Microchim Acta (2015) 182:1255–1261 DOI 10.1007/s00604-014-1438-8
ORIGINAL PAPER
Microwave-assisted synthesis of BSA-modified silver nanoparticles as a selective fluorescent probe for detection and cellular imaging of cadmium(II) Yu Gu & Nan Li & Mengmeng Gao & Zilu Wang & Deli Xiao & Yun Li & Huning Jia & Hua He
Received: 14 October 2014 / Accepted: 2 December 2014 / Published online: 14 January 2015 # Springer-Verlag Wien 2015
Abstract We have developed a microwave-assisted method for the synthesis of silver nanoparticles (AgNPs) whose surface is modified with bovine serum albumin (BSA). The reaction involves reduction of the BSA-Ag(I) complex by tyrosine in strongly alkaline solution to form BSA-AgNPs. The reaction takes a few minutes only owing to rapid and uniform microwave heating. The modified AgNPs were characterized by UV–vis and fluorescence spectroscopy, transmission electron microscopy and X- ray photoelectron spectroscopy. The BSA-AgNPs are yellow and display luminescence with a maximum at 521 nm if excited at 465 nm. They have a hydrodynamic diameter of 3–5 nm and possess good colloidal stability in the pH 4.6 to 12.0 range. The fluorescence of the BSA-AgNPs is enhanced by Cd(II) ion due to the formation of a stable hybrid conjugate referred to as Cd@BSA-AgNPs. The effect was exploited to quantify Cd(II) in spiked real water samples with a 4.7 nM detection limit, and also to fluorescently image Cd(II) in Hepatoma cells.
Keywords Fluorescence . Fluorescent probe . Silver nanoparticle . BSA . Microwave synthesis Electronic supplementary material The online version of this article (doi:10.1007/s00604-014-1438-8) contains supplementary material, which is available to authorized users. Y. Gu : N. Li : M. Gao : Z. Wang : D. Xiao : Y. Li : H. Jia (*) : H. He Division of Analytical Chemistry, China Pharmaceutical University, 24 Tongjia Lane, Nanjing 210009, China e-mail:
[email protected] H. He (*) Key Laboratory of Drug Quality Control and Pharmacovigilance, China Pharmaceutical University, Ministry of Education, Nanjing, China e-mail:
[email protected] H. He e-mail:
[email protected]
Introduction As one of the most toxic metal pollutants, cadmium (Cd) is used in many processes such as electroplating, metallurgy and nuclear industry, etc. These industries lead to a large amount of cadmium being released into the environment annually, which is harmful to human and environment. Many reports have exposed the serious toxicity of Cd2+ to plasma [1], bones, kidneys [2], nervous system [3] and liver [4]. Since cadmium can be accumulated in organisms, there is a great need for sensitive and selective methods of detecting and monitoring cadmium levels in Environmental samples. Although several approaches such as atomic absorption and inductively coupled plasma mass spectrometry have been previously developed for the detection of Cd2+ [5–7], these methods require either sophisticated and expensive equipment, or complex experiment procedures that only could be operated by well-trained professionals, which are timeconsuming and unsuitable for real-time monitoring especially for bio-application. In comparison, fluorescent probes are onsite methods for real-time detection of metal ions as their low cost, high sensitivity and high speed of analysis advantages [8, 9]. Currently the widely used fluorescent probes are small molecular probes [8], semiconductor quantum dots [10], noble metal nanoparticles. As small molecular probes are easily photobleaching, while quantum dots are usually toxic, noble metal nanoparticles [11] are good choices as sensing element based on their low toxicity, good biocompatibility [12]. Up to now, less attention has been focused on silver nanoparticles (AgNPs)-based assay compared with gold nanoparticles (AuNPs), as the fact that silver undergoes oxidization more readily than gold. However, AgNPs have shown some unique characters and advantages over AuNPs to some degree since their low cost and the extinction coefficients are higher than AuNPs of the same size [13, 14]. AgNPs can be modified by other groups to have luminescence [15, 16], enhance
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sensitivity [17, 18] and selectivity [19–21]. Typically, AgNPs were synthesized by the reduction of silver nitrate with sodium borohydride [22], which was applicable, simple for preparation of AgNPs and their colloidal dispersions in water or organic solvents. However, the whole synthesis process takes hours. The development of a rapid, facile and green approach to synthesis AgNPs with good stability is always a hot topic. Microwave irradiation is one of the most promising techniques for the preparation of nanomaterials due to its rapid and uniform heating. There have been reports on the synthesis of benzo-18-crown-6, soluble starch, polyvinyl alcohol modified AgNPs via microwave methods, but such methods were either toxic or low biocompatibility which limited their application on the analytical chemistry [23–25]. In this work, we first attempt a one-step microwave-assisted method to synthesis BSA modified AgNPs (BSA-AgNPs) in a few minutes without complicated experimental procedures and conditions, and the as-synthesised AgNPs were not easily aggregate. The BSA-AgNPs display green luminescence with a maximum emission at 521 nm and possess good colloidal stability in the pH 4.6 to 12.0 range, which is suitable for bio-application. To examine the feasibility of this approach, we select BSAAgNPs as a probe of Cd2+. These probe shows significant emission enhancement towards Cd2+ over Zn2+ and can be applied to detect Cd2+ in real water samples analysis (LOD= 4.7 nM) and liver cell imaging.
Material and methods Reagents In all experiments, ultrapure water (18.2 MΩ cm) was used. All glassware was washed with aqua regia and rinsed with ethanol and copious quantities of water. Bovine serum albumin (BSA), silver nitrate (AgNO3) were purchased from Sigma (http://www.sigmaaldrich.com/chinamainland.html). Dul- becco’s modified eagle media (DMEM) and fetal calf serum (FCS) were obtained from Thermo Fisher (http://www.thermo.com.cn/); other chemicals were reagent grade quality and used as received.
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finally obtained. The solution was then allowed to cool to room temperature before dialyzed using an 8–14 kDa dialysis bag in double-distilled water for 24 h. The BSA–AgNPs solution was kept in the dark at 4 °C. The solvent was removed by freeze-drying, and the BSA-AgNPs could be stored in solid form. Characterizations of BSA-AgNPs UV–vis and fluorescence spectra were recorded on a Shimadzu UV-1800 spectrometer (Shimadzu Corporation, Kyoto, Japan, http://www.risun-tec. com/en/index.aspx) and a RF-5301PC fluorescence spectrometer (http://www.risuntec.com/en/index.aspx), equipped with a SB-11water bath (Eyela) and 1.0 cm quartz cells, respectively. Highresolution transmission electron microscopy (HRTEM) images were obtained on FEI Tecnai G2 F20 microscope operating at 200 kV. X-ray photon spectrometry (XPS) was measured on a Kratos AXIS Ultra DLD spectrometer (Kratos Analytical Ltd.) with a mono Al Kα radiation source (hν= 1486.71 eV) and the charging shift was corrected by the binding energy of C1s at 284.6 eV. Determination of Cd2+ using BSA-AgNPs A typical Cd2+ detection procedure was conducted as follows. Cd2+ solutions at different concentrations were obtained by serial dilution of the stock solution. 10 μL of Cd2+ solutions with various concentrations were first mixed with 3 mL of BSA-AgNPs dissolved in pH 7.4 PBS for fluorescence spectra measurements after mixing about 10 min at room temperature. To examine the influence of BSA on the selectivity of the sensing system, the fluorescent spectra of BSA added Cd2+ was evaluated. To evaluate the selectivity of Cd2+ by using BSA-AgNPs, other metal ions such as Ca2+, Co2+, Cu2+, Mn2+, Ni2+, Pb2+, Al3+, Na+, K+, Ag+, Hg+, Fe3+and Zn2+ were also tested and the response recorded and analyzed.
Synthesis of BSA-AgNPs The BSA-AgNPs were prepared by the reduction of silver nitrate with sodium borohydride. Briefly, 5 mL of AgNO3 aqueous solution (15 mM) and 5 mL of BSA (50 mg·mL−1) were mixed together under vigorous stirring (1000 rpm). 3 min later, 0.30 mL of NaOH (1.0 M) solution were added to the mixture to form BSA-Ag+ complex. Afterwards, the resulting mixture was irradiated by microwaves for 60 s at 500 W in a domestic microwave oven (SIEMENS HF15G561W, Germany). The bright yellow AgNPs were
Fig. 1 The UV–visible absorption spectra of BSA-AgNPs (red line) and BSA (black line)
Silver nanoparticles as a probe for detection and cellular imaging of cadmium (II)
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Fig. 2 TEM images of the prepared BSA-Ag NPs
Imaging of AgNPs-stained hepatoma cells
Results and discussion
Human hepatocellular carcinoma Hep-G2 cells were cultured in DMEM supplemented with 10 % FCS, penicillin (100 μg· mL−1) in 8-well LabTek chambers (Nalgene Nunc, Rochester, NY, USA, http://www.gx-chem. cn/brand-57.html), allowed to adhere for 24 h in a humidified atmosphere with 5 % CO2 and 95 % air. After removing the medium by twice washing with PBS, cells were incubated with BSA-AgNPs (15 μg· mL−1) in serum-free DMEM at 37 C with 5 % CO2 and 95 % air for 2 h. Subsequently, cells were washed twice and bathed with either plain PBS or PBS containing Cd2+ (10 μM). Fluorescence imaging was performed using a fluorescent inverted microscope (Nicon Eclipse TE2000-s).
Synthesis and characterization of the BSA-AgNPs
Analysis of environmental water samples To demonstrate the applicability of the method, the developed method was applied to the determination of Cd2+ in real water samples. Samples were collected into pre-cleaned, lightpreserved glass bottles and 500 mL each sample was filtered immediately through 0.22 μm nylon membranes in order to remove suspended solids. The recoveries were determined from spiked Cd2+ to water samples.
Fig. 3 a Effect of Cd2+ concentration on emission of the AgNPs at 521 nm. Inset: The relative relationship of Ag NPs emission at 521 nm against the concentration of Cd2+ at pH 7.4. b Specificity of the Ag NPs for Cd2+ over others, the concentration of Cd2+ is 5 μM, Na+ and K+ are 250 μM, other metal ions are 50 μM
BSA could be both reducing agent as well as/or capping agent in alkaline medium in the process of synthesis BSA-AgNPs [26]. In general, BSA is composed of 583 amino acid residues, of which 35 are cysteine and 5 are methionine. When AgNO3 is added into BSA solution, Ag+ would coordinate to the various functional groups of BSA such as –NH, −OH, and – SH, which made Ag+ stable due to the bulky nature of BSA and prevented aggregation of Ag+. This reduction is initiated by the phenolic groups, and the pH should be greater than the pKa (10.46) of tyrosine. So adding NaOH to adjust the pH to 12, which enables the tyrosine residues reduce Ag+. Experiments conducted in neutral and acidic media did not form the nanoparticles. Microwave heating speed up rates of folding and unfolding for globular proteins in solution, and the whole reduction process was significantly accelerated [27]. In addition, microwave irradiation during synthesis provide a better control over the growth of AgNPs and correspondingly made the particle size of AgNPs more uniform [28]. The UV–visible absorption spectra prepared AgNPs are shown in Fig. 1. (red line). It could be seen that no obvious characteristic surface plasmon resonance peak in the range of 400–500 nm was
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Y. Gu et al. Comparison of the proposed fluorescent-based methods for determination of Cd2+
Materials used
Analytical LOD ranges (μM) (μM)
5-dimethylamino-2-(2-pyridinyl)-benzoimidazole
0.57–10
(4,4-difluoro-1,3,4,5,7-quantmethyl-4-bora-3a,4a-diaza-sindacene, 4-(bis(pyridin-2-ylmethyl)amino)-benzaldehyde S2−modified CdTe QDs
56.2–1000
2-(2,3-dihydroxylphenyl) benzoxazole
4–50
2,7-di-tert-butylpyrene-4,5,9,10tetraone BSA-modified silver nanoparticles
0.02–100
1.3–25
0.035–2.45
observed, indicating the formation of ultra small AgNPs or nanoclusters (NCs) rather than large nanoparticles. Our subsequent microwave-assisted synthesis of BSA–AgNPs is yellow in solution. Adding 15 mM AgNO3, MW heating 60 s was chosen in our experiment which described in the Electronic Supporting Information (ESM). The BSA–AgNPs are luminescent, exhibiting a maximum at 521 nm (excited at 465 nm). The solvent was removed by freeze-drying, and the BSA-AgNPs were stored in the solid form (inset of Fig. S1.a, Electronic Supplementary Material, ESM) for at least 2 months and redispersed by phosphate-buffered saline (PBS) when needed. Besides, our tests show that BSAAgNPs was pH-insensitive in the relevant pH 4.6 to 12.0 range. The morphology and size distribution of BSA-AgNPs was examined by high-resolution transmission electron microscope (HRTEM). As shown in Fig. 2, the BSA stabilized AgNPs are highly dispersed in aqueous solution without aggregation, and the average diameter of the nanoparticles is approximately 3–4 nm. As noble metal NCs are a subclass of NPs with a core size
