applications of microfluidics in quantitative biology

Quantitative Biology www.biotechnology-journal.com

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Applications of Microfluidics in Quantitative Biology

Yang Bai, Meng Gao, Lingling Wen, Caiyun He, Yuan Chen, Chenli Liu, Xiongfei Fu,*and Shuqiang Huang*

Quantitative biology is dedicated to taking advantage of quantitative reason-ing and advanced engineering technologies to make biology more predictable.Microfluidics, as an emerging technique, provides new approaches toprecisely control fluidic conditions on small scales and collect data in high-throughput and quantitative manners. In this review, the authors present therelevant applications of microfluidics to quantitative biology based on twomajor categories (channel-based microfluidics and droplet-based microflui-dics), and their typical features. We also envision some other microfluidictechniques that may not be employed in quantitative biology right now, buthave great potential in the near future.

1. Introduction

Traditional biology relies on descriptive characterization of thestructures and functions of basic components within livingorganisms, like genes, proteins, and pathways, which alwaysleads to qualitative pictures of biological systems. However, asmore information is deciphered, researchers realized that themyth of a living organism resides not only in the intricacies butthe interactions of these components.[1,2] The field of quantita-tive biology has emerged to go beyond qualitative understandingand unveil the rules that govern complex biological systems.Quantitative biology focuses on terms of modules and networksof living organisms from a quantitative approach: to understandthe biological modules and networks by analyzing quantitativedata and by modeling biological processes.[3,4] To this end,quantitative biologists demand the ability to finely controlenvironments and perform precise measurements. Thus, it is

Dr. Y. Bai, Dr. M. Gao, L. Wen, C. He, Y. Chen, Prof. C. Liu,Prof. X. Fu, Prof. S. HuangCenter for Synthetic Biology Engineering Research,Shenzhen Institutes of Advanced TechnologyChinese Academy of Sciences,Shenzhen 518055, People’s Republic of ChinaE-mail: [email protected]; [email protected]

The ORCID identification number(s) for the author(s) of this articlecan be found under https://doi.org/10.1002/biot.201700170.

© 2017 The Authors. Biotechnology Journal Published by Wiley-VCHVerlag GmbH & Co. KGaA. This is an open access article under theterms of the Creative Commons Attribution-NonCommercial License,which permits use, distribution and reproduction in any medium,provided the original work is properly cited and is not used forcommercial purposes.

DOI: 10.1002/biot.201700170

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imperative to apply advanced engineeringtechniques, among which microfluidics isproving to be of great value.

Microfluidics is the technique that canmanipulatefluids indimensionsof less thantens of micrometers.[5] First fabricated onsilicon substrates, microfluidics was startedin the 1950s, and was rapidly developed inthe 1980s because of the advances of siliconprocessing techniques. In the late 1990s,soft-lithography lowered the barrier ofmicrofluidic fabrication by reducing thedifficulty and cost,[6] and since then, micro-fluidics has beenwidely applied in biologicalresearches. Microfluidics allows biologicalresearchers to culture their objects at amuch

smaller scale, with higher resolution as well as more precisecontrol.[7,8] Integrated with external instruments, microfluidicsenables the studies from multi-cellular organisms to sub-cellularcomponents. Adapted to different biological purposes, thecapability of microfluidics continues being explored to satisfythe diverse applications.[9]

In general, microfluidics can be categorized into two majortypes by the location of cells: channel-based microfluidics anddroplet-based microfluidics. Performing both fluidic manipula-tion and cell culture in micro channels, channel-based micro-fluidics can provide a well-defined and long-term livingenvironment for real-time observation. Therefore, even subtlechange of cell behaviors can be captured for the purpose ofquantitative analysis. Droplet-based microfluidics can generatesamples (water droplets) at an extremely high throughput withcontrollable sizes. As each water droplet is protected by thesurrounding oil phase, they can be used as isolated reactors forcells living inside or for biochemical reactions.[10] Furthermore,the droplets can be employed as extracellular matrix, simulatingthree-dimensional micro-environments.

In this paper, recent applications of microfluidics inquantitative biology are reviewed, organized by category andtypical features of the applied devices. For the channel-basedmicrofluidics, the features are high resolution, flexible manipu-lation, and predefined function. For the droplet-based micro-fluidics, the features are high throughput, isolated environment,and three-dimensional platform (Figure 1). At the end of thispaper, we also discuss several emerging microfluidic techniquesthat have potential applications to quantitative biology.

2. Channel-Based Microfluidics

Channel-based microfluidics enables fluidic manipulation andcell culturing in channels and chambers of micro-scale. Fluidic

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Xiongfei Fu is a Professor at theCenter for Synthetic BiologyEngineering Research (CSynBER),Shenzhen Institutes of AdvancedTechnology (SIAT), Chinese Academyof Sciences. He received his PhD inphysics from University of Hong Kongin 2012. He undertook postdoctoraltraining at the Department ofMolecular, Cellular, and

Developmental Biology, Yale University. In 2016, hestarted his lab in CSynBER, focusing on the applicationsof quantitative analytics and advanced instrumentation to

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manipulations, such as flowing, mixing, confining, chemical/biological reacting, etc. can be performed to precisely control thebiological samples. For cell culturing, the channel-basedmicrofluidics allows both long-term and high-resolutionobservation at the same time. Using external controllers, onecan manipulate fluids down to pico-liter scale. With thisaccuracy, detailed behaviors of biological samples can bequantified. This method also allows the flexibility of tailoreddesigns for specific experiments. All these features cansignificantly assist quantitative biologists to interpret thebiological processes or results (Figure 2). Herein, we discussthree features of channel-based microfluidics and their respec-tive roles in quantitative biology.

biological studies, to facilitate our design ability of livingsystems.

Shuqiang Huang is an AssociateProfessor at the Center for SyntheticBiology Engineering Research(CSynBER), Shenzhen Institutes ofAdvanced Technology (SIAT), ChineseAcademy of Sciences. He finished hisdoctoral study in Dalian Institute ofChemical Physics in 2012. He was apostdoctoral associate in DukeUniversity from 2012 to 2016. His

research interests include microfluidics, synthetic biology,evolution study and material science, in particular, tointegrate microfluidics as a high-throughput, high-resolution, controllable and efficient tool to generate newmaterials and to answer specific biological questions.

2.1. High Resolution

Traditionally, researchers have used turbidostat or chemostat tomaintain a constant environment for long-term culture ofmicrobes. However, these devices often demand a large volumeof growth medium and normally have limited detectionresolution. Microfluidics can not only miniaturize these designsbut also enable high-resolution observations at small population,single-cell, and even sub-cellular levels.

By culturing different strains in one tiny chamber, micro-fluidics can serve as an excellent tool for analysis of microbialsocial ecology. For example, the You group co-cultured twosynthetic strains as prey and predator in a micro-chemostat chip.By long-term evolution and high-resolution observation, theysuccessfully observed extinction, coexistence, and oscillatorydynamics of the prey-predator system under different con-ditions. All these processes were predicted by a classical prey-predator model.[11] The Austin and Lambert group developed amicrofluidic device of connected chambers, where each chambercan be considered a small culturing environment. The authorsco-cultured wild-type bacterial cells with GASP cells (growthadvantage in stationary phase) that were evolved under aprolonged stress. Under different nutrition conditions, theydiscovered that the wild-type cells achieved maximum densityunder nutrient-limited conditions, while the GASP cellsdominated in the nutrient-rich conditions.[12] This phenomenonwas explained by evolutionary game theory under freedistribution.[13] One step further, they employed a similarmicrofluidic device to test the antibiotic resistance of the wild-type and GASP cells. In this case, the GASP cells were found toevolve antibiotic resistance faster.[14,15] In later work, they appliedthe device to a cancer cell model, from which they found thatboth the most substituted genes and the most highly expressedgenes of drug resistant cells were biased toward the most ancientgenes. This supports a model where cancer represents a revisionback to ancient forms of life adapted to high fitness underextreme stresses.[16]

Using devices called “mother machines,” the real-timeobservation can be promoted to the single-cell level. This kindof microfluidic chip confines a small quantity of microbial cells(one to few dozen) in a thin chamber. Connected to fluidicchannel to maintain the nutrient level, the mother machineallows researchers to continuously monitor the mother cells that

Biotechnol. J. 2017, 1700170 1700170 (2 of 9) © 2017 The

keep elongating and dividing while flushing away the daughtercells. Using such devices, several interesting aspects of cellgrowth or aging were discovered. For example, the Weitz grouptrapped yeast cells in lines, and analyzed the fluctuationsand patterns in protein expression within single cells overmultiple generations.[17] The Li group developed a microfluidicdevice that retained mother yeast cells by PDMS (Polydime-thylsiloxane) pillars.[18] They attached the yeast cells on a wallcoated by avidin to track individual mother cells throughout theirwhole lifespan, and found that the aging of cells wascharacterized by an increased general stress and a progressivelengthening of cell cycle for the last few cell divisions.[19] TheWalkmoto group demonstrated that growth noise amongsingle cells could cause clonal populations of Escherichia colito divide faster than the mean doubling time of their constituentsingle cells.[20]

Recently, with the help of a “mother machine” device, theJacobs-Wagner group discovered that microorganisms like E. coliand C. crescentus achieved cell size homeostasis by growing thesame volume or length between divisions.[21] This discovery wassummarized as the “adder model” to characterize microbial cellsize control. The same phenomenon was also observed by theJun group using a similar device that trapped single bacterialcells in tiny channels.[22–24] Interestingly, the You group analyzed

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Figure 1. A) Differences between conventional and microfluidic devices for biological researches. In conventional experiments, people usually useflasks, bottles or tubes for macro-scale culture and use pipettes for fluidic manipulation with the volume from microliters to millimeters. Whereas,microfluidic devices only need micro-scale space for cell culture and used micro-pump for fluidic manipulation with the volume from pico-liters tomicroliters. B) The way that microfluidics promote the development of quantitative biology. Microfluidics, which contains two major functionalcategories (channel-based and droplet-based) provides quantitative biologists a new tool to control, measure and analyze their objectives. The channel-based microfluidics can realize long-term culture and real-time observation for high-resolution data, flexible manipulation of diverse fluids to satisfy theexperiment control, and predefined function for special biological requirements. While the droplet-based microfluidics has some other particularcharacters, like high throughput for sample production, isolated environment to prevent from crosstalk or contamination, and three-dimensionalculturing micro-environment for in vivo simulation. All these features can help the development of quantitative biology, in terms of quantitative analysis,measurement, and environmental control.


the oscillating behavior in cell size and gene expressions, whichindicated a feedback mechanism in cell size control.[25]

Combining fluorescent microscopy to a “mother machine”device, the Elf grouppushed real-time observation precision to thesub-cellular level. Theyfirst labeled the replisome and ribosome ofE. coli in vivo. Bymonitoring the replisomes, they then confirmeddirectly thebacterial growth lawproposedbyDonachie,which saidthat the bacterial DNA replication starts at a constant cellvolume.[26,27] They also showed that only translating ribosomeswere excluded whereas free ribosomes could access the nucleoidfreely.[28] Microfluidics were also explored as in vitro platforms incharacterizing the transcription factor binding sites and theinteractions between transcription factors.[29,30]

Microfluidics has assisted inmonitoring and quantifying longterm cell behavior. For example, the Quake group monitoredsmall populations of bacteria and found unnatural cell densityoscillations programmed by a synthetic quorum sensing genecircuit.[31] Huang et al. developed a platform called “microbialswarmbot” with microfluidics, in which, biosafety control wasrealized through collective survival of engineered bacteria.[32]

2.2. Flexible Manipulation

By integrating particular units, such as micro-valves, micro-pumps, andmicro-mixers, microfluidics is capable to manipulatefluids flexibly at high precision. This feature is also enhanced byconnecting to external instruments like syringe pumps orprogrammed solenoid valves. The fluidic manipulation includesdiffusion, velocity adjustment, medium switching, and so on. It


allows researchers to apply defined stimulants or environmentalchanges to cells and furtherquantify the corresponding responses.

One simple example is a device that can maintain stablechemical gradients. This kind of device is widely used in cellmobility. For example, the Thierry group measured thequantitative relationship between phenotypic diversity andmotile performance of single bacterium in a chemotactic wave,which is realized within a microfluidic channel equipped withmicro-valves.[33] The Austin group actualized a three-dimen-sional visualization with confocal microscopy to study cancer cellinvasion through collagen matrix with constant chemicalgradients. By analyzing the front cells within such a microfluidicdevice, they proposed that cancer cells might minimize theirthermodynamic costs during invasion.[34] In these studies, thehighly stable gradient generated bymicrofluidics played a crucialrole in quantitative analysis of cell motility. As the moving tracksof single cells are highly stochastic, slight environmentalfluctuation can easily bury the quantitative differences in noise.To make the chemoattractant gradient more stable and theexperimental operation easier, a new chemotaxis microfluidicsdevice was designed by the Ouyang group recently.[35]

Microfluidics isnot only capableof creating stable environments,butcanalsoofcreating interchangeableenvironmentswhichmimicwell defined fluctuating nutrient conditions. The Hasty groupswitched thecarbonsource in thebacterialgrowthmediumbetweengalactose and glucose, showed that the wild-type strain gained agrowth advantage over mutant strains, and modeled the geneticselection within the fluctuating environment.[36] When a similarexperiment was repeated by the Kussell group, they discovered twotypes of non-genetic memory in E. coli.[37] Mathematical modeling




Figure 2. Applications of channel-based microfluidics in quantitative biology. According to the biological requirements, the fluidic flow withinmicrofluidic devices controlled by micro-valves and pumps could be designed as on-chip units or off-chip instruments, in a manner of flexible andprecise manipulation. More importantly, the microfluidic devices can be reshaped to realize different functions for diverse biological experiments, fromthe multi-cellular level to sub-cellular level. Taking advantage of long-term culturing and real-time observation, the channel-based microfluidics canfacilitate the acquisition of high-resolution results in an easier way.


showed that the memory mechanisms could improve long-termfitness under such fluctuating environments.[38]

The flexible and precise fluidic controlmakesmicrofluidics theideal method to introduce stimulants for quantitative biology.Chemical stimulants can be applied to cells at a pico-liter level, sothat the response of cells to specific stimulants can be preciselyanalyzed. For example, using the inducer IPTG (isopropyl-β-d-thiogalactoside) as a stimulant, the Elf group monitored theexpression dynamics of transcription factor LacI protein, andconfirmed the facilitateddiffusionmechanism that explains howatranscription factor searches its operator efficiently in vivo.[39–41]

Based on this observation, they later proposed a detailed model todescribe the binding dynamics between transcription factor andDNA,[42] andexcludedasimpleoperator occupancymodel for generegulation.[43] Using α-factor as a stimulant, the Hansen groupapplied different forms of α-factor (constant stimulation, singletransient pulse, and repeated short pulse) to induce genericperturbations on yeast cells and analyzed the responses of theMARK signaling system.[44] The Tu group modeled the bacterial


swimming behaviors in time-varying chemoattractant environ-ments.[45] Using the chemoattractant as stimulant, their modelwas then verified experimentally in microfluidics.[46]

Besides the chemical cues, osmotic pressure is alsoconsidered as a common stimulant to cellular behaviors.Microfluidics has proven to be a tunable and flexible technologyfor such research. By oscillating medium of different osmoticpressure in a Y-shape channel, the Ramanathan group studiedthe bandwidth of the HOG MAP kinase pathway of S. cerevisiaeresponding to high osmolality, and found that this prototypicalpathway acted as a low-pass filter.[47] The Hersen group usedsealed gasket chambers to experimentally regulate the osmoticpressure and found that severe osmotic compression couldtrigger a slowdown of intracellular signaling. They explained thatthis slowdown might be caused by molecular crowding.[48]

Similarly, the Luo group proposed a high-throughput methodfor the study of osmotic stress response of yeast cells.[49]

In combination with programmable optogenetic system, micro-fluidics could be used to induce real-time perturbations. The





McClean group also developed a culturing apparatus that couldgenerate flexible perturbations of intracellular protein concen-tration for the study of biological networks.[50]

2.3. Predefined Function

Besides traditional channels and chambers, microfluidics canalso be particularly designed for various purposes. For example,a brilliant design was made by the Ladoux group to map out theforce of epithelial cell migration. They placed the cells on a high-density array of elastomeric micro-fabricated pillars andmeasured the displacement of pillars during cell migration.[51]

Another example was the gut-like microfluidics designed by theHwa group. They studied the contraction effect to bacterialgrowth and showed that the bacterial density profile couldactually fit a simple reaction-diffusion model.[52]

Other than growth medium, the boundary effect andgeometries of microfluidic chamber may also affect cellbehaviors. The Hallatschek group designed a jamming chamberto map out the mechanic forces among jamming yeasts.[53] Afteranalyzing the experimental data, Gokhale and Gore realized thatthe jammed states in growing yeast populations sharedintriguing similarities with amorphous solids.[54] The Austingroup presented a microfluidic chamber with V-shape ratchets,and observed that swimming bacteria were enclosed instructures separated by a wall of funnels.[55–58] However, thebacteria could escape the ratchets under a chemoattractantgradient by collective cooperation.[59] Furthermore, the Ryugroup found that confined space could also help the movementof C. elegans.[60] Combined with fluidic visualization method, theZhang group even studied flow patterns generated by bacterialflagella movements.[61]

Microfluidics can also be designed for researches on multi-cellular systems like biofilms. For example, theSuel groupobservedan oscillating growth behavior of a monolayer bacterial biofilm in achemostat microfluidic chamber. Further tests and modelingsuggested communications between inner cells and outer cellswithin the biofilm, which was enabled by glutamate uptake andammoniumdiffusion.[62] In the samedevice, they foundout that ionchannels conducted long-range electrical signals within bacterialbiofilm communities through spatially propagating waves ofpotassium.[63] Later they realized that these electrical signals couldcoordinate bacterial nutrition uptake within the biofilm, and alsomodulate bacterial behaviors outside the community even fordifferent species.[64,65] Furthermore, numerous novel devices havebeendeveloped tostudyvarious livingorganismsincludingbacteria,yeast, algae, and C. elegan.[66–69]

Although the typical features of channel-based micro-fluidics have extended the research capabilities of quantitativebiology, there still exist several intrinsic limitations accordingto the experimental requirements. For example, the reducedsample volume and flexible manipulation features make itdifficult to produce high-throughput data, particularly whenthe biological library is large. With the high resolution andpredefined function, it is hard to simulate more complexbiological networks and monitor the corresponding dynamics.Additionally, due to the hydrophobic and permeable propertiesof PDMS, the mostly used material in microfluidics, cell


culturing and biological reactions may also confront criticalchallenges.

3. Droplet-Based Microfluidics

Microfluidic techniques are not only well recognized for theirabilities to handle, manipulate, and process small volumes offluids,[5] but also for their capabilities in producingmonodispersedroplets at high frequencies (Hz-KHz) with volumes rangingfrom pico-liter to micro-liter.[70] Droplets are small liquid-basedcompartments, partly or completely bounded by free interfaces.[71]

The single-emulsion droplets are normally either water-in-oil oroil-in-water, depending on the transition of fluidic phases. As cellsusually live in a hydrophilic environment, in this review we willonly focus on the water-in-oil droplets. There are several intrinsicand typical features for microfluidic droplets, which for biologicalapplications include high throughput, isolated environment andthree-dimensional platform (Figure 3).

3.1. High Throughput

Droplet generation andmanipulation can be easily modulated bythe microfluidic design, flow rate, fluid property, surfactant, andperturbing parameter. This tunability allows the high through-put feature to be applied in many fields, including biochemicalassays,[72] cell-based enzymatic assays,[73] DNA amplification,[74]

cell sorting,[75,76] microspheres,[77] nanoparticles,[78] core-shellmicrocapsule synthesis[79] so on.

As a detailed example, cellular heterogeneity arising from thestochasticity of gene expression, protein function andmetabolitequantity is crucial for cell behavior and evolution.With the abilityto generate diverse samples at high throughput, droplet-basedmicrofluidics is a good candidate for experiments on cellheterogeneity, which traditionally have been hard to perform.Biological samples can be randomly encapsulated in millions ofdistributed droplets in several hours.[80] As long as the randomsampling is assured, the probability to find a certain number ofsamples inside a droplet follows the Poisson distribution.[81] Inthis way, samples with a large diversity are generated for furtherinvestigations.

Using such methodology, the Huck group quantified thenoise of gene expression in vitro. They found that the DNA copynumber and macromolecular crowding were the key factors tothe heterogeneous gene expression, which directly increasedthe system’s stochasticity.[82] Moreover, they also used an agarosedroplet system to analyze the cellular heterogeneity of cytokinesecretion in cancer cells. This system encapsulates cells togetherwith functionalized cytokine-capture beads for subsequentbinding, and for following detection of secreted cytokines atsingle-cell level.[77] The Mazutis group used droplet-basedmicrofluidics to capture thousands of individual cells withunbiased diversity and performed the whole transcriptome andgenomic analysis successfully.[83] The Weitz group developed anultrahigh-throughput screening platform for directed evolutionto discover variants of the target enzyme. Their device is athousand-fold faster and a million-fold cheaper than traditionalones.[84]




Figure 3. Applications of droplet-based microfluidics in quantitative biology. There are three typical modes for droplet generation, as cross flow(Mode 1), flow focus (Mode 2), and co-flow within two glass capillaries (Mode 3). Due to the small size of microchannel and fast flow rate, the dropletscould be produced as a high-throughput manner. Since each droplet is surrounded by the outer oil, thus can be considered an isolated reactor forparticular biological assays. When biocompatible polymers are involved, each droplet can be solidified to form an independent three-dimensionalenvironment for cell culturing. By integrating the appropriate methods, the droplet could be detected, analyzed, or sorted to answer diverse questions inquantitative biology.


3.2. Isolated Environment

As the oil layer forms a barrier to water-soluble molecules,droplets are protected from crosstalk and contamination. Forthis reason, one droplet can be considered as an isolated reactor,and it allows researchers to perform complex biological reactionswith minimal sample consumption.[85–88]

For example, PCR and Real-time PCR are important technolo-gies to quantify gene copy numbers or genetic expression levels.Performing these kinds of reactions in droplets enables geneticmeasurements for small populations and even single cells, whichcannot be achieved using traditional methods. The Colston groupfirst built a lab-on-chip system for pico-liter droplet generation andPCR reactions. They used an off-chip valving system to stop thedroplets on-chip, allowing them to be thermally cycled throughthe PCR protocol without droplet motions. In comparison to thecommercial PCR systems, the microfluidic device reduced thereactor size by six orders ofmagnitude.[89] The Lee groupdesignedadroplet-basedPCRplatformofextremelyhigh throughput. Itwascapable to generate over one million monodispersed droplets of50 pico-liter in 2–7min, and then the nucleic acid contents ineach droplet were digitally quantified.[90] Similar improvementshave been reported by other groups, such as TaqMan PCRreaction, DNA synthetic reaction and even for mammalian cellsequencing.[80,91–93]


The traits of isolation can also benefit diverse biologicalquantifications, such as small molecules, nucleic acids, andproteins.[72,73,94] It allows researchers to quantify their targetmolecules with a very small amount of sample, sometimeseven a single cell. For example, the Anderson group developeda hollow-microcapsule based microfluidic platform forbiomolecular sensing.[95] Based on the laws of molecularadsorption and desorption of PDMS surface, they furtherexploited a contact-induced droplets dosing platform for DNAand ion quantification.[96] With droplet-based microfluidics,Abbsapourrad et al. developed a label-free protein analysis forsingle cells, which quantified the absolute amount, ratherthan relative amount, of proteins without antibody labelingnor spectrometry analysis.[97] The Shum group presented aone-step immunoassay of C-reactive proteins using thedroplet microfluidics. They promoted the limit of detectionto 0.01 μgmL�1, ten times more sensitive than the conven-tional assay.[98]

The ability to isolate population-level cells also makesmicrofluidics the perfect tool to analyze the myth of cell-cellcommunication, among which themost famous phenomenon isquorum sensing (QS). The Ismagilov group dispersed cells ofPseudomonas aeruginosa in droplets and confined them inmicrofluidic wells. They showed that droplets containing as fewas one to three cells were able to initiate quorum sensing and





achieve QS-dependent growth.[99] Using spatially extendedarrays of micro-droplets, the Simmel group studied the diffusionof cell–cell communication signals. They found that transferringsignal molecules among closely contacted droplets could affectthe spatial communication patterns.[100] Using addressabledroplets, the Lee group successfully analyzed the bacterialcommunications in response to the population ratio between thesignal senders and receivers.[101]

3.3. Three-Dimensional Platform

Thanks to the superior stability and large surface to volume ratio,droplets in microfluidics can be used as three-dimensionalplatform for cell culturing, particularly after being polymerizedas hydrogel micro-particles. The micro-particles are normallymade by biocompatible materials, which share similar chemicalproperties and physical stiffness with the extracellular matrix(ECM). For example, the Que group embedded the breast cancercells in collagen droplets and recorded themigration trajectories.They showed that the migration speed of breast cancer cellscould reach 3–6 μmh�1.[102]

Another application of droplet-based microfluidics werefound in tissue engineering and regenerative medicines, whereresearchers designed and fabricated organ-like three-dimen-sional structures to simulate the organ functions.[103] The three-dimensional micro-environment construction provides analternative between conventional two-dimensional methodsand animal models that are normally costly and unreproducible.The He group reported the generation of biomimetic ovarianmicro-tissue through microfluidics by using both harder(alginate) and softer (collagen) materials. They quantified theeffects of luteinizing hormone (LH) and epidermal growth factor(EGF) on ovulation, and discovered that the mechanicalheterogeneity of hydrogel droplets was crucial in regulatingfollicle development.[104] Using droplet-based microfluidics, theRevzin group cultured primary hepatocytes in microcapsules ofliquid core and polyethylene glycol (PEG) gel shell. Compared tothe two-dimensional culture, primary hepatocytes showedhigher production of albumin and other metabolites in thethree-dimensional micro-environment.[105] The development oforgan-like structures in droplets is also helping drug screeningThe Wang group constructed hydrogel bead-based tumorspheroids for testing anticancer drugs.[106] The Zuchowskagroup built tumor spheroid structures and observed the dynamicchanges in metabolic activity of the tumor cells after drugadministration.[107,108]

Similar to channel-basedmicrofluidics, droplet-basedmethodsare also limited by several confounding factors. For example, withthe high-throughput generation and sorting of tiny droplets, it isactually very challenging to address each one of them from thebeginning of a biological reaction to the end; With the feature ofisolated environment, there are still some hurdles to completelystop the diffusion of small chemical molecules. While thediffusion rate and quantity in the outer oil phase is typicallynegligible, this still might be quite detrimental to some biologicalsystems. Inaddition,while thedroplet-basedplatformallows threedimensional modeling, it remains difficult to mimic the multi-layer tissue environment sought e.g., in regenerative medicine.


4. Conclusions and Perspectives

The development of quantitative biology requires preciseenvironment control, quantitative measurement, and quantita-tive analysis supported by mathematical models. Novel toolsemployed in multidisciplinary research are providing newinsights into living creatures. Microfluidics sheds more light onquantitative biology from the angles of micro scale, fluidicmanipulation, and precise control. In this review, we have onlysummarized part of the biological applications of channel-basedand droplet-based microfluidics. However, the field of micro-fluidics is still growing, and there are more emerging techniqueswith the potential to be applied.

One example is the paper microfluidics. Here the device isconstructed out of paper, with fluidic flow driven by capillarywetting force and directed by channel boundaries of hydrophobicwax or polymer. Due to the low cost and handy operation, papermicrofluidics can be used as portable devices for metabolitesanalysis. With several drops of liquid, one can easily quantifycomponents like glucose, cholesterol, lactate in human plasmaand whole blood, or ethanol (or acetaldehyde) in aqueoussolution.[109] For example, the Whitesides group designed anelectrochemical paper-based analytical device for glucometer.[69]

The Tabeling group successfully performed isothermal ReverseTranscription and Recombinase Polymerase Amplification (RT-RPA) on synthetic RNA of Ebola virus, and applied it in Guineato detect the Ebola virus in humans.[110]

Another example is digital microfluidics (DMF), which wasproposed by the Fair group.[111] DMF uses programmed electro-field to manipulate droplets on a flat surface. The Fair groupdeveloped a series of glucose assays in physiological fluids.[111]

Besides the platform of controlled reactors, DMF can also beused for cell culturing and real-time analysis. TheWheeler grouptook the advantages of programmable droplet merging andsplitting to refresh culture medium automatically.[112] Further-more, they cultured and differentiated dopaminergic neurons,and successfully evaluated its dopamine uptake.[72] As anextension of droplet-based microfluidics, DMF has a higherpotential in programmable behaviors.

Other than the twomajor typesofmicrofluidicsmentioned, thereare still many aspects in microfluidics that are not covered in thisreview.Many are not currently employed in the field of quantitativebiology, but do carry great potential for future researches.

AbbreviationsGASP cells, growth advantage in stationary phase cells; PDMS,polydimethylsiloxane; IPTG, isopropyl-β-d-thiogalactoside; LIF, leukemiainhibitory factor; QS, quorum sensing; ECM, extracellular matrix; LH,luteinizing hormone; EGF, epidermal growth factor; PEG, polyethyleneglycol; DMF, digital microfluidics; RT-RPA, Reverse Transcription andRecombinase Polymerase Amplification.

AcknowledgementsY.B and M.G contributed equally to the manuscript. This paperwas supported by the National Natural Science Foundation ofChina (3157009, 11504399, 31770111, 31700090); Shenzhen ScienceTechnology and Innovation Commission (JCYJ20170307163830109;





JCYJ20170413153329565); the Guangdong Natural Science Funds forDistinguished Young Scholar Grant (S2013050016987); the ShenzhenPeacock Team Project (KQTD2015033117210153); the Shenzhen PeacockProject (KQCX2015033117354154); the SIAT Innovation Program forExcellent Young Researchers (2017003). We also thank Anita Hio KeiChong and Taku Tokuyasu for their suggestions and revisions of thismanuscript.

Conflict of InterestThe authors declare no commercial or financial conflict of interest.

Keywordslab-on-chip, micro/nanofluidics, quantitative biology, synthetic biology,systems biology

Received: July 14, 2017Revised: September 3, 2017

Published online:

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