Chip-Based Magnetic Solid-Phase Microextraction Online Coupled

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Chip-based Magnetic Solid Phase Microextraction On-line Coupled with MicroHPLC-ICP-MS for the Determination of Mercury Species in Cells Han Wang, Bei-Bei Chen, Siqi Zhu, Xiaoxiao Yu, Man He, and Bin Hu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03130 • Publication Date (Web): 25 Nov 2015 Downloaded from http://pubs.acs.org on November 26, 2015

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Analytical Chemistry

Chip-based Magnetic Solid Phase Microextraction On-line Coupled with MicroHPLC-ICP-MS for the Determination of Mercury Species in Cells Han Wang, Beibei Chen, Siqi Zhu, Xiaoxiao Yu, Man He, Bin Hu* Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, China

*

Corresponding [email protected]

author:

Fax:

+86-27-68754067;

Tel:

ACS Paragon Plus Environment

+86-27-68752162;

Email:


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ABSTRACT: Trace mercury speciation in cells is critical to understand its cytotoxicity and cell protection mechanism. In this work, we fabricated a chip-based magnetic solid phase microextraction (MSPME) system integrating cell lysis unit as well as sample extraction unit and on-line

combined

it

with

micro

high

performance

liquid

chromatography

(MicroHPLC)-inductively coupled plasma mass spectrometry (ICP-MS) for the speciation of mercury in HepG2 cells. Magnetic nanoparticles with sulfydryl functional group were synthesized and self-assembled in the microchannels for the preconcentration of mercury species in cells under an external magnetic field. The enrichment factors are ca. 10-fold, and the recoveries for the spiked samples are in the range of 98.3% - 106.5%. The developed method was used to analyze target mercury species in Hg2+ or MeHg+ incubated HepG2 cells. The results demonstrated that MeHg+ entered into the HepG2 cells more easily than Hg2+, and part of MeHg+ might demethylated into Hg2+ in HepG2 cells. Besides, comprehensive speciation of mercury in incubated cells revealed different detoxication mechanism of Hg2+ and MeHg+ in Hg2+ or MeHg+ incubated HepG2 cells.


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INTRODUCTION Mercury is a harmful element, but it has been widely used for centuries. In the past, mercury was a common constituent of many medicines and now it is still used as fungicides and dental products (amalgam)1. Even with the ban of using mercury in the medications, people can still contact mercury due to the environmental pollution (the burning of fossil fuel, refuse incineration, etc.) and the consumption of fish and other aquatic product2,3,4. It is well established that mercury entering in cells would increase the generation of reactive oxygen species (ROS), disturb oxidative phosphorylation and electron transport, and induce the unbalance of mitochondria homeostasis5-10. The high affinity of mercury for thiol group results in the depletion of glutathione (GSH), alters glutathione peroxidase (GPs) and glutathione reductase (GR)8,11-13 activities, which lead to lipid peroxidation and cell death14-16. The toxicity of mercury depends on their species (MeHg+>Hg2+). Burbacher et al.17 and Rodrigues et al.18 compared mercury levels and species distribution in infant monkeys (blood, brain) and rat tissues (blood, brain, heart, kidney and liver) which exposed to MeHgCl and thimerosal. The results indicated that the demethylation rate of MeHgCl is much slower than thimerosal which may explain the more potent toxicity of MeHg+. It should be noted that most works on the cytotoxicity of mercury are mainly focused on the total amount of mercury and its overall biological effect in cells19-22. Little consideration is given to mercury speciation which not only reveals cytotoxicity but also the cell protection mechanism of mercury intuitively, probably because of the lack of robust analytical methods for mercury speciation in cells currently. Conventional hyphenated techniques such as high performance liquid chromatography (HPLC) - inductively coupled plasma mass spectrometry (ICP-MS) 18 would consume cells with tremendous numbers which is self-explanatory troublesome (in some cases, the amount of cell samples are limited). While minusing17,20 methods have potential risk to cause inaccurate results due to possible untargeted mercury species in cell system. As a result, developing new analytical methods with high accuracy and sensitivity for mercury species in a small amount of cells is very urgent. However, it is a technically challenging task to achieve mercury speciation in cells. As mentioned above, hyphenated techniques such as HPLC-ICP-MS, which are the most effective methods for elemental speciation, is still faced with tremendous difficulties when directly applied



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in cells analysis: (1) the sample volume is limited which does not match the injection volume of normal HPLC-ICP-MS; (2) serious matrix effects and polyatomic interference from the cell matrix will cause potential inaccurate results; (3) instrumental sensitivity does not meet the requirements of ultra-trace mercury species levels in a small amount of cells (especially some species are in low abundance). MicroHPLC by using columns with smaller column dimension and stationary particle diameter has the features of smaller injection volume, higher chromatographic efficiency, and lower liquid flow rate. As a result, MicroHPLC combining with ICP-MS can solve the detrimental effects associated with the introduction of large amounts of organic solvents or salts into the plasma, and reduce consumption of reagent and sample introduction which matches well with cell analysis

23-26

. In order to improve method sensitivity and remove the cellular matrix effect, the

miniaturized sample pretreatment techniques prior to MicroHPLC-ICP-MS detection for the preconcentration of mercury species from cells is required. Magnetic nanoparticles (MNPs) have been widely used in microfluidic manipulation and analysis27-31. Microfluidic chip devices offer excellent spatial and temporal control over magnetic nanoparticles. In addition, magnetic field forces are stronger in a microfluidic chip than in conventional in-tube experiments because the closer proximity distance allows for large local gradients to be generated32. Microfluidic chips combined with magnetism have been applied in immunoassay29, nucleic acid analysis33, protein analysis34, cell sorting30 and so on. In our previous works, chip-based magnetic solid phase microextraction (MSPME) system was constructed by self-assembling MNPs in a magnetic zone to form a solid phase packed-column in a microfluidic chip channel. Such system was combined with electrothermal vaporization (ETV)-ICP-MS 35 and HPLC-ICP-MS36 for the analysis of trace heavy metals in HepG2 cells and selenoamino acids in selenium-enriched yeast cells. With high enrichment and low sample consumption, the number of cells for each run was reduced apparently. However, elaborate manual operations and possible contamination from exogenous reagents or employed vials still harassed us in these off-line systems. An automated chip-based on-line MSPME-ICP-MS system was developed for the analysis of trace heavy metals in our previous work37, which obviously overcame the problems associated with previous off-line systems. The purpose of this work is to build an on-line chip-based MSPME-MicroHPLC-ICP-MS system for the determination of Hg2+, MeHg+, EtHg+ and PhHg+ in cells. For this purpose, a


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microfluidic chip with integration of cell lysis unit, microextraction unit and microvalves was fabricated, and on-line combined with MicroHPLC-ICP-MS through a capillary tube and a six-way valve. The analytical performance of such on-line system was validated under the optimized conditions. Since liver is the main detoxication organ for heavy metal elements, and HepG2 cells are widely used for biochemical studies as a cell culture model of human hepatocytes due to they retain their morphology and most of their function in culture, we applied the proposed method for the speciation of mercury in HepG2 cells (incubated with HgCl2 or MeHgCl), and the obtained results is useful for understanding the cytotoxicity and cell protection mechanism of mercury.

EXPERIMENTAL SECTION Instrumentation and Reagents. An X Series II ICP-MS (Thermo Fisher Scientific, USA) and a Dionex ultimate 3000 HPLC (Dionex, Germering, Germany) were used in this work. The operation conditions for on-line MicroHPLC-ICP-MS are summarized in Table S1. Stock solutions (1 mg mL-1) of Hg2+, MeHg+, EtHg+ and PhHg+ were prepared from analytical reagent grade of HgCl2 (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China), MeHgCl (Acros, Geel, Belgium), EtHgCl and PhHgCl (Alfa Aesa, Massachusetts, USA), respectively. The information on other instruments and reagents is detailed in the Supporting Information.

Design of On-line Chip-based MSPME-MicroHPLC-ICP-MS system. The design of the microfluidic chip refers to our previous work 36 with some modifications. Figure 1(b) shows the design sketch of on-line chip-based MSPME-MicroHPLC-ICP-MS system. The photograph of the microfluidic chip is shown in Figure 1(c). The microfluidic chip integrates two cell sample pre-treatment systems together sharing the same outlet (blue line) which is connected with MicroHPLC-ICP-MS with a capillary tube. Each cell sample pretreatment system consists of a cell lysis unit (green line), an inlet of eluent (blue line), an inlet of PBS buffer (also as the inlet of MNPs, black line), a microextraction channel (black line), two “push up” controlling microvalves (red line) and an outlet (blue line). The width of the flow channels and the microvalves channels is 400 µm and 500 µm wide, respectively. All the channels are 50 µm high. In the microextraction



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zone, permanent magnets (1.0×0.5×0.2 cm, purchased from a local market, Wuhan, China) were used for the preparation of magnetic solid phase packed-column on the chip. A 50 µm ID fused silica capillary with 5 cm length was chosen to connect the outlet of the chip with the MicroHPLC-ICP-MS because it was characterized with little dead volume, good stability, large transfer efficiency, good leakproofness and easy-to-fabricate37. The fused silica capillary was plugged in a suitable PEEK tube which can easily connect with the six-way valve. Soft lithography and rapid prototyping with PDMS technology are employed for fabrication of microfluidic devices. The details on fabrication of microfluidic chips were shown in the Supporting Information.

On-line Chip-based MSPME-MicroHPLC-ICP-MS Procedure. As can be seen in Figure 1(b), two cell lysis units (green lines) and two microextraction channels (black lines) were fabricated on a

chip.

To

obtain

a

fast

analytical

speed,

a

sequential

on-line

chip-based

MSPME-MicroHPLC-ICP-MS procedure was processed. The details are listed in Table 1. From 0 to 10 min, a portion of cell sample was introduced into C1 inlet, at the same time the lysis solution was introduced into L1. The cells suffered lysis process in serpentine channel (green line) and flowed into the left microextraction channel (black line) to accomplish adsorption process (V1 was turned on to drain waste and V2, V3, V4 were turned off). From 10 to 20 min, the eluent was introduced into E1 (blue line) to accomplish desorption and the eluate was introduced into sample loop directly (the six-way valve is at LOAD state), at the same time another portion of cell sample and lysis solution were introduced into C2 and L2 inlet, respectively, to accomplish the lysis process and adsorption in the right serpentine and microextraction channel (V2, V4 turned on and V1, V3 turned off). Afterwards, the eluate in sample loop was determined by MicroHPLC-ICP-MS by switching the six-way valve to INJECT state. The separation took 20 min (from 20 to 40 min). In the first 10 min (20-30 min), the left microextraction channel was washed by PBS with only V1 turned on. From 30 to 40 min, the eluent was introduced to E2 to accomplish desorption in the right microextraction channel and the eluate was flowed into the sample loop (the six-way valve was at LOAD state), while the adsorption process for the next run was processed in system left (V1, V3 were turned on and V2, V4 were turned off) simultaneously. From 40 to 60 min, the eluate from system right was determined by MicroHPLC-ICP-MS. During


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this time, the right microextraction channel was washed by PBS (40-50 min), then the left system accomplished desorption while right system accomplished adsorption for the next run (50-60 min). Due to the limitation caused by the separation time in MicroHPLC (20 min), the sample throughput is 3 h-1. However, the on-line chip-based MSPME-MicroHPLC-ICP-MS is distinctly advantageous for cell sample analysis with the feature of integration and automation.

Preparation of the Magnetic Solid Phase Packed-column on the Chip. The magnetic Fe3O4 nanoparticles were prepared through a solvothermal reaction and then silylated by tetraethoxysilane (TEOS)32,40. The obtained Fe3O4@SiO2 nanoparticles were chemically modified with γ-mercaptopropyltrimethoxysilane (γ-MPTS). The details on preparation and characterization of Fe3O4@SiO2@γ-MPTS nanoparticles were described in the Supporting Information (see Figure S1-S3). The magnetic solid phase packed-columns on the chip were prepared by self-assembling Fe3O4@SiO2@γ-MPTS MNPs in the microﬂuidic channel under the magnetic field. The details on preparation of magnetic solid phase packed-column were shown in the Supporting Information.

Blank Controls. All the experiments were performed in a super-clean laboratory (ten-thousand class), and all the laboratory ware was made of polyethylene or Teflon material and thoroughly cleaned by soaking in 10% nitric acid for at least 24 h or disposable. The blank experiments were carried out as the method described above by using PBS buffer solution instead of the cell suspensions or the standard solutions. And all the analytical results were obtained by subtracting the blank values from the determined values.

RESULTS AND DISCUSSION On-line Chip-based MSPME-MicroHPLC-ICP-MS. The adsorption of target mercury species on the self-prepared Fe3O4@SiO2@γ-MPTS packed-column on chip is based on the high affinity between mercury species and thiol group on MNPs, therefore, sample pH plays a key role in the extraction of target mercury species. The effect of pH on the adsorption efficiency of target mercury species was studied with pH varying in the range of 2-9. As can be seen in Figure 2, a quantitative adsorption (efficiency higher than 85%) for the target mercury species was obtained in the pH range of 2-9. Due to the physiological environment in cells, pH 7.4 was selected for



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subsequent experiments. Other factors that will affect the extraction performance of chip-based MSPME, including sample flow rate and volume, eluent components and their concentrations, eluent volume and flow rate, were also systematically studied, and their detailed information is summarized in the Supporting information including Figure S4-S8. Finally, 0.1 mL sample solution was loaded with the flow rate of 10 µL min-1, while 0.01 mol L-1 HNO3 containing 4% (m/v) thiourea was employed as the eluent, and its optimal volume and flow rate are 10 µL and 1.0 µL min-1, respectively.

Interference Study. To investigate the interference of co-existing ions on the determination of target mercury species by the developed methodology, 0.1 mL solutions containing Hg2+, MeHg+, EtHg+ and PhHg+ each at 10 µg L-1 and a certain amount of foreign ions such as K+, Na+, Ca2+, Mg2+, Fe3+, Al3+, Cl-, NO3-, SO42-, HCO3- and PO43- were prepared and subjected to on-line chip-based MSPME-MicroHPLC-ICP-MS analysis. When the recoveries of the target mercury species were kept in the range of 85%-115%, the interference of the co-existing ions on target mercury species was considered as negligible. Table S2 is the tolerance limits for various co-existing ions. The tolerance folds for major cations and anions in this method is much higher than their common concentrations in human liver, indicating that this method has a good tolerance to the common co-existing ions.

Analytical Performance. The analytical performance of the developed on-line chip-based MSPME-MicroHPLC-ICP-MS system was evaluated under the optimized conditions and the results are shown in Table 2. The limits of detection were 18.8, 12.8, 17.4 and 41.8 ng L-1 for Hg2+, MeHg+, EtHg+ and PhHg+, with relative standard deviations (RSDs) of 6.4, 4.7, 3.1 and 6.7%, respectively. The enrichment factors (EFs), defined as the ratio of the calibration curve slope obtained before and after the chip-based MSPME, were found to be 9.4~9.9-fold for target mercury species. Since the theoretical EF is 10-fold, indicating that the extraction efficiency for target mercury species is higher than 94%. A comparison of the analytical performance of this method with other analytical approaches is shown in Table S3. Although the LODs of the developed method for target mercury species are relatively high, the absolute LODs (LODs (ng) = relative LODs (ng L-1) × sample volume (L)) are in the middle due to the lesser sample


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consumption in this work. Compared with other methods, the developed method consumed very small volume of sample (0.1 mL) and the microfluidic chip provided an integrated, automated analytical platform which favors the cells analysis. The

preparation

reproducibility

of

seven

prepared

Fe3O4@SiO2@γ-MPTS

packed

microcolumns on different chips was investigated and the RSDs of the extraction efficiencies for Hg2+, MeHg+, EtHg+ and PhHg+ are calculated to be 6.1, 4.8, 6.4 and 8.5% (cHg2+, MeHg+, EtHg+, PhHg+ = 2 ng mL-1, n=7), respectively. These results indicate that good preparation reproducibility was obtained for fabrication of MSPME (with Fe3O4@SiO2@γ-MPTS) chips. The regeneration of the chip-based MSPME packed-column was studied. It was found that the Fe3O4@SiO2@γ-MPTS packed columns could be regenerated by phosphate buffer solution (PBS, pH 7.4) after the whole microextraction procedure, and the chip-based MSPME packed-column (after regeneration) can be reused more than 10 times under the optimized conditions with good recoveries (102, 99.1, 99.6 and 97.8% for Hg2+, MeHg+, EtHg+ and PhHg+, respectively). The memory effect of the chip-based magnetic solid phase packed-column was also studied. And the results showed that memory effect for Hg2+, MeHg+, EtHg+ and PhHg+ was negligible (the memory signals are only 5.8, 3.6, 1.9 and 1.1% of the original signals for Hg2+, MeHg+, EtHg+ and PhHg+, respectively). The adsorption capacity of the packed Fe3O4@SiO2@γ-MPTS nanoparticles in each microextraction channel has been investigated. The results showed that the capacity of packed Fe3O4@SiO2@γ-MPTS nanoparticles for target Hg2+, MeHg+, EtHg+ and PhHg+ were 0.80, 0.73, 0.65 and 0.67 µg per channel, respectively. The accuracy of the developed method was tested by recovery experiment (four species were spiked into cell samples at the concentration of 1.0 ng mL-1). The recoveries were found to be 107, 103, 98.3 and 99.8% for Hg2+, MeHg+, EtHg+ and PhHg+, respectively. To validate the minimum cells needed for the analysis for each time in this work, HepG2 cells (provided by College of Life Sciences, Wuhan University, China) (incubated with MeHg+ at 500 µg L-1 for 24 h) with different density (800-10,000 per portion) were determined by on-line chip-based MSPME-MicroHPLC-ICP-MS. As can be seen in Table 3, the results of on-line chip-based MSPME-MicroHPLC-ICP-MS is of good consistency even with consumption of 800 HepG2 cells per sample. In other words, the proposed method is suitable for the analysis of mercury species with very small number of cells.



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Cell Analysis. HepG2 cells were used in this work. The details on sample preparation were described in the Supporting Information. Since Hg2+ and MeHg+ are the main mercury species existing in the environment, our work focus on the speciation of mercury in MeHg+ and Hg2+ incubated HepG2 cells. Cell viability was determined by using MTT assay. The details were shown in the Supporting Information, and the results in Figure S9 and S10 demonstrate that 2 mg L-1 MeHg+ and 5 mg L-1 Hg2+ induced a significant cell apoptopsis. To keep the cells viable in following incubation experiments, three concentration levels of 10, 100 and 500 µg L-1 was selected to study the mercury species in incubated cells. Speciation Analysis by On-line Chip-based MSPME-MicroHPLC-ICP-MS in Incubated Cells. The speciation of mercury in MeHg+ incubated HepG2 cells at 10, 100 and 500 µg L-1 for 12, 18 and 24 h have been conducted by on-line chip-based MSPME-MicroHPLC-ICP-MS and the results are listed in Table 4. As can be seen, Hg2+ has been found in MeHg+ incubated HepG2 cells, and the concentration of both Hg2+ and MeHg+ were increased with the increase of the incubation concentration or time, while no other two mercury species was found in all these samples. Since Hg2+ was found in MeHg+ incubated HepG2 cells, it’s necessary to rule out the possibility of that Hg2+ may come from MeHg+ decomposition in matrix, so the stability of MeHg+ in dulbecco’s modified eagle medium was studied by adding 10 µg L-1 MeHg+ in DMEM at the same environment for 24 h, and no Hg2+ was detected by chip-based MSPME-MicroHPLC-ICP-MS. It is reported that Hg2+ can be found in MeHg+ exposed monkeys18, rats19 and normal cells44 due to the demethylation of MeHg+. In this work, we found such demethylation existing in cancer cells as well. The mercury species in Hg2+ incubated HepG2 cells (10,000 cells in one portion of sample) at 10, 100 and 500 µg L-1 for 12, 18 and 24 h have also been determined by on-line chip-based MSPME-MicroHPLC-ICP-MS and the results are listed in Table 4. Only Hg2+ was found in all these cell samples, and its concentration were increased with the increase of the incubation concentration or time. HepG2 cells without incubation with MeHg+ or Hg2+ were used as negative control. It was found that only Hg2+ was found in negative control group, but the concentration was too low to be quantified. The chromatograms of mercury species standard solution, HepG2 cells, Hg2+ incubated HepG2 cells and MeHg+ incubated HepG2 cells are shown in Figure 3.


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Determination of Total Mercury by ETV-ICP-MS in Incubated Cells. The total mercury in incubated HepG2 cells was determined by ETV-ICP-MS without acid digestion (operating condition is shown in Table S4). The results are shown in Table 5. Without incubation with MeHg+ or Hg2+, the total Hg was found to be 5.5±0.2 fg per cell. With the increase of the incubation concentration or time of Hg2+ or MeHg+, the concentration of total mercury were increased. In addition, the content of total mercury in MeHg+ incubated HepG2 cells is much higher than that in Hg2+ incubated HepG2 cells, indicating that MeHg+ enters into HepG2 cells more easily than Hg2+, which is in accordance with the theory that MeHg+ is easier to penetrate the lipid bilayers and enter into the cells for better lipid solubility1-4. Such phenomenon was also found in mercury speciation as shown above. Compared the results obtained by on-line chip-based MSPME-MicroHPLC-ICP-MS system with the total mercury obtained by ETV-ICP-MS in incubated cells, it was found that the sum amount of Hg2+ and MeHg+ is only 0.34-6.6% of total mercury. Due to the extraction is based on the affinity between mercury and thiol group (shown in Figure 1(a)), we speculated that the determined Hg2+ and MeHg+ obtained by on-line chip-based MSPME-MicroHPLC-ICP-MS system may be dissociative or complexed with organic ligands (not so strong), whose concentrations are very low in the cells. Extractable Mercury Species. To figure out the extractable mercury species, the size exclusion chromatogram (SEC)-ICP-MS was used in this work, and the operating condition is shown in Table S4. The mercury species in cell lysis solution (cell incubated with 500 µg L-1 Hg2+ for 18 h) and the effluent after chip-based MSPME were analyzed with SEC-ICP-MS. The SEC-ICP-MS chromatograms are shown in Figure S11, and it can be seen that there are two main peaks for mercury in the cell lysis solution and the effluent with the retention time of 10.5 and 24.5 min (the peak at 29.0 min is weak), respectively, indicating these two main mercury fractionations were not extractable by the proposed method. The peak at 24.5 min has almost the same retention time as that of metallothionein (MT, ~24.7 min, obtained by monitoring Cu), indicating that it is contributed to mercury complexes with proteins (most likely MT). And on the basis of the principle of SEC, the peak at 10.5 min should be macro molecular mercury complex. Since all the peaks are almost the same before and after the microextraction, the results implied that the extractable species by the proposed method were dissociative mercury species (Hg2+, MeHg+,



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EtHg+ and PhHg+) and/or their weak complexes which are the minor portion of the mercury species in HepG2 cells and hard to be detected by conventional SEC-ICP-MS. Comprehensive Speciation of Mercury in Incubated Cells. The developed method demonstrates a portion of mercury species in the incubated cells but we are also interested in other species. To know more about them, the cells incubated with Hg2+ or MeHg+ at three different incubation concentrations (10, 100 and 500 µg L-1) for 12, 18 and 24 h were determined by SEC-ICP-MS and reversed phase (RP)-HPLC-ICP-MS. The results are detailed in the Supporting Information and the chromatograms are shown in Figure S12. From the results, Hg2+ and MeHg+ entered in the HepG2 cells shows different behaviors, and following items can be concluded: 1) Compared with Hg2+ incubated HepG2 cells, macro molecular mercury complexes was more likely accumulated in MeHg+ incubated HepG2 cells along with the increase of the incubation concentration and time; 2) The complex of MT and Hg2+/MeHg+ is the main mercury species in the cells incubated with Hg2+, but not in the cells incubated with MeHg+, demonstrating the significance of MT in the protection of cells against Hg2+ toxicity; 3) Complexation with cysteine (Cys) is the mainly mechanism for cell protection to MeHg+ toxicity especially in a relatively low incubation concentration, and the existence of both Cys-Hg and Cys-MeHg complex in MeHg+ incubated HepG2 cells further proves the demethylation of MeHg+ in HepG2 cells.

CONCLUSION In this work, an on-line chip-based MSPME-MicroHPLC-ICP-MS design concept was proposed. With a chip that integrated cell lysis unit, microextraction unit and microvalves combined with a six-way valve on MicroHPLC-ICP-MS, the on-line system was constructed. The developed method is featured with low sample/reagent consumption, good reproducibility, high integration, high accuracy, high sensitivity and has a good application potential for cell sample analysis. The method was used for speciation of mercury in Hg2+ or MeHg+ incubated HepG2 cells, and the demethylation of MeHg+ in HepG2 cells was found. It was found that more mercury accumulated in MeHg+ incubated HepG2 cells than Hg2+ incubated HepG2 cells and MeHg+ demethylation exists in HepG2 cells. Besides, this work also demonstrated the difference of cell protection mechanism in HepG2 cells to Hg2+ and MeHg+ toxicity.


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SUPPORTING INFORMATION This material is available free of charge via the Internet at http://pubs.acs.org. Additional information on Instrumentation and Reagents, fabrication of PDMS microfluidic devices, preparation of Fe3O4@SiO2@γ-MPTS, characterization of Fe3O4@SiO2@γ-MPTS nanoparticles (including Figure S1-S3), preparation of magnetic solid phase packed-column on the chip, On-line Chip-based MSPME-MicroHPLC-ICP-MS (Figure S4-S8), sample preparation, cell viability assay, effect of the incubated concentration of Hg2+ (Figure S9) and MeHg+ (Figure S10) on cell apoptosis, SEC-ICP-MS chromatograms (Figure S11), Comprehensive Speciation of Mercury in Incubated Cells (Figure S12), operating conditions for on-line MicroHPLC-ICP-MS (Table S1), and tolerance folds of coexisting ions to the target analytes (Table S2), Comparison of analytical performance (Table S3), and operating conditions for ETV-ICP-MS and SEC separation (Table S4) as noted in the text.

ACKNOWLEDGMENT This work is financially supported by the National Basic Research Program of China (973 Program, 2013CB933900), the National Nature Science Foundation of China (Nos. 21375097, 21175102, 21205090), the Science Fund for Creative Research Groups of NSFC (Nos. 20621502, 20921062), and the Fundamental Research Funds for the Central Universities (2015203020203), the MOE of China.



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Figure captions Figure 1. Diagram of magnetic packed column, microfluidic chip and on-line chip-based MSPME-MicroHPLC-ICP-MS system. (a) microextraction principle in the magnetic packed column; (b) design sketch of on-line chip-based MSPME-MicroHPLC-ICP-MS system: the chip consists of cell lysis unit (green lines), microextraction unit (black lines) and microvalves (red lines), the blue lines stand for the elution channels; C1 and C2 are the inlets of cell sample, L1 and L2 are the inlet of lysate solution, E1 and E2 are the inlets of eluent , B1 and B2 are the inlet of Fe3O4@SiO2@γ-MPTS and buffer solution, A is the outlet of eluent and connected with six-way valve by a capillary tube directly, W1 and W2 are the outlets of waste and V1, V2, V3, V4 are the gas inlets of microvalves. The width of microextraction channels is 400 µm and the microvalves channels are 500 µm wide, all the channels are 50 µm high; (c) photograph of the microfluidic chip. Figure 2. Effect of pH on the extraction of Hg2+, MeHg+, EtHg+ and PhHg+. (cHg2+, MeHg+, EtHg+. + PhHg =

10 µg L-1; sample volume: 0.1 mL; sampling flow rate: 2 µL min-1)

Figure 3. The chromatograms of mercury standard solution, HepG2 cells, Hg2+ incubated HepG2 cells and MeHg+ incubated HepG2 cells after on-line chip-based MSPME-MicroHPLC-ICP-MS.



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Figure 1


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2+

Hg + MeHg + EtHg + PhHg

100

80

Adsorption rate(%)

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60

40

20

0 1

2

3

4

5

6

7

8

pH

Figure 2


9

10


HepG2 cells 2+ Hg incubated HepG2 cells + MeHg incubated HepG2 cells Standard solution

14000 12000

2+

Hg

10000 +

8000

MeHg

EtHg

+

6000

+

PhHg

4000

CPS

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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2000

0 0

2

4

6

8

10

12

14

16

18

20

Time (min)

Figure 3


22

24

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Table 1 Procedure of chip-based MSPME-on-line-MicroHPLC-ICP-MS analytical system Time (min)

Operation

Valve control

0-10

Flow introduction: C1, L1 (system left: adsorption)

V1 turn on, V2, V3, V4 turn off Six-way valve: LOAD

10-20

Flow introduction: E1, C2, L2 (system left: desorption, system right: adsorption)

V2, V4 turn on V1, V3 turn off, Six-way valve: LOAD

20-30

Flow introduction: B1 (system left: wash)

V1 turn on, V2, V3, V4 turn off Six-way valve: INJECT

30-40

Flow introduction: E2, C1, L1 (system right: desorption, system left: adsorption)

V1, V3 turn on, V2, V4 turn off Six-way valve: LOAD

40-50

Flow introduction: B2 (system right: wash)

V4 turn on, V1, V2, V3 turn off Six-way valve: INJECT

50-60

Flow introduction: E1, C2, L2 (system left: desorption, system right: adsorption)

V2, V4 turn on, V1, V3 turn off Six-way valve: LOAD



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Table 2 Analytical performance for target analytes obtained by on-line chip-based MSPMEMicroHPLC-ICP-MS LOD

Linear range Linear equation

R2

RSD

(ng mL-1)

Hg2+

18.8

0.05-20

y=119581x+5463

0.9948

6.4

9.5

MeHg+

12.8

0.05-20

y=130458x+5017

0.9942

4.7

9.9

EtHg+

17.4

0.05-20

y=95427x+1957

0.9977

3.1

9.4

PhHg+

41.8

0.2-20

y=10825x+1101

0.9943

6.7

9.6

a：cHg2+, MeHg+, EtHg+, PhHg+=0.25 ng mL-1


(n=7, %)a

EF

(ng L-1)

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Table 3 Analytical results (mean±s.d., n=3) for mercury speciation in MeHg+ incubated HepG2 cells (500 µg L-1, 24 h) Cell number

Hg2+ (fg/cell)

MeHg+ (fg/cell)

10000

3.7±0.1

16.3±0.4

8000

3.1±0.1

15.3±0.2

5000

3.4±0.3

15.6±0.3

2500

4.1±0.2

16.2±0.5

1500

-

16.4±0.5

800

-

17.3±0.7



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Table 4 Analytical results (mean±s.d., n=3) for determination of mercury species in incubated HepG2 cells (10,000 cells) Incubation species / concentration (µg L-1)

Determined Incubation

MeHg+

Hg2+

MeHg+

Hg2+

MeHg+

Hg2+

(fg/cell)

/10

/10

/100

/100

/500

/500

Hg2+

NQa

0.53±0.01

1.2±0.2

1.0±0.1

1.7±0.1

1.9±0.1

2.5±0.1

NDb

6.5±0.4

ND

13±1

ND

0.8±0.1

0.85±0.04

1.7±0.1

1.7±0.1

2.2±0.1

6.5±0.2

3.2±0.2

ND

8.2±0.6

ND

14±1

ND

1.2±0.1

1.1±0.1

2.6±0.1

5.4±0.2

3.7±0.1

13±1

4.7±0.2

ND

10±1

ND

16±1

ND

results time (h)

MeHg+

12

Hg2+ MeHg+

18

Hg2+ MeHg+

24

a: not quantified; b: not detected


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Table 5 Analytical results (mean±s.d., n=3) for determination of total mercury in incubated HepG2 cells (10,000 cells) Incubation species / concentration (µg L-1) Incubation

Hg2+

MeHg+

Hg2+

MeHg+

Hg2+

MeHg+

/10

/10

/100

/100

/500

/500

12

8.0±0.2

64±2

71±7

568±89

562±37

2336±33

18

19±3

71±6

157±7

641±35

907±10

3348±149

24

21±1

112±7

228±34

824±53

1170±48

3704±111

time (h)

Total Hg (fg/cell)



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for TOC only


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Chip-Based Magnetic Solid-Phase Microextraction Online Coupled

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