Anisotropic Gold Nanostructures: Optimization via in Silico Modeling

Oct 26, 2018 - The bioinformatics tool i-TASSER predicts the effect of amino acid sequences on metal binding sites and the secondary structures of the...
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Anisotropic Gold Nanostructures: Optimization via In-Silico Modeling for Hyperthermia Ajay Vikram Singh, Timotheus Jahnke, Shuo Wang, Yang Xiao, Yunus Alapan, soheila Kharratian, Mehmet Cengiz Onbasli, kristen kozielski, Hilda David, Gunther Richter, Joachim Bill, Peter Laux, Andreas Luch, and Metin Sitti ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b01406 • Publication Date (Web): 26 Oct 2018 Downloaded from http://pubs.acs.org on October 27, 2018

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Anisotropic Gold Nanostructures: Optimization via In-Silico Modeling for Hyperthermia †, ζAjay

Vikram Singh*¥, ‡Timotheus Jahnke¥, †, ‡Shuo Wang¥, †, ‡Yang Xiao, †Yunus

Alapan, §Soheila Kharratian, ∥Mehmet Cengiz Onbasli, †Kristen Kozielski, #Hilda David, #Gunther Richter, ‡Joachim Bill, ζPeter Laux, ζAndreas Luch and †Metin Sitti †Physical

Intelligence Department, Max Planck Institute for Intelligent Systems, 70569

Stuttgart, Germany. ‡Institute

for Materials Science, University of Stuttgart, Heisenbergstr. 3, 70569 Stuttgart,

Germany §Department

of Materials Science and Engineering, Koç University, Sarıyer, 34450

Istanbul, Turkey ∥Department

of Electrical and Electronics Engineering, Koç University, Sarıyer, 34450

Istanbul, Turkey #CSF

Thin Films Group, Max Planck Institute for Intelligent Systems, Stuttgart 70569,

Germany. ζDepartment

of Chemical and Product Safety, German Federal Institute for Risk

Assessment (BfR), Max-Dohrn-Strasse 8-10, 10589, Berlin, Germany

ABSTRACT Protein- and peptide-based manufacturing of self-assembled supramolecular functional 1

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materials has been a formidable challenge for biomedical applications, being complex in structure and immunogenic in nature. In this context, self-assembly of short amino acid sequences as simplified building blocks to design Metal–Biomolecule Frameworks (MBioFs) is an emerging field of research. Here, we report a facile, bioinspired route of anisotropic nanostructure synthesis using gold binding peptides (10-15mers) secreted by cancer cells. The bioinformatics tool i-TASSER predicts the effect of amino acid sequences on metal binding sites and the secondary structures of the respective peptide sequence. Electron microscopy, x-ray, infrared, and Raman spectroscopy validated the versatile anisotropic gold nanostructures and the metal-bioorganic nature of this biomineralization. We studied the influence of precursor salt, pH, and peptide concentration on the evolution of nanoleaf, nanoflower, nanofiber, and dendrimer like anisotropic MBioFs. Characterization of photothermal properties using infrared laser (785 nm) revealed excellent conversion of light into heat. Exposure of bacterial cells in culture exhibits high rate of photothermal death using lower laser power (1.9 W/cm2) compared with recent reports. The MBioF’s self-assembly process shown here can readily be extended and adapted to superior plasmonic material synthesis with a promising photothermal effect for in-vivo biofilm destruction and cancer hyperthermia applications. KEYWORDS:

Metal–Biomolecule

Frameworks,

Biomineralization,

Photothermal effect, Surface Plasmon Resonance

2

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i-TASSER,

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* Corresponding author: [email protected] ¥ Equally

contributing authors

Metal and polymeric nanoparticles (NPs) can be utilized in a wide range of biomedical applications due to their size- and shape- dependent biophysical and optoelectronic properties

1-2.

Particularly, nanoparticles fabricated by using peptides containing short

sequences of amino acids can synthesize specific types of nanomaterials via controlled self-assembly. The physical and biochemical properties of these peptides are strongly dependent on constituent amino acids, which can be tailored for hydrophobicity, size, charge, and polarity. Non‐covalent interactions such as π-π orbital interactions, Van der Waals forces, ionic interactions, hydrogen bonding, and hydrophobicity predominantly control the self-assembly process of these short amino acid sequences. Manipulation of the environmental conditions as a function of covalent interactions has been used to fabricate versatile anisotropic protein/peptide nanostructures (e.g., nanocages3, nanogels4, nanovesicles5, nanoplates6, nanofibers7, nanotubes8). Using this approach of fabricating self-assembled metal hybrid nanomaterials, novel properties can also emerge as a collective function of individual amino acid sequences9. Biological functionality and stability of these self-assembled peptides can be further improved via ligand binding, enhanced additive functionality, and specificity of short amino acids10. However, the fabrication of nanomaterials based on self-assembly of short amino acid sequences for biomedical applications has not yet been achieved. This is due to the limited resources 3

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available to manipulate the peptide nanostructures via non‐covalent interactions and the limited availability of knowledge and bioinformatics tool to predict and control the process11. There are few reports based on ecofriendly or biological fabrication routes of such anisotropic structures for biomedical applications12. So far, reports adopting a biogenic synthesis route either did not elucidate the specific mechanism13, or the reported anisotropic structures have not been tested for biomedical applications14. Furthermore, there are no studies reporting systematical optimization of process parameters which could yield the largest anisotropy in such structures to promote their biomedical application. We have recently reported a cancer cell (MCF-7 breast cancer cell line) mediated biomineralization process of ionic gold to spherical nanoparticles and anisotropic microplate structures depending on serum composition of the respective cell culture media14. We have shown that specific defense proteins related to cellular stress in cancer cells are able to reduce ionic gold and subsequently bind to specific crystal surfaces. Upon addition of gold precursor to the cell culture medium, a sudden pH change occurs, which denatures the serum and cell membrane proteins. This triggers the reduction of ionic Au3+ to spherical Au0 nanoparticles, which leads to anisotropic growth of the gold NPs. As viewed from a molecular level, the instantaneous pH drop unfolds the 3D protein structures, which enables access to hydroxyl and thiol groups. Particularly; functional groups in tyrosine and tryptophan acts as electron donors and stabilizers for gold seeds and NPs, assisting in reduction of Au3+to metallic AuNPs 15-16. 4

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With this method, using slow reduction and seed-nucleated growth, we have been able to fabricate

triangular/hexagonal

microplates14,

17.

We

identified

novel

gold-binding/reducing polypeptide sequences from these secreted defense proteins by using high throughput proteomic approaches and a quartz crystal microbalance with dissipation (QCM-D) to screen gold-reducing proteins and peptide sequences. We hypothesized that solely these gold binding peptides (GBPs) that are found in these proteins

can

be

used

micro-to-nanostructures

with

as

bottom-up superior

self-assembly

plasmonic

of

properties.

the

anisotropic

These

anisotropic

nanoparticles can be applied in the photodynamic and photo-thermal therapy (PPTT) against tumors and for antimicrobial surface development 18-19. To test this hypothesis, we purchased commercially synthesized polypeptide libraries using 9‐fluorenylmethoxycarbonyl (FMOC) with short amino acid sequences (10-15 mers) based on the gold binding defense proteins found in our recent study14. By incubating the material at different gold ion and peptide concentrations, we observed dramatic changes in anisotropy. Furthermore, we studied the effect of different pH values on the growth of anisotropic nanofiber, nanoflower, nanoleaf, and dendrimers with different aspect ratios and geometries over time. Electron microscopy (SEM/TEM) was used to observe the high-resolution structures and their crystallinity. Various spectroscopic techniques (X-ray/FTIR/Raman/XPS) confirmed the metal-biogenic nature of the biomineralized hybrid MBioFs. Further biocompatibility and hyperthermia 5

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application were validated using suitable cellular assays, microscopy and NIR photothermal imaging and irradiations. RESULTS AND DISCUSSION In the previous work published by Singh et al., several specific peptide sequences, obtained from the cancer cells (MCF7), caused the anisotropic mineralization of gold. An in-vitro approach in the presence of cancer was used to investigate the influence of different peptide sequences on the biomineralization of gold. In order to explore this mechanism further without using live cancer cells, and to design plasmonic nanostructures with maximum anisotropy, we have used custom-made synthetic peptides. It is suggested that the secondary structure and metal ligand binding sites of the peptide sequences play a major role on the biomineralization of inorganic metals20. Therefore, we studied the secondary structures and ligand binding sites of the respective peptide sequences with an iterative threading assembly and refinement (i-TASSER) bioinformatics tool. We believe that it elucidates the fabrication conditions of anisotropic gold nanostructures. This, then, clarifies the synthesis mechanism and tuning of the morphology of the gold particles according to the desired applications. Ab initio modeling to predict the structure and function of peptide sequences Three peptide sequences obtained from the studying cancer cell secretions were selected based on their net positive charge to promote interaction with negatively charged gold ions (AuCl4). Based on their theoretical calculations using an online software 6

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(http://pepcalc.com/ Pepcal, Innovagen, Lund, Sweden), the isoelectric point and solubility were estimated (Table S1). The automated i-TASSER platform was used to predict the secondary structure and metal ligand-binding sites for these three different peptide sequences. In this method, the models of the peptide sequences are constructed by simulating the fragments’ structure. These bioinformatics modeling tools utilize input of amino acid sequences to predict the structure-function relation between gold-binding peptides. Table 1 and Figure 1 A-F display the i-TASSER results. Most amino acids presented in the peptide FWCYHAGHVL have the form of coils (C), with only three amino acids in form of sheets near the N-terminus of peptide. This is a similar secondary structure to VVAGSGGHTT, where most of the amino acids in the chain are coiled with only two amino acids in the form of sheets near the N-terminus. The third peptide sequence, MSRCWQPNPR, only includes coiled amino acids. Additionally, the metal binding sites on different peptide sequences were simulated using i-TASSER and are also shown in Table 1. On FWCYHAGHVL and MSRCWQPNPR, the metal binding sites are 7, 9 and 5, 6 respectively, which are close to the center site of peptide sequences, while the metal binding sites 8, 9, 10 on VVAGSGGHTT are close to C-terminus. In the Figure 1 A-C, each color corresponds to one type of amino acid. The twisted structures represent the secondary coil structure (C), whereas the wide arrow-like structure present the secondary sheet structure (S). In Figure 1 D-F, the metal ligand-binding sites are shown in purple and the green spheres represent immobilized metal ions. To check, the stability of polypeptides in solution, we performed Vibrational Circular Dichroism (VCD) on 7

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3 sequences studied herein, which vividly exhibit stable Amide I and II bonds originating from the polypeptides sequences as shown in figure S1, and confirm secondary structure of polypeptides. To experimentally validate the number of ligand binding sites predicted with i-TASSER based on their C-score on gold binding specificity, we performed Quartz Crystal Microbalance with Dissipation (QCMD) test via flowing the 3 peptides and a random control polypeptide with higher number of ligand binding sites on gold coated quartz crystal.

The

net

mass

addition

on

the

gold

QCM

crystal

due

to

polypeptide adsorption/desorption is studied with kinetic coefficients which reflected by drop in frequency (Δf) or more desorption of polypeptides with less ligand binding sites or less specificity as function predictions (Table S2).

Table 1. The predicted secondary structure and metal ligand-binding sites of peptide sequences (Note: C-Coil form, S-Sheet) Metal Predicted ligandName of Protein

Sequence

secondary binding structure sites

Cleavage and FWCYHAGHVL

CCSSSCCCCC

Polyadenylation

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7, 9

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specificity factor Polar subunit 3 (CPSF3) Insulin receptor related MSRCWQPNPR CCCCCCCCCC

5, 6

protein (INSRRP) ALG14-UDP-N-acetylglu cosamine transferase

VVAGSGGHTT

CSSCCCCCCC

8, 9, 10

(ALG14) There are three main factors that determine the formation of secondary structures and the interactions forming gold nuclei, which subsequently transform into anisotropic structures via a seed mediated growth: (1) Electrostatic interaction: Similar charges distributed along the peptide chain promote the formation of secondary coil structures (Figure 1.G), because the repulsion among neighboring amino acids impede the hydrogen bond formation21. The homogenous distribution of opposite charges on the peptide chain (Figure 1.H) is beneficial for the formation of the secondary sheet structures. The electrostatic attraction between different parts of the peptide sequence enhances hydrogen bond formation22. (2) Steric hindrance: An amino acid with large side groups (phenylalanine, tyrosine and tryptophan) prefers the formation of a coil secondary structure, because higher steric hindrance inhibits the formation of regular structures (helix and sheet)23. (3) Degrees of freedom regarding chain rotation: The side group is a five-membered ring

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(proline) including a secondary amine, where the nitrogen atom does not to connect with a hydrogen atom and the C-N bond cannot rotate in the amide group. The absence of glycine in the side groups is beneficial for the C-N rotation to form hydrogen bonds23. According to the explanation given above, most of the peptide chain in FWCYHAGHVL, MSRCWQPNPR and VVAGSGGHTT is in the form of coils because they include amino acids with large side groups (phenylalanine, tyrosine and tryptophan) except glycine or proline. These coils are beneficial to immobilize oppositely charged ions on the peptide chain and subsequently reduce them into nuclei, since most of the charges along the peptide sequences are not compensated in a coil form due to neighboring amino acids with similar charges. Except for the influence of the secondary structure, the metal ligand-binding sites also influence the formation of anisotropic nanoparticles. From the Table 1 and Figure 1 D-F, we have found that the metal ligand-binding sites are close to the C-terminus, because the end of peptide sequences can provide more space for the gold nuclei formation and gold nanostructures. Further, the availability of simpler amino acid such as glycine for metal binding could be argued in few sequences since amino acid is conjugated into a peptide and being integrated into an amide bond, not available for metal ion binding. However, the metal ligand interaction is multifactorial and complex process. We cannot rule out the likelihood of cooperative metal binding to glycine and other metal binding residue in the FWCYHAGHVL peptide sequence, which in turn could depend upon many factors such 10

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as water coordination, zwitterionic states of glycine or pH of surrounding medium (HAuCl4 gives H+ upon dissociation into water changing pH of medium)24. Since we consistently observed the origin of anisotropic nanoflower like structures when gold ion was added to FWCYHAGHVL peptide solution, also it could be possible that gold ion addition partially destabilizes few moieties initiating gold ion binding to these free moieties and further self-assembly with stabilized moieties in the solution. The metal-ligand distances, which depend upon metal-ligand coordination number (CN), may further act a key player in this context. Calculations of the molecular structure and relative stability of the Glycine with different metal ions indicate that in the complexes with monovalent metal cations the most stable species are the NO coordinated metal cations in non-zwitterionic glycine. On the other hand, divalent cations prefer coordination via the O-O bifurcated bonds of the zwitterion glycine. Stepwise addition of water molecules leads to considerable changes in the relative stability of the hydrated glycine species. These factors warrant further systematic experimental investigation. Scanning electron microscopy and energy-dispersive X-ray spectroscopy Scanning electron microscopy (SEM) was used to visualize the fabricated gold nanostructures at three different peptide concentrations. The SEM images reveal the influence that different amino acid sequences and their concentrations have on anisotropy. Figure 1 I-Q and S2 shows the morphology of fabricated gold particles. Here, we kept the gold ions concentration constant (1.0 mmol/L), and varied different concentrations of three

peptide

sequences

from

10 µmol/L,

50 µmol/L

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100 µmol/L.

The

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FWCYHAGHVL and MSRCWQPNPR peptide sequences show cauliflower-like structures (agglomerated spherical nanoclusters) at lower concentrations (10 µmol/L) (Figure 1.I-L). However, at middle and higher peptide concentrations (50 µmol/L and 100 µmol/L), we observed the evolution of anisotropy to agglomerated two-dimensional flower-like structures (compare Figure 1 J-M with Figure 1 K-N). We observed contrary results with the VVAGSGGHTT peptide sequence, which displayed more nanoleaf- and nanoflower-like anisotropic structures (Figure 1.O) at low concentrations. This can be explained with the few metal ligand-binding sites in the MSRCWQPNPR and FWCYHAGHVL peptide sequences, which initiated spherical nuclei at lower peptide concentrations. However, at a higher peptide concentration, there are plenty of two-dimensional nuclei formed and agglomerated into anisotropic structures. Contrary is the case for the peptide VVAGSGGHTT. The change in anisotropy is not clearly related to a variation in secondary structures, as peptide sequence VVAGSGGHTT and FWCYHAGHVL have similar coil- and sheet- like secondary structures (Table 1, Figure 1 A-C). Based on nanostructures formed by three different peptide sequences, VVAGSGGHTT was observed to be the most suitable peptide sequence for the generation of structures with maximal anisotropy. Therefore, we used only the VVAGSGGHTT sequence to study the influence of concentration (peptide versus gold ions), pH, and incubation time on anisotropy. In a recent report, authors demonstrated that Valine (V), Glycine (G) and Histidine (H) are able to reduce the Au3+ ions into nanoparticles25. Therefore in 12

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VVAGSGGHTT sequence even if Tryptophan is absent, other gold reducing amino acid makes the VVAGSGGHTT a favorite sequence for optimized nanoflower synthesis. In

Figure 1.R, we discuss schematically how the different concentrations of VVAGSGGHTT peptides play a role in anisotropic formation of gold nanostructures as compared with SEM micrographs (Figure 1.O-Q). At the first step, the AuCl4 ions are immobilized on the peptide backbone by electrostatic attraction and coordinate covalent bonds. The attached AuCl4 ions are reduced into gold nuclei by amino acids acting as electron donors. At this point, crystal growth occurs and two-dimensional platelets are formed, which are then assembled into dendrimer, nanoflower- or nanoleaf-like anisotropic structures. At a concentration of 50 µmol/L and 100 µmol/L VVAGSGGHTT, only gold nanospheres are formed (Figure 1.P-Q). The total amount of AuCl4 ions was the same in these three different VVAGSGGHTT concentrations; therefore, the peptide concentration of 50 µmol/L and 100 µmol/L VVAGSGGHTT was in excess corresponding to the 1.0 mmol/L HAuCl4. In this case, the excess peptide almost capped the whole surface of the formed gold nuclei26.

Since VVAGSGGHTT was positively

charged at neutral pH, the attached VVAGSGGHTT continued to attract

AuCl4 ions

and reduce them. Therefore, the anisotropic growth of gold particles was impeded. At the 10 µmol/L concentration of VVAGSGGHTT, gold nanoflowers and nanoleaves (Figure 1.O) were fabricated because there was no excess amount of the peptide. Therefore, only part of the gold nuclei surface was capped by the peptide sequences, which caused the anisotropic growth of gold nuclei into nanoleaves and nanoflowers. Subsequently, size 13

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uniformity of anisotropic gold nanostructures was gradually improved by Ostwald ripening. Spectroscopic analysis of anisotropic nanostructures Energy dispersive x-ray analysis To confirm the presence of gold and other biogenic elements (e.g. carbon, nitrogen and oxygen from peptides), we performed energy dispersive X-ray (EDX) analysis. Results demonstrated the presence of a strong gold signal, as shown in Figure 2.A-B. Other elements from peptides were also detected, but the signals were weak due to the low amounts of peptide and the entrapment of peptide sequences in the gold nanoflowers, since they act as the template. Raman and Fourier-transform infrared spectroscopy VVAGSGGHTT contains the polar and non-polar amino acid residues; hence, we used Raman spectroscopy to evaluate the polar and non-polar groups in the respective peptide residues. In Figure 2.C, the weak band at 349 cm-1 is assigned to the out-of-plane C-CH3 vibration from amino acids17. It verifies that VVAGSGGHTT contains valine and alanine residues, whose side groups include C-CH3 groups. The medium band at 423 cm-1 shown in Figure 2.C is associated with the carbon backbone vibration of VVAGSGGHTT17. The band at 755 cm-1 is related to the out-of-plane vibration of the imidazole ring, which belongs to the histidine residues in VVAGSGGHTT. The imidazole ring is a highly polar compound with a positive charge located on either of two nitrogen atoms present in the ring. Its existence is beneficial to attract HAuCl4 ions on VVAGSGGHTT template. The 14

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very weak and broad band at 1637 cm-1 is attributed to C=O stretching27, which corresponds to the amide groups in the peptide sequence. The detected peaks from the Raman spectra are summarized in Table 2. Table 2. The specific Raman bands for the gold conjugated peptide sequences Raman Sample

Assignment Bands[cm-1] Out-of-plane vibration of C-CH3 349 group 423

Skeleton vibration

VVAGSGGHTT Out-of-plane vibration of imidazole 755 ring 1637

C=O stretching (amide I)

In order to cross-check the existence of amino acid residues in VVAGSGGHTT Fourier-transform infrared spectroscopy (FTIR) was used to characterize the functional groups involved in the respective peptide – gold bonds (Figure S3a-S3b and Table S3). The stretching of the C=O band (amide I) exhibits a red-shift from 1603 cm-1 (black curve) to 1625 cm-1 (red curve). The band for N-H and C-N (amide II) also displays a red-shift from 1481 cm-1 (black curve) to 1522 cm-1 (red curve)28. These shifts are attributed to the formation of Au-VVAGSGGHTT complexes29. Gold is the electron acceptor because there are empty orbitals in valence shell, while the oxygen and nitrogen

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act as donors due to their lone pairs of electrons. Therefore, the Au-VVAGSGGHTT complexes can be formed. Additionally, in Figure S3b, the N-H (amide II) band shows a shift from 3221 cm-1 (black curve) to 3226 cm-1 (red curve)30. The O-H band is represented by a blue-shift from 3343 cm-1 (black curve) to 3333 cm-1 (red curve)30. This O-H band is attributed to the vibrations of the carboxyl moiety and alcoholic group. The shift is related to the hydrogen-bond formation in this sample. X-ray photoelectron spectroscopy The interaction between gold and amide group in VVAGSGGHTT as shown in previous FTIR spectra section, cannot demonstrate the valence state of the gold atoms. To confirm the reduction of Au (III) ions into metallic Au (0), X-ray photoelectron spectroscopy (XPS) was performed to characterize the valence state for gold. Moreover, XPS can comprehensively reveal the chemical composition for the sample. Figure 2.D shows the main elements (Au, C, O, N, Na and Si) are present in this sample. The sodium signals present in the XPS spectra correspond to the salts in the sample. The silicon signals are attributed to the silicon wafer, which was used as a substrate for XPS sample. In order to observe the characteristic peaks for the Au 4f, C 1s and O 1s more clearly, a narrow scan was performed. Figure 2.E displays the Au 4f peaks, which are located at the 90.3 eV and 86.6 eV, corresponding to metallic Au (0). It implies the reduction of Au (III) ions into metallic Au (0) by electron donors on the peptide sequence VVAGSGGHTT31 . In the Figure 2.F, the strong peak at 287.9eV from C 1s is assigned to the C=O bond in the amide group31. This is also confirmed by the two characteristic peaks for O 1s from 16

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C-O-H at 535.1 eV and C=O at 533.7 eV (Figure 2.G)32. The C-O-H group is attributed to serine residues containing hydroxyl groups, or the formation of hydroxyl groups during the reduction process. The C=O group is assigned to the amide group on the peptide chain. C 1s and O 1s spectra further verify the existence of amino acid residues and complement the EDX data. Transmission Electron Microscopy (TEM) Figure 2.H-I show TEM and selected area electron diffraction (SAED) patterns from nanoflowers obtained with VVAGSGGHTT polypeptide sequence shown in Figure 1.O. The image captured from the side of the nanoflower shows quasi-folded, petal-like structures with embedded nanoparticles, which could be seeds forming these anisotropic structures (Figure 2.H). The high-resolution image shown in Figure 2.I show crevice curves and local defects inside these petal-like structures. The SAED pattern shown in the inset of Figure 2.I exhibits the polycrystalline nature of these nanoflowers. Effect of HAuCl4 concentration on anisotropic growth It was previously noted that the HAuCl4 concentration influences the formation of anisotropic gold nanostructures26. The SEM measurements (Figure 3. A-C and Table S4) were used to investigate the influence of HAuCl4 concentrations on fabricated gold nanostructures. The concentration of the HAuCl4 solution was varied from 0.5 mmol/L to 1.5 mmol/L. At the 0.5 mmol/L concentration of HAuCl4 (Figure 3.A), we observed microspheres composed of gold nanoclusters, whose surface was covered by spherical gold nuclei. Figure 3.B shows the shapes of fabricated gold nanostructures, which are 17

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nanoflower- and nanoleaf-like at a concentration of 1.0 mmol/L HAuCl4. These nanoflower branches are constructed out of nanoleaves. With the increasing HAuCl4 concentration (1.5 mmol/L HAuCl4), thick gold nanosheets were fabricated with structures looking like large nanoleaves (Figure 3.C). This implies that the formation of nanosheets occurred through the stacking and ripening of nanoleaves. Therefore, they are thicker than the nanoleaves formed at 1.0 mmol/L. Figure 3.D represents a schematic for the formation of gold nanostructures at three different HAuCl4 concentrations. In the first and second steps, the mechanism is the same as mentioned in the schematic Figure 1.R. At 0.5 mmol/L concentration of HAuCl4, excess peptide VVAGSGGHTT is present. A few nuclei are formed initially and capped by the excess peptide VVAGAGGHTT, yielding spherical nanostructures (Figure 3.A). On the contrary, when the concentration of HAuCl4 was 1.0 mmol/L or 1.5 mmol/L, there were more nuclei present which were not capped, so growth of the nuclei was possible. However, at a concentration of 1.5 mmol/L HAuCl4, the peptide VVAGSGGHTT could only cap a small fraction of the gold nuclei’s surface, and free AuCl4 ions were continually attracted and reduced in-situ by VVAGSGGHTT in between the growing branches (Figure 3.C). At an AuCl4 concentration of 1.0 mmol/L, the formed gold nuclei grew into gold nanoflowers and nanoleaves (Figure 3.B) as discussed in previous schematic Figure 1.C. Effect of pH value on anisotropy Due to the influence of the pH value on the charge of the peptide sequence and the 18

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dissociation of HAuCl4 in the solvent, we studied the effect of different pH values on the shape of the fabricated gold particles by using SEM16 (Table S5). In acidic conditions (pH = 2, Figure 3.E), branched nanoleaves grew, which include a series of small side petals oriented parallel to one another. Their dendrimer-like branching or and improved anisotropy is better at lower pH values than higher pH values. At neutral pH, the nanoflower branch was composed of nanoleaves (Figure 3.F). While these nanoleaves were not parallel to each other, a random arrangement of the petals was observed. Comparing Figure 3.F with Figure 3.E, there are fewer nanoleaves with a midrib in Figure 3.F, and the aspect ratio (length/width) of the fabricated nanoleaves in Figure 3.F is smaller than in Figure 3.E. In alkaline conditions, only nanospheres (Figure 3.G) were fabricated by VVAGSGGHTT, whose surface was covered by spherical gold nuclei with similar size and shape. The optimum pH for anisotropic growth was determined to be pH = 2, as shown in the SEM images (Figure 3.E). Figure 3.H schematically describes the formation of gold nanostructures at these three different pH values. The isoelectric point (IEP) for VVAGSGGHTT is 7.78 (Table S1). When the pH value is lower than the 7.78, the VVAGSGGHTT is positively charged and these positive charges are homogenously distributed on the entire peptide sequences. The existence of positive charges on the coil form led to stronger repulsion among the neighboring amino acid residues

21, 33-34.

Thereby, more space is available to form gold nuclei and further anisotropic growth into nanoflowers and nanoleaves occurs. On the other hand, the acidic conditions inhibited the 19

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dissociation of HAuCl4. When the AuCl4 ions were consumed during the reaction, the new AuCl4 ions would be released by the further dissociation of HAuCl4. This process controls the amount of free AuCl4 ions in the solution, which provides more time for gold nuclei to arrange and grow, which is beneficial for optimum anisotropic growth at lower pH values. At pH = 7, the regions of positive charge are not as prevalent as shown for pH = 2 (IEP = 7.78) in coil form. However, their existence could lead to the repulsion among the neighboring amino acid residues. Further, there was limited space on coil form to arrange the gold nuclei as compared to pH = 2. Moreover, the dissociation of HAuCl4 was not inhibited or promoted at pH = 7. The amount of free AuCl4 ions was moderate at pH = 2, which provided time to arrange gold nuclei, resulting in formation of gold nanoflowers and nanoleaves (Figure 3.F). By contrast, at pH = 11 the peptide VVAGSGGHTT was negatively charged, since it is higher than the IEP. This situation also provided more space to form gold nuclei and anisotropic gold nanostructures due to repulsion among the neighboring amino acid residues. However, the existence of negative charges was a disadvantage to attract free AuCl4 ions on peptide sequences, which inhibited the anisotropic growth of the gold nuclei. Moreover, the higher pH value promoted the dissociation of HAuCl4, which led to the sufficient amount of free AuCl4 ions. In this case, limited time was provided to arrange gold into nuclei. Combining these two explanations, the gold nanospheres were formed by VVAGSGGHTT at pH = 11 (Figure 3.G). Effect of incubation time on anisotropy 20

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We optimized other synthesis parameters (VVAGSGGHTT: 10 µmol/L, HAuCl4: 1.0 mmol/L and pH = 2) according to the formation of most versatile gold nanostructure. Moreover, we were able to elucidate the formation mechanism for gold nanoflower- and nanoleaf-like structures. SEM images (Figure 4.A-E) show the different shapes of formed gold particles with the change of incubation time. Figure 4.A shows the gold nanofibers and their surface was covered by short, rod-like gold nuclei, which verifies that the gold nuclei grew and self-assembled anisotropically in the presence of VVAGSGGHTT peptide sequences. In Figure 4.B, the nanoleaves were formed through the further anisotropic growth of gold nuclei and arranged along a primary rod. With prolonged incubation time, they seem to be composed of a series of small nanoleaves arranged parallel to each other along a stem (Figure 4.C). When the incubation time reached 75 hours, we observed that a series of small nanoleaves were aggregating by Ostwald ripening (Figure 4.D). With further aggregation of nanoleaves, the larger nanoleaves were formed (Figure 4.E). The surface of these larger nanoleaves were smooth, and the midrib-like structures clearly seen at 60 hours had almost disappeared. In order to make a comparison with objects existing in nature, Table S6 presents the different SEM images for fabricated gold nanostructures at the different incubation time and pictures for objects existing in nature. The optimum incubation time (60 hours) was selected using the SEM images, (Figure 4.C). Figure 4.F shows in detail the scheme for the formation of anisotropic gold nanostructures. First, the AuCl4 ions were attached onto the peptide backbone by 21

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electrostatic attraction and coordinated covalent bonds. The electrostatic attraction is introduced by the opposite charges between VVAGSGGHTT and AuCl4 ions26. The coordinated covalent bonds were attributed to the complex-formation on the metal ligand-binding sites and amide groups35. Secondly, VVAGSGGHTT reduced the immobilized AuCl4 ions into gold nuclei, which were kinetically favored structures with lowest energy (111) facets26 . Here, the 10 µmol/L VVAGSGGHTT and 1.0 mmol/L HAuCl4 were the optimum concentrations, which were used to study the influence of incubation time. At this concentration, VVAGSGGHTT capped a moderate area of the gold nuclei, and then attracted free AuCl4 ions and reduced them in-situ. The short rod-like gold nuclei were formed and covered the self-assembled peptide backbone to generate one-dimensional nanofiber-like structures (Figure 4.A), which gradually grew into scale-like nanoleaves, arranged regularly to form cypress-like nanoleaves (Figure 4.B). Continued anisotropic growth of cypress-like nanoleaves generated pinnate-like nanoleaves with parallel branches along a central stem (Figure 4.C). We believe that branching from one-dimensional structures (compare Figure 4.A vs. Figure 4.B) to more asymmetric growth of branches (compare Figure 4.B vs. Figure 4.C) are initiated by homogeneously positive charge distribution on VVAGSGGHTT, which results in stronger repulsion among the neighboring amino acid residues. With an increase in incubation time, repulsion on VVAGSGGHTT was slightly mitigated in space to favor the growth over nuclei into nanostructures. Following further incubation, Ostwald ripening dominated, which transformed the pinnate-like nanoleaves into aggregated 22

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nanoleaves (Figure 4.D) and then to larger nanoflower/nanoleaf structures (Figure 4.E). The increase of the size of anisotropic structures led to decrease in the total surface energy (surface/volume) and simultaneously made this system stable26.

Photothermal effect of plasmonic nanoflowers Spherical NPs with 100 nm diameter exhibit surface plasmon resonance (SPR) around 570 nm, without a strong absorption peak in the NIR region. The branched features of nanoflower-like gold structures shown in this study display a strong optical absorption extending in NIR as shown by a broad peak in UV-VIS spectrum in Figure S4. As shown in the previous section, thin petal-like morphologies arranged around the midrib of the nanoflower-like structures (Figure 3.E) could exhibit very diverse plasmon transmission through corners to adjacent edges. The evolution and propagation of equidistant maxima of energy loss along the nanoflower petal’s edge, and corners make these anisotropic structures a strong candidate for photothermal applications36. These unique features could contribute to enhanced photothermal destruction of bacterial biofilms using these nanoflowers as long-range plasmonic wave-guides. Keeping this in mind, we performed simulation studies to understand the dispersion of a plasmon along the nanoflowers with finite-difference, time-domain calculations, which revealed the presence of wedge plasmon polaritons propagating along the nanoflower’s platelet like edges (Figure 5. A-D). Other nanoflower like structures also exhibited with high plasmonic effect in-and-around nanopetals as shown in Figure S5-7. We also experimentally investigated the rise in the 23

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temperature when nanoflowers were irradiated with an IR laser. For this, we purified the nanoflowers using overnight dialysis (340 kDa cut-off). Subsequently, the nanoribbons were irradiated with a 785 nm NIR laser at a power density of ~1.9 W/cm2 in DMEM medium, water and phosphate buffered saline (PBS) as controls. In contrary to the control group, where no obvious increase in temperature could be observed, nanoflowers exposed to NIR laser exhibited an increase in temperature of up to 61.1 ºC (Figure 5. E-F). Compared to the rise in temperature (up to 33 °C) of commercial spherical NPs, we recorded an excellent photothermal effect using nanoflowers

14, 37.

Bacterial biofilms are

a major problem and huge economic burden on the biomedical implant industry, as very often these grow on implants and catheters used in the clinic38. To test the effect of photothermal heating on bacterial biofilms, we mixed the nanoflowers with high density of E. coli and irradiated with NIR laser, where we found significant increase in bacterial dead cells compared with control or bacterial cells without Nanoflowers (student t-test, p = 0.03). As shown in Figure 5.K-N live-dead staining shows more red stained Propidium iodide (PI) uptake by dead cells in nanoflower treated samples compared to untreated control (Figure 5. G-J) or spherical gold NPs treated bacterial cells (Figure 5. O-R). We noticed 40% higher dead cells in nanoflower mediated hyperthermia samples compared to untreated bacterial cells irradiated with NIR laser (Figure S8). Therefore, these nanoflowers could be very effective for photothermal therapy not only for bacterial cells but also for the tumor cells. Conclusions 24

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We show here an in-silico and in-vitro model to fabricate versatile anisotropic nanoflower-like structures starting from a short sequence of amino acids as a bottom-up approach (schematic Figure 5.S). We investigated the optimum synthesis conditions that yield the maximum anisotropy with an excellent photothermal effect, which is in agreement with the simulations. The model demonstrated here could be extended to in-vivo applications of these nanomaterials, such as killing cancer cells. Alternatively, ionic gold can be directly injected into tumors in-vivo to homogeneously diffuse and in-situ reduction for enhancing photothermal effect in PPTT applications. The biogenic route for the synthesis of anisotropic NPs are highly advantageous for controlling microbial colonization over biomedical instruments such as catheters, stents and implants/prosthesis. We believe that the biogenic, peptide-based method described herein can be tuned to yield more varieties of anisotropic metal-biomolecule frameworks (MBioFs) via adding seed NPs and customizing short amino acid sequences39. In context with the amino acid sequences used herein, the hydrophobicity, polarity, charge, and size of the polypeptide side chains can be further used as control parameters to give rise to novel anisotropic nanomaterials. Materials and Methods Chemicals: HAuCl4·3H2O powder (99.9%, 5 g) was purchased from the Sigma Aldrich. Phosphate buffered saline (PBS: 99.9%, 500 mL) was purchased from the Gibco (Thermo Fischer Scientific, Germany) and dimethyl sulfoxide (DMSO: 99.9%, 3 mL) was purchased from the Life technologies (Thermo Fischer Scientific, USA). PBS was used to 25

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dissolve good water soluble peptides, while DMSO was used to dissolve poor water soluble peptide. In order to adjust the pH value of the sample solution, HCl solution (37%, 1 L) was purchased from Carl Roth (Karlsruhe, Germany); NaOH solution (50%, 1 L) was purchased from the Sigma Aldrich. Peptide sequences: FWCYHAGHVL (>65%, 7.6 mg), MSRCWQPNPR (>65%, 6.4 mg) and VVAGSGGHTT (>65%, 7.4 mg) powder were purchased from the EMC microcollections GmbH. They are manufactured by using the Fmoc solid-phase method on the PEG-polystyrene support resin. The peptide sequence is assembled step by step, which means the one type of amino acids is added per each time. The N-terminus of each amino acid is protected by a Fmoc (9-fluorenylmethoxycarbonyl) group. Only the active C-terminus of an amino acid can be coupled to the growing peptide chain. Then the Fmoc group is removed by the piperidine treatment to generate growing chains, used in the next reaction. When the assembly of peptide sequence has been finished, the peptide sequence is removed from the PEG-polystyrene support resin by trifluoroacetic acid (TFA). Simultaneously, the Fmoc group is removed from the N-terminus. At the end, the peptide sequence is purified by reverse phase high performance liquid chromatography (HPLC).

Synthesis of gold nanoflowers During the synthesis of gold nanostructures, the detailed information about synthesis parameters (e.g. the concentrations of peptide sequences and HAuCl4, pH values and incubation time) are shown in the supporting information. Integrated ab-inito modeling 26

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of

peptide

structure-function

analyses

using

Iterative Threading ASSEmbly Refinement (i-TASSER ) We used i-TASSER bioinformatics tools to predict metal ligand and polypeptide, and/or peptide-peptide interactions to understand initial events of gold ion binding, and in-situ reduction. All sequences were submitted to i-TASSER server at Dr. Zhang Lab at University of Michigan which returns detail analysis of predicted ligand binding residues, position and polypeptide secondary structures via a link to download the all necessary information.

Scanning electron microscope imaging A LEO 1530VP Gemini scanning electron microscope (SEM) was utilized to characterize the morphology of the synthesized particles. An accelerating voltage of 3 keV and SE detector was used for imaging. To prepare samples for SEM, the silicon wafer was treated by plasma treatment to remove the organic contamination and form a silicon dioxide layers with hydrophilic surface. As a result, the poly-L-lysine was coated onto the hydrophilic surface, upon which was possible to fix freshly synthesized gold particles onto the surface of silicon wafer. When the deionized water was used to wash away the remaining salts, the synthesized gold particles would not be removed. The SEM sample was dehydrated by the five ethanol solutions (25% ethanol, 50% ethanol, 75% ethanol, 90% ethanol and 100% ethanol) in order to remove the water. Wafers with Au-peptide sequences were sputtered by deposition of 5 nm nickel using a Leica coating system 27

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(Leica EM, ACE600). Raman spectroscopy The NanoRamanTM platform integrates an inverted optical microscope and near-field optical techniques (SNOM or NSOM) (HORIBA Scientific Germany). The instrument was equipped with a liquid nitrogen-cooled CCD detector were used to evaluate the imaging of nanoflowers. Our sample solution was directly dropped on the cover glass to avoid any influence of dried salt crystals. In order to reduce the background and obtain a baseline for the Raman measurement, the cover glass was scanned without sample. The light source is the green laser with a wavelength of 532 nm and a power of 100 mW.

Fourier-transform infrared spectroscope analysis Chemical imaging of peptide sequences with and without HAuCl4 was done with Bruker Tensor II spectrometer with ATR sensor. Before dropping the sample solution on the attenuated total reflectance (ATR) sensor, PBS and isopropanol were used to clean the ATR sensor respectively. In order to reduce background and obtain the baseline, firstly the solvent (the PBS and DMSO mixture) was scanned 512 times by the Tensor II spectrometer with 2 cm-1 resolution in transmittance mode. Subsequently the sample was scanned at the same conditions. Data analysis was performed with OPUS.

X-ray photoelectron spectroscopy 28

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A theta Probe Angle-Resolved X-ray Photoelectron Spectrometer (Thermo Fischer Scientific, Germany) measured one SEM sample. The incident source was monochromatic Al Kα whose energy was 1486.7 eV. The diameter of measured area was 400 μm2 on the sample surface. The base pressure of chamber was maintained below 10–7 Pa.

Transmission electron microscope analysis In-situ TEM observation of gold nanoflower was performed by a JEM-2100HR transmission electron microscopy (JEOL, Japan) operated at 100 kV equipped with an energy-dispersive X-ray (EDX) spectrum. Firstly the sample solution was centrifuged under 500 RPM and 10 minutes, in order to remove 80% supernatant. Then deionized water was added into sample solution which was centrifuged as the above condition to remove the 80% supernatant. This process was repeated twice to wash out salts in the sample solution. Following the 100% ethanol (200 µL) was added into the 20% remaining sample solution. Finally the washed sample solution was dropped on the TEM grid, which was dipped in the Osmium tetroxide solution and then dried overnight under room temperature. NIR triggered hyperthermia To test photothermal properties of anisotropic gold nanoflower structures and spherical nanoparticles (test or not) synthesized by peptide sequences, as well as commercial gold nanorods (test or not), samples were loaded in capillary tubes and irradiated under NIR 29

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(780 nm, ~0.6 W/cm2). Thermal Images were collected using an infrared thermal camera (ETS320, Flir Systems, Wilsonville, OR, United States).

For in vitro hyperthermia

demonstration, Escherichia coli (E. coli, CGSC number 8237, Strain MG1655) were incubated with gold nanoflowers and spherical nanoparticles as control. Next, cells (?) were irradiated to with NIR laser of (780 nm, ~0.6 W/cm2) for 5 min at three different spots. After BIR exposure, samples were recollected on microscopic imaging coverslip for fluorescent bacterial viability testing (LIVE/DEAD BacLight Bacterial Viability Kit, Thermo Fisher Scientific, Waltham, MA) with trypan blue and Calcein to quantify cell viability (?) .

Finite difference time domain simulations The 3D finite difference time domain (FDTD) simulations were performed using Maxwell`s solver of Lumerical software. The absorbing boundary conditions were assumed for FDTD solutions. Meshing was composed of 5 nm cubes for the nanoflowers (Figure S4). A circularly polarized plane wave at λ=780 nm was used for excitation of the samples. Optical properties of the gold NPs were taken from Palik`s Handbook (Handbook of Optical Constants of Solids)40. A Polystyrene (refractive index of 1.587 and extinction coefficient of 0 at 632.8 nm) and PBS (refractive index of 1.3325 and extinction coefficient of 7.2792e-9) were chosen as the substrate and medium, respectively. For extracting the features from the SEM images, an image processing with a specific threshold was applied to get a clear binary picture of the gold nanoribbons and 30

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then imported it to Lumerical software as a surface and finally extruded it to the desired thickness.

Statistical analysis The statistical analysis was performed using the software OriginPro 2016 (OriginLab, Northampton, USA). T-tests and one-way ANOVA were used to evaluate statistical significance, followed by post-hoc least significant difference tests. All data are presented as the mean ± standard deviation (SD). The error bars presented in this work are standard errors calculated using the Student’s t test at 95% confidence. All micrographs and images are representative of at least three independent samples imaged through three different regions of interest (ROIs). The differences were considered significant for p value < 0.05, and very significant for p value < 0.01.

ASSOCIATED CONTENT Supporting Information: SEM images of peptide sequences, FTIR spectrum and specific IR-bands, UV-Vis spectrum, Simulation results for high plasmonic effect, NIR triggered hyperthermia data, the detailed information of three peptide sequences, the description for synthesized gold nanostructures at different peptide concentrations, HAuCl4 concentrations, pH values and incubation time, the synthesis of gold nanostructures at different synthesis parameters.

31

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Acknowledgement. We thank Armin Schulz and Peter Schützendübe for the Raman and XPS spectroscopic analysis. AVS thanks Max Planck Institute for Intelligent Systems for the grass root project grants in 2017 (M10335) and 2018 (M10338). Y.A. thanks Alexander von Humboldt Foundation for the Humboldt Postdoctoral Research Fellowship.

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and food chemistry 2000, 48 (3), 631-635. 29. Wu, S.; Yan, S.; Qi, W.; Huang, R.; Cui, J.; Su, R.; He, Z., Green Synthesis of Gold Nanoparticles Using Aspartame and Their Catalytic Activity for P-Nitrophenol Reduction. Nanoscale Res. Lett. 2015, 10, 213. 30. Kamnev, A. A., Infrared spectroscopy in studying biofunctionalised gold nanoparticles. In Nanomaterials Imaging Techniques, Surface Studies, and Applications, Springer: 2013; pp 35-50. 31. Zou, C. e.; Bin, D.; Yang, B.; Zhang, K.; Du, Y., Rutin detection using highly electrochemical sensing amplified by an Au–Ag nanoring decorated N-doped graphene nanosheet. RSC Advances 2016, 6 (109), 107851-107858. 32. Liu, W.; Sun, D.; Fu, J.; Yuan, R.; Li, Z., Assembly of evenly distributed Au nanoparticles on thiolated reduced graphene oxide as an active and robust catalyst for hydrogenation of 4-nitroarenes. RSC Advances 2014, 4 (21), 11003-11011. 33. Maccallum, P. H.; Poet, R.; Milner-White, E. J., Coulombic interactions between partially charged main-chain atoms not hydrogen-bonded to each other influence the conformations of α-helices and antiparallel β-sheet. A new method for analysing the forces between hydrogen bonding groups in proteins includes all the Coulombic interactions. Journal of molecular biology 1995, 248 (2), 361-373. 34. Lyu, P. C.; Liff, M. I.; Marky, L. A.; Kallenbach, N. R., Side chain contributions to the stability of alpha-helical structure in peptides. Science (New York, N.Y.) 1990, 250 (4981), 669-673. 35. Ge, J.; Lei, J.; Zare, R. N., Protein–inorganic hybrid nanoflowers. Nature nanotechnology 2012, 7 (7), 428. 36. Xu, X. B.; Luo, J. S.; Liu, M.; Wang, Y. Y.; Yi, Z.; Li, X. B.; Yi, Y. G.; Tang, Y. J., The Influence of Edge and Corner Evolution on Plasmon Properties and Resonant Edge Effect in Gold Nanoplatelets. Phys. Chem. Chem. Phys. 2015, 17, 2641-50. 37. von Maltzahn, G.; Park, J. H.; Agrawal, A.; Bandaru, N. K.; Das, S. K.; Sailor, M. J.; Bhatia, S. N., Computationally Guided Photothermal Tumor Therapy Using Long-Circulating Gold Nanorod Antennas. Cancer Res. 2009, 69, 3892-900. 38. Singh, A. V.; Vyas, V.; Patil, R.; Sharma, V.; Scopelliti, P. E.; Bongiorno, G.; Podestà, A.; Lenardi, C.; Gade, W. N.; Milani, P., Quantitative Characterization of the Influence of the Nanoscale Morphology of Nanostructured Surfaces on Bacterial Adhesion and Biofilm Formation. PLOS ONE 2011, 6 (9), e25029. 39. Imaz, I.; Rubio-Martinez, M.; An, J.; Sole-Font, I.; Rosi, N. L.; Maspoch, D., Metal-biomolecule frameworks (MBioFs). Chemical communications (Cambridge, England) 2011, 47 (26), 7287-302. 40. Palik, E. D., Handbook of Optical Constants of Solids, Five-Volume Set: Handbook of Thermo-Optic Coefficients of Optical Materials with Applications. Elsevier: 1997; p 97-122.

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Figure Captions

Figure 1. Predicted 2ndry structures, ligand binding sites and SEM images of peptide sequences. (A) The secondary structure for FWCYHAGHVL, (B) The secondary structure for MSRCWQPNPR, (C) The secondary structure for VVAGSGGHTT. (D) The metal ligand-binding sites for FWCYHAGHVL, E) MSRCWQPNPR, F) and VVAGSGGHTT. G) The coil form of secondary structure, (H) The sheet form of secondary structure. (–I, L, O) The 10 µmol/L, 50 µmol/L, 100 µmol/L

concentration of FWCYHAGHVL. (–J, M, P) The 10 µmol/L, 50 µmol/L, 100 µmol/L 35

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concentration of MSRCWQPNPR. (–K, N, Q) The 10 µmol/L, 50 µmol/L , 100 µmol/L concentration of VVAGSGGHTT (scale bar: 200 nm). (R) The scheme for the different synthesized gold nanostructures at three different VVAGSGGHTT concentrations.

Figure 2. Spectroscopic and TEM analyses of anisotropic nanostructures formed by VVAGSGGHTT at 10 µmol/L.

(A) The overlay image of silicon, gold, carbon, and oxygen elements EDX-SEM for

synthesized gold nanoflower and nanoleaves. (B) The only gold elemental map of EDX image for gold nanoflower and nanoleaves (scale bar 200 nm). (C) The Raman spectrum for synthesized gold nanoflower and nanoleaves by VVAGSGGHTT at 10 μmol/L. (D) The XPS spectrum for VVAGSGGHTT-Au. (E) The XPS spectrum for the Au 4f. (F) The XPS spectrum for the C 1s, (G) 36

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The XPS spectrum for the O 1s. (H-I) TEM micrograph obtained from a part of nanoflower and inset show selected area electron diffractions pattern recorded through a thin petal of nanoflower.

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Figure 3. (A-C) The 0.5 mmol/L, 1.0 mmol/L, and 1.5 mmol/L concentration of HAuCl4 (scale bar 200 nm). (D) The scheme for the different synthesized gold nanostructures at three different HAuCl4 concentration. (E) The pH = 2, (F) The pH = 7, (G) The pH = 11. 38

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(H) The scheme for the different

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synthesized gold nanostructures at three different pH value

Figure 4. (A) The incubation time = 3 hours, (B) The incubation time = 30 hours, (C) The incubation 39

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time = 60 hours, (D) The incubation time = 75 hours, (E) The incubation time = 98 hours (scale bar 200 nm). (F) The scheme for the different synthesized gold nanostructures at five different incubation time.

Figure 5. Experimental and simulation validation of optical and NIR photothermal properties of nanoflowers for biofilm destructions. (A-D) simulated mode profiles of the plasmonic resonances from gold nanoflower as shown in SEM image. (E-F) Thermograms after laser power deposition to 40

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microcapillaries filled with nanoflowers obtained from VVAGSGGHTT-Au sequences and spherical gold NPs. (G-R) Photothermal effect based killing of untreated bacteria biofilms and their comparison with biofilms supplemented with nanoflowers (K-N) and spherical gold NPs (O-R). (S) Schematic illustration of the in-silico prediction of gold binding sites in biogenic peptides obtained from cancer cells (left panel). Bottom-up synthesis of gold nanoleaf/nanoflowers via the reaction of AuCl4 with the short peptide (middle panel) and their application in in-vitro anti-infection photothermal therapy.

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