Article pubs.acs.org/JPCC
Electrochemically Assembled Gold Nanostructures Platform: Electrochemistry, Kinetic Analysis, and Biomedical Application Rachna Sharma,†,∥ Md. Azahar Ali,† N. Rajan Selvi,‡ Vidya Nand Singh,§ Ravindra K. Sinha,∥ and Ved Varun Agrawal*,† †
DST Centre on Biomolecular Electronics, Biomedical Instrumentation Section, CSIR-National Physical Laboratory, New Delhi 110012, India ‡ Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560064, India § Electron and Ion Microscopy Section, CSIR-National Physical Laboratory, New Delhi 110012, India ∥ Department of Applied Physics, Delhi Technological University, New Delhi 110042, India S Supporting Information *
ABSTRACT: A novel one-step electrochemical method for controlled synthesis of electroactive gold nanoparticles (Au NPs) in an organic medium using an organometallic precursor Au(PPh3)Cl [Ph = phenyl] has been proposed. The hierarchical assembly of Au nanostructures has been tuned on indium tin oxide (ITO) surface during electrochemical reduction of Au(PPh3)Cl using cysteamine. The Au NPs act as building blocks to form secondary structures of Au that has been confirmed using transmission electron microscopic studies. The presence of triphenylphosphine in Au film enhances the electrocatalytic activity, resulting in higher charge transfer kinetics. The cholesterol oxidase (ChOx) as a model enzyme has been immobilized on various fabricated nanostructured Au films. Direct electron transfer properties of nanostructured Au films result in third-generation cholesterol biosensor. We have investigated the biosensing performance of different Au nanostructures toward cholesterol estimation at low operating potential (+0.3 V). The high sensitivity of 4.22 AM−1 cm−2 and low detection limit of 5.49 μM of this biosensor (ChOx-Glu/Cys-Au/ ITO) is due to higher current resulting from monodisperse Au NPs. In addition, this bioelectrode shows charge transfer rate constant as 247.27 s−1 and low Kapp m value as 0.57 mM. The biosensor shows good reproducibility, stability, and selectivity and thus can be utilized for health care diagnostics application.
1. INTRODUCTION The application of noble metal nanostructures such as gold (Au) in the development of biomedical diagnostic devices has remarkably increased.1,2 The unique properties of Au nanostructures like high quantum yield of Rayleigh scattering, photostability, plasmonic properties, etc., enable them to serve as molecular probes for biosensors and in vivo imaging of single live cells. 3−5 The potential use of bioconjugated Au nanostructures as a novel contrast agent for cancer cells imaging and photothermal therapy has been investigated.6 Chen et al. have reported the use of anti-EGFR functionalized gold nanocages as contrast agent for optical diagnostics of breast cancer and as potential thermal therapeutic agent for cancer treatment.7 In addition, the Au nanostructures are utilized in photovoltaics,8 optical sensing,9 drug delivery and discovery,10 electrocatalysis, and electrochemical biosensing applications.11,12 The building blocks of gold nanoparticles (Au NPs) to construct the electrochemical biosensor are promising platform owing to their extraordinary electrocatalytic activity. The electrochemical properties of Au can be tuned by controlling the size and shape of Au nanostructures in order to improve the efficacy of biosensor.13 The Au nanostructures provide unprecedented electron transfer between biomolecules © 2014 American Chemical Society
and electrode surface resulting in higher sensitivity, excellent detection limit, and fast response.14 The low redox potential of Au nanostructures may result in higher selectivity and reproducibility. It is known that most enzyme molecules are unable to directly exchange electrons with electrode surface due to the inaccessibility of the electroactive redox centers embedded in protein shells.15 This limits the performance of a biosensor. However, Au nanostructures have been found to possess excellent electrocatalytic activity and may thus result in efficient direct electron transfer between enzyme and electrode surface. Haruta et al. have demonstrated the high catalytic activity of Au nanoparticles (18.2 MΩ· cm was used for preparing all aqueous solutions. Indium−tin oxide (ITO) coated glass plates with a sheet resistance and transmittance of 25 Ω sq−1 and 90% were used for fabrication of Au films. The stock solution of cholesterol were prepared in 10% Triton X-100 and stored at 4 °C. 2.1. Instrumentation. High resolution transmission electron microscopy (HR-TEM) and scanning electron microscopy (SEM) studies were carried out using TecnaiiG2F30 STWIN and FEI Nova 600 microscopes, respectively. The optical absorption studies were conducted on a PerkinElmer Lambda 950 UV/vis/NIR spectrophotometer. Fourier transform infrared (FT-IR) studies were carried out on a PerkinElmer Spectrum BX II spectrophotometer. Contact angle measurements were carried out using OCA 15 EC (DataPhysics). The electrochemical experiments were conducted on an Autolab PGSTAT 302N System (Ecochemie, The Netherlands) in the three-electrode system with platinum as auxiliary, Ag/AgCl as reference, and ITO substrate as working electrode. 2.2. Synthesis of Au Nanostructures. Au nanostructures were deposited on ITO coated glass substrate using the chronoamperometric technique. During deposition, the Au− phosphine complex [Au(PPh3)Cl] (2.5 mM) and trichloroacetic acid (50 mM) dissolved in 15 mL of acetonitrile solvent 6262
dx.doi.org/10.1021/jp411797u | J. Phys. Chem. C 2014, 118, 6261−6271
The Journal of Physical Chemistry C
Article
Scheme 2. Surface Biofunctionalization of Au Nanostructure for Cholesterol Estimation
Figure 1. TEM and HRTEM micrographs of Au nanostructures electrochemically deposited with cysteamine concentrations of (a) 0, (b) 0.025, (c) 0.25, and (d) 2.5 mM.
on ITO surface. Higher cysteamine concentration in the precursor solution augments the reaction kinetics, leading to faster evolution of Au films with different nanostructures. Thus, the shape of Au nanostructures has been tuned and optimized using amphiphilic cysteamine molecules during electrochemical deposition. 2.3. Enzyme Immobilization. Prior to enzyme immobilization, the amine functionalized Cys-Au/ITO electrode was dipped in 0.5% glutaraldehyde (cross-linker) solution for 4 h and rinsed with deionized water. The modified electrode was incubated with ChOx solution (1 mg mL−1) for overnight at 4 °C. One terminal (CHO) of glutaraldehyde forms a covalent amide bond with amine functionalized Au surface while the other terminal forms an additional amide bond with amine groups of enzyme (ChOx)32 (Scheme 2). The loosely bound enzymes were washed off with pH 7.4 phosphate buffer saline (PBS). This fabricated ChOx-Glu/Cys-Au/ITO bioelectrode was kept in a refrigerator at 4 °C when not in use.
were used as electrolyte. The deposition potential has been optimized to −10 V at 25 °C. Further, we have introduced various cysteamine concentrations (0.025, 0.25, and 2.5 mM) in the electrolyte solution in order to control shape of Au nanostructures (Scheme 1). Onset of decomposition of Au(PPh3)Cl and liberation of Au3+ ions occur on applying voltage which is evident from the gradual emergence of yellow color in the electrolytic solution. Au3+ ions drift toward ITO electrode under the influence of electric field and electrochemical reduction of Au3+ ions results in Au0. Simultaneously, some Au−phosphine complex crystallizes on ITO surface, which gradually gets reduced.29 This is evident from characteristic UV absorption of the Au− phosphine complex at ∼390 nm.30 Nucleation of Au NPs occurs during electrochemical reduction of Au3+ to Au0, and various structures result from the assembly of NPs depending on cysteamine concentrations. In the absence of cysteamine, the rate of reaction is slow, which results in the formation of elongated nanostructures (rod shape).31 Cysteamine addition leads to higher current resulting in faster deposition of Au nanostructures (Figure 4A). Because of insufficient cysteamine molecules, the monodispersity of the Au NPs is unstable. They assemble themselves into various hierarchical nanostructures in order to attain minimum surface energy, and thus, the smaller amount of surfactant provide larger nanostructures.31 With increase of cysteamine molecules, Au NPs assembly gets restricted and finally results in well-spaced individual Au NPs
3. RESULTS AND DISCUSSION 3.1. TEM Studies. Figure 1a shows TEM micrograph of Au film prepared with Au(PPh3)Cl in acetonitrile solution. It can be seen that the rod-shaped Au (length ∼1 μm, aspect ratio ∼4.5) is randomly oriented on the ITO surface. The high resolution micrograph shows crystalline domains of Au with dspacing of 0.23 nm corresponding to (111) plane of fcc-Au (JCPDS- 040784). Inset shows a single cylindrical Au rod. It 6263
dx.doi.org/10.1021/jp411797u | J. Phys. Chem. C 2014, 118, 6261−6271
The Journal of Physical Chemistry C
Article
Figure 2. (A) UV−vis spectra and corresponding SEM micrographs of Au films electrochemically deposited with cysteamine concentration of (a) 0, (b) 0.025, (c) 0.25, and (d) 2.5 mM. (B) Contact angle of water with Au films synthesized under similar conditions.
Figure 3. FT-IR spectra of (a) Cys-Au/ITO electrode, (b) Glu/Cys-Au/ITO electrode, and (c) ChOx-Glu/Cys-Au/ITO bioelectrode.
Au:Cys = 1:1), no secondary structures resulted and monodisperse Au NPs were obtained due to complete coverage with the cysteamine molecules (Figure 1d). The average size of Au NPs has been estimated as ∼4 nm. Loading of these Au NPs on film surface is higher compared to other structures. 3.2. UV−vis and SEM Studies. Figure 2A shows the optical absorption spectra of the prepared nanocrystalline Au films with different concentrations of cysteamine and the corresponding SEM images. Films show characteristic plasmon bands from uncoupled Au nanocrystals at ∼560 nm alongwith bands at higher wavelength arising due to electronic coupling among Au NPs, elongated nanostructures, or assembly of NPs.34,35 The absorption intensity increases with increasing cysteamine concentration, suggesting higher loading of nanoparticles and formation of thicker films. A slight blue-shift has been observed in higher wavelength band, suggesting formation of smaller nanostructures with subsequent increase in cysteamine concentration, which is further confirmed by SEM studies. The higher wavelength band disappears with excess of cysteamine due to complete coverage of cysteamine molecules onto all the facets of Au NPs which restricts the electronic coupling among NPs.36 However, in the absence of cysteamine, the band at higher wavelength is due to elongated structure rather than assembly of NPs. The peak found at 390 nm corresponds to PPh3 stabilized Au clusters.30 3.3. Contact Angle Studies. Contact angle measurements have been carried out in sessile drop mode to measure the
has been observed that the slow deposition rate of Au due to the limited current across electrode−electrolyte system in organic solvent results in low loading of nanostructures on film surface. Owing to slow reaction kinetics, the Au−phosphine complex crystallizes into elongated structures on ITO surface and eventually reduces to form Au rods while few Au(PPh3)Cl crystals decompose and reduce to spherical shape Au NPs. On addition of cysteamine (0.025 mM), the microstructural transformation of Au rods has been observed (Figure 1b). These Au rods (length ∼600 nm, aspect ratio ∼9.2) have conical ends due to selective etching of rods at the ends.33 The enlarged view of this Au rod has been shown in image (Figure 1b, inset). The regular arrangement of monodisperse Au NPs (size
