Microfluidic Technologies for Synthetic Biology

Microfluidic technologies have shown powerful abilities for reducing cost, time, and labor, and at the same time, for increasing accuracy, throughput, and performance in the analysis of biological and biochemical samples compared with the conventional, macroscale instruments. Synthetic biology is an emerging field of biology and has drawn much attraction due to its potential to create novel, functional biological parts and systems for special purposes. Since it is believed that the development of synthetic biology can be accelerated through the use of microfluidic technology, in this review work we focus our discussion on the latest microfluidic technologies that can provide unprecedented means in synthetic biology for dynamic profiling of gene expression/regulation with high resolution, highly sensitive on-chip and off-chip detection of metabolites, and whole-cell analysis

Manipulation of magnetic microparticles in liquid phases for on-chip biomedical analysis methods

Magnetic microparticles and their application in bioanalytical microfluidic systems have been steadily gaining interest in recent years. This progress is fueled by the comparatively large and long range magnetic forces that can be obtained independently of the fluidic flow pattern. This thesis work presents new approaches for using magnetic microparticles in Lab-on-a-Chip systems. The first approach deals with the design of a magnetic droplet manipulation system and the second combines magnetic particle actuation with integrated optical detection. The applicability of both systems for miniaturized bioanalysis will be shown, demonstrating the potential of magnetic particle based Lab-on-a-Chip systems. The magnetic droplet manipulation system tackles the handling of small liquid volumes, which is an important task in miniaturized analytical systems. The careful adjustment of hydrophilic/hydrophobic surface properties and interfacial tensions leads to the design of a system, where small droplets are manipulated in a controllable fashion. The system's setup permits the direct implementation of bioanalytical protocols and two different procedures are in consequence examined. Based on a commercial laboratory kit, a platform for the on-chip extraction and purification of DNA will be designed. The miniaturized setup allows the user to capture and clean the DNA obtained from a raw cell sample containing as little as 10 cells, which is several orders of magnitude lower than known for macroscopic systems. A similar performance is observed for the colorimetric antibody detection further-on evaluated in the droplet manipulation system, where the small sample volumes permit a significant reduction of the reaction times. With the possibility of concentrating the biomolecules of interest on the particle surface, a sensitive and fast immunosorbent assay can be devised. A further miniaturization is examined in a CMOS system, which combines magnetic actuation and optical detection. The small dimensions of the actuation system allow the manipulation of single magnetic microparticles and the integration of Single Photon Avalanche Diodes (SPADs) enables their optical detection. An innovative detection algorithm permits hereby to distinguish the particles in size and, in combination with a velocity measurement, to evaluate the magnetic properties of the detected particles. In consequence, bioanalysis on a single magnetic particle using fluorescent measurements can be performed, as is shown by preliminary experiments

Development of microfluidic tools for cancer single cell encapsulation and proliferation in microdroplets

The role of microfluidics in liquid biopsy as a more capable solution to address the monitoring of cancer progression in patients is gaining increasing attention. One out of the several difficulties in can-cer monitoring resides with the offset between current cell growth techniques in vitro and the influence of the cellular microenvironment in proliferation. One application of microfluidics consists in the use of microdroplets to replicate the complex dynamic microenvironment that can accurately describe factual 3D models of cancer cell growth. The goal of this thesis was to develop a set of microfluidic-based tools that would enable the encapsulation, proliferation and monitoring of single cancer cells in micro-droplets. For this, a set of microfluidic devices made of PDMS for droplet generation and containment were developed by photo- and soft-lithography techniques, being tested and optimized to ensure single cancer cell encapsulation. After the optimization of the droplet generation parameters in terms of droplet size and long-term stability on-chip, the best performance conditions were selected for cell growth ex-periments. Different densities of MDA-MB-435S cancer cells were combined with various percentages of Matrigel®, an extracellular matrix supplement, to promote cell proliferation. As a result, it was possi-ble to monitor droplets with cancer cells for a range of 1-20 days. A preliminary observation showed signs of cell aggregation, indicating that the tools developed during the thesis have the potential of developing 3D cancer spheroids from cancer single cells

Continuous focusing and separation of microparticles with acoustic and magnetic fields

Microfluidics enables a diverse range of manipulations (e.g., focusing, separating, trapping, and enriching) of micrometer-sized objects, and has played an increasingly important role for applications that involve single cell biology and the detection and diagnosis of diseases. In microfluidic devices, methods that are commonly used to manipulate cells or particles include the utilization of hydrodynamic effects and externally applied field gradients that induce forces on cells/particles, such as electrical fields, optical fields, magnetic fields, and acoustic fields. However, these conventional methods often involve complex designs or strongly depend on the properties of the flow medium or the interaction between the fluid and fluidic channels, so this dissertation aims to propose and demonstrate novel and low-cost techniques to fabricate microfluidic devices to separate microparticles with different sizes, materials and shapes by the optimized acoustic and magnetic fields. The first method is to utilize acoustic bubble-enhanced pinched flow for microparticle separation; the microfluidic separation of magnetic particles with soft magnetic microstructures is achieved in the second part; the third technique separates and focuses microparticles by multiphase ferrofluid flows; the fourth method realizes the fabrication and integration of microscale permanent magnets for particle separation in microfluidics; magnetic separation of microparticles by shape is proposed in the fifth technique. The methods demonstrated in this dissertation not only address some of the limitations of conventional microdevices, but also provide simple and efficient method for the separation of microparticles and biological cells with different sizes, materials and shapes, and will benefit practical microfluidic platforms concerning micron sized particles/cells --Abstract, page iv

(R)evolution-on-a-chip

Billions of years of Darwinian evolution has led to the emergence of highly sophisticated and diverse life forms on Earth. Inspired by natural evolution, similar principles have been adopted in laboratory evolution for the fast optimization of genes and proteins for specific applications. In this review, we highlight state-of-the-art laboratory evolution strategies for protein engineering, with a special emphasis on in vitro strategies. We further describe how recent progress in microfluidic technology has allowed the generation and manipulation of artificial compartments for high-throughput laboratory evolution experiments. Expectations for the future are high: we foresee a revolution on-a-chip

Magnetic control of transport of particles and droplets in low Reynolds number shear flows

“Magnetic particles and droplets have been used in a wide range applications including biomedicine, biological analysis and chemical reaction. The manipulation of magnetic microparticles or microdroplets in microscale fluid environments is one of the most critical processes in the systems and platforms based on microfluidic technology. The conventional methods are based on magnetic forces to manipulate magnetic particles or droplets in a viscous fluid. In contrast to conventional magnetic separation method, several recent experimental and theoretical studies have demonstrated a different way to manipulate magnetic non-spherical particles by using a uniform magnetic field in the microchannel. However, the fundamental mechanism behind this method is not fully understood. In this research, we aims to use numerical and experimental methods to explore and investigate manipulation of microparticles and microdroplets in the microfluidics by using a uniform magnetic field. In the first part, rotational dynamics of elliptical particles in a simple shear flow is numerically investigated; then, lateral migration of elliptical particles in a plane Poiseuille flow is numerically investigated; The third part compares the rotational dynamics of paramagnetic and ferromagnetic elliptical particles particles in a simple shear flow; in the fourth part, particle-particle interactions and relative motions of a pair of magnetic elliptical particles in a quiescent flow are numerically investigated; magnetic separation of magnetic microdroplets by the uniform magnetic field is proposed in the fifth part. The methods demonstrated in this research not only develop numerical and experimental way to understand the fundamental transport properties of magnetic particles and droplets in microscale fluid environments, but also provide a simple and efficient method for the separation of microdroplets in microfluidic device, which can impact biomedical and bio-medicine technologies”--Abstract, page iv

DEVELOPMENT OF HIGH-THROUGHPUT IMPEDANCE SPECTROSCOPY-BASED MICROFLUIDIC PLATFORM FOR DETECTING AND ANALYZING CELLS AND PARTICLES

Impedance spectroscopy based microfluidics have the capability to characterize the dielectric properties of mediums, particles, cellular and sub-cellular contents in response to stimulating voltage signals over a frequency range. This label-free technology has broad ranges of applications in life sciences where there is a need for high-throughput, label-free, non-contact, and low-cost microsystems. To address these limitations, three innovative impedance spectroscopy microfluidic platforms have been developed and presented in this dissertation. The first platform was developed for detecting and characterizing the transverse position of a single cell flowing within a microfluidic channel using a single impedance spectroscopy electrode pair. Regardless of the cell separation methods used, identifying and quantifying the position of cells and particles within a microchannel are important, as these information indicate both the degree of separation as well as how many cells are separated into each position. Using a single pair of non-parallel surface microelectrodes, five different transverse positions of single cells flowing through a microfluidic channel were successfully identified at a throughput of more than 400 particles/s using the detected impedance peak height and width. The second platform utilizes the above technology to count and quantify cells flowing through multiple outlets of microfluidic cell separation systems. A single pair of step-shaped electrodes was developed by integrating five different electrode-to-electrode gaps within a single pair of electrodes. Using this platform, an overall misclassification error rate of only 1.85% was achieved. The result shows the technology’s capability in achieving efficient on-chip cell counting and quantification, regardless of the cell separation methods used, making it a promising on-chip, low-cost and label-free quantification method for cell and particle sorting and separation applications. The third platform was developed for counting cells and particles encapsulated in water-in-oil emulsion droplets using microfluidic based impedance spectroscopy systems. Impedance signal peak height and width were utilized to successfully quantify the number of cells encapsulated within a droplet, and was successfully applied for various cell types and growth media. In addition, the developed platform has been also successfully tested for identifying and discriminating filamentous fungal cell growth, where single fungal spores and filamentous fungi of different lengths could be discriminated inside droplets. Overall in this research, several impedance spectroscopy based microfluidic systems have been successfully developed to solve current limitations in technologies that need high-throughput, low-cost and label-free detection and characterization method for a broad range of cell/particle screening applications

Microdevices and Microsystems for Cell Manipulation

Microfabricated devices and systems capable of micromanipulation are well-suited for the manipulation of cells. These technologies are capable of a variety of functions, including cell trapping, cell sorting, cell culturing, and cell surgery, often at single-cell or sub-cellular resolution. These functionalities are achieved through a variety of mechanisms, including mechanical, electrical, magnetic, optical, and thermal forces. The operations that these microdevices and microsystems enable are relevant to many areas of biomedical research, including tissue engineering, cellular therapeutics, drug discovery, and diagnostics. This Special Issue will highlight recent advances in the field of cellular manipulation. Technologies capable of parallel single-cell manipulation are of special interest

Microfluidics for Biosensing and Diagnostics

Efforts to miniaturize sensing and diagnostic devices and to integrate multiple functions into one device have caused massive growth in the field of microfluidics and this integration is now recognized as an important feature of most new diagnostic approaches. These approaches have and continue to change the field of biosensing and diagnostics. In this Special Issue, we present a small collection of works describing microfluidics with applications in biosensing and diagnostics

Developing and Applying Microdroplet Co-Cultivation Technology for Elucidating Bacterial Interspecies Interactions in the Human Vaginal Microbiome

The role of the human vaginal microbiome (HVM) has gained increased recognition due to recent technological advancements that helped link community composition and women's health risks. However, the ecological roles of members of the HVM and microbe-microbe-host interactions remain unclear. Current approaches for investigating these mechanisms have been low-throughput, require large cultivation volumes and utilize chemically indistinct media diverging from the in vivo condition. Microdroplet-based co-cultivation is a new technology for overcoming these challenges by confining and performing sensitive assays at the nano-liter scale. This dissertation aims to: (i) develop new methods for elucidating interactions in the HVM through co-cultivation in microdroplets; (ii) test hypotheses that reduced iron limits the growth of L. iners and study interactions between lactobacilli in co-culture in laboratory media; and (iii) further extend the microdroplet technology for culturing vaginal bacteria in pooled cervicovaginal fluid (CVF). First, we adapted and extended a microdroplet co-cultivation technology pipeline to investigate the HVM and tested it using two pairwise model systems. In one case, Lactobacillus jensenii JV-V16, a lactic-acid bacterium, and Gardnerella vaginalis ATCC 49145, a putative pathogen, were cultured in microdroplets as pure cultures and co-cultures. Then, qPCR was used to quantify the bacteria in pooled microdroplets, and individual microdroplets were isolated and cells within each were plated on agar media. We demonstrated that L. jensenii inhibits G. vaginalis in microdroplets, which concurs with flask cultivation studies. We further demonstrated a second model system consisting of L. jensenii and another potential pathogen, Enterococcus faecalis. Our findings suggest that microdroplets can detect microbial interactions. Second, we determined the effects of iron on the growth of the most common lactobacilli, L. iners and L. crispatus, and investigated pairwise interactions between lactobacilli using laboratory media. We measured the growth of L. iners and L. crispatus in spent-media supplemented with Fe(II)SO4 or 2,2'-dipyridyl. Results show that higher concentrations of 2,2'-dipyridyl reduced the growth of L. iners, but not that of L. crispatus. We conducted serial dilutions on co-cultures of L. crispatus and L. iners, and L. crispatus and L. gasseri. As observed, one species became the most dominant in each co-culture. Spent-medium experiments indicated that no interference competition existed between these lactobacilli. Future investigation is needed to identify mechanisms for resource competition. Third, we extended our technology to cultivate vaginal bacteria in microdroplets using CVF and investigated whether L. crispatus, L. gasseri, or L. iners could grow in pooled CVF. We analyzed 16S rRNA genes of 49 vaginal samples collected from healthy reproductive-age women. Of them, 16 were selectively pooled to create L. crispatus (LC)-dominated CVF. Using microdroplets, we subsequently confined and axenically cultured L. crispatus, L. iners, and L. gasseri in LC-CVF. We observed that L. iners grew in LC-CVF at pH 7 but was killed at pH 4. Our results indicate how vaginal pH may influence L. iners growth. L. crispatus survived and L. gasseri decreased in viability in LC-CVF at pH 4. In conclusion, this dissertation demonstrates methods for investigating interactions in the HVM through co-cultivation in microdroplets in a high-throughput manner. We have also shown the utilization of small volumes of human samples in cultivating vaginal bacteria while simulating the natural condition of the vagina. Further extension of this approach and its future applications hold tremendous potential for elucidating microbial interactions and how they impact human health.PHDChemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/155166/1/corinemj_1.pd