Scientific paper Thiol Reactive Probe Based on Fluorescence Resonance Energy Transfer between Fluorescein and Au Nanoparticles Li Qi, Juan Song, Fang-Ying Wu* and Yi-Qun Wan Department of Chemistry, Nanchang University, Nanchang 330031, China * Corresponding author: E-mail: fywu@ncu.edu.cn Received: 13-09-2013 Abstract Sensitive and selective fluorescent probe of thiols with lower limit of detection based on fluorescence resonance energy transfer (FRET) between fluorescein and Au nanoparticles (AuNPs) is presented. The fluorescein-AuNPs complex emits weak fluorescence. Upon chemically binding to organosulfur compound that contains a carbon-bonded sulfhydryl (-C-SH or R-SH) thiols, a stable enhancement of fluorescence is observed due to the competitive binding on AuNPs between thiols and fluorescein. The magnitude of fluorescence enhancement is linearly proportional to the logarithm of the thiols concentration. We use cysteine as an example to show how this useful analytical assay works selectively, which is closely nonresponsive to 20 other amino acids even though they are in solution at a concentration 10 times greater than the thiols. The detection limit for cysteine is 7.27 x 10-9 mol L-1. The possible mechanism of this assay is discussed in details. The proposed method was successfully applied for the determination of Cys in urine. Keywords: Thiols, gold nanoparticles, fluorescein 1. Introduction Over the decades, particular interest has been focused on the chemosensing of sulfhydryl-containing amino acids and peptides, the thiols, as they play crucial roles in biological systems. For example, thiol group in cysteine (Cys) residues is the active site of proteins is related to oxidative stress.1 Cys is a member of essential amino acids in human body which also plays a vital role in food industry, pharmaceutical industry and cosmetics. It has a variety of important physiological functions, such as in metabolism, anti-aging and detoxification.1-3 The deficiency of cysteine causes many diseases, such as delayed growth in children, depigmentation of hair, edema, lethargy, liver damage, loss of muscle and fat, and skin lesions.4-5 In addition, glutathione (GSH) and homocysteine (Hcy) which belong to the thiol group, are also linked to various human diseases.6-11 Therefore, selective detection and quantification of thiols is of growing importance. The majority of the reported methods have been focused on capillary elec-trophoresis,12 high performance liquid chromatography,13-14 electrochemical analysis4 and mass spectrometry.15 In recent years, gold nanoparticles (AuNPs) have become highly studied materials and been widely used in a range of applications due to their excellent optical and electrical properties.16-18 One feature that makes them particularly appealing is that AuNPs possess high absorption coefficients in the visible region, which ensures them to function as efficient quenchers for most fluoropho-res.19,20 In order to take full advantage of the super-quenching ability of AuNPs, many high-performance fluorescence assays have been developed in recent years for optically sensing biologically important ions and molecules. Eunkeu Oh et al. designed an inhibition assay for avidin detection based on the modulation of FRET efficiency between biomolecule-conjugated QDs and AuNPs.21 Dong reported a simple fluorescent method for cyanide detection based on the dissolution of Rhodamine B-adsor-bed gold nanoparticles by cyanide.22 Such methods are also used to determine melamine,14 mercury ion,23 copper ion,24 cyanide,22 and fenamithion.25 It was reported that the FRET process between fluorescein and AuNPs was easily occurring because of a wide margin overlap between the absorption band of AuNPs and the emission band of fluorescein.26 Based on this, a novel and simple fluorescence assay for thiol determination was constructed, in which fluorescein acted as the energy donor and AuNPs served as acceptor. Upon addition of thiols such as Cys, Hcy and GSH, fluorescein was removed from the surface of AuNPs because of the stronger binding between the thiol group of Cys and gold to form Au-S bonds. As a result, the emission of fluorescein was restored. Herein, a simple and fast platform for the specific detection of thiols was presented and the proposed mechanism was depicted in Scheme 1. a) lin b) hr' ho S s' * x hu hp' Au NPs ^ Fluorescein Thiols gent Co. Ltd., L-Cys, L-Arginine, L-Histidine, L-Leucine, Glutathione (Reduced) were purchased from Beijing Solarbio Science & Technology Co., Ltd., other amino acids were purchased from Chengdu Gray West Chemical Technology Co., Ltd. All other reagents were of analytical grade and used without any further purification. All solutions were prepared using doubly distilled water. 2. 3. Preparation of AuNPs The monodisperse AuNPs were prepared through the classical citrate reduction method reported previously.27 All glassware used in this experiment were cleaned with aqua regia (3:1 HCl/HNO3) and rinsed with tap water and deionized water, and dried prior to use. Briefly, the mixture of 4.0 mL of 1% HAuCl4 and 96 mL deionized water was brought to boiling. Then 2.0 mL 5% sodium citrate solution was quickly added into the solution with stirring. The mixture was refluxed for an additional 5 min, during this time the color of solution became deep red. Then the solution was cooled to room temperature and then stored at 4 °C. Additionally, the concentration of the AuNPs in the testing solution was determined to be 15 nM according to the Beer's law using an absorption coefficient of 2.7 x 108 cm1 M1 at 520 nm for Au NPs of 13 nm diameter.27 Scheme 1: Schematic representations of fluorescent probe for thiols detection 2. Experimental 2. 1. Apparatus Fluorescence spectra were acquired on a F-4600 fluorescence spectrometer (Hitachi, Japan, www.hitachi-hitec.com) equipped with a xenon lamp source and a 1.0 cm quartz cell, and the scan speed was 1200 nm min-1. Absorption spectra were recorded on a UV-vis 2550 spectrophotometer (Shimadzu, Japan, www.shimadzu.com) using a 1.0 cm quartz cell. Size distribution of AuNPs was measured on a Hydrosol Nanoparticle Size Analyzer and Zeta Potential Analyzer (PSA NAN02590, Malvern Companies, UK, www.malvern.com). The surface morphology of the AuNPs was characterized using Transmission electron microscope (TEM, JEM-2010 transmission electron microscope (JEOL Ltd.)) All pH measurements were made using a pHS-3 digital pH-meter (Shanghai REX Instrument Corp., Shanghai, China, http://en.lei-ci.com) combined with a glass-calomel electrode. All optical measurements were carried out at room temperature. 2. 4. Detection Procedures for Cys 0.2 mL 3.0 x 10-5 mol L-1 fluorescein and 4.0 mL of prepared AuNPs were added to an amber bottle, and then the solution was diluted with 0.01 mol L-1 (pH 8.0) phosphate buffer solution to a final volume of 10 mL. The mixture was maintained at room temperature for 1 h in the dark. Afterwards, different amounts of Cys were added to 0.5 mL of the prepared mixture, and each sample solution was diluted with 0.01 mol L-1 (pH 8.0) phosphate buffer solution to 2.0 mL. After 5 minutes' reaction, the fluorescence spectra were collected. 2. 5. Pretreatment of Human Urine Samples Human urine sample (1.0 mL) collected from healthy adult female volunteers, and the analysis was conducted immediately after the sample collection. 1 mL of urine sample was added into a centrifuge tube, 1.0 mL of acetonitrile was added for removing proteins in urine. The mixture was centrifuged at 12,000 rpm for 10 min. The supernatant was filtered through a 0.22-pm filter, then dried by vacuum at 50 °C for 10 h, and diluted to 5 mL with deionized water before analysis. 2. 2. Materials Chloroauric acid tetrahydrate (AuCl3 ■ HCl ■ 4H20), trisodium citrate dihydrate (C6H507Na3 ■ 2H20), and fluo-rescein were purchased from Sinopharm Chemical Rea- 3. Results and Discussion 3. 1. Characterization of AuNPs The surface morphology of the prepared AuNPs was a) b)