Biosynthesis of Gold Nanoparticles using Osmudaria Obtusiloba Extract and their Potential use in Optical Sensing Applications

Research Article

Austin J Biosens & Bioelectron. 2015; 1(3): 1014.

Biosynthesis of Gold Nanoparticles using Osmudaria Obtusiloba Extract and their Potential use in Optical Sensing Applications

Rojas-Pérez A1, Adorno L2, Cordero M3, Ruiz A3, Mercado-DĂ­az Z1, RodrĂ­guez A4, Betancourt L1, Vélez C1, Feliciano I1, Cabrera C1 and DĂ­az- Vázquez LM1*

1Department of Chemistry, University of Puerto Rico-Rio Piedras Campus, Puerto Rico

2Nutrition Program, University of Puerto Rico-Rio Piedras Campus, Puerto Rico

3InterdisciplinaryProgram, University of Puerto Rico-Rio Piedras Campus, Puerto Rico

4Department of Environmental Science, University of Puerto Rico-Rio Piedras Campus, Puerto Rico

*Corresponding author: author: Diaz-Vazquez LM, Chemistry Department, University of Puerto Rico, Rio Piedras, PO Box 23346, San Juan, P.R. 00931, Puerto Rico

Received: September 02, 2015; Accepted: September 26, 2015; Published: September 29, 2015

Abstract

The optical properties of gold nanoparticles depend on particles size, shape, protective ligand and the synthesis method used for their production. The present study describes an eco-friendly method for the synthesis of gold Nanoparticles (AuNPs) utilizing the macro algae Osmundaria Obtusiloba. The optical properties of the obtained nanoparticles and whether the use of the macroalgae extract confers them with additional properties were evaluated and compared with nanoparticles obtained via traditional methods. AuNPs were physical and chemically characterized by UVVIS, FTIR, HRTEM, SEMEDS, XRD and Raman Spectroscopy. The UVVIS spectra showed a peak at 540 nm that confirmed the formation of AuNPs. XRD pattern confirmed a face centered cubic (fcc) crystalline structure for these nanoparticles. HRTEM images revealed several shapes including spherical, triangular, and diamond shaped nanoparticles with size ranges between 10 to 20 nm. The presence of 83.58 %wt. of elemental Au in the obtained nanoparticles was elucidated via EDS. Synthesized AuNPs SERS enhancement factor toward 4-nitrothiophenol concentration determination. Additionally, their utility as fluorescence quencher or enhancer was evaluated using methyl orange.

Keywords: Gold Nanoparticles; Biosynthesis; Osmundaria Obtusiloba; SERS; Fluorescence enhancement and quenching

Abbreviations

AuNPs: Gold Nanoparticles; UVVIS: Ultraviolet-Visible; SERS: Surface Enhanced Raman Spectroscopy; PL: Photoluminescence; AuNPs-Osm: Gold Nanoparticles Synthesized with Osmundaria Obtusiloba extract; AuNPs-NaBH4: Gold Nanoparticles Synthesized with NaBH4; SPR: Surface Plasmon Resonance; MO: Methyl Orange; FTIR: Fourier transform infrared spectroscopy; HR-TEM: High Resolution Transmission Electron Microscopy; SEM-EDS: Scanning Electron Microscopy- Energy Disperse Spectroscopy; XRD: X-Ray Diffraction; AEF: Analytical Enhancement Factor; NR: Normal Raman

Introduction

Gold Nanoparticles (AuNPs) have widely been used as an essential element in the fabrication of sensing and electronic devices, as well as for biomedical applications, because of their remarkable physical and chemical properties [1-5]. The use of AuNPs in spectrometric analysis is related to their Surface Plasmonresonance (SPR) properties, which cause that its solutions display a range of colors from red, purple, to violet with increasing particle size. This color is related to the strong absorption and scattering of radiation at 520 nm, which is the result of the collective oscillation of conduction electrons on the surface of AuNPs when they are excited by the incident light [6]. AuNPs SPR properties depend on particles size, shape, protective ligand, refractive index of the solvent, and temperature. Also AuNPs can enhance Raman radiation scattering, which leads to the development of Surface Enhance Raman Spectroscopy (SERS) based sensing. AuNPs can enhance or quench the fluorescence of a fluorophore depending on the AuNP-flourophoredistance [7]. All these mentioned AuNPs optical properties can be tailored during the synthesis of the nanoparticles through inducing a change in their size, morphology, and surface modifications.

During the last decade, many research efforts have being focused on the development of AuNPs’s synthesis methods to tailor their physical and chemical properties in order to expand their use for different research and industrial applications. Wet methods of synthesis are a bottom-up process in which the nanoparticles are produced by reducing gold salts in the presence of appropriate capping agents that minimizes particle aggregation. The method proposed by Turkevich and coworkers in 1951, in which tetrachloroauric acid (HAuCl4) is reduced in water by sodium citrate, and the reduction of gold salts with Sodium Borohydride (NaBH4) have been the most popular methods [8,9]. The top-down methods are used to produce AuNPs from bulk material via mechanical and/or physical processes to reduce it to the desired size such as milling, laser ablation [10],and digestive ripening [11].

Although there are a variety of AuNPs synthesis methods, they are fraught with many problems including the use of toxic solvents, generation of hazardous by-products, and high energy consumption [5]. Alternative routes for metallic nanoparticle synthesis stem from the use of natural resources in order to provide a cost effective and environmentally method [12]. Simple prokaryotes to complex

eukaryotic organisms, including higher angiospermic plants, are being used for the production of low-cost, energy-efficient, and nontoxic metallic nanoparticles [13]. Particular interest is given to the use of plants as a natural resource in the biosynthesis of metallic nanoparticles. Plants polyphenols, proteins, carbohydrates and other biomolecules allow them to act as reducing and stabilizing agents in nanoparticles synthesis [14,15].

The use of macroalgae is being researched as a possible new natural resource for AuNPs biosynthesis. Marine macroalgae are known as biofactories, because they are a rich source of biologically active compounds and antioxidants such as polyphenols, bromophenols, polysaccharides, photosynthetic pigments, proteins, vitamins, fatty acids, glycolipids, and protective enzymes [16]. Although macroalgae, like other photosynthesizing organisms, are constantly exposed to environmental conditions that lead to the formation of free radicals and other oxidizing agents, they are resilient to oxidative damage to their structural components. Their resistance to oxidation during photosynthesis and storage suggests that their cells have developed protective antioxidative defense systems [17]. Consequently, macroalgae chemical composition enables them to act as reducing and stabilizing agents in nanoparticles synthesis similarly to plants. The use of macroalgae extract could thus provide us with an ecofriendly and cost effective new method to synthesize metallic nanoparticles.

This research was directed towards determining whether the use of the macroalgae Osmundaria Obtusiloba, obtained from the north coast of Puerto Rico, is an appropriate resource to mediate the biosynthesis of AuNPs. Additionally, these biosynthesized nanoparticles were compared with nanoparticles synthesized using sodium borohydride as a reducing agent, in order to evaluate if the implementation of the macro algae extract provoked changes in functionality and/or the optical properties of the nanoparticles. The obtained nanoparticles were subjected to characterization methods in order to determine their size, composition, and morphology. The SERS enhancement properties of AuNPs produced via both synthesis methods were determined and compared. In addition AuNPs utility as a fluorescence enhancer or quencher was evaluated using Methyl Orange (MO) as a model compound.

Materials and Methods

Materials

Hydrogen Terrachloroaurate (III) trihydrate (HAuCl4.3H2O) 99.9 +%, sodium borohydride, methyl orange, and 4-nitrothiophenol (80%) were purchased from Sigma Aldrich and were used as received. All the glassware was analytically washed with sulfuric acid and nitric acid followed by nanopure water. All the solutions were prepared using nanopure water (18.2MΩ.cm) obtained from a Barnstead Nanopure Diamond™.

Preparation of macroalgae biomass and extract

The macroalgae Osmundaria Obtusiloba was collected from the north coast of Puerto Rico. It was washed and cleaned with distilled water to remove epiphytes and detritus attached to it. Then, it was dried at 65 °C, pulverized, sieved through< 0.5 mm, and stored at room temperature in a desiccator. A macroalgae extract was prepared by boiling 5.00g of grinded macroalgae in 100mL of anethanolic solution (1:1 ethanol and nanopure water) at 70°C for 20 min. The mixture was transferred to centrifuge tubes and subjected to 2,000 rpm for 20 minutes; the supernatant was filtered, evaporated to dryness, and reconstituted innanopure water. The extract was characterized by UVVIS and FTIR spectroscopy, and stored at 4°C for further use.

Biosynthesis of Gold Nanoparticles

An aqueous solution of 1mMHAuCl4was prepared. Reaction mixtures containing 5.0 mL of the algae extract and 20.0 mL of 1mMHAuCl4 were incubated at 60 °C, under a constant agitation of 200 rpm, for 2 hours. Changes in color of the reaction mixture were used to indicate the formation of nanoparticles. Aliquots of the reaction mixture were analyzed, using a UVVIS spectrophotometer (Hach Dr5000) with a resolution of 1 nm, every five minutes in order to determine whether nanoparticle formation had occurred by observing the absorbance at the characteristic band for AuNPs near 520 nm. In order to collect and purify the produced nanoparticles, the reaction mixture was centrifuged at 20,000rpm for 20 min, the precipitate was collected, and the supernatant was again centrifuged at 20,000rpm for 20 min. The collected solid pellets were washed with high purity ethanol to remove residues from the macroalgae extract and centrifuged at 20,000 rpm for 15 min. The supernatant was removed and the pellet was dried at 65°C under an inert nitrogen flow. The dried AuNPs were stored in desiccators for further analysis.

Chemical synthesis of AuNPs using NaBH4

AuNPs were synthesized using NaBH4as the reducing agent using the methodology suggested by Mulfinger, L. et al. [9]. Briefly, an aliquot of 10.00 mL of 1.0 mMGold (III) chloride hydrate was added dropwise (approximately 1 drop per second) to 30 mL of a 2.0 mMNaBH4 solution that had been chilled in an ice bath. The reaction mixture was stirred vigorously on a magnetic stir plate. The solution turned violet after the addition of 1 mL of gold solution. After the entire addition, the stirring was stopped, the stir bar removed, and the resulting solution stored for further use. The nanoparticles were recovered with the same procedure used for AuNPs biosynthesized with O. Obtusiloba extract.

Characterization of AuNPs

The size and morphology of AuNPs were evaluated using High Resolution Transmission Electron Microscopy (HRTEM) and Scanning Transmission Electron Microscopy (STEM). The analyses were done using a FEI Tecnai F20 microscope with a monochromator-energy filter. An 80 mm Oxford SDD detector was used for X-ray detection, and the images were analyzed using the Inca Software. Samples for HRTEM were prepared by drop-coating AuNPs solution onto carbon-coated copper grids. The crystal nature of AuNPs was determined by X-Ray Diffraction (XRD) analysis using a Rigaku ULTIMA III Diffractometer with a Cu-Kα radiation source (λ= 1.5406 Å) operating at 40 kV and 44 mA. The XRD patterns were recorded between 30o and 90o at scan rate of 4.0º/min. Gold concentration, conversion, and composition of nanoparticles were determined using disperse SEM-X-ray fluorescence spectrometer and Energy Dispersive Spectroscopy (EDS) at 20kv under 5,000x magnification with a Bruker S4 Pioneer Instrument. The presence of functionalization of the AuNPs surface was evaluated by Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATRFTIR) The ATR-FTIR spectra were recorded at a resolution of 1 cm 1between300 and 4000cm-1 at a Shimatzu IRAffinity-1 with an ATR 8000A accessory.

Optical properties of AuNPs

UV-Visible spectra of the AuNPs solution was recorded using a UVVIS spectrophotometer (Hach Dr5000) with a resolution of 1 nm over a wavelength range of 250 to 750 nm using 1cm path length quartz cuvettes. These cuvettes were cleaned before each use by sonicating them for five minutes in an acidic solution (H2SO4 0.10 M) and then rinsed with deonized water until the pH of the effluent was 7.0. The UV-spectra were used to compare the AuNPs in terms of their SPR properties and estimated the size using the peak maxima. For each type of AuNPs, UVVIS spectra at five different concentrations were recorded by directly diluting the as-prepared nanoparticles solution with nanopure water to the expected concentrations [18].

Quantum yield

The quantum yield of the synthesis AuNPs was determined using a procedure reported by Fery&Lavabre, 1999 [19]. Quinine sulfate acidic solution (0.05 M H2SO4) was used as a reference standard, for which quantum yield is yield = 0.54at an emission range 400-600 nm as reported in the literature. Absolute values of the quantum yield were calculated using the following equation (1)

Φ x = Φ std ( I x A x )( A std I std )( η x 2 η std 2 )            (1) [email protected]@[email protected]@+=feaaguart1ev2aqatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLnhiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq=Jc9vqaqpepm0xbba9pwe9Q8fs0=yqaqpepae9pg0FirpepeKkFr0xfr=xfr=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[email protected][email protected]

Were theΦx is the quantum yield of the AuNPsand Φstd of Quinine sulfate, A is the absorbance at the emission wavelength, I is the area under the corrected emission curve wavelength, and is the refractive index of the solvent of the corresponding solution. To reduce the reabsorption within the sample on the observed emission spectrum, the absorbance values of all the solutions were maintained under 0.05 [19,20].

Fluorescence properties

The effects of AuNPs synthesized with O. Obtusiloba on the fluorescence properties of Methyl Orange (MO) were studied and compared with the effect of AuNPs synthesized with NaBH4. All the analyses were performed in a Shimadzu RF-5301 Spectrophotofluorometer, using medium scanning speed, high sensitivity, and excitation and emission slit widths of 10 nm. After each sample run, the fluorometerquartz cuvettes were rinsed with sulfuric acid and distilled water until the effluent had a neutral pH and dried prior use with the next sample.

A 10 μM stock solution of methyl orange was prepared and adjusted to pH 4.0 with phosphate buffer solution. A set of aqueous solution was prepared for each type of NPs, maintaining the MO concentration constant at 1 μM and varying the concentration of nanoparticles from0.17mg/L to 17.9 mg/L. For each solution, the emission spectra were recorded using an excitation wavelength of 270 nm and an emission range of 250 to 800nm.

Surface enhancement raman spectroscopy (SERS) enhancement

The SERS enhancement of the AuNPs toward 4-Nitrothiophenol (4-NTP) was evaluated and compared with SERS of Au-NPs synthesized with NaBH4. For this purpose, the procedure reported by Seongmin Hong and Xiao Li was followed [21]. Briefly, 4-NTP samples were prepared using 1.00mL of gold colloidal solutions with different amounts of 4-NTP, ranging from 0.1 to 0.7 μM, then 1.00 mL of 1M NaCl was added to all solutions. The concentration of gold nanoparticles was kept constant for all solutions. All the SERS experiments were carried out using Raman Microscopy (Thermo Scientific, DXR), an excitation laser with a wavelength of 780nm was applied with 10mW of power, 3s of exposure time, and 3 accumulations. The spectrum grating was 700, and a 20X microscopic objective lens were used for the analysis. All the reported analyses were performed three times. Prepared samples were maintained in darkness for 10 minutes prior to the analysis to minimize fluctuations of SERS spectra by allowing the solutions to reach an equilibrium state. The SERS Analytical Enhancement Factor (AEF) was calculated using equation (2) as reported by Seongmin, where ISERS and INR are the intensity of the selected vibrational peak in SERS and Normal Raman (NR) measurements, respectively, and CNR and CSERS are the concentration of 4-NTP used for NR and SERS measurement, respectively.

EF= I SERS C NR I NR C SERS         (2) [email protected]@[email protected]@+=feaaguart1ev2aqatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLnhiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq=Jc9vqaqpepm0xbba9pwe9Q8fs0=yqaqpepae9pg0FirpepeKkFr0xfr=xfr=xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaaeaaaaaaaaa8qacaWGfbGaamOraiabg2da9maalaaapaqaa8qacaWGjbWdamaaBaaaleaapeGaam4uaiaadweacaWGsbGaam4uaaWdaeqaaOWdbiaadoeapaWaaSbaaSqaa8qacaWGobGaamOuaaWdaeqaaaGcbaWdbiaadMeapaWaaSbaaSqaa8qacaWGobGaamOuaaWdaeqaaOWdbiaadoeapaWaaSbaaSqaa8qacaWGtbGaamyraiaadkfacaWGtbaapaqabaaaaOWdbiaabccacaqGGaG[email protected][email protected]

Results

Biosynthesis and chemical synthesis of AuNPs

The synthesis of gold nanoparticles using Osmundaria Obtusiloba extract (AuNPs-Osm) was done successfully, as confirmed by the distinct color appearance and UVVIS spectrophotometric data. The SPR band of the reaction mixture was kinetically monitored from 300 to 750 nm. Initially, the reaction mixture exhibit a band near 325 nm, characteristic of the O. Obtusiloba extract as shown in Figure 1a; however, as the reaction proceeded, this band decreased, while a band centered at around 540 nm(characteristic of the SPR of AuNPs) was revealed and became more pronounced with time. This tendency confirms that the macroalgae extract is acting as a reducing and capping agent in the biosynthesis of AuNPs. Our results showed that the reaction was completed in approximately 20 minutes (Figure 1b), afterwards reaching a steady state. After recovering the synthesized nanoparticles, a 92% yield was determined by gravimetric analysis. The expounded results confirm that the biosynthesis method is timesuitable and efficient.