High-throughput microfluidic platforms applied to immunoassay-based diagnostics and environmental monitoring

The research presented in this thesis is motivated by the aim to employ microfluidic devices for high-throughput analysis. Two applications, in particular, have been explored: diagnostics and environmental sensing. Miniaturization and multiplexing of laboratory procedures leads to a decrease of the cost and response time of the test. Moreover miniaturization allows the system to become portable, enabling use in the field without bulky external equipment. This need is paving the way for innovative approaches and microfluidics can be a suitable substitute for standard bench top techniques. In order to be appealing, these new technologies should have the same or even better performances over the standard techniques, not only in terms of time and cost, but also quality of results. First, we developed a multilayer microfluidic platform for high-throughput immunoassay analysis. The device is composed of 384 units, each of them containing 4 mechanically induced trapping of molecular interactions (MITOMI) buttons, where the interaction of antibodies and antigens occurs. Therefore, the platform allows us to perform 1'536 tests, and we show that it can be useful for large scale screening in order to identify functional antibody pairs. However this system has two limitations: the experimental time (~10 hours) and the sensitivity (pM). In the second part, we presented another device modified in order to overcome the aforementioned drawbacks and to become more suitable for point of care applications. This new version of the device has 16 independent units, and the MITOMI buttons are patterned with femtoliter wells for digital analysis. We show how digital ELISA improves the sensitivity and allows us to detect down to fM concentrations in human serum. We also show how the combination of both digital and analog detection can broaden the dynamic range. A portable automatic controller has also been built to control the valves and the flow throughout the chip and a USB microscope has been exploited in order to measure the fluorescent signal. In the last part we present a different application for high-throughput microfluidic-based devices. A microfluidic device composed of 769 units has been developed for bacterial cell culture and environmental monitoring. Each unit can be considered as a pixel and the entire device as a biodisplay. Each unit contains a specific bacterial strain, which is deposited using microspotting techniques. We show how different bacterial strains can be cultured over time without contamination between pixels and how they can be monitored after induction. Cells can be spotted according to a specific pattern on the chip and express fluorescent protein reporters in the presence of specific molecules allowing an easy to interpret result of the test. For example, the image of a âskull and crossbonesâ appears if the analyzed sample is contaminated with arsenic. Overall, the presented work provides several microfluidic platforms for high-throughput analysis of clinical or environmental samples; these devices are useful tools for the study of a large number of proteins and molecular diagnostic applications and to characterize bacterial strains and environmental monitoring.

Integrating Gene Synthesis and MITOMI for Rapid Protein Engineering

Matthew Christopher Blackburn

The research described within this thesis is primarily motivated by two fields of science with reversed, yet complementary, approaches to addressing the same essential objective: manipulating and understanding the complex program of life. Whereas synthetic biology has to do with the bottom-up construction of living systems from a library of âpartsâ, systems biology takes a top-down, reverse-engineering analysis of the same processes within functioning cells to elucidate the crucial elements and interactions. Both perspectives have led to important contributions in learning how biological systems operate. Discoveries from these studies can have far-reaching implications, notably in medicine, manufacturing, energy, and the environment. One exciting outcome of the research efforts within synthetic biology is the ability to design genetic constructs encoding proteins with novel, optimized functions. Although advances have been made in predicting how a proteinâs amino acid sequence relates to its higher order structural form and behavior, experimental methods for protein engineering remain invaluable for evaluating computationally derived designs. Unfortunately, the design-build-test cycle for rational protein development is commonly a time, labor and resource intensive endeavor. This is primarily due to limitations in the ability to generate libraries of gene variants and bottlenecks in cell-based expression and quantitative screening of protein products. This thesis describes the development of a solid-phase gene assembly technique and its integration with microfluidic protein analysis to accelerate the prototyping of novel protein designs. The gene assembly method utilizes commodity-scale, chemically manufactured DNA, and operates without the need for ligase or restriction enzymes. As a bench top process amenable to scale up, the modularity of the assembly process allows the efficient and economical generation of expression-ready gene variants. We directly coupled this gene assembly pipeline to a microfluidic device to enable high-throughput, on-chip, cell-free protein expression, purification, and characterization. By circumventing molecular cloning and cell-based steps, the lag time between protein design and quantitative analysis was dramatically reduced. As a proof of concept, this protein engineering platform was applied towards the construction of over 400 artificial, engineered variants of C2H2 zinc finger (ZF) proteins. ZF protein domains are the most prevalent form of transcription factor (TF) in humans, and are found throughout the tree of life. They provide a convenient structure for refactoring due to their relatively small size and composability, and are an ideal model for exploring the biophysics of TF-DNA specificity. The ability to engineer ZF proteins makes them useful as programmable, DNA-targeting units for applications in biotechnology and synthetic biology. We demonstrate that although ZFPs can be readily engineered to recognize a particular DNA target, engineering the precise binding energy landscape remains a challenge. Additionally, we show that ZF-DNA binding affinity can be tuned independently of sequence specificity. Together, these results demonstrate the versatility of the coupled gene assembly and microfluidic analysis platform as a new tool for rational protein engineering.

EPFL2016

Functional single-cell hybridoma screening using droplet-based microfluidics

Christoph Merten

Monoclonal antibodies can specifically bind or even inhibit drug targets and have hence become the fastest growing class of human therapeutics. Although they can be screened for binding affinities at very high throughput using systems such as phage display, screening for functional properties (e.g., the inhibition of a drug target) is much more challenging. Typically these screens require the generation of immortalized hybridoma cells, as well as clonal expansion in microtiter plates over several weeks, and the number of clones that can be assayed is typically no more than a few thousand. We present here a microfluidic platform allowing the functional screening of up to 300,000 individual hybridoma cell clones within less than a day. This approach should also be applicable to nonimmortalized primary B-cells, as no cell proliferation is required: Individual cells are encapsulated into aqueous microdroplets and assayed directly for the release of antibodies inhibiting a drug target based on fluorescence. We used this system to perform a model screen for antibodies that inhibit angiotensin converting enzyme 1, a target for hypertension and congestive heart failure drugs. When cells expressing these antibodies were spiked into an unrelated hybridoma cell population in a ratio of 1∶10,000 we observed a 9,400-fold enrichment after fluorescence activated droplet sorting. A wide variance in antibody expression levels at the single-cell level within a single hybridoma line was observed and high expressors could be successfully sorted and recultivated.

2012

High-Throughput Screening of Enzymes by Retroviral Display Using Droplet-Based Microfluidics

Christoph Merten

During the last 25 years, display techniques such as phage display have become very powerful tools for protein engineering, especially for the selection of monoclonal antibodies. However, while this method is extremely efficient for affinity-based selections, its use for the selection and directed evolution of enzymes is still very restricted. Furthermore, phage display is not suited for the engineering of mammalian proteins that require posttranslational modifications such as glycosylation or membrane anchoring. To circumvent these limitations, we have developed a system in which structurally complex mammalian enzymes are displayed on the surface of retroviruses and encapsulated into droplets of a water-in-oil emulsion. These droplets are made and manipulated using microfluidic devices and each droplet serves as an independent reaction vessel. Compartmentalization of single retroviral particles in droplets allows efficient coupling of genotype and phenotype. Using tissue plasminogen activator (tPA) as a model enzyme, we show that, by monitoring the enzymatic reaction in each droplet (by fluorescence), quantitative measurement of tPA activity in the presence of different concentrations of the endogenous inhibitor PAI-1 can be made on-chip. On-chip fluorescence-activated droplet sorting allowed the processing of 500 samples per second and the specific collection of retroviruses displaying active wild-type tPA from a model library with a 1000-fold excess of retroviruses displaying a non-active control enzyme. During a single selection cycle, a more than 1300-fold enrichment of the active wild-type enzyme was demonstrated.

2010

Integrating Gene Synthesis and MITOMI for Rapid Protein Engineering

Matthew Christopher Blackburn

EPFL2016