Microroses on Levodopa Microtubes for SERS-Based

Subscriber access provided by EDINBURGH UNIVERSITY LIBRARY | @ http://www.lib.ed.ac.uk

Article

Gold Nano-/Micro-Roses on Levodopa Microtubes for SERS-Based Sensing of Gliomas Taru Dube, Nitin Kumar, Avneet Kour, Jibanananda Mishra, Manish Singh, Bhanu Prakash, and Jiban Jyoti Panda ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.9b00155 • Publication Date (Web): 21 Mar 2019 Downloaded from http://pubs.acs.org on March 25, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Nano Materials

Gold Nano-/Micro-Roses on Levodopa Microtubes for SERS-Based Sensing of Gliomas Taru Dube1, Nitin Kumar1, Avneet Kour1, Jibanananda Mishra2, Manish Singh1, Bhanu Prakash1 and Jiban Jyoti Panda1* 1Institute 2School

of Nano Science and Technology, Mohali, Punjab – 160062, India

of Bioengineering and Biosciences, Lovely Professional University, Phagwara,

Punjab – 144411, India * Corresponding

author

Taru Dube M. Pharm. (Pharmaceutics) PhD Scholar, INST, Mohali Nitin Kumar M. Tech. (Biotechnology) Intern, INST, Mohali Avneet Kour M. Pharm. (Pharmacognosy) PhD Scholar, INST, Mohali Dr. Jibanananda Mishra M. Phil, Ph.D Associate Professor, LPU, Punjab Dr. Manish Singh, Ph.D Scientist, INST, Mohali Bhanu Prakash, MSc. Scientist, INST, Mohali

1 ACS Paragon Plus Environment

ACS Applied Nano Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Dr. Jiban Jyoti Panda* Ph.D Scientist, INST, Mohali Tel: +91-172 - 2210075; Fax: +91-172-221107 Email ID: [email protected]


Page 2 of 49

Page 3 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ABSTRACT Anisotropic metal nanostructures with unique physicochemical properties are undeniable promising systems towards diverse applications, ranging from those in material science to biological fields. Burning examples of these are gold nano/microflowers, fabricated via reduction method. Here, a novel notion of assembly pathway dependent single-step synthesis of gold nano/micro “nested structures” with abundant gold nanoplates resembling petals is being reported, which involves mere manual mixing of chloroauric acid and self-build levodopa microtubes at room temperature (RT 25 °C). Compared to multifaceted, intricate methods reported in literature, the present system is facile, controllable, reproducible, and 100% aqueous with enhanced stability. Unique three dimensional nested structures of microroses accompanied by abundance of gold nanoplates coated with oxidized products of levodopa (biopolymer) may offer numerous interaction sites for the biomolecules as well as cells along with permeability across the blood brain barrier, thereby making the system a favorable bio-sensing platform. Application of rhodamine 6G capped levodopa gold microroses as surface enhanced Raman scattering (SERS) based bio-sensing probe has been demonstrated in C6 glioma cells towards in situ diagnostics of glioma or other neural disorders.

Keywords: self-assembly, levodopa microtubes, Au nano/microroses, single-step synthesis, biosensors



1. INTRODUCTION A highly driven researcher’s adoration for nano/microstructures generated from gold (Au), the yellow metal, may be credited to their excellent properties that ascertain utility in every field ranging from material science to biological applications.1-4 In past few years, apart from creating spherical Au structures, scientists are converging more and more towards developing non-spherical Au structures such as nanoclusters,5,6 prisms/plates,7,8 rods,9,10 bipyramids,11 cubes,12 bowls,13 stars,3,14,15 shells,16 toroidal,17 octahedral,18 polyhedral,19 jellyfish,20 tadpole,21 dentritic flowers,22 branched urchins,23-26 and many more. This is because of the fact that owing to the anisotropic distribution of electromagnetic field near their surface,24,27 the unsymmetrical structures exhibit unique optical and electronic properties in comparison to spherical ones. Optical properties of yellow metal nano/microstructures often extensively differ based on their surface features, size, and shape. In recent past, anisotropic yellow metal based nano/microstructures have arisen as promising candidates for diverse scientific applications. However, complex multi-step synthesis steps, poor stability, and high production cost, limits their utility. Flower shaped nano/microstructures can have widespread applications owing to their stability, low surface energy, inherent adsorptive properties, easy removal from the reaction mixture, high surface area to volume ratio, easy accessibility, presence of numerous active sites, and lastly reusability.28-31 In the last few years, there has been a tremendous increase in the number of methods reported for the synthesis of branched Au nanostructures, but most of them are multifaceted, involve many raw reagents which can make synthesis process tricky, intricate, and lengthy.24,27,32,33 Moreover, there are many factors which control the kinetics of such reactions. For example, apart from two main reagents, i.e. chloroauric acid (HAuCl4) and reducing agents such as sodium borohydride, sodium citrate, ascorbic acid etc., the metal reduction process also involves the presence of one or more additional agents, one of them being Au 4 ACS Paragon Plus Environment

Page 4 of 49

Page 5 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


seeds to enhance the uniformity of the resulting flowers. In fact, Au seeds serve as catalyst that promote the reduction of AuCl4ˉ, and facilitate selective growth of Au0 atoms onto the surface of metal clusters, thereby avoiding secondary nucleation.26,34-36 Other additional reagents are the surfactants such as cetyltrimethylammonium bromide,35,37 (1-hexadecyl)trimethylammonium chloride,32 poly(vinylpyrrolidone),38 bis(amidoethyl-carbamoylethyl) octadecylamine,39 gemini cationic surfactants (C14C2C14Br2),26 gelatin,23 which offer a flexible template for achieving the unique flower morphology, and also act as capping agents to control particle growth for obtaining a narrower size range. Furthermore, AgNO3 and other salt precursors are added to promote the growth of particles through preferential deposition of Au atoms onto a specific crystal plane for obtaining unique flower morphology.23,26,27,34 Suspending agents such as chitosan,27,40 thiolates,41 are being added to achieve long term colloidal stability. HCl/NaOH are added to obtain either acidic,22,27 or alkaline conditions42 in order to obtain flower morphology as opposed to globular microparticles. The yield and colloidal stability of anisotropic nanoparticles in solution can be manipulated by changing media pH. It is evidenced that the length of branches in Au flowers may be tuned by simply altering the pH of the growth media.22 Besides this, vigorous stirring and boiling are essential phases of general synthesis protocols to achieve particle uniformity. Thus, it is evident that the process for synthesizing different anisotropic Au nano/microstructures involves multiple steps along with many additional reagents. For a system to be robust, controllable as well as facile, single-step method which may generate particles with high yield and stability are the prerequisites. It is observed that the shape, size distribution, and stability of the nanoparticles depend on the reducing agents and stabilizers used43 and hence, the quest for an appropriate reductive agent and stabilizer is always solicited. Since last two decades, molecular self-assembly has emerged as an attractive tool for the fabrication of diverse range of advanced nanostructures with novel properties. Self5 ACS Paragon Plus Environment


assembled peptide or amino acid derived nanostructures have also been shown to act as excellent templates for the fabrication of metallic nanostructures with variable features.44,45 Levodopa, whose clinical usefulness in Parkinson’s disease was discovered by George Cotzias in 1967,46 is the immediate amino acid precursor to the neurotransmitter dopamine,47 and is capable of crossing the blood-brain barrier via facilitated diffusion through L-type amino acid transporter (LAT-1).48 The aromatic dipeptide diphenylalanine (Phe-Phe), its various derivatives, as well as the aromatic amino acid phenylalanine (Phe) have demonstrated magnificent self-assembly behavior to form fibrillar nanostructures.49,50 We assumed, levodopa, L-3, 4-dihydroxyphenylalanine, a derivative of Phe, could also selfassemble to form well-defined structures similar to Phe and these structures may act as templates for the fabrication of anisotropic Au nano/microstructures. Thus, in this study, we introduce a novel process for the fabrication of self-assembled microtubes from levodopa. We further report an assembly pathway dependent single-step synthesis scheme for the high yield fabrication of stable Au nano/microstructures molded like a rose flower with numerous sheet like petals (Au microroses) from these self-build levodopa microtubes. Gold nano/microstructures sculpted like flowers have been used for various applications, but so far the assembly pathway dependent single-step synthesis of Au ‘nano and microroses’ on self-assembled levodopa microtubes have not been demonstrated. The unique method reported in this study is 100% aqueous, and principally a single-step process simply involving manual mixing of chloroauric acid and self-assembled levodopa tubes (as both reducing and shape directing agent) at room temperature, eliminating the need of additional reagents, vigorous stirring, and heating; thereby making the process as simple as possible. The reported synthesis scheme being facile, single-step, and low cost is unique and can be very well differentiated from previous works reported on anisotropic Au nanostructures.24,27,34,36


Page 6 of 49

Page 7 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In addition, we report that the nature and morphology of Au structures could be significantly fine-tuned simply by exchanging the sequence of reagent addition in the singlestep manual mixing process, without altering raw reagents and their concentrations. Simply, reversing the addition of HAuCl4 solution to levodopa tubes instead of adding levodopa tubes to HAuCl4 aqueous solution resulted in the formation of 200 nm sized Au structures. This is an important observation, and can be tremendously helpful in designing hybrid nanostructures with varied morphology and tunable properties. We also explored the effect of various parameters like concentration of precursors, temperature, stirring etc. on nanostructure formation as well as on their resultant size, uniformity, and morphology. We further explored surface enhanced Raman scattering (SERS) based detection of rhodamine 6G (R6G) as analyte in C6 glioma cells using Au microroses. The current protocol stands out as a potentially interesting, although simplest approach for the fabrication of technologically important SERS-based sensors for cellular imaging applications. 2. RESULTS AND DISCUSSION 2.1. Fabrication and Characterization of Self-assembled Levodopa Microtubes Molecular self-assembly of levodopa, at a concentration of 5 mM in essentially aqueous environment (5% organic solvent and 95% water) as well as in pure water (double filtered Milli-Q water) without the traces of any organic solvent was investigated following a method similar to the one described in literature (by our group) for other peptides and amino acid mimetics.51 Dynamic light scattering (DLS) technique was employed to explore the selfassembling process of levodopa. Intensity based average particle size analysis revealed that levodopa could self-assemble in a mixture of 1,1,1,3,3,3-hexa-fluoro-2-propanol (HFIP)water to form discrete structures having average hydrodynamic size of 1465 ± 692.1 nm with a poly-dispersity index (PDI) of 0.847 ± 0.132 and zeta potential of -21.9 ± 1.85 mV. 7 ACS Paragon Plus Environment


Initial assembling solvent has been shown to play a crucial role in the process of molecular self-assembly.52 The nature of self-assembly varies depending on the polarity and dielectric constant of solvent molecules.53 Solvent dependent self-assembly has been explored in many peptide based systems such as Phe-Phe,54 hydrophobic pentapetides etc.55 Mason and group demonstrated that a change in the solvent environment represents a simple and convenient strategy to achieve structural and morphological control over peptide selfassembly.54 Based on these observations, the effect of various solvents on the self-assembly behavior of levodopa was further explored in other more biocompatible organic solvents like ethanol, dimethyl sulphoxide (DMSO) by keeping the ratio of organic solvent to water constant (5:95). For comparison, the self-assembly behavior of the molecule in pure water was taken as control. It was noticed that, similar to HFIP, the molecule was also capable of forming discrete structures in ethanol and DMSO. Average hydrodynamic size of the as formed discrete structures was found to be 597.9 ± 138.9 nm with a PDI of 0.47 ± 0.25 and zeta of -19.2 ± 0.45 mV in ethanol, 406.7 ± 52.8 nm with a PDI of 0.44 ± 0.02 and zeta of 17.4 ± 2.96 mV in DMSO. Fortunately, to our surprise, levodopa also demonstrated selfassembly behavior in pure water (double filtered Milli-Q water) and formed particulate structures having an average hydrodynamic size of 643.3 ± 497.9 nm with a PDI of 0.66 ± 0.24 and zeta potential of -18.6 ± 4.89 mV (Table S1, Supporting Information). To comprehend the morphological outcome of levodopa self-assembly in different solvents, scanning electron microscopy (SEM) of all the batches were carried out (Figure 1ad). It was observed that with HFIP as initial dispersing solvent (Figure 1a), levodopa could self-assemble to form tubular microstructures with mean diameters of 1.85 ± 1.08 µm. Results further revealed the formation of tubular structures, with mean diameter of 4.55 ± 1.56 µm in ethanol (Figure 1b). However, with DMSO as initial dispersing solvent (Figure 1c), micrographs pointed towards the formation of small aggregates instead of some specific 8 ACS Paragon Plus Environment

Page 8 of 49

Page 9 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


morphology. Similar behavior was observed in case of other peptides and amino acid mimetics that demonstrated a solvent dependent assembly behavior.53,56 SEM was employed to affirm the self-assembly behavior of levodopa in water. The morphological details of the fabricated tubular structures in water with mean diameter of 2.36 ± 0.78 µm (Figure 1d) were further compared to those formed by crude drug powder (Figure 1e). Micrographs revealed the presence of brick like structures for crude levodopa powder which transformed into tubular morphology on being dispersed in water. SEM micrographs further highlighted the tube shaped morphology of the self-assembled levodopa structures formed through molecular self-assembly in aqueous solution (Figure S1, Supporting information). Thus, all these observations clearly depicted the self-assembly behavior and microtube forming ability of the drug levodopa in pure water. Dopamine has been shown to undergo oxidation and self-assembly to form various types of nanostructures in aqueous media,57-59 but to the best of our knowledge, the tube forming ability of levodopa is being explored here for the first time. Though, assembly and fiber formation by fluorenylmethyloxycarbonyl chloride-modified levodopa has been reported.60 However, there have been no systematic reports demonstrating self-assembly and structure formation by levodopa in pure water and at near neutral pH. Owing to the simplicity of structure formation along with their biocompatibility, all further characterization studies in the present report were carried out on levodopa microtubes self-assembled in pure water. Fourier transformed infrared (FTIR) spectra for pure drug powder and freeze dried self-assembled levodopa tubes are shown in Figure 1f. The spectra for levodopa demonstrated the presence of all characteristic absorption peaks corresponding to various functional groups such as benzene ring, primary amine, hydroxyl, carboxylic, and other groups present in the structure.61,62 Absorption bands for levodopa drug at 3388, 3211, 3059, and 2603 cm-1 can be assigned to the (N−H) stretching vibrations due to primary amine, (O−H) stretching vibrations of the phenol ring, (Ar−H) stretching vibrations present in the aromatic ring, and (O−H) 9 ACS Paragon Plus Environment


stretching due to carboxylic group respectively. The absorption peaks observed at 1651 and 1572 cm-1 can be assigned to (N−H) bending due to primary amine, while absorption peak at 1274 cm-1 for the (C−O) stretching due to carboxylic group. The absorption bands at 1061 cm-1 and 1124 cm-1 can be assigned to (C−N) stretching and at 1439 cm-1 due to (C=C) stretching in the aromatic ring. The self-assembled levodopa tubes exhibited strong and highly broadened absorption band at 3435 cm−1 as compared to pure levodopa. The peak was also slightly shifted towards higher wavenumber, and could be attributed to (O−H) stretching vibrations of phenolic hydroxyl groups resulting due to the existence of hydrogen bonding between different levodopa molecules. As of now, it is a well-established fact that hydrogen bonding (together with hydrophobic and π-π interactions) is one of the crucial factor in mediating the self-assembly of molecules to supramolecular architectures.56,63 The peak corresponding to (N−H) bending was also slightly shifted to 1646 and 1533 cm−1 instead of 1651 and 1572 cm−1. For (C−O) stretching vibrations the peak shifted from 1274 cm−1 to 1293 cm−1, for (C=C) stretching from 1439 cm−1 to 1399 cm−1, and for (C−N) stretching from 1124 cm−1 to 1118 cm−1. These shifts may be attributed to intermolecular interactions between levodopa molecules as a result of the self-assembling process.61 Circular Dichroism (CD) spectroscopy is a valuable technique for interpreting the structural properties (secondary and tertiary structure composition) of biomolecules in solution64 and also an important tool to gain insight into the arrangement of molecules in assembled state. Hence, the molecular structure of levodopa tubes was also characterized by CD spectroscopy to gain deeper insight into the arrangement of molecules in assembled form (Figure S2, Supporting Information). Levodopa tubes exhibited an intense positive peak at 206 nm (n→π*) and a negative one around 191 nm pointing towards π→π* transitions.65 In the near UV region, levodopa tubes exhibited a peak at 279 nm pointing towards π→π* transitions in the aromatic groups. This peak might have arisen due to the 3, 4-


Page 10 of 49

Page 11 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


dihydroxyphenyl ring present in the structure, as other aromatic amino acids like tyrosine has been also shown to have a characteristic peak between 275 and 282 nm.66

2.2. Assembly Pathway Dependent Synthesis and Characterization of Au Microroses on Self-build Levodopa Microtubes (M1) Park and co-workers have recently exploited the role of levodopa in the synthesis of Ag nanoparticles and deciphered that the nanoparticle reducing ability could be conferred to the phenolic hydroxyl groups present in the molecule.67 Thus, next we aimed to explore the role of levodopa tubes in the fabrication of Au structures. DLS analysis revealed that Au structures synthesized using self-build microtubes in pure water had an intensity based average hydrodynamic size of 1.23 ± 0.14 µm with a tight PDI of 0.298 ± 0.06 and zeta potential of -28.5 ± 0.66 mV (Figure 2a). SEM micrographs showed that the Au structures formed from self-build levodopa tubes in water were extremely monodispersed structure having mean diameter of 1.28 ± 0.28 µm with numerous undulations on their surface, which at high resolution were resolved to be petal like structures with nanodimensions (Figure 2b). The main hierarchical components of the formed flower like Au structures are Au nanoplates (referred as petals) with observed diameter (thickness) of 62.8 ± 11.05 nm and approximate length of 963.2 ± 247.5 nm (Figure S3, Supporting Information). The micrometer sized structures formed resembled rose flower, hence named as Au microroses in this report. Similarly, the use of dopamine for the synthesis of dendritic Au nanoflowers has been reported during the course of present investigation.22 In another study, Ong and group reported a seed-mediated synthesis of multi-branched Au nanoparticles to confer active breast cancer targeting using the LAT-1 ligands such as L- and D-dopa.36 However generation of Au structures sculpted like microroses by levodopa and especially levodopa tubes has not been observed till date.



Elemental mapping of the microroses (Figure 2c-h), demonstrated that the flowers were primarily composed of Au with a trace amount of nitrogen randomly distributed throughout the particles indicating the presence of levodopa on the surface. UV-Vis absorption spectra of levodopa tubes and Au microroses after various time intervals are shown in Figure 2i. The spectra recorded for levodopa tubes (black line) showed only one peak at around 280 nm. Microroses (green line) demonstrated three distinct absorption peaks, a strong and narrow peak at around 270 nm, hump from 500-650 nm and a broad shoulder from 650900 nm. Hump observed at around 500-650 nm can be ascribed to the existence of transverse component of surface plasmon absorption band whereas the broader hump observed around 650-900 nm can be assigned to the longitudinal component of surface plasmon absorption band.43 The existence of two well-separated absorption bands is a characteristic feature of anisotropic structures.43 The number of surface plasmon absorption bands of the metallic nanoparticles have been shown to be sensitive to the particle shape than to their size, while position and intensity of absorption bands depends on the size only.43 The peak at 270 nm may be attributed to levodopa as the result of absorbance by the aromatic ring present in its structure.68,69 Stability is an important parameter to be studied in order to envisage any potential application of a particulate system. We observed that microroses were stable in solution at 48 h of post-incubation. Figure 2i shows a comparison between the UV-Vis spectra of the microroses taken at different time points after completion of the reaction i.e. after 2, 24, and 48 h. No new absorption peaks or any peak shift was observed during the time course of observation, suggesting that the microroses were stable within this time frame.16 The microroses even remained stable in solution and can be stored at 4 °C for weeks and further can be stored in dried state for months and re-dispersed whenever desired. Figure 2j shows the SEM micrograph of the microroses taken after 1 month of incubation in solution at 4 °C and Figure S4 (Supporting Information), shows the SEM micrographs of dried microroses taken 12 ACS Paragon Plus Environment

Page 12 of 49

Page 13 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


after 1 month of storage at RT. Even after 1 month Au microroses were observed to be monodispersed with mean diameter of 1.38 ± 0.28 µm and petal thickness of 65.8 ± 11.05 nm. There was no observable change in the flower morphology emphasizing the stability of the microroses under these conditions. Next, chemical composition and crystal structure of the Au microroses were recorded by X-ray diffraction (XRD). Typical XRD pattern of the synthesized Au microroses, as shown in Figure 2k, exhibited four peaks corresponding to diffractions from (111), (200), (220), and (311) planes of a face-centered cubic (FCC) lattice of Au.32 No other impurity was observed indicating high purity and poly-crystalline nature of microroses. These peaks confirmed the formation of (FCC) crystalline structure.24,32 High resolution transmission electron microscopy (HRTEM) was used to investigate different Au microrose’s features such as shape, structure, morphology, and crystallization. Interestingly, micrographs demonstrated the formation of nanocrystals having rose flower-like morphology with numerous petals. Figure 3a shows a typical TEM micrograph of Au microrose synthesized using self-build levodopa tubes. The synthesized microroses had an almost spherical shape with many whorls of Au nanoplates. The average diameter of these microroses was 1.5 ± 8.7 µm. The TEM micrograph exhibited regions of variable contrast noticeably illustrating a rough structure of the microroses. Agreeing to the contrast variation, it could be clearly comprehended that these sheet-like petals had many irregular pleats. Figure 3b shows a protruded single petal from the core of the microrose. An additional coating of the oxidized products of levodopa, i.e. 5, 6-dihydroxyindole, of about 2.5 nm in thickness on the petals, can be easily observed. Figure 3c,d shows selected area electron diffraction (SAED) and HRTEM to provide further insights into the structure of synthesized Au microroses. The SAED pattern (Figure 3c) indicated that the petals were polycrystalline in nature and randomly oriented. The observed diffraction rings were indexed as (111), (200), (220), and (311) reflections, respectively, corresponding to FCC structure of Au.32,70 A magnified 13 ACS Paragon Plus Environment


HRTEM image showing lattice fringes (Figure 3d) was further obtained from a petal of the microrose to determine lattice spacing (d). Crystallographic direction of growing branches in microroses was obtained via measuring the distance between lattice fringes (d) through Fast Fourier Transform (FFT) analysis of the fringes observed in HRTEM images. Au microroses exhibited branches towards the (111) plane as indicated by the obtained d value of 0.23 nm (Figure 3e). Figure 3f shows the Inverse Fast Fourier Transform (IFFT) profile of the HRTEM images (Figure S5, Supporting Information). In order to delineate the role of assembly process in triggering any difference in the microrose property such as change in morphology, size etc., the effect of levodopa tubes selfassembled in different solvents on the reduction process of Au microroses was studied. Though, literature shows the influence of various parameters like temperature,26,32,71 pH change,42 reducing agent concentration,26,27,32,38 type of reducing agents,40 and Au precursor concentrations,32 on the characteristic properties of synthesized Au nanoparticles, but no literature is available that depicts the role of solvent polarity on the process outcome. Though, a significant amount of literature has already been published focusing on the important role played by solvent polarity in controlling the morphology, size, and characteristic properties of self-assembled peptide nanoparticles.53,56 Figure 4a shows the corresponding SEM micrographs of microroses obtained by initiating self-assembly of levodopa microtubes in different solvents. The micrographs unveiled the formation of microroses in all chosen solvents, but with a variation in size and uniformity. Levodopa microtubes assembled in water and HFIP as the assembly initiating solvent demonstrated the formation of microroses in two size ranges. One minor population of micron sized structures sculpted like hibiscus flower with a mean diameter of 3.17 ± 0.19 µm and large population of microroses with a mean diameter of 1.26 ± 0.26 µm were observed. With ethanol as the initial dispersing agent, the micrographs again pointed towards the formation of structures in two size ranges. Briefly, a scanty population of microroses with mean diameter of 1.06 ± 0.10 µm and a huge population 14 ACS Paragon Plus Environment

Page 14 of 49

Page 15 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of Au nanostructures with some unclear undulations on the surfaces of each particle were observed. These crevices/undulations were not deep enough to give the appearance of petals, thus it can be concluded that the formed nanostructures were spherical in shape with rough surface having mean diameter of 570 ± 0.08 nm. With DMSO as the initial dispersing agent, SEM micrographs pointed towards the formation of Au nanostructures with mixed morphology such as presence of large microroses with less number of petals, large and small spherical nanoparticles, various aggregates etc. Here, we may recall that with DMSO as the initial solvent, levodopa did not displayed any self-assembly and no precise nanostructures of levodopa were observed. These results evidently indicate that the nature of self-assembled levodopa tubes in a particular solvent is crucial for the formation of microroses with a high density of petals. The morphology and growth of flowers were influenced by solvent polarity. The higher the polarity of the solvent, the higher the reduction rate.72 Thus, less polar solvents are expected to block the exposed Au surface more efficiently and may inhibit the growth of Au nanoparticles into flower shaped morphology. Encouraged by the significant difference exhibited by the nature of self-assembled levodopa tubes formed in different solvents on the final morphology of Au nano/microroses, we desired to explore the precise role of levodopa microtubes in the fabrication of Au microroses and nanoroses. To understand this, the reduction procedure of HAuCl4 was carried out using levodopa powder instead of self-assembled tubes. DLS analysis revealed that the structures obtained from levodopa powder as reducing agent were small with intensity based average hydrodynamic size of 456 ± 49.6 nm with a PDI of 0.47 ± 0.02. SEM micrographs (Figure S6 Supporting Information) reflected the formation of polydispersed Au nanostructures with almost spherical morphology. Thus, it was evident that the tubular morphology of self-assembled levodopa microtubes might be playing some crucial role in the transition of the architecture from sphere to microroses. A large difference, in both, size and 15 ACS Paragon Plus Environment


morphology of the formed Au structures was observed. This is a remarkable observation exemplifying the importance of molecular self-assembly towards templated growth of various types of metallic nanostructures.73,74 Further, we studied the effect of varying concentration ratio of levodopa tubes to Au precursor to fine tune the size and explore the morphological variation, if any in the resultant Au microroses. To understand this, the reduction procedure of HAuCl4 was carried out using levodopa microtubes self-assembled at varying concentration of 2.5, 5, 7.6, and 10 mM in 100% aqueous environment (Figure S7, Supporting information). SEM micrographs (Figure 4b-e) reflected the formation of varied shaped Au structures with almost spherical morphology at all concentrations. At a concentration of 2.5 mM, spherical shaped Au nanoparticles were observed with approx. 500 nm size range. Almost all nanoparticles were aggregated to form large clumps (Figure 4b). Further increasing the levodopa concentration from 5 mM (Figure 4c) to 7.6 mM (Figure 4d), increase in the number of overall Au nanoplates (petals) was observed with increase in microrose size upto 2 µm. Further increasing the concentration to 10 mM, Au structures resembling spiky balls were observed (Figure 4e). It is worth noticing that at higher concentrations a significant variation in the population size was observed. Thus, it was evident that the concentration of tubular shaped self-assembled levodopa microtubes might be playing some crucial role in the transition of the architecture from sphere to microroses to spiky balls. A large difference in morphology of the formed Au structures was observed. This is a remarkable observation again exemplifying the importance of concentration based change in molecular self-assembly towards templated growth in fine tuning various types of Au structures according to the need. Many reports suggest that during the formation of Au nanoparticles boiling or heating is essential for the quick reduction of Au26, 36 and stirring helps in the formation of uniform sized nanoparticles.25,27,32,36 Hence, effect of these parameters on the growth process of levodopa tubes mediated Au microroses was observed. Initially levodopa tubes were added to 16 ACS Paragon Plus Environment

Page 16 of 49

Page 17 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Au solution under vigorous stirring to achieve uniform size distribution. Here, we could obtain smaller flowers with a narrow size distribution of 800-900 nm range, however, the morphology of formed flowers was not as good as in the absence of stirring. Their shape was slightly disrupted and many aggregate like structures were visible (Figure 4f). We observed that the synthesis of uniform microroses did not require rigorous stirring, instead keeping the solution untouched till the end of reduction process led to the formation of full bloomed and uniform sized microroses. Effect of temperature on the levodopa tubes mediated synthesis of Au microroses was further investigated as many reports demonstrated the necessity of lower temperature for the synthesis of stable Au nanostructures.26,32 Herein; we carried out the synthesis at different temperatures i.e. 4, 25, and 50 ºC. At RT, monodispersed Au microstructures were formed with numerous nano-textured undulations on their surface; resembling rose flowers (Figure 4g). The average diameter of these flowers was 1.28 ± 0.28µm with average petal thickness of 62.8 ± 11.05 nm. At 4 ºC, SEM micrographs (Figure 4h) affirmed the formation of uniform Au microroses with higher petal density in slightly smaller size range, but with only a small population of fully bloomed flowers as compared to RT. Apart from this, a large population of flowers with shallow crevices was observed which gave the impression of the formation of microroses which were in the initial stage of their development. On the other hand, as compared to microroses at RT, SEM micrographs of microroses prepared at 50 ºC, demonstrated the formation of almost spherical nanoparticles with very few completely developed or distorted flowers (Figure 4i). This study encourages that it is not essential to raise/lower the temperature of HAuCl4 solution in order to achieve flower like morphology, as low temperature lowers the rate of microrose formation and high temperature results in spherical morphology. Hence, it is essential that optimum temperature should be provided for the formation of Au microroses.



2.3. An Insight into the Growth Kinetics of the Au microroses Furthermore, to get an insight into the growth kinetics of the as-prepared Au microroses, progression in morphology of the Au microroses was recorded via SEM imaging taken at different time interval during growth process, i.e. 0, 5, 10, 30 min, and 2 h (Figure 5). Au nanocrystals were obtained by terminating the synthesis at fixed time intervals and subsequent removal of the supernatant containing the unreacted reagents by centrifugation. It is evidently grasped from SEM micrographs that immediately after adding levodopa tubes (Figure 5a) to HAuCl4, Au nanostructures with an average size of 403 ± 33 nm were formed signifying the reduction of Au ions. Levodopa tubes and Au nanostructures co-existed in approximately the same quantity (Figure 5b). Magnified SEM image of the same field (insets) demonstrated a transition of levodopa tube morphology from tubes to ribbons at this stage while Au nanostructures formed were mostly spherical with non-uniform size. After a period of growth for 5 min (Figure 5c), numerous Au nanostructures with an average size range of 400-500 nm were observed with very few levodopa ribbons. Within next 10 min (Figure 5d), the Au nanostructures grew to 600-900 nm size range. Some indistinct, but regular depressions (undulations) were observed on the surface of each particle. Few deeply formed undulations giving the appearance of petals with a thickness of ~133 nm were observed. These indistinct troughs were the starting points for future formation of fully grown Au microroses. After continuous growth for 30 min (Figure 5e), previous troughs on the surfaces became deeper and denser giving the appearance of sheets protruding from a common center similarly to whorls in roses. The microrose had an almost round shape of petals with a flowering layer composed of more than 20-25 layers (approximately measured in SEM), depending on the size of the flower. As a result, the metal sheets (Au nanoplates) or petals further grew into denser protrusions with a thickness of 50-70 nm. Numerous petals were also observed in the background, as the growth process was yet not complete with the existence of many small flowers of approximately 500 nm in size. The growth process lasted for 18 ACS Paragon Plus Environment

Page 18 of 49

Page 19 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


approximately 120 min as evident in Figure 5f. After this, uniformly distributed Au microroses with a size of 1-1.5 µm along with the presence of larger and thinner petals were formed. It was observed that, after a period of reaction for 2 h, no obvious changes could be witnessed from the SEM images and UV-Vis spectra, which suggested the end of the growth process. In fact, in the typical synthesis procedure the products were further aged for 24 and 48 h in order to form more stable and uniform Au microroses. In relevance to all above experiments, a hypothesis of the microroses fabrication through the reduction of HAuCl4, via self-assembled levodopa tubes in aqueous solution is illustrated in Scheme 1. The mechanism of formation of Au microroses can be explained by the involvement of two steps; nucleation and growth. Initially Au3+ ions were reduced by levodopa tubes. Levodopa has a catechol (dihydroxybenzene) unit and zwitter-ionic amino acid part. It has been reported that dihydroxybenzene undergoes an oxidation reaction leading to the production of electrons (which will be used for the reduction of Au3+ ions) and oxidized products such as dopaquinone, dopachorme and other polymeric structures.75 The Au atoms produced after reduction, aggregates to small clusters i.e., nuclei after achieving supersaturation point in the reaction solution. As soon as the nuclei are formed, reduction of Au3+ ion on the surface of the nuclei will start. Since, heterogeneous nucleation is favorable than the homogeneous nucleation, this results in the rapid growth of nuclei to larger nanocrystals composed of different facets such as (111), (200), (220), and (311) planes in order to minimise the surface energy during the growth. When the nanocrystals grew to a critical size, certain crystal facets became large enough for selective binding of the oxidized products of levodopa, leading to alteration of free energies of different facets and finally formed Au microroses as a consequence of the unequal growth rate along different crystallographic directions. Thus it can be hypothesized that levodopa tubes preferentially accumulated on the crystal planes other than (111) and as a result, the growth rate of such facets was lowered and the released Au atoms were directionally added to (111) facets, 19 ACS Paragon Plus Environment


leading to an anisotropic growth. While more and more Au atoms got deposited onto these (111) facets, these facets protrude out creating crevices on the surface. Owing to the higher tendency of the protuberant edges to capture Au atoms, these protuberances overgrow to form thin, petal-like sheets (nanoplates), thus accelerating the fabrication of microroses.26,32,76 Our result of formation of Au microroses as a result of overgrowth of (111) facets is well consistent with the reported literature.32,75 2.4. Effect of Exchanging the Order of Adding Reducing Agent on the Characteristics of Au microroses By interchanging the sequence of addition of reducing agents, it was possible to elevate the reduction rate of Au, hence govern the morphology and dimensions of nanoparticles along with tuning the size and size distribution of the microroses.77 We also evaluated the impact of the sequence of the addition of self-assembled levodopa tubes on the diameters and morphology of the microroses formed. We just reversed the process of Au microroses synthesis i.e. in this case we added 0.5 mL of 10 mM HAuCl4 aqueous solution to the 0.6 mL of levodopa tubes (5 mM levodopa aqueous solution), henceforth this process will be termed as M2. A drastic change in both the size and morphology of nanostructures was observed. By just interchanging the sequence of addition of levodopa tubes, the diameters of Au structures were tunable directly from approximately 1 µm to 200 nm and a morphological deviation from flowers to spherical nanoparticles was observed. DLS analysis revealed that the Au nanoparticles synthesized using M2 had intensity based average particle size of 304.5 ± 61.1 nm with a tight PDI of 0.243 ± 0.109. Field emission scanning electron microscopy (FESEM) micrographs of Au microroses synthesized using M1 (Figure 6a) and M2 (Figure 6b) unveiled the formation of monodispersed spherical nanoparticles with an average size of 288.2 ± 40.84 nm as compared to Au microroses from M1. Figure 6b clearly revealed the presence of a rough surface topography of the nanoparticles. TEM micrograph showed flower 20 ACS Paragon Plus Environment

Page 20 of 49

Page 21 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


shaped morphology of synthesized Au microroses from M1 (Figure 6c) while TEM micrographs (Figure 6d) unveiled the formation of small flowers (nanoroses) with an average size of 286.25 ± 18.1 nm.24,36 Flower like morphology i.e. with distinct petals using M2 was evident in the TEM micrographs (Figure S8, Supporting Information) at higher magnification, but it was not evident in the FESEM micrographs. FESEM and TEM sizes of the nanoparticles were in good agreement with the size observed in DLS study. Using M2 method, small sized nanoroses with somewhat rough surface were achieved, while method M1 promoted a kinetically-driven deposition of Au atoms on the (111) lattice planes which resulted in the growth of petals imitating the rose flower type morphology. Profound analysis revealed that generation of Au microroses as well as their growing size strongly depended on the reactivity of Au ions, mainly controlled by the concentration of self-assembled levodopa tubes.24 We thereby conclude here that by simply interchanging the sequence of the introduction of levodopa microtubes as the reducing agent, it is not only possible to govern the dimensions of nanostructures, but also drastically tune their particle morphology.

2.5. Application of Au Microroses towards SERS based Sensing in Glioblastoma Surface enhanced Raman spectroscopy has been passionately discovered as a highly sensitive and powerful analytical tool to analyze even ultralow concentrations of various probe molecules for bio-imaging, cancer diagnosis, chemical and bio-sensing.26,76,78 SERS substrates with a high enhancement factor have always been a hunting ground for various diagnostic and biomedical applications. Stability and uniformity is also an important factor for the detection of probe molecules at trace levels.79 Metallic surfaces with sharp edges/roughness are one of the important materials for SERS substrates.38 It is worth noting that metallic nanoparticles are prone to photo-thermal reshaping26 which may affect SERS reproducibility. Three dimensional (3D) metallic SERS substrates holding nanoscale corners



or edges are promising systems in concentrating the plasmon induced electromagnetic field giving rise to large number of “hot spots”.80,81 Hence, SERS effect of Au microroses was assessed by measuring Raman spectra with R6G as the probe molecule. Figure 7a shows the Raman spectra of pure R6G, Au microroses and R6G capped Au nanospheres, and R6G capped Au microroses. Au microroses demonstrated an intense SERS spectrum upon being capped by R6G molecules even at a very low concentration of 5×10-3 mM. The increased intensities of bands at 1366 cm-1 and 1570 cm-1 in R6G capped microroses as compared to pure R6G are clearly evident from Figure 7a which indicate that microroses exhibited strong surface enhanced Raman scattering effects. The ability of the Au microroses to attain an enhancement in the Raman intensities of R6G molecules can be majorly attributed to small gaps present in between the petals on the scale of nanometers which can act as hot spots having high local electron densities. The above hypothesis was further proved by comparing the SERS performances of equivalent size Au microroses to Au nanospheres, approx. 1 µm (Figure S9, Supporting Information). For quantitative measurement of SERS activity, an enhancement factor (EF) was calculated using the following relationship: EF = (ISERS/IRaman)/(CSERS/CRaman).38 Where ISERS and IRaman are the measured intensities of the prominent SERS peak of R6G (here 1366 cm−1) in the SERS and the Raman spectra of R6G aqueous solution, respectively, and CSERS and CRaman are the concentration of R6G in the SERS sample (0.005 mM) and in the aqueous solution of R6G (0.25 mM). Significantly, the magnitude of the SERS response was highest for the Au microroses, and negligible for the Au nanospheres. The calculated EF for Au microroses was found to be 6.5 × 104 which is comparable to other R6G capped anisotropic structures reported, while the EF for the Au nanospheres were found to be 1.0 × 102. These results are in agreement with the presence of highly enhanced electric fields at hotspots associated with the flower shaped structures and are in accordance to the literature.82,83,84


Page 22 of 49

Page 23 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Also, theoretical simulations carried out earlier demonstrating the “lighting rod mechanism” explains that the enhancement of local electric field in metal nanoparticles intensely depends on the shape of the metal protrusions.82 Therefore, Au microroses with enhanced SERS signal exhibit promising future as more sensitive bio-sensing probes/biosensors as compared to spherical ones towards in situ diagnostics, where analytes of diverse nature need to be specifically detected in complex biological environment. This again signify the potential use of Au microroses as efficient biosensors with single step synthesis method compared to multifaceted, intricate methods reported in literature. Gold being an inert metal, its nano/microstructures offer good potency for biological applications. Hence SERS active Au microroses may be used in the Raman spectroscopy of living cells for targeting and assaying species of interest such as various cancer biomarkers. Further, levodopa coating would provide the structures with additional biocompatibility and may also facilitate the transfer of the particles across the blood brain barrier for potential imaging of neural disorders specifically brain cancers. Hence, the SERS based sensing ability of the Au microroses was investigated in C6 glioma cells (Figure 7b). C6 glioma cells were incubated with pure R6G, Au microroses and R6G capped Au microroses to investigate SERS effects. Figure 7b shows the intracellular SERS signals obtained from C6 glioma cells incubated with R6G capped Au microroses. Typical SERS bands of 1355, and 1570 cm−1, which correspond to the C-C stretching in the xanthene plane of R6G molecules, could be clearly observed in microroses treated C6 glioma cells signifying the ability of the composite as an intracellular detecting agent. Xie et al. in 2008 used denatured BSA stabilized HEPES capped and the Raman probe rhodamine capped Au nanoflowers (RhB@dBSA capped AuNFs) for in vivo SERS based imaging applications.76 However, in the present case the microroses are stable by their own and do not need any additional coating. Confocal images (Figure 7c) demonstrated that cells incubated with the R6G capped Au microroses exhibited red fluorescence owing to their efficient cellular internalization. It was also observed that the 23 ACS Paragon Plus Environment


fluorescence intensity of the C6 cells treated with the R6G capped Au microroses was significantly higher than those treated with the R6G solution alone (Figure S10, Supporting Information). This uptake study also signifies the role of Au microroses as drug carriers along with the efficient substrate for SERS based in vivo imaging.

3. CONCLUSION To conclude, we have developed a unique synthesis scheme for the fabrication of Au microroses which can be very well differentiated from previous works reported on anisotropic Au nanostructures. To the best of our knowledge, this is the first report that describes a singlestep aqueous synthesis of rose flower like Au nano/microstructures on self-build levodopa tubes at RT with minimum reagents. Herein, we also for the first time highlighted the role of self-assembled levodopa tubes in building the flower like morphology, as the absence of tubular structure did not result in the formation of microroses. In addition, the flower morphology is pathway dependent and can be tuned by altering the order of reagent addition as well as by varying the microtube assembly conditions. Formation of tubular structures by levodopa in pure water without the need of any organic solvent is worth noticing and could find unprecedented applications in nanomedicine, especially in the field of anti-Parkinsonian therapy. The tubular structures fabricated from levodopa may also work as ideal vehicles for delivering various drugs across the blood brain barrier for enhanced neurotherapy or as reducing agent in the fabrication of metallic nanostructures. Further, the application of R6G capped levodopa Au microroses can be potentially envisaged as surface enhanced Raman scattering active tags in C6 glioma cells towards in situ diagnostics of glioma or other neural disorders.


Page 24 of 49

Page 25 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4. EXPERIMENTAL SECTION Levodopa self-assembly in solution: Briefly, colloidal solution of levodopa as a stock was prepared by adding 1 mg of the drug in 50 µL of HFIP, followed by bath sonication for 10 min. Levodopa is sparingly soluble in HFIP. Spontaneous self-assembly was obtained by the swift addition of 950 µL of pure water to the stock to attain a final concentration of 5.0 mM. The sample was aged for 30 min before carrying out any experiment. Likewise, molecular self-assembly of levodopa was also explored in other solvents by dispersing same quantity of drug in 50 µL of ethanol, DMSO, and pure water followed by the addition of 950 µl of pure water, to attain a final concentration of 5 mM of the molecule. The self-assembly process for levodopa was carried out at room temperature (RT 25 ºC) unless specified otherwise. Synthesis of Au microroses on self-build levodopa microtubes (M1): For a single step aqueous synthesis of Au microroses, 0.6 mL of levodopa tubes in water (5.0 mM levodopa aqueous solution) were swiftly added to 0.5 mL of 10 mM HAuCl4 aqueous solution. Straightaway, color change from yellow to reddish brown and then brown indicated the formation of microroses. The suspension was set aside undisturbed for 2 h at RT for complete blooming of the microroses. Bloomed microroses settled down on their own. After 2 h of incubation, growth medium was removed and the fabricated Au microroses were re-dispersed in pure water. Resulting suspension was centrifuged at 1500 rpm for 5 min; pelleted microroses were collected, washed thrice, and finally re-dispersed in pure water. Synthesis of Au structures using powdered levodopa: To the Au solution (0.5 mL of 10 mM HAuCl4 aqueous solution), 0.6 mg of powdered levodopa was added, followed by aspiration for few seconds till complete dissolution of the drug. The color of the solution changed from yellow to reddish brown slowly over time indicating the formation of spherical Au nanostructures. The suspension was set aside undisturbed for 2 h at RT for complete reduction 25 ACS Paragon Plus Environment


of HAuCl4 to form Au nanostructures. After 2 h of incubation, the resulting suspension was centrifuged at 7000 rpm for 10 min; pelleted nanostructures were collected, washed thrice, and finally re-dispersed in pure water. Characterization: Intensity based average hydrodynamic size (Z. Avg.), size distribution, poly-dispersity index (PDI), and also the corresponding zeta potential for all the batches of self-assembled levodopa structures in different solvents, gold nano/microroses synthesized on or without self-assembled levodopa microtubes were obtained by Malvern Zetasizer Nano ZSP (Model-ZEN5600; UK). Average hydrodynamic diameter was acquired by dynamic light scattering (DLS) technique at an angle of 173º in 12 mm2 disposable polystyrene cuvettes (Model-DTS0012; Malvern). Zeta potential measurements were carried out in disposable folded capillary cells (Model-DTS1070; Malvern) using palladium electrodes by phase analysis light scattering (PALS) technique. All measurements were carried out at room temperature (RT

25 ºC) until specified. Average of the readings obtained from three

independent batches was presented as the actual figures. Scanning electron microscopy (JEOL JEM SEM, Tokyo, Japan) equipped with energy dispersive X-ray diffractometer and field emission SEM (JEOL 7600F FESEM) was employed to further investigate the morphological details and size of the fabricated structures. Self-assembled structures of levodopa fabricated at a concentration of 5 mM in different solvents (HFIP, ethanol, DMSO, and pure water) were drop casted (50-60 µL) on cleaned silicon wafers (washed thrice in acetone, methanol, and then water) followed by overnight drying in air. The dried structures were gold coated in JEOL JEC-3000FC sputter coater for 90 s at 30 pascals to enhance their visualization under the microscope by improving topographical contrast and minimizing thermal damage (Leica Microsystems). JSM IT300 software (version 1.032) was used to digitally record and process the obtained micrographs. Gold structures synthesized via various methods were drop casted (50-60 µL) on cleaned 26 ACS Paragon Plus Environment

Page 26 of 49

Page 27 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


silicon wafers followed by overnight drying in air. Before imaging the dried structures were Au coated for 200 s at 20 pascals. Fourier transformed infrared (FTIR) spectra of powdered levodopa and assembled levodopa tubes were collected using FTIR spectrophotometer (Agilent Cary 660 FTIR Spectrometer) via KBr pellet technique to elucidate any structural transitions that may occur during selfassembly process. Levodopa drug and powdered levodopa tubes were mixed with IR grade KBr in the ratio of 1:100 respectively. This mixture was then compressed in the form of a thin pellet in hydraulic press via applying 10 tons of pressure. Similar method was used to prepare the blank KBr pellet with 100% KBr for background subtraction. The pellets were scanned over a spectral range of 4000 to 400 cm-1 and spectral analysis was carried out via comparing the obtained spectra with standard group frequencies for levodopa using data analysis software Agilent Resolutions Pro. CD spectroscopic signals for levodopa tubes in water were collected on a JASCO J-1500 CD spectropolarimeter at a concentration of 1 mg/mL and 0.125 mg/mL in the range of 240-340 nm (near UV range) and 190 to 240 nm (far UV range). All spectra were recorded at 25 ºC in a quartz cuvette (1.0 mm path-length cell), at a scan rate of 50 nm min–1 with a 0.5 nm step size and at a bandwidth of 1.0 nm. Average of three scans was considered as final reading. UV-Vis Spectroscopy of levodopa tubes and Au microroses after various time intervals were recorded at room temperature with a Shimadzu UV-2600 spectrophotometer. Transmission electron microscopy (TEM) analysis was performed on a JEOL JEM-2100 transmission electron microscope operating at 200 kV under a tungsten filament (Tokyo, Japan). Gold microroses were drop casted (50-60 µL) onto a carbon-coated Cu grid (300 mesh, Beta Tech Equip. Pvt. Ltd., India), followed by overnight drying inside the desiccator



under vacuum for proper adsorption. Camera software Gatan was used to digitally record and process the obtained micrographs. To probe percentage crystallinity, atomic and molecular structure of Au microroses; X-ray powder diffraction (XRD) investigation was carried out on Bruker Eco D8 Advance X Ray Powder Diffractometer using Ni filtered with Cu Kα radiation (λ=1.54056 Å). Data was collected from 10˚< 2θ < 80˚ with increment of 0.0091˚. Self-assembly of levodopa at different concentrations: refer supporting information for further details. To Study the Effect of Levodopa Microtube Concentration on the Synthesis of Au structures: refer supporting information for further details. Effect of exchanging the order of adding reducing agent (M2): Briefly, 0.5 mL of 10 mM HAuCl4 aqueous solution was added to the 0.6 mL of levodopa tubes (50 mM Levodopa aqueous solution). The color of the solution changed from transparent to yellow and then slowly to reddish brown over the time indicating the formation of Au nanostructures. Then, the suspension was left undisturbed at RT. After 2 h of incubation the suspension was centrifuged at 1500 rpm for 5 min, and the precipitates were collected and washed with ultrapure deionized water three times and finally re-dispersed in ultrapure deionized water. Synthesis of spherical Au structures: refer supporting information for further details. SERS Measurements: Raman measurements of Au microroses, R6G capped Au microroses, R6G capped spherical AuNPs, R6G solution (1 mg/mL) were done by WITECH Raman spectrometer (514 nm Nd:YAG laser) using R6G as a probe molecule. SERS substrate was prepared by drop-casting 20 μL from the respective groups onto a cleaned silicon wafer and


Page 28 of 49

Page 29 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


air dried overnight.

The SERS spectra were recorded using a 10× objective with an

acquisition time of 30 s, 1 µm2 spot size and laser power of 75 mW. Cellular uptake of Au microroses in C6 glioma cells: C6 glioma cells were cultured in tissue culture flasks (T25) in DMEM media supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 U/mL) until 80% confluency, then trypsinized and seeded at a density of 50,000 cells per well on autoclaved coverslips placed inside 6-well plates. Cells were then allowed to grow for 24 h for cell adherence and spreading. Subsequently incubated with Au microroses, Rh6G capped Au microroses and R6G solution for 2 h in a humidified incubator at 37 ºC with 5% CO2. After treatment growth media was removed and cells were washed thrice with PBS. Thereafter, cells were fixed using 4% paraformaldehyde for 5 min. Fixed cells were treated for 5 min with DAPI (1 μg/mL) for nucleus staining, followed by washing with chilled PBS (4 ºC). Following this, coverslips were taken out from the wells, mounted onto glass slides (80% glycerol) and then visualized under confocal microscope (Carl Zeiss Microscope LSM 880) using 630 nm emission filter for R6G and 488 nm emission filter for DAPI. Confocal images were captured and processed using the ZEN software. Intracellular SERS Measurements: Raman spectra of C6 glioma cells incubated with Au microroses, Rh6G capped Au microroses and R6G solution was measured by WITECH Raman spectrometer. For Raman spectra of microroses, C6 glioma cells were grown in DMEM supplemented with 10% heat-inactivated bovine serum, penicillin (100 U/mL), and streptomycin (100 U/mL). The cells were cultured in a humidified incubator at 37 °C (95% humidity, 5% CO2), and 50,000 cells per well were seeded in a 6-well plate and incubated for 24 h. After 24 h, cells were treated with fresh medium containing different Au microroses for 2 h. The cells were then washed with PBS and fixed using chilled 4% paraformaldehyde solution, followed by PBS wash. The coverslip was removed and observed under the Raman 29 ACS Paragon Plus Environment


microscope for SERS measurements. Intracellular SERS measurements were recorded same as described above. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Materials; Hydrodynamic size and zeta potential of Levodopa self-assembled structures in various solvents; Tubular morphology of levodopa microtubes; CD spectroscopy of levodopa microtubes; Measurement of petal dimensions; Au microroses after 1 month of storage in dried form; FFT and IFFT profile; Role of self-assembled levodopa tubes in the synthesis of gold microroses; Self-assembled levodopa tubes at different concentrations; high resolution TEM images of Au nanoroses using M2; Spherical AuNPs; Cellular uptake of R6G capped Au microroses and R6G solution. ACKNOWLEDGEMENTS J.J.P. thanks the Department of Biotechnology, Ministry of Science and Technology (Award number: BT/PR17945/BIC/101/563/2016), Inspire Faculty Fellowship programme of Department of Science and Technology (DST), Govt. of India, and support from Institute of Nano Science and Technology, an autonomous institute supported by DST, Govt. of India, for funding. The authors acknowledge the help of Dr. Sonalika Vaidya, INST in analyzing HRTEM data.


Page 30 of 49

Page 31 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


REFERENCES 1. Niu, W.; Zhang, W.; Firdoz, S.; Lu, X. Dodecahedral Gold Nanocrystals: The Missing Platonic Shape. J. Am. Chem. Soc. 2014, 136, 3010-3012. 2. Qi, L.; Yuanyuan, J.; Rongcheng, H.; Xiaolan, Z.; Siyun, L.; Zhi-Yuan, L.; Yinlin, S.; Dongsheng, X. High Surface-Enhanced Raman Scattering Performance of Individual Gold Nanoflowers and Their Application in Live Cell Imaging. Small 2013, 9, 927-932. 3. Liu, Y.; Ashton, J. R.; Moding, E. J.; Yuan, H.; Register, J. K.; Fales, A. M.; Choi, J.; Whitley, M. J.; Zhao, X.; Qi, Y.; Ma, Y.; Vaidyanathan, G.; Zalutsky, M. R.; Kirsch, D. G.; Badea, C. T.; Vo-Dinh, T. A Plasmonic Gold Nanostar Theranostic Probe for In Vivo Tumor Imaging and Photothermal Therapy. Theranostics 2015, 5, 946-960. 4. You, J.; Zhang, G.; Li, C. Exceptionally High Payload of Doxorubicin in Hollow Gold Nanospheres for Near-Infrared Light-Triggered Drug Release. ACS Nano 2010, 4, 10331041. 5. Chunlei, Z.; Chao, L.; Yanlei, L.; Jingpu, Z.; Chenchen, B.; Shujing, L.; Qing, W.; Yao, Y.; Hualin, F.; Kan, W.; Daxiang, C. Gold Nanoclusters-Based Nanoprobes for Simultaneous Fluorescence Imaging and Targeted Photodynamic Therapy with Superior Penetration and Retention Behavior in Tumors. Adv. Funct. Mater. 2015, 25, 1314-1325. 6. Li, Z.; Peng, H.; Liu, J.; Tian, Y.; Yang, W.; Yao, J.; Shao, Z.; Chen, X. Plant ProteinDirected Synthesis of Luminescent Gold Nanocluster Hybrids for Tumor Imaging. ACS Appl. Mater. Interfaces 2018, 10, 83-90. 7. Shankar, S. S.; Rai, A.; Ankamwar, B.; Singh, A.; Ahmad, A.; Sastry, M. Biological Synthesis of Triangular Gold Nanoprisms. Nat. Mater. 2004, 3, 482-488. 8. Chen, L.; Ji, F.; Xu, Y.; He, L.; Mi, Y.; Bao, F.; Sun, B.; Zhang, X.; Zhang, Q. High-Yield Seedless Synthesis of Triangular Gold Nanoplates through Oxidative Etching. Nano Lett. 2014, 14, 7201-7206.



9. Huang, X.; El-Sayed, I. H.; Qian, W.; El-Sayed, M. A. Cancer Cell Imaging and Photothermal Therapy in the Near-Infrared Region by Using Gold Nanorods. J. Am. Chem. Soc. 2006, 128, 2115-2120. 10. Jang, B.; Park, J.-Y.; Tung, C.-H.; Kim, I.-H.; Choi, Y., Gold Nanorod-Photosensitizer Complex for Near-Infrared Fluorescence Imaging and Photodynamic/Photothermal Therapy In Vivo. ACS Nano 2011, 5, 1086-1094. 11. Fang, C.; Zhao, G.; Xiao, Y.; Zhao, J.; Zhang, Z.; Geng, B. Facile Growth of High-Yield Gold Nanobipyramids Induced by Chloroplatinic Acid for High Refractive Index Sensing Properties. Sci. Rep. 2016, 6, 36706. 12. Huang, Y.; Wu, L.; Chen, X.; Bai, P.; Kim, D.-H. Synthesis of Anisotropic Concave Gold Nanocuboids with Distinctive Plasmonic Properties. Chem. Mater. 2013, 25, 2470-2475. 13. Mo, A. H.; Landon, P. B.; Gomez, K. S.; Kang, H.; Lee, J.; Zhang, C.; Janetanakit, W.; Sant, V.; Lu, T.; Colburn, D. A. Magnetically-Responsive Silica-Gold Nanobowls for Targeted Delivery and SERS-Based Sensing. Nanoscale 2016, 8, 11840-11850. 14. Wang, S.; Huang, P.; Nie, L.; Xing, R.; Liu, D.; Wang, Z.; Lin, J.; Chen, S.; Niu, G.; Lu, G. Single Continuous Wave Laser Induced Photodynamic/Plasmonic Photothermal Therapy using Photosensitizer-Functionalized Gold Nanostars. Adv. Mater. 2013, 25, 3055-3061. 15. Chen, J.; Sheng, Z.; Li, P.; Wu, M.; Zhang, N.; Yu, X.-F.; Wang, Y.; Hu, D.; Zheng, H.; Wang, G. P. Indocyanine Green-Loaded Gold Nanostars for Sensitive SERS Imaging and Subcellular Monitoring of Photothermal Therapy. Nanoscale 2017, 9, 11888-11901. 16. Vankayala, R.; Lin, C.-C.; Kalluru, P.; Chiang, C.-S.; Hwang, K. C. Gold NanoshellsMediated Bimodal Photodynamic and Photothermal Cancer Treatment using Ultra-Low Doses of Near Infra-Red Light. Biomaterials 2014, 35, 5527-5538.


Page 32 of 49

Page 33 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


17. Yan, Y.; Padmanabha Pillai, P.; Timonen, J. V.; Emami, F. S.; Vahid, A.; Grzybowski, B. A. Synthesis of Toroidal Gold Nanoparticles Assisted by Soft Templates. Langmuir 2014, 30, 9886-9890. 18. Cao, C.; Park, S.; Sim, S. J. Seedless Synthesis of Octahedral Gold Nanoparticles in Condensed Surfactant Phase. J. Colloid Interface Sci. 2008, 322, 152-157. 19. Vutukuri, H. R.; Imhof, A.; Van Blaaderen, A. Fabrication of Polyhedral Particles from Spherical Colloids and Their Self-Assembly into Rotator Phases. Angew. Chem., Int. Ed. 2014, 53, 13830-13834. 20. Ma, X.; Huh, J.; Park, W.; Lee, L. P.; Kwon, Y. J.; Sim, S. J. Gold Nanocrystals with DNA-Directed Morphologies. Nat. Commun. 2016, 7, 12873. 21. Hu, J.; Zhang, Y.; Liu, B.; Liu, J.; Zhou, H.; Xu, Y.; Jiang, Y.; Yang, Z.; Tian, Z.-Q. Synthesis and Properties of Tadpole-Shaped Gold Nanoparticles. J. Am. Chem. Soc. 2004, 126, 9470-9471. 22. Yi, S.; Sun, L.; Lenaghan, S. C.; Wang, Y.; Chong, X.; Zhang, Z.; Zhang, M. One-Step Synthesis of dendritic Gold Nanoflowers with High Surface-Enhanced Raman Scattering (SERS) Properties. RSC Adv. 2013, 3, 10139-10144. 23. Lu, L.; Ai, K.; Ozaki, Y. Environmentally Friendly Synthesis of Highly Monodisperse Biocompatible Gold Nanoparticles with Urchin-Like Shape. Langmuir 2008, 24, 10581063. 24. Li, J.; Wu, J.; Zhang, X.; Liu, Y.; Zhou, D.; Sun, H.; Zhang, H.; Yang, B. Controllable Synthesis of Stable Urchin-like Gold Nanoparticles Using Hydroquinone to Tune the Reactivity of Gold Chloride. J. Phys. Chem. C 2011, 115, 3630-3637. 25. Wang, L.; Guo, T.; Lu, Q.; Yan, X.; Zhong, D.; Zhang, Z.; Ni, Y.; Han, Y.; Cui, D.; Li, X. Sea-Urchin-Like Au Nanocluster with Surface-Enhanced Raman Scattering in Detecting Epidermal Growth Factor Receptor (EGFR) Mutation Status of Malignant Pleural Effusion. ACS Appl. Mater. Interfaces 2014, 7, 359-369. 33 ACS Paragon Plus Environment


26. Vijayaraghavan, P.; Liu, C.-H.; Hwang, K. C., Synthesis of Multibranched Gold Nanoechinus using a Gemini Cationic Surfactant and its Application for Surface Enhanced Raman Scattering. ACS Appl. Mater. Interfaces 2016, 8, 23909-23919. 27. Tran, N. T.; Li, A.; Chen, P.; Wang, Y.; Li, S.; Liedberg, B. Gold Mesoflowers with a High Density of Multilevel Long Sharp Tips: Synthesis and Characterization. J. Mater. Chem. C 2017, 5, 4884-4891. 28. Zhang, K.; Wang, C.; Rong, Z.; Xiao, R.; Zhou, Z.; Wang, S. Silver Coated Magnetic Microflowers as Efficient and Recyclable Catalysts for Catalytic Reduction. New J. Chem. 2017, 41, 14199-14208. 29. Kim, E.; Zwi-Dantsis, L.; Reznikov, N.; Hansel, C. S.; Agarwal, S.; Stevens, M. M. OnePot Synthesis of Multiple Protein-Encapsulated DNA Flowers and Their Application in Intracellular Protein Delivery. Adv. Mater. 2017, 29, 1701086. 30. Tian, Q.; Tang, M.; Sun, Y.; Zou, R.; Chen, Z.; Zhu, M.; Yang, S.; Wang, J.; Wang, J.; Hu, J. Hydrophilic Flower-Like CuS Superstructures as an Efficient 980 nm laser Driven Photothermal Agent for Ablation of Cancer Cells. Adv. Mater. 2011, 23, 3542-3547. 31. Ge, J.; Lei, J.; Zare, R. N. Protein-Inorganic Hybrid Nanoflowers. Nat. Nanotechnol. 2012, 7, 428. 32. Song, C.; Zhou, N.; Yang, B.; Yang, Y.; Wang, L. Facile Synthesis of Hydrangea FlowerLike Hierarchical Gold Nanostructures with Tunable Surface Topographies for SingleParticle Surface-Enhanced Raman Scattering. Nanoscale 2015, 7, 17004-17011. 33. Fang, J.; Du, S.; Lebedkin, S.; Li, Z.; Kruk, R.; Kappes, M.; Hahn, H. Gold Mesostructures with Tailored Surface Topography and their Self-Assembly Arrays for Surface-Enhanced Raman Spectroscopy. Nano Lett. 2010, 10, 5006-5013. 34. Yuan, H.; Fales, A. M.; Vo-Dinh, T. TAT Peptide-Functionalized Gold Nanostars: Enhanced Intracellular Delivery and Efficient NIR photothermal Therapy using Ultralow Irradiance. J. Am. Chem. Soc. 2012, 134, 11358-11361. 34 ACS Paragon Plus Environment

Page 34 of 49

Page 35 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


35. Bardhan, M.; Satpati, B.; Ghosh, T.; Senapati, D. Synergistically Controlled NanoTemplated Growth of Tunable Gold Bud-To-Blossom Nanostructures: A Pragmatic Growth Mechanism. J. Mater. Chem. C 2014, 2, 3795-3804. 36. Ong, Z. Y.; Chen, S.; Nabavi, E.; Regoutz, A.; Payne, D. J.; Elson, D. S.; Dexter, D. T.; Dunlop, I. E.; Porter, A. E. Multibranched Gold Nanoparticles with Intrinsic LAT-1 Targeting Capabilities for Selective Photothermal Therapy of Breast Cancer. ACS Appl. Mater. Interfaces 2017, 9, 39259-39270. 37. Ahmed, W.; Kooij, E. S.; Van Silfhout, A.; Poelsema, B. Controlling the Morphology of Multi-Branched Gold Nanoparticles. Nanotechnology 2010, 21, 125605. 38. Jeong, G. H.; Lee, Y. W.; Kim, M.; Han, S. W. High-Yield Synthesis of Multi-Branched Gold Nanoparticles and their Surface-Enhanced Raman Scattering Properties. J. Colloid Interface Sci 2009, 329, 97-102. 39. Han, J.; Li, J.; Jia, W.; Yao, L.; Li, X.; Jiang, L.; Tian, Y. Photothermal Therapy of Cancer Cells using Novel Hollow Gold Nanoflowers. Int. J. Nanomed. 2014, 9, 517. 40. Wang, W.; Yang, X.; Cui, H. Growth Mechanism of Flowerlike Gold Nanostructures: Surface Plasmon Resonance (SPR) and Resonance Rayleigh Scattering (RRS) Approaches to Growth Monitoring. J. Phys. Chem. C 2008, 112, 16348-16353. 41. Bakr, O. M.; Wunsch, B. H.; Stellacci, F. High-Yield Synthesis of Multi-Branched Urchin-Like Gold Nanoparticles. Chem. Mater. 2006, 18, 3297-3301. 42. Zhao, L.; Ji, X.; Sun, X.; Li, J.; Yang, W.; Peng, X. Formation and Stability of Gold Nanoflowers by the Seeding Approach: The Effect of Intraparticle Ripening. J. Phys. Chem. C 2009, 113, 16645-16651. 43. Jena, B. K.; Raj, C. R. Synthesis of Flower-Like Gold Nanoparticles and their Electrocatalytic Activity Towards the Oxidation of Methanol and the Reduction of Oxygen. Langmuir 2007, 23, 4064-4070.



44. Acar, H.; Garifullin, R.; Guler, M. O. Self-Assembled Template-Directed Synthesis of One-Dimensional Silica and Titania Nanostructures. Langmuir 2011, 27, 1079-1084. 45. McMillan, R. A.; Howard, J.; Zaluzec, N. J.; Kagawa, H. K.; Mogul, R.; Li, Y.-F.; Paavola, C. D.; Trent, J. D. A Self-Assembling Protein Template for Constrained Synthesis and Patterning of Nanoparticle Arrays. J. Am. Chem. Soc. 2005, 127, 28002801. 46. Zhuo, C.; Zhu, X.; Jiang, R.; Ji, F.; Su, Z.; Xue, R.; Zhou, Y. Comparison for Efficacy and Tolerability among Ten Drugs for Treatment of Parkinson’s Disease: A Network MetaAnalysis. Sci. Rep. 2017, 7, 45865. 47. Salat, D.; Tolosa, E. Levodopa in the Treatment of Parkinson's Disease: Current Status and New Developments. J. Parkinsons Dis. 2013, 3, 255-269. 48. Kageyama, T.; Nakamura, M.; Matsuo, A.; Yamasaki, Y.; Takakura, Y.; Hashida, M.; Kanai, Y.; Naito, M.; Tsuruo, T.; Minato, N. The 4F2hc/LAT1 Complex Transports LDOPA Across the Blood–Brain Barrier. Brain Res. 2000, 879, 115-121. 49. Adler-Abramovich, L.; Vaks, L.; Carny, O.; Trudler, D.; Magno, A.; Caflisch, A.; Frenkel, D.; Gazit, E. Phenylalanine Assembly into Toxic Fibrils Suggests Amyloid Etiology In Phenylketonuria. Nat. Chem. Biol. 2012, 8, 701. 50. Adler-Abramovich, L.; Kol, N.; Yanai, I.; Barlam, D.; Shneck, R. Z.; Gazit, E.; Rousso, I. Self-Assembled Organic Nanostructures with Metallic-Like Stiffness. Angew. Chem., Int. Ed. 2010, 49, 9939-9942. 51. Dube, T.; Mandal, S.; Panda, J. J., Nanoparticles Generated from a Tryptophan Derivative: Physical Characterization and Anti-Cancer Drug Delivery. Amino Acids 2017, 49, 975-993. 52. Fu, I. W.; Markegard, C. B.; Nguyen, H. D. Solvent Effects on Kinetic Mechanisms of Self-Assembly by Peptide Amphiphiles via Molecular Dynamics Simulations. Langmuir 2015, 31, 315-324. 36 ACS Paragon Plus Environment

Page 36 of 49

Page 37 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


53. Ahmed, S.; Pramanik, B.; Sankar, K. N. A.; Srivastava, A.; Singha, N.; Dowari, P.; Srivastava, A.; Mohanta, K.; Debnath, A.; Das, D. Solvent Assisted Tuning of Morphology of a Peptide-Perylenediimide Conjugate: Helical Fibers to Nano-Rings and their Differential Semiconductivity. Sci. Rep. 2017, 7, 9485. 54. Mason, T. O.; Chirgadze, D. Y.; Levin, A.; Adler-Abramovich, L.; Gazit, E.; Knowles, T. P.; Buell, A. K. Expanding the Solvent Chemical Space for Self-Assembly of Dipeptide Nanostructures. ACS Nano 2014, 8, 1243-1253. 55. Kar, S.; Drew, M. G. B.; Pramanik, A. Formation of Vesicles Through Solvent Assisted Self-Assembly of Hydrophobic Pentapeptides: Encapsulation and pH Responsive Release of Dyes by the Vesicles. Protein & Peptide Lett. 2011, 18, 886-895. 56. Rissanou, A. N.; Georgilis, E.; Kasotakis, E.; Mitraki, A.; Harmandaris, V. Effect of Solvent on the Self-Assembly of Dialanine and Diphenylalanine Peptides. J. Phys. Chem. B 2013, 117, 3962-3975. 57. Li, H.; Jia, Y.; Feng, X.; Li, J. Facile Fabrication of Robust Polydopamine Microcapsules for Insulin Delivery. J Colloid Interface Sci. 2017, 487, 12-19. 58. Zheng, W.; Fan, H.; Wang, L.; Jin, Z. Oxidative Self-Polymerization of Dopamine in an Acidic Environment. Langmuir 2015, 31, 11671-11677. 59. Yu, X.; Fan, H.; Wang, L.; Jin, Z. Formation of Polydopamine Nanofibers with the Aid of Folic Acid. Angew. Chem., Int. Ed. Engl. 2014, 53, 12600-12604. 60. Saha, A.; Bolisetty, S.; Handschin, S.; Mezzenga, R. Self-Assembly and Fibrillization of a Fmoc-Functionalized Polyphenolic Amino Acid. Soft Matter 2013, 9, 10239. 61. Kura, A. U.; Ali, S. H. H. A.; Hussein, M. Z.; Fakurazi, S.; Arulselvan, P. Development of a Controlled-Release Anti-Parkinsonian Nanodelivery System using Levodopa as the Active Agent. Int. J. Nanomed. 2013, 8, 1103-1110. 62. Silverstein, R. M.; Bassler, G. C.; Morrill, T. C. Spectroscopic Identification of Organic Compounds. John Wiley & Sons, New York 1981. 37 ACS Paragon Plus Environment


63. Bhosale, R. S.; Al Kobaisi, M.; Bhosale, S. V.; Bhargava, S.; Bhosale, S. V. Flower-Like Supramolecular Self-Assembly of Phosphonic Acid Appended Naphthalene Diimide and Melamine. Sci. Rep. 2015, 5, 14609. 64. Greenfield, N. J. Using Circular Dichroism Spectra to Estimate Protein Secondary Structure. Nat. Protoc. 2006, 1, 2876. 65. Gupta, M.; Bagaria, A.; Mishra, A.; Mathur, P.; Basu, A.; Ramakumar, S.; Chauhan, V. S. Self-Assembly of a Dipeptide Containing Conformationally Restricted Dehydrophenylalanine Residue to Form Ordered Nanotubes. Adv. Mater. 2007, 19, 858861. 66. Kelly, S. M.; Jess, T. J.; Price, N. C. How to Study Proteins by Circular Dichroism. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics 2005, 1751, 119-139. 67. Narayanan, K. B.; Park, H. H. Unnatural Amino Acid-Mediated Synthesis of Silver Nanoparticles and their Antifungal Activity against Candida Species. J. Nanoparticle Res. 2014, 16, 2523. 68. Grewar, T.; Gericke, M. Synthesis and Characterization of Anisotropic Gold Nanoparticles. Adv. Nanoparticles 2012, 1, 15. 69. Teale, F. W. J.; Weber, G. Ultraviolet Fluorescence of the Aromatic Amino Acids. Biochem J. 1957, 65, 476–482. 70. Huang, P.; Pandoli, O.; Wang, X.; Wang, Z.; Li, Z.; Zhang, C.; Chen, F.; Lin, J.; Cui, D.; Chen, X. Chiral Guanosine 5’-Monophosphate-Capped Gold Nanoflowers: Controllable Synthesis, Characterization, Surface-Enhanced Raman Scattering Activity, Cellular Imaging and Photothermal Therapy. Nano Research 2012, 5, 630-639. 71. Xie, J.; Lee, J. Y.; Wang, D. I. Seedless, Surfactantless, High-Yield Synthesis Of Branched Gold Nanocrystals In HEPES Buffer Solution. Chem. Mater. 2007, 19, 28232830.


Page 38 of 49

Page 39 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


72. Kostin, G.; Mashukov, V.; Torgov, V.; Kal’chenko, V.; Drapailo, A. Reduction Kinetics of Gold (III) Complexes with Calix [4] Arenes Derivatized with Thioether Groups on the Upper Rim. Russian J Inorganic Chem 2006, 51, 488-494. 73. Banerjee, S.; Ghosh, H.; Datta, A. Lamellar Micelles as Templates for the Preparation of Silica Nanodisks. J. Phys. Chem. C 2011, 115, 19023-19027. 74. Sasidharan, M.; Senthil, C.; Kumari, V.; Bhaumik, A. The Dual Role of Micelles as Templates and Reducing Agents for the Fabrication of Catalytically Active Hollow Silver Nanospheres. Chem. Commun. 2015, 51, 733-736. 75. Sajitha, M.; Vindhyasarumi, A.; Gopi, A.; Yoosaf, K. Shape Controlled Synthesis of Multi-branched Gold Nanocrystals through a Facile One-pot Bifunctional Biomolecular Approach. RSC Adv. 2015, 5, 98318-98324. 76. Xie, J.; Zhang, Q.; Lee, J. Y.; Wang, D. I. The Synthesis of SERS-Active Gold Nanoflower Tags for In Vivo Applications. ACS Nano 2008, 2, 2473-2480. 77. Ojea-Jiménez, I.; Bastús, N. G.; Puntes, V. Influence of the Sequence of the Reagents Addition in the Citrate-Mediated Synthesis of Gold Nanoparticles. J. Phys. Chem. C 2011, 115, 15752-15757. 78. Huang, J.; Guo, M.; Ke, H.; Zong, C.; Ren, B.; Liu, G.; Shen, H.; Ma, Y.; Wang, X.; Zhang, H. Rational Design and Synthesis of γFe2O3@ Au Magnetic Gold Nanoflowers for Efficient Cancer Theranostics. Adv. Mater. 2015, 27, 5049-5056. 79. Jung, K.; Hahn, J.; In, S.; Bae, Y.; Lee, H.; Pikhitsa, P. V.; Ahn, K.; Ha, K.; Lee, J. K.; Park, N. Hotspot-Engineered 3D Multipetal Flower Assemblies for Surface-Enhanced Raman Spectroscopy. Adv. Mater. 2014, 26, 5924-5929. 80. Zhu,C.; Meng, G.; Huang, Q.; Wang, X.; Qian, Y.; Hu, X.; Tang, H.; Wu, N. ZnONanotaper Array Sacrificial Templated Synthesis of Noble-metal Building-block Assembled Nanotube Arrays as 3D SERS-substrates. Nano Res. 2015, 8, 957–966.



81. Hrelescu, C.; Sau, T. K.; Rogach, A. L.; Jackel, F.; Laurent, G.; Douillard, L.; Charra, F. Selective Excitation of Individual Plasmonic Hotspots at the Tips of Single Gold Nanostars. Nano Lett. 2011, 11, 402–407. 82. Tian, F.; Bonnier, F.; Casey, A.; Shanahan, A.; Byrne, H. Surface Enhanced Raman Scattering with Gold Nanoparticles: Effect of Particle Shape. Anal. Methods 2014, 6, 9116-9123. 83. Saini, A.; Medwal, R.; Bedi, S.; Mehta, B.; Gupta, R.; Maurer, T.; Plain, J.; Annapoorni, S. Axonic Au Tips Induced Enhancement in Raman Spectra and Biomolecular Sensing. Plasmonics 2015, 10, 617-623. 84. Saini, A.; Maurer, T.; Lorenzo, I.; Santos, A.; Beal, J.; Goffard, J.; Gerard, D.; Vial, A.; Plain, J. Synthesis and SERS Application of SiO2@Au Nanoparticles. Plasmonics 2015, 10, 791-796.


Page 40 of 49

Page 41 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Synthesis of self-assembled levodopa microtubes. Scanning electron microscopic (SEM) images affirming the formation of tubes like structures upon self-assembly of levodopa in various solvents. a) HFIP. b) Ethanol. c) DMSO. d) Water. e) Morphological details of crude levodopa powder.f) Fourier transformed infrared (FTIR) spectra of levodopa tubes formed in water and that of the raw drug.



Figure 2. Synthesis of gold (Au) microroses on self-assembed levodopa microtubes. a) Size distribution profile of gold microroses synthesized using self-build levodopa microtubes in pure water. b) SEM image of the gold microroses. c) SEM image of the gold microroses before elemental mapping. d) Representative EDS elemental mapping (Au, N, O) of microroses. e) mapped image. f) Gold. g) Nitrogen. h) Oxygen. i) UV-Vis absorption spectrum of levodopa tubes and gold microroses at different time points. j) SEM image of microrose after 1 month of incubation in solution at 4 °C. k) X-ray diffraction (XRD) pattern of the fabricated gold microroses.


Page 42 of 49

Page 43 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. a) Transmission electron microscopic (TEM) image of the Au microrose. b) Protruded single petal from the core of the microrose showing the uniform coating of levodopa and its oxidized products. c) Selected area electron diffraction (SAED) pattern from microrose. d) High resolution TEM image of the crystal lattice structure of the petal, from the core of microrose. e) Zoomed-in HRTEM image showing the obtained d value for (111) plane. f) Inverse Fast Fourier Transform (IFFT) profile of the HRTEM image.



Figure 4. Role of self-assembly and effect of various process parameters on the characteristic properties of synthesized Au microroses. a) Scanning electron microscopic (SEM) images of gold microroses obtained by varying the nature of the self-assembled levodopa tubes in HFIP, Ethanol, DMSO and Water. b,c,d,e) SEM images of gold structures obtained by varying the concentration of self-assembled levodopa tubes. f and g) SEM images showing the effect of stirring on the synthesized gold microroses. f) SEM image of floweres formed with stirring g) SEM image showing fully bloomed uniform sized microroses synthesized without vigorous stirring. h, i) SEM images showing the effect of temperature on the synthesized gold microroses at 4 and 50 °C.


Page 44 of 49

Page 45 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. Growth kinetics of the as-prepared gold microroses. a-f) Time evolution SEM images showing the growth process of microroses at 0, 5, 10, 30 min, and 2 h respectively. Insets (right): Magnified SEM images of the microroses.



Figure 6. a) Field emission scanning electron microscopy (FESEM) image showing the assembly pathway dependent single-step synthesis of gold microroses using method 1 (M1). b) FESEM image showing the single-step synthesis of gold nanoroses using method 2 (M2) which is done by merely interchanging the sequence of reagents addition. c) TEM image of gold microroses synthesized using M1. d) TEM image of gold nanoroses synthesized using M2.


Page 46 of 49

Page 47 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 1. Schematic illustration showing the proposed mechanism of assembly pathway dependent aqueous synthesis of gold microroses on self-build levodopa microtubes towards SERS based sensing applications in glioblastoma.



Figure 7. Surface enhanced Raman scattering (SERS) based detection of the gold microroses using rhodamine 6G (R6G) as analyte. a) SERS spectra of R6G capped gold microroses, R6G capped spherical gold nanoparticles, gold microroses, and pure R6G as a control. The increased intensities of bands at 1366 cm-1, and 1570 cm-1 in R6G capped microroses as compared to pure R6G indicated that the microroses exhibited strong SERS effects. b) Intracellular SERS signals detected from C6 glioma cells incubated with R6G capped gold microroses. c) Confocal laser scanning microscopy (CLSM) images of C6 glioma cells treated with Au microroses, R6G solution, R6G capped Au microroses showing cellular internalization of as synthesized microroses. Scale bar = 50 μm. 48 ACS Paragon Plus Environment

Page 48 of 49

Page 49 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table of Contents 82x44mm (300 x 300 DPI)

ACS Paragon Plus Environment

Microroses on Levodopa Microtubes for SERS-Based

Recommend Documents