ARTICLE pubs.acs.org/JPCC
Facile Synthesis of Bile Salt Encapsulated Gold Nanoparticles and Its Use in Colorimetric Detection of DNA S. Chandirasekar,† G. Dharanivasan,‡ J. Kasthuri,§ K. Kathiravan,‡ and N. Rajendiran†,* †
Department of Polymer Science, University of Madras, Maraimalai Campus, Guindy, Chennai-600 025, India Department of Bio-Technology, University of Madras, Maraimalai Campus, Guindy, Chennai-600 025, India § Department of Chemistry, Arignar Anna Government Arts College for Women, Walajapet- 632513, Vellore, Tamil Nadu, India ‡
ABSTRACT: A novel way to synthesize gold nanoparticles (AuNPs) using naturally occurring bile salts, namely, sodium cholate (NaC) and sodium deoxy cholate (NaDC), as reducing and capping agents at room temperature has been reported. Variations of NaC concentration provided good control over the size and shape of the AuNPs, allowing us to tune the color of NPs from green to red. While the hydroxyl group of bile salt reduced the Au3+ ions, the carboxylate group binds strongly to the surface of the NPs. The optical properties, size, and shape of the NPs were analyzed using UVvisible spectroscopy and transmission electron microscopy (TEM). The interaction of NaC on the AuNP surface was studied using cyclic voltammetry, FT-IR, and thermogravimetric analysis (TGA). The combination of steady-state and time-resolved quenching studies using fluorescent probes confirmed the hydrophobic interaction between NaC micelles and AuNPs. The color change properties associated with the aggregation of NPs were used for the colorimetric detection of plant-associated Gemini viruses using degenerate probes. The assay was completed within 20 min, and g600 pmol of the target DNA could be detected by UVvisible spectroscopy.
I. INTRODUCTION Surface functionalization of noble metal nanoparticles (MNPs) using a compound of biological origin is an emerging highlight of the intersection of nanotechnology and biotechnology, which has significantly promoted the cross-fertilization of research ideas to develop environmentally benign technologies in material synthesis.13 Among nanomaterials, gold (Au) NPs are especially attractive as they exhibit vibrant optical absorbance, high dispersibility in aqueous medium, chemical inertness, and biocompatibility.4,5 A small change in the AuNP size, shape, surface nature, and the distance between particles leads to tunable changes in their optical properties.6 These features have been employed for a number of applications including highresolution biomedical imaging,7 catalysis,8 drug delivery,9 as therapeutic agents,10 and for detection of biochemical substances.11 As a result of their uses in the medical arena, several research groups have made significant efforts to functionalize the AuNPs using biomolecules like carbohydrate,12 nucleic acids,13 proteins,14 biopolymers,15 and amino acid conjugated bile salts.16 In the present study, effort has been made to synthesize size- and shape-controlled AuNPs using sodium salts of cholic acid (NaC) and deoxycholic acid (NaDC) as capping and reducing agents. These bile salts are naturally occurring steroidal detergents in mammals, which aids in the digestion of fat and lipids.1719 In addition, they are amphiphatic in nature, possessing hydrophobic and hydrophilic faces. The interior of the bile salt micelles is more rigid and complex than micelles formed by conventional r 2011 American Chemical Society
aliphatic surfactants.20,21 Thus, the interaction mode of bile salt micelles with NPs is different from that of common alkyl chain surfactants. Furthermore, their chemically different functional groups, enantiomeric purity, unique amphiphilicity, easy availability, and low cost make them ideal building blocks for stabilization of NPs. Recently, AuNPs have been successfully employed as a colorimetric sensor for the detection of nucleic acid, enzymes, metal ions, and proteins and for screening of DNA binders.2224 The major advantages of AuNP-based assays are that the molecular recognition events can be directly observed by the naked eye with high sensitivity, simplicity, and low cost without the requirement of the sophisticated instruments.25 Immobilization of oligonucleotide on the AuNP surface was first established by Mirkin’s group for the colorimetric detection of DNA based on aggregation wherein DNA strands serve as recognition units and AuNPs serve as optical sensing elements.26,27 However, the disadvantage of this method is that it requires longer incubation time and an excess amount of oligonucleotides and is more expensive. Later, Alivisatos and co-workers demonstrated polymer-capped AuNPs with thiolated DNA where the irreversible coating on AuNPs hindered the diffusion and attachment of the probe on the particle surface.28 To simplify the detection process, Received: May 12, 2011 Revised: July 2, 2011 Published: July 06, 2011 15266
dx.doi.org/10.1021/jp2044465 | J. Phys. Chem. C 2011, 115, 15266–15273
The Journal of Physical Chemistry C developing a colorimetric method using unmodified AuNPs would be of considerable interest. Recently, Li and Rothberg reported the detection of a single base pair mismatch and target DNA using unmodified AuNPs based on electrostatic interactions.29 The steric repulsion of non cDNA with AuNPs in effectively controlling micro- and nanostructure assembly has also been reported.30 More recently, Rho and co-workers demonstrated the colorimetric detection of ssDNA in the presence of citrate-capped AuNPs against salt-induced aggregation.31 Though AuNPs have been used as colorimetric sensors with oligonucleotide probes for the detection of target DNA according to Chargaff’s rules,32 there are no reports on the use of degenerate probes for the detection of DNA by AuNPs. Degenerate probes are used to detect plant and human viruses and other microbes by polymerase chain reaction (PCR), Southern, and other hybridizations methods.33,34 They were designed based on the conserved region to overcome the nucleotide diversity in the genome of different isolates and base pair according to Wobble’s hypothesis.35 Whitefly transmitted Gemini viruses, like the Tomato leaf curl virus (ToLCV), Chilli leaf curl virus (CLCV), and Squash leaf curl virus (SqLCV), are reported to be prevalent in vegetables and horticultural and ornamental crops causing leaf curl, mosaic, and chlorotic stunt diseases36,37 causing total yield loss.3840 Detection of these viruses by molecular biology methods is time-consuming, is less accurate as in an immunoassay technique, requires skilled manpower and sophisticated instruments, and is too expensive.31,41,42 In this paper, we report a novel strategy for colorimetric detection of Gemini viral DNA using degenerate probes and their interaction with size-specific AuNPs synthesized by naturally occurring bile salts as reducing and capping agents.
II. EXPERIMENTAL METHODS a. Chemicals. Hydrogen tetrachloroaurate trihydrate (HAuCl4 3 3H2O), potassium nitrate (KNO3), potassium bromide (KBr), sodium cholate (NaC), sodium deoxycholate (NaDC), and pyrene were obtained from Sigma-Aldrich. Disodium hydrogen phosphate (Na2HPO4), monosodium dihydrogen phosphate (NaH2PO4), and sodium chloride (NaCl) were obtained from Loba-chemie Ltd., India. Ethylenediaminetetraacetic acid (EDTA) was obtained from HiMedia Laboratories Pvt. Ltd. (India). A lyophilized degenerate oligonucleotide probe and its complementary sequences were obtained from Bio Basic Inc. (Canada). Deionized double distilled water was used for all the experiments. b. Preparation of Bile Salt Capped AuNPs. Stock solutions of 1.0 103 M HAuCl4, 0.1 M NaC, and NaDC were prepared using deionized double distilled water, and the subsequent dilutions with the concentrations below and above CMC values were made from this stock solution. For all the experiments, the reaction mixture was adjusted to pH 7.0. To optimize the reaction conditions, 1 mL of HAuCl4 solution was added to different concentrations of NaC (0.00150.033 M), and the final volume was adjusted to 5 mL using deionized double distilled water. The addition of a HAuCl4 ion to the bile salt solutions was considered as the starting point of the reaction. The formation of AuNPs was observed by the gradual change in solution color from light yellow to colorless and finally green/red depending on the concentration of NaC in the reaction mixture. c. Characterization of AuNPs. UVvisible spectra over the range of 2001100 nm were measured using a Shimadzu UV-1601
ARTICLE
spectrophotometer. TEM was carried out using JEOL 3010, 300 kV, with an ultrahigh-resolution (UHR) pole piece. The samples were drop cast onto a carbon-coated copper grid sample holder and allowed to dry at room temperature. The FT-IR spectra measurements were carried out using a Perkin-Elmer FT-IR spectrometer. The pressed pellet was prepared by grinding lyophilized NaC capped AuNP powder with KBr in a 1:100 ratio and analyzed in the spectral range of 4000400 cm1. Cyclic voltammograms (CV) were recorded using a computer-controlled 400 A electrochemical analyzer at a scan rate of 25 mV/s. A conventional three-electrode electrochemical cell was used for the CV measurements, with a high surface area Pt counter electrode (area = 2 mm diameter), and Ag/AgCl wire was used as the reference electrode. Prior to use, the surface of the electrode was polished with alumina paper and rinsed with double distilled water. The TGA (Waters TA Instrument, model SDT Q600) of the pure NaC and NaCcapped AuNPs was carried out in the temperature range from 50 to 1000 °C under nitrogen atmosphere, with a heating rate of about 20 °C/min. d. Fluorescence Measurements. Steady state fluorescence measurements were carried out by using a fluorescence spectrophotometer (Fluoromax 4P, Horiba Jobin Yvon). Stock solutions of 3 105 M aqueous pyrene were prepared by gentle evaporation of pyrene solubilized methanol solution, followed by addition of an appropriate volume of double distilled water in a volumetric flask, and sonicated for 1 h. For quenching measurements, an excitation at 336 nm was used, and the emission spectrum was recorded from 360 to 410 at 90 nm/min. The variations observed in the first (I1) and third (I3) vibronic band intensity measured at 374 and 385 nm were used to determine the critical micelle concentration (CMC) and binding constant of the substrate with a micellar medium. The fluorescence decay curve and lifetime measurements were carried out using a timecorrelation single-photon counting spectrometer (IBH, model 5000U). The excitation source was 280 nm nanoLED (IBH) with a pulse width of
