Facile Chip-Based Array Monolithic Microextraction System Online

Article

A facile chip-based array monolithic microextraction system online coupled with ICPMS for fast analysis of trace heavy metals in biological samples Jing Zhang, Beibei Chen, Han Wang, Man He, and Bin Hu Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 25 May 2017 Downloaded from http://pubs.acs.org on May 29, 2017

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Analytical Chemistry

A facile chip-based array monolithic microextraction system online coupled with ICPMS for fast analysis of trace heavy metals in biological samples Jing Zhang, Beibei Chen, Han Wang, Man He, Bin Hu* Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, P R China Abstract Trace heavy metals have great impact on biological system, therefore, it is essential to develop suitable analytical methods for the determination of trace heavy metals in biological samples to elucidate their biochemical and physiological functions in organisms. Herein, we presented a chip-based array monolithic microextraction system and combined it with inductively coupled plasma mass spectrometry (ICPMS) for online analysis of trace Hg, Pb and Bi in real-world

biological

methacrylate-co-ethylene

samples. glycol

Six

ethylenediamine

dimethacrylate

(poly

modified

poly

(GMA-co-EDMA-NH2))

glycidyl capillary

monolithic columns were embedded parallelly in microchannels of a microfluidic chip for array monolithic microextraction. Various parameters affecting the chip-based array monolithic microextraction of target metals were investigated. The sample throughput of the proposed method was 16 h-1, with the limits of detection for Hg, Pb and Bi of 23, 12 and 13 ng L-1, respectively. The developed method was validated by the determination of trace Hg, Pb and Bi in HepG2 cells and human urine samples, and the recoveries for the spiked samples were in the range of 90.4-102%. This chip-based array monolithic microextraction system is easy to prepare, and the proposed online analytical system provides a new platform for trace elements analysis in biological samples with the merits of high sample throughput, high sensitivity and low sample/reagents consumption.

Keywords: microfluidic chips; array monolithic microextraction; online ICPMS analysis; trace elements; biological samples *

Corresponding author: Tel: 86-27-68752162, Fax: 86-27-68754067; email: [email protected] 1

ACS Paragon Plus Environment


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1. Introduction Continuously extensive use of heavy metals in various fields1,2 leads to prolonged exposure risks to human beings. Researches have shown that excessive heavy metals would induce side-effect on physiological functions and may even lead to the dysregulation of cellular pathways3,4. To investigate the mechanism of heavy metals in organism, it is important to develop analytical methods with high sensitivity, throughput and accuracy for trace heavy metals analysis in biological samples. Inductively coupled plasma mass spectrometry (ICPMS), as an elemental specific detection technique, has the advantages of low limits of detection (LODs), wide linear range, multielement and isotopes detection ability, etc. Thus, it has been widely used for the determination of trace elements in various samples. However, ICPMS still faces several shortcomings in the direct analysis of biological samples: complicated sample matrix may influence the accuracy of analysis results; the concentration of target trace/ultra-trace elements in biological samples may be even lower than the LODs of ICPMS; the most commonly used pneumatic nebulization (PN) ICPMS which demands sample consumption at sub-millilitre level which is hard to meet the requirement of the precious and limited biological samples analysis5. The last one can be resolved by using advanced miniaturized nebulizers such as PFA series microflow nebulizers (20~400 µL min-1, Elemental Scientific, NE, USA) and DS-5 microflow nebulizer (3~10 µL min-1, Cetac Technologies, NE, USA). While miniaturized sample pretreatment techniques prior to ICPMS detection have been proven to be effective to overcome the former two problems. Capillary monolithic microextraction, as an in-tube microextraction technique6, has multiple merits of high extraction efficiency, high mass transfer rate, low sample/reagent consumption, easy to online couple to other analytical techniques, etc. The dual-model porous structure of the monolithic column provides lots of pathways for the flow liquid resulting in low back pressure7, and the large specific surface area with abundant functional groups is beneficial for extraction. Therefore, capillary monolithic microextraction has been successfully used for the analysis of organic compounds 8,9, biomolecules10,11 and trace metals12-15. Liu et al.13 prepared a poly glycidyl methacrylate-co-ethylene glycol dimethacrylate (poly (GMA-co-EDMA)) capillary monolithic 2


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column with sulfydryl functional groups and developed an online capillary monolith microextraction-ICPMS method for the determination of trace Au and Pb in human urine and serum, the LODs were 5.8-8.3 ng L-1. The same authors also modified poly(methacrylic acid (MAA)-co-EDMA) capillary monolithic column with TiO2 nanoparticles14 and established a new method of online capillary monolithic microextraction-ICPMS for the analysis of free Gd3+ and Gd-based contrast agent in human urine with LODs of 3.2-4.5 ng L-1. Zhang et al.15 prepared aminopropyltriethoxysilane (APTES)-silica hybrid monolithic capillary and coupled it with ICPMS for online detection of trace metals in human urine and hair. All of the above works reveal that monolithic microextraction is a proper sample pretreatment technique with low sample consumption at sub millilitre level. However, when very limited biological samples such as cell samples are analyzed, more miniaturized and integrated sample pretreatment techniques are required. Microfluidic chips have numerous advantages including high integration, miniaturization, low cost, and easy to fabricate16. As a phenomenal functional platform, it is increasingly prevalent in the analysis of biological samples such as genes17, proteins18 and cells19. In our previous works, we have integrated different microextraction techniques such as liquid phase microextraction (LPME)20 and magnetic solid phase microextraction (MSPME)21-24 on microfluidic devices, and combined them with ICPMS and ICPMS based hyphenated techniques for trace elements and their species analysis in cell samples. Monolithic material is a promising alternative to be integrated in microfluidic chips, and chip-based monolithic microextraction can combine the merits of monolithic capillary microextraction and microfluidic chips. This technique does not need any additional chip design or external magnet to integrate monolith on chip, because the monolithic column can be either in situ polymerized in microchannel25 or embedded in the microchip (so called capillary assembled microchip (Cas-CHIP)26). Up to now, many attempts have been made in this area27, but reports focused on the chip-based monolithic microextraction for trace elements analysis in biological samples are rare. In 2014, Li et al.28 integrated poly (3-aminopropyl) triethoxy silane-co-ethylsilicate capillary monolithic columns into a microchip for speciation of Cr(III) and Cr(VI) in water. With a centrifugal motor, 8 samples can be conducted within 10 min. 3



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Although the time for sample pretreatment was reduced, a special chip design was required and the fabricated chip could not be further developed into an online analytical system. Very recently, we in situ prepared a polymer based monolith in chip channel and modified this polymer based monolith with ethanediamine for the analysis of Bi in cell samples followed by ICPMS detection29. The method has the merits of low sample/ reagent consumption, high extraction efficiency, good reproducibility and good anti-interference ability, but the analytical time is relatively long (ca. 1 h for each sample). An online chip-ICPMS system can avoid problems of the offline system, for example, laborious manual operations, possible contamination and sample loss. In addition, constructing online array microfluidic platform would offer higher throughput and better reproducibility22. Therefore, the purpose of this research is to develop a method of chip-based array monolithic microextraction online coupled with microconcentric nebulizer (MCN)-ICPMS for high throughput analysis of trace heavy metals in biological samples. For this purpose, poly glycidyl methacrylate-co-ethylene

glycol

dimethacrylate

monolithic

capillaries

modified

with

ethylenediamine (poly (GMA-co-EDMA-NH2)) were prepared, and a chip-based array monolithic microextraction system was fabricated by integrating six monolithic capillaries in one microfluidic chip. The experimental conditions for the extraction of Hg, Pb and Bi by online chip-based array monolithic microextractionwere carefully optimized, and the analytical performance of the established online chip-based array monolithic microextraction-ICPMS system was evaluated. The developed method was finally applied to the determination of trace Hg, Pb and Bi in cell and human urine samples for validation.

2. Experiment section 2.1 Apparatus A Thermo Fisher X Series II MCN-ICPMS (Thermo Fisher Scientific, USA) was used to determine target metals. The operating conditions for online ICPMS detection are summarized in Table S1. A KW-4A spin coater (Siyouyen Electronic Technology Co., Ltd, Beijing, China) and a PDC-M plasma cleaner (Mingheng Science and Technology Development Co., Ltd, Chengdu, 4


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China) were used for fabrication of microfluidic chips. TS2-60 syringe pumps (Baoding Longer Precision Pump Co., Ltd, Baoding, China) and sterile syringes were used to inject liquid into the microchannels of the microfluidic chips. Other instruments involved in the experiments include an XS105 balance for weighing and a 320-s pH meter for pH controlling (Mettler Toledo Instruments Co., Ltd, Shanghai, China). Fused silica capillary (520 µm i.d. × 630 µm o.d.) was purchased from Yongnian Optical Fiber Factory (Hebei, China). The prepared capillary monolithic column was characterized by Fourier transform infrared spectroscopy -3600 (FT-IR, Thermo, Madison, USA) and an X-650 scanning electron microscope (SEM, HITACHI, Japan).

2.2 Reagents and standard solutions Stock solutions (1 mg mL-1) of Hg, Pb and Bi were prepared by dissolving certain amount of HgCl2, Pb(NO3)2 and Bi(NO3)3 (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) in 2% (v/v) sub-boiled HNO3, respectively. 10 mmol L-1 phosphate-buffered saline (pH 7) was used as phosphate buffer solution (PBS). The oligomers (GE RTV 615, Component A) and crosslinking agents (GE RTV 615, Component B) were purchased from Momentive Performance Materials (NY, USA) to prepared polydimethylsiloxane (PDMS) based microfluidic chip. Glycidyl methacrylate (GMA), ethylene glycol dimethacrylate (EDMA) and azobisisobutyronitrile (AIBN) were obtained from Alfa Aesar Reagent Co., Ltd (Tianjin, China). Dodecanol and cyclohexanol were obtained from Aladdin Reagent Co., Ltd (Shanghai, China). All the reagents used were of at least analytical reagent grade without other statement. High purity deionized water was supplied by Milli-Q system (18.2 MΩ cm, Millipore, Molsheim, France). HepG2 cells were obtained from College of Life Sciences, Wuhan University (Wuhan, China). Human urine samples were provided by Zhongnan Hospital (Wuhan, China). The ethics committee reviewed and approved the informed consent forms provided by all participants according to ethics requirements.

2.3 Synthesis of poly (GMA-co-EDMA-NH2) capillary monolithic column Before the synthesis, the capillary (520 µm i.d. × 630 µm o.d.) was activated by 1 mol L-1 NaOH solution for 2 h, and then washed to neutral with hydrochloric acid and high purity 5



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deionized water. The activated capillary was dried with nitrogen and kept under 160 oC for 5 h. Poly (GMA-co-EDMA) was prepared via thermal-initiated polymerization30. Briefly, GMA (140 mg), EDMA (120 mg), dodecanol (70 mg), cyclohexanol (630 mg) and AIBN (9.6 mg) were blended well in an Eppendorf tube and sonicated for 10 min to obtain a homogeneous solution. The activated capillary was filled with the solution and heated in water bath at 65 oC for 8 h with both ends sealed. After the polymerization, the column was washed by plenty of ethanol and water to remove unreacted reagents and pore-forming agents. Then the capillary monolithic column was modified with 1 mol L-1 ethylenediamine aqueous solution at 80 oC for 4 h. After washed to neutral, the column was cut into 1 cm length segments prior to use.

2.4 Design and fabrication of microfluidic devices In this work, Cas-CHIP strategy26 was used to embed capillary monolithic segment in microchannel, and dual layer PDMS microfluidic chip with upper flow channels for parallel extraction and lower gas valve channels for programming controlling was designed to construct chip-based array monolithic microextraction system according to our previous report22 with some modifications. The layout and the photograph of the chip-based array monolithic microextraction system are shown in Figure 1 (a) and (b), respectively. The chip array system consists of six capillary monolithic columns (M1-M6) for parallel extraction, ten controlling microvavles (V1-V10), ten inlets (I1-I10), six outlets (W1-W6) for waste, and an outlet (CN) for eluent introduction into MCN-ICPMS for online detection. The width is 500 µm for microextraction channel and 800 µm for microvalve channel, and the cross section of microvalve channel was 1000 µm wide. All the channels except the yellow parts are 50 µm high. The microextraction channels, which have been marked in yellow in the blueprint, are 650 µm high and 650 µm wide in order to fit the size of capillary monolithic column (520 µm i.d. × 630 µm o.d., 1 cm length). On the chip, I1-I6 are the inlets for sample solution and elution solution, while W1-W6 are the outlets for waste solution. I7 and I8 are the inlets for the introduction of 5% (v/v) nitric acid, and I9 and I10 are the inlets of N2 gas. V1-V10 are the inlets of N2 gas for “push-up” gas microvalves. M1-M6 represent the capillary monolithic columns which were embedded into the chip channels. 6


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CN is the outlet which is connected to the MCN of ICPMS for subsequent detection. Since the introduction plastic tube of MCN cannot be directly inserted into the outlet CN with good leakproofness, a stainless steel capillary (400 µm i.d. × 640 µm o.d.) was used as the connector with the length of 1.5 cm. One end of the stainless steel capillary was punched into the outlet CN and the other end was inserted in the introduction tubing of MCN. Double layer PDMS based microfluidic chip with gas valves was prepared as follows. A transparent patterned mask was used to make a master on a silicon wafer with AZ-50XT photoresist. For the upper thick layer with flow channels, square quartz stick (1 cm long, 650 µm high and 650 µm wide) was used as the template for embedding capillary monolithic column. Six square quartz sticks were fixed in the location for microextraction channels (as shown in the yellow parts M1-M6 in Figure 1 (a)), and then the flow channels were fabricated by casting the mixture of PDMS oligomers and crosslinking agents of 10:1 (w/w) on the master. After being degassed, the PDMS sol was cured at 75 oC for 4 h. Then the solidified PDMS was peeled off and drilled on demand, and six prepared poly (GMA-EDMA-NH2) capillary monolithic column (1 cm long) was put into the microextraction channels. The gap between the capillary monolithic column and the chip channel was filled with a proper amount of liquid PDMS mixture (10:1 w/w) followed by curing at 105 oC for 30 min. The SEM of a typical capillary monolithic column embedded into the flow channel is shown in Figure 1 (c). For the lower thin layer with controlling channels, a mixture of PDMS (15:1 w/w) was prepared and degassed for 30 min. Afterwards, the PDMS gel was spin-coated onto the master at 800 rpm for 30 s and then solidified at 75 oC for 30 min. Then, two layers were treated with oxygen plasma for 2 min, and immediately irreversibly bonded at 75oC for 30 min. After the ensemble was peeled off and the holes (0.7 mm i.d.) for valves were frilled, the ensemble and a clean glass were exposed to oxygen plasma and then bonded together.

2.5 Chip-based array capillary monolithic microextraction procedure The developed chip-based array capillary monolithic microextraction system allows parallel analysis of six samples. The operating procedure for chip-based array capillary monolithic 7



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microextraction is listed in Table 1. Specifically, six portions of sample solution were simultaneously pumped into six array microextraction channels with the flow rate of 25 µL min-1 for 600 s through the inlets I1-I6, respectively. During the adsorption process, the microvalve V4 and V8 were open and the other valves were off, the liquid would flow out through W1-W6. After sample loading, V4, V8, V9 and V10 was turned off while V1 was opened, the elution solution was pumped into capillary monolithic column M1 through inlet I1 at 25 µL min-1 for 48 s and the eluent was injected into ICPMS through MCN directly. Then, V1 was turned off and V9 was opened, 5% nitric acid was injected from I7 and I8 to wash the system for 60 s. Later on, V9 was off and V10 was opened, it allowed the introduction of N2 gas into chip channel through I9 and I10 with the gas flow rate of 0.1 L min-1 for 10 s to separate the liquid. Then the elution and washing process for M1 extraction channel was completed. The whole elution and washing process was then repeated for another five monolithic columns in turn. A MATLAB interface (Matlab, The Maths Works Inc., USA) and a pressure controller as described in our previous works

21, 22

were used to control the opening/closure of different gas valves and realize the

chip-based array capillary monolithic microextraction.

2.6 Blank controls All the experiments were performed in a ten-thousand class super-clean room. The polyethylene or PTFE wares were used in all experiments. Before use, all the wares and vials were soaked in 10% nitric acid for 24 h and washed with high purity deionized water. For blank experiments, PBS buffer were used as sample solution. All the experiments were repeated for three times without other statement and the present data were the average results for the triplicate analysis by subtracting the blank values.

2.7 Samples treatment HepG2 cells and human urine were utilized as real-world biological samples. The HepG2 cells were collected and washed by PBS buffer (pH=7.0) for three times, and the density of HepG2 cells was diluted to 3×105 cell mL-1 with PBS buffer. 1 mL of the cell suspension solution 8


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was ultra-sonicated for 30 min, and then centrifuged at 12,000 rpm for 4 min. The upper cell lysate solution was collected for subsequent chip-based array monolithic microextraction-ICPMS online analysis. 1 mL human urine sample was diluted with sub-boiled nitric acid (4 mol L-1) to 3.5 mL. After 4 h standing, the urine samples were adjusted to pH 7.0 with NaOH solution (10 mol L-1) and diluted to 5 mL with PBS buffer (pH=7.0) prior to use.

3. Results and discussion 3.1 Design of online chip-based array monolithic microextraction-ICPMS system There are two main ways to integrate the monolithic microextraction into the microchip platform including in-situ synthesis of monolithic column in the chip channel25 and embedding capillary monolithic column in microfluidic devices26. Compared with in situ synthetic method, preparing the monolithic material in fused silica capillary is more sophisticated. The main points in embedding capillary monolithic column into a microchip are to promote leakproofness and downsize dead volume. In this work, a square quartz stick (1.0 cm long, 650 µm high and 650 µm wide) was used as the template, because it was much easier than cylinder to be fixed on the silicon wafer for PDMS chip fabrication. When a cylindrical capillary monolithic column (630 µm o.d., 1 cm length) was embedded into the microextraction channel, the gap between the monolithic column and the chip channel was filled with PDMS glue. Figure 1 (c) shows the SEM of a typical capillary monolithic column embedded into the chip. As clearly shown, there is no obvious gap between the capillary and the chip channel, and the microstructure of capillary monolithic column is well preserved. The length of the capillary monolithic columns was set as 1 cm, providing sufficient adsorption capacity and the easy operation at the same time. With the semi-automatic controlling microvalves system, ten gas paths can be controlled at the same time. This online chip array monolithic microextraction-ICPMS system is featured with high integration and sample throughput.

3.2 Characterization of poly(GMA-EDMA-NH2) capillary monolithic column The morphology of poly (GMA-co-EDMA) materials were characterized by SEM. As shown 9



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in Figure S1a, the prepared monolithic column was attached well to the inner wall of the capillary, and no shrinkage or crack was observed. As shown in Figure S1b (magnification of 5000×), abundant macrospores and flow-through channels were observed on the monolithic column, which offered large surface area and low back pressure. Besides, the prepared poly (GMA-co-EDMA) monolith with and without ethylenediamine modificationwas also characterized by FT-IR . Compared with the FT-IR spectrum of poly (GMA-co-EDMA) shown in Figure S2a, the N-H stretching vibrations at ~3100 cm-1and ~1650 cm-1 found in the spectrum of poly (GMA-co-EDMA-NH2) (Figure S2b) indicates that the modification of ethylenediamine was successful.

3.3 Optimization of microextraction conditions 3.3.1 Effect of pH The effect of pH (from 3 to 10) on the adsorption percentage of target metals on the prepared monolithic column was investigated with the sample volume of 250 µL at a loading rate of 0.15 mL min-1. As shown in Figure 2, the adsorption percentages of three target metals increased sharply to higher than 90% when the pH increased from 3 to 5. With further increasing pH from 5 to 10, their adsorption percentages remained constant. The high adsorption efficiency is due to the affinity of amino groups on the surface of monolithic column towards target metals. Since the physiological environment is usually neutral, pH 7.0 was selected for the subsequent experiments.

3.3.2 Effect of sample loading rate Considering that higher loading rate will accelerate the wastage of the microfluidic chips, the influence of sample loading rate in the range of 10~30 µL min-1 on the adsorption of target metals was studied with the sample volume and pH fixed as 250 µL and pH 7, respectively. As can be seen from Figure S3, the adsorption percentages higher than 90% were obtained for the target elements in the entire studied sample loading rate range, indicating that the monolithic column has relatively fast extraction kinetics. In the following experiments, 25 µL min-1 was used for the subsequent experiments. 10


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3.3.3 Effect of the elution conditions The experimental results obtained by the study of influence of sample pH on adsorption percentage as described in 3.3.1 demonstrated that low pH would lead to low adsorption percentage for target analytes, and reagents with thiol group would help to desorb the target analytes from monolithic material according to Lewis acid-base theory. Therefore, the mixed solution of nitric acid and thiourea was employed as the elution solution to destroy the strong affinity between target metals and amino groups. The extraction conditions for the study the effect of the elution conditions were fixed as follows: sample loading rate of 25 µL min-1; loading pH 7.0, sample volume of 250 µL, and eluent flow rate of 25 µL min-1. The influence of nitric acid concentration varying from 0.2 to 1 mol L-1 on the desorption efficiency was investigated with the thiourea concentration of 0.5% (m/v). As can be seen from Figure 3(a), a complete elution for all three target metals was obtained when the concentration of nitric acid was higher than 0.2 mol L-1. Therefore, 0.5 mol L-1 nitric acid was chosen for the elution of target metals. Then the influence of thiourea concentration ranging from 0 to 3% (m/v) on the desorption was studied with the nitric acid concentration at 0.5 mol L-1. The results in Figure 3(b) indicated that the concentration of thiourea had little effect on the desorption efficiency. Finally, 0.5 mol L-1 nitric acid containing 2% (m/v) thiourea were used as the elution solution to elute the target elements from the monolithic column. The eluent volume was also optimized. For this purpose, triplicates of 20 µL of 0.5 mol L-1 nitric acid containing 2% (m/v) thiourea was used to continuously elute the target elements from the monolithic column. As shown in Figure S4, the first portion is enough for the quantitative elution of all three target elements. Consequently, the elution volume was fixed at 20 µL.

3.4 Effect of sample volume For studying the effect of the sample volume on the adsorption of three target elements, 100, 250, 500 750, 1000 µL of sample solutions containing target metals each at 10 ng (pH 7) were passed through the prepared monolithic capillary embedded in microchip with 25 µL min-1 sample 11



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loading rate. The results in Figure S5 show that all three target metals can be quantitatively adsorbed on the monolithic column within the studied sample volume range. With the sample volume as 1000 µL and eluent volume as 20 µL, a theoretical enrichment factor of 50-fold can be achieved. However, considering the analytical speed and limited amount of real-world biological samples such as cells, 250 µL of sample solution was applied in real-world sample analysis, and the theoretical enrichment factor was 12.5-fold.

3.5 Effect of foreign ions Under the optimized condition, the effect of foreign ions on the extraction efficiency of target elements was investigated with 250 µL sample solution containing target metals each at 10 µg L-1 and a certain amount of different foreign ions. The studied foreign ions including K+, Na+, Ca2+, Mg2+, Cu2+, Zn2+, Fe3+, Al3+, Cl-, NO3-, SO42- are common coexisting ions in cell and human urine matrix. The interference of the foreign ions was considered to be negligible if the recoveries of the target metals were in the range of 85-115%. The tolerance limits for these foreign ions are listed in Table 2. As can be seen, the investigated foreign ions have no obvious effect on the recoveries of target metals. This indicates a good tolerance of the prepared poly (GMA-co-EDMA-NH2) monolithic column to common foreign ions.

3.6 Analytical performance In summary, the optimal microextraction conditions are as follows: 250 µL sample solutions (pH 7.0) were pumped into six microextraction channels parallelly at 25 µL min-1 for adsorption; then 20 µL of 0.5 mol L-1 nitric acid containing 2% (m/v) thiourea was used to elute the target metals with the flow rate of 25 µL min-1. Under the optimized conditions, the adsorption time was 600 s for six parallel samples, and the desorption time was 118 s (48 s for elution, 60 s for washing and 10 s for break) for each sample, so the sample throughput of about 16 h-1is obtained. Table 3 is the analytical performance of the developed method. The enrichment factor was 12.5 folds, and the limits of detection (LODs, 3σ) of Hg, Pb and Bi were 23, 12 and 13 ng L-1 with the relative standard deviations (RSDs) of 3.3, 12


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3.7 and 2.3% (cHg=0.20 µg L-1, cPb=0.12 µg L-1, cBi=0.13 µg L-1, n=9), respectively. The linear range was from 0.05 to 20 µg L-1 (r2>0.99) for all three target elements. The comparison of this method with other similar analytical methods15,21,22,29 is summarized in Table 4. As can be seen, the LODs of the proposed method are comparable with other analytical methods. The low consumption of sample/reagents makes the developed method very suitable for cell analysis. Compared with other chip-based analytical methods, the online chip-based array system has a relatively high throughput of 16 h-1, and avoids the possibilities of contamination and manual operation errors.

3.7 Evaluation of the performance of the prepared chip-based array capillary monolithic column The regeneration of the chip-based array capillary monolithic column was studied. The system can be easily regenerated by using PBS (pH 7.0) to wash the microextraction system. The chip-based array capillary monolithic microextraction system could be reused for more than 25 times with the extraction efficiency of all three target metals remained above 85%. The adsorption capacity of the chip-based array capillary monolithic column was also investigated. 80 ng mL-1 of Hg, Pb or Bi sample solution (one element at a time) was continuously injected into the monolithic column embedded in chip with the flow rate of 25 µL min-1. The maximum adsorption capacity was evaluated from the breakthrough curve. The obtained adsorption capacity of Hg, Pb and Bi was 188, 141 and 164 µg m-1, respectively. Since the capillary monolithic columns were synthesized outside the chip, the quality control of the material is simple and precise. The reproducibility of the capillary monolithic column (segments prepared in one batch and from different batches) embedded in chip was studied as well. For this purpose, the extraction efficiencies of eleven segments of monolithic capillary prepared in the same beach and seven segments of monolithic capillary prepared among different batches were investigated under the optimized conditions. It was found that the relative standard deviations (RSDs) were 3.6, 3.3 and 2.0% for Hg, Pb and Bi in one batch (n=11), and 4.0, 5.8 and 3.4% for Hg, Pb and Bi among different batches (n=7), demonstrating a good preparation reproducibility of 13



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the monolithic column.

3.8 Determination of Hg, Pb and Bi in real-world biological samples External calibration employing standard solutions instead of sample solution was used for the quantification analysis of Hg, Pb and Bi in HepG2 cell lysates and human urine samples. The analytical results are listed in Table 5. For HepG2 cells, Hg, Pb and Bi were detected to be 0.51, 0.44 and 0.34 fg per cell, respectively, with the cell consumption number of 30,000 for each analysis. These results are consistent with those obtained in our previous works22. In the healthy human urine samples, none of these three elements were detected. To validate the analysis accuracy, recovery test was conducted for these two biological samples. As can be seen in Table 5, the recoveries ranging from 90.4 to 98.1% were obtained for cells lysates with the spiking concentration of 0.06 µg L-1 for Hg, Pb and Bi; the recoveries in the range of 91.4~102% were obtained for human urine sample at two spiking levels of 0.08 µg L-1 and 5 µg L-1. These results indicate that the proposed method is suitable for the analysis of Hg, Pb and Bi in cells and urine samples.

4. Conclusions In this work, a chip-based array monolithic microextraction system was constructed by embedding six poly (GMA-co-EDMA-NH2) monolithic capillaries in one microfluidic chip. A novel online chip-based array monolithic microextraction-ICPMS method was developed for high throughput determination of trace heavy metals in different biological samples. The developed chip-based array monolithic microextraction system is easy to fabricate. The proposed method possesses the advantages of high sensitivity and sample throughput, good reproducibility and anti-interference ability, and low sample/reagent consumption, which exhibits a great potential in clinical applications.

Acknowledgement This work is financially supported by the National Nature Science Foundation of China (Nos 21575107, 21575108, 21375097, 21175102, 21205090), the National Basic Research Program of 14


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China (973 Program, 2013CB933900), the Science Fund for Creative Research Groups of NSFC (No. 20921062).

Supporting Information Available Additional information on fabrication of SEM of poly (GMA-co-EDMA) capillary monolithic column (Figure S1), FT-IR spectra of poly (GMA-co-EDMA) capillary monolithic column (before and after the modification of ethylenediamine) (Figure S2), effect of sample loading rate on the adsorption efficiency of Hg, Pb and Bi (Figure S3), effect of elution times on the desorption efficiency of Hg, Pb and Bi (Figure S4), effect of sample volume on the adsorption efficiency of Hg, Pb and Bi (Figure S5), and operating conditions for online ICPMS analysis (Table S1) were shown in supporting information. This material is available free of charge via the Internet at http://pubs.acs.org.

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Reference (1) Yang, N.; Sun, H. Z. Prog. Chem., 2009, 21, 856-865. (2) Wei, B. G.; Yang, L. S. Microchem. J., 2010, 94, 99-107. (3) Hernandez, L. E.; Sobrino-Plata, J.; Montero-Palmero, M. B.; Carrasco-Gil, S.; Flores-Caceres, M. L.; Ortega-Villasante, C.; Escobar, C. J. Exp. Bot., 2015, 66, 2901-2911. (4) Finney L. A.; O'Halloran, T. V. Science, 2003, 300, 931-936. (5) Trouillon, R.; Passarelli, M. K.; Wang, J.; Kurczy, M. E.; Ewing, A. G. Anal. Chem., 2013, 85, 522-542. (6) Shintani, Y.; Zhou, X.; Furuno, M.; Minakuchi, H.; Nakanishi, K. J. Chromatogr. A, 2003, 985, 351-357. (7) Fernandez-Amado, M.; Prieto-Blanco, M. C.; Lopez-Mahia, P.; Muniategui-Lorenzo, S. Prada-Rodriguez, D. Anal. Chim. Acta, 2016, 906, 41-57. (8) Zhao, M. Y.; Ma, X. D.; Zhao, F. J.; Guo, H. W. J. Mater. Sci., 2016, 51, 3440-3447. (9) Lirio, S.; Liu, W. L.; Lin, C. L.; Lin, C. H.; Huang, H. Y. J. Chromatogr. A, 2016, 1428, 236-245. (10) Vergara-Barberan, M.; Lerma-Garcia, M. J.; Simo-Alfonso, E. F.; Herrero-Martinez, J. M. Anal. Chim. Acta, 2016, 917, 37-43. (11) Zhang, W. P.; Li, Y. L.; Chen, Z. L. J. Sep. Sci., 2015, 38, 3969-3975. (12) Liu, D.; Lv, X. J.; Zhang, J. L.; Jia, Q.; Liao, W. P. Anal. Methods, 2012, 4, 2970-2976. (13) Liu, X. L.; He, M.; Chen, B. B.; Hu, B. Spectrochim. Acta B, 2014, 101, 254-260. (14) Liu, X. L.; Chen, B. B.; Zhang, L.; Song, S. Y.; Cai, Y. B.; He, M.; Hu, B. Anal. Chem., 2015, 87, 8949-8956. (15) Zhang, L.; Chen, B. B.; Peng, H. Y.; He, M.; Hu, B. J. Sep. Sci., 2011, 34, 2247-2254. (16) Mark, D.; Haeberle, S.; Roth, G.; von Stetten, F.; Zengerle, R. Chem. Soc. Rev., 2010, 39, 1153-1182. (17) Zhang Y. H.; Ozdemir, P. Anal. Chim. Acta, 2009, 638, 115-125. (18) Lin, X. X.; Leung, K. H.; Lin, L.; Lin, L. Y.; Lin, S.; Leung, C. H.; Ma, D. L.; Lin, J. M. Biosens. Bioelectron., 2016, 79, 41-47. 16


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(19) Espulgar, W.; Yamaguchi, Y.; Aoki, W.; Mita, D.; Saito, M.; Lee, J. K.; Tamiya, E. Sens. Actuators B-Chem., 2015, 207, 43-50. (20) Wang, H.; Wu, Z. K.; Zhang, Y.; Chen, B. B.; He, M.; Hu, B. J. Anal. At. Spectrom., 2013, 28, 1660-1665. (21) Chen, B. B.; Heng, S. J.; Peng, H. Y.; Hu, B.; Yu, X.; Zhang, Z. L.; Pang, D. W.; Yue, X.; Zhu, Y. J. Anal. At. Spectrom., 2010, 25, 1931-1938. (22) Wang, H.; Wu, Z. K.; Chen, B. B.; He, M.; Hu, B. Analyst, 2015, 140, 5619-5626. (23) Wang, H.; Chen, B. B.; Zhu, S. Q.; Yu, X. X.; He, M.; Hu, B. Anal. Chem., 2016, 88, 796-802. (24) Chen, B. B.; Hu, B.; He, M.; Huang, Q.; Zhang, Y.; Zhang, X. J. Anal. At. Spectrom., 2013, 28, 334-343. (25) Yu, C.; Davey, M. H.; Svec, F.; Frechet, J. M. J. Anal. Chem., 2001, 73, 5088-5096. (26) Hisamoto, H.; Nakashima, Y.; Kitamura, C.; Funano, S.; Yasuoka, M.; Morishima, K.; Kikutani, Y.; Kitamori, T.; Terabe, S. Anal. Chem., 2004, 76, 3222-3228. (27) Knob, R.; Sahore, V.; Sonker, M.; Woolley, A. T. Biomicrofluidics, 2016, 10, 032901. (28) Li, P.; Chen, Y. J.; Lian, H. Z.; Hu, X. J. Anal. At. Spectrom., 2014, 29, 1785-1790. (29) Zhang, J.; Chen, B. B.; Wang, H.; Huang, X.; He, M.; Hu, B. J. Anal. At. Spectrom., 2016, 31, 1391-1399. (30) Lv, Y.; Lin, Z.; Svec, F. Anal. Chem., 2012, 84, 8457-60.

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Figure captions Figure 1 Diagram of the integrated chip-based array monolithic microextraction analytical platform: layout (a), photograph (b), and SEM of the embedded poly (GMA-co-EDMA-NH2) capillary monolithic column (c). (I1-I10: inlets; W1-W6: outlets; V1-V10: microvavles; M1-M6: embedded capillary monolithic columns; CN: outlet to MCN-ICPMS) Figure 2 Effect of pH on the adsorption efficiency of Hg, Pb and Bi (cHg, Pb, Bi=10 µg L-1, sample volume: 250 µL; sample loading rate: 0.15 mL min-1) Figure 3 Effect of the concentration of nitric acid (a) and thiourea (b) on the desorption efficiency of Hg, Pb and Bi (cHg, Pb, Bi=10 µg L-1, sample loading rate: 25 µL min-1, loading pH: 7.0, sample volume: 250 µL; eluent flow rate: 25 µL min-1)

18


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Figure 1

19



100

Adsorption efficiency (%)

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80 202 Hg 208 Pb 209 Bi

60

40

20

0 2

3

4

5

6

7

8

9

pH

Figure 2

20


10

11

Page 21 of 27

Desorption efficiency (%)

120 100 80 202 Hg 208 Pb 209 Bi

60 40 20 0 0.1

0.2

0.5

0.8

1

Acid concentration (mol/L)

(a)

120

Desorption efficiency (%)

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


100 80

202 Hg 208 Pb 209 Bi

60 40 20 0 0.00

0.01

0.02

Concentration of thiourea (w/w)

(b) Figure 3

21


0.03


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Table 1 Controlling program of microvalves Microvalve (open)

Process

Solution

V4, V8

Sample introduction

Sample solution

V1

Elution of M1

Eluent

V9

Wash the channel

nitric acid of 5%(v/v)

V10

Separate liquid

N2

V2

Elution of M2

Eluent

V9

Wash the channel


V10

Separate liquid

N2

V3

Elution of M3

Eluent

V9

Wash the channel


V10

Separate liquid

N2

V5

Elution of M4

Eluent

V9

Wash the channel


V10

Separate liquid

N2

V6

Elution of M5

Eluent

V9

Wash the channel


V10

Separate liquid

N2

V7

Elution of M6

Eluent

V9

Wash the channel


V10

Separate liquid

N2

22


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Table 2 Tolerance concentration of foreign ions Foreign ions

Tolerance concentration (µg mL-1)

K+

5000

+

5000

2+

5000

2+

5000

Na Cu Zn

Ca2+

500

2+

500

3+

10

3+

10

Mg Fe Al

Cl

-

5000

NO3-

5000

SO42-

500

23



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Table 3 Analytical performance for target metals obtained by online chip-based array capillary monolithic microextraction-MCN-ICPMS Hg

Pb

Bi

23

12

13

Linear range (µg L )

0.05-20

0.05-20

0.05-20

Linear equation

y=5.0×104x+1.8×105

y=8.5×105x+7.3×105

y=2.5×106x+4.1×105

Correlation coefficient (R2)

0.9958

0.9970

0.9980

3.3

3.7

2.3

-1

LOD (ng L ) -1

RSD (n=9, %)

a

a: Hg 0.20 µg L-1, Pb 0.12 µg L-1, Bi 0.13 µg L-1

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Table 4 Comparison of analytical performance of this method with other analytical approaches Method

LOD (ng L-1) Volume Analytical On/off Throughput Hg Pb Bi

APTES-silica monolithic CME-ICP-MS Chip-based MSPME-ETV-ICPMS

-

14

-

0.82 1.16 -

(mL)

time (min)

line

(h-1)

1

21

on

2.9

0.5

35.8

off

-

Sample Ref. Urine, hair HepG2 cells

15 21

HepG2, Chip-based array MSPME-ICPMS

6.1 13 18

70 (for 5

0.5

samples)

on

5

Jurkat T, MCF-7

22

cells Chip-based monolithic microextraction-ICPMS

-

- 210

0.1

55

-

HepG2 cells

29

HepG2

Chip-based array monolithic

off

23

12 13

21.8 (for 6

0.25

samples)

microextraction-ICPMS

on

16.5

cells,

human work urine

ETV: electrothermal vaporization

25


This


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Table 5 Analytical results (mean ± s.d., n=3) for HepG2 cells and human urine samples HepG2 cell Elements

Hg

Pb

Bi

Added

Found

-1

-1

Human urine

Found

Recovery

-1

Added

Found

-1

-1

Recovery

(µg L )

(µg L )

(fg cell )

(%)

(µg L )

(µg L )

(%)

0

0.15±0.01

0.51±0.01

-

0

ND

-

0.06

0.21±0.02

0.71±0.06

90.4

0.08

0.07±0.01

91.4

5.0

4.9±0. 2

98.7

0

0.13±0.01

0.44±0.02

-

0

ND

-

0.06

0.19±0.01

0.63±0.02

98.1

0.08

0.08±0.01

94.9

5.0

5.1±0. 1

102

0

0.10±0.01

0.34±0.01

-

0

ND

-

0.06

0.15±0.01

0.51±0.01

95.3

0.08

0.08±0.01

96.3

5.0

4.9±0. 1

97.9

ND: not detected.

26


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For TOC only

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Facile Chip-Based Array Monolithic Microextraction System Online

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