B American Society for Mass Spectrometry, 2014

J. Am. Soc. Mass Spectrom. (2014) 25:861Y868 DOI: 10.1007/s13361-014-0825-z

RESEARCH ARTICLE

Ultrasensitive, Rapid, and Selective Detection of Mercury Using Graphene Assisted Laser Desorption/Ionization Mass Spectrometry Hani Nasser Abdelhamid,1,2 Hui-Fen Wu1,3,4,5,6 1

Department of Chemistry, National Sun Yat-Sen University, Kaohsiung, 804, Taiwan Department of Chemistry, Assuit University, Assuit, 71515, Egypt 3 School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, 806, Taiwan 4 Institute of Medical Science and Technology, National Sun Yat-Sen University, Kaohsiung, 804, Taiwan 5 Center for Nanoscience and Nanotechnology, National Sun Yat-Sen University, Kaohsiung, 804, Taiwan 6 Doctoral Degree Program in Marine Biotechnology, National Sun Yat-Sen University, Kaohsiung, 804, Taiwan 2

Abstract. We report an extremely sensitive and specific detection of mercuric ions 2+ (Hg ) based on graphene assisted laser desorption/ionization mass spectrometry (GALDI-MS). Combining the highly selective coordination interactions between 2+ thymine (T) and Hg , we present a simple, effective, and novel approach, based on 2+ π–π interactions of the T-Hg -T complex and G that can serve as a platform and matrix for GALDI-MS. The present sensor not only exhibits high selectivity and 2+ sensitivity (picomolar) to Hg in aqueous solution, but also can elucidate the chemical structures of the metal complexes. The significant advantage in the current approach is that there is no need for a sophisticated instrument, and no sample pretreatment is 2+ required to detect the Hg ions. Keywords: Graphene, Mass spectrometry, Mercury, Biosensing, Selective O

H 3C

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Received: 23 October 2013/Revised: 24 December 2013/Accepted: 26 December 2013/Published online: 4 March 2014

Introduction

M

ercury is highly noxious and most toxic of the heavy metals, whose bioaccumulation could lead to serious health problems. The accumulation of mercury ions (Hg2+) in living organisms can lead to death due to damage on DNA, inhibit biomolecules interactions, leads to liverkidney dysfunction, and bind to hemoglobin (Hb) [1–4]. Futhermore, Hg2+ emission, from various sources such as the mining, burning of fossil fuels, and volcanic activities, has been a pressing challenge [5]. Thus, it is important to develop a rapid analytical technique that can identify the Hg2+ contamination in order to provide the detailed information to support remediation decisions. Traditionally, Hg2+ has been detected by many conventional analytical instruments, such as atomic absorption spectrometry (AAS), fluorescence spectrometry (AFS), inductively coupled plasmaElectronic supplementary material The online version of this article (doi:10.1007/s13361-014-0825-z) contains supplementary material, which is available to authorized users. Correspondence to: Hui-Fen Wu; e-mail: [email protected]

optical emission spectrometry (ICP-OES) and inductively coupled plasma-mass spectrometry (ICP-MS) [6–9]. However, these tools suffer from limitations such as lack of selectivity, sensitivity, require complicated and expensive instrumentation, and tedious laboratory procedures that cannot be widely applied [10–12]. Liu et al. have developed a system that was not only selective and sensitive but also practical and convenient for colorimetric detection of Hg2+ ions at room temperature [13, 14]. They reported that thymine (T) could selectively capture Hg2+ to form T–Hg2+–T base pairs. Based on the intrinsic and specific interactions between Hg2+ and T, this strategy could provide a high selectivity toward Hg2+ ions than other heavy transition metals (HTM). Therefore, a series of Hg2+ biosensors based on T–Hg2+–T were developed afterwards [15–30], such as fluorescene [31–36], colorimetric [37–41], and electrochemical sensors [13, 15, 42–50]. Furthermore, mercury-specific aptamer (MSD) combined with colorimetric assay [38, 51, 52], electrochemical transducers [53] and surface plasmon resonance (SPR) spectroscopy [54] have been investigated. Also other selective approaches were also developed such as mercury-specific monoclonal antibody [55, 56] and DNA/oligonucleotides-conjugated dye tags to

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H. N. Abdelhamid and H.-F. Wu: Selective Biosensing of Mercury Using Mass

label the metallic nanoparticles [57–59]. Furthermore, the Tbased sensors were combined with reporters such as pyrene [60], gold (for SERS [61] and optical analysis [38]), photonic crystal (PC) [62], ionic imprinted polymers (IIPs) [63], phthalocyanine [64], biochip [65], and SiO2 [66]. Graphene (G) is a shining star in material science [67– 69]. It was utilized as a matrix for matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) to detect small molecules, such as amino acids, polyamines, peptides, steroids, nucleosides, nucleotides, and metallodrugs [70–77]. Conventional matrices of MALDIMS, such as organic acids, suffer limitations such as interferences at low molecular weight (M.Wt), destroy noncovalent interactions, and form clusters with metals [74, 75, 78–81]. Recently, we proposed a novel approach using graphene (G) to detect noncovalent interactions in metallodrugs [74, 75]. We claimed that there is no disturbance of these weak forces during the laser desorption/ionization process. Based on the T–Hg2+–T interactions, we further introduced the graphene assisted laser desorption/ionization mass spectrometry (GALDI-MS) as a selective and sensitive approach for Hg2+ detection. T interacts with G by the π–π interactions that can assist specific interactions between Hg2+ and T. The present method could offer high selectivity, sensitivity (picomolar), and rapid detection for Hg2+ ions.

Daltonics, Bremen, Germany) equipped with a nitrogen (N2) laser (wavelength 337 nm). The spectra were recorded in positive and linear mode using an acceleration voltage of 20 kV and 10 ns extraction delay time. Raman spectrum was collected using JOBIN-YVON T64000 Micro-PL/ Raman Spectroscopy, excitation source: He-Cd laser with wavelength 325 nm.

Experimental Preparation of Graphene Oxide (GO) and Graphene (G)

Zn(NO3)2, Fe(NO3)2, FeCl3.6H2O, Ni(NO3)2, Hg(NO3)2.2H2O, Cu(NO3)2.0.5H2O, and Cd(NO3)2 were purchased from Sigma-Aldrich (St. Louis, USA). Sulfuric and nitric acids were purchased from J.T. Baker (Gujarat, India). Natural graphite (–20 + 84 mesh, 99.9 %) was purchased from Alfa Aeser (Lancashire, Great British). Methanol (HPLC grade) and potassium permanganate were purchased from Merck (New Jersey, USA). The deionized water obtained from a Milli-Q Plus water purification system (Millipore, Bedford, MA, USA) was used for all experiments.

Graphene oxide was synthesized according to our previous publication [74, 75], which based on Hummer and Offeman’s method [82]. Briefly, a round flask containing natural graphite (1.0 g) was placed in an ice-bath (with sodium chloride), which was then subjected to magnetic stirring (Ciramec). After that, nitric acid (10.0 mL, 69 %– 72 %) was added to the flask, sulfuric acid (15.0 mL, 96.0 %) and subsequent potassium permanganate (3.0 g, ≥99 %) were added gradually to the mixture. The temperature was kept G0 °C by using ice-sodium chloride bath. After removing the ice bath, hydrogen peroxide (30 %– 32 %, 15 mL) was added dropwise to remove the excess of permanganate (till bubbles stop). After magnetic stirring for 2 h, distilled water (200 mL) was poured slowly into the mixture to obtain a dark-brown suspension. Subsequent to further stirring for another 30 min, the dispersion was filtered and then washed several times with a 5.0 wt% HCl solution to remove metal ions. To reduce GO to G, the above GONP colloidal dispersion (10 mL) was mixed with hydrazine solution (5 μL, 35 wt% NH2-NH2 in water) and then solution of ammonium hydroxide (35 μL, 28–30 wt%, J.T.Baker) was added to the flask. The mixture was subjected to magnetic stirring in an oil-bath at 100 °C for 2 h to reduce GONP to G. The synthesized G nanosheet was characterized using TEM, FT-IR, Raman, and UV spectroscopy.

Instruments

Solution Preparations

Transmission electron microscopic (TEM) observations were performed on a Philips CM-200, (Lausanne, Switzerland). TEM was operating at an accelerating voltage of 100 kV. The sample was prepared by placing a drop of homogeneous suspension (10 μL) on a copper grid and allowing it to dry in air. Fourier transform infrared (FT-IR) spectra were recorded on Perkin-Elmer (Rodgau, Germany) Spectrometer over wavenumber range of 450–7800 cm–1. UVvisible spectroscopy (Lambda 25) with a 1 cm quartz cell was carried out to characterize the optical properties of the synthesized nanomaterials. Origin V 6.0 was used to draw the experimental data and graphs. Matrix assisted laser desorption/ionization mass spectrometry (MALDI-TOFMS) spectra were obtained from Microflex (Bruker

Zn(NO3)2, Fe(NO3)2, FeCl3.6H2O, Ni(NO3)2, Hg(NO3)2.2H2O, Cu(NO3)2.0.5H2O, and Cd(NO3)2 were dissolved in distilled and deionized water to afford aqueous solution with concentration (1 × 10–3 mol.L–1 ).

Materials and Methods

Procedures for Hg2+ Detection For Hg2+ assays, the solution of (Hg2+, 10 μL, 1 × 10–3 mol.L–1) and T (20 μL, 1 × 10–3 mol.L–1) with molar ratio 1:2 were added to the G colloid (0.2 g in 50 mL) and then the solution was mixed well by pipette up-down three to four times to form homogenous solution. The above prepared solution (2 μL) was spotted on stainless steel plate (96 spots). Finally, the GALDI spectrum of the incubated solution was measured at 10 % above

H. N. Abdelhamid and H.-F. Wu: Selective Biosensing of Mercury Using Mass

the threshold ionization of the complex using N2 laser. The overall process of GALDI-MS is shown in Figure 1. For the sensitivity measurement, different volumes (1, 2, 3, 4, 5, 7, 10 μL) of Hg2+ (1 × 10–6 mol.L–1) were mixed with T and G in molar ratio (1:2:1). About 2 μL was spot in MALDI plate and allowed to dry before the MALDI analysis. For the selectivity measurement, various metal ions (Cd2+, Cu2+, Ni2+, Zn2+, Fe2+, and Fe3+, 1 × 10–3 mol.L–1) were mixed with thymine (1 × 10–3 mol.L–1 ) in molar ratio 1:2, then mixed, and 2 μL was spot in MALDI plate and allowed to dry before the MALDI analysis. In order to investigate the selectivity of other metals in the presence of Hg2+ ions, the other metals (Cd2+, Cu2+, Ni2+, Zn2+, Fe2+, and Fe3+, 1 × 10-3 mol.L–1, 10 μL) were mixed with 10 μL of Hg2+:thymine (molar ratio 1:2 ), and then 2 μL was spot on MALDI plate and allowed to dry before the MALDI analysis.

Results and Discussion Graphene (G) is a two-dimensional carbon nanomaterial. It has been prepared by Hummer and Offeman’s method [82], which was based on harsh oxidation of graphite to graphene oxide (GO) that was subjected to further reduction by hydrazine in alkaline media to G (Supplementary Figure S1A). The prepared material was characterized by TEM, UV, FTIR, and Raman spectroscopy (Supplementary Figure S1). The TEM image (Supplementary Figure S1B) shows that G is a transparent nanosheet with few layers. It displays continous UV-vis absorption (Supplementary Figure S1C)

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and shows maxinum at wavelength 295 nm. The UV-vis absorption property is sufficient for G to serve as an effective matrix for MALDI-MS analysis [70–78]. The preparation step is monitored by FTIR (Supplementary Figure S1D). The intense bands at 3450 and 1250 cm–1 are attributed to the stretching of the O–H band of C–O. The band at 1650 cm–1 is associated with stretching of the C = O bonds of the carboxyl groups. The FTIR spectrum (Supplementary Figure S1C) confirms the two steps of G synthesis i.eoxidation-reduction process due to the absence of the carbonyl group (C = O). The Raman spectrum of G is plotted in Supplementary Figure S1E. Generally, the Raman spectrum of G comprises three main distinctive peaks that are assigned as diamondoid (D, 1350 cm–1), graphitic (G, 1560 cm–1), and 2D bands (2500 cm–1). Mercury ion (Hg2+) can cause serious damage to human’s central nervous, DNA, mitosis, and endocrine systems [1–4]. Thus, selective and sensitive detection methods for mercury species have been received intensive attentions. The sensing principles of GALDI-MS are shown in Figure 1. Thymine (T, Thy, 5-methyluracil) is one of the four nucleobases in the nucleic acid of DNA that are represented by the letter ‘T’. Generally, Hg2+ can interact with T through specific T– Hg2+-T formation and promote T–Hg2+–T complex ions [13–66]. The ligand molecules (T) can interact with G by π–π interactions. These noncovalent interactions can assist the coordination between T and Hg2+ ions. T and Hg2+ are mixed in molar ratio 1:2. Metals such as Hg2+ can interact with G by electrostatic interactions among the positive charges of Hg2+ and the negative charge of G. This

Graphene Thymine Metals (Hg2+, Ni2+, Cu2+, Zn2+, …etc) H2O

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H 2O

Laser, 337 nm M.Wt = 488

Figure 1. The assumed complex formation between Hg+2 and Thymine to form Hg(T)2(H2O)2 which assemble on graphene nanosheet. Initially, Hg+2, T, and G mixed in molar ratio 2:2:1, respectively, then 2 μL of the mixture was spotted in MALDI plate and dry before analysis

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interaction also supports complexation on the G platform. Because the high selectivity of Hg2+–T interactions, they require only few minutes that are consumed during the drying time of MALDI spots. The rate of interaction may be high in the presence of G due to large surface areas, and the electrostatic interactions of Hg2+–G can assist metal complexation. Due to the specific T–Hg2+–T interactions with high selectivity and affinity, we aimed to develop a new approach based on T–Hg2+–T interactions, where T is used as a coordination ligand. Simply, T and Hg2+ were mixed in the molar ratios 1:2 in the presence of G. After that, 2 μL of the mixture was spotted onto the MALDI plate. When the drop was dried, it was detected by the MALDI-MS. Conventional MALDI-MS matrices such as 2,5-dihyroxy benzoic acid (DHB), fuoric acid, and mefenamic acid often

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produce many intense interferences at low mass regions [83], and they also can destroy the noncovalent bonding interactions among various metals and ligands [74, 75]. Thus, nanomaterials have been proposed in order to circumvent these drawbacks [78]. We have proposed a novel method to probe the noncovalent interactions among metals and nonsteroidal antiinflmmatory drugs (NSAIDs). It was based on π–π interactions of the drug and G [74, 75]. Therefore, we apply this approach to selectively detect Hg2+. Our primary goal is to improve the sensitivity and selectivity of Hg2+ ion detection from the aqueous solution. To achieve strong and highly specific binding to Hg2+ ions, T is proposed as ligand. The chemical formula of T is C5H6N2O2 with molecular weight 126.11 g mol–1. Desorption/ionization of T from the

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Figure 2. GALDI-MS spectra of (a) thymine, (b) Hg2+-T, (c) Cd2+-T, (d) Cu2+-T, (e) Zn2+-T, and (f) Fe2+-T. Metals were mixed with T in molar ratio 1:2

H. N. Abdelhamid and H.-F. Wu: Selective Biosensing of Mercury Using Mass

surface of G was plotted in Figure 2a. The GALDI spectrum of T (Figure 2a) shows weak peak at 126 that refer to [T + H]+. However, the dimmer peaks can be recognized at 2530 that was assigned as [2 T + H]+. This observation supports the general schematic representation that we proposed in Figure 1. The other peaks at 23, 39, 149, and 165 are assigned as Na+, K+, [T + Na]+, and [T + K]+, respectively. The assigned peaks are tabulated in Supplementary Table S1. In the presence of Hg2+, T coordinates and form complex at m/z 488 corresponding to [Hg(T)2(H2O)2 + H]+. Generally, any metal ions such as Hg2+ tend to complete the outer electronic configurations by coordination with suitable ligands. Hg2+ satisfies the electronic levels by coordination with ligands such as T and water molecules (H2O). Thus, Hg2+ coordinates with two molecules of T and H2O. Dehyration of the complex was observed from the peak at m/z 472 corresponding to [Hg(T)2(H2O) + H]+. We recorded the complex in the positive mode as it is easy to be protonated, whereas the negative ion mode needs electronegative groups that are able to hold negative charges. Generally, complexes lack this requirement. So the positive ion mode only is reported here. We noted that GALDI-MS showed low resolution of the complex that is attributable to the π–π interactions between G and T, which decreases desorption/ionization rate of the complex from the surface of MALDI plate. Furthermore, the present instrument (Micro Flex, Taiwan Micro Flex Suppliers, Taipei, Taiwan) has low resolution as shown in the isotopic pattern of the detected peaks m/z 488 and 472 (Supplementary Figure S2). However the simulated and practical patterns were matched well. We believe that the high resolution MALDI can be significant for the isotopic study. The two peaks at m/z 797 and 815 are Hg related peaks (Supplementary Figure S3). It is obvious as they share the same isotopic pattern of Hg-complex. To address the possibility of interference from other chelating chemicals, various metals such as Cd2+, Cu2+, Ni2+, Zn2+, Fe2+, and Fe3+ were added to the G solution in the presence and absence of Hg2+ (Figure 2). Data (Figure 2) reveal that only Hg2+ can coordinate with T to form complex at m/z 488. No detectable signal was observed for any of these metal ions (Supplementary Figure S4A). The effect of the selected metal ions was also tested in the presence of Hg2+, and no significant interference was observed on the GALDI signals (Supplementary Figure S4B). Incubation carried out with a different mixture of the metal ions or longer incubation times gave similar results. Masking agents are usually used to increase the selectivity in some assays [84]. In contrast, GALDI needs no masking agents or auxiliary chemical species. This probe shows excellent selectivity to Hg2+ detection based on GALDI-MS due to the advantage of T-Hg 2+ –T coordination chemistry (Figure 2). For the sensitivity measurement, different volume (1, 2, 3, 4, 5, 7, 10 μL) of Hg2+ (1 × 10-6 mol.L–1) were mixed with T and G in molar ratio (1:2:1). Then 2 μL was spotted on MALDI plate; the MALDI spectra were then

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Figure 3. Limit of detection of T-Hg2+-T complex; Hg2+:T (1 × 10–4 M) were mixed in molar ratio 1:2, respectively with different volumes (a) 10, (b) 8, (c) 7, (d) 5, (e) 4, (f) 3, (g) 2, (h) 1 and (i) 0.5 μL

investigated. MALDI spectra show 2 pmol is the limit of detection (LOD) (Figure 3). Compared with other sensors for Hg2+ detection, the novel probe possesses several attractive features: (1) simplicity, because sensing only needs mixing of Hg2+, T, and G before GALDI-MS detection without addition of extra

Table 1. Limit of Detection of the Present Approach in Comparison with Conventional Techniques Technique

LOD

Reference

XRF ICP-MS Fluorescence CVG-AFS GALDI-MS

20 mg/kg 0.4–0.8 ng g–1 4.7 nM 45 nM 2 pmol

[6] [8] [9] [10] Here

XRF, X-ray fluorescence; inductively coupled plasma mass spectrometry, ICP-MS; CVG-AFS, cold vapor generation atomic fluorescence spectrometry; GALD-MS, graphene assisted laser desorption/ionization mass spectrometry.

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reagents; (2) inexpensive, because the reagents such as DNA, aptamers,…etc., and other high selective recognition molecules, are not needed; (3) high selectivity, (4) it can clearly reveal the complex structure, (5) sensitivity, because the detection limit was reduced to 2 picomole, (6) low sample consumption, and (7) no reference is required. Classic analytical approaches to Hg2+ sensing, such as AAS, ICP-MS, ICPOES, and AFS, require expensive hardware, sophisticated sample treatment, and instruments operated by professionals in a centralized laboratory [6–9]. In contrast, our approach is very simple, fast, and easy operation with high sensitivity (Table 1). Recent literatures intensively demonstrate and develope sensitive and selective optical tests for Hg2+ [15–53]. Fluorescent chemosensors provide several advantages over other analytical methods, such as high sensitivity and selectivity [85, 86]. However, fluorescence or other spectroscopic techniques are pH, time, and temperature-dependent. Fluorescent chemosensor based on electron donor–acceptor are usually disturbed by protons in the detection of metal ions, so it is necessary to consider excluding the pH effect and finding optimal sensing conditions. Thus, general procedure of Hg2+ detection depends on buffer solution that makes the reaction between chromophore and metals feasible. Compared with other reported methods such as fluorescence methods that are also based on DNA tage dyes or nanomaterials, this proposed method had a superior detection limit for Hg2+ detection. Here, we detect Hg2+ directly without any buffer solution. Conventional MALDI analysis for metal–ligand interaction shares the same limitation. We already circumvent this limitation by apply G as a matrix [74, 75]. Most of fluorophores are water insoluble, so it is often investigated in organic solvents. These solvents usually have effects on the fluorophore intensity and response, and thus it should be optimized. Moreover, most of these sensors required complicated synthesis. In contrast, G is well-dispersed in water and has low or no response toward environment changes. Among the numerous nanomaterials that work as matrices, G has been widely used because it has many advantages, such as no toxicity, high water dispersion, and high capacity because of double sides [68, 69].

Conclusion We successfully combined molecular coordination with nanoparticle science to develop new tools in analytical chemistry. A new and rapid mass sensing method for selective and sensitive detection of Hg2+ based on GALDIMS was demonstrated. It offered many advantages such as simplicity, rapidity, high sensitivity (picomolar), and excellent selectivity for the response to Hg2+ in aqueous media. The novel approach does not require any expensive reagents, buffer solution, masking agents, or organic solvent. Owing to the advantages of high selectivity, sensitivity, and simplicity, we believe that GALDI-MS is a great potential platform for mercury detection in the near future.

Acknowledgment The authors thank the National Science Council of Taiwan for financial support.

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