Mercury speciation with fluorescent gold nanocluster as a probe

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Mercury speciation with fluorescent gold nanocluster as a probe Jian-Yu Yang, Ting Yang, Xiao-Yan Wang, Ming-Li Chen, Yong-Liang Yu, and Jian-Hua Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01222 • Publication Date (Web): 11 May 2018 Downloaded from http://pubs.acs.org on May 12, 2018

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Analytical Chemistry

Mercury speciation with fluorescent gold nanocluster as a probe

Jian-Yu Yang‡, Ting Yang‡, Xiao-Yan Wang, Ming-Li Chen, Yong-Liang Yu*, Jian-Hua Wang Research Center for Analytical Sciences, Department of Chemistry, College of Sciences, Box 332, Northeastern University, Shenyang 110819, China

Corresponding Author *E-mail: [email protected] (Y.-L. Yu) Tel: +86 24 83688944; Fax: +86 24 83676698

ABSTRACT: Fluorescent nanoparticles are widely used for sensing biologically significant species. However, it is rarely reported for the discrimination or speciation of metal species. In this work, we report for the first time the speciation of mercury (Hg2+) and methylmercury (CH3Hg+) by taking advantage of the fluorescence feature of folic acid-capped gold nanoclusters (FA-AuNCs). FA-Au NCs exhibit an average size of 2.08±0.15 nm and a maximum emission at λex/λem = 280/440 nm with a quantum yield of 27.3%. It is interesting that Hg2+ causes a significant quench on the fluorescence of FA-Au NCs, whereas CH3Hg+ leads to a remarkable fluorescence enhancement. Based on this discriminative fluorescent response between Hg2+ and CH3Hg+, a novel nanosensor for the speciation of CH3Hg+ and Hg2+ was developed,

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providing limits of detection (LOD) of 28 nM for Hg2+ and 25 nM for CH3Hg+ within 100-1000 nM. This sensing system is highly selective to mercury. Its practical applications were further demonstrated by the analysis of CH3Hg+ and the speciation of mercury (CH3Hg+ and Hg2+) in environmental water and fish samples.

KEYWORDS: gold nanoclusters, fluorescence quenching, fluorescence enhancement, mercury speciation.


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INTRODUCTION Heavy metals possess severe threat to the environment and human health, and their pollution has long been a global issue. Mercury is highly toxic,1 and it generally exists in three chemical states, i.e., elemental (Hg0), inorganic (Hg2+) and organic (mainly CH3Hg+) forms. Hg0 is volatile and can be easily oxidized to Hg2+ in the environment. Hg2+ is stable in aqueous medium and it can destroy the central nervous system and the endocrine system.2 In aquatic ecosystems, microorganisms can transform Hg2+ into CH3Hg+.3 This typically occurs in fish, wherein CH3Hg+ concentration is generally much higher.4 The biomagnified CH3Hg+ can be taken through humans’ daily meal, especially sea food, and accumulated in the internal organs, causing a variety of diseases, e.g., prenatal brain damage, cardiovascular problem, vision and hearing loss and Minamata disease.5 The determination of mercury and its speciation is closely dependent on spectrometric techniques, including atomic fluorescence spectrometry,6 cold vapor atomic absorption spectrometry,7 atomic emission spectrometry,8 and inductively coupled plasma mass spectrometry.9 However, these techniques require bulky and expensive instrumentations, and they are generally not suitable for field analysis. Recently, gold nanoclusters (AuNCs)-based fluorescent assay for Hg2+ is particularly attractive on account of their high sensitivity towards Hg2+. AuNCs are generally smaller than 3 nm in size, showing discrete electronic states due to strong quantum confinement effects, and exhibiting molecule-like properties in the absorption and fluorescence features.10 AuNCs with various protecting ligands were developed as



fluorescence probe for Hg2+ by virtue of the 5d10-5d10 interaction between Hg2+ and Au+ that altering electronic structures of AuNCs. In this respect, the assay of mercury (Hg2+) and its imaging in cells have been realized with BSA protected AuNCs,11 and dihydrolipoic acid capped fluorescent AuNCs.12 So far, the sensing of CH3Hg+ has been rarely described.13,14 A recent study reported the detection of Hg2+ and CH3Hg+ based on fluorescence quenching of lysozyme (Lys) type VI-stabilized-AuNCs.14 The content of Au+ on the surface of Lys VI-AuNCs (41%) was higher than that in BSA-AuNCs (17%), offering more Au+ to interact with Hg2+ and providing favorable sensitivities for both Hg2+ and CH3Hg+. It should be noted that up to now there is no report about the speciation of heavy metals based on fluorescent nanostructures. This is obviously restricted by the lack of discriminative features of fluorescent nanoparticles among various metal species. In this respect, it is highly necessary to thoroughly investigate the fluorescence responsive nature of a variety of nanoparticles to various metal species, especially the different chemical states of a single metal. In the present work, folic acid (FA), consisting of pteridine, p-aminobenzoic acid and L-glutamic acid, is adopted as a capping ligand for the preparation of fluorescent AuNCs with the reducing capability of NaBH4. The preparation is a fast process taking only 90 s under microwave agitation. A strong fluorescence is recorded for FA-AuNCs at λex/λem=280/440 nm along with a quantum yield of 27.3%. As shown in Scheme 1, FA-AuNCs exhibit completely different fluorescent response to Hg2+ and CH3Hg+, i.e., Hg2+ causes a significant quench on its fluorescence, whereas CH3Hg+ leads to a remarkable fluorescence enhancement. Based on this discriminative nature


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of the fluorescence response of FA-AuNCs, a novel approach for the speciation of mercury was developed with LODs of 28 nM for Hg2+ and 25 nM for CH3Hg+, respectively. A literature search found no similar mercury speciation protocols.

+

NaBH4

FL enhancement

Microwave

HAuCl4

FA

FA-Au NCs

FL quenching

Scheme 1. Schematic diagram of the preparation strategy of FA-AuNCs and the principle for Hg2+ and CH3Hg+ sensing.

EXPERIMENTAL SECTION Chemicals and Materials. Standard stock solutions of mercury (GSB 04-1728-2004) and methylmercury (GSB 08675) were obtained from the National Institute of Metrology (Beijing, China). Folic acid (FA) was supplied by Aladdin Chemical Reagent Co. Ltd (Shanghai, China). All the other reagents used in this study were obtained from Sinopharm Chemical Reagent Co. (Beijing, China) with purity of at least analytical reagent grade and were used without further purification (see details in the Supporting Information). T-rich DNA chain (5’-TTGTTTGTTGGCCCCCCTTCTTTCTT-3’)15 was synthesized by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). The river water sample was collected from Guanmen Mountain (Benxi, China). The lake water sample was



obtained from Nanhu River (Shenyang, China). The crucian carp sample was acquired from the local supermarket (Shenyang, China). Ultrapure water (18.2 MΩ cm) was used throughout. Apparatus. EM7KCGW3-NR microwave oven (Midea, China) was used for the preparation of FA-AuNCs. The microstructure of FA-AuNCs was characterized by a JEM-ARM 200F transmission electron microscope (JEOL Ltd., Japan) operating at 200 kV. FT-IR spectra were recorded on a Nicolet-6700 FT-IR spectrophotometer (Thermo Ltd., USA). XPS scanning curve was conducted on an ESCALAB 250 X-ray photoelectron spectrometer (Thermo Instruments Inc., USA). ZS90 Nano Zetasizer (Malvern, UK) was used to measure the surface charge properties and hydrodynamic diameter of FA-AuNCs. UV-vis absorption spectra were recorded on a U3900 UV-vis spectrophotometer (Hitachi High-Technologies Corporation, Japan). The fluorescence measurements were performed on an F-7000 fluorescence spectrophotometer (Hitachi High-Technologies Corporation, Japan) with a 1 cm optical path, a scan speed of 1200 nm min-1 and 10 nm slits of both excitation and emission. The quantum yield of FA-AuNCs was recorded on a Quantarus-QY absolute photoluminescence quantum yield measurement system (Hamamatsu Photonics, Japan). A 7500a inductively coupled plasma mass spectrometer (Agilent Technologies, USA) was used for the quantification of mercury in real sample matrixes. Preparation of FA-AuNCs. All the glassware was thoroughly washed with aqua regia and rinsed extensively with ultrapure water before use (Caution: aqua regia is a powerful oxidizing reagent and it should be handled with extreme care). FA-AuNCs


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were prepared with the assistance of microwave irradiation based on a previously reported procedure with minor modifications.16 Shortly, 1.0 mL of HAuCl4 solution (10 mM) was added rapidly into 1.0 mL of folic acid solution (2.5 mM, in 10 mM NaOH) under vigorous stirring. Then, 0.05 mL of sodium borohydride solution (100 mM, in ice water bath) was added into the reaction medium. The mixture was heated by microwave agitation at 300 W for 90 s and during this process it turned from transparent light yellow solution to dark brown suspension. After cooling down to room temperature, the supernatant was separated by centrifugation at 8000 rpm for 10 min followed by dialysis against ultrapure water (MWCO: 500 Da) to remove the unreacted species. The fluorescence response to Hg2+ and CH3Hg+. 100 µL of Hg2+ (0.1-5.0 µM) and CH3Hg+ (0.1-5.0 µM) solutions were separately added into 100 µL of FA-AuNCs solution (0.07 mg mL-1). PBS buffer solution (10 mM, pH 4.0) was used to control the acidity of the medium. The solutions were mixed thoroughly and allowed to react at room temperature for 2 min, followed by recording the fluorescence spectra to investigate the fluorescence response of FA-AuNCs to Hg2+ and CH3Hg+. The selectivity of this sensing system for Hg2+ and CH3Hg+ was thoroughly evaluated by measuring the fluorescence variation of FA-AuNCs in the presence of various coexisting cationic and anionic species, as well as some biological related species. These include 1000 µM of Ca2+, Mg2+, Na+, K+, Phe, His, GSH, Gly, glucose and anionic species, 100 µM of Zn2+, Al3+, Co2+, Ni2+, Cd2+, Mn2+, Cr3+, Cu2+, ONOO-, ·O2-, ·OH, ClO- and H2O2, 50 µM of Ag+, Fe3+ and HS-, 10 µM of Lys, BSA



and β-Lg. The solutions of the above species (100 µL) were prepared in PBS (10 mM, pH 4.0), and mixed with an equal volume of FA-AuNCs solution (0.07 mg mL-1). After reacting at ambient temperature for 2 min the fluorescence spectra were recorded. Hg2+ and CH3Hg+ analysis in real samples. Surface water samples were pretreated by centrifugation at 10000 rpm for 5 min followed by filtration through a 0.22-µm filter membrane to remove any suspended particulate matters. For demonstrating the sensing accuracy of Hg2+ and CH3Hg+, they were both spiked into the water samples at various levels before performing the sample pretreatment process. A classical acid leaching procedure was employed to release CH3Hg+ in biological samples.17 Briefly, 5 mL of hydrochloric acid (5 M) was added to 5 g of homogenized fish back muscle in a 10-mL centrifuge tube. The mixture was then placed in an ultrasonic bath for 15 min. After extraction, the suspension was transferred into a 50-mL flask. The residue was extracted again as described above. The two supernatant portions were combined, neutralized with NaOH solution and adjusted to pH 4.0 with PBS buffer (10 mM). The solution was then 10-fold diluted for the ensuing fluorescence measurement. For mercury speciation, the fluorescence spectrum in the presence of both Hg2+ and CH3Hg+ was first recorded. Afterwards, 0.25 µM T-rich DNA chain was added to mask Hg2+, and the fluorescence spectrum with only CH3Hg+ was then obtained. The change of fluorescence intensity attributed to Hg2+ was thus derived by subtracting the contribution of CH3Hg+ from the total


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fluorescence. The concentrations of both Hg2+ and CH3Hg+ were finally derived with their linear calibration curves.

RESULTS AND DISCUSSION Preparation and characterization of FA-AuNCs. In order to obtain stable AuNCs, it is highly important to select a suitable ligand capable of stabilizing AuNCs from aggregation, as the nature of ligands can obviously affect their optical properties. FA contains plenty of carboxyl and amino groups, which had been used as capping ligand for the preparation of AuNPs.18,19 Compared with its counterparts including proteins, peptides, DNA and dendritic polymers, its small molecular weight facilitates closer approaches between gold and mercury atoms. Therefore, FA is selected as capping ligand in this work. In the present study, fluorescent FA-AuNCs were readily obtained under microwave irradiation with FA as protecting ligand and sodium borohydride as reducing agent (as illustrated in Scheme 1). It is worth mentioning that this is a very fast process, the entire preparation process took only 90 s. High resolution transmission electron microscopy (HRTEM) images (Figure S1 A) illustrated that AuNCs were well dispersed spheres, with an average diameter of 2.08±0.15 nm. X-ray photoelectron spectroscopy of FA-AuNCs (Figure 1A-B) identified Au 4f7/2 and Au 4f5/2 peaks with binding energies of 84.4 eV and 88.2 eV, respectively. These are typical binding energies (BE) of Au atoms.20 The BE peak of Au 4f7/2 (84.4 eV) in between Au(0) (84.0 eV) and Au(I) (85.1 eV) illustrated the coexistence of Au(0) and Au(I) in FA-AuNCs. The content of Au(I) in FA-AuNCs was further calculated to be 29.8%. Meanwhile, C(1s), N(1s) and O(1s) core-level



photo-emission spectra (Figure 3A) were derived from the capping ligand, reflecting the fact that AuNCs were protected by FA.

A

B

O1s C1s

Au0 4f7/2 Au0 4f5/2

Counts (s)

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Au+ 4f5/2

Au+ 4f7/2

N1s

Au4f

1200

1000

800

600

400

Binding Energy (eV)

200

0 76

80

84

88

92

96

Binding Energy (eV)

Figure 1. (A) XPS spectra and (B) Au 4f XPS spectra of FA-Au NCs. FA contains amines (-NH2 and -NH-) and carboxyl groups (-COOH), they are both potential ligand for AuNCs conjugation. FT-IR spectrum helps to identify whether FA conjugates AuNCs through amine or carboxyl (Figure S2). For FA, the sharp twin absorptions at 3413 cm-1 and 3338 cm-1 belong to the stretching vibration of primary amine, and that at 3544 cm-1 is the characteristic stretching of the secondary amine. The band at 1697 cm-1 is due to C=O stretching vibration of carboxyl groups, whereas the absorptions in between 3200 and 2500 cm-1 are OH stretching vibration of carboxyl groups. After reacting with AuNCs, the absorptions of primary and secondary amines vanished while those of C=O and OH are still there, indicating the involvement of amine in the conjugation of FA with AuNCs via Au-N binding, while carboxyl group is not involved. This observation agrees with the variation of Zeta potentials in Figure S3. FA-AuNCs surface are negatively charged within the pH range of 5-8, due to the dominating terminal carboxyl groups directed into the surrounding solution.21 As expected, an increase of the negative charge on


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AuNCs surface was observed with the increase of pH due to the deprotonation of carboxyl groups. UV-vis absorption spectra for FA-AuNCs (Figure S4A) are unlike that of FA conjugated gold nanoparticles (FA-AuNPs), which display a strong surface plasmon resonance at 534 nm.18 The spectrum of FA-AuNCs rises sharply at

Mercury speciation with fluorescent gold nanocluster as a probe

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