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Despite the importance of droplet monodispersity, a universal methodology for high monodispersity droplet generation does not exist yet. We have recently demonstrated that unlike the conventional method of droplet generation, applying an identical pressure from a single source makes the microfluidic droplet system immune to the external fluctuations that originate from the imperfection of the flow source. In this work, we show that our method is universal and applicable to other common microfluidic devices and flow sources. We applied this method to flow-focusing and coflow devices that are commonly used for high-frequency microdroplet generation. In addition to the pressure pump, we used a syringe pump to show that our method is applicable to flow rate controllable systems as well. We compared the monodispersity of droplets formed by the conventional methods and the novel method explained in this work.

Variations in protein secretions between individual cells directly influences their functional activity in many applications. However, the inherent complexity associated with the accurate detection and quantification of secreted factors leaves these proteins vastly underutilized as phenotypic markers today. Here we report on the development, optimization, and application of novel, easy-to-use, droplet-based technologies for the detection and isolation of single viable immune cells based on their secreted factors. We first demonstrate that rapid compartmentalization significantly improves the resolution and accuracy of single-cell secretion studies. Using an ultra-high throughput microfluidic step emulsifier, we encapsulated 1,000 cells/second into uniformly sized 70 �m droplets, providing an environment to rapidly concentrate molecules and prevent intercellular diffusion. When combined with a commercially available cytokine capture reagent, droplet encapsulation reduced >99% of false positive signals generated during a screen of IL-2 production from primary T cells. Cells remained viable throughout analysis and could be sorted using standard fluorescence activated cell sorting, providing an easy method of isolating functionally unique clones for downstream analysis. Next, we detail the development of a novel lab-on-a-particle platform which enables the assessment of single cell secretions using only standard laboratory equipment and without the need for specialized knowledge of microfluidic device fabrication or operation. In this approach precisely engineered hydrogel nanovials are utilized as a surface to bind cells, partition them into uniform compartments, and capture their secreted products. The utility of this platform is initially highlighted by screening differences in the total antibody production levels of individual cells within a producer cell line (CHO-DP12), revealing that antibody titers can be increased by 50% through a single selection and expans

Immune-modulating biomaterials used to encapsulate cells and microtissue transplants can be engineered to dampen the immune reaction and increase treatment efficacy. Mucin-derived materials have gained attention for their ability to modulate macrophage and dendritic cell activity, and to trigger mild foreign body response when implanted in vivo. In this study, the potential of mucin hydrogels (Muc-gels) as cell-encapsulating materials is investigated. When placed in contact with blood, Muc-gels trigger significantly lower complement activation, compared to clinical grade alginate hydrogels. Muc-gel is a size-selective barrier strongly hindering the diffusion of molecules with a hydrodynamic radius larger than 6 nm such as immunoglobulins. Muc-gels support the growth of MIN6m9 insulin-secreting cells into islet-like organoids and the survival of primary human pancreatic islets, which maintained glucose responsiveness. Muc-gels can be shaped into microdroplets in which MIN6m9 cells or cell aggregates can be encapsulated without loss of viability. Microdroplet encapsulation will allow transplants to be easily injected and improve their survival by favoring mass transport through the capsule. The combination of strong immune modulatory properties, appropriate selective barrier profile, biocompatibility for embedded cells Muc-gels of particular value for microencapsulating cells or microtissues for transplantation.

Immune-modulating biomaterials used to encapsulate cells and microtissue transplants can be engineered to dampen the immune reaction and increase treatment efficacy. Mucin-derived materials have gained attention for their ability to modulate macrophage and dendritic cell activity, and to trigger mild foreign body response when implanted in vivo. In this study, the potential of mucin hydrogels (Muc-gels) as cell-encapsulating materials is investigated. When placed in contact with blood, Muc-gels trigger significantly lower complement activation, compared to clinical grade alginate hydrogels. Muc-gel is a size-selective barrier strongly hindering the diffusion of molecules with a hydrodynamic radius larger than 6 nm such as immunoglobulins. Muc-gels support the growth of MIN6m9 insulin-secreting cells into islet-like organoids and the survival of primary human pancreatic islets, which maintained glucose responsiveness. Muc-gels can be shaped into microdroplets in which MIN6m9 cells or cell aggregates can be encapsulated without loss of viability. Microdroplet encapsulation will allow transplants to be easily injected and improve their survival by favoring mass transport through the capsule. The combination of strong immune modulatory properties, appropriate selective barrier profile, biocompatibility for embedded cells Muc-gels of particular value for microencapsulating cells or microtissues for transplantation.

Immune-modulating biomaterials used to encapsulate cells and microtissue transplants can be engineered to dampen the immune reaction and increase treatment efficacy. Mucin-derived materials have gained attention for their ability to modulate macrophage and dendritic cell activity, and to trigger mild foreign body response when implanted in vivo. In this study, the potential of mucin hydrogels (Muc-gels) as cell-encapsulating materials is investigated. When placed in contact with blood, Muc-gels trigger significantly lower complement activation, compared to clinical grade alginate hydrogels. Muc-gel is a size-selective barrier strongly hindering the diffusion of molecules with a hydrodynamic radius larger than 6 nm such as immunoglobulins. Muc-gels support the growth of MIN6m9 insulin-secreting cells into islet-like organoids and the survival of primary human pancreatic islets, which maintained glucose responsiveness. Muc-gels can be shaped into microdroplets in which MIN6m9 cells or cell aggregates can be encapsulated without loss of viability. Microdroplet encapsulation will allow transplants to be easily injected and improve their survival by favoring mass transport through the capsule. The combination of strong immune modulatory properties, appropriate selective barrier profile, biocompatibility for embedded cells Muc-gels of particular value for microencapsulating cells or microtissues for transplantation.

Droplet microfluidic systems are becoming routine in advanced biochemical studies such as single cell gene expression, immuno profiling, precise nucleic acid quantification (dPCR) and particle synthesis. For all these applications, ensuring droplet monodispersity is critical to minimize the uncertainty due to droplet volume variation. Despite the wide usage of droplet-based microfluidic systems, the limit of monodispersity for droplet generation systems is still unknown. Here, we present an analytical approach that takes into account all the system dynamics and internal/external factors that disturb monodispersity. Interestingly, we are able to model the dynamics of a segmented two-phase flow system using a single-phase flow analogy, electron flow, in electrical circuits. We offer a unique solution and design guidelines to ensure ultra-monodisperse droplet generation. Our analytical conclusions are experimentally verified using a T-junction droplet generator. Equally importantly, we show the limiting experimental factors for reaching the theoretical maximum of monodispersity.

Immune-modulating biomaterials used to encapsulate cells and microtissue transplants can be engineered to dampen the immune reaction and increase treatment efficacy. Mucin-derived materials have gained attention for their ability to modulate macrophage and dendritic cell activity, and to trigger mild foreign body response when implanted in vivo. In this study, the potential of mucin hydrogels (Muc-gels) as cell-encapsulating materials is investigated. When placed in contact with blood, Muc-gels trigger significantly lower complement activation, compared to clinical grade alginate hydrogels. Muc-gel is a size-selective barrier strongly hindering the diffusion of molecules with a hydrodynamic radius larger than 6 nm such as immunoglobulins. Muc-gels support the growth of MIN6m9 insulin-secreting cells into islet-like organoids and the survival of primary human pancreatic islets, which maintained glucose responsiveness. Muc-gels can be shaped into microdroplets in which MIN6m9 cells or cell aggregates can be encapsulated without loss of viability. Microdroplet encapsulation will allow transplants to be easily injected and improve their survival by favoring mass transport through the capsule. The combination of strong immune modulatory properties, appropriate selective barrier profile, biocompatibility for embedded cells Muc-gels of particular value for microencapsulating cells or microtissues for transplantation.

The ability to detect and quantify viral nucleic acids is of paramount significance for diagnostics, therapeutics, and vaccine development. To combat globally spreading infectious diseases such as SARS-CoV-2, Ebola, and Zika, rapid and sensitive nucleic acid detection and quantification methods are in grave need for development. Compared with conventional tube-based assays, the digital format of bulk assays (e.g., digital PCR or digital LAMP) holds the potential of enabling absolute quantification of nucleic acids with no dependency on endogenous references or standards. Unlike hot-start digital PCR and LAMP, the implementation of digital quantification of the near-ambient-temperature assays using RPA and CRISPR may suffer from premature target amplification. Droplet microfluidics offers versatile yet controllable handling of samples and reagents, which provides confined compartments for enhanced assays kinetics and can be exploited to effectively circumvent premature amplification before droplet partition. In this thesis, we aim to develop effective yet facile microfluidic technology for reagent handling and manipulation with droplets. Firstly, we developed a Picoinjection Aided Digital reaction unLOCKing (PADLOCK) assay for ultrasensitive nucleic acid quantification. PADLOCK is realized through partitioning MgOAc-deprived RPA assays into picolitre water-in-oil droplets, the digital reaction unlocking is assisted by a downstream microfluidic injector loaded with the reaction initiator MgOAc. Hence, premature amplification in bulk is handily circumvented. To provide an in-depth view, we investigated three sensing schemes including CRISPR-Cas13a sensing, exo probe sensing, and EvaGreen sensing with PADLOCK-CRISPR and PADLOCK-EXO achieving sensitivity at single molecule level. We further demonstrated the utility of PADLOCK-CRISPR assay for HPV16 viral loads quantification in clinical samples, which generated 100% concordant results with qPCR. We believe the thorough i

We previously reported a new approach for micromanipulation and encapsulation of human stem cells using a droplet-based microfluidic device. This approach demonstrated the possibility of encapsulating and culturing difficult-to-preserve primary human hematopoietic stem cells using an engineered double-layered bead composed by an inner layer of alginate and an outer layer of Puramatrix. We also demonstrated the maintenance and expansion of Multiple Myeloma cells in this construction. Here, the presented microfluidic technique is applied to construct a 3D biomimetic model to recapitulate the human hematopoietic stem cell niche using double-layered hydrogel beads cultured in 10% FBS culture medium. In this model, the long-term maintenance of the number of cells and expansion of hHSCS encapsulated in the proposed structures was observed. Additionally, a phenotypic characterization of the human hematopoietic stem cells generated in the presented biomimetic model was performed in order to assess their long-term stemness maintenance. Results indicate that the ex vivo cultured human CD34+ cells from bone marrow were viable, maintained, and expanded over a time span of eight weeks. This novel long-term stem cell culture methodology could represent a novel breakthrough to improve Hematopoietic Progenitor cell Transplant (HPT) as well as a novel tool for further study of the biochemical and biophysical factors influencing stem cell behavior. This technology opens a myriad of new applications as a universal stem cell niche model potentially able to expand other types of cells.

Immune-modulating biomaterials used to encapsulate cells and microtissue transplants can be engineered to dampen the immune reaction and increase treatment efficacy. Mucin-derived materials have gained attention for their ability to modulate macrophage and dendritic cell activity, and to trigger mild foreign body response when implanted in vivo. In this study, the potential of mucin hydrogels (Muc-gels) as cell-encapsulating materials is investigated. When placed in contact with blood, Muc-gels trigger significantly lower complement activation, compared to clinical grade alginate hydrogels. Muc-gel is a size-selective barrier strongly hindering the diffusion of molecules with a hydrodynamic radius larger than 6 nm such as immunoglobulins. Muc-gels support the growth of MIN6m9 insulin-secreting cells into islet-like organoids and the survival of primary human pancreatic islets, which maintained glucose responsiveness. Muc-gels can be shaped into microdroplets in which MIN6m9 cells or cell aggregates can be encapsulated without loss of viability. Microdroplet encapsulation will allow transplants to be easily injected and improve their survival by favoring mass transport through the capsule. The combination of strong immune modulatory properties, appropriate selective barrier profile, biocompatibility for embedded cells Muc-gels of particular value for microencapsulating cells or microtissues for transplantation.

We previously reported a new approach for micromanipulation and encapsulation of human stem cells using a droplet-based microfluidic device We demonstrated the possibility of encapsulating and culturing difficult-to-preserve primary human hematopoietic stem cells using an engineered double layered bead composed by an inner layer of alginate and an outer layer of puramatrix constructed using a soft technology without the use of any external force. In this work, we use this micro manipulation technique to build a 3D scaffold as a biomimetic model to recapitulate the niche of patient-derived multiple myeloma cells (MM cell) using a multilayered 3D tissue scaffold constructed in a microfluidic device and cultured in 10% FBS culture medium. In the current study, we included the use of this biomimetic model comprising supporting human Mesenchymal stem cells to show the mid-term survival of MM cells in the proposed structures. We found that the generated microniches were suitable for the maintenance of MM cells with and without supporting cells. Additionally, cultured MM cells in droplets were exposed to both Bortezomib and Lenalidomide to test their toxicity in the cultured patient derived cells. Results indicate that the maintained MM cells were consistently responding to the applied medication, opening a wide field of possibilities to use the presented micro device as an ex vivo platform for drug screening.

Microdroplet formation by way of microfluidics has attracted great interest in the previous decade. This dissertation details the design, fabrication, development, and use of a low-cost lab-on-a-chip active on-demand microdroplet generator and cell encapsulation system. This system incorporates microfluidics, optics, and electronics into a single system where you can create tune-able microdroplets with precision and in isolation from other unwanted droplets.The polydimethylsiloxane (PDMS) microfluidic on-demand microdroplet generator system creates a microdroplet by overcoming the interfacial tension of an aqueous immiscible fluid in stable laminar flow with an oil-based continuous fluid by way of hydrodynamic forces created in the continuous fluid from a millisecond-scale impulse originating from a PZT-based acoustic actuator. The system created offers a significant increase in speed over existing tuneable on-demand microdroplet generators as well as increased flexibility in fluid velocities in the system.Additional optics to detect the location and velocities of fluorescently-labeled 15um beads, representing cells, are used in conjunction with the developed system to successfully capture beads in droplets as they flow through microfluidic cavities with single-bead purity much higher than Poisson-statistic-bound systems employed today.The development of tuneable and programmable microdroplet generators and accurate single-cell encapsulation can be of great use to many emerging biological assays, techniques, and applications such as droplet digital PCR (ddPCR), single-cell incubators, and drug discovery.

Microdroplet formation by way of microfluidics has attracted great interest in the previous decade. This dissertation details the design, fabrication, development, and use of a low-cost lab-on-a-chip active on-demand microdroplet generator and cell encapsulation system. This system incorporates microfluidics, optics, and electronics into a single system where you can create tune-able microdroplets with precision and in isolation from other unwanted droplets.The polydimethylsiloxane (PDMS) microfluidic on-demand microdroplet generator system creates a microdroplet by overcoming the interfacial tension of an aqueous immiscible fluid in stable laminar flow with an oil-based continuous fluid by way of hydrodynamic forces created in the continuous fluid from a millisecond-scale impulse originating from a PZT-based acoustic actuator. The system created offers a significant increase in speed over existing tuneable on-demand microdroplet generators as well as increased flexibility in fluid velocities in the system.Additional optics to detect the location and velocities of fluorescently-labeled 15um beads, representing cells, are used in conjunction with the developed system to successfully capture beads in droplets as they flow through microfluidic cavities with single-bead purity much higher than Poisson-statistic-bound systems employed today.The development of tuneable and programmable microdroplet generators and accurate single-cell encapsulation can be of great use to many emerging biological assays, techniques, and applications such as droplet digital PCR (ddPCR), single-cell incubators, and drug discovery.

Droplet microfluidics generates and manipulates microdroplets in microfluidic devices at high manufacturing efficiency and controllability. Microdroplets have proven effective in biomedical applications such as single-cell analysis, DNA sequencing, protein partitioning and drug delivery. Conventionally, a series of aqueous microdroplets containing biosamples is generated and controlled in an oil environment. One of the critical challenges in this system is that recovery of the aqueous samples from the oil phase is very difficult and often requires expensive and cumbersome post-processing. Also, the low Reynolds (Re) number characteristic of this system results in low throughput of droplet generation. To circumvent challenges and fully utilize microdroplets for practical clinical applications, this research aims to unpack the fundamental physics that governs droplet generation in oil-free systems including an aqueous two-phase system (ATPS) and a high inertial liquid-gas system., 2019, Includes bibliography., Degree granted: Dissertation (Ph.D.)--Florida Atlantic University, 2019., Collection: FAU Electronic Theses and Dissertations Collection

The conformation and mechanism of proteins that degrade and bind RNA, which has provided key insights into post-transcriptional gene regulation, is explored here. During the twentieth century's last decades, the characterization of ribonucleases and RNA binding domains revealed the diversity of their reaction mechanisms and modes of RNA recognition, and the bases of protein folding, substrate specificity and binding affinity. More recent research showed how these domains combine through oligomerization or genetic recombination to create larger proteins with highly specific and readily programmable ribonucleolytic activity. In the last 15 years, the study of the capacity of proteins, usually disordered, to pool RNAs into discrete, non-aqueous microdroplets to facilitate their transport, modification and degradation - analogous to cowboys herding cattle - has advanced our comprehension of gene expression. Finally, the current uses of RNA binding proteins and the future applications of protein/RNA microdroplets are highlighted.

This thesis presents a novel method to fabricate uniform metal microspheres by a custom-made programmable apparatus via controlled capillary. The programmable apparatus consisted of a programmable module, a robotic mechanical arm and a droplet-formation module comprised of an aluminum cylindrical core that encompassed (1) a material reservoir, (2) a buffer compartment (buffer part) with an embedded heating element, and (3) a cooling compartment (cooling part) with a meter-long glass tube filled with glycerol: DI water solution. The internal heating element could reach a temperature up to 250°C for accommodating a wide range of materials with different melting temperatures, including but not limited to eutectic metal alloys. The size of metal microsphere was determined by a set of programmable parameters executed by the programmable module, to control the air pressure exerted at the orifice of capillary via two independent electronic solenoid valves. The air pressure inside the capillary manipulated the handling time of the molten metal alloy. The molten metal alloy in liquid form was forced out of the capillary under specific air pressure. Molten metal alloy liquid was then segregated to form metal microdroplets when they emerged from the capillary. These microdroplets subsequently entered the meter-long glass tube filled with glycerol and water solution in which microdroplets solidified to form metal microspheres. Bismuth (Bi)-based 117 alloy which forms molten metal at melting temperature of 46.5°C was used to fabricate the metal alloy microspheres. The diameter of the fabricated microspheres ranged from 30 μm to greater than 500 μm. This fabrication technique offers a new prototyping method to produce metal microspheres with good uniformity and yield, with a much lower price tag and reasonable production throughput. Such method offers not only flexibility in production quantity but also versatility in material compatibility and on-demand production. The productio

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing >50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing >50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

In this work, a novel structure, high-aspect-ratio-induced (HIDS) structure, is developed for the micro-droplet generation based on self-breakup mechanism. Comparing with the traditional microfluidic-based methods, there are two main advantages. Firstly, in HIDS only one input is required for driving the droplet generation process, and hence, the design and fabrication is much more concise and cheap. Secondly, the droplet size is insensitive to the flow rates so that it has a high turbulence tolerance, and hence, is suitable for large-scale parallelization. However, for a single HIDS channel the maximum generation frequency is limited at around 30 Hz because of the polyemulsion. Therefore, trenches are utilized to optimize this structure. Upon the careful design of the structures of trenches and leading rails, the inlet channel with high aspect ratio can successfully lead the dispersed phase into individual droplets at relatively high frequency. This ensures its excellent suitability in the applications like integrated microsystems. Volume fraction of the emulsion droplets in the continuous phase can also be controlled simply through the adjustment of the applied pressure on the continuous phase. A very high volume fraction can be achieved, which can save a large amount of continuous phase compared to the traditional methods like flow focusing. Experiments also show the droplets size still weakly depends on the flow rate even when it’s relatively high. It is also proved that structure with trenches owns a lower sensitivity of the droplet size to the flow rate, compared to that without trenches, which is a significant advantage to enhance the stability of droplets generation. The droplet formation process is simulated and it is found that the flow rate of the continuous phase around the trenches is extremely low, based on which we propose a geometric theoretical model. This model predicts the droplet size in a great agreement with our experimental data. I believe thi

In this work, a novel structure, high-aspect-ratio-induced (HIDS) structure, is developed for the micro-droplet generation based on self-breakup mechanism. Comparing with the traditional microfluidic-based methods, there are two main advantages. Firstly, in HIDS only one input is required for driving the droplet generation process, and hence, the design and fabrication is much more concise and cheap. Secondly, the droplet size is insensitive to the flow rates so that it has a high turbulence tolerance, and hence, is suitable for large-scale parallelization. However, for a single HIDS channel the maximum generation frequency is limited at around 30 Hz because of the polyemulsion. Therefore, trenches are utilized to optimize this structure. Upon the careful design of the structures of trenches and leading rails, the inlet channel with high aspect ratio can successfully lead the dispersed phase into individual droplets at relatively high frequency. This ensures its excellent suitability in the applications like integrated microsystems. Volume fraction of the emulsion droplets in the continuous phase can also be controlled simply through the adjustment of the applied pressure on the continuous phase. A very high volume fraction can be achieved, which can save a large amount of continuous phase compared to the traditional methods like flow focusing. Experiments also show the droplets size still weakly depends on the flow rate even when it’s relatively high. It is also proved that structure with trenches owns a lower sensitivity of the droplet size to the flow rate, compared to that without trenches, which is a significant advantage to enhance the stability of droplets generation. The droplet formation process is simulated and it is found that the flow rate of the continuous phase around the trenches is extremely low, based on which we propose a geometric theoretical model. This model predicts the droplet size in a great agreement with our experimental data. I believe thi

In this work, a novel structure, high-aspect-ratio-induced (HIDS) structure, is developed for the micro-droplet generation based on self-breakup mechanism. Comparing with the traditional microfluidic-based methods, there are two main advantages. Firstly, in HIDS only one input is required for driving the droplet generation process, and hence, the design and fabrication is much more concise and cheap. Secondly, the droplet size is insensitive to the flow rates so that it has a high turbulence tolerance, and hence, is suitable for large-scale parallelization. However, for a single HIDS channel the maximum generation frequency is limited at around 30 Hz because of the polyemulsion. Therefore, trenches are utilized to optimize this structure. Upon the careful design of the structures of trenches and leading rails, the inlet channel with high aspect ratio can successfully lead the dispersed phase into individual droplets at relatively high frequency. This ensures its excellent suitability in the applications like integrated microsystems. Volume fraction of the emulsion droplets in the continuous phase can also be controlled simply through the adjustment of the applied pressure on the continuous phase. A very high volume fraction can be achieved, which can save a large amount of continuous phase compared to the traditional methods like flow focusing. Experiments also show the droplets size still weakly depends on the flow rate even when it’s relatively high. It is also proved that structure with trenches owns a lower sensitivity of the droplet size to the flow rate, compared to that without trenches, which is a significant advantage to enhance the stability of droplets generation. The droplet formation process is simulated and it is found that the flow rate of the continuous phase around the trenches is extremely low, based on which we propose a geometric theoretical model. This model predicts the droplet size in a great agreement with our experimental data. I believe thi

Microfluidic chips allow researchers to perform complex chemical and biological assays with less volume and in less time than comparable benchtop experimental formats. To date, many microfluidic chip designs have been developed that replicate macro-scale experimental techniques while increasing sensitivity and measurements density. Biochemical assays investigating the function of biological macromolecules have particularly benefitted from the ability to perform complex, micro-scale experiments that use expensive, often precious reagents in microfluidic devices; structural investigations of biological macromolecules also have benefitted from microfluidic approaches. For my doctoral work, I designed and fabricated microfluidic chips to carry out functional investigations of macromolecular interactions and for X-ray diffraction studies of microcrystalline samples. In the first phase of this work, microdroplet-based microfluidic devices were used to study the reaction kinetics of the protein DnaA with various DNA substrates. Despite successful device fabrication and calibration, attempts to study the rates at which DnaA can extend a single-stranded DNA substrate ultimately were unsuccessful due to technical issues involving poor sample behavior in-chip. These challenges led me to focus on a different problem; namely, how to deliver microcrystals to microfocus synchrotron and X-ray free electron laser beam sources. Using the knowledge of microfluidic technologies gained through designing microdroplet-based chips, I helped design a PDMS-based flow trap array for microcrystal capture and positioning, and I independently designed, fabricated, and tested a silicon nitride-based device for microcrystal harvesting and delivery to X-ray beam line endstations. Both chip designs provide a powerful new means for allowing data collection from challenging structural targets.

Microfluidics is a highly interdisciplinary research field which manipulates, controls and analyzes fluids in micro-scale for physical and bio/chemical applications. In this thesis, several aspects of fluid manipulations in micro-scale were studied, discussed and employed for demonstrations of practical utilizations. To begin with, mixing in continuous flow microfluidic was raised and investigated. A simple method for mixing actuation based on magnetism was proposed and realized via integration of magnetically functionalized micropillar arrays inside the microfluidic channel. With such technique, microfluidic mixing could be swiftly switched on and off via simple application or retraction of the magnetic field. Thereafter, in Chapter 3 we mainly focused on how to establish stable while tunable concentration gradients inside microfluidic network using a simple design. The proposed scheme could also be modified with on-chip pneumatic actuated valve to realize pulsatile/temporal concentration gradients simultaneously in ten microfluidic branches. We further applied such methodology to obtain roughness gradients on Polydimethylsiloxane (PDMS) surface via combinations of the microfluidic network and photo-polymerizations. The obtained materials were utilized in parallel cell culture to figure out the relationship between substrate morphologies and the cell behaviors. In the second part of this work, we emphasized on manipulations on microdroplets inside the microfluidic channel and explored related applications in bio/chemical aspects. Firstly, microdroplet-based microfluidic universal logic gates were successfully demonstrated via liquid-electronic hybrid divider. For application based on such novel scheme of controllable droplet generation, on-demand chemical reaction within paired microdroplets was presented using IF logic gate. Followed by this, another important operation of microdroplet - splitting - was investigated. Addition lateral continuous flow was applied at

Microfluidics is a highly interdisciplinary research field which manipulates, controls and analyzes fluids in micro-scale for physical and bio/chemical applications. In this thesis, several aspects of fluid manipulations in micro-scale were studied, discussed and employed for demonstrations of practical utilizations. To begin with, mixing in continuous flow microfluidic was raised and investigated. A simple method for mixing actuation based on magnetism was proposed and realized via integration of magnetically functionalized micropillar arrays inside the microfluidic channel. With such technique, microfluidic mixing could be swiftly switched on and off via simple application or retraction of the magnetic field. Thereafter, in Chapter 3 we mainly focused on how to establish stable while tunable concentration gradients inside microfluidic network using a simple design. The proposed scheme could also be modified with on-chip pneumatic actuated valve to realize pulsatile/temporal concentration gradients simultaneously in ten microfluidic branches. We further applied such methodology to obtain roughness gradients on Polydimethylsiloxane (PDMS) surface via combinations of the microfluidic network and photo-polymerizations. The obtained materials were utilized in parallel cell culture to figure out the relationship between substrate morphologies and the cell behaviors. In the second part of this work, we emphasized on manipulations on microdroplets inside the microfluidic channel and explored related applications in bio/chemical aspects. Firstly, microdroplet-based microfluidic universal logic gates were successfully demonstrated via liquid-electronic hybrid divider. For application based on such novel scheme of controllable droplet generation, on-demand chemical reaction within paired microdroplets was presented using IF logic gate. Followed by this, another important operation of microdroplet - splitting - was investigated. Addition lateral continuous flow was applied at

Microfluidic chips allow researchers to perform complex chemical and biological assays with less volume and in less time than comparable benchtop experimental formats. To date, many microfluidic chip designs have been developed that replicate macro-scale experimental techniques while increasing sensitivity and measurements density. Biochemical assays investigating the function of biological macromolecules have particularly benefitted from the ability to perform complex, micro-scale experiments that use expensive, often precious reagents in microfluidic devices; structural investigations of biological macromolecules also have benefitted from microfluidic approaches. For my doctoral work, I designed and fabricated microfluidic chips to carry out functional investigations of macromolecular interactions and for X-ray diffraction studies of microcrystalline samples. In the first phase of this work, microdroplet-based microfluidic devices were used to study the reaction kinetics of the protein DnaA with various DNA substrates. Despite successful device fabrication and calibration, attempts to study the rates at which DnaA can extend a single-stranded DNA substrate ultimately were unsuccessful due to technical issues involving poor sample behavior in-chip. These challenges led me to focus on a different problem; namely, how to deliver microcrystals to microfocus synchrotron and X-ray free electron laser beam sources. Using the knowledge of microfluidic technologies gained through designing microdroplet-based chips, I helped design a PDMS-based flow trap array for microcrystal capture and positioning, and I independently designed, fabricated, and tested a silicon nitride-based device for microcrystal harvesting and delivery to X-ray beam line endstations. Both chip designs provide a powerful new means for allowing data collection from challenging structural targets.

Microfluidics is a highly interdisciplinary research field which manipulates, controls and analyzes fluids in micro-scale for physical and bio/chemical applications. In this thesis, several aspects of fluid manipulations in micro-scale were studied, discussed and employed for demonstrations of practical utilizations. To begin with, mixing in continuous flow microfluidic was raised and investigated. A simple method for mixing actuation based on magnetism was proposed and realized via integration of magnetically functionalized micropillar arrays inside the microfluidic channel. With such technique, microfluidic mixing could be swiftly switched on and off via simple application or retraction of the magnetic field. Thereafter, in Chapter 3 we mainly focused on how to establish stable while tunable concentration gradients inside microfluidic network using a simple design. The proposed scheme could also be modified with on-chip pneumatic actuated valve to realize pulsatile/temporal concentration gradients simultaneously in ten microfluidic branches. We further applied such methodology to obtain roughness gradients on Polydimethylsiloxane (PDMS) surface via combinations of the microfluidic network and photo-polymerizations. The obtained materials were utilized in parallel cell culture to figure out the relationship between substrate morphologies and the cell behaviors. In the second part of this work, we emphasized on manipulations on microdroplets inside the microfluidic channel and explored related applications in bio/chemical aspects. Firstly, microdroplet-based microfluidic universal logic gates were successfully demonstrated via liquid-electronic hybrid divider. For application based on such novel scheme of controllable droplet generation, on-demand chemical reaction within paired microdroplets was presented using IF logic gate. Followed by this, another important operation of microdroplet - splitting - was investigated. Addition lateral continuous flow was applied at

A high-throughput method for single cell screening by microfluidic droplet sorting is applied to a whole-genome mutated yeast cell library yielding improved production hosts of secreted industrial enzymes. The sorting method is validated by enriching a yeast strain 14 times based on its a-amylase production, close to the theoretical maximum enrichment. Furthermore, a 105 member yeast cell library is screened yielding a clone with a more than 2-fold increase in a-amylase production. The increase in enzyme production results from an improvement of the cellular functions of the production host in contrast to previous droplet-based directed evolution that has focused on improving enzyme protein structure. In the workflow presented, enzyme producing single cells are encapsulated in 20 pL droplets with a fluorogenic reporter substrate. The coupling of a desired phenotype (secreted enzyme concentration) with the genotype (contained in the cell) inside a droplet enables selection of single cells with improved enzyme production capacity by droplet sorting. The platform has a throughput over 300 times higher than that of the current industry standard, an automated microtiter plate screening system. At the same time, reagent consumption for a screening experiment is decreased a million fold, greatly reducing the costs of evolutionary engineering of production strains., QC 20140214

The controlled movement of a chemical container by the light-activated expulsion of a chemical fuel, named here "photo-chemopropulsion", is an exciting new development in the array of mechanisms employed for controlling the movement of microvehicles, herein represented by lipid-based microdroplets. This "chemopropulsion" effect can be switched on and off, and is fully reversible.

The controlled movement of a chemical container by the light-activated expulsion of a chemical fuel, named here "photo-chemopropulsion", is an exciting new development in the array of mechanisms employed for controlling the movement of microvehicles, herein represented by lipid-based microdroplets. This "chemopropulsion" effect can be switched on and off, and is fully reversible.

The controlled movement of a chemical container by the light-activated expulsion of a chemical fuel, named here "photo-chemopropulsion", is an exciting new development in the array of mechanisms employed for controlling the movement of microvehicles, herein represented by lipid-based microdroplets. This "chemopropulsion" effect can be switched on and off, and is fully reversible.

This thesis mainly focuses on two topics: the electrorheological effect from dipolar water filaments and multiple droplet reaction and three dimension temperature mapping based on microfluidic method. In the first section, the field induced water filament is studied both theoretically and experimentally. Based on microscopic mechanism of the GER effect, a phenomenological model is raised to describe spontaneous and field-induced filaments formation at urea/water-silicone oil interface, giving the prediction that the field induced molecular filaments of water could become macroscopic in length, like mm. We have turned to use the molecular dynamics (MD) simulation for further examination. The simulated results have confirmed the existence of such filaments and resulted molecular electrorheological effect. Experiments have been conducted with parallel-plate channel for different systems: water-silicone oil and water decane. The measured results indicate that a pressure difference would occur under the applied electric field for water-silicone oil case while no such effect for water-decane case. The observed electrorheological effect gives a convincing demonstration of formation of water molecular filaments. An improved cartridge droplet generation device is introduced in the second section. The droplet volume, distance, number and droplet sequence can be precisely controlled by a programmed controller. Our experiments of droplet screening and aggressive droplet reaction imply that we have provided a simple model to develop a computer-aided sample-in, result-out system that utilizes nano- to picoliter droplets for low reagent consumption and high throughput. Moreover, a new functional material, RAP, has been developed for 3D temperature monitoring of microfluidic chip. Outstanding solvent, thermal stability and photo stability of this material permits accurate and rapid temperature measurement of local temperature surrounding a nanoliter droplet trapped inside a RAP cha

This thesis mainly focuses on two topics: the electrorheological effect from dipolar water filaments and multiple droplet reaction and three dimension temperature mapping based on microfluidic method. In the first section, the field induced water filament is studied both theoretically and experimentally. Based on microscopic mechanism of the GER effect, a phenomenological model is raised to describe spontaneous and field-induced filaments formation at urea/water-silicone oil interface, giving the prediction that the field induced molecular filaments of water could become macroscopic in length, like mm. We have turned to use the molecular dynamics (MD) simulation for further examination. The simulated results have confirmed the existence of such filaments and resulted molecular electrorheological effect. Experiments have been conducted with parallel-plate channel for different systems: water-silicone oil and water decane. The measured results indicate that a pressure difference would occur under the applied electric field for water-silicone oil case while no such effect for water-decane case. The observed electrorheological effect gives a convincing demonstration of formation of water molecular filaments. An improved cartridge droplet generation device is introduced in the second section. The droplet volume, distance, number and droplet sequence can be precisely controlled by a programmed controller. Our experiments of droplet screening and aggressive droplet reaction imply that we have provided a simple model to develop a computer-aided sample-in, result-out system that utilizes nano- to picoliter droplets for low reagent consumption and high throughput. Moreover, a new functional material, RAP, has been developed for 3D temperature monitoring of microfluidic chip. Outstanding solvent, thermal stability and photo stability of this material permits accurate and rapid temperature measurement of local temperature surrounding a nanoliter droplet trapped inside a RAP cha

This thesis mainly focuses on two topics: the electrorheological effect from dipolar water filaments and multiple droplet reaction and three dimension temperature mapping based on microfluidic method. In the first section, the field induced water filament is studied both theoretically and experimentally. Based on microscopic mechanism of the GER effect, a phenomenological model is raised to describe spontaneous and field-induced filaments formation at urea/water-silicone oil interface, giving the prediction that the field induced molecular filaments of water could become macroscopic in length, like mm. We have turned to use the molecular dynamics (MD) simulation for further examination. The simulated results have confirmed the existence of such filaments and resulted molecular electrorheological effect. Experiments have been conducted with parallel-plate channel for different systems: water-silicone oil and water decane. The measured results indicate that a pressure difference would occur under the applied electric field for water-silicone oil case while no such effect for water-decane case. The observed electrorheological effect gives a convincing demonstration of formation of water molecular filaments. An improved cartridge droplet generation device is introduced in the second section. The droplet volume, distance, number and droplet sequence can be precisely controlled by a programmed controller. Our experiments of droplet screening and aggressive droplet reaction imply that we have provided a simple model to develop a computer-aided sample-in, result-out system that utilizes nano- to picoliter droplets for low reagent consumption and high throughput. Moreover, a new functional material, RAP, has been developed for 3D temperature monitoring of microfluidic chip. Outstanding solvent, thermal stability and photo stability of this material permits accurate and rapid temperature measurement of local temperature surrounding a nanoliter droplet trapped inside a RAP cha

Characterization of polymers during polymerization is a challenge. Especially difficult is to study kinetics of gel formation inside tiny droplets. In many cases, viscosity of these fluids increases exponentially fast. In this paper, the rheological properties of fluids were evaluated by examining the behavior of magnetic nanorods in rotating magnetic field. We proposed a theory describing the rotation of a magnetic nanorod in a fluid when its viscosity increases with time exponentially fast. To confirm the theory, we studied the rheology of poly(2-hydroxyethyl-methacrylate) (HEMA)-based hydrogel undergoing photopolymerization and cross-linking with diethylene dlycol dimethacylate (DEGDMA). We showed that magnetic rotational spectroscopy provides rich physico-chemical information about the gelation process. . . . . Remarkably, one can analyze not only the liquid state of the polymer, but the gel as well. Since the probing nanorods are measured in nanometers, this method can be used for the in vivo mapping of the rheological properties of biofluids and polymers on a microscopic level at short time intervals when other methods fail short., The original document contains color images. Prepared in collaboration with the School of Materials Science and Engineering, Clemson University, Clemson, SC. Published in Langmuir, v28 n26 p10064-10071, 2012.

Iodineradical reactions insingle free-falling microdroplets of iodododecane, initiated using UV laser photolysis, are probed using Raman spectroscopy. Stimulated Raman spectra, with 532 nm laser excitation, are recorded at varying time delays from the UV pulse. I atom recombination reactions lead to I2 that changes the optical properties of the microdroplet ultimately quenching the Raman signal. This quenching is observed over ∼10 ns, which is about the time resolution of the two-laser experiment. Although the kinetics are too rapid to be measured in current laser configuration, it demonstrates that radical kinetics can be followed insinglemicrodroplets.

Iodineradical reactions insingle free-falling microdroplets of iodododecane, initiated using UV laser photolysis, are probed using Raman spectroscopy. Stimulated Raman spectra, with 532 nm laser excitation, are recorded at varying time delays from the UV pulse. I atom recombination reactions lead to I2 that changes the optical properties of the microdroplet ultimately quenching the Raman signal. This quenching is observed over ∼10 ns, which is about the time resolution of the two-laser experiment. Although the kinetics are too rapid to be measured in current laser configuration, it demonstrates that radical kinetics can be followed insinglemicrodroplets.

Iodineradical reactions insingle free-falling microdroplets of iodododecane, initiated using UV laser photolysis, are probed using Raman spectroscopy. Stimulated Raman spectra, with 532 nm laser excitation, are recorded at varying time delays from the UV pulse. I atom recombination reactions lead to I2 that changes the optical properties of the microdroplet ultimately quenching the Raman signal. This quenching is observed over ∼10 ns, which is about the time resolution of the two-laser experiment. Although the kinetics are too rapid to be measured in current laser configuration, it demonstrates that radical kinetics can be followed insinglemicrodroplets.

Iodineradical reactions insingle free-falling microdroplets of iodododecane, initiated using UV laser photolysis, are probed using Raman spectroscopy. Stimulated Raman spectra, with 532 nm laser excitation, are recorded at varying time delays from the UV pulse. I atom recombination reactions lead to I2 that changes the optical properties of the microdroplet ultimately quenching the Raman signal. This quenching is observed over ∼10 ns, which is about the time resolution of the two-laser experiment. Although the kinetics are too rapid to be measured in current laser configuration, it demonstrates that radical kinetics can be followed insinglemicrodroplets.

We present a novel homogeneous assay for detecting biomolecule interactions in microdroplets by fluorescence polarization (FP) for the first time. The FP assay allows the detection of target biomolecules directly after incubation without removing the detection reagent by separation or washing, making the assay amenable to automation. Using this assay we evaluate protein-protein and drug-DNA interactions. We detect these interactions at concentrations as low as 100nM and 69 pM respectively. This is a proof-of-concept homogeneous labeling assay in droplets for detecting bio-macromolecules., QC 20140815

Finding the few: Cell-surface proteins are useful disease biomarkers, but current high-throughput methods are limited to detecting cells expressing more than several hundred proteins. Enzymatic amplification in microfluidic droplets (see picture) is a high-throughput method for detection and analysis of cell-surface biomarkers expressed at very low levels on individual human cells. Droplet optical labels allow concurrent analysis of several samples., QC 20100525

A prominent characteristic of light scattered from a microparticle containing inclusions is fluctuating intensity due to changing positions of the inclusions with respect to each other and the host droplet. We calculate the magnitude of these fluctuations for a host sphere containing a single nonconcentric spherical inclusion and experimentally measure the fluctuation amplitudes for host spheres containing multiple inclusions. We find that for relatively small single inclusions, the amplitude of the scattering fluctuations increases approximately linearly with the area of the inclusion. For multiple inclusions, the fluctuation amplitude increases with concentration, with an approximate power-law dependence., Prepared in collaboration with California Univ., San Diego.

Review is made of the feedback and gain enhancement mechanisms unique to microdroplets. In particular, the growth of stimulated Raman scattering is considered in the presence of two-photon absorption as well as the usual optical losses inside the droplet because of scattering and leakage out of the droplet cavity.... Microparticles, stimulated Raman scattering, stimulated Brillouin scattering, and two-photon absorption.

A sample dispenser with electronic control of delivered volume has been designed and evaluated. With the new device, liquid samples are dispensed in the form of uniform droplets less than 4 nL in volume; total volumes as small as 40 nL can be delivered with 1.5% precision. Important features of the microdroplet formation process have been studied and the effect of sample solution concentration and viscosity have been examined. The new dispenser is expected to find application in several areas, including electrothermal atomization atomic absorption spectrometry, automated titrimetry, and high-precision, low-volume solution sampling. (Author)

Die Chemie kennt viele Methoden und Techniken, um chemische Reaktionen schneller ablaufen zu lassen. Seit langem ist bekannt, dass hohe Temperaturen, Drücke, große Reaktionsoberflächen und die Zugabe von Katalysatoren chemische Reaktionen beschleunigen können. In der chemischen Synthese kann zudem das Reaktorvolumen eine Rolle beim Reaktionsfortschritt spielen. Aus diesem Grund wurden in den vorhergehenden Jahrzehnten sogenannte Mikroreaktoren entwickelt. Wird nun die Größe des Reaktionsgefäßes auf mikroskopische Dimensionen reduziert, kann der Effekt auf die Reaktionsrate und Ausbeute enorm sein. So kann z.B. die Reaktionsgeschwindigkeit in geladenen Mikrotröpfchen jene im Inneren einer Lösung um das Millionenfache überschreiten. Diese extreme Reaktionsbeschleunigung wurde in den letzten Jahren mehrfach für organische Reaktionen und andere Anwendungen beschrieben. Über ein ähnliches Phänomen wurde in 2020 für den tryptischen Verdau von Proteinen unter Verwendung der ESSI Massenspektrometrie berichtet. Dies war die erste enzymatische Reaktion, für die eine vergleichbare Reaktionsbeschleunigung in geladenen Mikrotröpfchen nachgewiesen wurde. Hierbei wurde der komplette Verdau der Proteine in Millisekunden erreicht und sogar Protease resistente Oligopeptide konnten verdaut werden. Jedoch wird vermutet, dass diese Art der Reaktionsbeschleunigung systemspezifisch ist. Deshalb war es Ziel dieses Projektes zu untersuchen, ob ähnliche Effekte für den Proteinverdau mittels Pepsin erzielt werden können. Dabei wurde der zuvor beschriebene experimentelle Ausbau in mehreren Aspekten verbessert. Zudem konnten speziell entworfene Kontrollexperimente zweifelsfrei belegen, dass der enzymatische Verdau tatsächlich in den Mikrotröpfchen stattfindet und das in einer extremen Geschwindigkeit. Vorhergehende Studien konnten hierzu keinen eindeutigen Beweis liefern. Durch die Kombination vor reduzierter in-line Reaktionszeit mit Quenching Experimenten konnte die extreme Reaktionsbeschleun, In chemistry, multiple techniques and principles are used for increasing the rate of reactions. It is long known that high temperature, increased pressure, large surface area or the addition of catalysts can increase reaction rates drastically. Furthermore, the reaction volume can have a drastic effect on reaction behaviour in chemical synthesis. Hence the development of micro reactor in the recent year. If the size of the reaction vessel is reduced to a microscopic scale, the observed effect on reaction speed and yield can reach extreme levels. The reaction speed in microdroplets can be increased by up to a million times in comparison to the bulk phase reaction equivalent. This striking reaction acceleration effect was reported for various organic reactions and other applications in the recent decade. Astonishingly, a similar phenomenon was described in 2020 for the enzymatic digestion of proteins by trypsin using ESSI mass spectrometry. This was the first enzymatic reaction that was reported to show similar behaviour in charged microdroplets. Here, protein digestion was completed within milliseconds and was even achieved with protease resistant oligopeptides, highlighting the power of microdroplet reaction acceleration. As microdroplet reaction acceleration is believed to be system specific, the scope of this project was to assess whether comparable effects could be observed on the peptic digestion of proteins. In addition to improving the previously described experimental setup in several aspects, two specifically designed experiments unambiguously proved that the enzymatic digestion is indeed taking place within the microdroplet spray at an extreme rate. Previous studies did not address this question sufficiently. By combining quenching experiments with the reduction of in-line digestion time to a minimum, it was possible to conclusively demonstrate the extreme reaction acceleration, which is estimated to be approximately one million-fold. Furthermore, a compreh, eingereicht von Tobias Rainer, Kumulative Masterarbeit aus einem Artikeln, Zusammenfassung in deutscher Sprache, Masterarbeit Universität Innsbruck 2021

Die Chemie kennt viele Methoden und Techniken, um chemische Reaktionen schneller ablaufen zu lassen. Seit langem ist bekannt, dass hohe Temperaturen, Drücke, große Reaktionsoberflächen und die Zugabe von Katalysatoren chemische Reaktionen beschleunigen können. In der chemischen Synthese kann zudem das Reaktorvolumen eine Rolle beim Reaktionsfortschritt spielen. Aus diesem Grund wurden in den vorhergehenden Jahrzehnten sogenannte Mikroreaktoren entwickelt. Wird nun die Größe des Reaktionsgefäßes auf mikroskopische Dimensionen reduziert, kann der Effekt auf die Reaktionsrate und Ausbeute enorm sein. So kann z.B. die Reaktionsgeschwindigkeit in geladenen Mikrotröpfchen jene im Inneren einer Lösung um das Millionenfache überschreiten. Diese extreme Reaktionsbeschleunigung wurde in den letzten Jahren mehrfach für organische Reaktionen und andere Anwendungen beschrieben. Über ein ähnliches Phänomen wurde in 2020 für den tryptischen Verdau von Proteinen unter Verwendung der ESSI Massenspektrometrie berichtet. Dies war die erste enzymatische Reaktion, für die eine vergleichbare Reaktionsbeschleunigung in geladenen Mikrotröpfchen nachgewiesen wurde. Hierbei wurde der komplette Verdau der Proteine in Millisekunden erreicht und sogar Protease resistente Oligopeptide konnten verdaut werden. Jedoch wird vermutet, dass diese Art der Reaktionsbeschleunigung systemspezifisch ist. Deshalb war es Ziel dieses Projektes zu untersuchen, ob ähnliche Effekte für den Proteinverdau mittels Pepsin erzielt werden können. Dabei wurde der zuvor beschriebene experimentelle Ausbau in mehreren Aspekten verbessert. Zudem konnten speziell entworfene Kontrollexperimente zweifelsfrei belegen, dass der enzymatische Verdau tatsächlich in den Mikrotröpfchen stattfindet und das in einer extremen Geschwindigkeit. Vorhergehende Studien konnten hierzu keinen eindeutigen Beweis liefern. Durch die Kombination vor reduzierter in-line Reaktionszeit mit Quenching Experimenten konnte die extreme Reaktionsbeschleun, In chemistry, multiple techniques and principles are used for increasing the rate of reactions. It is long known that high temperature, increased pressure, large surface area or the addition of catalysts can increase reaction rates drastically. Furthermore, the reaction volume can have a drastic effect on reaction behaviour in chemical synthesis. Hence the development of micro reactor in the recent year. If the size of the reaction vessel is reduced to a microscopic scale, the observed effect on reaction speed and yield can reach extreme levels. The reaction speed in microdroplets can be increased by up to a million times in comparison to the bulk phase reaction equivalent. This striking reaction acceleration effect was reported for various organic reactions and other applications in the recent decade. Astonishingly, a similar phenomenon was described in 2020 for the enzymatic digestion of proteins by trypsin using ESSI mass spectrometry. This was the first enzymatic reaction that was reported to show similar behaviour in charged microdroplets. Here, protein digestion was completed within milliseconds and was even achieved with protease resistant oligopeptides, highlighting the power of microdroplet reaction acceleration. As microdroplet reaction acceleration is believed to be system specific, the scope of this project was to assess whether comparable effects could be observed on the peptic digestion of proteins. In addition to improving the previously described experimental setup in several aspects, two specifically designed experiments unambiguously proved that the enzymatic digestion is indeed taking place within the microdroplet spray at an extreme rate. Previous studies did not address this question sufficiently. By combining quenching experiments with the reduction of in-line digestion time to a minimum, it was possible to conclusively demonstrate the extreme reaction acceleration, which is estimated to be approximately one million-fold. Furthermore, a compreh, eingereicht von Tobias Rainer, Kumulative Masterarbeit aus einem Artikeln, Zusammenfassung in deutscher Sprache, Masterarbeit Universität Innsbruck 2021