Publications

2021
Yang, C. ; Chen, L. ; Zhang, R. ; Chen, D. ; Arriaga, L. R. ; Weitz, D. A. Local high-density distributions of phospholipids induced by the nucleation and growth of smectic liquid crystals at the interface. Chinese Chemical Letters 2021. Publisher's VersionAbstract

Amphiphilic molecules adsorbed at the interface could control the orientation of liquid crystals (LCs) while LCs in turn could influence the distributions of amphiphilic molecules. The studies on the interactions between liquid crystals and amphiphilic molecules at the interface are important for the development of molecular sensors. In this paper, we demonstrate that the development of smectic LC ordering from isotropic at the LC/water interface could induce local high-density distributions of amphiphilic phospholipids. Mixtures of liquid crystals and phospholipids in chloroform are first emulsified in water. By fluorescently labeling the phospholipids adsorbed at the interface, their distributions are visualized under fluorescent confocal microscope. Interestingly, local high-density distributions of phospholipids showing a high fluorescent intensity are observed on the surface of LC droplets. Investigations on the correlation between phospholipid density, surface tension and smectic LC ordering suggest that when domains of smectic LC layers nucleate and grow from isotropic at the LC/water interface as chloroform slowly evaporates at room temperature, phospholipids transition from liquid-expanded to liquid-condensed phases in response to the smectic ordering, which induces a higher surface tension at the interface. The results will provide an important insight into the interactions between liquid crystals and amphiphilic molecules at the interface.

Chen, L. ; Xiao, Y. ; Wu, Q. ; Yan, X. ; Zhao, P. ; Ruan, J. ; Shan, J. ; Chen, D. ; Weitz, D. A. ; Ye, F. Emulsion Designer Using Microfluidic Three-Dimensional Droplet Printing in Droplet. Small 2021, 17, 2102579. Publisher's VersionAbstract

Hierarchical emulsions are interesting for both scientific researches and practical applications. Hierarchical emulsions prepared by microfluidics require complicated device geometry and delicate control of flow rates. Here, a versatile method is developed to design hierarchical emulsions using microfluidic 3D droplet printing in droplet. The process of droplet printing in droplet mimics the dragonfly laying eggs and has advantages of easy processing and flexible design. To demonstrate the capability of the method, double emulsions and triple emulsions with tunable core number, core size, and core composition are prepared. The hierarchical emulsions are excellent templates for the developments of functional materials. Flattened crescent-moon-shaped particles are then fabricated using double emulsions printed in confined 2D space as templates. The particles are excellent delivery vehicles for 2D interfaces, which can load and transport cargos through a well-defined trajectory under external magnetic steering. Microfluidic 3D droplet printing in droplet provides a powerful platform with improved simplicity and flexibility for the design of hierarchical emulsions and functional materials.

chen2021.pdf
Shen, Y. ; Wu, H. ; Lu, P. J. ; Wang, D. ; Shayegan, M. ; Li, H. ; Shi, W. ; Wang, Z. ; Cai, L. ; Xia, J. ; et al. Effects of Vimentin Intermediate Filaments on the Structure and Dynamics of In Vitro Multicomponent Interpenetrating Cytoskeletal Networks. Physical Review Letters 2021, 127, 108101. Publisher's VersionAbstract

We investigate the rheological properties of interpenetrating networks reconstituted from the main cytoskeletal components: filamentous actin, microtubules, and vimentin intermediate filaments. The elastic modulus is determined largely by actin, with little contribution from either microtubules or vimentin. However, vimentin dramatically impacts the relaxation, with even small amounts significantly increasing the relaxation time of the interpenetrating network. This highly unusual decoupling between dissipation and elasticity may reflect weak attractive interactions between vimentin and actin networks.

shen2021.pdf
Parsa, S. ; Zareei, A. ; Santanach-Carreras, E. ; Morris, E. J. ; Amir, A. ; Xiao, L. ; Weitz, D. A. Unexpected scaling of interstitial velocities with permeability due to polymer retention in porous media. Physical Review Fluids 2021, 6 L082302. Publisher's VersionAbstract

Polymer retention from the flow of a polymer solution through porous media results in substantial decrease of the permeability; however, the underlying physics of this effect is unknown. While the polymer retention leads to a decrease in pore volume, here we show that this cannot cause the full reduction in permeability. Instead, to determine the origin of this anomalous decrease in permeability, we use confocal microscopy to measure the pore-level velocities in an index-matched model porous medium. We show that they exhibit an exponential distribution and, upon polymer retention, this distribution is broadened yet retains the same exponential form. Surprisingly, the velocity distributions are scaled by the inverse square root of the permeabilities. We combine experiment and simulation to show these changes result from diversion of flow in the random porous-medium network rather than reduction in pore volume upon polymer retention.

parsa2021.pdf
Aime, S. ; Sabato, M. ; Xiao, L. ; Weitz, D. A. Dynamic Speckle Holography. Physical Review Letters 2021, 127, 088003. Publisher's VersionAbstract

We introduce dynamic speckle holography, a new technique that combines imaging and scattering to measure three-dimensional maps of displacements as small as ten nanometers over several centimeters, greatly extending the capabilities of traditional imaging systems. We attain this sensitivity by imaging speckle patterns of light collected at three scattering angles and measuring the decay in the temporal correlation due to local motion. We use dynamic speckle holography to measure the strain field of a colloidal gel undergoing fracture and establish the surprising role of internal tension in driving the fracture.

aime2021.pdf
Mohammed, D. ; Park, C. Y. ; Fredberg, J. J. ; Weitz, D. A. Tumorigenic mesenchymal clusters are less sensitive to moderate osmotic stresses due to low amounts of junctional E-cadherin. Nature 2021, 11, 16279. Publisher's VersionAbstract

The migration of tumorigenic cells is a critical step for metastatic breast cancer progression. Although the role of the extracellular matrix in breast cancer cell migration has been extensively described, the effect of osmotic stress on the migration of tumor breast cohorts remains unclear. Most of our understanding on the effect of osmotic stresses on cell migration comes from studies at the level of the single cell in isolation and does not take cell–cell interactions into account. Here, we study the impact of moderate osmotic stress on the migration of cell clusters composed of either non-tumorigenic or tumorigenic cells. We observe a decrease in migration distance and speed for non-tumorigenic cells but not for tumorigenic ones. To explain these differences, we investigate how osmotic stress impacts the mechanical properties of cell clusters and affects their volumes. Our findings show that tumorigenic mesenchymal cells are less sensitive to osmotic stress than non-tumorigenic cells and suggest that this difference is associated with a lower expression of E-cadherin. Using EGTA treatments, we confirm that the establishment of cell–cell adhesive interactions is a key component of the behavior of cell clusters in response to osmotic stress. This study provides evidence on the low sensitivity of mesenchymal tumorigenic clusters to moderate osmotic stress and highlights the importance of cadherin-based junctions in the response to osmotic stress.

mohammed2021.pdf
Chen, L. ; Yang, C. ; Xiao, Y. ; Yan, X. ; Hu, L. ; Eggersdorfer, M. ; Chen, D. ; Weitz, D. A. ; Ye, F. Millifluidics, microfluidics, and nanofluidics: manipulating fluids at varying length scales. Materials Today Nano 2021, 16, 100136. Publisher's VersionAbstract

Manipulating fluids at varying length scales and understanding their underlying mechanisms are significant for interdisciplinary studies of physics, chemistry, biology, and engineering; fluid manipulation plays an important role in both scientific research and industrial applications. Here, we systematically review millifluidics, microfluidics, and nanofluidics with dimensions ranging from millimeters to nanometers. The major physics associated with flow property and interactions of millifluidics, microfluidics, and nanofluidics are discussed in detail, especially the differences arising from different length scales. Device fabrication techniques are summarized including additive and nonadditive manufacturing methods for millifluidics and microfluidics and top-down and bottom-up strategies for nanofluidics. Recent developments and applications of millifluidics, microfluidics, and nanofluidics are described to provide an overview on current researches. Outlooks of millifluidics, microfluidics, and nanofluidics are discussed at the end.​​​​​​

Wu, B. ; Yang, C. ; Xin, Q. ; Kong, L. ; Eggersdorfer, M. ; Ruan, J. ; Zhao, P. ; Shan, J. ; Liu, K. ; Chen, D. ; et al. Attractive Pickering Emulsion Gels. Advanced Materials 2021, 33, 2102362. Publisher's VersionAbstract

Properties of emulsions highly depend on the interdroplet interactions and, thus, engineering interdroplet interactions at molecular scale are essential to achieve desired emulsion systems. Here, attractive Pickering emulsion gels (APEGs) are designed and prepared by bridging neighboring particle-stabilized droplets via telechelic polymers. In the APEGs, each telechelic molecule with two amino end groups can simultaneously bind to two carboxyl functionalized nanoparticles in two neighboring droplets, forming a bridged network. The APEG systems show typical shear-thinning behaviors and their viscoelastic properties are tunable by temperature, pH, and molecular weight of the telechelic polymers, making them ideal for direct 3D printing. The APEGs can be photopolymerized to prepare APEG-templated porous materials and their microstructures can be tailored to optimize their performances, making the APEG systems promising for a wide range of applications.

wu2021.pdf
Sun, Z. ; Yan, X. ; Xiao, Y. ; Hu, L. ; Eggersdorfer, M. ; Chen, D. ; Yang, Z. ; Weitz, D. A. Pickering emulsions stabilized by colloidal surfactants: Role of solid particles. Particuology 2021. Publisher's VersionAbstract

Pickering emulsions are emulsions stabilized by colloidal surfactants, i.e. solid particles. Compared with traditional molecular surfactant-stabilized emulsions, Pickering emulsions show many advantages, such as high resistance to coalescence, long-term stability, good biocompatibility and tunable properties. In recent years, Pickering emulsions are widely applied in scientific researches and industrial applications. In this review, we focus on the influences of particle properties on Pickering emulsions, including particle amphiphilicity, concentration, size and shape, and summarize the strategies developed for the preparation of amphiphilic Janus particles. The applications of Pickering emulsions in food industry, cosmetic industry, material science, drug delivery, biomedical research and vaccine adjuvant will also be covered. Pickering emulsions are a unique system for multi-disciplinary studies and will become more and more important in the future.

Chowdhury, M. S. ; Zheng, W. ; Singh, A. K. ; Ong, I. L. H. ; Hou, Y. ; Heyman, J. A. ; Faghani, A. ; Amstad, E. ; Weitz, D. A. ; Haag, R. Linear triglycerol-based fluorosurfactants show high potential for droplet-microfluidics-based biochemical assays. Soft Matter 2021, 17, 7260-7267. Publisher's VersionAbstract

Fluorosurfactants have expanded the landscape of high-value biochemical assays in microfluidic droplets, but little is known about how the spatial geometries and polarity of the head group contribute to the performance of fluorosurfactants. To decouple this, we design, synthesize, and characterize two linear and two dendritic glycerol- or tris-based surfactants with a common perfluoropolyether tail. To reveal the influence of spatial geometry, we choose inter-droplet cargo transport as a stringent test case. Using surfactants with linear di- and triglycerol, we show that the inter-droplet cargo transport is minimal compared with their dendritic counterparts. When we encapsulated a less-leaky sodium fluorescent dye into the droplets, quantitatively, we find that the mean fluorescence intensity of the PFPE-dTG stabilized PBS-only droplets after 72 h was ∼3 times that of the signal detected in PBS-only droplets stabilized by PFPE-lTG. We also demonstrate that the post-functionalization of PFPE-lTG having a linear geometry and four hydroxy groups enables the ‘from-Droplet’ fishing of the biotin–streptavidin protein complex without the trade-off between fishing efficiency and droplet stability. Thus, our approach to design user-friendly surfactants reveals the aspects of spatial geometry and facile tunability of the polar head groups that have not been captured or exploited before.

chowdhury2021.pdf
Shiba, K. ; Li, G. ; Virot, E. ; Yoshikawa, G. ; Weitz, D. A. Microchannel measurements of viscosity for both gases and liquids. Lab on a Chip 2021, 21, 2805-2811. Publisher's VersionAbstract

Quantifying the viscosity of a fluid is of great importance in determining its properties and can even be used to identify what the fluid is. While many techniques exist for measuring the viscosity of either gases or liquids, it is very challenging to probe both gases and liquids with a single approach because of the significant difference in their nature, and the vast difference in the values of their viscosities. We introduce a facile approach to measuring the viscosity of a Newtonian fluid, either a gas or a liquid, by flowing it through a deformable microchannel where the deformation depends on the pressure required to induce the flow, which, in turn, depends on the fluid viscosity. A strain gauge embedded just above and across the microchannel transduces the flow-induced deformation into strain. The strain is proportional to the square of the flow-induced deformation enabling us to precisely discriminate not only gases but also liquids based on their viscosities with the same device.

shiba2021.pdf
Yang, G. ; Liu, Y. ; Hui, Y. ; Tengjisi, ; Chen, D. ; Weitz, D. A. ; Zhao, C. - X. Implications of Quenching-to-Dequenching Switch in Quantitative Cell Uptake and Biodistribution of Dye-Labeled Nanoparticles. Angewandte Chemie 2021, 133, 15554-15563. Publisher's VersionAbstract

A general strategy to carry out cell uptake and biodistribution studies is to label nanoparticles (NPs) with a fluorescent dye. However, the comparative study of different dye‐loaded NPs remains difficult owing to uncontrolled dye quenching and de‐quenching. Here we compared two types of dye‐labeled NPs and demonstrated their distinct properties. NPs with dye molecules at a solid state suffer from dye quenching, so the dye release and/or NP degradation in biological environments leads to a several‐fold increase of fluorescence intensity despite the same amount of NPs, owing to the state switch from quenching to de‐quenching. In contrast, NPs with dye molecules at a soluble state exhibit no quenching effect. To standardize the comparative study, we propose two possible solutions: using lower dye loading or using medium analysis for quantifying cell uptake of NPs. This work provides valuable insights into selecting valid quantification methods for bio‐nano studies.

yang2021.pdf
Ko, J. ; Wang, Y. ; Sheng, K. ; Weitz, D. A. ; Weissleder, R. Sequencing-Based Protein Analysis of Single Extracellular Vesicles. ACS Nano 2021, 15, 5631-5638. Publisher's VersionAbstract

Circulating extracellular vesicles (EVs)—biological nanomaterials shed from most mammalian cells—have emerged as promising biomarkers, drug delivery vesicles, and treatment modulators. While different types of vesicles are being explored for these applications, it is becoming clear that human EVs are quite heterogeneous even in homogeneous or monoclonal cell populations. Since it is the surface EV protein composition that will largely dictate their biological behavior, high-throughput single EV profiling methods are needed to better define EV subpopulations. Here, we present an antibody-based immunosequencing method that allows multiplexed measurement of protein molecules from individual nanometer-sized EVs. We use droplet microfluidics to compartmentalize and barcode individual EVs. The barcodes/antibody-DNA are then sequenced to determine protein composition. Using this highly sensitive technology, we detected specific proteins at the single EV level. We expect that this technology can be further adapted for multiplexed protein analysis of any nanoparticle.

ko2021.pdf
Xia, J. ; Cai, L. ; Wu, H. ; Mackintosh, F. C. ; Weitz, D. A. Anomalous mechanics of Zn2+-modified fibrin networks. PNAS 2021, 118. Publisher's VersionAbstract

Fibrin is the main component of blood clots. The mechanical properties of fibrin are therefore of critical importance in successful hemostasis. One of the divalent cations released by platelets during hemostasis is Zn2+; however, its effect on the network structure of fibrin gels and on the resultant mechanical properties remains poorly understood. Here, by combining mechanical measurements with three-dimensional confocal microscopy imaging, we show that Zn2+ can tune the fibrin network structure and alter its mechanical properties. In the presence of Zn2+, fibrin protofibrils form large bundles that cause a coarsening of the fibrin network due to an increase in fiber diameter and reduction of the total fiber length. We further show that the protofibrils in these bundles are loosely coupled to one another, which results in a decrease of the elastic modulus with increasing Zn2+ concentrations. We explore the elastic properties of these networks at both low and high stress: At low stress, the elasticity originates from pulling the thermal slack out of the network, and this is consistent with the thermal bending of the fibers. By contrast, at high stress, the elasticity exhibits a common master curve consistent with the stretching of individual protofibrils. These results show that the mechanics of a fibrin network are closely correlated with its microscopic structure and inform our understanding of the structure and physical mechanisms leading to defective or excessive clot stiffness.

xia2021.pdf
An, P. ; Cantalupo, P. G. ; Zheng, W. ; Saenz-Robles, M. T. ; Duray, A. M. ; Weitz, D. A. ; Pipas, J. M. Single-Cell Transcriptomics Reveals a Heterogeneous Cellular Response to BK Virus Infection. Journal of Virology 2021, 95. Publisher's VersionAbstract

BK virus (BKV) is a human polyomavirus that is generally harmless but can cause devastating disease in immunosuppressed individuals. BKV infection of renal cells is a common problem for kidney transplant patients undergoing immunosuppressive therapy. In cultured primary human renal proximal tubule epithelial (RPTE) cells, BKV undergoes a productive infection. The BKV-encoded large T antigen (LT) induces cell cycle entry, resulting in the upregulation of numerous genes associated with cell proliferation. Consistently, microarray and transcriptome sequencing (RNA-seq) experiments performed on bulk infected cell populations identified several proliferation-related pathways that are upregulated by BKV. These studies revealed few genes that are downregulated. In this study, we analyzed viral and cellular transcripts in single mock- or BKV-infected cells. We found that the levels of viral mRNAs vary widely among infected cells, resulting in different levels of LT and viral capsid protein expression. Cells expressing the highest levels of viral transcripts account for approximately 20% of the culture and have a gene expression pattern that is distinct from that of cells expressing lower levels of viral mRNAs. Surprisingly, cells expressing low levels of viral mRNA do not progress with time to high expression, suggesting that the two cellular responses are determined prior to or shortly following infection. Finally, comparison of cellular gene expression patterns of cells expressing high levels of viral mRNA with those of mock-infected cells or cells expressing low levels of viral mRNA revealed previously unidentified pathways that are downregulated by BKV. Among these are pathways associated with drug metabolism and detoxification, tumor necrosis factor (TNF) signaling, energy metabolism, and translation.

an2021.pdf
Tang, J. ; Fan, B. ; Xiao, L. ; Tian, S. ; Zhang, F. ; Zhang, L. ; Weitz, D. A. A New Ensemble Machine-Learning Framework for Searching Sweet Spots in Shale Reservoirs. SPE Journal 2021, 26, 482-497. Publisher's VersionAbstract
Knowing the location of sweet spots benefits the horizontal well drilling and the selection of perforation clusters. Generally, geoscientists determine sweet spots from the well-logging interpretation. In this paper, a group of prevalent classifiers [extreme gradient boosting (XGBoost), unbiased boosting with categorical features (CatBoost), and light gradient boosting machine (LightGBM)] based on gradient-boosting decision trees (GBDTs) are introduced to automatically determine sweet spots based on well-log data sets. Compared with linear support vector machines (SVMs), these robust algorithms can deal with comparative scales of features and learn nonlinear decision boundaries. Moreover, they are less influenced by the presence of outliers. Another prevailing approach, named generative adversarial networks (GANs), is implemented to augment the training data set by using a small number of training samples. An extensive application has been built for the field cases in a certain oilfield. We randomly select 73 horizontal wells for training, and 13 features are chosen from well-log data sets. Compared with conventional SVMs, the agreement rates of interpretation by XGBoost and CatBoost are significantly improved. Without special preprocessing of the input data sets and conditional tabular GAN (CTGAN) model fine tuning, the fake data set could still bring a relatively low agreement rate for all detections. Finally, we propose an ensemble-learning framework concatenating multilevels of classifiers and improve agreement rate. In this paper, we illustrate a new tool for categorizing the reservoir quality by using GBDTs and ensemble models, which further helps search and identify sweet spots automatically. This tool enables us to integrate experts’ knowledge to the developed model, identify logging curves more efficiently, and cover more sweet spots during the drilling and completion treatment, which immensely decrease the cost of log interpretation.
tang2020.pdf
Werner, J. G. ; Lee, H. ; Wiesner, U. ; Weitz, D. A. Ordered Mesoporous Microcapsules from Double Emulsion Confined Block Copolymer Self-Assembly. ACS Nano 2021, 15, 3490-3499. Publisher's VersionAbstract

Polymeric microcapsules with shells containing homogeneous pores with uniform diameter on the nanometer scale are reported. The mesoporous microcapsules are obtained from confined self-assembly of amphiphilic block copolymers with a selective porogen in the shell of water-in-oil-in-water double emulsion drops. The use of double emulsion drops as a liquid template enables the formation of homogeneous capsules of 100s of microns in diameter, with aqueous cores encapsulated in a shell membrane with a tunable thickness of 100s of nanometers to 10s of microns. Microcapsules with shells that exhibit an ordered gyroidal morphology and three-dimensionally connected mesopores are obtained from the triblock terpolymer poly(isoprene)-block-poly(styrene)-block-poly(4-vinylpyridine) coassembled with pentadecylphenol as a porogen. The bicontinuous shell morphology yields nanoporous paths connecting the inside to the outside of the microcapsule after porogen removal; by contrast, one-dimensional hexagonally packed cylindrical pores, obtained from a traditional diblock copolymer system with parallel alignment to the surface, would block transport through the shell. To enable the mesoporous microcapsules to withstand harsh conditions, such as exposure to organic solvents, without rupture of the shell, we develop a cross-linking method of the nanostructured triblock terpolymer shell after its self-assembly. The microcapsules exhibit pH-responsive permeability to polymeric solutes, demonstrating their potential as a filtration medium for actively tunable macromolecular separation and purification. Furthermore, we report a tunable dual-phase separation method to fabricate microcapsules with hierarchically porous shells that exhibit ordered mesoporous membrane walls within sponge-like micron-sized macropores to further control shell permeability.

werner2021.pdf
Tiribocchi, A. ; Montessori, A. ; Lauricella, M. ; Bonaccorso, F. ; Succi, S. ; Aime, S. ; Milani, M. ; Weitz, D. A. The vortex-driven dynamics of droplets within droplets. Nature Communications 2021, 12, 1-10. Publisher's VersionAbstract

Understanding the fluid-structure interaction is crucial for an optimal design and manufacturing of soft mesoscale materials. Multi-core emulsions are a class of soft fluids assembled from cluster configurations of deformable oil-water double droplets (cores), often employed as building-blocks for the realisation of devices of interest in bio-technology, such as drug-delivery, tissue engineering and regenerative medicine. Here, we study the physics of multi-core emulsions flowing in microfluidic channels and report numerical evidence of a surprisingly rich variety of driven non-equilibrium states (NES), whose formation is caused by a dipolar fluid vortex triggered by the sheared structure of the flow carrier within the microchannel. The observed dynamic regimes range from long-lived NES at low core-area fraction, characterised by a planetary-like motion of the internal drops, to short-lived ones at high core-area fraction, in which a pre-chaotic motion results from multi-body collisions of inner drops, as combined with self-consistent hydrodynamic interactions. The onset of pre-chaotic behavior is marked by transitions of the cores from one vortex to another, a process that we interpret as manifestations of the system to maximize its entropy by filling voids, as they arise dynamically within the capsule.

tiribocchi2021.pdf
2020
Ko, J. ; Wang, Y. ; Carlson, J. C. T. ; Marquard, A. ; Gungabeesoon, J. ; Charest, A. ; Weitz, D. A. ; Pittet, M. J. ; Weissleder, R. Single Extracellular Vesicle Protein Analysis Using Immuno-Digital Droplet Polymerase Chain Reaction Amplification. Advanced Biosystems 2020, 4 1900307. Publisher's VersionAbstract

There is a need for novel analytical techniques to study the composition of single extracellular vesicles (EV). Such techniques are required to improve the understanding of heterogeneous EV populations, to allow identification of unique subpopulations, and to enable earlier and more sensitive disease detection. Because of the small size of EV and their low protein content, ultrahigh sensitivity technologies are required. Here, an immuno‐droplet digital polymerase chain reaction (iddPCR) amplification method is described that allows multiplexed single EV protein profiling. Antibody–DNA conjugates are used to label EV, followed by stochastic microfluidic incorporation of single EV into droplets. In situ PCR with fluorescent reporter probes converts and amplifies the barcode signal for subsequent read‐out by droplet imaging. In these proof‐of‐principle studies, it is shown that multiplex protein analysis is possible in single EV, opening the door for future analyses.

ko2020.pdf
Ding, R. ; Hung, K. - C. ; Mitra, A. ; Ung, L. W. ; Lightwood, D. ; Tu, R. ; Starkie, D. ; Cai, L. ; Mazutis, L. ; Chong, S. ; et al. Rapid isolation of antigen-specific B-cells using droplet microfluidics. RSC Advances 2020, 10, 27006-27013. Publisher's VersionAbstract
Monoclonal antibodies are powerful tools for scientific research and are the basis of numerous therapeutics. However, traditional approaches to generate monoclonal antibodies against a desired target, such as hybridoma-based techniques and display library methods, are laborious and suffer from fusion inefficiency and display bias, respectively. Here we present a platform, featuring droplet microfluidics and a bead-based binding assay, to rapidly identify and verify antigen-binding antibody sequences from primary cells. We used a defined mixture of hybridoma cells to characterize the system, sorting droplets at up to 100 Hz and isolating desired hybridoma cells, comprising 0.1% of the input, with a false positive rate of less than 1%. We then applied the system to once-frozen primary B-cells to isolate rare cells secreting target-binding antibody. We performed RT-PCR on individual sorted cells to recover the correctly paired heavy- and light-chain antibody sequences, and we used rapid cell-free protein synthesis to generate single-chain variable fragment-format (scFv) antibodies from fourteen of the sorted cells. Twelve of these showed antigen-specific binding by ELISA. Our platform facilitates screening animal B-cell repertoires within days at low cost, increasing both rate and range of discovering antigen-specific antibodies from living organisms. Further, these techniques can be adapted to isolate cells based on virtually any secreted product.
ding2020.pdf

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