Article pubs.acs.org/cm
ZnO Binding Peptides: Smart Versatile Tools for Controlled Modification of ZnO Growth Mechanism and Morphology Marion J. Limo, Rajesh Ramasamy, and Carole C. Perry* Biomolecular and Materials Interface Research Group, Interdisciplinary Biomedical Research Centre, School of Science and Technology, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, U.K. S Supporting Information *
ABSTRACT: Material binding peptides are proving to have great potential in improving material synthesis and advancing device fabrication; however, their specificity and interaction mechanisms with target surfaces remain largely elusive. This study contributes to the developing understanding of fundamental principles through which ZnO binding peptides (ZnO-BPs) interact with and modify ZnO growth/morphology. The ZnO-BPs used were the reported phage display (PD) identified sequence (G-12 (GLHVMHKVAPPR) and its derivative, GT-16 (GLHVMHKVAPPR-GGGC)) as well as novel sequences generated from postselection modifications including alanine mutants of G-12 (G-12A6, G-12A11, and G-12A12) chosen on the basis of peptide stability calculated in silico. ZnO growth was monitored in the absence and presence of ZnO-BPs during solution synthesis using two different growth routes: the Zn(NO3)2·6H2O−HMTA system and the Zn(CH3COO)2−NH3 system. The outcomes of the ZnO synthesis studies demonstrate that a single ZnO-BP can utilize different sequence and concentration dependent mechanisms to control ZnO growth and generate different morphologies. The specific synthesis system used dictated the species present in solution and the solid phases formed, some of which ZnO-BPs could interact with and consequently modify ZnO growth and resultant morphologies. The role of histidine within ZnO-BPs in interaction with ZnO and stabilization of LBZs is also demonstrated.
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INTRODUCTION
matics and computational modeling tools to identify inorganic binding proteins/peptides.7−10 The development of ZnO, specifically for optoelectronic and electrical device application, relies on control over its properties such as purity, size, structure, morphology, and chemical composition.11−13 A number of studies have reported on PD identified peptide sequences that bind specifically to ZnO, and some have successfully been used to modify its morphology.14−21 EM-12 (EAHVMHKVAPRP) was among the first ZnO binding peptides (ZnO-BPs) to be identified using PD.14 EM-12 has been shown to suppress crystal growth of ZnO in the (0001) direction.18 More interestingly, growth of ZnO along the (0001) direction was suppressed much more in the presence of dipeptides from the EM-12 sequence, i.e., M5H6 and H6K7 in comparison to the entire EM-12 sequence. Its derivative, EM-12 fused to a GGGSC tag at its C-terminus, was shown to lead to the formation of flower-like ZnO microparticles when incorporated in ZnO synthesis.14 EM-12 fused to a collagen triple helix structure has also been used to template the growth of monodisperse single crystalline ZnO nanowires.15 Tomczak and co-workers isolated another ZnOBP, G-12 (GLHVMHKVAPPR), that has 67% homology to the
Over the past decade, complex materials with specific composition, size, shape, and hierarchical organization have been synthesized as a direct result of the exploration and application of bioinspired approaches.1,2 Biomimetic bottom-up synthesis strategies are being employed with the desire of achieving materials with covetable properties such as those made by living organisms. Proteins undoubtedly play a significant role in the formation of biological structures and in controlling the assembly of biominerals.3 Their unique properties of recognition, specificity, self-assembly, and biofabrication have captured scientists’ research interests validating their inclusion in synthesis, function, and/or assembly of inorganic materials.3,4 Although there have been improvements in extraction and identification techniques for proteins involved in biomineralization processes,5 combinatorial biology techniques such as cell-surface display (CSD) and phage display (PD) have become the preferred alternative strategy for faster identification of peptide sequences that can bind specifically to a broader spectrum of commercially relevant materials including metals and metal oxides.3,6 Nevertheless, because combinatorial biology protocols have some disadvantages and biases associated with their use, multiple parallel screening techniques need to be used in combination with newer rational and/or random approaches such as bioinfor© 2015 American Chemical Society
Received: September 24, 2014 Revised: March 3, 2015 Published: March 5, 2015 1950
DOI: 10.1021/acs.chemmater.5b00419 Chem. Mater. 2015, 27, 1950−1960
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Chemistry of Materials EM-12 peptide.16 When incorporated in the synthesis of ZnO, both G-12 and its derivative GT-16 (GLHVMHKVAPPRGGGC) were shown to modify the morphology of ZnO via a peptide adsorption−growth inhibition mechanism.16,21 G-12 was shown to inhibit the anisotropic growth of ZnO crystals by adsorbing nonspecifically to both the (0001) and (101̅0) planes decreasing the growth of the crystals along both the a- and caxes compared to higher aspect ratio twinned ZnO rods formed without additive.21 GT-16 was shown to adsorb to both (0001) and (101̅0) planes of ZnO; however, its sequence inherent properties predisposed it to preferentially adsorb to the (0001) plane causing a greater growth inhibition effect along the c-axis leading to the formation of low aspect ratio twinned ZnO platelets.21 Molecular dynamics was used to predict possible binding moieties of G-12 and GT-16 to ZnO planes; however, a challenge remained to establish a detailed mechanism of interaction at the molecular level.21 The plane selectivity of GT16 to the (0001) plane of ZnO (which has the highest density of zinc atoms compared to other planes of ZnO) may have been facilitated by the flexible spacer in GT-16 between histidine and cysteine, similar to the motif in zinc finger nucleases.21,22 In the preceding studies using EM-12 and G-12, different ZnO solution synthesis methods were used which employed different growth conditions and precursors precluding a direct comparison to determine the link or the dominating factor (to control ZnO growth and morphology) between the reaction system and the ZnO-BP used. Additionally, further studies are needed to understand the nature of complex phenomena governing peptide−inorganic interfacial interactions, specifically, elucidation of the roles of individual amino acid functionalities in inorganic binding peptide sequences, the significance of sequence order, and the mechanisms through which peptides interact with inorganic surfaces leading to morphology modification. In a recent contribution, an in-depth study of ZnO growth mechanisms was reported23 using two known hydrothermal growth routes: (i) synthesis using zinc nitrate hexahydrate (Zn(NO3)2·6H2O) and hexamethylenetetramine (HMTA),16 henceforth referred to as the Zn(NO3)2·6H2O−HMTA system, and (ii) synthesis using zinc acetate (Zn(CH3COO)2) and ammonia solution (NH3),16 hereafter referred to as the ZnAc2−NH3 system. Both systems support the anisotropic growth of elongated wurtzite structured twinned hexagonal rods without additives.16,24 ZnO growth was demonstrated to occur through multistep pathways involving the formation of intermediates, layered basic zinc salts (LBZs).23 The growth conditions were shown to dictate the products formed during the syntheses and influenced the progression of solid phase transformation of intermediates to ZnO.23 In this contribution a detailed investigation is described comparing ZnO formation using the two aforementioned growth systems in the presence of G-12 peptide and its mutant peptides generated using computational alanine scanning mutagenesis and selected on the basis of in silico determined peptide stability. ZnO-BPs are presented as smart, versatile tools able to control ZnO formation processes, operating through different mechanisms that are governed by the intrinsic properties of the peptide but also influenced by the synthesis system used.
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acquired from Novabiochem. Piperazine, diisopropyl ethylamine (DIEA), thioanisole (TIS, C7H8S), trifluoroacetic acid (TFA, C2HF3O2), and 3,6-dioxa-1,8-octanedithiol (DODT, C6H14O2S2) were all acquired from Sigma-Aldrich. Zinc nitrate hexahydrate (Zn(NO3)2·6H2O), zinc acetate (Zn(CH3COO)2), and 1,3-hexamethylenetetramine (HMTA, C6H12N4) were purchased from SigmaAldrich. Concentrated ammonia solution (35%) was purchased from Fisher Scientific. All reagents were used in their purchased form. Where required, distilled−deionized water (ddH2O) having conductivity measurement of less than 1 μS cm−1 at 25 °C was used. Computational Alanine Scanning Mutagenesis and Molecular Dynamics. The roles of amino acid functional groups in an inorganic binding peptide sequence can be elucidated using computational alanine scanning mutagenesis coupled to experimental alanine mutagenesis.25 The software Tripos SYBYL 8.0 (Tripos, St. Louis, MO, USA)26 was used to build G-12 peptide in a random conformation and its mutant sequences generated by single substitution of an alanine amino acid into each position in the original sequence. In addition to G-12 sequence, 11 new sequences were generated (all positions substituted except A9 in G-12). Molecular dynamics (MD) was used to monitor the conformation and stability of each peptide sequence at 300 K over a total simulation period of 2.0 ns with a time step of 2 fs. The initial structures of the original peptide and each of the mutants (all random conformation) were built 11 times, and energy minimization was repeated 11 times both in vacuum and after solvation in water. The simple point charge (SPC) water model (for solvated analyses) and the force field AMBER were used to simulate and minimize G-12 and its alanine mutant sequences. Microwave Assisted Solid Phase Peptide Synthesis and Peptide Characterization. Peptides were synthesized Fmoc chemistry using a Liberty1 single channel automated peptide synthesizer (CEM Corp.) as previously described, details in section S1 of the Supporting Information (SI). Peptide purity of >80% for each sequence was ascertained using a Dionex reverse phase high performance liquid chromatography (RP-HPLC) instrument, and the Mr values of the peptides were determined using a Bruker Ultraflex III matrix-assisted laser desorption/ionization time-of-flight (MALDITOF) mass spectrometer (Table S1a and Figure S1a−g of the SI). The physicochemical properties of the synthesized peptides and the amino acids in the sequences were determined using the Innovagen online peptide property calculator and are also shown in Table S1b of the SI.27 Synthesis of ZnO with Peptides: Zn(NO3)2·6H2O−HMTA and ZnAc2−NH3 Systems. The two hydrothermal ZnO synthesis methods used in this study have previously been described.16,21,23,24 Here, G-12 peptide and its mutants selected after computational studies on peptide stability were incorporated as additives. In syntheses following the Zn(NO3)2·6H2O−HMTA system, stock solutions were first prepared: 104.17 mM Zn(NO3)2·6H2O, 100 mM HMTA, and 30 mM of each peptide. The reactions were carried out by mixing 9.6 mL of Zn(NO3)2·6H2O with 10 mL of HMTA and 0.4 mL of ddH2O/peptides (to achieve final concentrations of 0.1, 0.3, and 0.5 mM) in a glass vial while stirring using a magnetic stirrer. The reactions were incubated at 20 °C for 24 h (±2 min) after which they were transferred to a water bath set at a temperature of 65 °C for an additional 48 h, 72 h in total. In syntheses following the ZnAc2−NH3 system, stock solutions were also prepared: 30 mM Zn(CH3COO)2, 31.25 mM NH3 solution, and 30 mM of the selected peptides. For precise preparation of the NH3 stock solution concentration, it was made just prior to syntheses from a 5% NH3 solution whose exact concentration was ascertained by carrying out back-titrations. For all reactions, 0.4 mL of ddH2O (control) or peptide (to the desired concentration: 0.1, 0.3, and 0.5 mM) and 9.6 mL of 31.25 mM NH3 were stirred into 10 mL volumes of 30 mM Zn(CH3COO)2 heated to 50 °C in glass vials. The mixtures were retained in a 50 °C water bath up to 72 h. The difference in concentration of precursor and base used between the two systems (50 mM for the Zn(NO3)2·6H2O−HMTA system and 15 mM for the ZnAc2−NH3 system) was necessary for each reaction to be carried out at circumneutral pH where elongated
EXPERIMENTAL SECTION
Materials. Fmoc-protected amino acids and 2-(1H-benzotriazole1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) were purchased from CEM Corp. Amino acid preloaded Wang resins were 1951
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Chemistry of Materials ZnO rods are formed without additives.16,21,23,24 Each reaction was carried out in triplicate, and the pH values of the solutions were monitored throughout the reactions. Aliquots containing some of the formed solid materials were collected at chosen time points (±2 min). The supernatants were separated from precipitates after centrifugation at 13000 rpm for 3 min. The supernatant was again recentrifuged, and the precipitates collected were washed three times in ddH2O and then lyophilized at −70 °C using a Virtis-110 freeze-dryer. Kinetic Studies from Supernatants and Characterization of Precipitates. The supernatant was prepared for inductively coupled plasma atomic emission spectroscopy (ICP-OES) analysis to determine the concentration of zinc ions, [Zn2+], using a PerkinElmer Optima 2100DV. Precipitates were characterized using several techniques. A JEOL JSM-840A scanning electron microscope was used to analyze the morphology of precipitates sputter coated with gold using an Edwards sputter coater S150B. From scanning electron microscopy (SEM) images, the aspect ratio (length/diameter, L/D) of crystals (n = ≥ 50) was measured using ImageJ software (a Java-based program). The SEM was coupled to an energy dispersive X-ray (EDX) system with a light element detector which was used for elemental analysis of carbon coated precipitates. The crystallinity of precipitates was characterized using a PANalytical X’Pert PRO X-ray diffractometer having Cu Kα radiation operating at a wavelength of 1.54056 Å. The samples were scanned over a range between 3° and 90° of 2θ with a step size of 0.02° s−1. Experiments were carried out at room temperature with an acceleration voltage of 45 kV and 40 mA filament current. The X’Pert-HighScore Plus (Version 2.0a) program was used to analyze and identify peaks in the diffraction patterns. FTIR-KBr analysis was carried out using a Nicolet Magna IR-750 instrument for the identification of functional groups present in different samples: reagents, peptides, and precipitates formed during syntheses. Samples were scanned 500 times at a resolution of 2 cm−1 and wavenumber range of 400−4000 cm−1. TGA (Mettler Toledo TGA/SDTA 851e with STARe software for data analysis) was used to determine the peptide, intermediate (LBZs), and inorganic (ZnO) content of samples. To decompose all organic matter, samples were heated at 30 °C for 10 min and then the temperature was raised to 900 °C at a steady rate of 10 °C/min and held constant at 900 °C for a 10 min period. The optical band gap of the precipitates was studied using a JASCO V-670 spectrophotometer with an integrating sphere. UV−vis absorbance of precipitates was measured over a wavelength range of 200−800 nm and converted to diffuse reflectance spectra.
investigation and comparison of the computational outcomes to experimental findings. Peptide Directed ZnO Morphology Modification: G12, G-12A6, G-12A11, and G-12A12. ZnO synthesis was carried out in the absence of peptide (control) and in the presence of 0.3 mM (the concentration reported to lead to aspect ratio reduction of ZnO rods16,21) G-12 and each of the selected mutants: G-12A6, G-12A11, and G-12A12. As the reactions progressed, precipitates and supernatants were collected for analysis at chosen time points. The microstructures of precipitates were analyzed using SEM (Figure 1).
Figure 1. SEM micrographs of 24 and 48 h precipitates synthesized using the Zn(NO3)2·6H2O−HMTA system in the absence of peptide (control) and in the presence of 0.3 mM of G-12 and G-12A6. Scale bars are 5 μm except insets: (c) 30 μm and (e) 1 μm.
During the nucleation period (24 h = t20°C), in the control reaction, a gel-like precipitate was formed which in SEM appeared as continuous sheets with rough surfaces (Figure 1a).21,23 This intermediate compound was confirmed using FTIR and XRD analysis to be a layered basic zinc nitrate (LBZN), Zn5(OH)8(NO3)2·2H2O.23 When synthesis was carried out in the presence of G-12 and its alanine mutants, reaction solutions remained clear during the nucleation period. However, SEM analysis of dried aliquots revealed the presence of precipitates with morphology such as the LBZN observed in the control reaction (Figure 1b) with the exception of the synthesis reaction with G-12A6 (Figure 1c) where unique variably sized (10−50 μm) microspheres with rough porous surfaces were observed. Some of the microspheres appeared to be fused together, and others were broken apart (Figure S3 of the SI). When the reactions were transferred to a water bath set at a higher temperature, 65 °C, all solutions became cloudy within an hour and solid white precipitates were formed. Without peptide, anisotropic growth (along the c-axis) of crystals took place; thus 48 h precipitates (24 h, t20°C + 24 h, t65°C) consisted of elongated twinned hexagonal rods with an aspect ratio of 8.92 ± 3.26 (Figure 1d) confirmed to be wurtzite structured ZnO (XRD; JCPDS card No. 36-1451).21,23 As expected, short twinned hexagonal rods with a lower aspect ratio of 2.16 ± 0.86 were formed in the presence of G-12 (Figure 1e). Similar short twinned rods were observed in 48 h precipitates synthesized in the presence of G-12A11 and G12A12 with aspect ratios of 1.89 ± 0.77 and 2.25 ± 0.72, respectively (Figure S4 of the SI). Distinctively different, 48 h precipitates synthesized with G-12A6 contained microspheres with porous rough surfaces, some of which had hexagonal rods attached to their surface (Figure 1f). Short hexagonal rods (L/ D = 2.05 ± 0.82) that were not bound to the microspheres
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RESULTS AND DISCUSSION In Silico Conformational Stability Studies: G-12 Peptide and Its Alanine Mutants. In silico conformational stability studies were used as a simple way to identify which peptides should be used for the interaction studies. The average conformational energy of G-12 and its mutants were computed after several rounds of energy minimization in solvated and nonsolvated states using MD (Table S2 and Figure S2 of the SI). In accordance with the literature, sequences with the lowest values of conformational energy are thought to be more stable than those with higher energy values and are also thought to be the most plausible inorganic binding configurations.28,29 In vacuum, the most stable sequences with the lowest conformational energies were G-12A10 (GLHVMHKVAAPR) followed by G-12A6 (GLHVMAKVAPPR) and then G-12A3 (GLAVMHKVAPPR) while in water the most stable sequences were G-12A11 (GLHVMHKVAPAR) followed by G-12A6 and then G-12A5 (GLHVAHKVAPPR). Scoring the overall stability of the sequences in both solvated and nonsolvated states indicated that G-12A6 and G-12A11 may have been the most stable sequences whereas the G-12A12 (GLHVMHKVAPPA) sequence may have been the most unstable. Based on these findings, G-12A6, G-12A11, and G12A12 were selected together with G-12 peptide for further 1952
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Chemistry of Materials were also present. Like the ZnO rods formed, some microspheres appeared to be twinned, which is a common feature in ZnO structures, thought to reduce the surface energy of crystals, lowering the ΔG of the system.30,31 EDX analysis confirmed the presence of atomic zinc and oxygen in both the rods and the microspheres (Figure S5 of the SI). Intrigued that a single amino acid substitution of H6 in the sequence of G-12 with A6 had resulted in a great modification in ZnO growth and morphology, further investigation was carried out. ICP-OES analysis of supernatants collected during the progress of the reaction showed that the peptides did not significantly alter the kinetics of Zn2+ consumption in ZnO synthesis (Figure S6a of the SI). The formation of microspheres in the presence of G-12A6 could therefore not be attributed to modification of reaction kinetics. The decrease observed in solution pH from ∼6.9 at the start of the reaction to ∼6.2 at 72 h (Figure S6b of the SI) in syntheses without and with peptide could also not be correlated to the morphology changes observed. Irrespective of the differences in the morphology formed in the presence of the different peptides, the crystal structure of the 48 h precipitates was predominantly wurtzite structured (Figure 2a), having characteristic diffraction peaks at 31.65°, 34.32°, and 36.14° of 2θ that can be indexed to the (101̅0), (0002), and (101̅1) planes of ZnO, respectively.32 Previous TGA studies of ZnO platelets synthesized using GT16 showed that the crystals formed had significantly more adsorbed organic matter than the short rods synthesized in the presence of G-12.21 The high organic content of ZnO platelets synthesized using GT-16 was correlated to the peptides’ ability to adsorb to and modify the morphology of growing ZnO.21 In this study, thermal decomposition of 48 h precipitates heated to 900 °C resulted in complete degradation of organic material while retaining the structure of the rods and microspheres (Figure S7 of the SI). Sintering and phase transformation require temperatures much higher than 950 °C and above 1300 °C, respectively.33,34 The weight loss in 48 h precipitates formed in the presence of G-12, GT-16, and the different G-12 alanine mutants was significantly higher (p value < 0.05, 95% CI, n = 3) than that of the control reaction (Figure 2b). The weight loss in the control reaction was attributed to the presence of LBZN while the weight loss observed in the precipitates synthesized with peptide was attributed to the presence of LBZN and adsorbed peptide. The observed reduction of the aspect ratio of ZnO rods synthesized with the peptides corroborated the adsorption growth inhibition mechanism.21 However, the mechanism through which ZnO microspheres were formed in the presence of G-12A6 peptide was not understood. Modification of the synthesis process was carried out to further understand the ZnO microsphere formation in the presence of G-12A6. First, synthesis was carried out using the Zn(NO3)2·6H2O−HMTA system with different concentrations of G-12A6. Second, synthesis was carried out following an alternative synthesis route: the ZnAc2−NH3 system also known to result in the formation of elongated wurtzite structured twinned hexagonal rods. The outcomes would determine if the microsphere formation process with G-12A6 was solely a peptide sequence and/or peptide concentration dependent process. The findings would also shed light on the question of whether the reaction conditions of the system used, which have been shown to direct the growth process and dictate the solid phases formed, also influenced the process of morphology
Figure 2. (a) X-ray diffractrograms of 48 h precipitates synthesized using the Zn(NO3)2·6H2O−HMTA system. Four-digit Miller−Bravis indices designate the crystal planes of ZnO. Where visible, low intensity peaks in the region from ∼13° to ∼30° of 2θ and a broad peak at ∼42° of 2θ correspond to polypropylene peaks from the sample holder. (b) TGA determined weight loss of the 48 h precipitates and schematic representations of ZnO crystals formed (drawn to scale). Precipitates synthesized without peptide are labeled as the control, and those synthesized in the presence of 0.3 mM of the different peptides are labeled with the peptide used.
modification in the presence of peptides. For comparison, parallel experiments were also carried out with G-12. Zn(NO3)2·6H2O−HMTA System with Different Peptide Concentrations: G-12A6 and G-12. From syntheses incorporating peptide (G-12 and G-12A6), solid state characterization was carried out for 25 h (24 h, t20°C + 1 h, t65 °C) precipitates because the amounts present in 24 h samples was too little for most characterization techniques used. The microstructures and crystallinity of precipitates were examined using SEM and XRD (Figure 3). In agreement with previously reported findings,21,23 25 h precipitates synthesized in the control reaction consisted of a mixture of short twinned rods (L/D = 2.63 ± 1.32) and layered structures (Figure 3a). The 25 h precipitates synthesized with 0.1 mM G-12A6 consisted of continuous rough aggregates with some microspheres that were ∼2 μm in diameter (Figure 3b). At a higher concentration of 0.3 mM G-12A6, 25 h precipitates consisted of porous microspheres ∼ 4 μm in diameter (Figure 3c). Aggregated structures were present in 25 h precipitates synthesized with the 0.5 mM G-12A6 (Figure 3d). Similar aggregates were present in precipitates synthesized with G-12 (Figure 3e−g). XRD analysis of 24 and 25 h precipitates from the control 1953
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precipitates had previously been established using FTIR marked by the NO3− vibration at 1384 cm−1. The LBZN weight loss in the third stage may be associated with decomposition of anhydrous Zn(NO3)2 formed as LBZN is decomposed to form ZnO. A similar weight loss in stage 3 was observed in the weight loss curve of Zn(NO3)2·6H2O (Figure S8 of the SI) which may occur as anhydrous Zn(NO3)2 and is thermally decomposed to form ZnO. The major weight loss of 25 h precipitates synthesized with G-12A6 and G-12 fell within the second stage of weight loss, except precipitates synthesized with 0.5 mM G-12 (Figure 4b−d). This suggests that, at this stage, much of the LBZN formed during the nucleation period had not yet undergone phase transformation to ZnO, especially those synthesized with G-12A6 (Figure 4b−d). The greater weight loss in stage 3 of precipitates synthesized with 0.5 mM G-12 suggests a greater adsorption of the peptide to the precipitates (Figure 4c). The differences observed in weight loss from precipitates synthesized with G-12A6 and G-12 may have been a consequence of different growth processes resulting in different morphologies. SEM analysis showed that 48 h precipitates synthesized with 0.1 mM G-12A6 consisted of ZnO hexagonal rods (L/D = 5.54 ± 1.89) and continuous networks of porous sheets (Figure 5a). As previously described, with 0.3 mM G-12A6, microspheres that were ∼10 μm in diameter and ZnO rods (L/D = 2.05 ± 0.82) were present in 48 h precipitates (Figure 5b). Small aggregated rod-like structures (L/D = 1.65 ± 0.61) were present in 48 h precipitates synthesized with 0.5 mM G-12A6 (Figure 5c). Only ZnO rods were formed in 48-h precipitates synthesized with the three concentrations of G-12 peptide (Figure 5d−f). The aspect ratio of ZnO rods in 48 h precipitates synthesized with both peptides decreased with an increase in peptide concentration (Figure 5g). XRD analysis confirmed that the major component of all 48 h precipitates synthesized using the Zn(NO3)2·6H2O−HMTA system was wurtzite structured ZnO (Figure 5h). The formation of microspheres with G-12A6 in this system was evidently a peptide sequence and peptide concentration dependent process. Evidence of strong peptide adsorption on precipitates was obtained from TGA (Figure 6) and FTIR (Figure 7) analysis. Weight loss that can be attributed to the decomposition of adsorbed peptide was observed in the third stage of weight loss from 250 to 700 °C (Figure 6a). The weight loss observed was greater than that in the control precipitates which only had weight loss from LBZN (Figure 6b). FTIR analysis of precipitates (Figure 7) supported TGA data showing the adsorption of peptide to 48 h precipitates. Peptides characteristically have an FTIR amide I band in the region of 1600−1700 cm−1 and an amide II band between 1500 and 1600 cm−1. The band characteristic of a ZnO stretching mode was observed in the region of 430−550 cm−1 (Figure S9 of the SI). A shift in amide I band, i.e., ∼1673 to ∼1646 cm−1, was observed in the spectra of the precipitates synthesized with peptides in comparison to spectra of pure peptides. This was also observed in previous studies of ZnO synthesis using G-12 and GT-16 in this system.21 The energy of vibration of the amide I may have been altered by the adsorption of peptide to ZnO surfaces.21 Also noted, the NO3− vibration at 1384 cm−1, indicating the presence of LBZN, was present in all of the precipitates and increased with an increase in peptide concentration. The general growth mechanism of twinned hexagonal ZnO rods synthesized in aqueous solution without any additive is
Figure 3. SEM micrographs of 25 h precipitates synthesized using the Zn(NO3)2·6H2O−HMTA system: (a) synthesis without peptide (control) and (b−f) synthesis incorporating peptide, with (b) 0.1 mM G-12A6, (c) 0.3 mM G-12A6, (d) 0.5 mM G-12A6, (e) 0.1 mM G-12, (f) 0.3 mM G-12, and (g) 0.5 mM G-12. All scale bars are 5 μm. (g) XRD diffractogams of 24 and 25 h precipitates synthesized without peptide (control) and 25 h precipitates synthesized in the presence of different concentrations of G-12A6 and G-12. Four-digit Miller−Bravis indices designate the crystal planes of ZnO while three-digit Miller indices correspond to crystal planes of LBZN, highlighted using down facing arrows.
syntheses revealed the presence of the LBZN peaks at ∼9° and ∼32.9° of 2θ attributed to the (200) and (021) planes, respectively (Figure 3h).23 The (021) plane was most prominent in the 24 h control precipitate which only consisted of layered structures (Figure 1a) of LBZN.21,23 Although ZnO was the dominant phase detected in 25 h precipitates synthesized with peptide, LBZN was still present, marked by the (021) plane (Figure 3h). Thermal decomposition (TGA) of the precipitates (Figure 4a−c) resulted in weight loss that was divided into three stages. Loss of physisorbed water occurred up to ∼100 °C in the first stage.35,36 The second stage had weight loss between 100 and 250 °C which in some profiles could be distinguished into two overlapping regions, i.e., from 100 to 168 °C normally associated with the dehydration of intercalated water in LBZs, here known to be LBZN, and the second region from 168 to 250 °C usually correlated to dehydroxylation of LBZs to form ZnO.23,35,36 The weight loss observed at the third stage from 250 to 500 °C may be attributed to the presence of LBZN from control precipitates and both LBZN and organic weight loss from precipitates synthesized with peptide. For the control precipitates (Figure 4a), weight loss was mainly observed in stage 2 and stage 3 in the 24 h precipitates and decreased but was still present in 25 h precipitates and was much less in 48 h precipitates. The LBZN content of 24, 25, and 48 h control 1954
DOI: 10.1021/acs.chemmater.5b00419 Chem. Mater. 2015, 27, 1950−1960
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Figure 4. (a−c) TGA first derivative weight loss curves of precipitates synthesized using the Zn(NO3)2·6H2O−HMTA system: (a) 24, 25, and 48 h precipitates synthesized without peptide (control), with inset showing total weight loss (n = 3), (b, c) comparison of 25 h control and weight loss of precipitates synthesized with different concentrations of G-12A6 and G-12, respectively, and (d) graph showing total weight loss and weight loss in the third stage (250−500 °C) for the 25 h precipitates.
Figure 6. (a) TGA first derivative weight loss curves of 48 h precipitates synthesized using the Zn(NO3)2·6H2O−HMTA system without peptide (control) and with different concentrations of G-12 and G-12A6. (b) Graph showing total weight loss and third stage weight loss of 48 h precipitates (n = 3).
Figure 5. (a−f) SEM micrographs of 48 h precipitates synthesized using the Zn(NO3)2·6H2O−HMTA system and different concentrations of peptide: (a) 0.1 mM G-12A6, (b) 0.3 mM G-12A6, (c) 0.5 mM G-12A6, (d) 0.1 mM G-12, (e) 0.3 mM G-12, and (f) 0.5 mM G12. All scale bars are 5 μm. (g) Graph showing measured aspect ratio (L/D, n = 50) of ZnO rods in 48 h precipitates formed without peptide (control) and with different concentrations of peptide. (h) XRD diffractogams of 48 h precipitates.
summarized as follows: anisotropic crystal growth is thought to occur because oppositely charged (0001) Zn-terminated and (0001)̅ O-terminated polar planes of ZnO result in a dipole moment and have higher surface energy than nonpolar planes such as the (101̅0) plane that is both Zn-/O-terminated.32,37 Therefore, to minimize the total free energy of the system, 1955
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of the reaction, observed up to 72 h, the microspheres continued to grow and secondary nucleation of hexagonal rods occurred on the surface of some microspheres (Figure S10 of the SI). A schematic depiction of the plausible mechanisms of ZnO growth and morphology modification in this system in the presence of G-12 and G-12A6 is shown in Scheme 1. The Scheme 1. ZnO Growth Process and Morphology Modification in the Presence of G-12 and G-12A6 in the Zn(NO3)2·6H2O−HMTA System
Figure 7. FTIR characterization of 48 h precipitates synthesized using the Zn(NO3)2·6H2O−HMTA system without peptide (control) and with different concentrations of (a) G-12 and (b) G-12A6. Spectra of pure peptides are also shown in gray.
incoming precursor molecules favorably adsorb to the polar planes resulting in growth along the c-axis.37,38 The twinning mechanism, also a device thought to reduce the surface energy of crystals,30,31 occurs through (0001̅) as the common plane of connection.37,39 Thus, the main planes exposed to possible interaction with peptide may be the (0001) Zn-terminated polar plane and the (101̅0) Zn-/O-terminated nonpolar plane of ZnO. Both G-12A6 and G-12 were able to adsorb to and modify the morphology of ZnO rods using the adsorption growth inhibition mechanism. Under the experimental conditions used in this study, pH below the pI of ZnO (∼9.5),37 hydroxylated ZnO may react with protons from solution due to surface amphoteric reactions to become a positively charged surface with ZnOH2+ groups.37,40 This proton accepting behavior of the ZnO surface may have attracted the positively charged peptides used in this study to the surface of ZnO. Alternatively, peptide adsorption may have occurred using specific amino acid residues (i.e., through complexation of histidine residues with divalent zinc ions such as in zinc finger dependent proteins and metalloproteins41,42) or via groups of residues, “hot spot” regions17 within the sequences. There was no evidence of peptide incorporation into the lattice of ZnO from a comparison of XRD determined d-spacing values for the (0001) plane (for growth along the caxis) and the (101̅0) plane (for growth along the a-axis) of 48 h precipitates: the control and precipitates synthesized with the examined peptides. Only G-12A6 at a concentration of 0.3 mM was able to support the isotropic growth of ZnO. Previous studies have reported that, in solution, peptides may be able to interact with each other through peptide−peptide interactions.9,43,44 Substitution of H6 in G-12 with A6 to form G-12A6 may have, under the specific synthesis conditions and peptide concentration, modulated the conformational organization of G-12A6 molecules into micelle-like assemblies which may have then acted as a template for the nucleation and growth of ZnO. The larger microspheres observed in 24 h precipitates synthesized with 0.3 mM G-12A6 may have consisted of an intermediate phase, plausibly LBZN with a modified morphology. With an increase in temperature to 65 °C, as in the control reaction, phase transformation may have taken place while retaining the structure of LBZN. The phase transformation of LBZs into ZnO without changing the morphology has been reported elsewhere via heat treatment generally in the range of 70−140 °C.35,45 The sudden increase in temperature after the nucleation period (24 h, t20°C) to 65 °C could have disintegrated the larger microspheres (10−50 μm in diameter) formed during the nucleation period into smaller microspheres (∼4 μm) observed in 25 h (24 h, t20°C + 1 h, t65°C) precipitates. With progression
synthesis of ZnO whole and hollow spherical structures have been reported using other structure directing agents including poly(vinylpyrrolidone), poly(ethylene glycol), and citrate.31,46 Similar ZnO microspheres have also been synthesized using amino acid based surfactants with different headgroup functionalities: lauroyl chloride coupled to aspartic acid, glutamic acid, or alanine.47 ZnAc2−NH3 System with Different Peptide Concentrations: G-12A6 and G-12. The formation of ZnO using the ZnAc2−NH3 system has been reported to occur through the formation of a large amount of the intermediate layered basic zinc acetate (LBZA) due to the abundance of hydroxide ions (OH−) from the onset of the reaction.23,35 Acetate anions within the structure of LBZA may then be gradually replaced by OH− to form Zn(OH)2 and ZnO.23,48 Layered basic zinc acetate carbonate (LBZAC) was identified as an additional product formed in this system as a consequence of the replacement of some acetate groups in LBZA with carbon dioxide dissolved in the synthesis solution.23,49 The formation of ZnO eventually occurred through phase transformation of intermediates through a dissolution/reprecipitation process.23 In the control synthesis, 48 h precipitates consisted of twinned hexagonal rods (L/D = 4.52 ± 1.75) some of which were asymmetrically twinned forming grenade-like structures (Figure S11 of the SI). Asymmetrical twinning may be attributed to slower growth along one axis that can later become symmetrical in solutions with adequate precursor growth units.30,50 There was no evidence of microsphere formation in syntheses using the ZnAc2−NH3 system incorporating G12A6 and G-12 (Figure 8a−f). Where ZnO rods were formed in the presence of G-12 and G-12A6, aspect ratio reduction was observed with an increase in peptide concentration. Interestingly, few to no ZnO rods were present in 48 h precipitates synthesized with 0.5 mM G-12 (Figure 8f). The precipitates mainly consisted of layered structures confirmed to be LBZA 1956
DOI: 10.1021/acs.chemmater.5b00419 Chem. Mater. 2015, 27, 1950−1960
Article
Chemistry of Materials
Figure 9. (a) TGA first derivative weight loss curves of 48 h precipitates synthesized using the ZnAc2−NH3 system without peptide (control) and with different concentrations of G-12 and G-12A6. (b) Graph showing total weight loss and insets showing weight loss in the third stage from 48 h precipitates (n = 3).
Figure 8. (a−f) SEM micrographs of 48 h precipitates synthesized using the ZnAc2−NH3 system and different concentrations of peptide: (a) 0.1 mM G-12A6, (b) 0.3 mM G-12A6, (c) 0.5 mM G-12A6, (d) 0.1 mM G-12, (e) 0.3 mM G-12, and (f) 0.5 mM G-12. All scale bars are 5 μm. (g) Graph showing aspect ratio (L/D, n = 50) of ZnO rods in 48 h precipitates formed without peptide (control) and with different concentrations of peptide. (h) XRD diffractogams of 48 h precipitates. Four-digit Miller−Bravais indices designate the crystal planes of ZnO while three-digit Miller indices correspond to crystal planes of LBZA, highlighted using down facing arrows.
using XRD (Figure 8h) with the diffraction peak of highest intensity at 6.61° of 2θ (d-spacing = 13.37 Å), attributed to the (001) plane and a broad peak at ∼33.2° of 2θ corresponding to the (100) plane. 45,51 LBZA was also detected in the diffractogram (peak at 6.64° of 2θ, d-spacing = 13.29 Å) of 48 h precipitates synthesized using 0.5 mM G-12A6, although ZnO peaks were also present (Figure 8h). With an increase in concentration, both peptides were able to suppress the growth of ZnO with this effect being greater with G-12 where growth was almost entirely inhibited using 0.5 mM of the peptide. Further characterization of 48 h precipitates using TGA (Figure 9) and FTIR (Figure 10) was carried out to understand how the peptides suppress and/or inhibit the growth of ZnO. Assignments of the main FTIR adsorption peaks used to identify the components in the precipitates are shown in Table 1. The total weight loss of 48 h precipitates synthesized using 0.5 mM G-12 was 31.62 ± 2.45% (Figure 9b) which is close to the reported weight loss of LBZA (32.6 ± 1.4%) identified in the control sample collected not long after mixing of reagents (
