Facile Design of Phase Separation for Microfluidic Droplet-Based

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Facile design of phase separation for microfluidic droplet-based liquid phase microextraction as a front end to electrothermal vaporization-ICPMS for the analysis of trace metals in cells Xiaoxiao Yu, Beibei Chen, Man He, Han Wang, Songbai Tian, and Bin Hu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03078 • Publication Date (Web): 24 Jul 2018 Downloaded from http://pubs.acs.org on July 24, 2018

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Analytical Chemistry

Facile design of phase separation for microfluidic droplet-based liquid phase microextraction as a front end to electrothermal vaporization-ICPMS for the analysis of trace metals in cells Xiaoxiao Yu, Beibei Chen, Man He, Han Wang, Songbai Tian, Bin Hu* Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, China

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Analytical Chemistry 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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Abstract The issue of quantifying trace metals in cells has drawn widespread attention but is threatened with insufficient sensitivity of the instruments, complex cellular matrix and limited cell consumption. In this study, microfluidic droplet-based liquid phase microextraction (LPME), as a miniaturized platform, was developed and combined with

electrothermal

vaporization

(ETV)-inductively

coupled

plasma

mass

spectrometry (ICPMS) for the analysis of trace Cd, Hg, Pb and Bi in cells. A novel and facile design of phase separation region was proposed, which made the phase separation very easily for subsequent ETV-ICPMS detection. Mechanism of the phase separation was carefully discussed using the incompressible formulation of the Navier-Stokes equations. The developed microfluidic droplet-based LPME system exhibited much higher extraction efficiency to target metals than microfluidic stratified flow-based LPME. Under the optimized conditions, the limits of detection of the proposed microfluidic droplet-based LPME-ETV-ICPMS system were 2.5, 3.9, 5.5 and 3.4 ng L-1 for Cd, Hg, Pb and Bi, respectively. The accuracy of the developed method was well validated by analyzing the target metals in Certified Reference Materials of GBW07601a human hair. Finally, the proposed method was successfully applied to the analysis of target metals in HeLa and HepG2 cells with the recoveries for the spiked samples ranging from 83.5 to 112.3%. Overall, the proposed design is a simple and reliable solution for the phase separation on droplet-chip and the microfluidic droplet-based LPME-ETV-ICPMS combination strategy shows great promise for trace elements analysis in cells. Keywords: microfluidic droplet-based liquid phase microextraction; electrothermal vaporization-inductively coupled plasma mass spectrometry; trace heavy metals; cell analysis


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Introduction Cadmium (Cd), mercury (Hg), lead (Pb) and bismuth (Bi) are acknowledgedly toxic elements. Cell is the basic structural and functional unit of the organism and the cell-level research is increasingly regarded as a gateway to get more information on the roles of elements in the organism. To name but a few, Cd down-regulates the expression of apolipoprotein E and induces malignant transformation of rat liver cells1; methyl mercury (MeHg) is the most toxic species of Hg that can induce an oxidative stress responsible for apoptosis2 and there exists the demethylation of MeHg in cells3; Pb interferes with DNA-binding of transcription specificity protein 1 which is important for the transcription of genes involved in growth and differentiation of the nervous system4; Bi can be methylated in cells and the methylated bismuth compound is able to engender DNA damage and cell death5. Therefore, to figure out how heavy metals damage cells, it is a prerequisite to determine these trace metals in cells. However, some challenging problems stem from: (1) extremely low concentration of interest metals in cells which is far below the limits of detections (LODs) of analytical instruments; (2) serious matrix interference resulting from complex intracellular components will affect the accuracy of the analytical results; (3) limited amount of available cell sample which is incompatible with the sample consumption of conventional analytical methods. Inductively coupled plasma mass spectrometry (ICPMS) has been regarded as one of the most powerful instruments allowing multi-elemental detection over a wide linear dynamic range together with very low LODs. Electrothermal vaporization (ETV), as a miniaturized sample introduction technique of ICPMS, has advantages of high efficiency of sample introduction, low sample consumption (a few of microliter) and alleviating matrix effects6,7. Sample preparation is necessary for the analysis of ultra trace metals in samples with very complicated matrix. Solvent extraction is one of the most widely used sample preparation technique, and the newly developed liquid phase microextraction (LPME) with the consumption of microliter level organic solvents is considered as highly enriched and environmental-friendly sample preparation 3 ACS Paragon Plus Environment


technique. The combination of LPME with ETV-ICPMS is perfectly compatible and straightforward. In this combination, the organic extractant phase can be in situ vaporized by employing appropriate heating program in ETV, and the chelating reagent used in LPME for extraction of target metals can also play a role of the chemical modifier in ETV to improve the vaporization behavior of target metals as their metal-chelates forms. For instance, Xia et al. used benzoylacetone (BZA)8, sodium diethyldithiocarbmate (DDTC)9 and 1-(2-pyridylazo)-2-naphthol (PAN)10 as both the chelating reagent for LPME and the chemical modifier for low-temperature ETV-ICPMS determination of trace metals in real-world samples. Although LPME is a kind of miniaturized sample preparation techniques, its sample consumption is still at several millilitre level, which makes LPME ill-suited for cell sample analysis. The integration of LPME into the microfluidic chip enables a marked reduction in reagent volume due to the structural characteristics of a network in a microfluidic chip11,12. Besides, high surface-to-volume ratio and short diffusion distance can boost extraction efficiency. A host of early studies13-15 were undertaken by Kitamori and his colleagues who fabricated a laminar flow glass chip involving a Y-shaped inlet and outlet coupling with sensitive thermal lens microscopy (TLM) to quantify target metals. However, one of the major limitations of laminar flows is insufficient mixing of fluids (only by diffusion). In addition, TLM is less selective towards trace metals when compared to elemental specific detector, such as ICPMS, which limited its applicability in real-world samples16-19. Droplets, most frequently generated by a flow focusing geometry in microfluidic device20-22, have larger surface-to-volume ratio in addition to recirculation inside and outside the droplets, which can accelerate the extraction process significantly. Moreover, the droplets travel at different velocities relative to the wall in curved channels, which result in unsteady flows to disturb a pair of symmetrical eddies within the droplets and achieve chaotic advection23,24.

Therefore, the extraction efficiency of microfluidic

droplet-based LPME is usually higher than the microfluidic stratified flow-based LPME25. However, except for only a few studies involving in situ fluorescence 4 ACS Paragon Plus Environment

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detection of metals (such as Al- 2,2-dihydroxyazobenzene26 or Hairpin labelled Hg27), phase separation is necessary to collect the droplet-phase together for subsequent detection, which is the cumbersome step inhibiting its further application in trace metals analysis. Thereby, to combine microfluidic droplet-based LPME with ICPMS detection for better performance, the issue of phase separation is thrown into sharp focus. Much efforts have been dedicated to deal with phase separation, including the use of surface modification28,29, a membrane phase separator30,31 or special channel designs on basis of density32-35, gravity36 and capillary forces37,38. Nevertheless, surface modification is only available at the expense of low flow rate which runs contrary to the goal of high enrich factor and high-throughout. Besides, for phase separation and stabilization, complicated structures, such as porous hydrophobic membranes, extra syringe pumps or elaborate chips, are usually required. The work described here aims to address the challenges in operating an effective phase separation and developing a method of microfluidic droplet-based LPME technique with enhanced extraction efficiency for the determination of Cd, Hg, Pb and Bi in cells followed by ETV-ICPMS detection. A hydrophilic surface modification was applied for obtaining oil-in-water droplet system, and a facile design at the outlet was employed for phase separation of droplet organic phase from aqueous phase. Several parameters of the dimensions and structure of the droplet chip was investigated to get the best performance. For microfluidic droplet-based LPME of target metals, 1-octanol was chosen as appropriate organic phase in consideration of low solubility in water, less volatile, low boiling point as well as low toxicity, while DDTC played a dual role of chelating reagent for extraction of metal ions and chemical modifier for the subsequent ETV-ICPMS detection according to our previous work18. Various factors affecting the extraction of Cd, Hg, Pb and Bi in the process of microfluidic droplet-based LPME were also adequately discussed and the analytical performance of the developed method was evaluated under the optimized 5 ACS Paragon Plus Environment


conditions. The developed method was validated by the analysis of a Certified Reference Material of GBW07601a human hair and further applied to the analysis of trace target metals in HeLa and HepG2 cells. Experimental Apparatus An Agilent 7500a ICP-MS (Agilent, Tokyo, Japan) was equipped with a modified commercial WF-4C graphite furnace (Beijing Second Optics, Beijing, China) as an electrothermal vaporizer with a home-made connecting interface. A concentric glass tube and a polyethylene tube (6 mm id) with a total length of 70 cm were used as connecting tubes. The operation conditions for ETV-ICP-MS are listed in Table S1. An inverted fluorescence microscope (Axio Observer.A1, Zeiss, Germany) in conjunction with a charge-coupled device (RETIGA R3, Q Imaging, USA) was used to observe the microfluidic chips in situ. The contact angle was measured by a drop shape analysis system (DSA100, Krüss, Germany). A KW-4A spin coater (Siyouyen Electronic Technology Co., Ltd., Beijing, China) and PDC-M plasma cleaner (Mingheng Science and Technology Development Co., Ltd., Chengdu, China) were used in the preparation of microfluidic chips. TS2-60 syringe pumps (Baoding Longer Precision Pump Co., Ltd., Baoding, China) and sterile syringes were applied for sample introduction into the microfluidic chips. A Mettler Toledo 320-s pH meter (Mettler Toledo Instruments Co., Ltd., Shanghai, China) was used to adjust pH. The sample was operated with a WX-3000 microwave accelerated digestion system (EU Chemical Instruments Co. Ltd., Shanghai, China) for acid digestion. Reagents and materials The standard stock solutions (1 g L-1) of Cd, Hg, Pb and Bi were prepared by dissolving CdCl2 •2.5H2O, HgCl2, Pb(NO3)2 and Bi(NO3)2 (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) in 2 % HNO3, respectively. Sodium diethyldithiocarbamate (DDTC) (ACS grade) and n-octanol (≥99%) were both 6 ACS Paragon Plus Environment

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purchased from Aladdin Reagent Inc. (Shanghai, China). Dopamine hydrochloride (DA) ( ≥ 98%, Aladdin Reagent Inc. Shanghai, China) was dissolved in tris(hydroxymethyl)aminomethane buffer (Tris, 10 mM) at pH 8.5. Phosphate buffer solution (PBS, 10 mmol, pH 7.4) consisted of 137 mmol L-1 NaCl, 2.7 mmol L-1 KCl, 1.9 mmol L-1 KH2PO4, and 8.1 mmol L-1 Na2HPO4. Bovine serum albumin (BSA) was dissolved in PBS solutions. Polydimethylsiloxane (PDMS) was made by mixing oligomers (component A) with crosslinking agents (component B) (GE RTV 615, Momentive Performance Materials, NY, USA) at a mass ratio of 10:1. Sub-boiled HNO3 was used in all experiments. All other reagents were of at least analytical reagent grade. Highly purity deionized water obtained from a Milli-Q system (18.2 MΩ·cm, Millipore, Molsheim, France) was used throughout this work. All laboratory ware was made of polyethylene or Teflon material and thoroughly cleaned by soaking in 10% nitric acid for at least 24 h. HeLa and HepG2 cells were provided by Cell bank of Chinese Academy of Sciences (Shanghai, China). Design and preparation of PDMS microfluidic droplet-based devices As shown in Figure 1, three functional sections were designed to perform monodisperse droplet-based LPME: (a) droplets generation; (b) extraction zone and (c) phase separation. The design of microfluidic droplets formation was referred to the flow-focusing geometry and the channels narrowed down in the focusing part towards generating stable oil phase droplets. To be specific, the width of organic phase channel, aqueous phase channel, droplet generation channel and flow focusing channel were 400, 400, 400 and 150 µm, respectively (Figure 1(a)). Furthermore, the length of organic phase channel, aqueous phase channel and droplet generation channel were 1.5, 2 and 2 mm, respectively. At the extraction zone, serpentine channel (Figure 1(b)) was designed for better extraction performance, and the length was 185 cm taking the size of the glass substrate into account. For phase separation (Figure 1(c)), the site of 7 ACS Paragon Plus Environment


roundness was drilled by a 0.6 mm o.d. hole punch and a 0.8 mm diameter stainless steel bar was plugged into the drilled hole of phase separation region. The channel dimensions of the oil outlet were 100 µm wide and 40 µm long, respectively. As for aqueous outlets, the channel dimensions were 400 µm wide and 0.9 mm long. The height of all microchannels was 50 µm. Fabrication and hydrophilic modification of PDMS microfluidic devices Referring to our previous work39, soft lithography and rapid prototyping with PDMS technology were employed for fabrication of microfluidic devices. A transparent mask patterned with a high-resolution laser printer was used to make a master on a silicon wafer with AZ 50XT photoresist. Before PDMS casting, the master was exposed to trimethylchlorosilane vapor for 10 min to avoid the adhesion between PDMS and silicon wafer. For the preparation of the flow channels, GE RTV 615 (PDMS) component A and B were mixed at a ratio of 10:1 and poured onto the master after degassing. After incubating at 75 ºC for 3 h, the solidified PDMS was peeled off and drilled on demand. Afterwards, the ensemble and a clean glass substrate were exposed to oxygen plasma and then bonded together. The procedure of hydrophilic surface modification was essential when oil-in-water droplets were required to generate in an inherent hydrophobic PDMS microfluidic chip. Referring to Ref.40 with slight modification, 50 µL DA solution (2 mg mL-1) was pumped into the microchannel with the flow rate of 2 µL min-1 and then maintained for 2 h at room temperature to form polymerized dopamine coating (PDA). 50 µL PBS buffer solution (pH 7.4) with the flow rate of 5 µL min-1 was used as the washing solution to clean the device successively. Afterwards, 50 µL BSA solution (1 mg mL-1) was injected into the microchannel at the rate of 2 µL min-1 and then kept for 4 h at 4 oC. At last, the channel was washed with PBS to remove the unbonded residues as stated. It should be noteworthy that the phase separation region was independent of hydrophilic modification which was attributed to the hole A without the stainless-steel bar letting the modified solution out smoothly. 8 ACS Paragon Plus Environment

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Contact angle studies The wettability of microfluidic chip surfaces was evaluated by contact angle measurements. A drop water of 2 µL was dripped on the native and coated PDMS surface and allowed to rest on the surface for 30 s. The image of the droplet was captured by a high-resolution optical microscopy. Then, the drop profile was able to calculate the contact angle by the software. Microfluidic droplet-based LPME procedure Firstly, the hole A was plugged with the stainless-steel bar. Subsequently, n-octanol flow at 1 µL min-1 was pumped into the aqueous stream containing 0.5% (m/v) DDTC with the flow rate of 35 µL min-1. It can be seen that the oil stream was broken into droplets due to the shear force of the aqueous phase at the flow focusing section. Before the extraction process officially started, it should be ensured that stable droplets were generated at the indicated flow rates by visually inspect under a microscope. Then, the winding channel was used to induce convective recirculation within each oil droplet and achieve the extraction of target metals. When oil droplets moved through the phase separation region, they switched to side-by-side flow rapidly. Nevertheless, the droplets were segmented to reach the separator, which made the formative distinct laminar flow fluctuant. It took about 10 min to collect 5 µL of the extract into the Eppendorf tube through an inverted U shaped stainless-steel tube which one end was inserted in the oil outlet and the other end was inserted into the Eppendorf tube. Finally, the collected n-octanol was off-line introduced into a graphite furnace with a microsyringe for subsequent ETV-ICPMS determination. After each microextraction process,100 µL ethanol, 100 µL 0.5 mol L-1 HNO3 as well as 100 µL PBS solution was used as the washing solution to wash the system at the flow rate of 10 µL min-1 successively in order to eliminate the memory effect of the whole system. Sample preparation 9 ACS Paragon Plus Environment


Certified Reference Materials The Certified Reference Materials of GBW07601a human hair was analyzed to validate the accuracy of the proposed method. For this purpose, 0.20 g of GBW07601a human hair was weighed into PTFE digestion vessels. The vessels were then placed on an electric hot plate (80 ºC) for 1 h after adding 4 mL of HNO3 before they were placed on a turntable and subjected to microwave digestion. The microwave system was operated at firstly 150 ºC, 18 atm for 4 min and then 180 ºC, 22 atm for 4 min. After the microwave treatment, the vessels were self-cooling towards room temperature, and then placed on an electric hot plate (85 ºC) again. When the digest got nearly dryness, it was transferred to EP tube and diluted with 2% HNO3 to 4 mL as the prepared sample solution. Then the aliquots were diluted by 100 times with highly purity deionized water. After adding an appropriate amount DDTC (concentration of 0.5% (m/v)) and adjusting of pH to 9.0, the samples were served to subsequent analysis. Cell culture and ultrasonic lysis procedure HepG2 and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U mL-1 penicillin and 100 U mL-1 streptomycin in 5% CO2 at 37 °C, respectively. Cells were detached by trypsinization with 0.25% trypsin and 0.02% EDTA in PBS buffer. The collected cells were washed by PBS solution twice with the aid of centrifuge at 1500 rpm for 5 min. After the number of cells was counted by using a hemocytometer, the number of cells suspension was diluted to 2000,000 cells in 400 µL highly purity deionized water. Subsequently, the cells were subjected to thirty-minute ultrasonication and centrifuged at 12,000 rpm for 5 min. Afterwards, the supernatant was filtered by using a filter membrane (0.45 µm) and added to 1.6 mL DDTC solution (0.625%, m/v) which guaranteed that the final concentration of DDTC is 0.5% (m/v). At last, the sample solution was adjusted to pH 9.0 and then introduced into the aqueous inlet on the chip. 10 ACS Paragon Plus Environment

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Results and Discussion Surface characterization of PDA/BSA-coated PDMS microfluidic chips DA tended to self-polymerization at a weak alkaline pH and became to form PDA films as an adhesion layer onto PDMS surface. As shown in Figure S1, the contact angle of the native PDMS was 119° (Figure S1a), which was thoroughly in tune with the inherent hydrophobicity of PDMS. Compared with the native one, the contact angle of PDA coating decreased dramatically to 73° (Figure S1b) with the aid of the abundant active hydroxyl groups of PDA. For further BSA decoration based on Michael addition and/or Schiff-base reaction between the quinone/catechol groups of PDA and the amine of lysine in BSA, the contact angle reduced sequentially to 41° (Figure S1c). Aforementioned results revealed that PDA/BSA-coated PDMS surfaces was modified successfully and improved the hydrophilicity of the microchannel. Last but not least, the relative hydrophilicity of PDMS surface could be kept at least within a week stored at 4 oC (evidenced by stable oil droplets observed in the extraction microchannel shown in Figure S2). Mechanism of the phase separation In this work, we found that the hole plugged with the stainless steel bar (hole A) governed either success or failure of phase separation in the experimental process. Therefore, the location of hole A was carefully investigated. As shown in Figure 2(a), when the edge of the hole A was not in the main channel, it maintained droplet flow. When the hole A occupied almost the whole width of the main channel (Figure 2(b)), it transformed the droplets into the water-oil-water laminar flow which did not facilitate oil phase collection. When the distance of the stainless-steel bar’s bottom edge and the top of the main channel was from 100 to 200 µm (occupy 1/4-1/2 of the microchannel), the droplet flow was converted to oil-water laminar flow (Figure 2(c)). We characterized the position of the end of the stainless-steel bar in microfluidic chip by SEM. As shown in Figure 3(a), the height of the gap between the bottom of the bar 11 ACS Paragon Plus Environment


and the glass slide was similar to the height of the channel. It indicates that the droplet flow flowed through the bottom of the stainless-steel bar. The change of the flow channel and the difference of the surface property between stainless steel bar and the PDMS might be the reasons for phase separation. To illustrate the behavior of the droplets in the phase separation region, the flow field of the microfluidic chip around the stainless steel was modelled on the basis of measured data as set forth in 3D using commercial Finite Element Method (FEM) package Comsol Multiphysics (version 5.3). The Laminar Multiphase Flow and Level Set using the incompressible formulation of the Navier-Stokes equations is as follows: ρ

∂ + ρ ∙ ∇ = ∇ · −p + μ∇ + ∇ + ∂t

∇∙=0 ρ denotes density (kg/m3), u is velocity (m/s), t is time (s), p is pressure (Pa), µ is the dynamic viscosity (Pa · s), and Fst is the surface tension force (N/m3). In our theoretical model, water and n-octal was used as aqueous and organic phase, respectively, relevant parameters and input values were listed in Table S2. According to the theoretical calculation, the process for phase separation is illustrated in Figures 4 (a-p) with the video shown in Supporting Information. An oil droplet was induced to pass through and stand in the area of the stainless-steel bar firstly and then another one headed-on into the previous one. They collided, and more and more oil droplets merged together subsequently. At last, when the whole area of stainless steel bar was filled with oil phase, the droplet flow was made to be oil-water laminar flow. In this theoretical modelling system, it was found that phase separation only succeeds when the contact angle of the stainless-steel bar was lower than 40°. To verify it, the contact angle of stainless steel bar was characterized. For practical reasons, the stainless-steel bar intended for use was too small to be measured its surface’s contact angle. Therefore, the contact angle (θ) on the smooth surface of the same stainless-steel material was measured, and the value is 61° (Figure 3b). According to the principle of wetting action on actual solid surfaces outlined decades ago by 12 ACS Paragon Plus Environment

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Wenzel41, cosθW of the actual contact angle of rugged surfaces was calculated by multiplying cosθ with the roughness factor γ. And the γ equals to the ratio of the area of the actual surface to that of a smooth surface having the same geometric shape and dimensions42. The SEM of the stainless-steel bar’s surface was shown in Figure S3(a-b), then the profile was simplified to Figure S3(c) and the roughness factor γ was evaluated as 1.6. Given the above, the contact angle of stainless steel bar was lower than 35°. It demonstrates that the phase separation process could be well explained by this theoretical model using the incompressible formulation of the Navier-Stokes equations. After phase separation, the oil phase was ready to be collected. It is known that the wider oil outlet might be more likely to jeopardise the purity of the collected oil phase. Therefore, the width of oil outlet was optimized to collect oil phase maximumly and prevent aqueous phase from flowing into oil outlet. It was found that when the width of oil outlet was fixed at 100 µm, the collection efficiency of organic phase was about 50%, and no aqueous stream entered into narrow oil outlet. Conclusively, 100 µm was finally selected as the width of the oil outlet. Effect of pH DDTC was unstable in weak acidic solutions, meanwhile, hydrolysis of metal ions was a probable occurrence under the strong alkali condition. For these reasons, the effect of pH on the extraction was investigated with pH varying in the range of 7-10. As shown in Figure 5, the maximum signal intensity could be obtained at pH 9.0. Thereby, pH 9.0 was used for the further experiments. Effect of the DDTC concentration The concentration of DDTC was investigated with its concentration ranging from 0 to 1.0% (m/v). The experimental results shown in Figure S4 indicated that the signal intensity of all target metals increased with the addition of DDTC and then maintained nearly constant when the concentration of DDTC was higher than 0.5% 13 ACS Paragon Plus Environment


(m/v). Accordingly, 0.5% (m/v) was employed as the concentration of DDTC in subsequent experiments. Effect of the size of oil droplets and extraction conditions Besides the extraction pH value and the DDTC concentration, there are several other potential factors affected the extraction efficiency, such as the size of oil droplets and extraction time. In a sense, the contribution of the droplets size to the extraction efficiency was strongly dependent on the parameters of the flow rate ratio of aqueous phase to oil phase and geometrical parameters of the microchannel. Owing to the fact that these parameters were interrelated and interdependent, an L9 (33) orthogonal test was applied to optimize these parameters. The stability of phase separation was susceptible to higher flow rate ratio of aqueous phase to oil phase. It happened to generate stable droplets under the circumstances that the flow rate ratio of aqueous phase to oil phase equal to or exceeded 20:1. However, it would make the oil stream spin out of control when the flow rate ratio was over 40:1. Thus, the flow rate ratio of aqueous phase to oil phase ranging from 20:1 to 35:1 was adopted for the orthogonal experiments with the flow rate of oil phase fixed at 1 µL min-1. The orthogonal experiment and other parameters were listed in Table 1. The orthogonal experimental results were analyzed by analysis of variance (ANOVA). Then the Type III sum of square (Type III SS) and the optimal solutions of all variables were displayed in SPSS statistic software. As we know, the value of Type III SS was greater and the influence of each factor was more important. Thus, the order of factors and the optimal combination for all target elements were listed in Table 1. It was deduced that the flow rate ratio of aqueous phase to oil phase was the most important factor for target metals except for Hg. Theoretically, the higher flow rate ratio of aqueous phase to oil phase could give rise to the higher enrichment factor. Hence, 35:1 was chosen as the flow rate ratio of aqueous phase to oil phase. As for Hg, the principal factor was the length of extraction channel due to its own slower mass transfer rate between the aqueous phase and the organic phase. In view of the optimal combination for each 14 ACS Paragon Plus Environment

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analyte, there existed a uniform rule that the higher extraction efficiency arose from the longer extraction channel. Therefore, 1850 mm was selected as the length of extraction channel. As to the width of main channel, its effect on extraction efficiency was intertwined. A relatively narrower width would cause a shorter diffusion distance which benefits to accelerate the extraction, while it could also lead to a higher linear velocity, correspondingly a shorter contact time, which is negative to promote extraction efficiency. The experimental results demonstrated that the main channel of 400 µm wide is better for Cd and Hg, while 200 µm wide is better for Pb and Bi. Considering that the width of main channel is not the most important factor and a relatively wider channel facilitated the operation of drilling the Hole A precisely, the main channel was fixed as 400 µm. Overall, the optimal combination was summarized as follows: the length of extraction channel was 1850 mm; the flow rate ratio of aqueous phase to oil phase was 35:1 and the width of main channel was 400 µm. Subsequently, the flow rate of the oil phase was examined with the flow rate varying from 0.5 to 4 µL min-1 by keeping the flow rate ratio of aqueous phase to oil phase was 35:1. As can be seen in Figure S5, the signal intensity of Hg as well as Bi decreased obviously once the flow rate of the oil phase was more than 1.5 µL min-1, and the signal intensity of Cd and Pb kept constant when the flow rate of the oil phase was less than 3 and 2 µL min-1, respectively. Consequently, 1 µL min-1 was used as the flow rate of the oil phase in further experiments. Effect of co-existing ions Under the optimized conditions, the sample solutions with Cd, Hg, Pb and Bi each at 1 µg L-1 and a certain amount of foreign ions (including K+, Na+, Ca2+, Mg2+, Cu2+, Zn2+, Fe3+, Al3+, NO3-, Cl-, SO42- and PO43-) were subjected to the microfluidic droplet-based LPME-ETV-ICPMS, and the interference caused by coexisting ions was evaluated. It was regarded as no adverse effect of coexisting ions within this range when the recoveries of target metal ions maintain in the range of 85-115%. As shown in Table S3, a relatively high tolerance of the developed method to matrix interference was 15 ACS Paragon Plus Environment


found, indicating this method was capable of analyzing target elements in biological samples. Reproducibility and regeneration of the droplet-based chip Under the optimized conditions, the extraction efficiencies of five prepared microfluidic chips for target Cd, Hg, Pb and Bi were studied to observe the reproducibility of the self-made microfluidic droplet-based LPME device. The relative standard deviations (RSDs) of the signal intensity for Cd, Hg, Pb and Bi were calculated to be 7.4, 6.8, 11.0 and 6.3% (cCd, Hg, Pb, Bi=1 µg L-1, n=5), respectively. The experimental results demonstrated that the prepared microfluidic chips had good reproducibility. The regeneration of the self-fabricated microfluidic droplet-based LPME chip was also investigated. It was manifested that the PDMS platform could be regenerated by washing with 100 µL ethanol, 100 µL 0.5 mol L-1 HNO3 as well as 100 µL PBS buffer solution at the flow rate of 10 µL min-1 successively after each LPME procedure. With the assistance of this process, the prepared microchip could be reused for about 20 times without re-modification of PDMS surface and about 100 times until it was affected by physical fissuration and leakage. Analytical performance Under the optimal experimental conditions, the analytical performance of the developed method of microfluidic droplet-based LPME-ETV-ICPMS for target metals was investigated and the analytical results are shown in Table 2 and Figure 6. In accordance to the IUPAC definition, the LODs were 2.5, 3.9, 5.5 and 3.4 ng L-1 for Cd, Hg, Pb and Bi with RSDs (n=7; cCd, Hg, Pb, Bi=0.2 µg L-1) of 7.8, 7.1, 7.5 and 6.2%, respectively. The enrichment factors (EFs) were defined as the ratio of the calibration curve slopes after and before the chip-based LPME, and calculated to be 33.5, 13.7, 12.8 and 8.7-fold for Cd, Hg, Pb and Bi, respectively, while the theoretical EF was 35-fold. The analytical performance of the developed method was compared with other 16 ACS Paragon Plus Environment

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analytical approaches for the analysis of Cd, Hg, Pb or Bi. The results are displayed in Table S4. As compared with other relevant chip-based microextraction-ICPMS methods, the LODs of Cd, Hg, Pb and Bi obtained by the proposed microfluidic droplet-based LPME-ETV-ICPMS method were located at the lower level. Compared with our previous chip-based LPME work18, the extraction efficiency of Cd, Hg, Pb and Bi obtained by the proposed microfluidic droplet-based LPME (95.7, 38.9, 36.6 and 24.9%) was remarkably higher than that reported in our previous work based on the laminar flow (20.2, 3.4, 8.6 and 15.9%), which means the pattern of droplets excelled laminar flow in extraction efficiency. Admittedly, the LODs were marginally inferior by comparison with chip-based magnetic solid phase microextraction (MSPME)ETV-ICPMS43. The chip-based MSPME leaned heavily on tedious procedure including preparing magnetic solid-phase packed-column and desorption process. In contrast, the proposed microfluidic droplet-based LPME system was easy to operate and save analysis time extensively. Sample Analysis The proposed method was validated by analyzing target heavy metals in Certified Reference Materials of GBW07601a human hair. As can be seen in Table 3, the determined values were in good agreement with the certified ones, indicating a good accuracy of the proposed method. The cells subjected to ultrasonication for wall breaking were analyzed by the proposed microfluidic droplet-based LPME-ETV-ICPMS method. As shown in Table 4, the values for Cd, Hg, Bi and Pb were determined at the level of sub-fg or fg per cell, respectively. The spiking test was also conducted to validate the accuracy of the proposed method, and good recoveries in the range of 83.5-112.3% were obtained. Conclusions In this work, a novel and elegant design of a microfluidic chip was devised to achieve rapid phase separation and a method of microfluidic droplet-based LPME combined 17 ACS Paragon Plus Environment


with ETV-ICPMS was developed for analysis of trace Cd, Hg, Pb and Bi in cells with a consumption of 350 µL sample of cell lysate and an analytical time of less than ten minutes. The superiority of this approach is its ability to make the oil droplets convert into the laminar flow rapidly. Besides, the proposed method has high extraction efficiency and low LODs. The approach also satisfies the demands of rare sample consumption and has a great application potential in cells analysis. There is no doubt that it is more attractive to quantify trace elements in a single cell even thousands of cells, but this work does not meet the requirement of single cell analysis. Some further research works on the determination of trace elements in a single cell even thousands of cells are under way in our laboratory. Associated Content Supporting Information Additional information on the profile of 2 µL water droplets (Figure S1), microscope images of stable droplets (Figure S2), The SEM of the stainless steel bar’s rough contour’s and a simplified graphic of stainless steel bar’s surface (Figure S3), effect of DDTC concentration on the signal intensity of Cd, Hg, Pb and Bi (Figure S4), effect of flow rate of organic phase on the signal intensity of Cd, Hg, Pb and Bi (Figure S5), line flow rate of the section of inlets (Figure S6), operating parameters of ETV-ICP-MS (Table S1), relevant parameters in our theoretical model (Table S2), tolerance limits of coexisting ions (Table S3) and comparison of analytical performance of this method with other analytical methods (Table S4) as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Author Information Corresponding Author *Tel.: 0086-27-68752162. Fax: 0086-27-68754067. E-mail: [email protected]. 18 ACS Paragon Plus Environment

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Notes The authors declare no competing financial interest.

Acknowledgments This work is financially supported by the National Nature Science Foundation of China (Nos. 21775113, 21575107, 21575108, 21375097), the National Basic Research Program of China (973 Program, 2013CB933900), the Science Fund for Creative Research Groups of NSFC (No. 20921062), the MOE of China, and the Large-scale Instrument and Equipment Sharing Foundation of Wuhan University.



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Figure captions Figure 1 Schematic diagram of the design of a droplet-based microfluidic chip. (a) droplets generation; (b) droplet-based LPME; (c) phase separation. Figure 2 The effect of position of Hole A on the phase separation efficiency. (a) Hole A beyond the main channel leads to the droplets maintained discontinuous; (b) Hole A in the middle of the main channel leads to the droplets turned into water-oil-water laminar flow; (c) Hole A in the right position (occupy 1/4-1/2 of the microchannel) leads to the droplets changed into oil-water laminar flow. Figure 3 (a) The SEM of the gap between the bottom of the bar and the glass slide; (b) The profile of 2 µL water droplets on the smooth surface of the same stainless steel material. Figure 4 Simulation of the flow field in the region of phase separation. (a)-(p) detailed pictures of process for phase separation. The width of the main channel was set as 0.4 mm, the diameter of stainless steel bar was set as 0.6 mm and the contact angle of the stainless steel bar’s surface was set as 30°. Figure 5 Effect of pH on the signal intensity of Cd, Hg, Pb and Bi. (cCd, Hg, Pb, Bi = 2 µg L-1; extraction channel length: 470 mm; main channel width: 200 µm; aqueous phase flow rate: 20 µL min-1; organic phase flow rate: 1 µL min-1; 0.5% (m/v) DDTC) Figure 6 Calibration curves of Cd, Hg, Pb and Bi.



Figure 1


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Figure 2



Figure 3


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Figure 4



Cd Hg Pb Bi

6

1.4x10

6

Signal intensity(counts)

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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1.2x10

1.0x106 8.0x105 5

6.0x10

5

4.0x10

5

2.0x10

0.0 7.0

7.5

8.0

8.5

9.0

9.5

pH

Figure 5


10.0

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Cd Hg Pb Bi Y=222086X+6967 R2=0.992 Y=108252X+3754 R2=0.993 Y=448924X+64478 R2=0.993 Y=620817X+22427 R2=0.998

14000000 12000000 10000000

Counts

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


8000000 6000000 4000000 2000000 0 0

10

20 2+

30 2+

2+

40 2+

50 -1

Concentration of Cd , Hg , Pb and Bi (µg L )

Figure 6



Table 1 The design and experimental results of orthogonal array L9（33） No. Aa (mm) 1 2 3 4 5 6 7 8 9

1850 1850 1850 470 470 940 470 940 940

Ba

Ca (µm)

20:1 300 30:1 200 35:1 400 30:1 400 20:1 200 30:1 300 35:1 300 20:1 400 35:1 200 Order Optimal combination

Counts Cd Hg Pb Bi 62348 17786 936860 800370 102842 81160 1774040 2013370 161309 63122 1523330 2156420 95251 17029 1290810 1550470 79663 31320 989520 1566650 114308 43906 985130 1530200 56943 28854 1263610 1016830 51527 84264 820300 898510 94648 30727 2179230 1958410 B>A>C A>C>B B>C>A B>A>C 1850/35:1/400 1850/30:1/400 1850/35:1/200 1850/35:1/200

a: A, B and C represent the length of extraction channel, the ratio of aqueous phase flow rate to organic phase flow rate and the width of main channel, respectively.


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Table 2 Analytical performance of the microfluidic droplet-based LPME method Analytes Cd Hg Pb Bi a

LODs (ng L-1) 2.5 3.9 5.5 3.4

Linear range (µg L-1) 0.05-20 0.05-50 0.05-20 0.05-20

Linear Equation y=222x+7 y=108x+4 y=449x+64 y=621x+22

Determination coefficient (R2) 0.992 0.993 0.993 0.998

n=7; cCd, Hg, Pb, Bi = 0.2 µg L-1.


RSDa (%) 7.8 7.1 7.5 6.2

EF 33.5 13.6 12.8 8.7


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Table 3 Analytical results (mean±s. d., n=3) for the determination of heavy metals in Certified Reference Materials of GBW07601a human hair Elements

Found (µg g-1）

Certified (µg g-1）

T-test value (t0.05,2=4.30)

Cd Hg Pb Bi

0.010±0.001 0.040±0.003 0.92±0.045 0.032±0.004

0.011±0.003 0.036±0.008 0.88±0.011 0.034±0.002

1.73 2.31 1.54 0.87


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Table 4 Analytical results (mean±s. d., n=3) for the determination of heavy metals in cells Elements HeLa Cd Hg Pb Bi HepG2 Cd Hg Pb Bi a

Found (µg L-1)

Average (fg cell -1)

Add (µg L-1)

Found (µg L-1)

Recovery (%)

0.31±0.05a 0.30±0.04a 2.73±0.42a 0.20±0.01a

0.31±0.05 0.30±0.04 2.73±0.42 0.20±0.01

0.2 0.2 2.0 0.2

0.50±0.04 0.50±0.08 4.86±0.60 0.42±0.06

95.1 101.1 106.7 112.3

0.67±0.01a 0.13±0.03a 5.40±0.31a 0.78±0.07a

0.67±0.01 0.13±0.03 5.40±0.31 0.78±0.07

0.5 0.5 5.0 0.5

1.23±0.11 0.58±0.07 10.04±0.83 1.19±0.18

110.6 88.5 92.6 83.5

: determined value for target elements in 350,000 cells in 350 µL.



For TOC only


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Facile Design of Phase Separation for Microfluidic Droplet-Based

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