Microfluidics for biology

We develop microfluidic devices to very precisely control small drops of one fluid in a second carrier fluid. The volume of each drop is between about a picoliter and a nanoliter. We use each drop as a carrier or reaction vessel. We are able to very precisely control these reaction vessels and can mix, add, divide and sort these drops at rates of 1 to 100 kHz. This enables us to carry out very large numbers of reactions in short times with very small quantities of reagents. Many of the applications of this technology are for the study of biology, and we, therefore, typically use aqueous drops in an inert carrier oil. We use the drops to do ultra-high-throughput screening of different biomolecules. We also use the drops to encapsulate single cells, enabling us to do very high-throughput studies of populations of cells, all at the level of single cells. We are exploring a wide range of potential applications from fundamental studies of evolution to single cell selection to applications in drug discovery and diagnostics. Several of the concepts have led to the formation of start-up companies based on this technology.

brouchon-julie-icon-fig1.jpg Microfluidics for single cell analysis of human antibody-secreting cells. Identification of new monoclonal antibodies able to target a specific epitope, for example on a virus capsid or on a cancer cell, is crucial for immuno-therapy development and vaccine design. The human body is an amazing source of antibodies: each human immune system contains >100 million different B cells, each producing many copies a unique antibody. Because each cell encodes only one type of antibody, an ability to test the activity of antibodies secreted by single cells will dramatically speed the identification of useful antibodies. To accomplish this, we have developed droplet microfluidic assay in which individual cells are encapsulated in 50pL micro-reactors containing all reagents to test the function of the secreted antibody. This assay allow us to isolate relevant B cells and to sequence the mRNA encoding the antibody. We are now increasing the accuracy and throughput of this assay by combining the single cell resolution of droplet microfluidics with the advanced optics and high-speed sorting of Fluorescence Activated Cell Sorting (FACS), to facilitate interrogation of a large repertoire of individual, non-immortalized cells. Using this technology we seek to identify monoclonal antibodies that could improve immuno-therapy in the fields of AIDS and cancer. Julie Brouchon

Drop-Seq: High-throughput Single-cell RNA-Seq: Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated dropIVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (10^3–10^6). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution. Naiwen Cui

Microfluidic droplets for rapid isolation of individual cells with desired activity: We develop droplet-microfluidics assays to identify and isolate individual cells of interest. We have used a droplet-based fluorescence-concentration assay to isolate, from an input comprised primarily of cells that secrete irrelevant antibody, a fraction of living cells comprised almost entirely of cells secreting antibody against antigen of interest. We are currently developing assays to identify and select individual, living T-cells that recognize and kill specific target cells. Our ultimate goal is to commercialize these microfluidic methods. John Heyman.

 Droplet-based digital PCR: Digital PCR uses partitioning to provide a digital readout of the number of DNA strands present in a sample. Here, digital PCR is performed in droplets to increase sensitivity and reduce reagent use. In particular, the droplet digital PCR technique will be applied to studying low abundance gene mutations. Huidan Zhang

Low-Concentration Quantification: Detection and quantification of low-concentration substances or microorganisms is difficult using conventional methods. We leverage the sequestration capabilities of droplets to create localized high concentrations in order to create an accurate measurement method. At present, we are able to perform accurate measurements in the attomolar range, and are working at increasing the sensitivity to improve this considerably. Jonathan Didier

Single-Cell Polymerase Chain Reaction (PCR): High-throughput single cell-based screening can benefit considerably from drop-based microfluidics. This technique addresses the need for lower cost, shorter time, and higher sensitivity by using water-in-oil emulsion droplets to compartmentalize reactants into picoliter volumes. In our current research work, we test the biological reactions in drops at all the levels of single cells, including single-cell PCR to detect genomic variation, single-cell RT-PCR to analyze gene expression, single-cell surface protein marker analysis, and long-term cell culture. This promising platform provides a powerful tool to study single cells, thus allows us to explore the heterogeneity in development and tumorigenesis. Huidan Zhang

Drops to Help Study the Brain: How do emotions, thoughts, or behaviors relate to physically observable properties of neurons? Fully answering this question may require first solving a technical challenge in studying the brain. How can we simultaneously measure the activity of billions (or more) individual neurons separated by distances from microns to centimeters? We apply micro- and milli-fluidic emulsions to this problem. In one project, we seek to engineer a DNA polymerase that could be used to record the voltage of many individual neurons simultaneously, a task that may be beyond the scope of optical, electronic, and magnetic recording methods. The recording medium would be DNA, and sequencing the DNA would read back the time series of neuronal pulses. We screen 100s-1000s of drops per second and confirm that we can detect fluorescent drops containing a single DNA polymerase gene, a step towards screening for a polymerase mutant that could record neuron activity into DNA. In another project, we encapsulate 0.1-1 mm clusters of human stem cells in drops that can gel and contain growth and differentiation factors. The stem cells differentiate into neurons, resulting in organoids, clusters of cells that resemble neocortical regions of the brain. It would be interesting to test in such organoids how a Ca2+ sensitive DNA polymerase records neuron activity by comparing recorded nucleotide sequences with microscopy recordings of Ca2+ dye fluorescence from the same organoid. Jesse Collins

Cell Assays in Alginate Hydrogel Microparticles: I develop the high throughput method to encapsulate different cellular systems into alginate hydrogel microparticles. The alginate hydrogel platform has high biocompatibility allowing continuous cell culturing and high adaptability with high speed sorting using FACS. I further apply this technology platform to address 3 biomedical challenges: engineering a model of transmitted M. tuberculosis for vaccine testing, T cell receptor identification for cancer immunotherapy and cortical neuron differentiation for drug screening. Xu Zhang

High throughput screening of active compounds using droplet microfluidics: We use droplet microfluidics to screen large libraries of compounds. Few picoliters of each compound is encapsulated in aqueous droplets and interacted with a target molecule or organism. By reducing the reagent volumes and reaction time, we can perform extremely high throughput screens at lower costs. ilke akartuna

Elucidating Lipid Domains Function by Combinatorial Screening of Protein-Lipids Interactions: Membrane proteins are believed to require certain lipid compositions in order to incorporate in the lipid membrane, yet to date, we have limited tools to determine with which lipid domains a protein associates with. By the use of a large liposome library and microfluidics we can now determine the lipid mixtures a protein associates with. Most of the membrane proteins we studied so far, have shown to be highly specific to the lipid content in the liposomes. Roy Ziblat

Next Generation Cell Purification Platform Using Acoustic Switching: Separating individual cells of interest from a mixed population of cells is of great importance and used in numerous applications in our daily life. For example, in planned parenthood; enabling sex selection of the child. Despite of the pure selective aspect; cell purification is a powerful tool in combination with i.e. adoptive cell therapy; optimizing the therapeutic outcome by selecting only the most vital and effective immune cells for cancer treatment. However, current standard methods such as fluorescence activated cell sorting (FACS) have their limitations in terms of biosafety, cost efficiency and the handling of specific types of samples. The focus of my work lies on the development of a microfluidics platform, which can overcome these limitations. I am particularly interested in using acoustics as a switching technology offering a universally usable, non-invasive and cost effective method to sort particles of interest, such as cancer cells, in a fluid stream of a microfluidic chip. Pascal Spink

Novel Biomedical Applications Using Surface Acoustic Wave Microfluidics. Just as smart phone apps speak to our interests and desires for customization; providing a range of tools aggregated to a singular device, microfluidics, has become the mobile app analog for the biotechnology field. Despite providing applications such as cell sorting, reagent mixing, diagnostics, and more; microfluidics still remains limited. To circumvent these limitations microfluidics will need to be combined with other technologies. One promising union is that of surface acoustic wave technology and microfluidics. Dubbed acoustic microfluidics, this growing technology spans a number of disciplines in an attempt to address the shortcomings of microfluidics. My focus is to investigate different applications that may be possible for acoustic microfluidics such as developing microfluidic sensors, high-speed cell sorting, and single cell analysis applications. Kirk Mutafopulos

High-throughput and high-sensitivity single-cell RNA sequencing. Single-cell RNA sequence (scRNA-seq) is becoming a powerful technology to profile cell types in single-cell level, because the profile of the heterogeneous cells would provide invaluable information for diagnosis and treatments. Current scRNA-seq suffers only 10% recovery of total transcripts from a single cell and the inefficiency to recover low-abundance transcripts, such as T cell receptor (TCR) from T cell. My research goal is to develop a high-throughput and high-sensitivity scRNA-seq to increase the recovery of total transcripts and identify low-abundance transcripts of interest. To achieve my goal, droplet microfluidics together with template-switching reverse transcription polymerase chain reaction (TS-RT-PCR) are developed to recover the TCR transcripts from a single T cell. This technology would provide higher throughput and sensitivity to profile low-abundance transcripts without lose the sequencing power to sequence whole transcriptome. Kuo-Chan Hung

Merging microfluidicis and metagenomics for novel high throughput virus discovery: This project is focused on developing the microfluidic platform for the detection, isolation, and complete sequencing of multiple types of viral genomes for use in standard molecular virology laboratories. For the experiments, total virion populations are isolated from the environmental sample, and deep sequencing and subsequent computational analysis detect the viral sequences present. We are aiming to isolate novel viruses of possible significance to human health and discover their whole genome sequences. Hee-Sun Han

Evolution of small viral populations: A human viral disease can emerge when a single bird flu virus is able to infect a human host; characterizing the propagation of viral lineages is difficult but crucial for understanding, predicting and preventing the outburst of viral epidemics. A standard approach to probe this genetic propagation is by mutation accumulation experiments, in which a single lineage is propagated by plating a population of viruses, randomly picking a single colony to propagate, and repeating this process over time. Unfortunately, it is difficult to extract from these experiments enough information for a reliable characterization of an evolving species due to the small number of replicates collected and to the insufficient duration of the experiments. Using microfluidics we can perform millions of parallel mutation accumulation experiments in drops. To accomplish this, I developed an “evolution chip”, which passes millions of populations per hour from one drop where they replicated in the previous generation to a new drop containing fresh nutrients and hosts, where they will replicate in the next generation. When we co-encapsulated viruses and host cells in the same drop, viruses readily replicate in drops and smaller populations yield a broader clonal distribution. After propagating lineages of small populations in a new environment for 4 generations we find a much broader distribution of adapted clones compared to conventional passaging of large populations. Assaf Rotem

High throughput Single Cell Labeling (Hi-SCL): Cells of identical genetic origin develop to perform a variety of different functions in our body. The proteins encoded in the genome are not all expressed at the same level in each cell, leading to this cell-to-cell variation. Characterizing the cellular modifications that lead to this variation is key to understanding cellular differentiation in our body. However, the measurements for such modifications are currently performed over a population of cells and it is impossible to use them to study variations between cells in the same population. To perform these assays at the resolution of a single-cell we developed a new technique, called High-throughput Single Cell Labeling (Hi-SCL), where each cell is uniquely labeled in a drop. After labeling, drops are pooled and the emulsion is broken to make a single sample, which undergoes an epigenomic assay and deep-sequencing. Upon sequencing, the labels attached to the cellular fragments are used to associate each fragment with the cell of origin, enabling deconvolution of the epigenomic data to 100 profiles of single cells. These profiles are sufficient to identify two cell types that were pre-mixed in the sample, as described in Figure 3. Thus, Hi-SCL allows a sample of many cells to be assayed simultaneously, yet analyzed at single-cell resolution. Assaf Rotem

Isolation of antigen-specific B-cells from primary cells. B-cells are immune cells that secrete antibody to fight pathogens in animals. The antibodies secreted by a single clone of B-cells, called monoclonal antibodies, are highly useful for both research and clinical applications. However, isolating B-cell clones that secrete antibodies that binds to a specific antigen using the traditional method of clonal expansion is inefficient and laborious. We develop a drop microfluidic platform to isolate B-cells based on the antibody they secrete. Using this platform we can isolate tens of different B-cells that secrete specific antibodies against a given antigen within one day. The antibody sequence can be subsequently retrieved by single-cell RT-PCR. This work will greatly improve the efficiency of antibody development and give better insights in antigen-antibody interactions. Ruihua Ding

 

Low-abundance mutant KRAS detection. Constitutively active KRAS mutations have been found to be involved in various processes of cancer development. The poor detectable level of mutant type KRAS maintaining around 20% results from several problems like high background of view field under fluorescence light, and taqman hydrolyzes. Mutation detection methods with higher sensitivity will increase the possibility of choosing the correct individual therapy, and trying to realize low-abundance mutant KRAS detection is our goal. We test a new fluorescent probe for gene mutation detection using drop-based digital PCR and design a series of mixing experiment between KRAS mutations and wild type, and the minimum detectable ratio comes to 0.01%. Lexiang Zhang

 

Drug susceptibility detection using double ddPCR. The goal of this project is to develop fast, sensitive and quantitative diagnostic methods for the identification of low-abundance bacterial pathogen in a bacterial infection where currently no diagnosis can be made. For the detection of low abundance mRNA to indicate drug susceptibility, we use a two rounds of drop-based digital PCR to perform a reliable detection. After the first round of drop-based digital PCR, pooled amplicons are re-encapsulated using a Poisson distribution to ensure <30% of droplets contain templates, are subjected to digital PCR and the bright droplets are counted by fluorescence detection. This method can expand the starting material without changing the original expression profile. Lexiang Zhang