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A Microwell Array Method for Rapid Generation of Uniform Agarose Droplets and Beads for Single Molecule Analysis Xingrui Li, Dongfeng Zhang, Huimin Zhang, Zhichao Guan, Yanling Song, Ruochen Liu, Zhi Zhu, and Chaoyong James Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04040 • Publication Date (Web): 19 Jan 2018 Downloaded from http://pubs.acs.org on January 19, 2018

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Analytical Chemistry

A Microwell Array Method for Rapid Generation of Uniform Agarose Droplets and Beads for Single Molecule Analysis Xingrui Li1, Dongfeng Zhang1, Huimin Zhang1, Zhichao Guan1, Yanling Song1,2, Ruochen Liu3, Zhi Zhu1*, Chaoyong Yang1

1

The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, the Key

Laboratory of Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Collaborative Innovation Center of Chemistry for Energy Materials, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Pen-Tung Sah Institute of Micro-Nano Science and Technology, Xiamen University, Xiamen 361005, P.R. China. 2

The MOE Key Laboratory of Analysis and Detection Technology for Food Safety, State Key Laboratory of Photocatalysis on Energy and Environment, College of

Biological Science and Engineering, Fuzhou University, Fuzhou 350116, P. R. China. 3

Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey, USA.

* To whom correspondence should be addressed. Tel: (+86) 592-218-7621, E-mail: [email protected]

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Abstract Compartmentalization of aqueous samples in uniform emulsion droplets has proven to be a useful tool for many chemical, biological, and biomedical applications. Herein, we introduce an array-based emulsification method for rapid and easy generation of monodisperse agarose-in-oil droplets in a PDMS microwell array. The microwells are filled with agarose solution, and subsequent addition of hot oil results in immediate formation of agarose droplets due to the surface-tension of the liquid solution. Because droplet size is determined solely by the array unit dimensions, uniform droplets with pre-selectable diameters ranging from 20 µm to 100 µm can be produced with relative standard deviations less than 3.5%. The array-based droplet generation method was used to perform digital PCR for absolute DNA quantitation. The array-based droplet isolation and sol-gel switching property of agarose enable formation of stable beads by chilling the droplet array at -20° C, thus maintaining the monoclonality of each droplet and facilitating the selective retrieval of desired droplets. The monoclonality of droplets was demonstrated by DNA sequencing and FACS analysis, suggesting the robustness and flexibility of the approach for single molecule amplification and analysis. We believe our approach will lead to new possibilities for a great variety of applications, such as single-cell gene expression studies, aptamer selection, and oligonucleotide analysis.


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Introduction The invention of the polymerase chain reaction (PCR)1 has revolutionized bioscience because of its ability to amplify DNA templates efficiently and specifically from very low starting concentrations2-6. Due to highly efficient exponential amplification7-11, theoretically PCR can amplify a DNA template originally present at the single-molecule level12,13. However, typical PCR is performed in microliter volumes (~10-5 L)8, resulting in extremely low concentrations (~10-18 M) to approach the single-molecule limit4. Consequently, conventional single-molecule PCR often requires accurate optimization of critical parameters to maximize product yield14, minimize false incorporations15 and avoid dimerization of primers16. The development of droplet microfluidics allows rapid generation of uniform pico/nanoliter emulsion droplets, which can serve as reaction compartments for a variety of applications17-21. Performing PCR in these tiny reactors effectively reduces reaction volume by a factor of 106, thus enabling efficient amplification of DNA molecules down to the single-molecule level.20,22-24 More importantly, large scale generation of discrete emulsion droplets allows parallel monoclonal amplification of single-copy DNA molecules, which find wide applications in chemical, biological, and biomedical fields25-28. For example, massively parallel amplification of single-copy DNA templates in monodispersed droplets has been used for digital detection of nucleic acids23,27,29,30, sensitive analysis of rare mutations within a large population26,31, efficient screening of functional aptamers9,25,32, and preparation of DNA libraries for next generation sequencing33-35. Recently, we developed an agarose droplet emulsion approach for single template amplification.9,10,36 The agarose-in-oil droplets were generated by a flow-focusing microfluidic chip for highly parallel single-copy PCR. The thermally responsive sol-gel transition of agarose ensures that the liquid form is maintained at all PCR temperatures for high PCR efficiency (~95%)10. After cooling to form the gel, the monoclonality of the amplified DNA is maintained even after removing the oil phase, thus affording flexible downstream processing for washing and storage, flow



cytometry analysis, easy retrieval of amplified product, or DNA sequencing. Thus, our agarose droplet approach could be applied for highly efficient evolution of aptamers, high throughput single cell gene expression studies, as well as sensitive detection of rare cells in a background of normal cells. For producing monodisperse droplets, T-junction37,38 and flow-focusing systems39-41 are two widely applied techniques. The aqueous phase is intercepted by one or two continuous oil phases41,42, and droplets are generated in the nozzle of a T-junction or cross-junction. Because the uniformity of droplet size is sensitive to fluid ratio,43,44 a costly syringe device is necessary to achieve an accurately controlled continuous fluid.29,45-48 Moreover, the poor controllability of the droplet diameter makes simultaneous generation of droplets with different sizes difficult.45 With regard to throughput, although the generation rate can be as high as 2.4 x 103 droplets/s,38 the accumulation of a large number of droplets is a time consuming process49. As a popular method, increasing the number of nozzles can easily improve the throughput.50,51 Nonetheless, the nozzle numbers still limit the throughput due to T-junction and flow focusing principles, in which one nozzle generates one droplet in a unit time. In addition, a large volume of sample is usually wasted before and after droplet generation to ensure uniform droplet formation.52,53 As a result, simple, rapid, and efficient droplet generation methods are still highly desirable. Herein, we introduce an array-based emulsification method for rapid, efficient and easy generation of monodisperse agarose-in-oil droplets for highly parallel single copy DNA amplification in a PDMS microwell array. After the microwells are filled with agarose solution, hot oil is dropped onto the microchip, leading to immediate formation of agarose droplets due to the difference in intermolecular forces of the two liquids. Uniform droplets with pre-selectable diameters ranging from 20 µm to 100 µm can be produced with relative standard deviations of less than 3.5%. The droplet size depends on the array unit dimension (depth, width) only, thus eliminating the need for a constant fluid ratio and other complications. The array-based droplet generation method was used to perform digital PCR for absolute quantitation of DNA molecules. ACS Paragon Plus Environment

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The sol-gel switching property of agarose enables formation of stable beads by rapid cooling after amplification, thus ensuring effective maintenance of monoclonality of each droplet for downstream processing. Retrieval of monoclonal beads for downstream DNA sequencing and FACS analysis was further demonstrated, suggesting the robustness and flexibility of the approach for single molecule amplification and analysis. We believe our approach will lead to new possibilities for a great variety of applications such as single-cell gene expression studies, aptamer selection, and nucleic acid analysis.

Experimental Section Materials and reagents Glass slides (1 mm thickness with size of 2cm×2cm) were employed as a support for the microwell array chip (Sail, China). SU8-2015 photoresist was purchased from MicroChem (Newton, MA, USA). The pattern blueprint of the microwell array on 2-inch cleaned silicon wafer using film photomask was produced by Qingyi Precision Maskmaking Co. Ltd. (Shenzhen, China). Polydimethylsiloxane (PDMS, RTV 615, GE) was obtained from CChip Technology CO., Ltd (Suzhou, China). Type IX-A agarose with ultra-low gelling temperature and Silicone oil AR 20 were purchased from Sigma-Aldrich (Shanghai, China). EasyTaq® DNA polymerase was purchased from TransGen Biotech (Beijing, China). SYBR Green I (10000X) was purchased from Gen-bio (Beijing, China). The lambda DNA (0.3 µg/µL) was purchased from Fermentas China (Shenzhen, China). The other DNA sequences as listed in Table S1 were purchased from Sangon Biotech (Shanghai, China). Microchip fabrication We used a soft lithography technique54 to fabricate the micromold with a high-density array of microwells. First, photoresist SU-8 2015 was spin-coated (2000 rpm) on a silicon wafer. After soft baking for 5 min at 95 °C, the microwell pattern on a film photomask was transferred onto the photoresist via UV exposure (365 nm) for 20s. After post-exposure baking for 5 min at 95 °C, the patterned silicon wafer



appeared in the photoresist coating. After 5 min development, unexposed photoresist was washed away from the silicon wafer. The mold was replicated using the silicone elastomer polydimethylsiloxane (PDMS) to create a 50 µm thickness layer by spincoating (Easy Coater 6, Jiatu-Tech, China) at 1500 rpm for 35s, followed by 5 min baking at 90 °C (Scheme 1A). To enhance the chip physical strength and reduce the gas permeability of the microchip, we used a PDMS ratio of 5A:1B (polymer base: curing agent) instead of the normal ratio of 10A:1B55. To further enhance the physical strength, the backside of the PDMS microchip was bound to a clean glass slide56. After peeling the PDMS chip from the SU-8 mold, 30s plasma exposure under oxygen was necessary for bonding the PDMS to the glass slide. It was necessary to cover the microarray with another PDMS layer during PCR processing. To reduce evaporation during the PCR thermal cycles, an emulsion of fluorosilane polymer (EGC-1720, 3M) was spread over the surface of the cover which contacted microwells, and dried immediately after coating55. Rapid generation of agarose beads We have chosen ultra-low gelling agarose10, which has a melting point at 56 °C and gelling point around 16 °C. Scheme 1B shows the bead generation procedure. First, an aqueous solution with 2% (w/v) agarose was pre-heated to 70°C and added to the microchip surface. Then the PDMS cover with one side painted with a fluorosilane polymer layer was pressed on the chip. To ensure that agarose solution filled the entire microchamber, we use a home-built negative pressure device (Scheme 1A). After the sample was evenly dripped onto the chip surface in the form of multiple drops, a cover slide was pressed onto the microwell array chip and the drop sample were flattened and enclosed in the narrow gap between the cover and the chip. The flattened drops were connected to each other to form a rough, thick water film in the gap. Then, the device was degas by pump for a few seconds to make a negative air pressure around the chip (~-8.7 psi or -0.59 atm) to ensure solution fill into mcirowells. A screw was then used to press the cover with a positive pressure (7 psi). A force-sensing piezoresistor (Interlink Electronics, Shenzhen) was glued to the ACS Paragon Plus Environment

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bottom of the homemade device for pressure monitoring. As a result, the sample was uniformly distributed on the chip surface, and the negative pressure inside the device made the liquid sample enter the microwells more easily. The detailed steps of the loading process were shown in Figure S2. The temperature-changing reaction including PCR can be performed at this stage. Afterwards, the entire chip was frozen at -20 °C for 5 min to solidify the agarose and generate the agarose gel array, which can be stored without any loss of moisture in a freezer. Insufficient freezing time can cause agarose loss during subsequent transfer and storage. After chilling, the chip was placed on a petri dish, and the PDMS cover was peeled away carefully using tweezers. Then, 70 °C oil was added dropwise to the surface of the microchip to melt the agarose gels in the microwells. Agarose gels melted and converted to agarose droplets in 20s, due to the difference in intermolecular forces. Finally, by chilling the entire chip with petri dish for 5 min at 4 °C, the agarose droplets were converted to agarose beads in the microwells for subsequent analysis.

Scheme 1. (A) The fabrication procedure of PDMS microchip. Two layers, one generated through photolithography binding on glass, one preformed on slab coating fluorosilane polymer, are pressed together using a homemade screw device in negative surrounding pressure. (B) Rapid generation of the agarose droplet array in microchip with uniform size and high throughput.

Single molecule PCR in agarose droplet and digital detection All PCR reaction components were first mixed in 2% agarose solution. The mixture was supplemented with 0.5mg/mL BSA to prevent reaction components from being adsorbed onto the PDMS surface during the reaction. For single molecule PCR, lambda DNA was chosen as the template. In order to ensure single distribution of lambda DNA to individual droplets, the stock DNA solution (10 nM) was further ACS Paragon Plus Environment


diluted to 0.1 pM. The reaction mixture of 20 µL was prepared consisting of 0.8 µL of both forward primer 1 and reverse primer 1 (1 µM), 1 µL of 20×SYBR Green I, 1 µL BSA (10 mg/mL), 1 µL dNTP (2.5 mM), 10 units EasyTaq® DNA Polymerase, 2 µL of 10×EasyTaq® buffer, 1.1 µL DNase-free water, 10 µL agarose solution (4% w/v) and 1 µL diluted template lambda DNA. The reaction mixture was added to the PDMS microchip, as described above. A conventional PCR machine with a flat heating block was used for microchip thermocyling. The thermocycling protocol included 8 min heating step at 94 °C, 35 cycles of 30 s at 94 °C, 25 s at 56 °C, 28 s at 70 °C; and 3 min at 72 °C to finish the reaction. Finally, the microchip was cooled to 4 °C for agarose gelation. The entire experiment was completed in approximately 1 hour. After microchip cooling and agarose gelation, the PDMS cover was peeled away, and hot oil was dropped onto the peeled chip to generate agarose droplets in the microwells. All these processes were observed under an inverted fluorescence microscope (Ti-U, Nikon). SYBR Green I in the beads was used to detect the PCR reaction product. A significant fluorescence signal was observed in agarose beads where single copy DNA was amplified, while beads with zero copy of target exhibited only a weak fluorescence signal (due to the PDMS autofluorescence and the background SYBR Green I). Fluorescence images were acquired with a scientific CCD camera, and each field of view contained 420 beads. The fluorescence positive beads were recorded to calculate the ratio in all beads. The relationship between the DNA concentration and the percentage of positive beads was tabulated using Poisson distribution. Every experiment was repeated three times to ensure the accuracy and reproducibility. Agarose bead retrieval and storage To retrieve specific beads, the general method was capillary or pipet suction57. Agarose with a low gelling temperature is softer and more fragile than conventional agarose, so blunt capillaries are required for the retrieval process to prevent breakage of the beads. We expect to retrieve the positive droplets specifically. The fluorescent beads with monoclonal PCR products were imaged using a fluorescence microscope ACS Paragon Plus Environment

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and marked by software. Monitoring the marked beads in bright field, a capillary tip with inner diameter of 70 µm was used for agarose bead retrieval. During recovery of the marked beads, the proximate unmarked beads were not affected. After retrieval, the agarose beads were transferred to a PCR tube using a positive pressure from rubber suction bulb and stored at 4 °C for further analysis. The operation of recovery one bead was completed in 1 min. Sequencing of monoclonal products Two different sequences of DNA were mixed to test our methodology in segregation and selective recovery of monoclonal DNA, and DNA sequencing was used to verify the monoclonal nature of the product. The mixture was prepared with equal amounts of EpCAM protein binding aptamer SYL2, and a random DNA sequence, which has no binding to EpCAM protein. For DNA sequencing, the monoclonal PCR product in agarose beads needed to be further amplified. 23 fluorescent agarose beads were retrieved and were individually mixed with 25 µL PCR mixture containing 2 µL forward and reverse primer (5 µM), 3 µL dNTP (2.5 mM), 5 units EasyTaq® DNA Polymerase, 2 µL 10×EasyTaq® buffer, and 17 µL DNase-free water. A PCR machine with a tube heating block was used to carry out the monoclonal product enrichment. The thermocycling protocol began with a heating step of 8 min at 95 °C; then 30 cycles: 30 s at 95 °C, 30 s at 56 °C, 30 s at 72 °C; and finally 3 min at 72 °C to finish the reaction, which can be finished within 1 hr. The monoclonal PCR product was subjected to DNA sequencing (Sangon Biotech, Shanghai) and the sequencing data were analyzed by Chromas 2.0. Flow cytometry analysis To verify the binding capability of the DNA products from 23 fluorescent (positive) beads to EpCAM protein, they were individually amplified with fluorophore labeling for evaluation using EpCAM+ cell line A549. The same protocol to amplify monoclonal PCR product was applied but with 5’-FAM labeled forward primers and 5’-biotin labeled reverse primers. The amplified monoclonal PCR products with biotinylation were enriched on streptavidin-covered beads. Then, the fluorescent PCR products were separated from the complementary DNA and streptavidin beads under ACS Paragon Plus Environment


alkaline conditions. After desalination and spin drying, the fluorescent PCR products were dissolved in water and the concentration was determined. The amplified products of 100 nM were incubated with A549 cells in binding buffer at 37 °C for 30 min individually, and FAM-labeled synthetic DNA sequence SYL2 and random sequence was applied as positive and negative standards. Cells were washed twice with 400 µL washing buffer and resuspended in 200 µL binding buffer. The fluorescence intensity of the cells was recorded by FACS cytometer (Becton Dickinson Immunocytometry Systems) by counting 5000 events. Confocal imaging of cells stained with synthetic DNA and monoclonal products. To further characterize the prospect of cell imaging using monoclonal products, we chose typical samples (#8 and #17) and synthetic DNA (SYL2 and random sequence) of 100 nM each to incubate with A549 cells in binding buffer at 37 °C for 30 min. The cells adhering to the dish were washed twice with washing buffer to remove the excess DNA. The washed cells were observed by confocal imaging.

Results and discussion Fabrication of large-scale agarose microwell array for droplet array generation A uniform microwell array PDMS membrane was fabricated as a micro-mold for high-throughput monodisperse agarose droplet and agarose bead generation. Melted agarose solution with 2% (w/v) was poured onto the PDMS chip. A cover slide was used to press the microwell array chip. After a brief degassing step, the whole chip was frozen at -20 °C to solidify agarose and generate agarose gel array for storage. Addition of hot oil onto the array resulted in immediate melting of agarose and formation of droplets due to the surface-tension of the liquid solution. Single-copy DNA amplification can take place in droplets because of the excellent bio-compatibility of agarose11. The sol-gel switching property of agarose enables formation of stable beads by rapid cooling after amplification57, thus ensuring effective maintenance of monoclonality of each droplet for downstream processing. Figure 1 shows the transformation of agarose gels into droplets within 20 s. With a


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microwell size of 100×100×35 µm, droplets with diameters of 85 ± 5 µm were obtained. More than 120,000 droplets were generated in 20 s on a 2-inch silicon wafer with total volume of 42 µL. In order to improve the quality of the agarose droplet array, the entire chip needs to be covered with agarose solution during the production. Therefore, a certain amount of excess sample (50 µL) was used. When the microcell dimensions were decreased to 20×20 µm, the throughput of droplet generation increased to 53,000/cm2. Droplets obtained were very uniform in diameter, with a relative standard deviation (RSD) of 3.2%.

Figure 1. Rapid generation of agarose droplets on chip. (A) Agarose gels melt to form agarose droplets within 20 s. (B) Large-scale agarose droplet array with uniform drop diameter. The average size of the droplets was 85 µm in diameter. The throughput of droplet generation was more than 62 000/cm2 within 20 s. The scale bar was 200 µm. (C) Size distribution of agarose droplet generated with a standard deviation of 3.2%. All droplet sizes were analyzed using ImageJ software.

The ability to rapidly generate large-scale microarray agarose droplets with controllable diameter offers an attractive approach to single copy DNA amplification and monoclonal product analysis. The droplet diameter can be predetermined by the dimensions of the microwells. The areas of microwells from 40 µm2 to 10000 µm2 can be designed accurately by mask, and molds with different depths ranging from 10 µm to 40 µm can be easily controlled by the speed of spin coating on the silicon wafer. The final volumes of different microwells range from 0.4 pL to 400 pL. In addition, uniform droplets with variable volumes can be generated simultaneously in one chip with the same method. Figure 2A shows the droplet images from microwells with the same depth of 12 µm but different sizes: 20×20 µm, 40×40 µm, 60×60 µm, 70×70 µm, ACS Paragon Plus Environment


and 90×90 µm. The volumes were designed to be 5 pL, 20 pL, 45 pL, 60 pL, and 100 pL. Accordingly, average volumes of droplet obtained were 4.8 pL, 18 pL, 41.5 pL, 57.8 pL, and 94.3 pL, with a relative standard deviation of 3.8%, 4.2%, 5.3%, 4.5%, 4.8% respectively. In Figure 2B, the different diameters of droplets are shown in a histogram, and the normal distribution of each diameter of droplet was calculated and shown in red. The droplets were uniform with slight variation caused by the mold size variation from the precision limitation of soft lithography. The appearance of this deviation is a stochastic process, and we did not find a clear correlation between drop size and deviation.

Figure 2. Simultaneous generation of uniform droplets with different diameters. (A)Agarose droplets with controllable diameters (from 20 µm to 60 µm) generated in different sizes of microwells (20×20, 40×40, 60×60, 70×70, 90×90 µm) with same depth (12 µm). The scale bar is 200 µm. (B) Uniform size distribution of droplets.

Single-molecule PCR in agarose droplet array To demonstrate the feasibility of our agarose droplet array approach for single copy DNA amplification, a series of lambda DNA solutions was prepared with final concentrations ranging from 2.5 fM to 60 fM in droplet array with 50 µm in diameter. SYBR Green I was used to stain the amplified single DNA product, so that droplets with successful amplification could be visualized and counted. Under the single molecule condition, increasing numbers of positive droplets were observed with increasing DNA concentrations (Figure 3A). Specifically, 4.58%, 11.9%，36.5% of droplets were found brightly fluorescent for DNA concentrations of 2.5, 5, and 15 fM, respectively. The percentage of positive droplets confirmed the good reproducibility and was agreed well with the ratio predicted by Poisson statistics (Figure 3B), establishing the capability of amplifying single copy DNA in agarose droplets. ACS Paragon Plus Environment

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One of the applications of single copy PCR in droplets is digital PCR. To test the accuracy of the current method, Lambda DNA solution was quantified independently by the agarose droplets method and by q-PCR. As shown in Figure 3C, the final concentration in solution was measured to be 13.4 ± 1.0 fM from agarose droplet digital PCR which matches closely with the 13.6 ± 1.0 fM from q-PCR, and the concentration and expanded uncertainty was at a confidence level of 95%, demonstrating the capability of the large-scale agarose droplet array platform for accurate digital PCR. Limited to the soft lithography and PDMS chip fabrication process, the slight mold dimension deviates from the design, resulting in slight changes in the droplet diameters (deviation 3.2%), which also induced the bias of the quantitative results of digital PCR. We used chi-square test to evaluate the calculated concentration of different chips and obtained a confidence level of 95%. Thus, our method showed a potential of single-molecule amplification for quantification of trace nucleic acids.

Figure 3. Digital detection of different concentrations of lambda DNA. (A) Digital PCR experimental results: A region containing ~420 droplets after PCR. Three repeats of experiments of digital PCR with 3 different concentrations of templates. The scale bar was 100 µm. (B) Experimental percentage of positive droplets plot vs DNA concentration compared to theoretical result based on a Poisson distribution. The error bars represent the standard deviation based on triple measurement. (C) Comparison of quantitation result for lambda DNA concentration obtained by quantitative PCR and the droplet array method.

Flexible monoclonal product retrieval and downstream functional analysis



The agarose droplet array method offers a flexible way to retrieve and store the amplified DNA products for further analysis and sequencing.9,57 In the agarose droplet array, the droplets were generated in each microwell and isolated and immobilized by physical barriers (PDMS walls). Thus, the uncertainty problem of droplet floating and moving in the oil phase by other droplet microfluidic methods was easily overcome. Moreover, the conversion of agarose droplets into agarose beads allows the beads to be retrieved sequentially by micro-machine or even manual manipulation. Micromanipulator has certain advantage in retrieving very few positive droplets in large amount of negative background without droplet losing and cross-contamination. FACS device is faster and easy for large amount of droplet retrieval, but it requires high cost instrument. In our experiment, only a small amount of positive monoclonal amplified beads are needed, where precise micromanipulation approach is preferred. As schematically shown in Figure 4A, a capillary can be used to specifically retrieve the desirable beads with DNA products for further analysis. In this process, the beads containing monoclonal products with bright fluorescence were first observed and marked under the fluorescence microscope (Figure 4B). Because the restriction by microwells avoids beads floating in oil, the marked beads observed under bright field were still kept in the same place. A capillary with 70 µm diameter tip controlled by a microoperator was moved toward the marked bead. The agarose bead could be easily picked up by applying negative pressure to the capillary. Such a retrieval process does not affect other surrounding beads. Finally, by blowing the capillary, the retrieved agarose bead is released and can be stored for further analysis.


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Figure 4. The positive agarose bead can be easily picked out individually from the chip for subsequent analysis and manipulation. (A) The scheme of agarose bead retrieval using capillary and storage in PCR tubes. (B) The positive beads were marked using red circles under fluorescence microscope via the help of computer software. Specially marked beads were retrieved in bright field without any disturbance of other beads close to it. The scale bar was 100 µm. (C) Characterization of the binding capacity of amplified products. Flow cytometry was used to monitor the binding of 23 samples with A549. The dotted red ling shows a standard SYL2 binding with A549; the dotted green line shows a synthetic random DNA binding with A549. (D) Confocal images of two synthetic DNA sequences and two monoclonal samples bound with A549. The final concentration of FITC-labeled DNA sequences was 100 nM

When amplified at the single molecule level, each droplet containing a DNA template will produce millions of copies of identical DNA amplicons, thus creating a monoclonal droplet/bead. Each monoclonal bead can be flexibly processed for downstream analysis. To demonstrate the feasibility of our new method for isolating single clonal DNA product for downstream functional analysis, a pre-mixed PCR solution with two sequences, EpCAM aptamer SYL2 and random sequence R in a 1:1 ratio, was diluted to a final concentration of 0.2 copy per microwell (cpw), and then used to fill the microwell chip for amplification. SYL2 aptamer has been proven to have a convincing ability to bind EpCAM protein,58,59 which is widely accepted as a tumor biomarker. After amplification and agarose bead formation, 23 fluorescent (positive) beads were retrieved and the binding capability of the DNA product to EpCAM protein was evaluated using EpCAM+ cell line A549. As shown in Figure 4C, the 23 samples were divided into two main groups as compared to SYL2 standard.



The fluorescence values of the 10 amplified samples were similar to that of synthetic SYL2, while 13 samples had lower fluorescence values than SYL2, suggesting the capability of using this droplet approach to identify strong binding sequences from an enriched library. These binding results were further confirmed by cell imaging (Figure 4D), where cells incubated with a strong binding bead sample were brightly fluorescent, while a weak binding bead sample led to weakly fluorescent cells. To further confirm the single-molecule amplification ability of the agarose droplet array method, all 23 beads were sequenced, where 10 beads were found to contain only SYL2 sequence, 12 beads contained random sequence R, and 1 bead contained both sequences. Theoretically, at the concentration of 0.2 cpw, the probabilities of a well containing no DNA, single type of DNA (A, B, AA, or BB), and two types of DNA (AB) are 81.8%, 17.5% and 0.6%. Thus theoretically, the ratio of droplets containing a single type of DNA to droplets containing two types of DNA is about 29:1 (17.5%: 0.6%). In our experiment, among 23 positive beads sequenced, the ratio of single type DNA to mixed DNA sequences was 22:1 (Figures S1). Considering the limited sample number, the experimental value matched well with the theoretical prediction, establishing the capability of the agarose droplet array method for amplification of a single type of DNA. Conclusions In conclusion, we have developed an array-based emulsification method for producing monodisperse agarose-in-oil droplets for highly parallel single copy DNA amplification in a PDMS microwell array. There are several advantages of the new array-based agarose droplet generation methods. First of all, the method allows for rapid, efficient and easy generation of monodisperse droplets. More than 100,000 uniform and size-tunable droplets with relative standard deviations of less than 3.5% can be generated in less than 1 min using a preformed agarose well array. Second, single-copy DNA can be efficiently amplified in the pL droplets due to the excellent biocompatibility of agarose, enabling accurate and absolute quantitation of DNA molecules. More importantly, the agarose droplet array method offers a flexible yet


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robust way to retrieve and store the amplified DNA products for further analysis and sequencing. Due to the capability of rapid droplet generation, accurate absolute detection of ultra-low concentration of DNA, and facile monoclonal product retrieval, the array-based emulsification approach offers great potential for great variety of applications, such as rare mutant detection, aptamer selection, and single-cell gene expression studies.

Acknowledgments We thank the National Natural Science Foundation for Excellent Youth Scholars of China (21422506), National Natural Science Foundation for Distinguished Young Scholars of China (21325522), National Natural Science Foundation of China (21435004, 21521004) and Program for Changjiang Scholars and Innovative Research Teams in University (IRT13036) for their financial support.

Supporting Information DNA sequence information, sequencing results of two positive retrieved beads, and the scheme of sample loading process.

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