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Dec 28, 2016 - Zhi Zhu received her Bachelor degree in chemistry from Peking University (China) ..... single-cell protein analysis has posed a major c...
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Hydrogel Droplet Microfluidics for High-Throughput Single Molecule/Cell Analysis Zhi Zhu and Chaoyong James Yang* MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Collaborative Innovation Center of Chemistry for Energy Materials, Key Laboratory for Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China CONSPECTUS: Heterogeneity among individual molecules and cells has posed significant challenges to traditional bulk assays, due to the assumption of average behavior, which would lose important biological information in heterogeneity and result in a misleading interpretation. Single molecule/cell analysis has become an important and emerging field in biological and biomedical research for insights into heterogeneity between large populations at high resolution. Compared with the ensemble bulk method, single molecule/cell analysis explores the information on time trajectories, conformational states, and interactions of individual molecules/cells, all key factors in the study of chemical and biological reaction pathways. Various powerful techniques have been developed for single molecule/cell analysis, including flow cytometry, atomic force microscopy, optical and magnetic tweezers, single-molecule fluorescence spectroscopy, and so forth. However, some of them have the low-throughput issue that has to analyze single molecules/cells one by one. Flow cytometry is a widely used high-throughput technique for single cell analysis but lacks the ability for intercellular interaction study and local environment control. Droplet microfluidics becomes attractive for single molecule/cell manipulation because single molecules/cells can be individually encased in monodisperse microdroplets, allowing high-throughput analysis and manipulation with precise control of the local environment. Moreover, hydrogels, crosslinked polymer networks that swell in the presence of water, have been introduced into droplet microfluidic systems as hydrogel droplet microfluidics. By replacing an aqueous phase with a monomer or polymer solution, hydrogel droplets can be generated on microfluidic chips for encapsulation of single molecules/cells according to the Poisson distribution. The sol−gel transition property endows the hydrogel droplets with new functionalities and diversified applications in single molecule/cell analysis. The hydrogel can act as a 3D cell culture matrix to mimic the extracellular environment for long-term single cell culture, which allows further heterogeneity study in proliferation, drug screening, and metastasis at the single-cell level. The sol−gel transition allows reactions in solution to be performed rapidly and efficiently with product storage in the gel for flexible downstream manipulation and analysis. More importantly, controllable sol−gel regulation provides a new way to maintain phenotype-genotype linkages in the hydrogel matrix for high throughput molecular evolution. In this Account, we will review the hydrogel droplet generation on microfluidics, single molecule/cell encapsulation in hydrogel droplets, as well as the progress made by our group and others in the application of hydrogel droplet microfluidics for single molecule/cell analysis, including single cell culture, single molecule/ cell detection, single cell sequencing, and molecular evolution.

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INTRODUCTION Scientists have for many years been aware of molecular and cellular heterogeneity.1,2 However, this information is generally lost in traditional bulk assays, which assume the outcome to be homogeneous. Because of the heterogeneity, use of an average response as representative of a typical population may result in a misleading interpretation. Therefore, single molecule/cell analysis has become a key technique in biological and biomedical research to gain insight into heterogeneity between large populations at high resolution.3 In the past decades, various powerful techniques have been developed with single-molecule or single-cell resolution, including flow cytometry,4 atomic force microscopy,5 optical and magnetic tweezers,6 single-molecule fluorescence spectroscopy,7 and so forth. However, most of them have the throughput issue for large population analysis. While flow cytometry is successful as a high-throughput single cell © 2016 American Chemical Society

measurement system, its application in the study of intercellular interactions and control of the local environment is limited. Droplet microfluidic techniques have recently been recognized as powerful tools because single molecules/cells can be individually encased in monodisperse microdroplets, allowing high-throughput analysis and manipulation with precise control of the local environment.8 To support the controlled manipulation of droplets in a high-throughput manner, a wide suite of methods has been developed for droplet generation, fusion, mixing, analysis and sorting. 9 The advantages and technique development have enabled the applications of droplet microfluidics in high-throughput single molecule/cell analysis, including single molecule amplification, Received: July 18, 2016 Published: December 28, 2016 22

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Figure 1. Chemical structures of natural and synthetic hydrogels. (a) Agarose, (b) alginate, (c) hyaluronic acid, (d) chitosan, (e) poly(ethylene glycol), (f) poly(acrylic acid), (g) poly(vinyl alcohol), and (h) poly(acrylamide).

below its gelling temperature (15−30 °C). Because agarose is bioinert and nonadhesive to proteins and cells, it finds many applications in biomedical areas, including cell culture and tissue engineering.19 The anionic polysaccharide alginate, which is obtained from brown seaweed and bacteria, chelates with alkaline earth ions (Ca2+, Ba2+, or Sr2+) to form hydrogels. Collagen, the major component of skin and bone, is widely used for cell encapsulation and tissue engineering. Unlike agarose, collagen undergoes an opposite temperature-responsive gelation process that is stable in acidic solution at low temperature and is capable of forming hydrogels at body temperature and neutral pH.20 Although natural hydrogels have the advantage of innate biocompatibility, it is usually difficult for controllable chemical modification. Alternatively, hydrogels with desirable and finetuned properties can be prepared from synthetic polymers, including poly(ethylene glycol), poly(acrylic acid), poly(vinyl alcohol), poly(acrylamide), and their derivatives (Figure 1e− h).18 Functional groups, such as peptides, oligonucleotides, and degradable linkages, can be easily incorporated as part of the synthesis process. Commonly, the aqueous monomer solutions are mixed with initiator and cross-linker, followed by polymerization by a number of methods, such as photoinitiated polymerization. Synthetic hydrogels are usually mechanically stronger than natural hydrogels, but they are not inherently biodegradable. Since synthetic hydrogels have common features, poly(ethylene glycol) (PEG) was taken as an example. It can be chemically modified to generate the UV-sensitive PEG-diacylate that allows photoinitiated polymerization.21 PEG is bioinert to protein absorption and cell adhesion. However, when covalently grafting RGD peptides on PEG, human cells can attach to the RGD-PEG gel.22 The synthetic hydrogels can be simply modified with desired properties or even incorporated with the features of natural hydrogels to possess the advantages of both.

directed evolution, rare cell detection, drug screening, and so forth.10 Hydrogels are cross-linked polymer networks that are swelling but not soluble in water.11 The physical, mechanical, and diffusive properties of hydrogels are generally determined by the type of polymer and cross-link, the degree of crosslinking, and the water content. Recently, hydrogels have been introduced into microfluidics, and these hybrid systems generate some novel and significant properties. For example, stimuli-responsive hydrogels with volumetric change capability in response to local chemical stimulation have been used in microfluidic channels as valves to regulate flow without external control.12 With a porous structure, hydrogels functionalized with capture molecules can be fabricated in the microfluidic channel for specifically and efficiently binding analyte molecules13 or capturing cellular secretions.14 Moreover, the biophysical similarity of hydrogels to soft biological tissues makes them widely applicable in hydrogel-microfluidics for 3D cell culture, tissue engineering, and regenerative medicine.15,16 In particular, using monomer or polymer solutions as the aqueous phase, the hydrogel droplets can be generated rapidly using a microfluidic device. As an upgraded version of normal aqueous droplet microfluidics, hydrogel droplet microfluidics provides more controllable and flexible manipulation, which makes it attractive for single molecule/cell analysis. In this Account, we will review the progress made by our group and others in the applications of hydrogel droplet microfluidics in this area.

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HYDROGELS Both natural and synthetic hydrogels have been widely used in droplet microfluidics.17,18 These hydrogels can be cross-linked by various methods, including use of chemical chelators, ultraviolet light, and temperature changes. Hydrogel morphology and function are highly dependent on the nature of the starting materials as well the polymerization method. Natural hydrogels are either polysaccharides (e.g., agarose, alginate, hyaluronic acid, and chitosan) (Figure 1a−d) or proteins (e.g., collagen, gelatin, and fibrin), which are extracted from cells and polymerize under physiological conditions by electrostatic or other physical means. For example, agarose is obtained from the cellular walls of agarophyte seaweed. It is a neutral polysaccharide that forms thermally reversible hydrogels

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MICROFLUIDICS FOR GENERATING HYDROGEL DROPLETS Similar to liquid droplet generation, hydrogel droplets can be formed by microfluidics via emulsification of aqueous monomer or polymer solutions in an immiscible nonpolar liquid followed by polymerization. As the most frequently used microfluidic devices for liquid droplet generation,23 flow-focusing and T23

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Figure 2. Three principle microfluidic geometries available for hydrogel droplet generation: (a) T-junction, (b) flow focusing, and (c) microcapillary. Reproduced with permission from ref 24. Copyright 2015 Royal Society of Chemistry.

heterogeneity of cellular proliferation, differentiation, and drug cytotoxicity. Hydrogel droplets provide a highly controllable, reproducible, and flexible supporting microenvironment that allows individual cells to be independently stabilized, packaged, cultured, monitored, or manipulated. A microgel with precise internal structure is important for stable entrapment of cells in a controllable microenvironment.29 In order to produce homogeneous microgels, Utech et al. developed a new method to control the gelation process of alginate hydrogel droplets (Figure 3a).29 To prevent unin-

junction geometries (Figure 2a,b) can also be used for precursor droplet formation. The dimensions of the microfluidic-chip channel and the flow-rate ratio of the two phases can fine-tune the sizes of the droplets. The precursor droplets can be solidified into hydrogel droplets (gel beads or microgels) upon gelation. Once solidified, the gel beads are stable and remain as solids at room temperature. Alternatively, microcapillary device (Figure 2c) can also be used for hydrogel droplet formation. The aqueous and oil phases are supplied into the inner and outer capillaries, respectively, and droplets are formed from the breakage of aqueous phase by the shear stress of oil phase.

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HYDROGEL DROPLETS FOR SINGLE MOLECULE/CELL ENCAPSULATION Single molecule/cell encapsulation by emulsification of an aqueous suspension in an immiscible liquid is the primary step for high-throughput hydrogel droplet-based single molecule/ cell analysis. In general, passive encapsulation methods are used to produce single molecule/cell-encapsulated droplets. The droplet occupancy is governed by the Poisson distribution,24 where the average number of molecules/cells per droplet is determined by the concentration of molecules/cells in the feed solution. The Poisson distribut ion is g iven by λk

P(x = k) = k ! e−λ , where P is the probability of obtaining k molecules/cells in a droplet and λ is the average number of molecules/cells per droplet. By statistically diluting the feed solution, the proportion of single molecule/cell encapsulation can be statistically estimated. For example, if the λ is 0.5 copies per droplet (cpd), P(x=0) is 0.606, P(x=1) is 0.303, and P(x≥2) is 0.091, predicting that 60.6% of droplets contain no molecules, 30.3% contain 1 molecule, and 9.1% contain 2 or more molecules. However, the vast majority of droplets generated by Poisson distribution are empty (in the case λ ≪ 1). A more efficient means to obtain a large number of purified single molecule/cell emulsions is via postencapsulation sorting, in which molecules/ cells are sorted by their physical or chemical properties. Various methods have been developed for postencapsulation sorting, including fluorescence-activated dielectrophoresis,25 acoustic actuation with laser-based detection,26 and size-based hydrodynamic methods.27,28 These active methods can greatly improve the sorting efficiency and speed for generating a large number of single molecules/cells-encapsulated droplets.

Figure 3. (a) Microscopic image of fabricating alginate microgels on a flow-focusing device (scale bar: 50 μm). (b) Schematic illustration of the cross-linking process. The additional acid in the continuous phase dissociates the calcium−EDTA complex and releases the calcium ions to cross-link alginate. (c) Images of cell-containing alginate gels after being cultured for 0, 3, 6, 12, and 15 days, respectively (scale bar: 25 μm). Encapsulated cells are stained using calcein for cell viability analysis (insets). Reproduced with permission from ref 29. Copyright 2015 Wiley-VCH.

tended gelation due to direct contact of Ca2+ with alginate chains, they delivered Ca2+ as Ca2+-EDTA complex, which was dissociated by adding acetic acid in the continuous oil phase, thereby releasing Ca2+ to react with alginate chains (Figure 3b). By this method, the alginate microgels were formed in a highly controlled manner with excellent structural homogeneity. The authors further demonstrated the encapsulation of single mesenchymal stem cells in RGD-functionalized alginate, in which RGD offers integrin binding sites for cell attachment. Because of the mild polymerization process, the encapsulated cells with high viability could grow and proliferate inside the microgels for 2 weeks (Figure 3c).

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HYDROGEL DROPLETS FOR SINGLE CELL CULTURE 3D Cell microencapsulation is a promising strategy applicable to tissue engineering, drug screening, cell therapy, and regenerative medicine.18 In particular, single-cell 3D culture enables long-term single cell analysis, such as single-cell 24

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Figure 4. (a) Schematic illustration of the agarose emulsion droplet microfluidic method for single nucleic acid analysis. Statistically diluted templates were encapsulated in uniform agarose droplets, which were then thermally cycled for PCR amplification. Afterward, the droplets were cooled to form agarose beads for subsequent analysis. (b,c) Fluorescence microscope images of agarose microbeads after amplification from (b) 0 and (c) 1.5 copy/bead. (d) Percentage comparison of microbeads containing PCR product between the theoretical value using Poisson distribution and the observed value from experimental data. Reproduced with permission from ref 33. Copyright 2010 Royal Society of Chemistry.

a single DNA template, agarose solution, and PCR mix. The thermally responsive sol−gel transition of agarose ensured that the liquid form was maintained at all PCR temperatures for high PCR efficiency (∼95%). After cooling to form the microbeads, the monoclonality of the amplified DNA was maintained even after removing the oil phase, thus affording flexible downstream processing for flow cytometry, DNA sequencing, long-term storage, and so forth. In subsequent work, Yang et al. further demonstrated the capability of the agarose droplet microfluidic method for single RNA molecule detection34 in which reverse transcription and PCR were performed in one step. Using this method, single-cell transcriptome analysis was carried out to display a clear differentiation in gene expression level of the EpCAM cancer biomarker gene between two different cell lines (Kato III and MDA-MB-231 cells). The agarose droplet microfluidic method was also applied to single-cell multiplex PCR for highly sensitive, specific and quantitative detection of single Escherichia coli O157:H7 cells in a high background of 105 excess normal K12 cells.35 In these applications, the rapid sol− gel phase transition of agarose ensured high PCR efficiency and maintained the monoclonality of the PCR product for downstream processing. Tamminen et al.36 reported another emulsion-based procedure to encapsulate single microbial cells and amplify entire genomes in hydrogel beads with multiple layers of polyacrylamide and agarose. Individual microbial cells were trapped in the rigid polyacrylamide droplets that supported their genomes after cell lysis. The droplets were further converted into picoliter agarose reactors for the multiple displacement amplification (MDA) reaction. The picoreactors containing target genes were fluorescently labeled by PCR and analyzed by flow cytometry, and a single gene of individual microbial genomes could be distinguished from a mixture of microbial cells. E. coli strain XL1 genomes present at 0.1% of

The 3D culture of epithelial cells is a valuable tool for acinar formation, tubulogenesis, angiogenesis, and so forth. Dolega et al. applied flow-focusing droplet microfluidics to generate Matrigel beads containing single cells for 3D single epithelial cell culture.30 They demonstrated that a single prostate cell could proliferate and differentiate into a single acinus per bead without interacting with neighboring cells. Compared with traditional bulky 3D culture, the single-cell method exhibited similar growth rates, and formed acini with a more homogeneous size distribution. Moreover, it was easy to record acinar development from the very first division to the final development. This method provides a high-throughput approach for 3D culture preparation, recovery, analysis and screening in studying epithelial development, homeostasis, and cancer.

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HYDROGEL DROPLETS FOR SINGLE MOLECULE/CELL DETECTION There is a pressing need for sensitive and selective detection of single molecules, especially single DNA and proteins, as well as single cells, in chemistry, biology, medicine, and environmental science.8,31 The sol−gel transition property of hydrogel droplets enables flexible manipulation and detection after single molecule/cell encapsulation. The ability to selectively and sensitively detect nucleic acids at the single-molecule level is of great significance for basic biomedical research, medical diagnostics, and drug discovery.32 Polymerase chain reaction (PCR) is one of the most promising technologies for single nucleic acid analysis, due to its dramatic amplification capability to generate a large number of copies even from a single sequence. Yang et al. proposed an agarose droplet microfluidic method for emulsion PCR, which is highly efficient for detection of single copies of nucleic acids (Figure 4).33 The agarose-in-oil droplets were generated by a flowfocusing microfluidic chip at a rate of ∼500 Hz, each containing 25

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Figure 5. (a) Schematics of the workflow for single-cell encapsulation and detection of secreted cytokines using agarose droplet microfluidics. (b−e) Flow cytometry analysis of T cell coreceptor CD3 molecules expressed on the cell membrane: (b) empty agarose beads; (c) beads encapsulating Jurkat cells; (d) beads with Jurkat cells and control antibodies; (e) beads with Jurkat cells and anti-CD3 antibodies. Reproduced with permission from ref 37. Copyright 2013 Royal Society of Chemistry.

strain MC 1061 genomes could be differentiated. The first layer of polyacrylamide droplets was used to maintain genome integrity, while the second layer of agarose droplets provided picoreactors for amplification and facilitated downstream analysis. The method is especially useful for the study of highly complex environmental microbial communities by screening and capturing the genetic material of interest. Cellular heterogeneity also occurs at the protein level, but while single-cell genetic analysis has advanced significantly, single-cell protein analysis has posed a major challenge. Chokkalingam et al. used an agarose droplet microfluidic system to analyze the cellular heterogeneity of cytokine secretion in cancer cells (Figure 5).37 They used agarose droplets to encapsulate stimulated Jurkat T cells with 500 nm polystyrene capture beads functionalized with antibodies specific for IL-2, TNF-α, or IFN-γ. During the incubation at 37 °C, the secreted cytokines from single activated Jurkat T cells were confined in the same droplet and captured by

antibody-functionalized beads, followed by droplet solidification at 4 °C. After breaking the emulsion, the microbeads were incubated with fluorophore-labeled second antibodies and subjected to flow cytometry analysis for determination of the cytokine secretions of individual cells. This study demonstrated the successful use of agarose droplet microfluidics for protein analysis at the single-cell level.

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HYDROGEL DROPLETS FOR SINGLE CELL SEQUENCING In recent years, single cell sequencing38−40 has become a frontier edge for quantifying genetic expression variability between individual cells, especially for circulating tumor cells,41 prenatal testing samples,42 and so forth. There are many challenges for single-cell genomic sequencing, such as timeconsuming parallel processing of large numbers of cells,43 inefficient handling of limited and precious single-cell starting material, and amplification bias with low initial numbers of 26

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Accounts of Chemical Research single cells.44 Hydrogel droplet microfluidics is an ideal technique to address these challenges.39 In addition to the advantages of high-throughput, small sample volume, easy manipulation, and time and reagent savings, hydrogel droplets with 3D networks also provide rigid support for maintaining the genetic materials from the lysed cell while allowing access to reagents for amplification. Novak et al. proposed a robust agarose droplet platform for single-cell multiplex genetic detection and sequencing.45 As shown in Figure 6, single cells were encapsulated in agarose

PCR and subsequent analysis.45 Later on, the same group further used this system for single-cell forensic short tandem repeat (STR) typing.46 They conducted large-scale parallel single-cell multiplex droplet PCR to transfer the desired STR targets onto microbeads, followed by STR fragment size analysis by capillary electrophoresis. The agarose matrix enabled uniform cell lysis and DNA release, capture and amplification within the droplets, thus providing a robust platform for single-cell genomic analyses, including single cell expression studies, rare mutation detection, and forensic analysis. Based on alignate hydrogel droplets, Bigdeli et al. developed a robust method for single cell encapsulation and whole genome amplification.47 Cells were diluted in an alginate solution and sprayed into droplets. The encapsulated cells were lysed, followed by two-step whole genome amplification (WGA). The amplified products with high molecular weight could be trapped inside the alginate hydrogel beads, preventing genome cross-contamination. The DNA was extracted from the single beads for sequencing using the Illumina MiSeq platform. Sequencing results matched with the NCBI database, demonstrating the feasibility of this method for highthroughput single-cell sequencing. This work could be further improved with a more compatible genome amplification protocol, such as MALBAC, for higher and more uniform genome coverage.

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HYDROGEL DROPLETS FOR MOLECULAR EVOLUTION In natural systems, Darwinian evolution serves to develop or improve functional biomolecules. For decades, biotechnologists have tried to mimic nature and have explored various technologies for high-throughput molecular evolution to obtain novel functionalities from a large population library.48 Compartmentalization of individual biomolecules in hydrogel droplets has become a powerful tool for high-throughput molecular evolution in chemistry and biology, because the method maintains the monoclonality of each droplet and provides a simple retrieval method for evolving biomolecules. SELEX (Systematic Evolution of Ligands by EXponential enrichment) is one of the in vitro evolution methods for generating functional nucleic acids, known as aptamers, which

Figure 6. Workflow diagram of agarose droplets for single-cell genetic analysis. (a) Single cells and primer-functionalized beads are coencapsulated in agarose droplets. (b) Genomes of single cells are released inside droplets upon SDS lysis. (c) Agarose droplets are solidified into agarose beads, equilibrated in the PCR mix, emulsified with oil, and thermally cycled for PCR amplification. (d) Primer beads are then released and analyzed by flow cytometry or subjected to PCR and sequencing. Reproduced with permission from ref 45. Copyright 2011 Wiley-VCH.

droplets with primer-modified beads. After solidification, the cell-containing agarose beads were subjected to cell lysis to release the DNA. Because of the relatively small pore size of 1.5% agarose, the genomic DNA was effectively trapped in the agarose beads. After equilibration with PCR mix, the agarose droplets were reemulsified for large-scale parallel single-cell

Figure 7. Schematic overview of aptamer evolution by agarose droplet microfluidics. Single DNA sequences from a pre-enriched library are compartmentalized individually into agarose droplets for high-throughput single DNA amplification. The agarose droplets are then solidified to form agarose beads and stained with SYBR Green to select the DNA-containing fluorescent beads. The binding affinity of the DNA from each selected bead is screened against the target molecule. DNA sequences with high binding affinity and good selectivity can be directly used as aptamers or further subjected to sequencing. Reproduced with permission from ref 51. Copyright 2012 American Chemical Society. 27

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Figure 8. Directed evolution in biomimetic GSBs. (a) Emulsion droplets containing agarose and alginate were produced on microfluidics and allowed to gel into solid beads as GSBs. (b) For directed evolution in GSBs, single E. coli cells capable of expressing target enzyme were delivered in individual droplets. Cell lysis (1), catalysis (2), and GSB formation (3) were performed, followed by FACS sorting (4) and recovery of the coding plasmid (5). Further randomization can be carried out as a new library for next round of evolution (6). Reproduced with permission from ref 53. Copyright 2014 Nature Publishing Group.

library without their exact sequence information, thus making the aptamer selection process more rapid, efficient, and costeffective. Maintaining phenotype−genotype linkage is essential throughout in vitro directed evolution to mimic natural selection so that the sequences of the protein variants with the desired property can be retrieved.52 In addition, having large numbers of library members is also crucial for screening to be successful. Hydrogel droplet microfluidics provides an excellent, high-throughput platform to mimic cells by compartmentalizing both genes and proteins. Fischlechner et al. demonstrated this capability by evolving enzyme catalysts in biomimetic gel-shell beads (GSBs).53 As shown in Figure 8a, the hydrogel droplets were first generated by a flow-focusing microfluidic chip with the aqueous stream containing agarose and the alginate polyanion. The agarose was then solidified to form the gel core by decreasing the temperature. When breaking the emulsion, alginate and poly(allylamine hydrochloride) (PAH) formed a polyelectrolyte shell surrounding the agarose core as the GSBs. Such core−shell structures can retain molecules with molecular weights larger than 2 kDa, compared with 250 kDa for agarose beads. Taking the directed evolution of phosphotriesterase (PTE) as an example (Figure 8b), the authors generated monoclonal GSBs containing a single E. coli

can bind to their target molecules with high affinity and specificity.49,50 The SELEX process involves progressive enrichment of aptamer sequences from an initial library containing 1014−1016 ssDNA molecules by 8−30 rounds of partitioning and amplification, followed by screening of aptamer candidates from the enriched pool by cloning, sequencing, synthesis and characterization. Such a process is labor-intensive, time-consuming, inefficient and expensive. Yang et al. have employed agarose droplet microfluidics for more efficient aptamer screening.51 As the workflow in Figure 7 shows, a pre-enriched ssDNA library against Shp2 protein, a cancer biomarker, was statistically diluted and compartmentalized into individual uniform agarose droplets on a flow-focusing microfluidic chip. Then single-molecule emulsion PCR was performed in the agarose droplets, followed by temperature reduction to form monoclonal agarose beads with entrapped PCR amplicons. Upon staining, the bright gel beads containing DNA were harvested to retrieve the encapsulated DNA. The binding ability of ssDNA from each clonal bead was then screened via high-throughput flow cytometry. Only the ssDNAs with high binding affinity and high selectivity were selected as aptamers for sequencing. This method utilized compartmentalization in hydrogel droplets to allow rapid molecular evolution of individual DNA sequences from an enriched 28

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cell that retained the expressed PTE and its encoding plasmid as well as PTE substrate and lysis agent. The cells were lysed to release the PTE and allow it to react with substrate. To maintain the reaction product in GSBs, the substrate was linked to an oligonucleotide to increase its molecular weight for retention. Thus, the genotype and phenotype were preserved together in same GSB. The active clones were identified by fluorescence-activated cell sorting (FACS) at rates >107 GSBs per hour. They isolated a mutant PTE with 20-fold faster kinetics than the wild-type enzyme in less than 1h. The GSB method provides a practical and straightforward approach for high-throughput directed evolution of enzymes and functional proteins, as well as enzyme cascades and synthetic gene circuits.

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AUTHOR INFORMATION

Corresponding Author

*Tel: (+86) 592-218-7601. E-mail: [email protected]. ORCID

Zhi Zhu: 0000-0002-3287-4920 Chaoyong James Yang: 0000-0002-2374-5342 Notes

The authors declare no competing financial interest. Biographies Zhi Zhu received her Bachelor degree in chemistry from Peking University (China) in 2006 and her Ph.D. in analytical chemistry from the University of Florida (USA) in 2011. She joined Xiamen University (China) as an Assistant Professor in 2011 and was promoted to Associate Professor in 2012 and Professor in 2015. She was awarded with National Excellent Young Investigator Award in 2014. Her current research is focused on molecular recognition, singlecell analysis, and point-of-care testing.

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CONCLUSION AND OUTLOOK Through examples described in this Account, we have demonstrated hydrogel droplet microfluidics as a powerful tool for single molecule/cell analysis. By replacing an aqueous phase with a monomer or polymer solution, hydrogel droplets can be generated on microfluidic chips for encapsulation of single molecules/cells according to the Poisson distribution. The sol−gel transition endows the hydrogel droplets with new functionalities and diversified applications. First, the hydrogel can act as a 3D cell culture matrix to mimic the extracellular environment, and the porous structure allows the transport of oxygen, nutrients, growth factors, and waste. Therefore, the hydrogel droplets can support the single cell 3D culture up to 2 weeks, a key factor for proliferation heterogeneity studies, drug screening, and metastasis studies at the single-cell level. Second, the sol state of hydrogel droplets allows reactions, such as amplification and molecular recognition, to occur in solution at rapid rates with high efficiency. Afterward, the transition from sol to gel allows retention of the reaction products inside without diffusion. Then, the oil phase can be removed and flexible downstream analysis and processing can be performed, such as flow cytometry, sequencing, long-term storage, and so forth. Finally, because reaction products can be trapped in the hydrogel matrix, the phenotype and genotype can be easily maintained in the same hydrogel bead, providing a new platform for high-throughput molecular evolution. Although hydrogel droplet microfluidics for single molecule/ cell analysis is a very promising area of research in polymer sciences, analytical chemistry and cell biology, it still faces several challenges. First, the method to encapsulate one molecule/cell per hydrogel droplet is still limited by the Poisson distribution. New creative solutions are highly desired to avoid the vast majority of empty droplets as waste. Second, microchannel wetting due to cell secretions or cell debris deposited on microchannel walls15 is another problem affecting the robustness and reliability of molecule/cell encapsulation in hydrogel droplets. This problem could be improved with the development of efficient surfactants. Third, although a large number of hydrogel materials have been used for cell culture and tissue engineering, the number of polymers used in hydrogel droplet microfluidics is limited. Finally, most of hydrogels currently rely on temperature change, pH adjustment, or the addition of ions to induce the sol−gel transition. More effort is needed to develop biocompatible hydrogels that can switch phase by addition of molecules or physical parameters such as light or magnetism. With these challenges addressed, it is expected that hydrogel droplet microfluidics will find wide application in this exciting area of single molecule/ cell analysis.

Chaoyong James Yang received his Ph.D. from the University of Florida (USA) in 2006 and conducted his postdoctoral research at the University of California, Berkeley (USA) from 2006 to 2007. In 2008, he joined Xiamen University (China), where he is now the Lu Jiaxi Professor of Chemistry in the Department of Chemical Biology. He is the recipient of a CAPA Distinguished Faculty Award in 2012, a National Outstanding Young Investigator Award in 2013, a Chinese Young Analyst Award in 2015, and a Chinese Chemical Society-Royal Society of Chemistry Young Chemist Award in 2016. His current research is focused on molecular engineering, molecular recognition, high-throughput evolution, single-cell analysis, and microfluidics.

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ACKNOWLEDGMENTS We thank the National Science Foundation of China (21325522, 21422506, 21435004, 21275122, 21521004), the National Basic Research Program of China (2013CB933703), and Program for Changjiang Scholars and Innovative Research Team in University (IRT13036) for their financial support.

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