Article pubs.acs.org/bc
Phage-Directed Synthesis of Photoluminescent Zinc Oxide Nanoparticles under Benign Conditions Kamila Ż elechowska,†,# Joanna Karczewska-Golec,‡,# Jakub Karczewski,† Marcin Łoś,‡ Andrzej M. Kłonkowski,§ Grzegorz Węgrzyn,‡ and Piotr Golec*,∥ †
Solid State Physics Department, Faculty of Applied Physics and Mathematics, Gdansk University of Technology, Narutowicza 11/12, 80-233 Gdansk, Poland ‡ Department of Molecular Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland § Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland ∥ Laboratory of Molecular Biology (affiliated with the University of Gdansk), Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Wita Stwosza 59, 80-308 Gdansk, Poland ABSTRACT: Biological systems, especially bacteriophages and peptides, are an attractive green alternative to other known methods of nanoparticle synthesis. In this work, for the first time, bacteriophages were employed to synthesize a specific peptide, capable of producing nanoparticles (NPs). Derivatives of M13 bacteriophage exposing a ZnO-binding peptide (TMGANLGLKWPV) on either pIII or pVIII phage coat protein were constructed and used as a biotemplate. The exposition of the ZnO-binding peptide, synthesized by phages during their propagation in bacteria, on M13 virions provided a groundwork for growing ZnO nanostructures. Depending on the recombinant phage type used (M13-pIII-ZnO or M13-pVIII-ZnO), well separated ZnO NPs or complex 3D structures of ZnO NPs of ca. 20−40 nm were synthesized at room temperature. The synthesized ZnO nanoparticles served as a luminescent material that emitted light near the short wavelength end of the visible region (at ca. 400 nm). The next very low intensity emission band at 530 nm demonstrated that the ZnO material obtained is characterized by a low concentration of surface defects.
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stabilizers.13 Hence, chemical synthesis methods give rise to the presence of toxic chemicals adsorbed on the surface of NPs, hindering the use of the final product in medical applications.25 For these reasons, and due to the environmental concerns, new green synthesis methods of ZnO nanostructures are desired.13 Recently, a biogenic synthesis of ZnO nanoparticles (NPs), with the use of collagen, plant extracts, macromolecules, bacteria, and amino acid sequences, has been proposed.13,26−32 Peptides and proteins were demonstrated to be particularly useful in this approach, even when applied to materials not commonly found in biological systems, such as semiconductors.33 Production of inorganic solids by biological systems, including those genetically engineered, is an attractive alternative to other known methods, and gains more and more interest in the development of bottomup manufacturing of nanometer-scale devices. Peptide-directed growth of zinc structures was reported for the first time by Umetsu et al.,34 when a phage display library was employed to identify a unique peptide sequence that specifically binds ZnO particles. The identified peptide (ZnO-1) with a glycine linker (GGGS) and a cysteine residue at the C-terminus
INTRODUCTION Considerable attention is currently being paid to zinc oxide (ZnO) of a hexagonal wurzite structure. As an n-type II−VI semiconductor, ZnO is characterized by a wide band gap of 3.37 eV and a high exciton binding energy of 60 meV.1 It is nontoxic, generally recognized as safe (GRAS) by the Food and Drug Administration, and shows an optical transparency in the visible range. Its defect centers were identified as oxygen vacancies on the surface, zinc interstitials, or complex defects.2−6 Nanostructures of zinc oxide are among the most studied materials in nanotechnology as they exhibit unique medical, optical, acoustic, electronic, and diagnostic properties.7−11 They are known in a variety of morphology types, which may be obtained with the use of diverse methods.12,13 Physical and chemical methods used to produce ZnO nanostructures are widely described in the literature;14−23 however, they have several crucial disadvantages. Nanoparticles (NPs) obtained in physical processes are generally of lower quality as compared to their counterparts produced by chemical methods. Moreover, to obtain NPs with the use of physical methods, expensive vacuum systems, or equipment (plasmas) are usually required.24 On the other hand, the major drawback of chemical methods is the low stability and self-aggregation of the final product, which necessitates the addition of surfactants and © XXXX American Chemical Society
Received: April 18, 2016 Revised: July 12, 2016
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DOI: 10.1021/acs.bioconjchem.6b00196 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry was then chemically synthesized and used to generate ZnO crystals. Three days after addition of the GGGSC-conjugated ZnO-1 peptide to the Zn(OH)2 sol, the authors observed a precipitated material containing wurtzite ZnO that assembled into highly ordered flower-type structures. The phage display technique was also used by Tomczak et al.,35 who identified a ZnO-binding peptide, called Z1, nearly identical to the one reported previously.34 The authors observed precipitation of ZnO particles after 72 h incubation of GGGC-Z1 peptide with Zn(NO3)2 and 1,3-hexamethylenetetramine (HMTA) at 65 °C. Following these publications, other research groups described various morphologies of ZnO NPs obtained with the use of peptide sequences as biotemplates.36−42 Additionally, an interesting application of ZnO-binding peptides was described by Vrelus et al.43 The isolated inorganic-binding peptides44 were implemented as quality control tools for detecting defects on inorganic surfaces of any shape.43 More recently, a high variability in amino acid sequences and isoelectric points of peptides that are able to specifically bind ZnO particles have been demonstrated.39,45 In this report, we focus on green synthesis of ZnO nanoparticles. We used genetically modified phages as a machinery for the synthesis of both the previously selected and identified45 ZnO-binding peptide and ZnO NPs. The exposure of ZnO-binding peptide on a virion laid a groundwork for growing unique ZnO nanostructures. It should be underlined that, in contrast to previous reports, only phages presenting the peptide sequence and a zinc oxide precursor were required for the synthesis of ZnO NPs, and the material obtained revealed luminescent properties.
Figure 1. TEM images of M13 phages bound to ZnO NPs. Images A and B show M13-pIII-ZnO phages binding ZnO NPs at one virion end. The M13-pVIII-ZnO phages binding ZnO NPs along the phage virons are presented in images C and D. ZnO NPs are marked with white arrows.
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RESULTS AND DISCUSSION Construction of Bacteriophages Exposing ZnO-Binding Peptides. Recombinant M13 bacteriophages, exposing a ZnO-binding peptide (TMGANLGLKWPV) attached to either pIII (phage M13-pIII-ZnO) or pVIII (phage M13-pVIII-ZnO) proteins, were constructed and propagated as described in the Experimental Section. The ability of M13-pIII-ZnO and M13pVIII-ZnO phages to specifically bind ZnO NPs was analyzed by TEM. M13 phage exposing the ZnO-binding peptide on pIII protein was able to bind one ZnO NP at the phage virion end in contrast to M13-pVIII-ZnO that was able to bind many ZnO NPs along its phage virion (Figure 1). The control phage M13KE did not bind ZnO NPs (data not shown). Additionally a Western blot analysis was performed to estimate the ratio of recombinant pIII and pVIII proteins to their wild-type counterparts on M13pIII-ZnO and M13-pVIII-ZnO phages, respectively (Figure 2). We observed that more than 80% of pIII protein occurred in a recombinant form that was able to bind and synthesize ZnO NPs (Figures 1 and 2). The M13-pVIII-ZnO phage contained ca. 45% of recombinant pVIII protein. This means that ca. 1700 copies out of ca. 3900 copies of pVIII were displaying ZnO-binding peptides. Phage-Assisted Synthesis of ZnO Nanoparticles. First, the M13-pVIII-ZnO phages suspended in TBS buffer were used as a biotemplate, and the synthesis of ZnO NPs was performed using various concentrations of zinc oxide precursor, as described in the Experimental Section. SEM images, showing morphology of the nanostructures obtained along the phage capsids, are presented in Figure 3. Synthesis of separate ZnO NPs was observed only when the lowest concentration of the ZnO precursor, Zn(OH)2, was applied. At higher concentrations of Zn(OH)2, it seems likely that phages were covered with a ZnO
Figure 2. Quantitative analysis of Western-blotting results with protein bands corresponding to pVIII and pIII proteins (wild-type (WT)) in relation to recombinant pIII-ZnO and pVIII-ZnO proteins exposed on M13-pIII-ZnO and M13-pVIII-ZnO phages, respectively. Total protein value was calculated as a summation of signal values from recombinant and WT bands.
film, forming a three-dimensional porous structure, or a structure of bundled wires (data not shown). Figure 3B presents an image of ZnO NPs at a higher magnification, revealing their average size of 20−40 nm. No ZnO NPs were observed when M13KE was used as the biotemplate (data not shown). ZnO Nanoparticle Characterization by UV−vis Absorption Spectroscopy. In order to investigate the optical properties of the synthesized material, UV−vis and photoluminescence spectroscopy measurements were carried out. Figure 4 presents UV−vis absorption spectra of zinc oxide nanostructures synthesized by M13-pVIII-ZnO phage (ZnOM13), when the ratio of Zn2+ per 1 M13-pVIII-ZnO phage (Zn/ M13) was equal to 0.05 × 10−19 mol. Spectra of M13 phages and a zinc oxide precursor [Zn(OH)2] were also registered for comparison. The absorption spectra of the prepared ZnO NPs (Figure 4, curve a) and M13-pVIII-ZnO phage (Figure 4, curve b), both peaked at 345 nm, were similar except a shoulder at about 330 nm in the former spectrum. However, the spectrum of M13-pVIII-ZnO phage had significantly lower absorbance in comparison with synthesized ZnO NPs. ZnO Nanoparticles Characterization by Luminescence Spectroscopy. The excitation and emission photoluminesB
DOI: 10.1021/acs.bioconjchem.6b00196 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Figure 5. Excitation spectra of (a) ZnO NPs synthesized by M13-pVIIIZnO phage, when Zn/M13 ratio was 0.05 × 10−19 mol/phage, (b) M13pVIII-ZnO phage, and (c) Zn(OH)2.
band at 235 nm and a broad, strong one at 330 nm. Both bands decrease in intensity for M13 phage (Figure 5, curve b) and Zn(OH)2 (Figure 5, curve c), respectively. Simultaneously, the strong band of M13 is shifted to a shorter wavelength and it vanishes in the Zn(OH)2 case. In addition, another small band peaked at ca. 280 nm is present in the spectrum of M13. The excitation band of ZnO NPs peaked at 330 nm is located near the position of the absorption band of the same sample (Figure 5, curve a). It is known that these bands of ZnO NPs are of excitonic origin.46 Besides, a band at 235 nm, repeated in all three samples (Figure 5, curves a−c), probably originates from the glass substrate. Photoluminescence (PL) spectra of the samples are presented in Figure 6A,B. Only ZnO NPs exhibited a spectrum consisting Figure 3. SEM images of ZnO nanostructures obtained in the phageassisted synthesis when the ratio of Zn2+ per one M13-pVIII-ZnO phage (Zn/M13) was equal to 0.05 × 10−19 mol/phage. Magnifications: A 10 000-fold; B - 100 000-fold.
Figure 6. (A) Photoluminescence spectra (λexc = 330 nm) of (a) ZnO NPs (Zn/M13 ratio was 0.05 × 10−19 mol/phage), (b) M13-pVIII-ZnO phage, and (c) Zn(OH)2. (B) PL intensity changed with ZnO NPs concentration in the reaction mixture.
of a main, broad emission band at around 400 nm corresponding to excitonic recombination.46 However, the second ZnO-specific band, located at around 530 nm, was noticeable as a weak shoulder. Obviously, the characteristic band for ZnO at ca. 400 nm is absent in the spectrum of Zn(OH)2 that presents a very low PL intensity (Figure 6A, curve c). At the same time, M13 spectrum has an edge shape with some weak bands (Figure 6A,
Figure 4. UV−vis absorption spectra of (a) ZnO synthesized in the presence of M13-pVIII-ZnO phage (Zn/M13 was 0.05 × 10−19 mol/ phage) in comparison to (b) M13-pVIII-ZnO phage and (c) Zn(OH)2.
cence spectra were recorded. The excitation spectra of samples are shown in Figure 5. The spectrum of ZnO NPs synthesized by M13-pVIII-ZnO (Figure 5, curve a) consists of a small, sharp C
DOI: 10.1021/acs.bioconjchem.6b00196 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry curve b). The photoluminescence intensity increased continuously with increasing concentration of ZnO NPs (the phage synthesis product), as shown in Figure 6B, indicating that the studied ZnO NPs concentration range was below the range observed in the concentration quenching effect. Synthesis of ZnO Nanoparticles by Peptides Exposed on Phage Virion without the Need for Any Additional Reagents or Conditions. As mentioned in Experimental Section, repeated washing of the samples was necessary after the synthesis of ZnO NPs. Otherwise, a thick layer of solids precipitated from buffer was formed, masking the synthesized nanoparticles. However, during exhaustive washing, a part of the nanoparticles, especially the smallest ones, may be removed with supernatant. Moreover, the buffer components may influence the morphology of the nanoparticles obtained. Therefore, in the next step, experiments similar to those described in the Phage-Assisted Synthesis of ZnO Nanoparticles section were performed using phages M13-pVIII-ZnO, M13pIII-ZnO, and M13KE suspended in water. SEM images of NPs synthesized in the presence of M13-pVIII-ZnO and M13-pIIIZnO (0.05 × 10−19 mol Zn2+ per one phage) are shown in Figure 7A,B. For comparison, SEM image of the sample with M13KE, confirming that no nanoparticles were synthesized, is also presented (Figure 7, inset). It is evident that phages M13-pVIIIZnO and M13-pIII-ZnO were able to synthesize ZnO NPs contrary to the wild type M13KE. If phages modified on pVIII were used as biotemplates, the synthesized nanoparticles were aligned along the phage capsid. The nanoparticle dimensions were in the range of 20−40 nm. The ZnO NPs obtained in M13pIII-ZnO phage-assisted synthesis revealed a similar size (Figure 7B). However, they did not form aggregates as observed for M13-pVIII-ZnO. Additionally, the ability of phages exposing peptides of similar length, but with different amino acid sequences, to synthesize ZnO NPs was analyzed. Two phages exposing HLYLNTASTHLG and SRTGNWTRIDQS peptides (M13-pIIIHLYLNTASTHLG and M13-pIII-SRTGNWTRIDQS, respectively) were used. The reaction parameters were the same as in the case of the analysis described above. SEM images, revealing morphology of the structures obtained in the presence of M13pIII-HLYLNTASTHLG and M13-pIII-SRTGNWTRIDQS, are presented in Figure 8. No synthesis of nanoparticles was observed. The chemical composition of ZnO NPs synthesized by M13pVIII-ZnO and M13-pIII-ZnO was evaluated by EDX spectroscopy. Additionally, the chemical composition of the amorphous structures obtained in experiments with M13-pIII-HLYLNTASTHLG and M13-pIII-SRTGNWTRIDQS was analyzed. The results are presented in Table 1. It is clear that the observed nanostructures, synthesized by M13-pVIII-ZnO and M13-pIIIZnO, are ZnO nanoparticles, which was additionally verified by other methods. The absorption UV−vis, as well as emission and excitation PL spectra, also confirmed the formation of ZnO NPs (data not shown). They are very close to spectra presented in Figures 4−6, revealing a high quality of the synthesized nanoparticles. In both negative control tests, the mol ratio of O/Zn is higher in comparison to ZnO NPs and it can be assumed that the observed amorphous structure is mainly composed of Zn(OH)2. Thus, the mineralization of the precursor did not occur in the negative control. The ZnO NPs and the amorphous structures were then analyzed using photoluminescence spectroscopy. The results (Figure 9) confirmed the assumption that the amorphous
Figure 7. SEM image of (A) ZnO NPs synthesized by M13-pVIII-ZnO and (B) ZnO NPs synthesized by M13-pIII-ZnO. Inset: wild type M13KE phage. In all cases the Zn/M13 ratio was 0.05 × 10−19 mol/ phage.
Figure 8. SEM image of (A) amorphous structure obtained in the presence of M13-pIII-HLYLNTASTHLG and (B) amorphous structure obtained in the presence of M13-pIII-SRTGNWTRIDQS.
structure obtained with the use of M13-pIII-HLYLNTASTHLG phage is Zn(OH)2. The spectrum obtained is nearly identical to the spectrum of Zn(OH)2 solution when no phages were present (Figure 9b,c). In the case of M13-pIII-ZnO, a broad emission band at around 400 nm corresponding to excitonic recombination confirmed the presence of ZnO (Figure 9, curve a). Crystallinity of the synthesized ZnO nanomaterial was evaluated by X-ray diffraction. For all analyzed samples the D
DOI: 10.1021/acs.bioconjchem.6b00196 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Table 1. Elemental Composition of ZnO NPs Synthesized by M13-pVIII-ZnO and M13-pIII-ZnO Phages in Comparison to the Amorphous Structures Obtained, When M13-pIII-HLYLNTASTHLG and M13-pIII-SRTGNWTRIDQS Phages Were Used M13-pIII-ZnO
M13-pVIII-ZnO
M13-pIII-HLYLNTASTHLG
M13-pIII-SRTGNWTRIDQS
element
wt %
at. %
wt %
at. %
wt %
at. %
wt %
at. %
OK Zn K
20.041 79.958
50.598 49.401
20.039 79.960
50.595 49.404
32.05 56.45
59.38 28.13
48.81 47.66
78.63 18.79
synthesize ZnO nanostructures is demonstrated in Figures 3 and 7. Morphology of ZnO nanostructures obtained in the phageassisted synthesis was zinc precursor- and phage type-dependent (Figures 3 and 7). The pIII protein is present on the phage particle (virion) end, and the aggregates could only be formed within NPs which are attached to different virions, which is an unlikely effect in diluted suspensions. Thus, ZnO NPs remained well dispersed. When M13-pVIII-ZnO phage was used, it was able to produce ZnO NPs along the phage capsids. Depending on the concentration of the zinc oxide precursor [Zn(OH)2], we observed various morphologies of ZnO NPs obtained. Thus, bacteriophages exposing the ZnO-binding peptide proved useful to produce both separate ZnO NPs and intricate, threedimensional porous structures of ZnO NPs. The ZnO NPs obtained and M13-pVIII-ZnO phage had similar UV−vis absorption spectra (Figure 4). In our opinion the spectrum presented in Figure 4 (curve a) is a superposition of the overlapped M13-pVIII-ZnO phage and ZnO NPs spectra, with the shoulder referring to the trace of the ZnO NPs band exhibiting an excitonic character.47 The band is blue-shifted in comparison to the pure bulk zinc oxide (band gap of 3.37 eV, 368 nm),48 indicating that ZnO NPs synthesized by M13-pVIII-ZnO are in the nanosize range, and that the blue shifting is caused by the quantum confinement effect. Consequently, we concluded that ZnO NPs were present in the analyzed material, indeed. Their size and shape are shown in SEM images (see Figure 3). PL spectra showed the characteristic bands for ZnO at ca. 400 and 530 nm only when phage-assisted synthesized ZnO NPs were analyzed (Figure 6). The green−yellow emission is commonly referred to the deep-level emission, which is generally attributed to interstitial oxygen impurities or defects in ZnO.49,50 Therefore, ZnO NPs synthesized by the phages reveal rather low concentration of surface defects, according to the interpretation in a previous report.51 Previously, several ZnO-binding peptides were described;34,35,38,39,43,44 however, only a few of the abovementioned papers reported the synthesis of zinc oxide nanostructures in the presence of novel chemically synthesized ZnO-binding peptides.34,35,38 Flower-like ZnO nanostructures were obtained with the use of Zn(OH)2 as a zinc precursor and chemically synthesized EAHVMHKVAPRP peptide with the GGGSC linker as a biotemplate.34 It should be underlined that, without the GGGSC linker, the isolated ZnO-1 peptide showed a high affinity for ZnO crystals, but no ability to synthesize ZnO nanostructures. Moreover, the obtained flower-like ZnO structures were of micrometer size, and were built of aggregated ZnO particles. Similarly, Tomczak et al.35 described the use of a very similar peptide (GLHVMHKVAPPR conjugated with GGGSC tail) in the synthesis of ZnO nanocrystals; however, 1,3-hexamethylenetetramine (HMTA), an elevated temperature, and a longer time were required. The ZnO hexagonal platelets obtained were formed in the peptide-assisted synthesis, while control (no peptide) samples contained ZnO hexagonal bipyramid structures. Thus, the presence of the peptide was
Figure 9. Photoluminescence spectra (λexc = 330 nm) of (a) ZnO NPs synthesized by M13-pIII-ZnO, (b) a material obtained in the presence of M13-pIII-HLYLNTASTHLG, and (c) Zn(OH)2.
characteristic reflexes of wurtzite hexagonal ZnO (that is 2θ = 31.7°; 36.0°; 47.6°; 56.6°; and 68.2°) were found (Figure 10). Particle size estimated from the diffraction spectrum by using half-maximum widths were in the range of 25−35 nm, which is in agreement with SEM images.
Figure 10. X-ray diffraction spectrum fo ZnO wurtzite type nanocrystals synthesized by M13 phage.
Significance of the Results. In this study, we focused on designing a new eco-friendly method of synthesizing ZnO NPs, based on a phage display technique. For the first time in ZnO NPs fabrication, phages were used not to identify ZnO-binding peptides, but to synthesize ZnO NPs. We proved that the synthesis itself actually occurred on phages which exposed ZnObinding peptides on either pIII or pVIII phage proteins. The ability of genetically engineered bacteriophages, exposing recombinant pVIII or pIII proteins, to not only bind but also E
DOI: 10.1021/acs.bioconjchem.6b00196 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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respectively) overnight at 4 °C. Next, membranes were probed with appropriate peroxidase-conjugated secondary antibodies and visualized with Pierce ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL). Each protein was detected two or three times from independently prepared lysates. Densitometric analysis was carried out using Quantity One software. Synthesis of ZnO NPs by Peptides Exposed on Virions. Experiments were performed in three replicates to confirm the ability of isolated peptides to mineralize ZnO nanostructures. Zn(NO3)2·6H2O, LiOH, NaCl, and tris(hydroxymethyl)methylamine-HCl (Tris-HCl), all of p.a. purity, were purchased from Sigma−Aldrich. Buffered (Tris-HCl, NaCl) aqueous suspensions of phages were prepared in the concentration of (8 × 1012) to (1 × 1013) plaque forming units (pfu)/cm3. Equal volumes of 0.1 M zinc nitrate and 0.2 M lithium hydroxide solutions were mixed, giving Zn(OH)2 sol. Different volumes of Zn(OH)2 sol were added to 20 mL of phage suspension to obtain the ratio of 0.05 × 10−19 mol Zn2+, 10−19 mol Zn2+, and 5 × 10−19 mol Zn2+ per 1 M13pVIII-ZnO phage, respectively. The samples were left overnight. Simultaneously, blank experiments were performed with wildtype phage M13KE, M13-pIII-HLYLNTASTHLG, and M13pIII-SRTGNWTRIDQS, using the same ratio of zinc ions per one phage. Similarly to Umetsu et al.,34 after that time we observed a white precipitate formed in the samples containing M13-pVIII-ZnO phages. No precipitate was formed in the presence of M13KE. The samples were centrifuged (12 600 g, 10 min) and pellets were washed several times with deionized water. The exhaustive washing was crucial to remove buffer components before analyzing the nanoparticles by microscopy and spectroscopy. Additionally, after the propagation procedure, the phages (M13-pIII-ZnO, M13-pVIII-ZnO, and M13KE) were suspended in water with no buffer solution. Various volumes of Zn(OH)2 sol were added to the phage suspensions as described above. The reaction mixtures were left overnight and centrifuged (12 600 g, 10 min). Nanoparticles obtained were analyzed using scanning electron microscopy (SEM), UV−vis, and photoluminescence (PL) spectroscopy. Transmission Electron Microscopy Analysis. M13-pIIIZnO, M13-pVIII-ZnO, and M13KE phages suspended in water (at a concentration of ca. 1 × 1010) were incubated with 1000× diluted ZnO NPs (Sigma-Aldrich) for 1 h. For TEM, samples were placed on grids coated with a 2% collodion solution and carbon. Phages were negatively stained with 2% uranyl acetate and examined using a Philips CM100 electron microscope at 80 kV. Scanning Electron Microscopy Analysis. For microscopy analysis (SEM, FEI Quanta FEG 250), aqueous solutions of the samples were dropped onto a carbon conducting support and left overnight to dry at room temperature (RT). If necessary, samples were covered with thin films of gold, using a high vacuum sputter coater (Leica EM SCD 500). Energy-Dispersive X-ray (EDX) Spectroscopy. For chemical characterization of the samples, EDX spectroscopy was performed using the EDAX Genesis APEX 2i with Apollo X SDD spectrometer at 10 kV. UV−vis Absorption Spectroscopy Measurements. UV−vis spectra were measured using Lambda 10 (PerkinElmer). Measurements were carried out in the wavelength range of 200− 900 nm. Absorption spectra of aqueous solutions in quartz cuvettes were recorded.
not necessary to synthesize ZnO nanostructures in the method reported, and only the influence of the peptide on the ZnO morphology was observed. Recently, Moon et al.38 described synthesis of ZnO NPs with the use of ZnO NPs precursor and 8mer (VPGAAEHT) suspended in TBS buffer. Tris(hydroxymethyl)methyl-amine-HCl (Tris-HCl), the main component of TBS buffer, is a polydentate ligand which adsorbs strongly on one or more ZnO surfaces, directing nanoparticle growth.52 Therefore, the presence of Tris-HCl in phage or peptide suspensions could also influence the synthesis processes of ZnO structures. Contrary to the studies discussed above, we performed experiments both with and without buffer, observing the influence of buffer components on the morphology of the structures obtained. Furthermore, we carried out blank experiments with no peptide, and with the wild-type peptide. Our results clearly show that phages with ZnO-binding peptides exposed on either pIII or pVIII possess high affinity for the ZnO surface. It has to be emphasized that their ability to mineralize ZnO from Zn(OH)2 precursor was demonstrated and ZnO NPs of ca. 20−40 nm were synthesized at room temperature, proving that a bioassisted synthesis is a benign bottom-up approach for manufacturing of novel nanostructures with unique properties. Moreover, in our method, green synthesis was utilized at both crucial steps: dual-function phages served both as a machinery for the synthesis of desirable peptides and as a biotemplate for the synthesis of nanoparticles. Frequently used chemical methods of peptide synthesis, such as Boc and Fmoc solid-phase syntheses, require special equipment and/or costly reagents. Additionally, they are time-consuming and the final product is prone to aggregation and diminished solubility.53 Our approach overcomes these challenges.
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EXPERIMENTAL PROCEDURES Construction and Production of M13 Virions Exposing ZnO-Binding Peptides. The ZnO-binding peptide (TMGANLGLKWPV) exposed on pIII protein of M13 phage was identified previously with the use of Ph.D.-12 Phage Display Peptide Library (New England Biolabs, NEB).45 The M13 phage exposing the ZnO-binding peptide on pVIII protein was constructed with the use of filamentous phage display vector f88 (GenBank, Accession AF218363), kindly donated by George P. Smith (University of Missouri, Columbia, MO, USA). We designed and commercially synthesized a dsDNA fragment encoding the ZnO-binding peptide and including HindIII and PstI cloning sites used to ligate the fragment into the f88 vector. Next, Escherichia coli MC106154 was transformed with the constructed F88-ZnO vector. The correctness of the sequence obtained was confirmed by a commercially available sequencing service. Both phages (M13-pIII-ZnO and M13-pVIII-ZnO) were then propagated in E. coli ER2738 (NEB), according to the NEB protocol, and were suspended in TBS buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl) or in water. In the control experiments, we used M13KE (NEB) phage with wild-type pIII and pVIII proteins. Western Blotting. A clear phage lysate (at a concentration of ca. 1 × 1012 pfu/mL) was mixed with Laemmli buffer and boiled at 95 °C for 5 min. The phage proteins were separated by SDSPAGE and electrotransferred onto PVDF membranes. Membranes were blocked with 5% nonfat dry milk in TBS Tween 1% and incubated with primary antibodies (Mouse monoclonal antiM13/fd/F1 Filamentous Phages antibody, PROGEN, and AntiM13 pIII Monoclonal Antibody, NEB specific for pVIII and pIII, F
DOI: 10.1021/acs.bioconjchem.6b00196 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry
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Luminescence Spectroscopy Measurements. The same glass slides covered with samples were used for photoluminescence (PL) measurements. Luminescence excitation and emission spectra were measured with a Perkin−Elmer LS 55B luminescence spectrometer with a reflection spectra attachment. None of the excitation spectra were corrected for the lamp and photomultiplier response. X-ray Diffraction Analysis. X-ray diffraction patterns (XRD) were recorded on a X-ray diffractometer (Xpert PROMPD, Philips) with Cu target Kα-ray (λ = 0.154 04 nm) in the range 2θ = 20−68°.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel.: +48 58 523 6041. Fax: +48 58 523 6025. Author Contributions
Kamila Ż elechowskaa and Joanna Karczewska-Golec contributed equally to this work/paper. #
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We would like to thank Dr. Aleksandra Hać for her assistance with Western blotting and Dr. Magdalena Narajczyk for her help with TEM. This work was supported by the National Science Center (Poland) project grant no. 1814/B/P01/2010/39.
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DOI: 10.1021/acs.bioconjchem.6b00196 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.bioconjchem.6b00196 Bioconjugate Chem. XXXX, XXX, XXX−XXX
