Microfluidic Synchronizer Using a Synthetic Nanoparticle-Capped

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Letter

Microfluidic synchronizer using a synthetic nanoparticle-capped bacterium Chenli Liu, Zhiguang Chang, Yue Shen, Qi Lang, Hai Zheng, Taku A. Tokuyasu, and Shuqiang Huang ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.9b00058 • Publication Date (Web): 09 Apr 2019 Downloaded from http://pubs.acs.org on April 10, 2019

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ACS Synthetic Biology

Microfluidic synchronizer using a synthetic nanoparticle-capped bacterium Zhiguang Chang†,††,‡,⊥, Yue Shen§,¶,#,||,⊥, Qi Lang†,††,‡,⊥, Hai Zheng†,††,‡, Taku A. Tokuyasu†,††, Shuqiang Huang†,††,‡ and Chenli Liu†,††,‡,* †

Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen

518055, People’s Republic of China ††

Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences,

Shenzhen 518055, People’s Republic of China ‡ University

of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China

§ BGI-Shenzhen, ¶ Shenzhen

Shenzhen, 518083, China

Engineering Laboratory for Innovative Molecular Diagnostics, Shenzhen, 518120, China

# Guangdong

Provincial Key Laboratory of Genome Read and Write, Shenzhen, 518120, China

|| Guangdong

Provincial Academician Workstation of BGI Synthetic Genomics, BGI-Shenzhen, Guangdong, China

⊥

equal contribution

* To whom correspondence may be addressed. Email: [email protected]

ABSTRACT: Conventional techniques to synchronize bacterial cells often require manual manipulations and lengthy incubation lacking precise temporal control. An automated microfluidic device was recently developed to overcome these limitations. However, it exploits the stalk property of Caulobacter crescentus that undergoes asymmetric stalked and swarmer cell cycle stages and is therefore restricted to this species. To address this shortcoming, we have engineered Escherichia coli cells to adhere to microchannel walls via a synthetic and inducible “stalk”. The pole of E. coli is capped by magnetic fluorescent nanoparticles via a polar-localized outer membrane protein. A mass of cells is immobilized in a microfluidic chamber by an externally applied magnetic field. Daughter cells are formed without the induced stalk and hence are flushed out and to yield a synchronous population of “baby” cells. The stalks can be tracked by GFP and nanoparticle fluorescence; no fluorescence signal is detected in the eluted cell population, indicating that it consists solely of daughters. The collected daughter cells display superb synchrony. The results demonstrate a new on-chip method to synchronize the model bacterium E. coli and likely other bacterial species, but also foster the application of synthetic biology to the study of the bacterial cell cycle. KEYWORDS: Microfluidic synchronizer, baby machine, AIDA, magnetic fluorescent nanoparticles, cell cycle

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The cell cycle continues to be a central topic in studying bacterial physiology and hence possible applications. Synchronized cultures can be particularly valuable to study dynamics of the cell cycle such as DNA/plasmid replication1,2, cell wall and membrane synthesis3,4, and gene expression5. Synchronization methods have been under development since the first report6 in 1956. One approach uses physical or chemical means to block cells at a certain phase of the cell cycle7, but this interference may introduce unrelated phenomena8. In the early 1960s, Charles Helmstetter developed a bacterial membrane-elution method termed the “baby machine” for producing minimally disturbed, normally growing synchronous E. coli B/r cells9. In this method, “mother” cells are attached to a nitrocellulose membrane, and newly divided “baby” cells are eluted by the flow of media through the inverted membrane. Synchrony of a wider range of bacterial strains was later realized by immobilizing them to the membrane with the adhesive poly-D-lysine10. Limitations of the method include possible retention of both cells after cell division, and variation of machine performance with the composition of the culture medium. The “baby column” of Bates et al uses an inducible flagellar protein to attach mother cells to microscopic glass beads inside a chromatography column11, thus permitting the production of greater volumes of synchronized cells than the membrane elution method. Microfluidic systems provide significant advantages to study cell biology. They can provide more control over the environment on cellular length scales, while integrating sample preparation and analysis on the same device. Prominent recent examples include devices to study the dynamics of single cell growth12–14. Sophisticated devices based on physical methods such as dielectrophoresis15, spiral fluid flow16, or laminar flow in a chamber with holes17 have been developed to synchronize eukaryotic cells in particular. An exquisite device synchronizes E. coli cells by length sorting through microfluidic channels, minimizing the mechanical stress experienced by the cells18. Synchronization in such devices often relies on a correlation between cell size and position within the cell cycle, which may not always hold. Another common limitation is that both mothers and their progeny may be collected together but cannot be distinguished, weakening the synchronization signal and limiting downstream analyses. A baby machine was recently developed for Caulobacter crescentus19, which undergoes asymmetric binary fission resulting in two morphologically different progenies: a mature stalked cell and a motile swarmer cell. This baby machine can highly enrich for swarmer cells, and synchrony quality can be determined by inspecting the fraction of motile cells. This baby machine was integrated into an automated lab-on-chip to produce synchronized cell populations for three days continuously20. This approach decreased incubation time and media consumption while improving synchrony quality compared to the conventional plate-release technique used for this species. The reliance on specific features of this species’ life cycle however clearly limits the applicability of the approach. To circumvent these drawbacks, we explored the idea of using magnetic bacteria. The discovery of magnetotactic bacteria has inspired numerous studies and new fields of investigation such as synthetic swimmers21,22. A variety of biomedical applications take advantage of selective coating of bacteria by magnetic particles to facilitate diagnosis and therapy23–25. These applications typically do not emphasize the cell surface localization of the magnetic moiety, rather seeking to coat the entire surface with magnetic particles. Here, we describe a new method that can be used with diverse strains based on synthetic biology. Our basic insight was to design magnetic capped bacteria (MCBs) as mother cells. As a proof of concept, we loaded these cells into a customized microfluidic device that attached MCBs to a surface via a permanent magnet. As the mother cells grew, daughters were eluted at short intervals to collect cells that remain synchronous over multiple generations. In our design, daughter cells cannot revert back to mothers, even in the presence of leaky gene expression. This approach gives access to the full range of microfluidic capabilities, including tight control of flow rates, precise and quick changes in medium composition, and downstream analyses such as sorting by size and single cell transcriptomics. In addition to baby cells, mothers can also be collected with the device simply by removing the magnetic field while remaining distinguishable. This device is easy to fabricate, and the MCB model can be widely used in any bacterial species that permits similar engineering of exposed polar proteins.


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RESULTS AND DISCUSSION Overview of synchronizer

Figure 1. Microfluidic synchronizer working principle. (A) MCBs have an induced stalk (not drawn to scale) made up of a polar chimeric fusion protein (eGFP-AIDA-I) and a streptavidin-labeled MFN attached by a biotinylated monoclonal GFP antibody (αGFP). (B) MCBs are bound to the top of a microfluidic channel by a magnet. Daughter cells are born without the induced stalk and are eluted out of the device at regular intervals.

Our microfluidic synchronizer is designed to bind the MCBs (Figure 1A) to the top of a fluid channel through magnetic forces, with newborn baby cells eluted out from the device by flowing medium (Figure 1B). MCBs are created by gene induction with IPTG (see Methods) and initially have magnetic fluorescent nanoparticles (MFNs) at one or both poles. These cells were introduced to the device and immobilized within a working region covered by a permanent magnet. After incubation in the device for several cell generations, most of the cells were either immobilized via nanoparticles at only one pole or were washed out. Since there is no inducer in the medium, continued division of the immobilized MCBs produced baby cells without the nanoparticle "stalk”, as ensured by the polar localization of the MFNs. These baby cells were eluted into the outlet chamber by a constant slow perfusion of cell culture medium. Transport of the newborn cells from the channel to the chamber (Figure 3) took no more than 30 s. We describe further how synchronization is achieved with this design in the Supplement. Development of magnetic capped bacteria We used strain E. coli BL21 (DE3) (lab stock) to build MCBs. To assemble a magnetic stalk, we began by constructing a chimeric fusion protein made up of eGFP and AIDA-I, a 132-kDa autotransporter adhesin from an enteropathogenic E. coli (EPEC) that localizes to the bacterial pole26–28. AIDA-I is composed of an N-terminal signal peptide (5.3 kDa), a middle passenger domain, and a C-terminal autotransporter domain (53.0 kDa), with the passenger domain displayed on the pole surface. In this study, a 23.9-kDa eGFP replaced the passenger domain of AIDA-I (see Supplement). The recombinant protein eGFP-AIDA-I was encoded in a plasmid and driven by a Plac promoter upon IPTG induction. The plasmid was then transformed into the E. coli cells. To complete this first assembly step, cells were induced with IPTG for two hours, then cultured overnight. The localization of the protein to the outer membrane was confirmed by SDS-PAGE and Western blot, and localization specifically to the poles was confirmed by fluorescence (see Supplement). To prepare MCBs after IPTG-induced expression of eGFP-AIDA-I, a two-step approach was used. A



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biotinylated monoclonal antibody was employed against the surface-displayed eGFP protein, and cells were then incubated with streptavidin-coated MFNs. MFNs were found almost exclusively in the polar regions of E. coli cells and colocalized with the GFP (Figure 2). Before arriving at the above protocol, we examined both the order of combining the various ingredients (GFP antibodies, MFNs, and cells). These efforts, and further examination of MFN localization by electron microscopy, are detailed in the Supplement.

Figure 2. Confirmation of MCB assembly. Representative fluorescent micrographs of GFP-MFN colocalization. Cells are visualized by differential interference contrast microscopy (D.I.C.). eGFP-AIDA-I fusion protein (green) and MFNs (red) colocalize at cell poles.

Device design A schematic top view of our device is shown in Figure 3A. To avoid cell adhesion at sharp corners, a spiral channel was used to load MCBs. The width of the polydimethylsiloxane (PDMS) channel was 200 μm and the height of the channel was 50 μm. The total volume of the magnetized working region was 4 μL. The outlet was designed as an open chamber to decrease the pressure, facilitating timely collection of synchronized cells. A small piece of PDMS with punched holes functioned as a cover to reduce medium evaporation (Figure 3B). With a flow rate of 0.5 ml/hr, MCBs took 30 s to travel from the inlet to the outlet of the device.


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Figure 3. Microfluidic synchronizer design. (A) Schematic of the microfluidic device. The channel was continuous, and the two colors represent the inward and output spirals. The synchronized cells can be collected from the outlet chamber by pipette. (B) Photograph of the microfluidic device on a 37 °C hot plate. The permanent neodymium ferroboron magnet was placed on top of the spiral channel.

Microfluidic synchronizer performance In order to perform as designed, our device must satisfy two main criteria. The mother MCBs should be captured by the magnetic field, whereas newborn baby cells should not adhere to the microfluidic channel but be washed away immediately. Fulfilling these conditions, our device should produce synchronized baby cells. The magnetic induction intensity applied in the microfluidic channel was 278 mT. The MCBs were pumped into the device at a flow rate of 1 ml/h, before application of the magnetic field. Figure 4A shows typical results of capturing MCBs to the magnetic working region of the device. Green fluorescence indicates eGFP-AIDA-I, red indicates MFN, and orange indicates MCBs. The results show abundant trapping of MCBs after 20 minutes at the flow rate of 1 ml/h. Conversely, to determine a sufficient flow rate to wash out the baby cells, we first pumped MCBs at high concentration (109 cells/ml) into the channel, incubated them for two hours, then washed the cells with culture medium at a flow rate of 0.5 ml/h. Figure 4B depicts two sequences of events separated by 0.1 s under the washing flow. In the top sequence, the cells were not washed away, while in the bottom sequence, the group of cells were washed away successfully. We observed that most of the baby cells could be eluted and concluded that 0.5 ml/h is a sufficient flow rate for the device to produce synchronized newborn cells.

Figure 4. Device performance assessment. (A) Trapping of MCBs by the device after pumping cells for 5 minutes, 10 minutes, and 20 minutes. The orange (stacked green and red) fluorescence shows the distribution of MCBs. (B) Example of cells that are washed (top sequence) and not washed (bottom sequence) away by the culture medium with the flow rate of 0.5 ml/h, as captured by video frames separated by 0.1 s.

Synchrony validation To determine the synchrony quality, fluorescence microscopy was first used to analyze the eluted cells. At times 1, 6, 12, and 24 hours after bacterial loading, neither red nor green fluorescence was detected in the media ACS Paragon Plus Environment


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or daughter cells. We note here that the same medium was used throughout these experiments (rich defined medium, see Methods). In order to characterize the performance of our device on synchronizing cells, flow cytometry was applied to quantify the cell number concentration29. The cells were stained with DAPI, and the DAPI positive partials were deemed as cells. The accuracy of the cell count methodology was verified by counting serially diluted samples (Figure 5A). The cell number concentration decreases in accordance with the dilution ratio, and the reproducibility of the measurement is within 3%. These results suggest that the flow cytometry-based cell count methodology is quite reliable and could be used to validate our baby machine. It is essential to verify whether the cells in our device were in the same physiological states as cells in batch culture. The doubling time of cells cultured in flasks, measured by OD600 measurement and cell count, yield a doubling time of 44 minutes (Figure 5B, 5C). To validate the device, the cells were collected from the outlet chamber for 2 minutes, then inoculated into 3 ml pre-warmed fresh medium, and cell concentration was determined every 10 minutes (Figure 5D). As our device is designed to select the newborn cells, there should be no significant cell division upon inoculation. Cell concentration indeed remained constant for over 30 minutes. On the other hand, cell number precisely doubled within a relatively short time period, indicating good synchronization. Furthermore, the time interval between cell divisions was comparable to the doubling time of the cells in flask culture, implying that physiological state was preserved in the newborn daughter cells. As growth rates vary between different cells at the single cell level even when cultured in the same medium30, the synchronized cell population could not be maintained indefinitely. In this study, cell synchronization was maintained for two cell cycles and then began to decay. Taken together, we conclude that our device generates well-synchronized cells and can be used in further investigations.


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Figure 5. Evaluation of synchrony of collected cell populations. (A) Validation of the cell count methodology by serial dilution. A set of bacterial cell samples was prepared by serial 2-fold dilution, and flow cytometry applied to characterize the cell concentration of each sample. The cell concentration decreased at the same rate as the increase in dilution factor, confirming that the cell count methodology was quite reliable for samples with 1 × 105 to 2 × 106 cells per ml. Each diluted sample was characterized by flow cytometry three times; error bars ( ± S.D.) are smaller than the plot symbols. (B) Representative mass growth curves of magneticcells in batch culture. Data for three independent biological replicates are displayed, shifted by 20 minutes between replicates for clarity. (C) Representative cell number growth curves of magnetic-cells before loading into the baby machine. Three independent biological replicates are displayed, shifted by 30 minutes between replicates. (D) Cell number growth curves from three replicate collections of daughter cell obtained from baby machine; theoretical curves are doublings every 44 minutes.

Concluding remarks We propose MCBs and microfluidics as a new platform on which to perform precision studies of bacterial cell physiology, in particular of the cell cycle. The presented microfluidic baby machine successfully discriminates mother and daughter cells by using magnetic forces to trap mother cells on the device. This technique avoids damaging chemicals, does not rely on cell size fractionation, and is not limited to cells with specific chemical surface properties. Any leaky expression of the chimeric protein does not affect the operation of the device, as it does not contain antibodies nor MFNs. The growth curves of eluted baby cells exhibit excellent synchrony. While the overall yield of the device is low relative to the membrane elution method, it is sufficient for many biological experiments, including transcriptomic analysis. The mother cells can be conveniently collected and studied by simply removing the externally applied magnetic field. In this sense, the device can be termed a “family machine.” Variations of the machine to allow study of the mother cells in situ are also easily envisioned. Future versions of the device will seek to increase the daughter cell yield, e.g. by increasing the mother cell density, which will allow even shorter cell collection times. The mechanism and the construction of the synchronization apparatus was unambiguous, rapid and efficient. The device may assist the development of more complex and flexible microfluidic systems.



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METHODS Plasmid constructs The original AIDA-I plasmid pET-23(a)-AIDAc was kindly provided by Dr. M. Alexander. The (AIDA-I native) N terminal signal peptide was incorporated into the plasmid vector as an oligo. A DNA fragment containing the eGFP gene was amplified by PCR (94 oC 30 s, 55 oC 30 s, 72 oC 60 s, 25-30 cycles) from plasmid pEGFP (lab stock) using the forward primer 5’-ATGGAATTCATGGTGAGCAAGGGCGAGG-3’ containing an EcoRI restriction site and reverse primer 5’-TTGAGCTCCTTGTACAGCTCGTCCATG-3’ containing a SacI restriction site. Both the pET-23(a)-AIDAc plasmid and the eGFP fragment were digested by EcoRI and SacI at 37℃ for 4 h. Then the digested products were recovered with Gel Extraction Kit (Qiagen) respectively and then ligated with T4 ligase at 16 oC for 4 h to construct the pEA plasmid. Outer membrane protein isolation and Western blot Outer membrane proteins were isolated using N-lauroylsarcosine methods as described previously.31 Briefly, 5 × 109 IPTG-induced bacteria were collected by 3,000 g centrifugation for 10 minutes, 4°C, suspended in 10 ml of 20 mM Tris-HCl, pH 7.2 and lysed by sonication. After 16,000 g centrifugation for 10 minutes, 4°C, and supernatant was 80,000 g ultracentrifuged for 2 h, 4°C. Pellets (Containing total membrane proteins, TM) were suspended in 5 ml 0.5% Sarkosyl in 20 mM Tris-HCl and incubated at 37 oC for 30 minutes. and 80,000 g ultracentrifuged for 2 h, 4°C again. After centrifugation, pellets containing outer membrane proteins were homogenized in 200 μL of 1% (w/v) Sodium N-lauroylsarcosinate, 20 mM Tris-HCl. The supernatant mark as TM-r. The outer membrane proteins were run on an SDS–PAGE gel and analyzed via western blotting. Briefly, the protein samples were prepared using 5× loading buffer (Sangon Biotech, Shanghai, China) and electrophoresed on 12% SDS–PAGE gels, proteins transferred to a 0.2-mm PVDF membrane using Semi-Dry blotting method. Mouse anti-GFP mAb (Santa Cruz, sc-9996B) was used as the primary antibody, and an HRP-labelled goat antirabbit IgG (Abcam, Shanghai, China) was used as the secondary antibody. Preparation of biotinylated anti-GFP antibody attached bacteria AIDA-I E.coli cells were cultured in rich defined medium32 with glucose (0.4% w/v) as the carbon source (RDM+glucose) at 37 °C containing 100 mg/ml ampicillin, eGFP expression was induced by the addition of 0.1 mM IPTG when the OD600 reached 0.4. After overnight culture at 37 °C, the cells were harvested by centrifugation at 3,000g, 4℃, for 10 minutes. The cells was resuspended in PBS, pH7.4 and re-pelleted by centrifugation at 3,000g, 4 °C, for 10 minutes. After washing with PBS twice as described above, the pellet was resuspended in PBS and the OD600 of the suspension was adjusted to 0.5. 1 ml of bacteria suspension was transferred into a 1.5 ml microcentrifuge tube. Biotinylated anti-GFP antibody (Abcam ab6658) was added into the tube to reach a final concentration of 1 μg/ml. The antibody-bacteria mixture was incubated at room temperature for 60 minutes. After that, the bacteria were washed with 1 ml PBS twice as described above to remove free antibody. Finally, the bacteria were resuspended in 1 ml PBS. Immobilization of MFNs and optimization 1 ml of biotinylated anti-GFP antibody attached bacteria were pelleted in 1.5 ml microcentrifuge tube by centrifugation at 3,000 g, RT, for 10 minutes. After carefully removing the supernatant, 1 ml of Streptavidin-coated nanoparticles (nano-screenMAG/R-Streptavidin, 100 nm, Chemicell) (1x1010 particles/ml) were added into the tube. The bacteria were resuspended and mixed with the nanoparticles. The suspension was gently mixed at room temperature for 60 minutes while avoiding light. Then the bacteria were preferentially concentrated by a magnetic frame. After carefully removing the supernatant, the bacteria were resuspended in 100 μL PBS, and the assembled MCBs were diluted to 109 c.f.u./ml and used immediately. Fabrication


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Our microfluidic device was fabricated with classic soft lithography technology. The SU-8 mold for PDMS layer were fabricated using photolithography. The washed 3 inch silicon wafer was firstly plasma treated for 10 minutes in medium power before spin coating photoresist. The negative photoresist (SU-8, 3050, MicroChem) was spun in the wafer by a spin coater at 500 rpm for 15 s and then at 3000 rpm for 45 s. The SU-8 was soft-baked for 15 minutes at 95 °C and selectively exposed by a film photomask designed with the AutoCAD software. Then, the photoresist was heated to 95 °C for 5 minutes as post exposure bake. After post exposure bake, the SU-8 resist was developed in MicroChem’s SU-8 developer. Finally, the wafer with SU-8 pattern was hard baked to 150 °C in the oven for 1 h. The PDMS curing agent was combined with the elastomer base (DOW Corning) at a ratio of 10:1 (w/w) to create a mixture that was then applied to the exposed photoresist mold and dried through degassing in a vacuum chamber for 30 minutes. The thickness of PDMS channel layer was around 1 mm. Then the PDMS mixture was cured in an oven at 80 °C for 1 h. Another thick PDMS layer (1 cm) was cured as outlet chamber. The two PDMS layers were bonded together using an oxygen plasma. After punching holes of inlet and outlet chamber, the assembled PDMS layer was bonded with a glass slide by plasma cleaner. Before loading MCBs, the device was sterilized by soaking in 75% ethanol for 30 minutes, then subsequently apply UV irradiation for 60 minutes. Device operation To prevent adhesion of newborn cells, the PDMS surface was passivated by soaking with 10 mg/ml BSA in culture medium for 1 h, then washed three times with PBS. Highly concentrated MCBs (109 cells per ml) were gently pumped into the channel at a flow rate of 0.5 ml/h by syringe pump (Harvard Apparatus). The device was moved onto a 37 °C hot plate and a permanent neodymium ferroboron magnet placed on top of the spiral channel. After standing for 30 minutes to load cells to the top of the channel, the culture medium was perfused with the flow rate of 0.5 ml/h for 3 h to activate cells. The magnet was kept on top of the device throughout the experiment. Before collecting synchronized cells, the outlet chamber was washed with PBS 3 times by pipette. The synchronized cells could then be collected from the outlet chamber in 2 or 3 minutes. Cell collection and count Cell solution that collected in the outlet chamber after two minutes was transferred to 3 ml medium in 12 ml culture tube. The tube was subsequently placed in a 37 °C shaking bath to continue culturing. After 10 minutes, 100 μL cell solution was gently collected and placed into 100 μL fixation medium (4% paraformaldehyde with 1 μg/ml DAPI) every 5 minutes, for a total of 15 times. The cell number was counted by flow cytometry (Beckman), with DAPI+SSChigh particles counted as bacteria. ASSOCIATED CONTENT Supporting Information. Details on MCB assembly and synchronization mechanism, supporting Figures S1−S4. AUTHOR INFORMATION Corresponding Author Tel: +86-755-86585245. Fax: +86-755-86585245. E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was financially supported by Major Research Plan of the National Natural Science Foundation of China (91731302, 81803084), Strategic Priority Research Program (XDPB0305) and the Key Research Program (KFZD-SW-216) of the Chinese Academy of Sciences, Shenzhen Grants (JCYJ20170818164139781, KQTD2015033117210153, Engineering Laboratory [2016]1194). China Postdoctoral Science Foundation Project (2016M600683) National Postdoctoral Program for Innovative Talents (BX201600180), Guangdong Provincial



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Microfluidic Synchronizer Using a Synthetic Nanoparticle-Capped

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