Simultaneous Quantification of Hg2+ and MeHg+ in Aqueous Media

Article pubs.acs.org/ac

Simultaneous Quantification of Hg2+ and MeHg+ in Aqueous Media with a Single Fluorescent Probe by Multiplexing in the Time Domain Ziqian Zhang,‡ Baoyan Zhang,‡ Xuhong Qian,†,‡ Zhong Li,‡ Zhiping Xu,*,‡ and Youjun Yang*,†,‡ †

State Key Laboratory of Bioreactor Engineering, and ‡Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology, Shanghai, 200237 China S Supporting Information *

ABSTRACT: Development of a molecular probe for selective detection of MeHg+ in the presence of Hg2+ is a mission impossible to accomplish. Speciation analysis of two substrates with a single kinetic trace exploiting their differential reactivity toward a single probe, i.e., multiplexing in the time domain, is a cost-effective and powerful alternative. We have developed such a probe (Hg410) for simultaneously quantification of Hg2+ and MeHg+ in aqueous media. Hg410 is designed via the “covalentassembly” approach, displays a zero background, and bears a very concise molecular construct. It has harnessed proximitybased catalysis to achieve high reactivity toward Hg2+ and MeHg+. An unprecedentedly low detection limit of ca. 4.6 pM and 160 pM was measured for Hg2+ and MeHg+, respectively.

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ercury, in forms of Hg0, Hg2+, and MeHg+, is widely present in the environment, mainly as a combined result of both natural and human activities, including oceanic volcanic eruptions, fossil fuel combustion, and solid-waste incineration.1,2 Discharge from industrial applications of mercury constitutes another source of mercury emission due to human activities, in chlor-alkali industry, pulp and paper industry, gold mining, electric switches, medical devices, fluorescent lamps, arc lamps, infrared sensitive semiconducting HgCdTe alloys, industrial catalysts, dental amalgam, antifouling and antimildew paints, cosmetics, pesticides, and antiseptics.1−3 Environmental Hg2+ mainly comes from oxidation of Hg0 vapor in the atmosphere by ozone or reactive radicals and subsequent deposition to the ground with precipitation.4−6 In fact, anthropogenic mercury emission has contributed to the Arctic and Antarctic ozone depletion.7,8 Biomethylation of Hg2+ to MeHg+ occurs in ground-surface water bodies by various aquatic microorganisms, mainly sulfate-reducing bacteria, and accumulates through the trophic chain.1,2,9−16 So, all living organisms including human beings are unfortunately and inevitably exposed to Hg0, Hg2+, and MeHg+ through respiration, drinking water, or diet, albeit to different extent. Liquid elementary Hg0 is of limited toxicity due to poor absorption via either dermal contact or through the human gastrointestinal tract.3 However, its vapor is readily absorbed through alveolar membrane upon inhalation and subsequently oxidized to Hg2+ by catalases and H2O2 in erythrocytes or liver.17 In vivo, Hg2+ and MeHg+ unleash high neurological toxicity via inhibition of selenoenzymes including thioredoxin reductases (TRxR), glutathione peroxidases (GPx), and thioredoxin glutathione reductases (TGR), which play fundamental roles in cell redox hemeostasis.18−23 Notably, MeHg+ in the form of a cysteinyl complex readily passes © XXXX American Chemical Society

through the placenta and is particularly harmful to developing fetuses.23−26 Dietary selenium sacrificially protects those critical enzymes and hence effectively combats mercury toxicity.9−16 Mercury species may also inhibit Na+/K+-ATPase to disrupt cell energy hemostasis.27,28 Chronic (or low dose) mercury poisoning may induce gingivitis, vomiting and diarrhea, thyroid enlargement, inflammation, respiratory distress, psychological changes, sensory disturbances, neurobehavioral disturbances, circulatory disorders, or renal failure, while acute (or high dose) mercury poisoning often leads to deaths.17 Major epidemics occurred in Japan, Iraq, Pakistan, Guatemala, Ghana, Yugoslavia, Argentina, etc.17 Restricting the industrial use and anthropogenic emission of mercury have reached global consensus and been an ongoing effort.1−3,29,30 For reliable risk assessment, close monitoring of environmental mercury levels is essential and a robust and convenient mercury speciation analysis is warranted.1−3,17,31,32 The total mercury is readily quantified with an elementspecific detector.33,34 In comparison, speciation of both Hg2+ and MeHg+ is much more complicated because separation of the two species is required prior to individual detection by inductively coupled plasma-mass spectrometry (ICP-MS) or cold vapor atomic fluorescence spectrometry (CV-AFS).33,34 An alternative speciation method, which does not require physical separation the two species, can greatly simplify instrumentation, can reduce laboratory efforts, and is clearly advantageous. Considering the paramount sensitivity of fluorescence-based detection techniques, it is our goal to Received: October 18, 2014 Accepted: November 6, 2014

A

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develop a practical small-molecule fluorescent probe for simultaneous determination of Hg2+ and MeHg+ in an aqueous sample.

Scheme 1. (a) Generic Scheme of the “Covalent Assembly” Probe Design Principle. (b) Chemical Structures of the Probe Hg410 and Its Detection Product (1)

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EXPERIMENTAL SECTION MeHgCl was purchased from Aladdin Industrial Corporation. All other chemicals were purchased from Energy Chemicals, Ltd. Solvents of analytical grade including dichloromethane (CH2Cl2), ethyl acetate (EtOAc), petroleum ether, dimethylformamide (DMF), and toluene were purchased from Titan Scientific. All chemicals and solvents were used without further purification. Milli-Q water was used for preparation of sample and buffer solutions. The 1H NMR and 13C NMR spectra were acquired on a Bruker AV-400 spectrometer. Chemicals shifts were referenced to the residual solvent peaks and given in ppm. HRMS was acquired on a Micromass GCT spectrometer. UV− vis absorption spectra were acquired on a Shimadzu UV-2600 UV−vis spectrophotometer. Fluorescence emission spectra were acquired on a PTI-QM4 steady-state fluorimeter equipped with a 75 W xenon arc-lamp and a R928 PMT. All emission spectra were corrected with respect to the PMT sensitivity at different wavelengths. Fluorescence titration studies were performed by addition of an aliquot of the stock solution of Hg2+ or other metal ions into a Hg410 solution in HEPES solution (10 mM at pH = 7.4) with 5% DMSO using a microsyringe. The excitation and emission slits were set to 2 nm for all experiments except lower detection sensitivity measurements, in which emission slits were adjusted to 5 nm to achieve a higher instrumental signal/noise ratio. The relative fluorescence quantum yield of 1 was measured with anthracene in cyclohexane (ϕ = 0.36) as the reference.

large spectral red-shift, concise molecular construct, and convenient synthesis. The key to the design of Hg410 is the 2-aminophenyl group installed ortho to the Hg2+/MeHg+reactive 1,3-dithiolane moiety. Upon desulfurization of 1,3dithiolane to a formyl group, a condensation with the nearby amino group (NH2) will occur to generate a phenanthridine, which is a polyaromatic hydrocarbon with a conjugation more extensive than that of the biphenyl precursor. Also, NH2 can coordinate to a mercury center and thereby brings it in close proximity to 1,3-dithiolane. The dialkylamino group is introduced onto the phenanthridine backbone to promote internal charge transfer (ICT), i.e., electronic push−pull, and further shift the optical wavelengths to a longer wavelength spectral region. The methyl ester in Hg410 enhances the water solubility of the probe compared to a simple alkyl group and is also a chemical handle for further derivatization when necessary. Both the probe Hg410 and the expected detection product 1 were synthesized following Scheme S1 (Supporting Information), and their spectral properties in neutral HEPES buffer with 5% DMSO (Figure 1) were studied. Probe Hg410 has an

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RESULTS AND DISCUSSION Theoretically, any existing Hg2+ probe35−49 may be employed to quantify both Hg2+ and MeHg+ in a sample using a single kinetic trace, as it has been repeatedly exemplified that Hg2+ reactive probes based on thiocarbonyls, dithioacetals, and vinyl ethers are also reactive toward MeHg+ only with much poorer kinetics. However, to render such supposition practically viable, the following three prerequisites should be fulfilled: (1) reaction kinetics of the probe toward Hg2+ should be so fast that the reaction is completed before MeHg+ can interfere; (2) reaction of the probe with MeHg+ should also be fast enough to allow measurements to be completed in a reasonable amount of time; (3) detection could be carried out in an aqueous media containing a minimum amount of organic cosolvent. Only a fraction of all Hg2+ probes in the literature have been tested against MeHg+ detection, partly due to limited access to the toxic MeHg+ especially in US-based research institutions.50−55 Still, a general conclusion can be reached that MeHg+ detection in aqueous media with the existing probes would be impractically slow. We have judiciously employed the concept of temporary intramolecularity, which is a form of proximity-based catalysis, to achieve high detection kinetics.56,57 Herein, we report Hg410 as a result of these considerations. Probe Hg410 was designed via the “covalent assembly”58−62 principle (Scheme 1). The hallmark of the covalent assembly principle is that two fragments are assembled via a substratetriggered chemical cascade to furnish the conjugative backbone of a push−pull fluorescent dye. Advantages of an assembly type probe include zero background, optimal detection sensitivity,

Figure 1. Absorption and emission spectra of Hg410 and 1 in neutral HEPES buffer with 5% DMSO.

absorption band in the ultraviolet region with a maximum at 273 nm (ε = 1.52 × 104 cm−1·M−1). Excitation at its absorption maximum did not result in any noticeable fluorescence emission. Therefore, unreacted Hg410 does not yield any background signal to compromise the detection of a weak signal. This is a highly desired property for any probe because even a weak signal can be sensitively detected from a dark background. In comparison, the absorption band of detection B

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absorption and emission intensities. Remarkably, detection kinetics of Hg2+ by Hg410 was so fast that signal enhancement was saturated essentially instantaneously (Figure 2c). High reactivity of Hg410 allowed sensitive detection of very low levels of Hg2+ (Figure 2d). A lower detection limit (LDL = 3σ) toward Hg2+ was determined to be ca. 4 pM. We note that an LDL at a low nanomolar level is typical for most existing probes for Hg2+.49 Hg410 is the first small molecule probe to display an LDL at a low picomolar level, to our knowledge. Responses of probe Hg410 to MeHg+ were spectrally analogous to those induced by Hg2+ except that 2 equiv of MeHg+ was required to saturate the signal enhancements (Figure 2e,f). Signal enhancement by 0.6 equiv of MeHg+ took ca. 600 s to reach saturation (Figure 2g). Detection of MeHg+ by Hg410 is comparatively much slower than Hg2+, as expected. However, Hg410 is still likely the most reactive probe for MeHg+ so far, and a lower detection limit of ca. 160 pM was estimated (Figure 2h). The 2-aminophenyl moiety in Hg410 has a profound influence on its detection kinetics toward Hg2+ and MeHg+ as clearly exhibited by a side-by-side comparison with a model probe 2, which has the 1,3-dithiolane moiety for recognition but lacks the 2-aminophenyl moiety for catalysis (Scheme 2).

product (1) is located at a much redder spectral region with a maximum at 337 nm (ε = 1.74 × 104 cm−1·M−1). Such a feature allows selective excitation of the detection product in the presence of any unreacted probe. Upon photoexcitation of compound 1 with light at 350 nm, an intense blue fluorescence emission peak at 410 nm was observed with a quantum yield of 0.26. Probe Hg410 (10 μM) was titrated with Hg2+ and MeHg+ in HEPES buffer (10 mM at pH = 7.4) containing 5% DMSO. Upon addition of an aliquot of Hg2+ stock solution, an absorption band at 337 nm appeared, while the absorption at 273 nm from Hg410 decreased, in agreement with the expected conversion of Hg410 to 1 (Figure S1, Supporting Information). Emission from 1 at 410 nm was also observed to rise from a zero background upon Hg2+ addition (Figure 2a). The

Scheme 2. Structures of a Control Probe (2) and Its Detection Product (3), for Comparison with Hg410

Figure 3. Kinetic traces of probe 2 by various amounts of Hg2+ (a) and MeHg+ (b).

The reactions between probe 2 with both Hg2+ (Figure 3a) and MeHg+ (Figure 3b) were unbearably slow. Within 1 h, a dose of Hg2+ as high as 1.0 equiv only converted ca. 10% of the probe 2 (10 μM in neutral HEPES buffer with 10% DMSO) to 3. When 5 equiv of Hg2+ was applied, deprotection of probe 2 was not completed within ca. 1 h. Similarly, detection kinetics of MeHg+ by probe 2 is also much more inferior than that of Hg410. As much as 10 equiv of MeHg+ converted no more than 5% of probe 2 within 1 h, under the same conditions. The sharp contrast between detection kinetics of Hg410 and probe 2 clearly exhibited how dramatically the 2-aminophenyl moiety has improved an otherwise sluggish receptor, i.e., a simple 1,3dithiolane or 1,3-dithiane, for Hg2+/MeHg+ detection. A detection mechanism was proposed to explain the catalytic effect of the 2-aminophenyl moiety in the probe Hg410 (Scheme 3). Presumably, the NH2 group in Hg410 can reversibly coordinate to Hg2+. This brings Hg2+ into close proximity of the 1,3-dithiolane moiety, promotes the effective

Figure 2. (a) Fluorescence titration of Hg410 by Hg2+. Spectra collected 100 s after Hg2+ addition. (b) Dose-dependent fluorescence enhancement with respect to Hg2+ equivalence. (c) Fluorescence titration of Hg410 by a low dose of Hg2+. (d) Fluorescence detection kinetics of Hg410 against Hg2+. (e) Fluorescence emission titration of Hg410 by MeHg+. Spectra collected 1200 s after Hg2+ addition. (f) Dose-dependent fluorescence enhancement with respect to MeHg+ equivalence. (g) Fluorescence titration of Hg410 by a low dose of MeHg+. (h) Fluorescence detection kinetics of Hg410 against MeHg+.

enhancements of both absorbance intensity at 337 nm (Figure S1, Supporting Information) and emission intensity at 410 nm (Figure 2b) were linearly proportional to the added Hg2+ dose until the signal enhancement was saturated upon addition of a stoichiometric amount of Hg2+. Further addition of Hg2+ (tested up to 2 equiv) did not induce any fluctuation of both C

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Scheme 3. Proposed Detection Mechanism of Hg410 toward Various Mercurial Species with Hg2+ as an Example

molarity56 of Hg2+, and catalyzes the subsequent ring-opening of the 1,3-dithiolane moiety to yield intermediate a. Such a mechanism has been previously termed as “temporary intramolecularity”.57 The viability of this mechanism is supported by a few existing Hg2+ probes 4,47 5,48 and 6,42 each of which has also displayed favorable detection kinetics and importantly has one (or more) heteroatom(s) (N in probe 4, and O in probes 5 and 6) near the sulfur atom for coordination to Hg2+, reminiscent of the same structural feature of Hg410 (Figure 4). However, this does not seem to fully account for the still-

As mentioned previously, reaction between Hg2+ and Hg410 was completed in ca. 2 s, during which time the reaction between MeHg+ and Hg410 proceeded to a negligible extent. In other words, the signal enhancements for Hg2+ and MeHg+ are kinetically orthogonal. Therefore, it seems feasible to simultaneously quantify Hg2+ and MeHg+ in a sample by means of a single kinetic trace, i.e., the signal enhancement in the first ca. 2 s originates from Hg2+ and signal enhancement thereafter from MeHg+. This idea was tested by reacting a Hg410 solution (20 μM) in neutral HEPES buffer (10 mM at pH = 7.4) with 5% DMSO with an array of samples containing varying amounts of Hg2+ and MeHg+ (Figure 5). With samples containing only MeHg+, a steady signal enhancement was observed and the intensity was proportional to the MeHg+ level (Figure 5a). When Hg2+ was present in these MeHg+ samples, an abrupt jump in the first ca. 2 s occurred, which was apparently from Hg2+ (Figure 5b−f). Steady signal enhancement from MeHg+ followed thereafter. The height of the initial abrupt signal jump was indicative of the Hg2+ level, and the signal enhancement thereafter was proportional to the MeHg+ level. Signal enhancements, corresponding to Hg2+ and MeHg+ from each kinetic curve, were extrapolated, and reliable calibration curves for both Hg2+ (0−7.5 μM) and MeHg+ (0−30 μM) were established (Figure 5g). In case a wider range of either [Hg2+] or [MeHg+] is necessary, the concentration of Hg410 may be increased accordingly. Alternatively, the sample may be diluted to avoid an inner filtering effect. This experiment has proven that Hg410 is capable of simultaneous quantification of Hg2+ and MeHg+ in a sample, which is a practically important yet chemically challenging task.63 Potential interference from heavy transition metal ions were tested. Interference from Fe3+, Cd2+, Zn2+, Mn2+, Fe2+, Cu2+, Cu+, Co2+ Ni2+, and Pb2+ was not noticeable (Figure 6a). Only Ag+ gave a significant interfering signal (Figure 6a). However, because the signal enhancement from Ag+ was not instantaneous, like that from Hg2+ (Figure 6b), interference from Ag+ with the detection of Hg2+ can be easily circumvented by a kinetic scan, the way that MeHg+ could be distinguished from Hg2+ by multiplexing in the time domain. However, in cases where Hg410 is used for quantification of MeHg+, the presence of high levels of Ag+ will cause serious interference.

Figure 4. Chemical structures of a few previously reported probes 4, 5, and 6 for Hg2+.

superior detection kinetics of Hg410, which only requires a few seconds, while probe 4, 5, or 6 requires 1 min or more. For example, probe 4 by He and Guo et al. was shown to be fully deprotected by 2 equiv of Hg2+ within 3 min in neutral HEPES buffer containing 20% CH3CN. Probes 5 and 6 by Wang et al. were used in near pure aqueous media, and 2 equiv of Hg2+ fully deprotected the probe within 1 min. This led us to assume that the NH2 group in Hg410 had also assisted in the desulfurization of the sulfonium moiety of intermediate a. Ahn et al. has demonstrated that hydrolysis of this sulfonium moiety is the rate-limiting step of the Hg2+/MeHg+-triggered desulfurization cascade of a 1,3-dithiolane moiety.46 In Hg410, the need for a water molecule to intermolecularly attack the electrophilic sp2-hybridized carbon atom of the sulfonium moiety is obviated. Instead, the NH2 group, which is more nucleophilic than H2O in the first place and in closer proximity to the electrophilic sulfonium moiety, is believed to have attacked the sulfonium moiety and dramatically enhanced the kinetics of the desulfurization of intermediate a as indicated in Scheme 3. D

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Figure 5. (a) Kinetic traces upon addition of various amounts of MeHg+ into a solution of Hg410 (20 μM) in neutral HEPES buffer with 5% DMSO. (b−f) Kinetic traces upon addition of various amounts of Hg2+ and MeHg+. (g) Calibration curves for simultaneous quantifications of Hg2+ and MeHg+. Note: Irregular intensity jumps likely caused by an unstable power supply were removed in three curves in Figure 5, parts a, e, and f, respectively.

type probes for biological imaging applications, and related works are underway.

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ASSOCIATED CONTENT

S Supporting Information *

Synthetic scheme, procedures, 1H/13C NMR and MS data of new compounds, HPLC data, and additional spectral data. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +86-21-64251781. Fax: +86-21-64252603. *E-mail: [email protected].

Figure 6. (a) Cross-reactivity study from potentially interfering transition metal ions toward Hg410. (b) Kinetic traces of the reactions upon addition of 1 equiv or 10 equiv of Ag+ to a solution of Hg410, respectively.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work is supported by the Natural Science Foundation and of Shanghai Municipality (no. 11ZR1408800), Shanghai RisingStar Program (no. 13QA1401200), Innovation Program of Shanghai Municipal Education Commission (no. 12ZZ047), Doctoral Fund of Ministry of Education of China (no. 20110074120008), and the National Natural Science Foundation of China (nos. 21106043, 21372080, and 21236002).

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CONCLUSIONS A novel fluorescent probe (Hg410) was designed and synthesized for Hg2+ and MeHg+ quantification via the “covalent assembly” principle. The probe exhibits a favorable fluorescence turn-on signal from a zero background, unparalleled reactivity, and hence picomolar level detection sensitivity in aqueous media for both Hg2+ and MeHg+. Additionally, Hg2+ and MeHg+ levels in a sample can be simultaneously quantified via a single kinetic curve. This approach may also be applicable for differentiation of other pairs of substrates, i.e., cysteine/ homocysteine64 and H2O2/OONO−,65 against each of which development of a specific fluorescent probe has been proven difficult. Hg410 may be covalently attached to a dedicated longwavelength fluorophore to construct “dye−linker−receptor”

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