Article pubs.acs.org/ac
Nanomolar Hg2+ Detection Using β‑Lactoglobulin-Stabilized Fluorescent Gold Nanoclusters in Beverage and Biological Media Jiachen Zang,† Changan Li,† Kai Zhou,† Haisheng Dong,‡ Bin Chen,‡ Fudi Wang,† and Guanghua Zhao*,† †
Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Key Laboratory of Functional Dairy, Ministry of Education, Beijing, 100083, China ‡ Key Laboratory of Space Nutrition and Food Engineering, State Key Lab of Space Medicine Fundamentals and Application, China Astronaut Research and Training Center, No. 26, Beiqing Road, Beijing 100094, China S Supporting Information *
ABSTRACT: Owing to diverse functionalities and metal binding abilities, proteins have been proven to be promising ligands in the synthesis of gold nanoclusters (Au NCs). In this work, we explored β-lactoglobulin (β-Lg), a protein byproduct generated during cheese processing, as a biotemplate for fabrication of Au NCs by a facile and green method for the first time. The as-prepared Au NCs are water soluble and highly fluorescent and exhibit high sensitivity and selectivity for Hg2+ detection in aqueous solution. Interestingly, we found that the fluorescence of these Au NCs is stable either in a variety of complex matrixes or over a broad pH range (5.0−13.0) and therefore can be explored as a cell and animal imaging agent. More importantly, we demonstrated that the β-lactoglobulinstabilized Au NCs (β-Lg−Au NCs) could serve as a sensor for the detection and quantification of Hg2+ in beverages, urine, and serum with high sensitivity. synthesized fluorescent sensors for Hg2+ detection have been suffering low sensitivity and selectivity, metal ion interference, the turn-on or turn-off response, and poor water solubility,7 and all of these shortcomings limit their application. Recently, proteins have been explored as a variety of platforms for the synthesis of various inorganic nanomaterials under benign experimental conditions. This is most likely derived from the fact that the functional groups such as amine, carboxyl, and thiol from proteins can help stabilize different inorganic ions through binding or noncovalent interactions.11−14 Proteins as templates for the preparation of noble metal nanoclusters have several advantages over other fluorescent nanomaterials: good water solubility, low toxicity, and facile synthesis, excluding harsh reducing agents or organic solvents. Following the first reported case of bovine serum albumin (BSA)-templated Au NCs,15 several protein-stabilized Au NCs have been synthesized,16−18 two of which have been developed for Hg2+ detection in aqueous solution.15,16 However, the application of these protein-stabilized Au NCs toward beverage and biological samples like urine and blood is hampered most likely because of complexity of detected matrix
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ecently, nanotechnologists have made great achievement in the applications of noble metal nanoclusters (NCs) in chemical sensing and biological imaging because of their intrinsic fluorescent properties.1−3 The NCs usually serve as the missing link that exists between isolated metal atoms and plasmonic metal nanoparticles and are typically ∼1 nm in size which approaches the Fermi wavelength of the conduction electrons.4 Consequently, the ultrasmall size of the NCs leads to the formation of a variety of new and fascinating molecularlike characteristics, such as discrete electronic states, sizedependent fluorescent feature, and biomedical properties that larger metal nanoparticles lack.5,6 So far, many types of noble metal NCs including Au, Ag, Cu, Pt, and Pd NCs have been developed by different research groups. Among them, Au NCs are most preferable due to their low toxicity, ease of preparation, high photoluminescence property, and extraordinary chemical stability. Mercury is a toxic and dangerous element to human beings and other living organisms when ingested or inhaled. Accumulation of mercury in organs can cause serious damage to the central nervous system seriously because Hg2+ can bind strongly to the −SH group in protein. Therefore, the design and synthesis of sensors of mercury species have received considerable attention. Accordingly, fluorescent sensors as a valuable means of detecting Hg2+ have been synthesized in various chemical systems.7−10 However, these chemically © 2016 American Chemical Society
Received: August 4, 2016 Accepted: September 15, 2016 Published: September 28, 2016 10275
DOI: 10.1021/acs.analchem.6b03011 Anal. Chem. 2016, 88, 10275−10283
Article
Analytical Chemistry
Alto, CA). Fluorescence images of the β-Lg−Au NCs were obtained under UV light. The quantum yield (QY) of β-Lg−Au NCs was determined by measuring the integrated fluorescence intensities of β-Lg−Au NCs and the reference compound (rhodamine 6G in basic ethanol, QY = 95%).24 Transmission electron micrographs (TEM) were obtained at 80 kV using a Hitachi S-5500 scanning electron microscope. The oxidation state of the Au clusters was determined by X-ray photoelectron spectroscopy (XPS). Narrow-scan XPS spectra of Au 4f were deconvoluted by the XPSPEAK software (version 4.1) using adventitious carbon to calibrate the binding energy of C 1s (284.5 eV).24 Dynamic light scattering (Wyatt, DynaPro NanoStar, U.S.A.) was used for hydrodynamic size determination of β-Lg−Au NCs. Laser scanning microscope (Leica, TCS SP5II, Germany) was used for cell imaging. The excitation/emission wavelengths were 488 nm/650 nm for βLg−Au NCs. In vivo fluorescence images of the mice were obtained with a Maestro in vivo spectrum imaging system.24 Au contents in different tissues of mice were determined by inductively coupled plasma optical emission spectroscopy (ICP-OES) (Shimadzu, ICPE-9800, Japan). Synthesis of β-Lg−Au NCs. β-Lg−Au NCs were synthesized according to a reported method with some modification.12 In a typical experiment, 200 μL of 10 mM aqueous NaAuCl4 solution was added to a 200 μL of water solution containing 10 mg of β-Lg with vigorous stirring at 37 °C. Two minutes later, 80 μL of 1.0 M NaOH solution was introduced with further vigorous stirring for 10 min. After that, the mixture solution was incubated at 37 °C for 2−3 h. The reaction was stopped by dropwise addition of 1.0 M acetic acid to neutral pH value. The products were stored in dark for further use. Cell Cytotoxicity and Imaging. Caco-2 cells were seeded at a density of 6 × 104 cells/well in 96-well plates for 12 h. The medium was replaced with 400 μL of medium containing a series of concentrations from 0.1 to 100 μM of β-Lg−Au NCs. The numbers of viable and total cells were quantified using a CCK-8 kit. To monitor the cellular uptake of β-Lg−Au NCs by Caco-2 cells, the cells were incubated with β-Lg−Au NCs for 4 h. The cells were washed with PBS (10 mM, pH 7.40) three times before imaging. The β-Lg−Au NCs were excited with a red excitation source. Small-Animal Imaging. Nu/nu female mice (8 weeks, 20 ± 2 g, Beijing Vital River) were injected via the tail vein with βLg−Au NCs (100 μM, 100 μL) diluted in 10 mM PBS. In vivo fluorescence images of mice were obtained with excitation filter set as 500 nm, emission filter as 650 nm. Then the mice were sacrificed for hematoxylin−eosin (HE) staining to check if the β-Lg−Au NCs were safe for organs. All animal experiments were approved by the Animal Ethics Committee of the Medical School, Beijing University. Hg2+ Detection Using β-Lg−Au NCs. The typical Hg2+ detection procedure was conducted as follows. Aqueous solution of HgCl2 (100 mM) was freshly prepared before use. Different amounts of stock solution were added to PBS buffer containing 100 μM β-Lg−Au NCs to make the final concentration of Hg2+ ranging from 10 nM to 500 μM. After 2 min of incubation, the fluorescence spectra were recorded on Cary-50 fluorescence spectrophotometer with an excitation wavelength of 500 nm. Selectivity Analyses of β-Lg−Au NCs for Various Metal Ions. To measure the selectivity of metal ions, the following inorganic salts were used: potassium chloride, sodium
in chemical composition and fluorescent interference with other ligands. On the other hand, cheese whey is the liquid remaining following the precipitation and removal of milk casein during cheese production. It has been known that during production of 1 kg of cheese, ∼9 kg of whey is generated, and then discarded, leading to a severe environmental problem based on its high organic matter content.19,20 About 20% of the milk proteins are represented in whey proteins, in which βlactoglobulin (β-Lg) is the most abundant and accounts for 50−60% of the total whey. β-Lg has been extensively studied for its binding ability for hydrophobic ligands such as fatty acids or vitamins. The most common variants of β-Lg, namely, A and B, both contain 162 amino acids, in which Cys 121 is a free thiol that lies buried in the center.21 The reactivity of the free sulfhydryl has attracted a number of experiments over the years,22 reminding us of the synthesis of nanoclusters in which free sulfhydryl is regarded necessary. Here we report a facile synthesis of highly fluorescent Au NCs in an economical and fast way by mixing β-Lg and NaAuCl4 under basic conditions, and their application as Hg2+ sensors, with high sensitivity and selectivity (Figure 1).
Figure 1. Schematic representations of the synthesis and Hg2+ detection of β-Lg−Au NCs.
Compared with the reported proteins used for the preparation of Au NCs, β-Lg is much cheaper and easier to be obtained. More importantly, we found that this newly synthesized Au NCs can be used for Hg2+ detection in commercially available beverages and even biological samples for the first time. This represents not only an upstream of nanotechnology in the practical application, but also a downstream of effective utilization of waste products from food processing.
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EXPERIMENTAL DETAILS Materials. All the chemicals used are at least of analytical grade. Milli-Q water was used throughout. Sodium tetrachloroaurate (NaAuCl4·2H2O), other metal salts, small bioactive molecules, PBS (phosphate buffer saline), CCK-8 kit, and other chemicals were all obtained from Solarbio (Beijing, China). All the mice were purchased from Beijing Vital River Laboratories. β-Lg was purified from whey proteins as previously described.23 Instrumentation. UV−vis spectra were recorded on a UV spectrophotometer (Varian, 50 Bio, U.S.A.), and fluorescence studies of the β-Lg−Au NCs in a sealed cuvette were carried out with a fluorescence spectrometer instrument (Varian, Palo 10276
DOI: 10.1021/acs.analchem.6b03011 Anal. Chem. 2016, 88, 10275−10283
Article
Analytical Chemistry
Figure 2. (a) Photographs of β-Lg and β-Lg−Au NCs in aqueous and solid state (from left to right, respectively) under visible light (1) and UV light (2). (b) UV−vis absorption spectrum of β-Lg−Au NCs. (c) Excitation fluorescence spectrum of β-Lg−Au NCs. (d) Emission fluorescence spectrum of β-Lg−Au NCs.
NCs.15−18 β-Lg contains two disulfide bonds (Cys66−160 and Cys106−119) and one free thiol group at Cys121 based on amino acids sequence analyses,26 so it was chosen as a ligand for stabilizing Au NCs. Initially, chloroauric acid (HAuCl4) was used as starting materials as recently reported.15−18 However, the fluorescence quantum yield of prepared Au NCs is too low in the presence of β-Lg. Instead, we found that NaAuCl4 was more preferable for β-Lg to cooperate, suggesting that neutral pH is more suitable for the initial binding of AuCl4− ion to βLg. Consistent with this idea, Volden et al. found that no obvious fluorescence was observed when HAuCl4 was used to react with β-Lg.27 Subsequently, reaction conditions of protein and NaAuCl4 were optimized. As shown in Figure S1, when equal volumes of β-Lg (50.0 mg/mL) and NaAuCl4 (10.0 mM) in ddH2O were mixed at 37 °C, the fluorescence intensity of the resulting solution was the strongest among four different experimental conditions. Without appropriate ratio of protein and Au3+, the ion would accumulate in an inhomogeneous way. TEM results were similar to Figure S2d. As an important reducing agent, NaOH was added 2 min after mixing of NaAuCl4 with β-Lg. We found that the amount of NaOH used has a pronounced effect on reaction time, namely, more NaOH shortened reaction time that the mixture of NaAuCl4 and β-Lg reached the highest fluorescence intensity. In a typical reaction, we added 80 μL of NaOH (1.0 M) to a 400 μL mixture of NaAuCl4 and β-Lg. This reaction takes 2−3 h for completion, with the color changing from light yellow to light brown. It is noteworthy that all the reactions need to be processed under vigorous stirring. Only with good distribution and coherent reduction can the size of gold be controlled within the range of NCs (2 nm), finally 10278
DOI: 10.1021/acs.analchem.6b03011 Anal. Chem. 2016, 88, 10275−10283
Article
Analytical Chemistry
Figure 5. (a) Cell viability of Caco-2 cells upon incubated with various concentrations of β-Lg−Au NCs (from 0 to 100 μM). (b) In vitro fluorescent imaging of Caco-2 cells incubated with β-Lg (10 μM) and β-Lg−Au NCs (10 μM). (c) In vivo fluorescent imaging of Nu/nu mice injected with or without β-Lg−Au NCs (100 μM, 100 μL).
point of β-Lg. Moreover, it was observed that the luminescence of the as-prepared Au NCs at pH 7.0 or lower is relatively stronger than that in basic conditions (Figure 4b). This might be owing to the association characteristic of β-Lg. It has been established that β-Lg molecules exist in monomers below pH 3.5 or above pH 7.5, while they associated into dimers at other pH values.29 This association property might enhance the fluorescence of β-Lg−Au NCs through energy transfer.30,31 When the β-Lg−Au NCs were dissolved in various buffers such as Tris, HEPES, and PBS, nearly identical fluorescence was observed, indicating that these buffers has no effect on the luminescence of the β-Lg−Au NCs (Figure S3). After being lyophilized, the light brown solid powder of β-Lg−Au NCs was obtained, and it maintained highly fluorescent under UV light (Figure 2a). Upon long storage (about 1 month), the powder can be redispersed in water or the above buffer solutions. Although protein stabilized fluorescent Au NCs usually exhibit high water solubility, to keep the stability of these Au NCs in the complicated matrix remains a challenge. To shed light on the stability of the β-Lg−Au NCs in the presence of various molecules, we added different components such as sodium chloride, D-xylose, sucrose, galactose, vitamin C, sorbitol, lactic acid, biotin, BSA, and lysozyme to a β-Lg−Au NCs diluted system (500 μM in PBS), respectively. These components represent salt, sugar, protein, and some small bioactive molecules which are widely distributed in various matrixes. According to Figure 4c, there was no obvious change in the fluorescence intensity of β-Lg−Au NCs in the presence of these molecules. Since β-Lg can act as a natural vehicle for a wide range of bioactive nutrients such as oleic acid, linoleic acid, sodium oleate, epigallocatechin gallate (EGCG), folic acid, carotene and sodium dodecyl sulfate (SDS) with high affinity,32 we also evaluated the effect of these bioactive compounds on the fluorescence of the synthesized β-Lg−Au NCs. Similar results were obtained as above (Figure 4c), indicating that the Au NCs are protected well by β-Lg from interference with these compounds. Subsequently, the fluorescence intensity was also checked after 24 h of incubation with the above-mentioned compounds. According to Figure S4, there was no obvious
luminescence profile was observed with its solid form. In contrast, no such red fluorescence was visualized with β-Lg alone under the same experimental conditions (Figure 2a). The UV−vis absorption spectrum of the β-Lg−Au NCs exhibited a peak at around 400 nm (Figure 2b) due to quantum confinement effects. The photoluminescence profiles of the βLg−Au NCs are shown in Figure 2, parts c and d. Although their excitation spectrum had two peaks, one at 420 nm and the other at 500 nm (Figure 2d), the emission maximum was 650 nm (Figure 2c). By comparison with the UV−vis absorption and fluorescence spectra of rhodamine 6G, the quantum yield (QY) of β-Lg−Au NCs is ∼8%,28 similar to the classical cases like BSA and lysozyme. Thus, by using simple, time-saving procedures presented here, highly fluorescent β-Lg−Au NCs can be fabricated. To characterize the core size, β-Lg−Au NCs were analyzed by TEM. Results revealed that the β-Lg−Au NCs had spherical shape and were homogeneous in size, which was defined in the range of nanoclusters (
