Stable Colloidosomes Formed by Self-Assembly of Colloidal

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Stable Colloidosomes Formed by Self-Assembly of Colloidal Surfactant for Highly Robust Digital PCR Kun Yin, Xi Zeng, Weizhi Liu, Yakun Xue, Xingrui Li, Wei Wang, Yanling Song, Zhi Zhu, and Chaoyong James Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00470 • Publication Date (Web): 27 Mar 2019 Downloaded from http://pubs.acs.org on March 28, 2019

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Analytical Chemistry

Stable Colloidosomes Formed by Self-Assembly of Colloidal Surfactant for Highly Robust Digital PCR Kun Yina, Xi Zenga , Weizhi Liua, Yakun Xuea, Xingrui Lia, Wei Wangb, Yanling Songb, Zhi Zhua, Chaoyong Yang*ab a 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 Engineering, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Pen-Tung Sah Institute of Micro-Nano Science and Technology, Xiamen University, Xiamen 361005, China b Institute of Molecular Medicine, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200127, China E-mail: [email protected]

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT As third-generation nucleic acid amplification technology, digital PCR has been widely adopted in the analysis of nucleic acids. However, further application of this powerful technology is hindered due to the limitation of surfactants. Here, for the first time, we propose the use of colloidosomes self-assembled from fluorinated silica nanoparticle for digital PCR to address this limitation. A one-step fluorinated silica nanoparticle synthesis method is proposed, which is much more convenient and reproducible compared with the synthesis of conventional fluorine-based surfactants. Fluorinated silica nanoparticles facilitate the formation of colloidosomes with excellent stability capable of enduring the rapid temperature changes associated with the polymerase chain reaction (PCR) and avoiding material exchange (cross-talk) between droplets for high-fidelity analysis. The colloidosome digital PCR (cdPCR) method was developed using these colloidosomes as highly parallel reactors for single-copy nucleic acid amplification and rare mutant detection. The method is robust and accurate, and it offers possibilities for a great variety of applications, such as gene expression studies, single cell analysis, and circulating tumor DNA detection.

KEYWORDS: Colloidosome, Self-assembly, Digital PCR, Colloidal Surfactant


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Digital PCR is a third-generation nucleic acid amplification technology, which amplifies compartmentalized targets in numerous micro-reactors and quantifies DNA concentrations using Poisson statistics.1,

2

Since Kinzler and Vogelstein et al first

reported the principle of digital PCR,2 this powerful tool has been widely used in research on circulating tumor DNA,3-5 copy number variation,6, 7 gene expression,8, 9 rare mutant detection,10, 11 single cell analysis,9, 12and 14

trace nucleic acid detection,13,

etc., because of its superior features, including absolute quantification of nucleic

acids, as well as higher reproducibility and lower susceptibility to inhibition than realtime PCR. Currently, digital PCR can be classified into two types: one distributes nucleic acids into discrete micro wells15, 16 or micro chambers,17-19 and the other dilutes targets into massive independent uniform pico- or nanoliter droplets7, 20. Generally, the second method, droplet digital PCR (ddPCR) can realize higher throughput reactions with a lower cost compared with the former.7 Unfortunately, there are several key problems limiting the further application of ddPCR, among which is the use of surfactant. It is highly challenging for surfactants to stabilize massively monodispersed droplets, because droplets must endure critical conditions, such as rapid temperature cycling and complex aqueous samples throughout the entire PCR process. Currently, these surfactants are primarily dispersed in three types of carrier oil.21 The first type is a silicone oil system (complex oil mix including DC 5225C, DC 749, Ar20 etc.), which was initially reported for emulsion PCR.22 However when applied in microfluidics chips, it is incompatible with the commonly used material PDMS, which restricts its use to glass devices.23-25 The second type is a hydrocarbon oil system (ABIL EM90 etc.)26, 27, which solved the incompatibility issue with PDMS material. However, the droplets are not sufficiently stable in critical environments like high temperature and extremely compressed conditions. In addition, the hydrophobic substances in droplets can readily diffuse into the oil phase, leading to cross-talk between droplets.28, 29

The third type is a fluorinated oil system, which

overcomes the issue of stability in critical conditions. Currently, many droplet-based systems rely on fluorinated oils and a copolymer surfactant PFPE-PEG-PEG triblock



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copolymer (EA surfactant) ,30, 31 but the complex synthesis and purification of these compounds significantly restricts their use.32 In addition, although the fluorinated oils minimize the transport of molecules between droplets, it has been observed that some small hydrophobic molecules in droplets tend to leak

33-35due

to the solubilization by

reverse micelles formed by the surfactants in continuous phase36. The cross-talk between droplets can result in inaccurate detection. Moreover, there are few commercially available fluorine-based surfactants (by Bio-Rad etc.). Although those surfactants can stabilize droplets effectively, their incompatibility with commercially available PCR Master Mix reagent limits their further applications. Therefore, it is of great interest to develop new surfactants that are easy-to-synthesize, cost-effective, and cross-contamination free. Compared to an emulsion stabilized by surfactant molecules, the Pickering emulsion,37 which is stabilized by self-assembly of nanoparticles at the water/oil interface, 38 shows superior stability39. Due to their liposome-like structures, Pickering emulsion droplets are often termed as colloidosomes.40 Nanoparticle adsorption at the interface can be considered as irreversible, because it requires high energy (estimated 105kBT, 100 nm nanoparticle) to overcome the barriers and desorb from the water/oil interface, while the desorption of small molecule surfactant requires only several kBT.39 In addition, nanoparticles as surfactant do not form micelles in continuous phase,41 which can avoid escape of molecules from colloidosomes because of the micelle solubilization effect. Previous studies have demonstrated that Pickering emulsions function nicely in preventing the leakage of contents from the colloidosomes.32,

41

Hence, the Pickering emulsion is a potential solution to prevent the false positives due to droplet fusion and crosstalk between each droplet unit, which is of great importance in digital PCR. In this study, for the first time, we report the use of self-assembled nanoparticles for robust colloidosome digital PCR (cdPCR). A one-step method was developed to synthesize colloidal surfactant fluorinated SiO2 nanoparticles (F-SiO2 NPs41). The method is highly convenient and reproducible compared to the synthesis of conventional fluorine-based surfactants. The excellent stability of colloidosomes under


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rapid thermocycling conditions ensures robust PCR, and the capability of avoiding cross-talk guarantees high-fidelity in assays. In addition, the F-SiO2 NPs can stabilize the commercially available PCR Master Mix reagent, providing an open digital PCR platform. Using colloidosome digital PCR, we have achieved the amplification of single-copy nucleic acids and the detection of rare mutants. With the advantages of robustness and accuracy, cdPCR is expected to find use in a variety of applications, such as gene expression and single cell analysis, as well as circulating tumor DNA and trace nucleic acid detection.

EXPERIMENTAL SECTION Materials and Reagents All chemicals used in this research were purchased and used without purification. PDMS (RTV 615, GE) was obtained from CChip Technology CO. Ltd. SU8-2015 photoresist was obtained from MicroChem (Newton, MA, U.S.A.). Film photomask and silicon wafer were purchased from Qingyi Precision Maskmaking Co. Ltd. (Shenzhen, China). Perfluorodecyltriethoxysilane (96%) was purchased from Aladdin Biochemical Technology Co. Ltd (Shanghai, China). Fluorinated oil GH135 was obtained from Lianqun Co. Ltd. (Shenzhen, China). Fluorinated oil HFE-7100 was purchased from 3M (Shanghai, China). Ammonium hydroxide solution (NH3 ·H2O, 25%), absolute ethanol (EtOH, 99%), and tetraethyl orthosilicate (TEOS, SiO2% ≥ 28.4%), were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Resorufin was purchased from J&K Scientific. Amplitaq DNA polymerase and TaqMan Universal Master Mix were produced by Thermo-Fisher (Shanghai, China), Premix Ex TaqTM qPCR was purchased from Takara (Beijing, China). AceQ qPCR Probe Master Mix was obtained from Vazyme (Nanjing, China). Luna Universal Probe qPCR Master Mix was purchased from NEB (Beijing, China). 2× Real Time PCR Mix was purchased from Aidlab (Beijing, China). 5× One Step RT- qPCR buffer and 25×One Step RT- qPCR RTase mix were obtained from Solarbio (Beijing, China). TaqProbe 2× qPCR Master Mix was provided by abm (Jiangsu, China). TransStart Top Green qPCR SuperMix was obtained from TransGen Biotech (Beijing, China). DNA



sequences (listed in Table S1) were purchased from Sangon Biotech. E.Z.N.A.® F-SiO2 NPs synthesis F-SiO2 NPs were prepared with a modified Stôber method.41, 42 First, 3.6 mL TEOS and 1.2 mL perfluorodecyltriethoxysilane were added to an aqueous solution containing 50 mL of EtOH, 2 mL NH3·H2O, and 1 mL H2O . Stirring vigorously overnight, the F-SiO2 NPs were obtained. To disperse the F- SiO2 NPs in fluorocarbon oil, they were washed by centrifugation at 10000 rpm for 30 min and redispersed in HFE-7100. The washing was repeated three times, and the last time HFE-7100 was replaced with GH135.43 The aggregates were removed by filtration before use. Microchip fabrication T-junction microfluidic chips44 were produced using soft lithography techniques.45 The pattern was designed by AutoCAD software, and then transferred on the photomask. Next, photoresist (SU-8 2015) was coated onto silicon wafers and the specific area which was protected by the designed photomask solidified via UV exposure. Finally, the mold was produced following photoresist development and post baking. The mold was replicated on silicone elastomer polydimethylsiloxane (PDMS) by pouring precursor (10:1)19 onto the mold and curing at 100 °C for 15min. Then, the patterned PDMS was peeled off and punched to form two inlets and one outlet, before bonding the patterned PDMS onto a flat bottom PDMS layer to seal the channels. Finally, the patterned PDMS module was bonded polished glass slide to provide high rigidity. After bonding, the three-layer structure (PDMS-PDMS-Glass side) T-junction chip was obtained and EGC-1720 solution was injected into the channels, followed by drying it at 75 °C for 30 min to obtain fluorinated surfaces. Generation of droplets The continuous phase GH-135 containing 3% (w/w) 100 nm F-SiO2 NPs or 3%(w/w) EA-surfactant (homemade) and dispersal phase were injected from two separate inlets to generate monodispersed droplets in the T-junction according to the fixed flow ratios of the two phases. The sizes of the droplets can be controlled by adjusting the flow ratios of the continuous phase and dispersed phase or by changing the structure of the T-junction.


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Resorufin cross-talk detection In order to compare molecular leakage between micro units stabilized by F-SiO2 NPs or EA surfactant, we chose resorufin as a molecular leakage model and generated two kinds of droplets containing different resorufin concentrations. For F-SiO2 NPs ， positive colloidosomes (12 µM resorufin in PBS) and negative colloidosomes (3.5 µM resorufin in PBS）were produced from T-junction chips and collected in Eppendorf tubes, respectively. Until all colloidosomes floated on the upper layers, the lower layers of GH-135 were removed and added clean fluorocarbon oil GH-135 was added. This step was repeated three times. After that, equal positive and negative colloidosomes were mixed together and incubated at room temperature. These colloidosomes were observed by the fluorescence microscope (Nikon Eclipse Ti−U, Japan) at different times. For the droplets stabilized with EA surfactant, the operation was the same. Positive and negative droplets were mixed at the ratio of 1:1 and detected at different times. Nucleic acid extract and reverse transcription The PSA cDNA was chosen as the templated model. It was synthesized based on the reverse transcription of RNA. Total RNA was extracted from about 106 LNCaP cells using E.Z.N.A.® Total RNA Kit I, according to the manufacturer’s protocol. Then, 3.6µg of total RNA was reverse-transcribed into cDNA and stored in -20 °C. The process of Digital PCR Each digital PCR reaction volume was 20 µL, including 10 µL TaqMan Universal Master Mix, 7.5 U Amplitaq DNA polymerase, 1 µM forward primer and reverse primer, 0.3 µM TaqMan probe, 1mg/mL BSA and 1μL desired DNA template concentration. No template control used ddH2O instead of templates. The digital PCR reaction mixture was loaded into a 1 mL syringe and injected by the micro syringe pump. The flow rates of the dispersed phase and continuous phase were constant at 0.15 mL/h and 0.6 mL/h, respectively. The generated colloidosomes were 95 µm in diameter. Then, the colloidosomes were collected and PCR was performed by thermocycling. The cycling program included a 10 min heating step at 94 °C, 48 cycles of 20 s at 95 °C, 1 min at 60°C, finally storage at 4°C. After amplification, a fluorescence microscope was



for rapid signal reading. Then, positive droplets and negative droplets were counted using ImageJ software, and the concentrations of templates were quantified with Poisson statistics. Bio-Rad7 digital PCR: The reaction mixture contained 10 µL 2× QX200 ddPCR Supermix for Probes (Bio-Rad, Hercules, CA), 1 µM forward primer and reverse primer, 0.3 µM TaqMan probe, 1 μL desired DNA template concentration and appropriate ddH2O to give a total volume of 20 μL. The premixed digital PCR reaction mixture was loaded into the Bio-Rad QX200 system used for droplet generation. After that, droplets were transferred to a 96-well plate and incubated in a thermal cycler. The procedure of thermocycling was 94 °C for 10 min, 48 cycles of 95 °C for 15 s and 60°C for 1 min, finally storage at 4 °C. Then, the amplified droplets were analyzed according to QX200 reader, and. the copies of template were counted using Quanta Soft (Bio-Rad, Hercules, CA) Complex sample detection with cdPCR The target mutant and wildtype KRAS gene templates were extracted from the SW480 cell line and the 293T cell line. A dilution 0.2% mutant KRAS genes (224 copies) was prepared in a high background of wildtype genes (11200 copies). The DNA sequences are listed in Table S1. The detailed process of cdPCR is described above.


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RESULTS AND DISCUSSION Working Principle of cdPCR In this study, for the first time, we introduced Pickering emulsions for digital PCR. The workflow is shown in Fig.1. First of all, the colloidal surfactant F-SiO2 NPs are synthesized by the Stôber method by co-hydrolysis of TEOS and the fluorosilane reagent perfluorodecyltriethoxysilane. When the aqueous solution is mixed with F-SiO2 NPs, the nanoparticles immediately self-assemble at the water/oil interface forming extremely stable colloidosomes, as shown in Fig. 1.a. Using a T-junction microfluidic chip, we can compartmentalize nucleic acids into massively uniform colloidosomes. After PCR amplification, the fluorescence intensities of the target-containing colloidosomes are significantly higher than the intensities of the empty ones, as shown in Fig. 1.b. Therefore, nucleic acid concentrations can be quantified by counting the number of light and dark colloidosomes. The cdPCR system offers several advantages: (1) the colloidal surfactant F-SiO2 NPs can be synthesized with a low cost, facile and highly reproducible method; (2) colloidosomes are extremely stable under rapid

Fig.1 (a) Principle of F-SiO2 NPs self-assembly at the interface of oil and aqueous solution to stabilize the colloidosomes. (b) Workflow of cdPCR. Monodispersed colloidosomes are generated with T-junction chip, followed by 48 cycles of amplification, and the fluorescence intensities of droplets are read by fluorescence microscopy. ACS Paragon Plus Environment


thermocycling, long-term storage, and compressed conditions, avoiding fusion of droplets and the resulting mixing of droplet contents; (3) crowded F-SiO2 NPs can be anchored at the water/oil interface rigidly, preventing molecular diffusion and leakage; (4) colloidosomes are compatible with a wide range of commercially available PCR Master Mix reagents, making it a generic platform for digital PCR. Synthesis of F-SiO2 NPs for Uniform Colloidosomes In this study, we first developed a facile and highly reproducible approach to synthesize F-SiO2 NPs (Fig.2a). Colloidal surfactant F-SiO2 NPs were synthesized using the Stôber method by co-hydrolysis of TEOS and fluorosilane reagent perfluorodecyltriethoxysilane. As the stability of colloidosomes is closely related to the wettability of the nanoparticle surface,46 we performed a series of optimizations on the initial input of TEOS and fluorosilane reagents. When the volume ratio TEOS:fluorosilane was 3:1, the F-SiO2 NPs could be well dispersed in the fluorocarbon oil avoiding clogging the chip and effectively stabilizing the colloidosomes. As shown in Fig.2b and video 1, the colloidosomes were generated continuously by the T-junction chip. To characterize the degree of uniformity, approximately 750 colloidosomes were imaged and analyzed. The colloidosomes presented good uniformity with a coefficient of variation (CV) of 2.2%, which is similar to the value for droplets stabilized by conventional surfactants, as shown in Fig.2c. In addition, it is convenient to control the size of colloidosomes by adjusting the flow ratios of two phases or by changing the dimensions of the T-junction. Fig.S1 shows the variation of colloidosome diameters from 50 µm to 150 µm. To further characterize the morphology of colloidosomes, they were dropped onto a glass slide before visualization with an optical microscope. Following water vaporization, the colloidosomes collapsed, as clearly shown in Fig.2e, suggesting that their membrane was formed by numerous nanoparticles anchored on the interface, as shown in the model of Fig.2d. To directly visualize the configuration of colloidosomes, we fixed nanoparticles in situ by a freeze-drying method and performed SEM imaging. The self-assembled nanoparticles formed a rigid shell as clearly observed in Fig.2f.


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Fig.2. (a) SEM image of the F-SiO2 NPs, scale bar, 500 nm; (b) Generation of colloidosomes with T-junction chip; (c) Size distribution of the colloidosomes. Scale bar, 250 µm; (d) Model of F-SiO2 NPs adsorbed at the oil/aqueous interface;(e) Microscope image showing the collapsed colloidosomes due to water vaporization, scale bar, 200 µm; (f) SEM image showing the nanoparticle selfassembled at the oil/aqueous interface, the scale bar is 500 nm Characterization of the Stability of Colloidosomes For digital PCR, it is critical to guarantee that no fusion occurs between micro-reactor units under high temperature, rapid thermal cycles and compressed environment. To assess the stability of colloidosomes stabilized with F-SiO2 NPs, we placed colloidosomes in various conditions. We first performed a week-long investigation on colloidosomes at room temperature. Fig.2a shows the colloidosomes stored for 0 h and one week, respectively. No apparent changes could be observed in the diameter of the colloidosomes, indicating that these colloidosomes stabilized with F-SiO2 NPs remain uniform during long-term storage. This property is very beneficial for experiments like single cell culture in droplets, which requires a long period of incubation time. During the process of digital PCR, colloidosomes need to withstand temperatures as high as 95 °C and rapid temperature changes. We tested their stabilities in PCR thermal cycling, including holding at 94 °C for 10 min, 48 cycles of 95 °C for 18 s and 60 °C for 1 min, followed by storage at 4 °C. Fig3.b shows the unchanged diameter of colloidosomes before and after the PCR cycles, demonstrating that these colloidosomes



are sufficiently stable to endure the conditions of high temperature and rapid temperature changes. In addition, during the process of digital PCR, droplets usually need to be transferred from chips, and thus highly compressed droplets may lead to coalescence. To demonstrate the stability of colloidosomes under compressed conditions, massive colloidosomes were injected into a special reservoir where the height was lower than the diameter of colloidosomes, resulting in highly compressed colloidosomes, as shown in Fig.3c. When colloidosomes flowed out of the reservoir, the pressure was released, and colloidosomes recovered to spherical form and remained uniform, suggesting their excellent robustness to withstand compressed treatment. In addition to the robustness of the colloidosomes, another important factor is to provide an open digital PCR platform that is compatible with a wide range of commercial PCR Master Mix reagents. To test the stability of colloidosomes with commercial PCR Master Mix reagents, we encapsulated 8 kinds of reagents in colloidosomes and compared them with the commercial surfactant produced by Bio-Rad. As shown in Fig.S3 and Table S2, all of the colloidosomes encapsulated with different commercial PCR Master Mix reagents remained uniform after PCR cycles, while the droplets synthesized by the Bio-Rad surfactant could only stabilize 2 types of commercial PCR Master Mix reagents,

Fig.3 (a) Size distribution of colloidosomes after storage for 0 h (i) and one-week (ii); (b) Size distribution of colloidosomes (i) before and (ii) after PCR cycle; (c) Microscope images show colloidosomes (i) under compression and (ii) with compression released. Scale bar is 150 µm


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suggesting the superior compatibility of colloidosomes with commercial PCR reagents. Leakage test To ensure the accuracy of digital assay, cross-talk must be avoided. To test the leakage, we selected resorufin as leakage model to determine whether the molecules leak from the colloidosomes. Resorufin is an ideal model to study cross-contamination between colloidosomes, because small hydrophobic substances tend to leak from the liquid-liquid interface33,

34.

We generated two kinds of colloidosomes containing

different concentrations of resorufin. The same amount of “positive” (12 µM resorufin in PBS) and “negative” colloidosomes (3.5 µM resorufin in PBS）were mixed together enable monitoring the fluorescence intensity evolution in real time. From 0h to 24 h, the fluorescence intensity of neither positive nor negative colloidosomes changed (Fig.4a), suggesting that F-SiO2 NPs were close-packed on the water/oil interface, thus effectively preventing resorufin diffusion. As a comparison, we also tested the leakage of droplets stabilized with EA surfactant, which is frequently used for droplet-based assays. Fig.4b shows the fluorescence intensities of positive and negative droplets after incubation for 0h, 2h, 24h. The fluorescence intensity of negative droplets increased, while the intensity of positive droplets dropped significantly after 2 h of incubation. After 24 h, the fluorescence intensities of all droplets were the same. Some research

Fig.4: (a) The fluorescence intensities of droplets stabilized with F-SiO2 NPs. Time: (i) 0 h, (ii) 2 h, (iii) 24 h; (b) The fluorescence intensities of droplets stabilized with EA. Time:0 h, 2 h, 24 h. Positive droplet:12 µM resorufin; negative droplet: 3.5 µM resorufin.



suggested that EA surfactants form reverse micelles in continuous phase, thus assisting resorufin transport between droplets.36 In contrast, F-SiO2 NPs were unable to form micelles41 and difficult desorb at the water/oil interface39. Therefore, in preventing cross-talking, colloidosomes stabilized with F-SiO2 NPs perform much better than those stabilized with EA surfactants. This property is very beneficial in droplet-based assays. The feasibility of colloidosome PCR To test the feasibility of colloidosomes as robust micro-reactors for digital PCR, we further investigated the adsorption of DNA at the water/oil interface. To investigate the distribution of DNA in droplets, we encapsulated 1 µM FAMtagged random DNA sequence in colloidosomes, and then analyzed the fluorescence intensity to estimate DNA distributions in colloidosomes. If DNA tended to adsorb at the water/oil interface, the fluorophore would be enriched on the interface, leading to a bright rim around the droplet. On the contrary, if the maximum fluorescence intensity appeared in the center of the colloidosome and gradually decreased to the rim, it would suggest that DNA uniformly distributes in colloidosomes due to their spherical shape. As shown in Fig 5a, fluorescence intensity at the edge of droplets was weakest and increased to a maximum at the center of the droplet, suggesting that DNA did not enrich at the interface.


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To test whether DNA could be amplified in the colloidosomes, we encapsulated 20

Fig.5: (a) (i) Fluorescence image showing the droplets containing DNA-FAM solution. Scale bar, 250 µm; (ii) The fluorescence intensity of droplets through the dotted line; (b) (i) PCR control with no templates (ii) with 10 ng templates. Scale bar, 200 µm 50 µm µL PCR reaction mixture with 10 ng DNA templates in 44600 colloidosomes followed

by 48 amplification cycles. As shown in Fig. 5b, the fluorescence intensities of positive samples were higher than those of the controls with no templates, suggesting the feasibility of performing PCR in the colloidosomes. To further demonstrate that the templates were amplified, colloidosomes were demulsified and samples were collected for gel electrophoresis. A clear band appeared at the position of 100 bp sequence as shown in Fig. S2, proving that the targets were indeed amplified in colloidosomes. Colloidosome in digital PCR To test the feasibility of using colloidosomes for digital PCR, we amplified a series of diluted nucleic acids at single molecule concentrations in colloidosomes to verify whether different concentrations of nucleic acids fit the values predicted by Poisson statistics and compared it with ddPCR performed on a Bio-Rad QX200 system. First of all, we used PSA cDNA as the template, which was synthesized by reverse transcription of RNA extracted from LNCaP cells and quantified with ddPCR on the



Bio-Rad QX200 system. To test whether a single copy template could be amplified in a colloidosome, the PSA genes were diluted to a series of concentrations (5×102~5.5 × 104copies) and amplified in 44600 colloidosomes (95 µm). The fluorescence intensities of colloidosomes are shown in Fig6. a. Seven thousand colloidosomes were analyzed and the results of DNA copies agreed with the theoretical Poisson statistics (Fig. S4) and showed a linear fit at low template concentration (R2=0.996), as shown in Fig6.b(i). Moreover, ddPCR performed on the Bio-Rad QX200 system with the same template concentrations showed similar results（Fig6.b）, verifying that cdPCR could function for absolute quantification of nucleic acids. To test whether the cdPCR system could be used for complex sample detection, we applied it in the detection of rare mutations. We selected the KRAS gene as a model, which serves as a biomarker for cancer diagnosis. Conventional method Real-time PCR TaqMan assay can detect only approximately 1% mutant templates in the background

Fig6: (a) Fluorescence imaging of droplets after cdPCR reaction, template copies (i) Negative control, (ii) 517 copies, (iii) 13275 copies, (iv) 27280 copies; (b) comparison of digital PCR with different methods; (c) cdPCR in detection of point mutation with KRAS gene, template copies (i) Negative control, (ii) 112000 copies of wildtype templates, (iii) 224 copies of mutant templates and 112000 copies of wildtype ACS Paragon Plus Environment templates, (iv) 224 copies of mutant templates; (d) qPCR in detection of point mutation with KRAS gene as the same template concentration as (c). Scale bar, 500µm

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of wildtype templates11, and the sensitivity of this method has limited its application in clinics. However, for digital PCR, all templates are compartmentalized in massively independent micro-reactors, which greatly increases the ratio of mutant templates to wildtype templates in certain reactors.47 In this study, we compartmentalized 224 copies of mutant templates and 112000 copies of wildtype templates in 44600 colloidosomes. On average, each colloidosome encapsulated 2.5 copies of wildtype templates and no more than one mutant template. The bright colloidosomes demonstrated that mutant templates were successfully amplified, as shown in Fig6.c(iii). Compared with Fig6.c(iv), which amplified the same concentration of mutant templates in colloidosomes, the amplified result is similar. It showed that our cdPCR approach could accurately detect 0.2% mutant templates in wildtype templates. In contrast, real-time PCR could not distinguish mutant templates with the same ratio, as shown in Fig6. d, strongly demonstrating that the cdPCR system can be applied for detection of a rare mutation in a complex sample. We expect cdPCR can be widely applied in other fields of DNA detection.

CONCLUSIONS In summary, for the first time, we report the use of the Pickering emulsion for digital PCR. Taking advantage of the properties of Pickering emulsions, massively superstable monodispersed colloidosomes were generated. Our results demonstrated that the cdPCR system has the ability to amplify a single copy of nucleic acid and achieve absolute quantification. The success of the cdPCR system resolved several key problems in digital PCR. First, it is a facile, low-cost and highly reproducible approach to synthesize colloidal surfactant. In addition, colloidosomes stabilized with F-SiO2 NPs are extremely stable even in extreme environments, including long-term storage, rapid thermal cycling, and compressed conditions. Moreover, colloidosomes performed better in preventing cross-contamination than droplets stabilized with conventional surfactants. In addition, the cdPCR system is compatible with a wide range of commercial PCR Master Mixes, thus providing an open and generic digital PCR platform. We believe our cdPCR system has great potential in various clinical



diagnostic and scientific research settings, such as the detection of circulating tumor DNA, copy number variation, gene expression, single cell analysis, etc.

ASSOCIATED CONTENTS Supporting Information: The Supporting Information is available free of charge on the ACS Publications website Primer sequences information, characterization of the stability of micro units with commercial PCR Master Mix reagent, controlling the size of colloidosomes from 50 µm to 150 µm, electrophoresis gel verifying the feasibility of colloidosome PCR, the uniformity of micro units with different commercial PCR Master Mix reagents, the measured results fit well with theoretical Poisson statistics (PDF) Video file: colloidosomes generation

Notes The authors declare no competing financial interest. 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 Chang Jiang Scholars and Innovative Research Teams in University (IRT13036) for their financial support.

(ii)


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REFERENCES 1. Sykes, P.; Neoh, S.; Brisco, M.; Hughes, E.; Condon, J.; Morley, A., Quantitation of targets for PCR by use of limiting dilution. BioTechniques 1992, 13 (3), 444-449. 2. Vogelstein, B.; Kinzler, K. W., Digital Pcr. Proc. Natl. Acad. Sci. U.S.A. 1999, 96 (16), 9236-9241. 3. Dawson, S. J.; Tsui, D. W.; Murtaza, M.; Biggs, H.; Rueda, O. M.; Chin, S. F.; Dunning, M. J.; Gale, D.; Forshew, T.; Mahler-Araujo, B.; Rajan, S.; Humphray, S.; Becq, J.; Halsall, D.; Wallis, M.; Bentley, D.; Caldas, C.; Rosenfeld, N., Analysis of circulating tumor DNA to monitor metastatic breast cancer. N Engl J Med 2013, 368 (13), 1199-209. 4. Gevensleben, H.; Garcia-Murillas, I.; Graeser, M. K.; Schiavon, G.; Osin, P.; Parton, M.; Smith, I. E.; Ashworth, A.; Turner, N. C., Noninvasive detection of HER2 amplification with plasma DNA digital PCR. Clin. Cancer. Res. 2013, 19 (12), 3276-84. 5. Diaz, L. A., Jr.; Bardelli, A., Liquid biopsies: genotyping circulating tumor DNA. J Clin Oncol 2014, 32 (6), 579-86. 6. Whale, A. S.; Huggett, J. F.; Cowen, S.; Speirs, V.; Shaw, J.; Ellison, S.; Foy, C. A.; Scott, D. J., Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation. Nucleic Acids Res. 2012, 40 (11), e82. 7. Hindson, B. J.; Ness, K. D.; Masquelier, D. A.; Belgrader, P.; Heredia, N. J.; Makarewicz, A. J.; Bright, I. J.; Lucero, M. Y.; Hiddessen, A. L.; Legler, T. C.; Kitano, T. K.; Hodel, M. R.; Petersen, J. F.; Wyatt, P. W.; Steenblock, E. R.; Shah, P. H.; Bousse, L. J.; Troup, C. B.; Mellen, J. C.; Wittmann, D. K.; Erndt, N. G.; Cauley, T. H.; Koehler, R. T.; So, A. P.; Dube, S.; Rose, K. A.; Montesclaros, L.; Wang, S.; Stumbo, D. P.; Hodges, S. P.; Romine, S.; Milanovich, F. P.; White, H. E.; Regan, J. F.; Karlin-Neumann, G. A.; Hindson, C. M.; Saxonov, S.; Colston, B. W., High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal. Chem. 2011, 83 (22), 8604-10. 8. Hindson, C. M.; Chevillet, J. R.; Briggs, H. A.; Gallichotte, E. N.; Ruf, I. K.; Hindson, B. J.; Vessella, R. L.; Tewari, M., Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat. Methods 2013, 10 (10), 1003-5. 9. Hashimoto-Torii, K.; Torii, M.; Fujimoto, M.; Nakai, A.; El Fatimy, R.; Mezger, V.; Ju, M. J.; Ishii, S.; Chao, S. H.; Brennand, K. J.; Gage, F. H.; Rakic, P., Roles of heat shock factor 1 in neuronal response to fetal environmental risks and its relevance to brain disorders. Neuron 2014, 82 (3), 560-72. 10. Zhong, Q.; Bhattacharya, S.; Kotsopoulos, S.; Olson, J.; Taly, V.; Griffiths, A. D.; Link, D. R.; Larson, J. W., Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR. Lab Chip 2011, 11 (13), 2167-74. 11. Taly, V.; Pekin, D.; Benhaim, L.; Kotsopoulos, S. K.; Le Corre, D.; Li, X.; Atochin, I.; Link, D. R.; Griffiths, A. D.; Pallier, K.; Blons, H.; Bouche, O.; Landi, B.; Hutchison, J. B.; Laurent-Puig, P., Multiplex picodroplet digital PCR



to detect KRAS mutations in circulating DNA from the plasma of colorectal cancer patients. Clin. Chem. 2013, 59 (12), 1722-31. 12. Zhang, H. F.; Jenkins, G.; Zou, Y.; Zhu, Z.; Yang, C. J., Massively Parallel Single-Molecule and Single-Cell Emulsion Reverse Transcription Polymerase Chain Reaction Using Agarose Droplet Microfluidics. Anal. Chem. 2012, 84 (8), 3599-3606. 13. Persaud, D.; Gay, H.; Ziemniak, C.; Chen, Y. H.; Piatak, M., Jr.; Chun, T. W.; Strain, M.; Richman, D.; Luzuriaga, K., Absence of detectable HIV-1 viremia after treatment cessation in an infant. N Engl J Med 2013, 369 (19), 1828-35. 14. Mu, D.; Yan, L.; Tang, H.; Liao, Y., A sensitive and accurate quantification method for the detection of hepatitis B virus covalently closed circular DNA by the application of a droplet digital polymerase chain reaction amplification system. Biotechnol. Lett 2015, 37 (10), 2063-73. 15. Morrison, T.; Hurley, J.; Garcia, J.; Yoder, K.; Katz, A.; Roberts, D.; Cho, J.; Kanigan, T.; Ilyin, S. E.; Horowitz, D.; Dixon, J. M.; Brenan, C. J., Nanoliter high throughput quantitative PCR. Nucleic Acids Res. 2006, 34 (18), e123. 16. Li, X.; Zhang, D.; Zhang, H.; Guan, Z.; Song, Y.; Liu, R.; Zhu, Z.; Yang, C., Microwell Array Method for Rapid Generation of Uniform Agarose Droplets and Beads for Single Molecule Analysis. Anal. Chem. 2018, 90 (4), 2570-2577. 17. Ottesen, E. A.; Hong, J. W.; Quake, S. R.; Leadbetter, J. R., Microfluidic digital PCR enables multigene analysis of individual environmental bacteria. science 2006, 314 (5804), 1464-1467. 18. Warren, L.; Bryder, D.; Weissman, I. L.; Quake, S. R., Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR. Proc. Natl. Acad. Sci. U.S.A. 2006, 103 (47), 17807-17812. 19. Zhu, Q.; Qiu, L.; Yu, B.; Xu, Y.; Gao, Y.; Pan, T.; Tian, Q.; Song, Q.; Jin, W.; Jin, Q.; Mu, Y., Digital PCR on an integrated self-priming compartmentalization chip. Lab Chip 2014, 14 (6), 1176-85. 20. Dressman, D.; Yan, H.; Traverso, G.; Kinzler, K. W.; Vogelstein, B., Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations. Proc. Natl. Acad. Sci. U.S.A. 2003, 100 (15), 8817-8822. 21. Baret, J. C., Surfactants in droplet-based microfluidics. Lab Chip 2012, 12 (3), 42233. 22. Margulies, M.; Egholm, M.; Altman, W. E.; Attiya, S.; Bader, J. S.; Bemben, L. A.; Berka, J.; Braverman, M. S.; Chen, Y. J.; Chen, Z.; Dewell, S. B.; Du, L.; Fierro, J. M.; Gomes, X. V.; Godwin, B. C.; He, W.; Helgesen, S.; Ho, C. H.; Irzyk, G. P.; Jando, S. C.; Alenquer, M. L.; Jarvie, T. P.; Jirage, K. B.; Kim, J. B.; Knight, J. R.; Lanza, J. R.; Leamon, J. H.; Lefkowitz, S. M.; Lei, M.; Li, J.; Lohman, K. L.; Lu, H.; Makhijani, V. B.; McDade, K. E.; McKenna, M. P.; Myers, E. W.; Nickerson, E.; Nobile, J. R.; Plant, R.; Puc, B. P.; Ronan, M. T.; Roth, G. T.; Sarkis, G. J.; Simons, J. F.; Simpson, J. W.; Srinivasan, M.; Tartaro, K. R.; Tomasz, A.; Vogt, K. A.; Volkmer, G. A.; Wang, S. H.; Wang, Y.; Weiner, M. P.; Yu, P.; Begley, R. F.; Rothberg, J. M., Genome sequencing in microfabricated high-density picolitre reactors. Nature


Page 20 of 23

Page 21 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2005, 437 (7057), 376-80. 23. Kumaresan, P.; Yang, C. J.; Cronier, S. A.; Blazej, R. G.; Mathies, R. A., High-throughput single copy DNA amplification and cell analysis in engineered nanoliter droplets. Anal. Chem. 2008, 80 (10), 3522-3529. 24. Zeng, Y.; Novak, R.; Shuga, J.; Smith, M. T.; Mathies, R. A., Highperformance single cell genetic analysis using microfluidic emulsion generator arrays. Anal. Chem. 2010, 82 (8), 3183-3190. 25. Leng, X.; Zhang, W.; Wang, C.; Cui, L.; Yang, C. J., Agarose droplet microfluidics for highly parallel and efficient single molecule emulsion PCR. Lab Chip 2010, 10 (21), 2841. 26. Schaerli, Y.; Stein, V.; Spiering, M. M.; Benkovic, S. J.; Abell, C.; Hollfelder, F., Isothermal DNA amplification using the T4 replisome: circular nicking endonuclease-dependent amplification and primase-based whole-genome amplification. Nucleic Acids Res. 2010, 38 (22), e201. 27. Mary, P.; Dauphinot, L.; Bois, N.; Potier, M.-C.; Studer, V.; Tabeling, P., Analysis of gene expression at the single-cell level using microdroplet-based microfluidic technology. Biomicrofluidics 2011, 5 (2), 024109. 28. Ghadessy, F. J.; Ong, J. L.; Holliger, P., Directed evolution of polymerase function by compartmentalized self-replication. Proc. Natl. Acad. Sci. U.S.A. 2001, 98 (8), 4552-4557. 29. Courtois, F.; Olguin, L. F.; Whyte, G.; Theberge, A. B.; Huck, W. T.; Hollfelder, F.; Abell, C., Controlling the retention of small molecules in emulsion microdroplets for use in cell-based assays. Anal. Chem. 2009, 81 (8), 3008-3016. 30. Holtze, C.; Rowat, A. C.; Agresti, J. J.; Hutchison, J. B.; Angile, F. E.; Schmitz, C. H. J.; Koster, S.; Duan, H.; Humphry, K. J.; Scanga, R. A.; Johnson, J. S.; Pisignano, D.; Weitz, D. A., Biocompatible surfactants for water-influorocarbon emulsions. Lab Chip 2008, 8 (10), 1632-1639. 31. Mazutis, L.; Baret, J.-C.; Treacy, P.; Skhiri, Y.; Araghi, A. F.; Ryckelynck, M.; Taly, V.; Griffiths, A. D., Multi-step microfluidic droplet processing: kinetic analysis of an in vitro translated enzyme. Lab Chip 2009, 9 (20), 2902-2908. 32. Pan, M.; Lyu, F.; Tang, S. K., Fluorinated Pickering Emulsions with Nonadsorbing Interfaces for Droplet-based Enzymatic Assays. Anal. Chem. 2015, 87 (15), 7938-43. 33. Woronoff, G.; El Harrak, A.; Mayot, E.; Schicke, O.; Miller, O. J.; Soumillion, P.; Griffiths, A. D.; Ryckelynck, M., New generation of amino coumarin methyl sulfonate-based fluorogenic substrates for amidase assays in droplet-based microfluidic applications. Anal. Chem. 2011, 83 (8), 2852-7. 34. Gruner, P.; Riechers, B.; Semin, B.; Lim, J.; Johnston, A.; Short, K.; Baret, J.-C., Controlling molecular transport in minimal emulsions. Nat. Commun 2016, 7. 35. Scheler, O.; Kaminski, T. S.; Ruszczak, A.; Garstecki, P., Dodecylresorufin (C12R) Outperforms Resorufin in Microdroplet Bacterial Assays. ACS Appl Mater Interfaces 2016, 8 (18), 11318-25. 36. Skhiri, Y.; Gruner, P.; Semin, B.; Brosseau, Q.; Pekin, D.; Mazutis, L.;



Goust, V.; Kleinschmidt, F.; El Harrak, A.; Hutchison, J. B.; Mayot, E.; Bartolo, J.-F.; Griffiths, A. D.; Taly, V.; Baret, J.-C., Dynamics of molecular transport by surfactants in emulsions. Soft Matter 2012, 8 (41), 10618-10627. 37. Pickering, S. U., CXCVI.—emulsions. J. Am. Chem. Soc 1907, 91, 2001-2021. 38. Subramaniam, A. B.; Abkarian, M.; Stone, H. A., Controlled assembly of jammed colloidal shells on fluid droplets. Nature Mater 2005, 4 (7), 553-556. 39. Binks, B. P., Particles as surfactants—similarities and differences. Curr. Opin. Colloid Interface Sci 2002, 7 (1-2), 21-41. 40. Dinsmore, A.; Hsu, M. F.; Nikolaides, M.; Marquez, M.; Bausch, A.; Weitz, D., Colloidosomes: selectively permeable capsules composed of colloidal particles. Science 2002, 298 (5595), 1006-1009. 41. Pan, M.; Rosenfeld, L.; Kim, M.; Xu, M.; Lin, E.; Derda, R.; Tang, S. K., Fluorinated pickering emulsions impede interfacial transport and form rigid interface for the growth of anchorage-dependent cells. ACS Appl Mater Interfaces 2014, 6 (23), 21446-53. 42. Stöber, W.; Fink, A.; Bohn, E., Controlled growth of monodisperse silica spheres in the micron size range. J Colloid Interf Sci 1968, 26 (1), 62-69. 43. Guan, Z.; Zou, Y.; Zhang, M.; Lv, J.; Shen, H.; Yang, P.; Zhang, H.; Zhu, Z.; James Yang, C., A highly parallel microfluidic droplet method enabling singlemolecule counting for digital enzyme detection. Biomicrofluidics 2014, 8 (1), 014110. 44. Nisisako, T.; Torii, T.; Higuchi, T., Droplet formation in a microchannel network. Lab Chip 2002, 2 (1), 24-6. 45. Xia, Y.; Whitesides, G. M., Soft lithography. Annu. Rev. Mater. Sci 1998, 28 (1), 153-184. 46. Binks, B. P.; Lumsdon, S., Influence of particle wettability on the type and stability of surfactant-free emulsions. Langmuir 2000, 16 (23), 8622-8631. 47. Baker, M., Digital PCR hits its stride. Nat. Methods 2012, 9 (6), 541-544.


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Table of Contents Graphic and Synopsis


Stable Colloidosomes Formed by Self-Assembly of Colloidal

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