the addition of 0.5 mL of NaBH4 (0.025 M). In both cases, the mixture was maintained with constant agitation (400 rpm) at 25 °C throughout the entire experiment. UV−vis spectra (Agilent 8453 Hewlett-Packard) were used to monitor the reaction by measuring the absorbance (at 400 nm for 4-NP and 664 nm for MB) at regular time intervals using the kinetic mode of the software (Agilent ChemStation). The reusability of the catalyst was tested for the reduction of 4-NP after each run. The catalyst was recovered by centrifugation, washed with water, and finally dried in vacuum at 80 °C for 2 h for subsequent reactions. The degradation (reduction) reaction with MB was carried out with two different samples, and each experiment was run in triplicate.

with ∼2700 units distributed along the body of the phage, encapsulating the single-stranded DNA genome.8 Either wildtype (wt) or genetically engineered capsid proteins have been used to produce nanomaterials. For instance, Avery et al. produced Rh, Pd, and Ru NPs on the surface of wt M13.9 Furthermore, Belcher’s group, using M13 libraries, found binding motifs that are able to nucleate and direct the growth of a great variety of nanomaterials, such as ZnS nanocrystals10 and Au NPs.11 In particular, the M13 phage has been genetically engineered to display the motif Glu-Glu-Glu (EEE) at the N-terminus of each protein p8, showing great versatility in biomineralizing distinct materials such as amorphous iron phosphate FePO412 and perovskite-type structures such as BaTiO313 and Pb[ZrxTi1−x]O3.14 Chung et al. showed that M13 viral particles are able to selfassemble in complex hierarchical liquid-crystalline phases. In fact, they found that a phage concentration of between 4 and 6 mg/mL resulted in a pseudohexagonal structure.15 Recently, Mao et al. used the fd phage (M13 phage variant) to synthesize mesoporous silica structures. They also used some genetically engineered phages to investigate the importance of the surface charge of the virion in the final mesoporous structure.16 Li et al. used the residues of wt protein p8 to screen their reactivity for further chemical modification.17 They showed that amine groups were able to react with other groups. In view of such results, Vera-Robles et al. used amine groups to introduce sulfhydryl groups (SH) on the phage surface, as the p8 protein has no cysteine residues in its sequence. Subsequently, SHcontaining M13 (thiolated phage, SHM13) was employed as a scaffold to nucleate and biomineralize Au, Pt, and Ag NPs, which were then assembled into electrodes and tested as electrocatalysts for methanol oxidation.18 Consequently, it is easy to acknowledge that the M13 phage represents a versatile viral particle that is able to nucleate and biomineralize inorganic materials. Herein, we report the preparation of mesoporous silica materials employing the M13 phage as a biotemplate. In this work, we manipulate protein p8’s amine groups by chemical bioconjugation with either N-succinimidyl S-acetylthioacetate (SATA) or Nsuccinimidyl S-acetylthiopropionate (SATP) in order to add thiol groups to the surface of the virion. Thus, using nonfunctionalized M13, we obtained mesoporous silica in the presence of a mixture of tetraethyl orthosilicate/(3aminopropyl)triethoxysilane (TEOS/APTES). Furthermore, employing thiol-functionalized M13, we were able to produce (for the first time to the best of our knowledge), in a one-pot synthesis, mesoporous structures decorated with gold and platinum NPs, without detriment to the self-assembly of the phage particles. Moreover, Au and Pt NPs were encrusted in the silica walls instead of being placed in the interior of the pore. This structure potentially offers advantages compared to traditional mesoporous structures, in which the NPs occupy the pore cavity and can impede the free flow of molecules through the mesoporous system. These gold-containing mesoporous structures were tested as catalysts in the reduction of 4nitrophenol (4-NP) and methylene blue (MB). Finally, this approach is a green process because the gold and platinum reduction was induced by the chemical groups on the structure of the proteins without any external reducing agent.

3. RESULTS AND DISCUSSION 3.1. Formation of Silica Mesostructures Biotemplated by an M13 Phage. M13 has been shown to be a viral particle able to self-assembly in different liquid-crystal structures as a function of concentration.15,19 Accordingly, we tested M13 concentrations between 0.5 and 6.0 mg/mL which, in the presence of a mixture of TEOS/APTES, allowed the precursor polymerization to form amorphous silica white powders. The progress of these reactions was observed by the naked eye: immediately after the TEOS/APTES mixture was added to the phage M13 suspension, it became turbid. A white gel was

2. EXPERIMENTAL SECTION 2.1. Synthesis of M13 Mesoporous Systems. In a typical synthesis, 120 μL of a fresh mixture of TEOS/APTES (10:1 molar B


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Figure 1. Characterization of mesoporous systems obtained with the M13 phage. (a) SAXS patterns of the materials before and after annealing at 600 °C. (b) Infrared spectra of as-synthesized and calcined materials. (c) Longitudinal and (d) cross-sectional TEM images of the calcined mesostructures.

Additionally, the bands appearing at 1655 and 1636 cm−1 are consistent with the vibrations belonging to the amide I group. The band at 1539 cm−1 can be ascribed to the bending vibrations of NH2 groups. Additionally, bands at 2852 and 2960 cm−1 are observed, which are typical of the symmetric and asymmetric vibrations of CH2 groups. All of these bands come from the viral capsid. After calcination, the spectra showed important differences; the signals from the M13 phage disappeared, indicating that it was effectively removed. Accordingly, bands corresponding to the silica framework are still present. It must be noted that the typical bands of silanol groups are absent. This absence could be due to the presence of multiple chemical groups from the phage, which can hinder the direct interaction between the silica network and water molecules. The mesoporous silica structures biotemplated by the M13 phage (M13@SiO2C) were confirmed by transmission electron microscopy (TEM). Figure 1c shows a typical image of the mesostructures, which consist of bundles several hundred nanometers in length having a fringe pattern of a lamellar structure that corresponds to the longitudinal view left by the phage (white stripes), whereas dark stripes are related to the siliceous wall. Additionally, the length of the bundles matches the phage length (∼1 μm), indicating a possible nematic liquidcrystal structure according to previous reports.16 The TEM image in Figure 1d displays a cross-sectional view of the mesostructure, where the pore arrangement is clearly in a hexagonal phase. From the TEM image, the size of the pore before annealing is difficult to measure due to the poor contrast in the proteinaceous−inorganic interface. For annealed samples the pores are clearly visualized, having a size of ∼4.5 nm and a wall thickness of ∼1.8 nm, which are similar to those obtained by Mao et al.16 The porous structure was analyzed by N2 adsorption−desorption measurements. From the BET and BJH methods a surface area of 160 m2/g and a size pore of 4.8 nm were obtained (Supporting Information Figure S1), in agreement with TEM observations.

formed after 30 min, and the mixture was left unperturbed for 4 days. Subsequently, the gel was immersed in a bath at 45 °C for 2 days to promote the complete hydrolysis−condensation of the precursors and then washed with water and dried at room temperature. Finally, the resulting white powder was calcined at 600 °C for 4 h. In this approach, we found that using an initial concentration of M13 phage of ∼4 mg/mL guaranteed a hexagonal liquid-crystal array15 which acts as a template for the formation of a silicon oxide mesoporous network. The formation of mesoporous structures was confirmed by small-angle X-ray spectroscopy (SAXS). Figure 1a shows the pattern of the sample before and after annealing. For the assynthesized mesophase, a peak at q = 0.74 nm−1 is observed, which corresponds to the planar distance of d100 = 8.5 nm, confirming that the phage (diameter ∼6.5 nm) acted as a biotemplate and that the Si−O framework was forming on its surface. After heat treatment, a sharper peak can be detected at q = 0.85 nm−1, indicating a reduction of the distance to d100 = 7.4 nm due to the removal of the biotemplate molecules and network shrinkage (which is typical of mesostructures). Additionally, the intensity of the peak increases as a result of the scattering contrast due to the empty pores. Two more small peaks are clearly observed at q = 1.52 and 1.75 nm−1 which correspond to (110) and (200) planes, respectively, which are characteristic of hexagonal phases. From this pattern, the parameters of the unit cell (a) and thickness of the wall (b) are calculated, giving values of a = 8.5 nm and b = 1.9 nm. These measurements were made as is reported in the literature20 (Supporting Information) Infrared spectra of the as-made and calcined mesostructures are shown in Figure 1b. For the former, the peaks observed at ∼797, and ∼1065 cm−1 correspond to the symmetric and asymmetric stretching bonds of Si−O−Si groups. The band at ∼956 cm−1 can be ascribed to the stretching of Si−OH; in some cases, this band has also been assigned to Si−O−R.21 The peak at 455 cm−1 is due to bending vibrations of the Si−O−Si group. All of these peaks are characteristic of silica structures. C


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Figure 2. TEM images of gold-containing mesoporous structures using SHAM13 (a and d), SHPM13 (b and e), and M13 (c and f) phages as biotemplates. The right images were taken without any special preparation, and the left images were trapped in resin and cut into thin slices to facilitate TEM visualization.

3.2. Effect of the Thiol Moiety in the Formation of Silica Mesostructures Decorated with NPs. Once the effectiveness of the M13 phage to act as a template was demonstrated, we investigated its ability to produce mesoporous silica materials decorated with Au and Pt NPs. Thiol groups (SH) are known to possess a strong affinity for metal ions such as Au, Ag, and Cd, among others. So, they can serve as nucleation sites in the metal NPs formation. With this in mind, we introduced the SH functionality using the most external NH2 groups of the p8 protein in the M13 phage by bioconjugation techniques.18 In order to insert the SH group, two molecules with different arm lengths were utilized: SATA and SATP, producing SHprotected groups, following a deacetylation reaction to produce the thiolated phages (SHAM13 and SHPM13, respectively). Both thiolated phages were tested to prepare in situ Au and Pt NPs inside the mesoporous framework. When the silicon precursors (TEOS/APTES) were added to SHAM13 or SHPM13 a gel was formed. Polymerization reactions were allowed to proceed to guarantee SiO2 network formation. In order to find the best moment for adding the metal precursors, these were supplemented at different stages: (i) when the framework was formed (1 or 2 days, respectively). In this way, the sample turned yellow (Au) or pallid orange (Pt), indicating that the ions have migrated inside the gel. However, at the end of the synthesis the ions were not homogeneously distributed as is shown in Figure S2 (Supporting Information). (ii) The addition of Au or Pt precursors immediately after gel formation provoked the material to turn yellow or orange, indicating that Au and Pt ions have migrated inside the gel to reach the outermost (accessible) SH groups without disrupting the liquid-crystal arrangement or hampering subsequent hydrol-

ysis−condensation reactions. Consequently, 1 day after the gel was held at 45 °C, it changed from yellow to violet in the case of Au3+ ions. At the end of the synthesis, the samples were homogeneously colored, indicating that the ions were reduced under these conditions without any reducing external agent; a similar effect was detected for the case of Pt ions. The progress of the reactions was recorded by taking images at different stages (Supporting Information Figure S3). In separate experiments, the different gold-containing gels were washed and dried at room temperature, producing powders that were resuspended in water. To these mixtures, aliquots of NaBH4 solution were supplied, and no change in the color was detected to the naked eye. For this reason, we assumed that the reduction of Au ions was complete and that NaBH4 was not necessary. We suggest that the Tyr (Y) and/or Phe (F) residues coming from the capsid, particularly from the p8 protein, should originate in the reduction of Au3+ ions. This assumption is supported by previous reports in which the authors have reported the reduction of Ag1+ and Au3+ ions by aromatic residues of proteins; even amine groups could reduce silver ions, according to the literature.22−24 3.3. Characterization of the Mesoporous Silica Materials Decorated with Au NPs. In order to investigate the effect of the formation of Au NPs inside the mesostructures using thiolated phages, characterization with SAXS, XRD, TEM, and UV−vis spectroscopy was conducted and compared with the mesoporous structures obtained with the nonfunctionalized M13 phage under the same conditions. The three as-prepared materialsAuM13@SiO2, AuSHAM13@ SiO2, and AuSHPM13@SiO2showed a red-violet coloration, indicating that the protein groups were most likely responsible for the reduction of Au3+ ions, as noted above. Therefore, the D


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Figure 3. X-ray diffraction patterns of the distinct gold-containing mesoporous silica materials: (a) as-prepared and (b) calcined.

Figure 4. Absorption spectra of the obtained materials using the distinct M13, SHAM13, and SHPM13 phages (a) before and after calcination at 600 °C for 4 h. (b) Development of the SPR of the Au NP-containing mesoporous structures as a function of temperature.

main function of the thiol groups is to anchor the Au3+ ions and serve as nucleation sites. After annealing, the samples turned red. Subsequently, the porous structure was investigated by SAXS, and diffraction patterns are shown in Supporting Information Figure S4. The resulting materials AuSHAM13@ SiO2C and AuSHPM13@SiO2C show peaks at q values of 0.89 and 0.91 nm−1, respectively, corresponding to interplanar distances (100) of 7.0 and 6.9 nm, respectively, which are smaller than that of AuM13@SiO2C with d100 = 7.4 nm. Additionally, we observed that the peak intensity diminished for AuSHAM13@SiO2C and AuSHPM13@SiO2C with respect to AuM13@SiO2C as a consequence of the effect of the partial filling of pores by Au NPs or, most likely, due to a less ordered structure.25 TEM images are shown in Figure 2. When thiolated phage M13 was used (either SHAM13 or SHPM13), the Au NPs were homogeneously distributed inside the structure and no agglomeration was observed. Au NPs were ∼5 nm in size when SHM13 was used, regardless of which thiol-containing molecule was used in the bioconjugation reaction (Figure 2a,b). On the contrary, when nonfunctionalized M13 phage was employed as a biotemplate, it resulted in larger Au NPs, which are located near of the edges of the mesoporous structure as shown Figure 2c. The yield of mesostructures and the distribution size were estimated from TEM images (Supporting Information Figure S5), producing around 70% of the mesostrucutres with lengths and widths of 600−1200 and 35−50 nm, 500−1500 and 30−60 for nonthiolated and thiolated phages, respectively. Interestingly, the images of Figure 2a,b reveal that the NPs reside primarily within the wall, thus releasing the pore canal. To gain insight into the position of Au NPs in the frameworks, the samples were trapped in a resin and cut with a microtome for further TEM analysis. As shown in Figure 2d,e, the hexagonal arrangement of the silica network left by the phage is evident; no important distortion of

the network due to the formation of Au NPs is obvious, confirming the SAXS results. It is also clear that particles are implanted in the silica wall of the framework, instead of the pores (Supporting Information Figure S6). Thus, when the SHM13 phage is used, Au NPs are observed to be embedded in the silica wall and the pores are free. It must noted that NPs are thicker than the silica wall; consequently, part of their surface is exposed to the void. In contrast, when M13 alone is utilized, Au NPs are larger and they are situated in the pore channel near the edge or on the surface’s framework (Figure 2c,f and Supporting Information Figure S6). This feature can be very advantageous for catalysis applications because the reactants could travel freely inside the pore, avoiding obstruction and the poisoning of the support. In addition, this characteristic should allow us to design doped-silica mesostructures with enhanced properties. As was noted above, all as-synthesized gold-containing mesoporous systems were violet, indicating the reduction of Au3+ ions to form the NPs. This assumption was verified by XRD analysis, and from the diffractogram shown in Figure 3a, we distinguished three peaks at 2θ = 38.1, 44.3, and 64.6°, corresponding to distances d111 = 2.36, d200 = 2.04, and d220 = 1.44 nm, respectively (typical of a face-centered-cubic gold lattice). After being annealed, the samples became red, and the diffraction pattern was measured again. No changes in the position of the peaks were detected, confirming that the red color is coming from the Au NPs (Figure 3b). Interestingly, the width of the peaks has similar values for gold-containing mesoporous structures before and after calcination, indicating that the nanoparticle size remained almost constant and sintering has been prevented. The SPR of the Au NPs inside the mesoporous structure was evaluated by UV−vis spectroscopy. Typically, for Au NPs in an aqueous medium, the SPR appears between 500 and 600 nm as a function of the size and shape of the particle and temperature E


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Langmuir treatment, among other parameters. Spectra of the as-prepared mesoporous systems are shown in Figure 4. For AuM13@SiO2 a peak with a maximum at approximately 540 nm is evident, whereas samples obtained using thiolated phages AuSHAM13@SiO2 and AuSHPM13@SiO2 present wider SPR bands shifted to higher wavelengths. In all cases, after heat treatment, the materials showed a blue shift of the SPR band and more defined peaks. The maximum absorption is practically the same for materials biotemplated with the thiolated phage (∼521 nm), and a slight shift (∼4 nm) is detected for the mesoporous structure obtained by nonfunctionalized M13. Particle size is a very well known parameter that affects the position of the SPR. For Au NPs dispersed in water, a displacement to longer wavelengths is observed with an increase in size; however, our particles are trapped in a silica matrix instead, with a refractive index higher than that of water (nH2O = 1.33, nSiO2 = 1.45), which might explain the observed red shift for the as-prepared materials. From the XRD results, we noticed that the size did not change significantly with the temperature; thus, the blue shift is likely due to gold-matrix interactions. In the as-prepared materials, the Au NPs are enclosed by amine acid residues, which must influence the peak position of the SPR. Additionally, in thiolated phage the SH groups have a strong affinity for Au NPs, changing the medium surrounding the NPs; this result most likely explains the larger red shift. In fact, Feng et al.26 observed a similar effect. They synthesized Au nanorods (NRs) where the longitudinal SPR peak appeared at 752 nm. Then, they covered the Au NRs with a silica shell, and the SPR peak was shifted to 762 nm; a red shift was also reported for Au NPs embedded in polystyrene (n = 1.58).27 On the other hand, it is well known that heat treatment is responsible for the voiding of the pores with the concomitant elimination of the phage. For that reason, the SPR was evaluated for a sample treated at different temperatures. Interestingly, when the sample was held at 300 °C for 1 h, the SPR band was evident at approximately 558 nm; when the temperature was increased to 400 °C, a blue-shifted peak at 521 nm was observed. Higher temperatures or longer time did not produce any significant change in the intensity, width, or displacement of the band (Figure 4b). On the basis of thermogravimetric analysis (Supporting Information Figure S7), the main weight loss occurs at approximately 300 °C due to the decomposition of the viral capsid. Therefore, at such a temperature the environment that covers the Au NPs changes drastically from organic to inorganic. Moreover, when the temperature was increased a new pore (hole)−gold interface was created, accompanied by a decrease in the refractive index of the silica mesostructure. Thus, the blue shift of the SPR band could be explained by the changing interactions among the Au NPs, the phage, and the silica. To clarify this behavior, more experiments are in progress and will be the subject of a future report. 3.4. Mechanism of Formation of the Mesostructures Biotemplated by an M13 Phage. Taking all of this information together (a mechanism is proposed and depicted in Scheme 1), we suggest that thiolated phage particles are selfassembled in hexagonal phases when the TEOS/APTES mixture is added. Therefore, silicate species are adsorbed on the phage surface and begin to form an inorganic phase (gel “state”). At this stage, Au3+ ions are supplied and they pass through the gel (incipient framework), binding to the thiol

Scheme 1. Proposed Assembly of the M13 Phage in a Hexagonal Liquid-Crystal Phase, Followed by Silica Network Formationa

a

Arrows pointing right represent the formation of Au NPs on the surface of mesoporous silica using M13. The arrow pointing down represents the formation of Au NPs within the walls of the mesoporous silica using SHM13.

groups of protein p8, which reside in the boundary between the inorganic and proteinaceous phases. It should be noted that neither is the hexagonal phase disturbed nor are the hydrolysis−condensation reactions hampered by the Au3+ ion addition. During temperature treatment at 45 °C, the hydrolysis−condensation becomes complete and Au3+ ions are reduced to Au0 particles by the aromatic (F and T) and amine groups of the surrounding proteins; in fact, these groups are able to tolerate reactions17 because they are in/or next to the external domain.28 This assumption that protein groups are able to reduce Au3+ to Au0 is supported by similar results found by other authors.29 After annealing, the phage is removed, which is accompanied by silica shrinkage. Au NPs with a narrow size distribution are mainly encrusted in the silica wall instead of the pore, most likely due to the SH anchor effect and lattice shrinkage. Conversely, for a nonfunctionalized M13− silica framework, when Au3+ ions are added, they are not able to bind directly to the phage due to the lack of strong binder and stay adsorbed primarily on the external wall of silica without affecting the hexagonal phase arrangement. Thus, after phage elimination by heat, the resulting Au NPs are larger than those obtained with a thiolated phage and are found on the edges of the pores and the surface of the mesostructure. Hence, metal particles have no effect on the size of the porous canal or the width of the wall. This mechanism agrees with the SAXS results; the lattice parameter is practically the same for M13@ SiO2C and AuM13@SiO2C, whereas both AuSHAM13@ SiO2C and AuSHPM13@SiO2C feature a reduction in the lattice parameter. Notably, from XRD we observe that annealing has no important effect on the size and/or agglomeration of the particles when they grow anchored to an SH moiety; however, some aggregation is observed in the absence of the SH moiety as shown in Figure S6 (Supporting Information). F


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Figure 5. (a) SAXS patterns of platinum-containing mesostructures with different phages. The inset shows the peaks for thiolated phages (dotted line). (b) X-ray patterns of the final mesoporous materials decorated with Pt NPs. TEM images of (c) PtSHAM13@SiO2C and (d) PtSHPM13@ SiO2C mesoporous frameworks.

Figure 6. UV−vis absorbance spectra of the reduction of (a) 4-NP and (c) MB using catalysts AuSHPM13@SiO2C and AuSHAM13@SiO2C, respectively. Plot of ln(Ct/C0) versus reaction time using the three calcined catalysts for (b) 4-NP and (d) MB.

In fact, Setyawati et al. have recently shown that the M13 phage can adsorb and reduce gold ions in the presence of water alone, producing Au NPs with an SPR peak at 558 nm.29 However, the NPs obtained by them were very irregular in shape and size. In our present study, we also demonstrate that a thiolated phage can (1) act as a biotemplate to produce

mesoporous silica networks and (2) serve as a scaffold to anchor, nucleate, and grow Au NPs with controlled size. To prove that this method can be extended to the synthesis of mesoporous structures decorated with different metal NPs, we tried with a platinum precursor instead of a gold precursor. Therefore, mesoporous structures decorated with Pt NPs were produced using SHAM13, SHPM13, and M13 as biotemplates. G


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details in the Supporting Information). In the absence of a gold-containing catalyst no reduction takes place, at least at observable rates (no color change was observed). From these results it is apparent that gold-containing catalysts derived from thiolated phage have better performance and are able to reduce MB in a few minutes. Similar results were obtained in the reduction of 4-NP; however, in that case catalyst AuSHPM13@ SiO2C was slightly better than AuSHAM13@SiO2C. This behavior could be explained by the different size of 4-NP with respect to that of MB and the distinct mechanisms of reduction. In fact, Sahiner et al. found k values of 0.43 and 0.68 min−1 for the reduction of 4-NP and MB, respectively, although the catalyst they used was Co or Cu NPs supported in poly(tannic acid) microgels.31 Catalyst activity is affected by several parameters such as temperature, aging, pH, water quality, and oxidation state, among others. To investigate the influence of the oxidation state of gold, XPS experiments were performed. The survey spectra of AuM13@SiO2C and AuSHPM13@SiO2C catalysts are shown in Figure S8 of the Supporting Information. Peaks from Si, O, and Au are observed, as well as a C signal coming from carbonaceous species adsorbed on the surface as contaminants. A closer inspection shows a doublet corresponding to Au 4f5/2 and 4f 7/2 with maxima at 84.54 and 88.13 eV (binding energy, BE) (Figure S8). These values are shifted to higher BE with respect to metallic gold foil, indicating that gold is present as the Au0 species on the nanometer level, as reported by other authors.32,33 Specifically, this value has been found when Au NPs are supported in oxides such as SiO2, CeO2, and TiO2,34 confirming, in this way, the existence of Au0 NPs as indicated by our XRD and plasmon surface results. It must be noted that both catalysts, i.e., prepared using thiolated and nonthiolated phages, produced very similar curves, indicating that the oxidation state of gold is quite similar; this in turn implies that the increase in catalytic activity is probably due to the position of the NPs inside the structure and not to the presence of different gold species.

SAXS patterns are shown in Figure 5a; when the M13 phage is utilized, mesoporous structures are produced, with a welldefined peak at q = 0.87 nm−1 which corresponds to d100 = 7.2 nm, in agreement with the mesostructures alone and decorated with Au NPs. However, sample PtSHAM13@SiO2C coming from a thiolated phage shows a diminished peak at a value of q = 0.86 nm−1, and the same effect is observed for PtSHPM13@ SiO2C, corresponding to d100 = 7.3 nm. This value is slightly higher than those obtained with Au NPs. This finding confirms that Pt ions are attached to the surface of the thiolated phage and that Pt NPs have grown inside of the mesoporous framework. In order to confirm the crystallinity of Pt NPs, XRD experiments were conducted. Figure 5b displays peaks appearing at 2θ = 39.8, 46.2, and 67.5° corresponding to planes (111), (200), and (220) of the face-centered cubic of the platinum lattice. The formation of ordered phases decorated with Pt NPs was verified by TEM analysis. Typical images of PtSHAM13@SiO2C and PtSHPM13@SiO2C are observed in Figure 5c,d, respectively. TEM pictures show bundles of rodshaped particles, where a strip pattern that corresponds to a silica wall−pore framework is clearly observed. Pt NPs are relatively monodisperse and homogeneously distributed along the sample. 3.5. Application of the Catalysts in the Reduction Reactions. As discussed previously, sintering and agglomeration of Au NPs have been prevented using the thiolated phage as a biotemplate, most likely as a consequence of the NPs trapped inside the silica wall. Catalytic applications can take advantage of this characteristic. Two reduction reactions were studied: the reduction of 4-NP and MB. The progress of both reactions with respect to time can be evaluated by UV−vis spectroscopy. 4-NP presents an absorption maximum at 317 nm, but this peak is displaced to 400 nm in the presence of NaBH4 (alkaline medium). When catalyst AuSHPM13@SiO2C is added, the peak at 400 nm decreases and a new peak at 315 nm appears. As the reaction advances, the band at 400 nm diminishes still further with a concomitant increase of the band at 315 nm; this behavior is due to the initial formation and subsequent increment of the 4-aminophenol (4-AP) amount, as main product in the 4-NP reduction (Figure 6a). Because the reaction occurs in excess NaBH4, the concentration of BH41− can be considered to be constant, so the data can be fitted to a pseudo-first-order reaction with regard to the amount of 4-NP. The concentrations of 4-NP at times t (Ct) and 0 (C0) can be measured from the relative values of the absorbance At and A0 at 400 nm. A linear relationship between ln(Ct/C0) versus the reaction time is observed. From the slope of Figure 6b, the observed rate constant is calculated to be k = 0.026 ± 0.010 s−1 for AuSHPM13@SiO2C, confirming that this material has good catalytic activity.30 We also tested the catalytic performance of the other gold-containing catalysts and carried out control reactions in which original mesoporous silica M13@SiO2C, with and without the support, was tested as a catalyst. While the reaction for AuSHPM13@SiO2C was relatively rapid (a few minutes), for AuSHAM13@SiO2C and AuM13@SiO2C the reaction was slightly slower, resulting in k values of 0.019 ± 0.008 and 0.004 ± 0.002 s−1, respectively, as shown in Figure 6b. No evident changes are observed for control reactions, demonstrating that Au NPs are responsible for the catalytic activity. The three catalysts were tested in the reduction of MB, and their k values were calculated to be 0.093 ± 0.013, 0.035 ± 0.006, and 0.007 ± 0.003 s−1 for AuSHAM13@SiO2C, AuSHPM13@SiO2C, and AuM13@SiO2C, respectively (more

4. CONCLUSIONS In this work, we show that the M13 phage can be used as a versatile biotemplate to create regular mesoporous structures in the presence of silicon precursors. Moreover, it is conceivable that the great chemical diversity of the phage can be exploited to attach different chemical groups, besides our SH groups, that can still allow self-assembly and silica framework formation. In fact, several works have described the M13 phage as a multipurpose platform for performing organic synthesis and combinatorial chemistry on the nanoscale.35,36 Thus, the thiolated phage can be successfully employed to produce mesophases decorated with metal NPs. Au and Pt NPs were located partially within the silica wall, leaving the pore free and becoming a rather promising catalyst. More importantly, these NPs can be considered to be dopant agents that can modify the properties of the silica wall. The AuSHPM13@SiO 2C presented good catalytic performance in the degradation of 4NP and MB. Both thiolated-phage-derived materials had better catalytic activities than that of AuM13@SiO2C, supporting the proposal that Au NPs inside the mesoporous systems improve the activity. In addition, the catalyst can be easily recovered while retaining their catalytic activity. This method seems to be easily extended to other metals, quantum dots, and core−shellfunctionalized mesoporous structures. Furthermore, this bioconjugation approach could be substituted by genetic H


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Langmuir

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engineering routes to use M13 mutants containing targetspecific binding peptides and produce new, interesting, and convenient technological mesoporous materials.

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ASSOCIATED CONTENT

* Supporting Information S


The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b01741. Bioconjugation reaction, thiol quantification, images of the development of the metal-containing mesoporous silica materials, N2 adsorption−desorption data, thermogravimetric analysis, catalysis and XPS setup and results, and additional TEM images (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

The manuscript was written through the contributions of all authors. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS J.G.-G. thanks SEP-PROMEP from Mexico for a scholarship. This work was partially supported by SEP-PROMEP “Apoyo a la Incorporación de Profesores de Tiempo Completo” (grant 12411858 SEP-Promep). We thank Dr. Roberto Olayo-Valles for help with SAXS characterization at the CBI-UAMI SAXS facility. We thank Laboratorio Central de Microscopıá Electrónica UAM-I for TEM images, Laboratorio de Difracción de Rayos-X (T-128) UAM-I for XRD measurements, and Laboratorio Central de Biologı ́a Molecular and Laboratorio de Docencia Quı ́mica for allowing us to use their equipment. Dr. Marcos Esparza-Schulz is acknowledged for the N2 adsortion− desorption experiments. We are grateful to Dr. Luis Lartundo for XPS experiments. We especially thank Dr. Andres Hernández-Arana and Dr. J. L. Hernández-Pozos for helpful discussions during the preparation of this article.

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REFERENCES

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