Droplet Microarrays in Biomedical High-Throughput Research

Yifeng Chen; Cong Wang; Kefeng Ren; Jian Ji

doi:10.7536/PC200667

Progress in Chemistry ›› 2021, Vol. 33 ›› Issue (4) : 543-554 DOI: 10.7536/PC200667

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Droplet Microarrays in Biomedical High-Throughput Research

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Abstract

In order to handle a large number of experiment conditions and outputs and further accelerate the material development process, high-throughput screening has become a more and more important experimental method, which has been widely used in many fields to improve experimental efficiency. And the high-throughput platform is the basic condition for carrying out high-throughput experiments. Existing high-throughput platforms, like microtiter plates, still suffer from problems such as high consumption and low experimental throughput when dealing with precious samples and reagents and further optimization is still required. As an emerging miniaturized and integrated high-throughput platform, droplet microarrays have the advantages of low consumption of reagent and sample, short reaction time, high integration and strong operability, and have been widely studied and applied in the field of biomedicine. In this paper, the construction methods of droplet microarrays are summarized and divided into two categories: surface chemistry driving and physical morphology assisted. The advantages and disadvantages of different construction methods are briefly analyzed. Then the applications of droplet microarrays in biomedical high-throughput research are also briefly introduced from the four directions of 2D cell screening, 3D cell culture, single cell analysis and whole organism screening. Finally, the problems existing in the application process of droplet microarrays and its future development direction are also summarized and analyzed.

Contents

1 Introduction

2 Construction methods

2.1 Surface chemistry

2.2 Surface morphology

3 Applications in biomedical high-throughput research

3.1 2D cell screening

3.2 3D cell culture

3.3 Single cell analysis

3.4 Whole organism screening

4 Conclusion and outlook

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Keywords

droplet microarrays / construction methods / biomedicine / high-throughput

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Yifeng Chen, Cong Wang, Kefeng Ren, Jian Ji. Droplet Microarrays in Biomedical High-Throughput Research. Progress in Chemistry, 2021, 33(4): 543-554 DOI:10.7536/PC200667

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1 Introduction

By means of parallel and integrated technology, high-throughput experiments can rapidly prepare materials with different compositions and structures, and obtain their performance parameters, which can greatly improve the efficiency of experiments and is one of the effective tools to obtain the optimal parameter combination of complex material systems. In the past decades, the concept of miniaturization has been popularized as a means of high throughput and has been widely used in the field of biological and chemical analysis^[1⇓~3]. Miniaturized devices can further greatly improve the preparation and characterization throughput of materials, so they have great development potential and application value. In the field of biomedicine, microplate is a relatively mature high-throughput experimental platform. However, compared with the difficulty of obtaining cells or reagents and the diversity of factors to be explored, the consumption of reagents and samples in microplates is still large, and the throughput of experiments is also low^[4]. Therefore, a high-throughput experimental platform for further miniaturization remains to be developed.

Micro-droplet array technology realizes the splitting and capturing of droplets by constructing hydrophilic-hydrophobic patterned areas on the surface or physical intervals such as regularly distributed micropores and microcolumns, thus forming an array of tiny droplets with specific geometric shapes (Fig. 1). The difference of surface wettability in different regions, especially the extreme superhydrophobic-superhydrophilic contrast or the volume repulsion and shear effect caused by dense physical spacing make the split droplets closely arranged, which greatly improves the throughput that can be processed in a parallel operation. With the technology of surface wettability difference, researchers can control the morphology and size of micro-droplet arrays more freely without worrying about cross-contamination or fusion between different micro-droplets, which ensures the reliability and reproducibility of high-throughput experiments. As a new high-throughput experimental platform, the volume of a single droplet in the array is very small, even up to 80 nL or less, which saves 20 times the volume of the 1536-well microplate with the smallest hole, and at least 125 times the volume of the 384-well microplate^[5]. Imultaneously, due to that small volume of the liquid drop, the reaction time is effectively shortened; As an integrated chip, the microdroplet array can greatly improve the experimental throughput. The openness of the droplets on the array is conducive to the operation of adding liquid, taking liquid and analyzing in the subsequent experiments^[6]. Because of these advantages, microdroplet arrays have great potential for high-throughput screening. In this paper, the preparation methods of microdroplet arrays are briefly summarized, and their applications in biomedical fields are introduced, and some existing problems and future development directions are summarized and analyzed.

2 Micro-droplet array preparation technology

The preparation process of micro-droplet array is the process of formation, separation, capture and stabilization of many micro-droplets. In general, the preparation methods can be divided into two categories: surface chemical-driven and physical morphology-assisted. The former is more widely used and has a clear theoretical basis, but it is more suitable for liquids with high surface tension (such as water). The preparation process of the latter has no clear theoretical rules, but it also has good applicability to liquids with low surface tension (such as most organic solvents).

2.1 Surface Chemical Drive

the development of surface chemistry provides a very effective means for The regulation of surface properties,such as coating,layer-by-layer assembly and photochemical reaction^[9]^[10]^[11]。 Patterned surfaces with different wettability are widely used in the preparation of micro-droplet arrays。

Fang et al.prepare a hydrophilic silicon dioxide layer on that surface of a silicon WAF by thermal oxidation treatment,and the silicon dioxide layer is silanized to become hydrophobic,and then the silanized coat is removed by ultraviolet light irradiation and wet etching under the action of a photoresist and a photomask,Thus obtaining a hydrophilic-hydrophobic dot array,and a micro-droplet array can be obtained by dripping water droplets containing different food colorants on the hydrophilic dots,and the volume of a single droplet is in a nanoliter scale(fig.2)^[12]。 Liu et al.Prepared microdroplet arrays by using polydimethylsiloxane(PDMS)stamp method^[13]。 They first Used soft lithography to prepare patterned PDMS stamps,then attached them to glass slides,and stripped PDMS after oxygen plasma treatment.A layer of hydrophobic PDMS nano-film remained in the area of glass slides in contact with PDMS,while the exposed area of glass slides was hydrophilic(Fig.3A).When an aqueous solution is dropped on the surface,a micro-droplet array can be obtained due to the repelling effect of the hydrophobic region and the wetting effect of the hydrophilic region.Lee et al.used laser direct writing technology to replace the above soft lithography technology,prepared micro-droplet arrays without mask,and then cast PDMS prepolymer on the arrays to obtain PDMS stamps with complementary surface topography^[14]。 In turn,the stamp can be used for preparing the micro-droplet array chip,thereby realizing the mutual conversion between the two.Huang et al.used soft lithography to fabricate a microfluidic chip with parallel microchannels,which can efficiently distribute droplets to the target substrate,while using wet etching and lithography to fabricate a micropore array with hydrophilic inside and hydrophobic outside,thus avoiding cross-contamination of droplets between adjacent sites^[15]。

However,the hydrophilic-hydrophobic patterned surfaces are prepared by the above processes,and due to the small difference in wettability between the hydrophilic-hydrophobic patterned surfaces,the formation of the micro-droplet array is often realized by dropping through a dropper or by means of specialized liquid processing equipment,and the distance between adjacent droplets is difficult to further reduce,otherwise,it is easy to cause droplet fusion.in order to further simplify the experimental operation,the preparation of superhydrophilic-superhydrophobic patterned surfaces has been studied in depth.Levkin et al.Obtained a superhydrophilic porous polymer layer by photoinitiated copolymerization,and then prepared a superhydrophobic region by photografting under the coverage of a photomask(Fig.3B)^[16]。 They built on this by taking advantage of the extreme wettability differences of the superhydrophilic-superhydrophobic regions,when rolling a liquid over the patterned surface or pulling the substrate out by immersing it in an aqueous solution,Discontinuous dewetting occurs,resulting in an array of tens of thousands of picoliter to microliter volumes in one step(fig.4A)^[7]^[17]。 Subsequently,they applied the thiol-alkyne photoclick reaction to the preparation of superhydrophilic-superhydrophobic patterned surfaces,which greatly shortened the preparation time^[18]。 Song et al.prepared a uniform porous micro-nano composite structure,modified it by chemical vapor deposition to obtain a super-hydrophobic region,then irradiated it by deep ultraviolet under the cover of a photomask to obtain a super-hydrophilic pattern,and split the droplets by using the difference of surface wettability to prepare a uniform micro-droplet array of picoliter to femtoliter volume^[19]。 At the same time,the effects of the size of the super-hydrophilic pattern,the contact force and the relative sliding speed on the droplet volume were investigated.Wang et al.Dispersed Ag-ZnO nanorods with high photocatalytic activity into octadecyltrichlorosilane(OTS)matrix,then deposited on indium tin oxide substrate to form a superhydrophobic Ag-ZnO-OTS coating,and further obtained a superhydrophilic pattern under ultraviolet radiation(UV)(Fig.4B)^[20]。 Through the combination of photolithography and other technologies,the super-wettability difference of silicon wafer surface is successfully obtained,thus forming a micro-droplet array for the realization of transfer technology^[21,22]。 Superhydrophilic-superhydrophobic patterning greatly simplifies the operation of microdroplet array formation And improves the throughput of the operation,but it requires higher chemical modification.and because of the further reduction in size,new requirements are placed on the precision of the overlay alignment when other compounds are subsequently added by the sandwich method using the overlay of two microdroplet arrays^[23]。

Between the hydrophilic-hydrophobic and superhydrophilic-superhydrophobic wettability differences described above,hydrophilic-superhydrophobic patterns have also been extensively studied^{[24⇓⇓⇓~-28]}。 Mano et al.used a polyvinyl chloride patch to protect the local polystyrene substrate,and then performed phase separation to prepare a superhydrophobic surface,so that after removing the patch,the original area retained wettability^[26]。 Zhao et al.,inspired by the uneven texture and wettability of the beetle,directly used the natural superhydrophobic surface,the morpho butterfly wing,to prepare the micro-droplet array^[28]。 Aft that wings of the morpho butterfly are pretreat,the wings are soaked with a pre-gel solution,and then the wings are irradiate with ultraviolet light under the cover of a photomask to initiate polymerization,so that hydrophilic gel sites are obtained.As an alternative to hydrophilic sites,Mano and others have developed a new way to prepare micro-indentations on super-hydrophobic surfaces and use capillary force to fix micro-droplets,thus forming micro-droplet arrays^[29]。 Although hydrophilic-superhydrophobic patterning technology is lower in throughput than superhydrophilic-superhydrophobic technology,it is more tolerant to surface chemical modification defects。

2.2 Physical Morphology Assistant

In addition to the above ways of obtaining wettability differences by surface chemistry,the capture of micro-droplets by surface physical topography is also a very direct and effective means to prepare micro-droplet arrays。

the microwell array can be use to capture microdroplets to form an array.Blainey et al.fabricated a microwell array chip by means of soft lithography.Emulsified sample droplets can be loaded onto The chip by a micropipette.each microwell can randomly capture two droplets,each with a volume of 1 nL,and each pair of droplets can be fused by applying a high-voltage AC electric field^[30]。 To prevent evaporation and chemical cross-contamination,the microwell array was encapsulated with a glass substrate.Zhang et al.fabricated an array of microcavities using photolithography and other techniques.Each microcavity is composed of a hydrophilic bottom and a hydrophobic barrier,which can capture femtosecond volumes of microdroplets^[31]。 In order to prevent the evaporation of micro-droplets,perfluoroether oil is used to seal the micro-cavity.Lee et al.First cured the monomer layer by UV light under the cover of a photomask to obtain a polymer cavity array,and then formed a layer of conductive polymer film on the array^[32]。 When the array is pressed onto a pre-prepared polyvinyl chloride gel,a microdroplet is obtained in each cavity,and the formed microdroplet array can be used as a microlens array。

Physical spaces such as microcolumns can also be used to capture microdroplets.Compared with the micropore array,the micro-column gap is used to capture the micro-droplets without blocking the flow of the liquid,so it is more efficient.Fang et al.Fabricated a microcage array chip using soft lithography for rapid formation of large-scale two-dimensional microdroplet arrays^[8]。 Each microcage is surrounded by four microcolumns,and the oil phase can spread rapidly through the gaps between the microcolumns,while the microcages can efficiently capture the droplets in the oil phase,and an array containing about one million droplets can be formed in less than 90 s.Jeon et al.Prepared microcolumn arrays on polystyrene surfaces by solvent-assisted molding(fig.5)^[33]。 Unlike the previous work,this work does not require the preparation of micro-droplets in advance.When the droplet body slides over the surface of the microstructure,the micro-droplets are trapped in the area surrounded by micro-pillars due to shear effect,thus directly forming a micro-droplet array.At the same time,different microdroplets can be obtained by adjusting the height and spacing of the microcolumns。

the microdroplets may not only be captured between the microcolumns,but may also be loaded onto the microcolumns.Zhang et al.Used perforated stainless steel plates as templates for casting to obtain PDMS microcolumn arrays^[34]。 Due to the high adhesion of the microcolumns,each microcolumn can be loaded with a microdroplet,and the microdroplet can still exist stably even if the array is rotated and deformed.In combination with an integrated circuit,the array is used for the electrochemical determination of glucose concentration.Inspired by the adherent Collembola skin and gecko feet,Gu et al.Prepared a miniature double concave corner array with high resolution using 3D printing technology based on two-photon polymerization(TPP)(Fig.6a)^[35]。 the compact arrangement of microstructures on the array can effectively capture microdroplets,and three different Cassie adhesion behaviors can be achieved by reasonably adjusting the arrangement.The array is suitable for both water and different organic solvents,and the ambient phase can be either air or liquid。

Microhole and microcolumn arrays provide clear physical boundaries to limit the diffusion of microdroplets and avoid the fusion of droplets to the greatest extent,but the corresponding preparation costs are generally high,such as relying on photolithography technology.the technology of micropore and microcolumn arrays to capture microdroplets usually does not require chemical modification,so it has higher durability,but with the continuous reduction of microdroplet size,the corresponding requirement for the accuracy of physical spacing is gradually increasing,and the cost problem is becoming more and more prominent。

in addition to the above methods,other physical mechanisms have also been explored for the formation of microdroplet arrays.Mann et al.Used acoustic standing waves to capture pH-responsive polymer agglomerate droplets in aqueous solution and form patterned arrays in situ^[37]。 Under the induction of pH,the molecules assemble on the surface and core of the microdroplets to form nanofilaments,and the nanofilaments are entangled to form micro-patterned hydrogels.Song et al.Utilized demulsification to achieve patterning of the microdroplet array(fig.6B)^[36]。 Firstly,a hydrophilic texture substrate is prepared by using a traditional photolithography technology and deep reactive ion etching,then an oil-in-water emulsion is dripped on the substrate,and due to local capillary difference,A plurality of emulsion patterns will be formed,and as the water and oil evaporate,demulsification will occur radially from the outer boundary to the center of the emulsion pattern,forming a domino like array of microdroplets.the technology of forming micro-droplet arrays by using physical effects such as acoustics provides researchers with a wider range of choices,but its applicability is smaller,and it is difficult to adjust the shape,size and other parameters of micro-droplets flexibly。

3 Applications in biomedical high-throughput research

Due to the advantages of low sample consumption, short reaction time and good compatibility with various analytical methods, microdroplet arrays have been developed and applied in many fields, such as microsensor chips, microparticle assembly and so on^[38⇓~40]^[41⇓~43]. In the biomedical field, it also has great application potential.

3.1 2D cell screening

cell culture and screening is a very important research method in the field of biomedicine,and it is the basic operation in biomedical experiments.At present,Cell culture and screening are mainly carried out in microplates.However,compared with some precious samples,the amount of microplates is large and difficult to apply.in order to further reduce the sample consumption and improve the experimental throughput,the micro-droplet array is a good choice.Each droplet in the array is equivalent to a separate micro-culture container,and different cells and reagents can exist in isolation in these droplets,effectively avoiding cross-contamination。

Mano et al.Used biodegradable PLLA to prepare superhydrophobic surfaces and treated them with Ar plasma for different times to make the surface hydrophilicity adjustable in the range of superhydrophobic and superhydrophilic(Fig.7)^[44]; At the same time,cell culture experiments showed that there was almost no cell adhesion on the superhydrophobic surface,while the enhancement of hydrophilicity was conducive to cell adhesion.This provides a guarantee for the division of cell culture micro-regions based on the difference of hydrophilicity and hydrophobicity of micro-droplet arrays in different regions of the surface.Huang et al designed a polydimethylsiloxane microchannel chip for forming and distributing microdroplets to the micropore array,and successfully realized the operations of cell inoculation,passage,drug stimulation,etc.the application of peristaltic pump enabled the accuracy of liquid manipulation to reach 5 nL,which provided a guarantee for the accuracy of high-throughput cell experiments^[45]。 Xi et al.Coated a layer of poly(N-isopropylacrylamide)on a glass substrate,formed a patterned surface by mask plasma etching,and printed miRNA or siRNA solution on the patterned surface sites to form a microarray^[46]。 Ince cell are difficult to grow on poly(N-isopropylacrylamide),

But grow well on a glass surface,that cell can be confined to each site when the cell suspension is entirely coat on the array.at the same time,they studied the behavior of miRNA in cell migration regulation by using the temperature-sensitive characteristics of poly(N-isopropylacrylamide),which can be dissolved and eliminated,and cells can move freely.Levkin et al.Further developed a method for preparing complex patterns of multiple types of cells,and demonstrated that the spatial arrangement and geometry of multiple different cell types could be controlled on hydrophilic-superhydrophobic patterned surfaces,and could be maintained for At least 3 days^[47]。 A micropipette is use to drop different cell suspensions into that correspond hydrophilic regions,respectively,thereby obtaining a plurality of isolated micro-culture vessel.the shape of the droplet depends on the geometry of the hydrophilic region,which is surrounded by a superhydrophobic boundary.the results showed that the superhydrophobic boundary of 50μm could effectively prevent the fusion of adjacent droplets and the migration of cells even at a high initial cell concentration,thus greatly improving the throughput of the platform。

the way of adding liquid drop by drop to the array by using a micropipette is too complicated to operate and has little significance For practical application.for the case of adding the same liquid,Levkin et al.Proposed a one-step formation method of ultra-high density micro-droplet array by using discontinuous dehumidification:when sliding the liquid on the superhydrophobic-superhydrophilic patterned surface or pulling the platform out of the liquid,Due to the extreme difference in wettability between the superhydrophobic and superhydrophilic regions,the liquid film will break down,resulting in the formation of numerous microdroplets with specific geometries in the superhydrophilic region^[17]^[7]。 This greatly simplifies the operation of adding liquid to the micro-droplet array and improves the efficiency of the experiment.At the same time,the volume uniformity of the formed droplets is good,which can meet the requirements of most experiments.When different reagents need to be added simultaneously in the experiment,they proposed a sandwich method(Fig.8A),in which the corresponding chemical library array and the microdroplet array inoculated with cells are stacked up and down like a sandwich,so as to realize the transfer and addition of chemicals^[48]。

In order to further verify the low consumption of reagents and samples by microdroplet arrays,Levkin et al.Prepared microdroplet arrays with square hydrophilic regions^[50]。 When the side length of the square site is 1 000,500 and 350μm,the droplet volume is 80,9 and 3 nL,respectively,which can save 600,5 000 and 15 000 times of the medium and reagents compared with the commonly used 384 well microplate.using these arrays,They performed reverse cell transfection.they also conducted a high-throughput screening experiment of cell transfection enhancers Using a microdroplet array(Fig.8B)to study the effect of 774 drugs approved by the Food and Drug Administration of the United States on the transfection efficiency of CHO-K1,Jurkat and HEK293T cells^[49]。 At the same time,this experiment shows that the consumption of cells and reagents by the microdroplet array can be reduced by 2500 times compared with the 384-well microplate.Song et al.Successfully prepared picoliter to femtoliter microdroplets with uniform volume,and systematically explored the effects of superhydrophilic pattern size,contact force and relative sliding speed on the volume of microdroplets^[19]。

In addition,microdroplet arrays have been used for high-throughput screening of mouse embryonic stem cells and cancer stem cells^[4,51]。 for example,Levkin et al.Cultured mouse embryonic stem cells on a microdroplet array and found that it had the ability to inhibit the differentiation of mouse embryonic stem cells,and the inhibition lasted for 72 H,which provided a system for high-throughput culture and screening of mice embryonic stem cells without mouse embryonic fibroblasts^[5]。 the difference in the behavior of mouse embryonic stem cells is caused by the special micro-nano topology of the polymer surface^[52]。 Ying et al.Used microdroplet arrays as a miniaturized screening platform to study the drug resistance of human cancer stem cells^[51]。 Because the number of stem cells is often extremely rare,in order to efficiently capture these rare cells in a large number of cell mixed samples,Fuchs et al.Thereby realizing the capture of the circulating tumor cells pre-labeled by the biotinylated antibody in the blood sample of the tumor patient,and realizing the capture of different cells by selecting different antibodies^[53]。

3.2 3D cell culture

Some specific tissue properties of cells are often related to their specific spatial structure,and 2D cell culture only gets a layer of cells,which often leads to the loss of these tissue properties.in order to better study the growth of cells In vivo,3D cell culture is very necessary.In different 3D cell culture methods,hanging drop method can effectively avoid the adhesion of cells to the culture dish,thus forming spheroids with specific size,cell number and composition,which is widely used in the culture of embryonic stem cells,tumor cells and so on^[54⇓~56]^[57,58]^[59]。 For example,Zhang et al.designed and 3D printed a hanging drop carrier ring device matched with 96 and 384 well plates,in which cell suspension can be dropped onto the carrier ring and form a hanging drop at the lower part of the through hole in the carrier ring^[60]。 They confirmed that the device could be used for drug detection and other operations in 3D tumor spheroids。

Mano et al.patterned the superhydrophobic polystyrene(PS)surface by microindentation to obtain a microdroplet array(Fig.9a)^[29]。 Due to the capillary force,the liquid drops can penetrate into the micro indentation,so that the liquid drops are fixed and cannot drop even if the surface is turned down,and the cell culture of the hanging drop method is realized.When the cell suspension array was cultured downward for 24 H,the cells in the droplet aggregated and formed spheroids at the liquid-gas interface under microgravity,so that drug screening experiments could be carried out in a single droplet.in another work,they obtained spheroids of different sizes and densities using suspensions with different initial cell concentrations(Fig.9 B)^[61]。

In addition to the above micro-indentation method,they prepared hydrophilic patterns on the surface of super-hydrophobic PS,and formed micro-droplet arrays by one-step method with the help of discontinuous dehumidification,which is more time-saving and efficient than the traditional dripping method^[26]。 In this work,they drilled holes at different positions on the platform(Fig.10a)to facilitate the subsequent operation of adding and taking liquid in the hanging drop culture.Levkin et al.Also used the microdroplet array to culture tumor cells by hanging drop method,which realized the rapid formation of tumor spheroids and high-throughput screening^[62]。 Zhao et al.Used morpho wings with natural superhydrophobic surfaces as substrates,patterned them with hydrogels to prepare microdroplet arrays,and then realized the hanging drop method to culture 3D cell spheroids(Fig.10B)^[28]。 This method makes full use of the original biological materials in nature without superhydrophobic surface modification。

Hydrogel is also a common means of 3D cell culture,which can provide soft space support for cells and better simulate the microenvironment of cells in vivo.Mano et al.Deposited natural biomaterials containing cells on the hydrophilic sites of the array,ionically crosslinked to obtain hydrogels,and explored the effects of different material combinations on two cell types^[24]。 Cytoskeleton and nuclear staining,fluorescence microscopy and image analysis were used to determine the local non-destructive cytotoxicity and cell number,and the results were consistent with those of traditional destructive tests。

Levkin et al.fabricated microdroplet arrays using superhydrophilic-superhydrophobic patterned surfaces to obtain microgel arrays encapsulated with living cells(Fig.11A)^[7]。 They first deposited polyethylene glycol crosslinkers on superhydrophilic sites by discontinuous dehumidification,dried them in air,and then prepared cell suspension arrays containing maleimide-functionalized polyvinyl alcohol by the same method,which were crosslinked to form microgel arrays.Therefore,the non-adherent cells are fixed in each microgel,so that the non-adherent cells are prevented from being washed away in the subsequent processes of medium exchange and the like,and the screening of the non-adhesive cells is effectively promoted.in addition to the above way of pre-depositing crosslinking agents on super-hydrophilic sites to achieve subsequent crosslinking operations,they also use the sandwich method to fuse two micro-droplet arrays to achieve gelation.and the conversion between the hydrogel array and the hydrogel free particles can be realized by exchanging the upper and lower positions of the cross-linking agent array and the cell suspension array^[63]。 Magnetic hydrogel particles containing living cells were also successfully prepared in this work。

Liu et al.Proposed a method for preparing heterogeneous 3D cell microenvironment arrays^[13]。 A patterned hydrophilic and hydrophobic surface is prepare by using a PDMS stamp method,and then a hydrogel prepolymer is adde to that surface to form a microdroplet array,and the microgel array is obtained after gelation.Since the microgel array still retains the hydrophilicity of the hydrophilic region,the layer-by-layer assembly of different cells can be performed by using the same method and using two different cell suspensions for gelation(fig.11B);the side-by-side assembly of the two types of cells was achieved by microdroplet fusion(fig.11C).by using the method,the shape,size,chemical substance concentration,cell density,multi-component 3D spatial distribution and the like of the cell microenvironment can be regulated and controlled to realize high-throughput screening.This shows that microgel arrays with complex 3D microenvironments have great potential in the study of cell-cell and cell-matrix interactions。

In addition,similar to hydrogel arrays,porous polymer scaffolds can also be prepared on patterned arrays by freeze-drying to achieve 3D cell culture^[25]。

3.3 Single Cell Analysis

Single cell analysis is of great significance for the study of cell growth,differentiation and migration mechanism^[64,65]。 The microdroplet array can fix single cells at a specific position for subsequent analysis。

Fang et al.prepared a low-density nanoliter droplet array on a chemically modified silicon chip for quantitative reverse transcription-polymerase chain reaction(PCR)detection.The minimum input of RNA for each detection was 1 pg,which could achieve gene quantification at a single cell scale^[12]。 Compared with commercial instruments,the sensitivity of this system is similar,and the dynamic range is larger,reaching 6 orders of magnitude.Subsequently,they further improved the above system,integrating a series of operations such as single cell encapsulation,cell lysis,reverse transcription and quantitative PCR with real-time fluorescence detection(Fig.12)^[66]。 Firstly,hydrophilic sites are prepared on a silicon wafer by using a photolithography method and a wet etching technology,then cell suspension liquid is dripped onto the sites to obtain a micro-droplet array containing cells,and a layer of mineral oil is covered on the micro-droplet array to prevent the droplets from evaporating^[12]; Cell counting and thermal lysis were then performed and a reverse transcription mixture was added to convert the RNA to complementary DNA;The PCR mixture was then added for amplification and the gene expression levels were quantified using real-time fluorescent PCR.In order to obtain the highest proportion of single cells in the micro-droplet array,the concentration of cell suspension and droplet volume need to be optimized.In the experiment,when the droplet volume was 2 nL and the cell suspension concentration was 5×10⁵cells/mL,the highest single cell fraction of 35%was obtained.In order to verify the accuracy and uniformity of liquid handling,the droplet robot was used to print 2 nL,18 nL and 30 nL liquid in continuous printing mode,respectively,and then 10×10 arrays were obtained.The relative standard deviations of fluorescence intensity images were 2.68%,2.73%and 2.67%,respectively,which showed that the droplet operation had high accuracy and reliability.In addition,the chip can be reused hundreds of times in a few months through cleaning,which effectively reduces the cost of the experiment.However,due to the relatively high detection limit,the system still needs to be further optimized 。

Whole-genome amplification is a key step in most single-cell genome studies,but the preferential amplification of certain genomic regions by this technique can affect the reliability of the results.multiple displacement amplification can effectively reduce amplification bias.Hansen et al combined it with non-contact printing technology to evaluate the effectiveness of Multiple displacement amplification technology in nano-volume droplet single cell array^[67]。 They first spin-coated a layer of PDMS film on the surface of a glass substrate,whose hydrophobicity was specially adjusted to ensure the fixation of printed droplets and avoid mutual contamination,obtained droplets containing single cells through limited dilution,and printed them on the substrate through a non-contact printer,and then coated a layer of oil film on the printed microarray to reduce volatilization.Subsequent studies on normal diploid cells and ovarian cancer cells demonstrated the accuracy of the multiple displacement amplification technique for large data sets。

Song et al.Obtained living single cell arrays on patterned superhydrophilic-superhydrophobic surfaces by droplet splitting^[19]。 A patterned super-hydrophilic-super-hydrophobic surface is prepare by electrochemical etching,chemical vapor deposition(CVD)and photolithography,and a culture medium contain cells is slid on that patterned super-hydrophilic-super-hydrophobic surface to obtain a single-cell micro-droplet array with controllable volume and good uniformity.Single cell arrays were obtained on a 100μm hydrophilic surface by sliding a 10μL drop of human breast cancer cell(MCF7)culture medium at a concentration of 10⁷cells/mL.Cells were stained with acridine orange/propidium iodide(AO/PI),which was green under 488 nm fluorescence and almost invisible under 546 nm fluorescence,indicating successful isolation of a single viable cell.Fuchs et al.Also successfully captured specific rare single cells using a microarray platform of streptavidin micropatterns and performed single cell analysis^[53]。

Levkin et al.optimized the distribution of single cells on microdroplet arrays of different hydrophilic site sizes by limiting dilution and varying cell density and seeding time(fig.13)^[68]。 First,the cell suspension is added to the surface of the patterned chip,and after a certain period of time,the chip is tilted to remove excess liquid,so as to obtain a micro-droplet array containing cells.the results showed that the highest single cell ratio of about 20%could be obtained under the conditions of superhydrophilic site size of 500μm,inoculation time of 60 s and cell concentration of UNK 1 cell/mL.the survival rate and doubling time of single cells on the microdroplet array were about 100%,and the doubling time was similar to that of HeLa cells under traditional culture conditions。

3.4 Whole-body screening

Cells and tissues are physiologically interconnected,and this interaction plays an important role in the expression of certain properties and functions^[69]。 Therefore,some successful experiments at the cell level are difficult to achieve the expected results when they are carried out at the whole body level.to solve this problem,whole-body high-throughput screening has been developed and studied.Among several whole-body models,such as Caenorhabditis elegans and Drosophila melanogaster,zebrafish has attracted extensive research in recent decades because it is more similar to humans and its embryos are optically transparent。

Levkin et al.Applied microdroplet arrays to miniaturized whole body screening(fig.14)^[27]。 Hydroxyethyl methacrylate-ethyl dimethacrylate(HEMA-EDMA)copolymer film was prepared on the surface of glass slide,and the patterned superhydrophobic-superhydrophilic surface was obtained by thiol-alkyne click reaction^[18,50]。 Dispersing the zebrafish embryos on the super-hydrophilic sites by means of discontinuous dehumidification to obtain a micro-droplet array containing a single zebrafish embryo in each droplet,the organ specificity of several fluorescently labeled peptides and the cytotoxicity of zinc chloride and silver nitrate in a certain concentration range were studied directly on the array,and the results were consistent with those of standard microplate experiments^[17]。

4 Conclusion and prospect

From the above series of introductions, we can see that as a new high-throughput platform, micro-droplet array has great potential in biomedical fields because its miniaturization greatly saves sample consumption and its high-throughput greatly improves experimental efficiency.

But at the same time, there are still many challenges in promoting the further application of micro-droplet arrays: for micro-droplet arrays driven by surface chemistry,Its formation depends on the wettability difference between hydrophilic and hydrophobic regions on the surface. How to establish a rapid preparation method for stable and repeatable interfacial chemical composition and topology is one of the bottlenecks for large-scale application of micro-droplet arrays. Although exist microdroplet array technology can obtain multiple parallel samples by a parallel drag method, that multivariable control require in high-throughput experiments,It is still necessary to use the printer to add or construct the gradient of the compound library, and how to establish a differentiated and integrated micro-droplet array technology is the key issue to further improve the variable control ability of the platform. In some recent works, domestic and foreign researchers have explored the rapid construction of differentiated droplets by using gradient templates, photocoupler cleavage, solvent exchange and so on^[13]^[70]^[71]. On the basis of these studies, the development of miniaturization, integration and multi-functionality of micro-droplet arrays will provide a new possibility for the construction of a high-throughput experimental platform for materials.

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