A Facile Droplet-Chip-Time-Resolved Inductively Coupled Plasma

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A facile droplet-chip-time-resolved ICPMS online system for determination of zinc in single cell Han Wang, Beibei Chen, Man He, and Bin Hu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00134 • Publication Date (Web): 11 Apr 2017 Downloaded from http://pubs.acs.org on April 13, 2017

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Analytical Chemistry

A facile droplet-chip-time-resolved ICPMS on-line system for determination of zinc in single cell Han Wang, Beibei Chen, 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 author: Fax: +86-27-68754067; Tel: +86-27-68752162; Email: [email protected]

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ABSTRACT: Single cell analysis is a significant research field in recent years reflecting heterogeneity of cells in biological system. In this work, a facile droplet chip was fabricated and on-line combined with time-resolved inductively coupled plasma mass spectrometry (ICPMS) via a micro-flow nebulizer for the determination of zinc in single HepG2 cells. On the focusing geometric designed PDMS microfluidic chip, the aqueous cell suspension was ejected and divided by hexanol to generate droplets. The droplets encapsulated single cells remain intact during the transportation into ICP for subsequent detection. Under the optimized conditions, the frequency of droplet generation is 3-6×106 min-1, and the injected cell number is 2500 min-1, which can ensure the single cell encapsulation. ZnO nanoparticles (NPs) were used for the quantification of zinc in single cells, and the accuracy was validated by conventional acid digestion-ICPMS method. The ZnO NPs incubated HepG2 cells were analyzed as model samples, and the results exhibit the heterogeneity of HepG2 cells in the uptake/adsorption of ZnO NPs. The developed on-line droplet-chip-ICPMS analysis system achieves stable single cell encapsulation and has high throughput for single cell analysis. It has the potential in monitoring the content as well as distribution of trace elements/NPs at single cell level.


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INTRODUCTION Cells are the basic unit in organism consisting of DNA, RNA, proteins, small organic molecules, and inorganic ions1. Zinc, as an essential trace element, is the cofactor to more than 300 enzymes, and its major role in cells is to stabilize the structure of proteins. Zinc has critical effect in homeostasis, immune function, oxidative stress apoptosis, aging and chronic disease such as cancer, diabetes, depression, Wilson’s disease, Alzheimer’s disease and other age-related diseases2,3. In recent years, plenty of evidence has shown that cellular heterogeneity is commonly existing and can affect the physiological property of cells. Therefore, to figure out how zinc affects the cell functions, it is prerequisite to develop suitable analytical method for the detection of zinc in single cells. So far, much effort has been made in single cell analysis4-6, and several techniques have been applied for the biological and chemical analysis of single cell. Microscopic imaging is the most commonly used technique for visualization of single cells, but assays on single cells are difficult to perform. The patch-clamp technique enables to measure the change in ion channels of single cell, but it requires high skills to perform. Flow Cytometry is a high throughput technique for single cell analysis and is widely used in detection, sorting and collection of interested cells7. In addition, microfluidic chips are the powerful platforms for single cell analysis with the advantages of high throughput, comparable size for single cell and ease to combine with many detection techniques (such as fluorescence and electrochemistry) 7-10. Droplet-chip is one of the most widely used chip for single cell analysis which allows the isolation of single cell in picoliter liquid at high throughput of thousands droplet per second, and is easy to perform and control. Recently, droplet chips have been used for enzyme analysis11,12, genetic analysis13,14 and drug screening15 of single cell, featuring with high throughput16,17 and time-resolved ability18,19. However, no such application has been reported for quantitative analysis of zinc in single cell. Inductively coupled plasma mass spectrometry (ICPMS) is the most powerful technique for trace elemental analysis and has been applied for single cell analysis20-23. Li et al.24 first reported the single cell analysis by ICP-magnetic sector-MS and observed U+ spikes of individual Bacillus subtilis. Commercial ICP-quadrupole (Q)-MS merits lower cost and higher popularity than other modes of ICPMS, and has also been used for the analysis of trace elements in single cell. Ho et al.25,26 applied time-resolved ICPMS for 3 ACS Paragon Plus Environment


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the analysis of trace elements in unicellular alga and Helicobacter pylori cell. Besides, laser ablation (LA)-ICPMS could also be used in single cell analysis28,29. Based on the elemental labeling, Bendall et al.29 combined transition element isotopes as chelated antibody tags with ICP-time of flight (TOF)-MS for the analysis of 31 antibodies, viability, DNA content and relative cell size of single cell. Above works demonstrate that ICPMS is a powerful technique for trace elemental analysis in single cell as well as biomolecules by elemental tagging strategy. However, since the generation of aerosol in pneumatic nebulization (PN) system is random, it is hard to make sure whether a signal peak represents single cell or multiple cells when PN is employed as the sample introduction system in ICPMS measurement. For example, Ho et al.24 controlled the probability of the presence of an algal cell in an aerosol droplet as low as 6×10-5 (by the cell density), but the chance of having two algal cells in one peak was still 0.08%. Therefore, it is impossible to make sure whether some unusual spikes are the signals of two or more cells or the evidence of cellular heterogeneity. To solve the problem, the droplets generator was combined with ICPMS for the single cell analysis30,31. Compared with direct PN of cell suspension solution, the droplets help to isolate cells and ensure the spikes come from single cells. Microfluidic chips are proved to be excellent platforms for the generation and manipulation of droplets. Recently, Verboket et al.32 combined droplet-chip with ICPMS for single cell analysis: a crosschannel droplet chip was fabricated for the generation of 50 µm droplets, the highly volatile perfluorohexane was used as organic phase on the chip; after the generation of droplets, perfluorohexane was removed before ICPMS detection through the custom-built transport system of heater and membrane desolvator. The aqueous phase droplets containing single cells were determined by ICPMS and the content of Fe in single red blood cell was investigated. However, the chips as well as transport system are complicated and difficult to fabricate which make the approach hard to perform. Therefore, an on-line droplet chip-ICPMS single cell analysis system with the merits of simplicity and easy-to-operate is in urgently needed. In this work, a droplet microfluidic chip was fabricated and on-line combined with time-resolved ICPMS for the analysis of zinc in single HepG2 cells. The droplet chip is easy to fabricate and could be directly connected with micro-flow nebulizer of ICPMS. 4 ACS Paragon Plus Environment

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What is more, by using miniaturized nebulization system and optimizing experimental parameters of droplet-chip-ICPMS analysis system, the instability of the plasma caused by introduction of large amount of organic solvent can be avoided. Several parameters such as the kinds of organic phase, the dimensions and structure of droplet chip, the liquid flow rates on the chip, the interface between droplet chip and ICPMS as well as the detection conditions of ICPMS have been systematically investigated. The signal peaks of single HepG2 cells were obtained by the developed method, and droplets containing ZnO nanoparticles (NPs) was used for the quantification of zinc in single cell. The ZnO NPs incubated HepG2 cells were also used as the model samples to demonstrate the potential of the developed method for metal-containing NPs study at single cell level.

EXPERIMENTAL SECTION Apparatus. An X Series II ICPMS (Thermo Fisher Scientific, USA) with a micro-flow nebulizer (PFA Micro-Flow Nebulizer, Elemental Scientific, Omaha, USA) was used in this work. The operation conditions for ICPMS are summarized in Table S1. According to the user’s manual, the self-aspiration flow rate (1L/m Ar) of PFA nebulizer is 20-35 uL/min, and the nebulizer efficiency is ca. 10%. 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), microbore autoanalysis tubing (Tygon, Cole-Parmer, USA) and sterile syringes were applied for liquid introduction on the chips. An XS105 balance (Mettler Toledo Instruments Co., Ltd., Shanghai, China) was used for weighing. The commercial ZnO NPs were characterized by dynamic light scattering (Zetasizer Nano ZS, Malvern, UK). Bright-field microscopy was performed on a Nikon microscope (Ti-U, Tokyo, Japan) with a CCD camera (Nikon DS-Ri1). Reagents and standard solutions. Suspension solution of ZnO NPs (1 mg mL-1 of ZnO, 0.1% (m/v) sodium pyrophosphate (Merck, Germany), pH=9.0) was prepared from analytical reagent grade of ZnO (200±10 nm, Aladdin, Shanghai, China). The phosphate buffer solution (PBS, pH 7.4) consists of 0.01 mol L-1 phosphate-buffered saline. The 5 ACS Paragon Plus Environment


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suspension solution consists of 1% hydroxypropyl methyl cellulose in PBS was used for the dispersion of HepG2 cells and dilution of ZnO NPs as aqueous phases on droplet chips. Hexyl alcohol (Aladdin, Shanghai, China) with 1% Span 80 (Aladdin, Shanghai, China) was used as organic phase on droplet chips. Polydimethylsilicone (PDMS) was prepared by mixing oligomers (component A) with crosslinking agents (component B) (GE RTV 615, Momentive performance materials, NY, USA) at a ratio of 1:10. DMEM culture medium and fetal bovine serum (FBS) were obtained from Gibco Invitrogen Corporation. Sub-boiled HNO3 was used in all experiments. All other reagents used were at least analytical reagent grade. Highly pure 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.

Design of microfluidic devices. Flow focusing geometric design was used to construct the droplet chip in this work. The width of cell suspension channel, organic phase channel, droplet generation channel and flow focusing channel were 200, 200, 150 and 75 µm, respectively. The height of channel was 50 µm. The design sketch of the droplet chip is shown in Fig. 1A. Fabrication of PDMS microfluidic devices was illustrated in Supporting Information.

The operation on the droplet chip. In the developed system, all fluids were supplied by TS2-60 syringe pumps and 1 mL sterile syringes. The aqueous sample solution was introduced into the chip at a flow rate of 5 µL min−1, and two organic phase flow for the generation and separation of droplets was delivered at flow rate of 12.5 µL min−1 with two syringes. The generation of droplets (as shown in Fig. 1B) was stabilized in 3 min, and then the chip was connected to the micro-flow nebulizer for ICPMS analysis. To investigate whether the single cell encapsulated in one droplet can be well maintained during its transportation from the outlet of droplet chip to the micro-flow nebulizer, droplets from the nebulizer (without adding nebulizer gas) were collected on glass slides and then observed by microscopy measurement immediately. Briefly, droplets on the slides were first observed by a microscope with 10× magnification. Droplets contain6 ACS Paragon Plus Environment

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ing cells were further observed by 40× magnification to record the cell number in them. After 100 droplets containing cells was randomly found and recorded, the proportion of droplets containing single cell was calculated by the number of droplets containing single cell divided by 100.

Design of the interface between a droplet chip and ICPMS. A 75 µm ID fused silica capillary was chosen to connect the outlet of the chip with the micro-flow nebulizer assorted in ICPMS. The length of fused silica capillary was chosen as 3 cm, one end of the fused silica capillary was fixed to the outlet of the chip with cyanoacrylate glue, the other end was directly connected to the micro-flow nebulizer via Teflon tubing (250 µm ID, Thermo, USA). The schematic diagram is shown in Fig. 1C.

Cell culture and sample preparation. HepG2 cells were maintained in a humidified incubator containing 5% CO2 and 95% air at 37 °C, and incubated in DMEM medium, supplemented with 10% FBS. After the cell density reached approximately 1.0×106 mL-1, HepG2 cells were detached by trypsinization using a 0.25% trypsin-EDTA solution, and collected by centrifugation (1500 rpm, 5 min), the supernatant was removed and the cells were washed with PBS twice. Then the number of cells was counted, and the density of cells suspension was diluted to 5.0×105 HepG2 cells per milliliter. As a comparison, the cell samples were also analyzed by conventional ICPMS with acid digestion. 1.0×106 HepG2 cells were dispersed in 0.25 mL PBS, then cell suspension was put into PTFE digestion vessels. After adding 2.0 mL of HNO3, the vessels were put on an electric hot plate (120 ºC) for 2 h and maintained at 80 ºC. When the samples were nearly dryness, the digest was transferred and diluted with 5% HNO3 to 1.0 mL. The obtained solution was then subjected to ICPMS measurement directly. HepG2 cells incubated with ZnO NPs were used as the model samples to validate the applicability of the developed on-line droplet chip-ICPMS single cell analysis system. The cells with the number of 1.0×106 were first seeded in one well of a six-well plate, after all



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the cells were adhered, the cells were incubated with 1.0 µg mL-1 of ZnO NPs (200 nm) for 24 h. The stability of ZnO NPs in culture medium was also investigated. The culture medium contained 1.0 µg mL-1 ZnO NPs were prepared in six-well plates and put in the humidified incubator containing 5% CO2 and 95% air at 37 ºC for 24 h. Then the culture medium was subjected to centrifugation (15,000 rpm for 15 min) and the supernatant were introduced into electrothermol vaporization (ETV)-ICP-MS for the determination of Zn. The results showed that only 0.92 % Zn was release from ZnO NPs in the cell culture medium for 24 h, indicating an acceptable stability of ZnO NPs in culture medium.

Data processing. The Origin Pro 8 and Microsoft Excel 2010 were used for the analysis of data. For Time-resolved ICPMS, an iterative algorithm based on the three times standard deviation (3σ) was used to distinguish single cell/ZnO NPs events from background signal. The average signal value and standard deviation of the entire dataset in 2 min were first calculated and the data 3σ above the average signal value are collected. Then the reduced dataset was recalculated with the same method until no data points were higher than the sum of 3σ and average signal value. The number and intensity of signals higher than the sum of 3σ and average signal value were recorded and then analyzed by Gaussian Fitting.

RESULTS AND DISCUSSION On-line Droplet Chip-ICPMS system. To obtain stable single cell encapsulated droplets for on-line ICPMS determination, several factors should be taken into consideration. 1) The organic phase used in droplet chip should be carefully studied to be compatible for on-line ICPMS detection. 2) The geometric structure of the chip should be designed for a stable generation and isolation of droplets on chip. 3) The interface between the droplet chip and ICPMS greatly affects the stable transportation of droplets into ICPMS. 4) The total flow rate of the effluent from the droplet chip needs to match the sample uptake rate of ICPMS detection, and a stable baseline of ICPMS detection should be ensured.


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Choice of the Fluids on the Droplet Chip. On the droplet chip, organic phase is always used for the generation of aqueous phase droplets which leads to a match problem between the effluent of droplet chip and ICPMS detection. Generally, the effluent from the droplet chip should be suitable for subsequent ICPMS detection. On droplet chip, high ratios of organic/ aqueous phase are often applied for the generation of stable and small droplets (for the benefit of single cell encapsulation). However, for conventional PNICPMS measurement, the tolerable limit of organic phase is low (normally < 10%). To solve this problem, a micro-flow nebulizer and the addition of O2 gas were employed for ICPMS detection in this work. And for droplet chip system, the kinds of organic solvent should be optimized. Frequently used organic solvents in droplet chip system include silicon oil, hydrocarbon oil, and fluorinated oil33. However, most of them are not suitable for ICPMS measurement due to relatively high carbon content. Alcohol with the merits of high viscosity and low carbon content is a good alternative for coupling droplet chip to ICPMS. With the higher viscosity of alcohol, lower flow rate was required for stable generation of droplets, resulting in less amount of organic phase introduced into ICPMS. As a result, five kinds of alcohol: amyl alcohol, hexyl alcohol, heptyl alcohol, capryl alcohol and dodecyl alcohol were investigated. Results demonstrate that hexyl alcohol, heptyl alcohol, capryl alcohol and dodecyl alcohol were capable of generating stable droplets in chip channel. Finally, hexyl alcohol was chosen as the organic phase for the lowest carbon content. In addition, to stabilize the aqueous phase droplets in hexyl alcohol during the transportation to ICPMS, 1% (v/v) Span 80 was added into the organic phase.

Droplet Microfluidic Device. After fixing the organic phase, the geometric design of chip and the flow rate of fluids are the key points for single cell encapsulation. According to the reported droplet chip system8, a simple focusing geometric design was employed and the geometric design of the chip is shown in Fig. 1A. The channels narrow down in the focusing section helps to generate stable aqueous phase droplets in chip channel. The geometric parameters of chip channels were carefully optimized, and the details of the parameters are listed in Table S2. By fixing the total flow rate of 2.2 µL min-1 with the ratio of aqueous/organic phase as 1/10, different width of the chip channels was employed to 9 ACS Paragon Plus Environment


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investigate the generation of droplets, and the microscopy images are shown in Fig. S1. As can be seen, along with the decrease of channel width, smaller droplets were generated which would be benefit for single cell encapsulation. Therefore, the width of flow focusing channel, cell suspension channel, organic phase channel and droplet generation channel were chosen to be 75, 200, 200 and 150 µm, respectively. Then the length of chip channels was optimized with the aqueous and two organic phases flow rates of 5, 12.5 and 12.5 µL min-1, respectively. Since the droplets in the channel are produced in jetting regime (shown in Fig. 2A and 2B), a certain length of droplet generation channel is required for droplet generation. The results indicate that length of 3 mm is enough for the stable generation of droplets after flow focusing. To reduce the pressure of the whole chip system, the length of the aqueous, organic phase and droplet generation channels were chosen to be 2, 2 and 3 mm, respectively. Interface. The interface between droplet chip and ICPMS should meet the following requirements: size compatibility between the connecting tube and the chip channel to avoid droplets merging, good mechanical stability, good leakproofness and easy-to-fabricate. Among these requirements, the suspension stability of droplets is the most significant. In this work, fused silica capillary was used to connect the outlet of droplet chip with the micro-flow nebulizer assorted in ICPMS, and the effect of different inner diameter of capillary (i.d. 50, 75 and 150 µm, respectively) on system stability was investigated (the length of the capillary was 3 cm and the total fluid flow rate ranged from 20 to 30 µL min-1). Results demonstrated that when the i.d. of the capillary was 50 µm, the interface would be damaged because of high systematic pressure, while stable droplets could be obtained when the silica capillary was 75 and 150 µm i.d., respectively. To obtain good mechanical stability and control the whole systematic pressure, a 3 cm long fused silica capillary with 75 µm i.d. was plugged into a Teflon tubing to interface the droplet chip and micro-flow nebulizer of ICPMS.

Flow Rate of Organic and Aqueous Fluids. The flow rate of organic and aqueous fluids is one of the important factors. It not only influences the generation of stable droplets on the chip, but also influences the size of droplets which is of great significance for single cell encapsulation. What is more, the flow rate should match the sample uptake rate of micro10 ACS Paragon Plus Environment

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flow nebulizer to obtain a stable baseline in ICPMS detection. Thus, different flow rates of aqueous and organic phase were investigated for the on-line droplet chip-ICPMS system, and both microscopy and ICPMS measurements were performed to optimize the generation of droplets and record ICPMS signal. Results of ICPMS measurement demonstrate that the stable baseline could be obtained when the total flow rate reached 20 µL min-1 with the flow rate of aqueous phase equal to or more than 6 µL min-1 (Fig. S2A, B and C). However, in such cases, the flow rate ratio of organic phase to aqueous phase is too small to form droplets in the chip channel. Therefore, the total flow rate was raised and the flow ratio of aqueous and organic phase was adjusted. When the aqueous and two organic phases flow rates were 5, 12.5 and 12.5 µL min-1, respectively (total flow rate is 30 µL min-1), both stable droplets (Fig. 2A, B and C) and baseline of ICPMS (Fig. S2D) was obtained. As can be seen from the image of collected droplets after nebulization (Fig. 2C), the average diameter of the formed droplets is 25 µm. In such conditions, droplets could be generated stably in chip channel with the frequency of ~3-6×106 droplets per minute. Therefore, 5, 12.5 and 12.5 µL min-1 were chosen as the aqueous and two organic phase flow rates.

Single cell encapsulation. After the on-line droplet chip-ICPMS system was established, the following issue is how to obtain single cell encapsulated droplets for single cell analysis. In droplet chips, the process of loading cells into droplets is purely random, and the distribution is dictated by Poisson statistics. It means that to decrease the number of droplets containing more than one cell, low cell density (relative to the density of generated droplets) should be employed. To investigate the single cell encapsulation in the droplet chip, the microscopy measurement was performed. With the fixed flow rate of aqueous, organic phase and the design of chip channel, the parameter influences single cell encapsulation is the density of HepG2 cell suspension. The cell density was then optimized with the cell suspension of 2.0×106, 1.0×106, 5.0×105 and 2.0×105 cells per milliliter. The proportions of droplets contained single cell and more than one cell (obtained by microscopy measurement) are shown in Table S3. As can be seen, with the cell density equal to or lower than 5.0×105 cells per milliliter, no droplet containing more than one cell was observed. The theoretic calculation was also conducted. Since the frequency of 11 ACS Paragon Plus Environment


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droplet generation is 3-6×106 min-1, the injected cell number is 2500 min-1, the probability of droplets contained two cells is lower than 3.4×10-5 %. To ensure the single cell encapsulation and gain as many single cell encapsulated droplets as possible in a certain period of time, the cell density of 5.0×105 cells per milliliter was chosen. Under such conditions, there were ca. 100 droplets that contain one cell in about 3 seconds. Theoretically, the injected cell number is 2500 min-1 (the cell density is 5×105 cells per mL and the flow rate is 5 µL min-1), there would be 125 droplets containing one cell in 3 s. So the real recovery of cells in droplets should be higher than 80%. The microscopy image for the collected droplets after ICPMS nebulizer is shown in Fig. 2D. No merging of droplets was observed during the transportation, the average diameter of HepG2 cells is 8.0 µm, and the average diameter of droplets is 25 µm.

Single cell analysis and quantitation of zinc in HepG2 cells. The developed system was then applied for the single cell analysis of zinc in HepG2 cells. In order to obtain sensitive and accurate signals of single droplet/cell by ICPMS, the dwell time (tdwell) need to be investigated. Figure 3 presents the temporal profile of zinc in single cells by ICPMS with different tdwell (1, 2.5, 5 and 10 ms), and the number of peaks and detected efficiency were calculated and listed in Table S4. The detected efficiency is mainly influenced by two factors in the developed system: integration time and transport efficiency. When the Dwell time is 10 ms, the obtained detected efficiency is obviously lower than others, indicating a long integration time would cause the decrease of the detected efficiency. When the Dwell time was equal to/lower than 5 ms, the detection efficiency almost kept the same. It indicates that the Dwell time ≤ 5 ms is short enough and has no significant influence on detected efficiency. In such conditions, the detection efficiency is mainly ascribed to the transport efficiency which is determined by the nebulization efficiency of PFA nebulizer and other ICP-MS parameters. Since lower Dwell time means higher scan frequency which would lead to more loading capacity of ICP-MS, 5 ms was chosen to be the Dwell time for single cell analysis, and the detection efficiency is 2.96%. Compared with the reported approaches30-32, this work has a lower detected efficiency but very high throughput (frequency of droplet generation is 3-6×106 droplets per minute) (detailed comparison of detected efficiency and throughput is shown in Table S5). 12 ACS Paragon Plus Environment

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The data obtained by the developed on-line droplet chip-single cell analysis system shown in Fig. 4 provide a distribution pattern of zinc in individual HepG2 cells and reflect heterogeneity of cells (ICPMS temporal profile of zinc in single HepG2 cell are shown in Fig. S4). The relative frequency of different signal intensity of single cell follows the Gaussian distribution, and the mean of the distribution is 17429 CPS. According to Ho’s work24, calibration using particles would correct the errors due to difference in diffusion loss of elemental atoms originated from aqueous standards and the cells. When one cell was encapsulated into a droplet of 25 µm, the matrix in the droplet is PBS buffer, and zinc atoms converged in the cell. Compared with a zinc ions solution droplet which is homogeneous, a droplet contained a ZnO nanoparticle is much more similar to a droplet contained a cell because they possessed the same matrix and the same character that the zinc atoms converged in a certain section in the droplets. In addition, since particle sublimation time θ which is defined as the time to increase the temperature of ~200 nm ZnO NPs from an average particle temperature to final temperature of 4200 k (Tf) in ICP is just 2.54×10-8 s, ZnO NPs (~200 nm) could be well ionized in the ICP (detailed discussion shown in supporting information). Therefore, ZnO NPs were used as the standard for the quantitation of zinc in single HepG2 cells. The suspension stability and average size of ZnO NPs were determined by dynamic light scattering, and the results are shown in Fig. S3. As can be seen, the average size of ZnO NPs is 222 nm which demonstrates that all ZnO NPs are monodispersed. Since the density of cell samples was 5×105 cells per milliliter, 20 ng L-1 ZnO NPs dispersion (almost 5×105 particles per milliliter, the detection efficiency of ZnO NPs is 2.85%) was applied as the aqueous phase on the droplet chip for the quantitation of zinc in single HepG2 cells. The signal peaks of ZnO NPs determined by time-resolved ICPMS were obtained and calculated. As can be seen in Fig. 4, the distribution of ZnO NPs also follows the Gaussian distribution, and the mean of the distribution is 20653 CPS. Considering the average diameter of ZnO NPs is 222 nm, the number of zinc atoms in one ZnO NP is estimated to be 2.4 × 108, assuming spherical particles density of 5.606 g cm-3. Therefore, the mean of the Gaussian distribution of ZnO NPs as 20653 CPS represents 2.4×108 zinc atoms. Correspondingly, the content of zinc in single HepG2 cells (17429 CPS) could be estimated by the calibration of ZnO NPs, and the number of zinc atoms in 13 ACS Paragon Plus Environment


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single HepG2 cell was estimated to be 2.0 × 108 (corresponding to 21.7 fg). For a comparison, the cell samples were also analyzed by conventional ICPMS after acid digestion. It demonstrated that there are 2.3×108 (25.0 fg) of zinc atoms in single HepG2 cell, which is in a good agreement with the results obtained by the developed method. The results verified the accuracy of the developed on-line droplet chip-ICPMS single cell analysis system.

Detection of zinc in ZnO NPs incubated HepG2 cells by time-resolved ICPMS. The ZnO NPs incubated HepG2 cells were used as the model samples for the developed online droplet chip-ICPMS single cell analysis system and the results of cells incubated with ZnO NPs are shown in Figure 5a. As can be seen, a main peak with the means of 19766 CPS of Gaussian distribution representing 2.3 × 108 zinc atoms is similar to that obtained for HepG2 cells without incubation with ZnO NPs (Fig. 4a), indicating that these cells did not uptake/adsorb ZnO NPs. To reveal the cells uptaken/adsorbed ZnO NPs more clearly, a subtraction of numbers of HepG2 cells incubated with ZnO NPs from numbers of HepG2 cells without incubation with ZnO NPs has been conducted, and the results was shown in Figure 5b. As can be seen, there are three peaks with the means of 49374, 67464 and 81739 CPS of Gaussian distributions, which represent 5.6 × 108, 7.6 × 108 and 9.4 × 108 zinc atoms. In other words, there are four different concentration levels of zinc in HepG2 cells, which represent the cells without ZnO NPs (contained 2.3 × 108 zinc atoms), uptaking/adsorbing one ZnO NP (contained 5.6 × 108 zinc atoms), uptaking/adsorbing two ZnO NPs (contained 7.6 × 108 zinc atoms) and uptaking/adsorbing three ZnO NPs (contained 9.4 × 108 zinc atoms). Among them, the proportion of the cells without ZnO NPs is about 71%, and proportion of the cells uptaken/adsorbed NPs is about 29%. However, it should be mentioned that since this method is for total amount determination of Zn in one cell, it is hard to differentiate whether a certain higher signal (for example higher than 60,000 CPS) comes from the atypical cells containing high amount of zinc or from cells uptaken/adsorbed one NP or two ZnO NPs. For comparison, the ZnO NPs incubated cell samples were also analyzed by conventional ICPMS with acid digestion and the results show that the average amount of zinc per cell is 46.7 fg with the average number of ZnO NPs of 1.13 per cell. It is worth noting 14 ACS Paragon Plus Environment

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that the determination of total zinc in the cells can only provide average information of cells, but the developed method is able to analyze the uptake/adsorption behavior of NPs on single cell level which is more valuable than conventional ICPMS detection. These results also show the heterogeneity of HepG2 cells. As can be seen, in one group of cells with the same incubation conditions, some cells would not uptake/adsorb NPs, some cells would uptake/adsorb one NPs and some cells would uptake/adsorb more NPs, which reveals the different characteristics of individual cell. Compared with direct time-resolved PN-ICPMS method for single cell analysis, any possibility that more cells in one aerosol during PN is avoided in the proposed on-line system which is very important for cell heterogeneity study.

CONCLUSION In this work, single cell encapsulation was realized on a facile droplet chip with high throughput. The fabricated droplet chip was directly on-line connected to time-resolved ICPMS for the quantification of zinc and ZnO NPs uptake/adsorption in single HepG2 cells without any special instruments or apparatus. Compared with the reported approaches30-32, this work has a lower detected efficiency (2.96%) but very high throughput (frequency of droplet generation is 3-6×106 droplets per minute). What is more, the proposed method has a unique feature that all the cells were single encapsulated and detected by the developed on-line droplet chip-ICP-MS system. No incorrect single cell information is essential for heterogeneity analysis of cells. Besides, the developed on-line droplet chip-ICPMS single cell analysis system is facile and easy-to-operate, exhibiting good application potential in monitoring the content as well as distribution of trace elements/NPs in single cell.

SUPPORTING INFORMATION Additional information on fabrication of PDMS microfluidic devices, microscopy images of the generation of droplets (Figure S1), ICPMS signal diagrams of on-line droplet chipICPMS analysis system (Figure S2), size distributions of ZnO NPs (Figure S1), optimized operating conditions for ICPMS (Table S1), parameters of investigated droplet chips (Table S2), proportion of droplets contained single cell and more than one cell (Ta15 ACS Paragon Plus Environment


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ble S3), peak numbers and detection efficiencies determined by ICPMS at various tdwell (Table S4) were shown in the supporting information, and comparison of detected efficiency and throughput of the developed method and other published work (Table S5) were shown in supporting information. This material is available free of charge via the Internet at http://pubs.acs.org.

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


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Figure captions Figure 1. Schematic diagrams of chip design (a), droplets generation (b) and on-line droplet chipICPMS single cell analysis system (c). Figure 2. Microscopy images of generated droplets (A, B), collected droplets after nebulization (C) and collected droplets containing single cell after nebulization (D). Figure 3. ICPMS temporal profile of zinc in HepG2 cells determined by developed on-line droplet chip-ICPMS system at different tdwell (1-10 ms). Figure 4. Intensity distributions of zinc signal peaks in HepG2 cells (a) and ZnO NPs (b) (Baseline intensity has been deducted, the background signals are 6275 CPS for HepG2 cells and 7001 CPS for ZnO NPs. The detection time is 24 min). Figure 5. Intensity distributions of zinc signal peaks in HepG2 cells incubated with ZnO NPs (Baseline intensity has been deducted, the background signal is 9106 CPS, and the detection time is 36 min) (a) and intensity distributions of zinc signal peaks after subtraction of data for HepG2 cells without incubation with ZnO NPs (b).



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Figure 1.

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Figure 2.

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Figure 3.

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HepG2 Cell

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for TOC only

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A Facile Droplet-Chip-Time-Resolved Inductively Coupled Plasma

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