3D Droplet-Based Microfluidic Device Easily Assembled from

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A 3D Droplet-Based Microfluidic Device Easily Assembled from Commercially Available Modules Online Coupled with ICPMS for Determination of Silver in Single Cell Xiaoxiao Yu, Beibei Chen, Man He, Han Wang, and Bin Hu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04844 • Publication Date (Web): 17 Jan 2019 Downloaded from http://pubs.acs.org on January 22, 2019

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Analytical Chemistry

A 3D Droplet-Based Microfluidic Device Easily Assembled from Commercially Available Modules Online Coupled with ICPMS for Determination of Silver in Single Cell Xiaoxiao Yu, Beibei Chen, Man He, Han Wang, 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] 1

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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

Abstract More recently, single-cell analysis based on ICPMS has made considerable headway while a challenge remains to differentiate single cell from doublets during the analysis. One burgeoning solution is to encapsulate single cell into droplets on the platform of the microfluidic chip. However, the manufacture of the droplet-based microfluidic chip requires sophisticated fabrication, and limit its potential application. In this paper, we presented an off-the-shelf 3D microfluidic device by assembling commercial available parts without any proficient manufacturing process. Uniform monodisperse microdroplet was generated from the 3D microfluidic device with a size variation of 1.5 % and the innner diameter of 3D microfluidic device was the same as the nebulizer (150 μm). The proposed 3D microfluidic device-time resolved ICPMS system was applied to detect silver in single AgNPs (51 nm), and the result is in good agreement with conventional acid digestion method, demonstrating the accuracy of the method. Silver uptake behaviors in HepG2 cells were then studied by incubating with Ag+ or AgNPs under biocompatible conditions. The results revealed the cell-to-cell variability in terms of that the diversity of cells incubated with AgNPs was wider than those cells incubated with Ag+ from the aspect of the content distribution of silver at single-cell level. Keywords: 3D microfluidic device; co-flowing; inductively coupled plasma mass spectrometry; single cell; silver

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Introduction A plethora of substantial evidences of cell heterogeneity have led to a consensus that the mean value derived from the bulk measurement could not represent the actual behaviour of either subpopulation1,2. Thus, single-cell analysis is extremely valuable and becomes acting as the fuel to drive developments in many fields. However, grand challenges remain in single-cell analysis3: (1) tiny size of single cells; (2) scarce absolute amount of target analytes; (3) serious matrix effects caused by complex intracellular components. As a consequence of these difficulties, we starve for a reliable method possessing the feature of single-cell resolution, high sensitivity, high tolerance to matrix interference as well as high throughput. Due to the ability of broad linear dynamic range and extremely low detection limits for many elements, inductively coupled plasma mass spectrometry (ICPMS) has been generally acknowledged as one of the most robust tools in the field of single-cell analysis4,5. Admittedly, there has been an enormous escalation in single-cell analysis based on ICPMS over recent years. The drawback associated with direct introduction of cells into ICP is that the possibility of two or more cells co-existing in one ion plume cannot be excluded completely in their works. In the last decades, microfluidic systems with the characteristic of micrometer-scale channel network has emerged as an ideal platform to manipulate single cells and minimizes the consumption of reagents6. Particular attention has been devoted to droplet-based microfluidic devices owing to remarkable advantages7,8 including an independent reactor without cross-contamination, precise manipulation and fast mass transfer kinetic. In order to circumvent the chance of several cells co-existing in one aerosol droplet, making single cell compartmentalized in one droplet is generally accepted as one of the most sensible solutions. As already demonstrated by Verboket et al.,9 and Wang et al.,10 online droplet chip-ICPMS single-cell analysis systems were fabricated successfully to investigate the content of Fe and Zn in single cells, 3



respectively. However, the generation of droplets is strongly dependent on the wettability of the chip channels in quasi-two-dimensional (2D) microfluidic device, because it is inevitable for droplets to contact with the walls of the channels. Increasing the flow rate of continuous phase circumvents the direct contact between droplets and channels with the sacrifice of an increase in possibility of leaking in chips. Unfortunately, the most common substrate material used for microfluidic chips, such as polydimethylsiloxane (PDMS), usually has limited ability to suffer high pressure. Besides, an extra design of interface between microfluidic chip and the nebulizer is generally required. Distinct from 2D chips, the dispersive phase in 3D chips is surrounded by continuous phase, avoiding the touch of channels’surface11. In addition, the tolerance of pressure will be improved intensively in the whole system of 3D chip, which benefits from used commercial materials, such as poly(ether ether ketone) (Peek), Teflon, glass. From these vantage points, much efforts have been dedicated to fabricate 3D microfluidic device and the prevalent approaches are summarized as follows12: (1) micro electro mechanical systems (MEMS) technology; (2) moding; (3) 3D printing technology; (4) modular assembly. For powerful MEMS technology, just like femtosecond laser irradiation, expensive apparatus with great precision is required13. Based on the principle of soft lithography that the height of channels can be adjusted by the types of photoresists and the rotational speeds of spin-coating14,15, needless to say, moding is complicated and time-consuming16,17. As for 3D printing technology, the size of droplets is always more than 50 μm18,19, suffering from either low resolution or opaque and coarse printing materials20,21. In recent years, great efforts have been devoted to modular assembly by using off-the-shelf 3D micro structure, which can be classified into four main categories. (1) Fused silica capillary was embedded in the internal channel of polymethyl methacrylate (PMMA), PDMS or 3D printed substrates to assemble a microfluidic device, following different droplets generation principles, such as co-flowing22-26, flow-focusing27 or break-up at a T-junction28,29. (2) Customized 4


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adapters and T-shaped connectors were applied to generate microdroplets30,31. However, it is lack of universality. (3) In some works, although the microfluidic device was assembled by commercial off-the-shelf modules, it was required to post-manufacture by a micropipette puller32-36, a diamond lapping machine37, a laser cutting instrument38, a diamond scratch39 or a radius cutter40. (4) The demanded skilful operations above intrigued people to achieve assembling by full-commercial off-the-shelf modules. To date, more and more commercial components are involved in the assembling. Off-theshelf dispensing needles, mini cross-links and tee-links were used to assemble a microfluidic device that can control the size and structure of droplets precisely41. A Tshaped capillary junction was served to produce uniform pico-droplets in a digital PCR system42. Micro-cross, several fluidic fitting parts and capillary tubes were integrated to assemble a droplet generator and the size of droplets can be easily adjusted by altering the flow rate ratio43. Throughout this study, we aimed to fabricate a facile 3D droplet-based microfluidic device by assembling full-commercial off-the-shelf modules. It was cost-effective and no need for any special manufacturing technologies and extra interface connecting the outlet with the nebulizer to ICP. What is more, fused silica capillary was used as outer tubing with no sacrifice of observability. Monodisperse droplets were generated by coflowing of two immiscible fluid sample and introduced into ICP. Several key parameters, such as the design of microfluidic device, the kinds of oil and aquatic phase, the total flow rates of oil phase from two inlets (Qo), the flow rate of water phase (Qw) and the detection conditions of ICPMS were optimized. The signal peaks of single HepG2 cells were obtained by the time-resolved ICPMS, and droplets encapsulating Ag nanoparticles (NPs) were used to quantify silver in single cells. The developed method was applied to analyze HepG2 cells incubated with Ag+ and AgNPs at single cell level. Experimental Apparatus 5



The details are given in the Supporting Information(SI). Reagents and materials The details are given in SI. Design and fabrication of microfluidic droplet-based devices The schematic and profile diagram of microfluidic device online coupled with ICPMS are shown in Figure 1(a) and (b). The involved commercial components were displayed in Figure 1(c) and the actual photo of the developed microfluidic device is displayed in Figure 1(d). The detailed fabrication steps are as follows. (1) Two peek tubes (I) were placed at the oil inlet of the four-way connector by tightening two peek screws. (2) The innermost peek tube (III) was screwed by peek tubing sleeve (II) positioning at the water phase inlet of the four-way connector and inserted in the fused silica capillary (V). (3) The front and rear ends of fused silica capillary (V) were both secured by tightening peek sleeves (IV) and fixed in the exit of the four-way connector and the back of the nebulizer, respectively. All inlets of peek tubes were connected with 1 mL sterile syringes via microbore autoanalysis tubings. Taking advantage of the symmetry of the four-way connector, peek tube (III) was aligned with the fused silica capillary (V). Water and oil phase were introduced into the 4-way connecting system by syringe pumps at a flow rate of 0.1 and 15 μL min-1, respectively. Overall, this microfluidic device played a dual role of droplet generation by co-flowing and the interface coupling with the nebulizer. Results and Discussion Design of microfluidic droplet-based devices and the interface The size of droplets decreases with the increasing of the capillary number Ca, (Ca=μU/γ, where μ is the viscosity of the continuous phase (Pa s), U is the shear velocity of the continuous phase (U=Volume flux/Sectional area, m s-1) and γ is the interfacial tension of the two phases (N m-1)). The interspace between inner and outer tube is the decisive 6


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factor of U, and a higher value of U can be achieved by decreasing the interspace between inner and outer tube. To this end, as for the finest commercial peek tube, nozzle size with 0.1 mm O.D. was definitely to be selected as the inner tube in this work. As an external tube, fused silica capillary with an inner diameter of 0.15 mm and outer diameter of 0.365 mm best correspond to the narrowest interspace between inner and outer tube and also had a good match for the nebulizer with 0.15 mm inner diameter. Then, peek tubing sleeves F225 (for tubes with O.D. ranging from 70 to 110 μm) and F230 (for tubes with O.D. ranging from 350 to 390 μm) were elected to fix peek tube with 0.1 mm O.D. and fused silica capillary with 0.365 mm O.D., respectively. Due to the symmetry of the four-way connector, coaxial geometry was formed and shown by the optical microscope and SEM in Figure 2. Herein, droplets generation and the interface between microfluidic device and the nebulizer were integrated on the unified module. The sizes of droplets were measured to investigate the reproducibility of the assembled microfluidic device. We adopted 5% Span 80 in hexyl alcohol as oil phase (15 μL min-1) and 1% (m/v) HPMC in PBS solutions as water phase (0.1 μL min-1). The sizes of droplets obtained by five microfluidic devices were 27.0±0.5, 25.6±1.4, 28.3±0.6, 26.1±1.3 and 24.4±1.9 μm, respectively. The relative standard deviations (RSDs) of the droplet sizes was calculated to be 1.5 % (n=5). Overall, the microfluidic device was fabricated easily by full-commercial off-the-shelf modules and capable to withstand fluid pressure up to 4000 psi, implicating that the generator meets a broad range of needs. Besides, the fabricated device is robust with long lifespan and good reproducibility. Compared with other 3D microfluidic devices assembling by full-commercial modules (Table S2), the proposed microfluidic device has a merit of visible droplets generation owing to transparent fused silica capillary, relatively small droplets achieved with the consumption of oil phase with low carbon content and low flow rate, reducing carbon deposition on the sampling cone in ICPMS system. Choice of the fluids of oil and water phase 7



As a suspending agent, HPMC was used to keep the cells evenly dispersed during the experiment. The effect of HPMC concentration (0.1-1.0 %, m/v) on the cell dispersibility and stability was investigated by counting cells number at the same height of the suspension with hemacytometer. The initial cell number was obtained for cell suspensions right after mixing with a certain concentration of HPMC, and the existing cell numbers was obtained for cell suspensions with the presence of different concentrations of HPMC at different standing time. The effect of HPMC concentration was evaluated by the ratio of existing cell numbers / initial cell numbers. The experimental results (Figure S1) reflected that 1% (m/v) HPMC was enough to maintain cells dispersed evenly in 2 h. Besides, the morphology of cells in PBS solution containing 1% HPMC was observed in 30 min, and no obvious variation in cell integrity was observed. Accordingly, PBS solution with 1% (m/v) HPMC was selected as the water phase in further experiments. The oil phase benefits the formation of water-in-oil droplets but high carbon content of oil phase plays a sinister role to ruin the cone of ICPMS. Thus, as mentioned in our previous work22, we elected hexyl alcohol as the oil phase with a view of the lowest carbon content as well as the ability of generating stable droplets. Moreover, Span 80 was added in outer continuous oil phase to provide both steric stabilization to the droplets and increase the viscosity of the external oil phase. For the carbon content limit, the effect of Span 80 on the formed droplets size was studied in the content range of 1-5% (v/v) (Qw=1 μL min-1). As can be seen in Figure 3, the size of droplets decreased with the increase of Span 80 percentage. We would likely prefer low Qo whilst maintaining as small sizes of droplets as possible. Thereby, 5% (v/v) Span 80 in hexyl alcohol was adopted as the oil phase for further experiments. What is more, how droplets were pinched off from its tip could be observed clearly. As shown in Figure 3, the droplets were generated in dripping (above the dotted line) or jetting type (under the dotted line) within the range of investigation. Optimization of Qo and Qw 8


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Qo and Qw play a crucial role on the generation of the droplets. When Qo increases or Qw decreases, the droplet size decreases. The effect of Qo and Qw on the size of the droplet was shown in Figure 4 and the smaller size of droplets were preferable in consideration of the efficiency of single cell encapsulation. Besides, the total flow rate is also dependent on the suited sample uptake rate of the nebulizer to achieve the stable baseline. Thus, the sizes of droplets were initially investigated under different combinations of Qo (10-60 μL min-1) and Qw (0.1-2 μL min-1), considering that the minimum flow rate of the syringe pump is 0.1 μL min-1 and the maximum Qo within the toleration of ICPMS is 60 μL min-1. For much the same size of droplets, the combination of Qo and Qw within the dashed box in Figure 4 were preferable for subsequent hyphenation with ICPMS, from the perspective of the carbon content as low as possible. In addition, it should be noted that the flow rate within the dashed box all matched well the uptake rate of employed nebulizer, which is suggested to operate with the flow rate of 0.005-1.0 mL min-1. As an endogenous element in cells, the peak numbers of zinc represent the detected cell numbers. The detected efficiency of cells is defined as the ratio of the peak numbers to total cells introduced in ICPMS. Detected efficiency = Peak numbers/Total cells numbers introduced in ICPMS×100% 5×105 mL-1 was fixed as the cell density and total cells numbers introduced in ICPMS equalled the density of cell suspension multiply by the product of measurement time and Qw. Zinc peak numbers of the cells sample were obtained by time-resolved ICPMS with data processing (details in SI) under eight proper combinations of Qo and Qw and the detected efficiencies of cells were all listed in Table 1. Obviously, Qo was inclined to be as low as possible and we elected 15 and 0.1 μL min-1 for Qo and Qw respectively in subsequent experiments. Single cell encapsulation As we know, encapsulating cells into droplets is intrinsically governed by Poissonian 9



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statistics and the formula is shown as follows. λk × e ―λ

The probability of containing k cells in one droplet =

k!

, where λ equals the average

number of cells per droplet. Under the optimized conditions, cell density ranging from 5×105 mL-1 to 2×106 mL-1 was investigated to enable droplets only containing one or none of cells. The proportion of droplets containing cells was calculated with the method shown in SI and listed in Table S3, there were no droplets containing more than one cell if the cell density was equal or less than 5×105 mL-1. The microscopy images for the collected droplets with different cell densities was displayed in Figure S2. By the use of cell density of 5×105 mL-1, the theoretical calculation was also conducted and the probability of droplets containing two cells is 1.2×10-3 %. Furthermore, too low cell density would lead to low throughout dilemma. Thus, we selected 5×105 mL-1 as the cell density loading into the droplets. Effect of dwell time in ICPMS Under the optimized conditions above mentioned, the dwell time (tdwell) was examined to obtain the accurate and efficient results of single cell analysis. The temporal profile of Zn in single cells by time-resolved ICPMS with different tdwell (1, 2, 5, and 10 ms) was presented in Figure S3 (the limit of detection of Zn obtained by time-resolved ICPMS is 0.209 fg), and the number of peaks and the efficiency of detection were calculated and listed in Table S4. The efficiency of detection is massively dependent on two main factors in our developed approach: transport efficiency and integration time. Due to a relatively long integration time, dwell time of 10 ms would cause signals loss of single cells and decrease of detected efficiency. In addition, the detection efficiencies were much the same when tdwell was equal to or lower than 5 ms. It was indicative of the fact that 5 ms as dwell time was enough in this work. Detailed comparisons of other single droplets introduction - ICPMS systems for cell analysis are shown in Table S5. As can be seen, the developed system is lower cost than the commercial microdrop dispenser and has a higher detection efficiency than other single 10


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cells introduction systems. In addition, high encapsulation efficiency and low probability of containing two cells in one droplet are contradictory but these two indicators of our developed method are both at the intermediate level and we strike a good balance between them. Detection of silver in HepG2 cells by time-resolved ICPMS Before the analysis of silver in HepG2 cells, the feasibility of the proposed off-the-shelf 3D microfluidic device-time-resolved ICPMS system was evaluated by determining silver in Ag NPs. The size of AgNPs was characterized by TEM, and Figure S4 reveals that AgNPs are uniform and monodispersed particles with a diameter of 51±6 nm. AgNPs dispersion (5×105 particles per milliliter) was used as the water phase and the signal peaks of AgNPs were measured by time-resolved ICPMS. As shown in Figure S5, the distribution of AgNPs followed the Gaussian distribution and the mean value of the distribution was 13656 CPS. Given that the average diameter of AgNPs was 51 nm and the density of silver was 10.5 g cm-3, the number of silver atoms in one silver nanoparticle was estimated to be 4.1×106 under the assumption that AgNPs was spherical particles. In other words, the value of 13656 CPS represented 4.1×106 silver atoms. AgNPs was also digested and quantified by conventional ICPMS measurement which is provided in SI. The quantification result of AgNPs by single-cell analysis was 0.87 fg/particle, which was in good agreement with the results (0.69 fg/particle) obtained from a conventional acid digestion with silver nitrate standard solution as calibration series. It indicated a good accuracy of the developed approach. The viability of cells incubated with Ag+ or AgNPs was investigated by MTT assays and calculated by setting the viability of the control cells as 100%. The details of cell sample preparation and MTT assays were displayed in SI. As shown in Figure S6, an obvious decrease was observed in the cell viability when the incubation concentration of Ag+ or AgNPs was more than 1.5 and 5 μg mL-1, respectively. Thus, 1 μg mL-1 was chosen to be the incubation concentration for Ag+ or AgNPs. 11



Under the optimized conditions as mentioned earlier, HepG2 cells incubated with 1 μg mL-1 Ag+ or AgNPs for 6, 12, 18 and 24 h were determined at single cell level by our developed system and the results of intensity distributions of silver content in HepG2 cells were charted in Figure 5. We found that the diversity of cells incubated with AgNPs was wider than those cells incubated with Ag+ from the aspect of the content distribution of silver at single-cell level. It revealed obviously the cell-to-cell variability. The zinc signals of single cells in the same samples were also monitored to reveal the numbers of detected cells. In combination of the peak numbers of silver, the percentages of the cells uptaking silver were calculated (Table 2). The results of the silver intensity were processed on average and quantified by using the quantitative relationship mentioned above. As shown in Table 2, almost all cells uptake Ag+ after 6 h and uptake AgNPs after 12 h. For a comparison, cell samples incubated with 1 μg mL-1 Ag+ or AgNPs for 6, 12, 18 and 24 h respectively were also analyzed via conventional ICPMS after acid digestion. The acid digestion procedure is shown in SI in detail and the results are displayed in Table 2 as well. Yet, the results obtained by conventional ICPMS after acid digestion did not correspond to the values obtained by the proposed single cell analytical method. A few femtograms of silver per cell was obtained by the proposed single cell analytical method, which was an order of magnitude lower than that acquired by conventional ICPMS after acid digestion. It should be noted that a few of weird extreme high peaks appeared in the process of detecting cells samples incubated with Ag+ as well as AgNPs by single cell ICPMS analysis, as shown in Figure S7. If the extreme high peaks were included in data processing, the results of Ag in cells obtained by 3D microfluidic device-time resolved ICPMS and acid digestion-ICPMS measurement were comparable. Such high peaks were probably due to the formation of silver chloride precipitates (the concentration of Cl- in culture medium is about 7000 mg L-1, and the reaction quotient (Q)=1.8×10-6 ＞ Ksp, AgCl= 1.8×10-10) or the aggregation of AgNPs. These signal was excluded in our data processing. The results indicate that single cell 12


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ICPMS analysis can eliminate the influence of silver chloride precipitates or the aggregation of AgNPs on cell detection by removing abnormal high peaks, while the precipitation of Ag+ and the aggregation of AgNPs would be detrimental to quantify the amount of silver in cells by conventional ICPMS after acid digestion. Conclusions In this work, we made full use of commercially available components to assemble a 3D droplet-based microfluidic device with the merits of easy-to-operate without any extra manufacturing, low-cost, and high pressure tolerance. In addition, our device is able to monitor the generation of droplets and couple with ICPMS for detection of target element in single cells directly. Important factors on the design and analytical performance of this microfluidic device were adequately discussed. Under the optimized conditions, the developed method was applied to analyze cells samples incubated with Ag+ and AgNPs. In view of single-cell level, it demonstrated clearly that HepG2 cells had different uptake behaviors of Ag+ and AgNPs. The effect of precipitation of Ag+ and the aggregation of AgNPs can be eliminated by single cell ICPMS analysis when compared with conventional ICPMS after acid digestion. With the advent of industrialization production technology, the use of smaller diameter of peek or other material tubing would reduce the sectional area between inner and outer tubes, favoring the formation of smaller size of droplets, and boosting the efficiency of single cell encapsulation. Latterly, single-cell analysis using droplet-based microfluidic chip has gained momentum and became a booming field owing to its tiny volume and facile manipulation. To this point, our developed microfluidic device shows a promising potential to be a valuable platform for single-cell analysis in the future. Associated Content Supporting Information Additional information on explanation of apparatus, reagents and materials, sample preparation, data processing, calculation method of the percentage of droplets 13



containing only single cell, MTT assay, acid digestion of AgNPs and cells, effect of time on the percentages of existing cell numbers to initial cell numbers with different concentrations (m/v) of HPMC (Figure S1), the images of collected droplets containing single cell and more than one cell (Figure S2), time-resolved scans of 66Zn obtained for the HepG2 cells with different dwell time (Figure S3), the TEM image of AgNPs (Figure S4), intensity distributions of silver signal peaks in 51 nm AgNPs (Figure S5), effect of AgNPs and Ag+ on cell viability (Figure S6), intensity of weird extreme high peaks during the detection of cells samples incubated with Ag+ and AgNPs (Figure S7), optimized operating conditions for ICPMS (Table S1), the comparison with other 3D microfluidic devices assembling by full-commercial modules (Table S2), proportion of droplets contained single cell and more than one cell (Table S3), peak numbers and detection efficiencies determined by ICPMS at various tdwell (Table S4) and the comparison with other single droplets introduction - ICPMS systems for cell analysis. (Table S5) 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]. 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 14


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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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(19) Zhang, J. M.; Aguirre-Pablo, A. A.; Li, E. Q.; Buttner, U.; Thoroddsen, S. T. Droplet generation in cross-flow for cost-effective 3D-printed “plug-and-play” microfluidic devices. RSC Adv. 2016, 6, 81120-81129. (20) Shallan, A. I.; Smejkal, P.; Corban, M.; Guijt, R. M.; Breadmore, M. C. Cost-effective three-dimensional printing of visibly transparent microchips within minutes. Anal. Chem. 2014, 86, 3124-3130. (21) Au, A. K.; Lee, W.; Folch, A. Mail-order microfluidics: evaluation of stereolithography for the production of microfluidic devices. Lab Chip 2014, 14, 1294-1301. (22) Lan, W. J.; Li, S. W.; Xu, J. H.; Luo, G. S. Rapid measurement of fluid viscosity using coflowing in a co-axial microfluidic device. Microfluidic. Nanofluidic. 2009, 8, 687-693. (23) Lan, W.; Jing, S.; Guo, X.; Li, S. Study on “interface – shrinkage – driven” breakup of droplets in co-flowing microfluidic devices. Chem. Eng. Sci. 2017, 158, 58-63. (24) Dong, P.-F.; Xu, J.-H.; Zhao, H.; Luo, G.-S. Preparation of 10μm scale monodispersed particles by jetting flow in coaxial microfluidic devices. Chem. Eng. J. 2013, 214, 106-111. (25) Cordero, M. L.; Gallaire, F.; Baroud, C. N. Quantitative analysis of the dripping and jetting regimes in co-flowing capillary jets. Phys. Fluids 2011, 23, 8. (26) Meng, Z. J.; Wang, W.; Liang, X.; Zheng, W. C.; Deng, N. N.; Xie, R.; Ju, X. J.; Liu, Z.; Chu, L. Y. Plug-n-play microfluidic systems from flexible assembly of glass-based flowcontrol modules. Lab Chip 2015, 15, 1869-1878. (27) Xu, X.; Song, R.; He, M.; Peng, C.; Yu, M.; Hou, Y.; Qiu, H.; Zou, R.; Yao, S. Microfluidic production of nanoscale perfluorocarbon droplets as liquid contrast agents for ultrasound imaging. Lab Chip 2017, 17, 3504-3513. (28) Li, Y. K.; Liu, G. T.; Xu, J. H.; Wang, K.; Luo, G. S. A microdevice for producing monodispersed droplets under a jetting flow. RSC Adv. 2015, 5, 27356-27364. (29) Li, Y. K.; Wang, K.; Xu, J. H.; Luo, G. S. A capillary-assembled micro-device for monodispersed small bubble and droplet generation. Chem. Eng. J. 2016, 293, 182-188. (30) Yu, Z.; Zhou, L.; Zhang, T.; Shen, R.; Li, C.; Fang, X.; Griffiths, G.; Liu, J. Sensitive Detection of MMP9 Enzymatic Activities in Single Cell-Encapsulated Microdroplets as an Assay of Cancer Cell Invasiveness. ACS sensors 2017, 2, 626-634. (31) Shen, R.; Liu, P.; Zhang, Y.; Yu, Z.; Chen, X.; Zhou, L.; Nie, B.; Zaczek, A.; Chen, J.; Liu, J. Sensitive Detection of Single-Cell Secreted H2O2 by Integrating a Microfluidic Droplet Sensor and Au Nanoclusters. Anal. Chem. 2018, 90, 4478-4484. (32) Utada, A. S.; Lorenceau, E.; Link, D. R.; Kaplan, P. D.; Stone, H. A.; Weitz, D. A. Monodisperse double emulsions generated from a microcapillary device. Science 2005, 308, 537-541. (33) Chu, L. Y.; Utada, A. S.; Shah, R. K.; Kim, J. W.; Weitz, D. A. Controllable monodisperse multiple emulsions. Angew. Chem.-Int. Edit. 2007, 46, 8970-8974. (34) Wang, W.; Xie, R.; Ju, X. J.; Luo, T.; Liu, L.; Weitz, D. A.; Chu, L. Y. Controllable microfluidic production of multicomponent multiple emulsions. Lab Chip 2011, 11, 15871592. (35) Zhao, Y.; Gu, H.; Xie, Z.; Shum, H. C.; Wang, B.; Gu, Z. Bioinspired multifunctional Janus particles for droplet manipulation. J. Am. Chem. Soc. 2013, 135, 54-57. 17



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Figure captions Figure 1 (a) Schematic diagram of online assembled microfluidic device - ICPMS single cell analysis system. (b) A profile schematic of the 3D Droplet-Based Microfluidic Device. (c) The assembled microfluidic device including five peek screws, a four-way connector, (I) peek tubing with 1/16 inches O.D. and 0.5 mm I.D., (II) peek tubing sleeve with 1/16 inches O.D. and 0.125 mm I.D (Part No. F225), (III) peek tubing with 0.1 mm O.D. and 0.05 mm I.D., (IV) peek tubing sleeve with 1/16 inches O.D. and 0.405 mm I.D (Part No. F230), and (V) fused silica capillary with 0.365 mm O.D. and 0.15 mm I.D.. (d) Image of the actual assembled microfluidic device and the interface between the microfluidic device and the nebulizer. Figure 2 (a) The micrograph of lateral view and (b) the SEM of the section of the coaxial microfluidic device. Figure 3 The droplet size versus the flow rate of oil phase with different concentrations (v/v) of Span 80 in continuous phase. (the water phase was PBS solution consisting of 1% (m/v) HPMC and the flow rate of water phase was 1 μL min-1) Figure 4 The droplet size versus the flow rate of oil phase with different flow rate of water phase. (the water phase is PBS solution consisting of 1% (m/v) hydroxypropyl methyl cellulose, the oil phase is 1-Hexano consisting of 5% (v/v) Span 80) Figure 5 Intensity distributions of silver signal peaks in HepG2 cells incubated with 1 μg mL-1 Ag+ and Ag NPs for 6, 12, 18 and 24 h, respectively.

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Figure 1

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Figure 2

21



Figure 3

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Figure 4

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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

Figure 5

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Table 1 Peak numbers and detection efficiencies determined by ICPMS at various flow rates of oil phase and water phase Qoil

Qwater

Size of droplets

Total cell numbers

Peak numbers

Detection efficiency

(μL min-1)

(μL min-1)

(μm)

in 3 min

in 3 min

(%)

15

0.1

26

150

36±4

24.0±2.7

20

0.1

21

150

39±4

26.0±2.7

20

0.2

30

300

74±2

24.7±0.7

30

0.2

27

300

75±4

25.0±1.3

30

0.5

30

750

182±15

24.3±2.0

40

0.5

22

750

179±11

23.9±1.5

40

1

27

1500

201±13

13.4±0.9

40

2

28

3000

209±19

7.0±0.6

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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

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Table 2 Results of Ag for HepG2 cells incubated with Ag+ or AgNPs with different incubation time

Percentage of cells Incubation time (h)

uptaking Ag+ or AgNPs

Average CPS per cell

(%)

Time-resolved ICPMS

Acid digestion

Average mass

Average mass

per cell (fg)

per cell (fg)

6

92.5±6.9

17430±1751

0.9±0.1

14.4±2.5

12

93.9±5.2

35317±2550

1.9±0.1

32.3±3.5

18

98.8±3.5

72766±6206

3.9±0.3

58.3±11.4

24

100±1.2

113978±10699

6.1±0.6

95.4±4.2

6

86±7.1

53102±3894

2.9±0.2

41.5±5.2

12

92.1±5.3

79888±6387

4.3±0.3

73.7±3.3

18

97.1±2.9

94706±10852

5.1±0.6

85.7±10.1

24

100±1.5

140582±17101

7.6±0.9

117±11

Ag+

AgNPs

26


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For TOC only

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3D Droplet-Based Microfluidic Device Easily Assembled from

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