The objective of this work is to develop a micro/nanofluidic-based single molecule detection (SMD) scheme, which would allow us to inspect individual protein or protein complex study protein-protein interactions and their dynamics. This is a collaboration work with MD Anderson Cancer Center and we applied this scheme to study functions of various proteins related to cancer progression in hope to shed new light on cancer research. State-of-the-art micro/nano-fabrication technology is used to provide fused silica micro/nano-fluidic channel devices as our detection platform. Standard contact photolithography, projection photolithography and advanced electron-beam lithography are used to fabricate micro/nano-fluidic channel with width ranging from 100nm to 2?m. The dimensions of these miniaturized biochips are designed to ensure single molecule resolution during detection and shrinking the detection volume leads to increase in signal-to-noise ratio, which is very critical for SMD. To minimize surface adsorption of protein, a fused silica channel surface coating procedure is also developed and significantly improved the detection efficiency. A fluorescent-labeled protein sample solution is filled in the fluidic channel by capillary force, and proteins are electro-kinetically driven through the fluidic channel with external voltage source. Commercial functionalized Quantum Dots (Qdots) are used as fluorescent labels due to its various advantages over conventional organic dyes for single molecule multi-color detection application. A fluorescence correlation spectrometer system, equipped with a 375nm diode laser, 60x water immersion objective with N.A. of 1.2 and two avalanche photodiodes (APD) is implemented to excite single molecules as well as collect emitted fluorescence signals. A two-dimensional photon burst analysis technique (photon counts vs. burst width) is developed to analyze individual single molecule events. We are able to identify target protein or protein complex directly from cell lysate based on fluorescence photon counts, as well as study the dynamics of protein-protein interactions. More importantly, with this technique we are also able to assess interactions between three proteins, which cannot be done with current ensemble measurement techniques. In summary, the technique described in this work has the advantages of high sensitivity, short processing time (2-3 minutes), very small sample consumption and high resolution quantitative analysis. It could potentially revolutionize the area of protein interaction research and provides us with more clues for the future of cancer diagnostics and treatments.

In the cancer research, it is important to understand protein dynamics which are involved in cell signaling. Therefore, particular protein detection and analysis of target protein behavior are indispensable for current basic cancer research. However, it usually performed by conventional biochemical approaches, which require long process time and a large amount of samples. We have been developed the new applications based on microfluidics and Raster image Correlation spectroscopy (RICS) techniques. A simple microfluidic 3D hydrodynamic flow focusing device has been developed for quantitative determinations of target protein concentrations. The analyte stream was pinched not only horizontally, but also vertically by two sheath streams by introducing step depth cross junction structure. As a result, a triangular cross-sectional flow profile was formed and the laser was focused on the top of the triangular shaped analyte stream. Through this approach, the target protein concentration was successfully determined in cell lysate samples. The RICS technique has been applied to characterize the dynamics of protein 53 (p53) in living cells before and after the treatment with DNA damaging agents. P53 tagged with Green Fluores-cent Protein (GFP) were incubated with and without DNA damaging agents, cisplatin or eptoposide. Then, the diffusion coefficient of GFP-p53 was determined by RICS and it was significantly reduced after the drug treatment while that of the one without drug treatment was not. It is suggested that the drugs induced the interaction of p53 with either other proteins or DNA. This result demonstrates that RICS is able to detect protein-protein or protein-DNA interactions in living cells and it may be useful for the drug screening. As another application of microfluidics, an integrated microfluidic platform was developed for generating collagen microspheres with encapsulation of viable cells. The platform integrated four automated functions on a microfluidic chip, (1) collagen solution cooling system, (2) cell-in-collagen microdroplet generation, (3) collagen microdroplet polymerization, and (4) incubation and extraction of the microspheres. This platform provided a high throughput and easy way to generate uniform dimensions of collagen microspheres encapsulating viable cells that were able to proliferate for more than 1 week.

Nanopores have emerged as a simple but effective tool to investigate the behavior of polymers in solution. They have shown great potential to simplify expensive and time consuming procedures like DNA sequencing, protein detection, and disease biomarker detection. With the development of in situ fabrication of solid-state nanopores by controlled breakdown (CBD) of a dielectric material, nanomanufacturing of nanopore-based technologies became feasible. However, there are still a lot of challenges to overcome for these applications to become reality. One of the major problems with solid-state nanopores is the rapid passage time of analytes going through the pore, complicating detection and reliable identification of molecules. In this thesis molecular structures are proposed that increase passage times due to increased interactions between analyte and pore wall, and at the same time increase signal amplitude due to increased blockage of the pore. These structures are short, branched DNA molecules that were assembled with built-in modifications and matching sequences to assume their structure. Nanopore experiments reveal that these structurally defined DNA produce higher detection rates than their linear DNA counterparts, making them better candidates for labels in single-molecule sensing experiments.

The miniaturization afforded by the integration of microfluidic technologies within lab-on-a-chip devices has greatly enhanced analytical capabilities in several key applications. Microfluidics has been utilized in a wide range of areas including sample preparation and analysis, DNA microarrays, cell detection, as well as environmental monitoring. The use of microfluidics in these applications offer many unique advantages: reduction in the required sample size, reduction in analysis time, lowered cost through batch fabrication, potentially higher throughput and the vision of having such devices used in portable systems. Nanopore sensors are a relatively new technology capable of detection and analysis with single-molecule sensitivity, and show promise in many applications related to the diagnosis and treatment of many diseases. Recently, some research groups demonstrated the integration of nanopores within microfluidic devices to increase analytical throughput. This thesis describes a methodology for integrating nanopore sensors within microfluidic devices with the aim of enhancing the analytical capabilities required to analyze biomolecular samples. In this work, the first generation of an integrated nanopore-microfluidic device contained multiple independently addressable microfluidic channels to fabricate an array of nanopore sensors using controlled breakdown (CBD). Next, for the second generation, we added pneumatic microvalves to manipulate electrical and fluidic access through connected microfluidic channels. As a proof-of-concept, single molecules (single- and double-stranded DNA, proteins) were successfully detected in the devices. It is also demonstrated that inclusion of the microfluidic via (microvia) limited the exposed area of the embedded silicon nitride membrane to the solution. This helped in localizing nanopore formation by confining the electric field to specific regions of the insulating membrane while significantly reducing high frequency noise in the ionic current signal through the reduction of chip capacitance. The devices highlighted in this thesis were designed and fabricated using soft lithography techniques which are available in most biotechnology laboratories. The core of this thesis is based on two scientific articles (Chapters 3 and 4), which are published in peer-reviewed scientific journals. These chapters are preceded by an introductory chapter and another chapter detailing the experimental setup and the methods used during the course of this study.

Thesis advisor: John T. Fourkas / One can extract a tremendous amount of information about the organizational and dynamic states of molecules, in situ and in real-time, through highly sensitive and noninvasive single particle optical probing. The highly efficient, multi-photon excited luminescence from stabilized metal nanoclusters renders these species useful as optical probes that can be used in detecting single particle and molecular dynamics. We generate stable, and monodisperse samples of Ag nanoclusters as small as 1 nm in diameter, and find that through substitution of various stabilizer molecules we can precisely tune the size of the clusters over a 1-6 nm range of diameters, ensuring monodispersity and stability at every stage. These clusters also exhibit highly efficient, polarized luminescence upon two photon excitation at 800 nm and remain highly photostable, not exhibiting the deleterious blinking that occurs with many single-molecule fluorophores. In order to demonstrate the utility of these clusters as single-molecule probes, we track their emission polarization over long periods in deeply supercooled liquids such as 4'(octahydro-4,7-methano-5H-inden-5-yliden) bisphenol dimethyl ether (ODE). Our results suggest that these clusters can detect nanoscale dynamics with high sensitivity. / Thesis (BS) — Boston College, 2004. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Chemistry. / Discipline: College Honors Program.

<p>Improved methods are needed to interrogate the genome and the proteome. Methods with high selectivity, wide dynamic range, and excellent precision, capable of simultaneously analyzing many biomolecules are required to decipher cellular function. This thesis describes a molecular and microfluidic toolbox designed with those criteria in mind. It also presents a tool for graphical representation of nucleic acid sequences.</p><p>Proximity ligation is a novel protein detection method that requires dual and proximate binding of two oligonucleotide-tagged affinity reagents to a protein or protein complex in order to elicit a signal. The responses from such recognition reactions are the formation of specific nucleic acid reporter molecules that are subsequently amplified and quantitatively detected. </p><p>A scalable microfluidic platform suitable for fluorescence detection, cell culture, and actuation is also described. The platform uses rapid injection molding to produce microstructures in thermoplastic materials. By applying a thin layer of silica to the structures, a lid made of silicone rubber coated onto a thermoplastic support can be covalently bonded to generate enclosed channels.</p><p>A method is presented for precise biomolecule counting, termed “amplified single-molecule detection”. The method preserves the discrete nature of biomolecules, converting specific molecular recognition events to fluorescence-labeled micrometer-sized objects that are enumerated in microfluidic channels. </p><p>I also present a novel microarray-based detection method. To attain high selectivity and a wide dynamic range, the method is based on dual recognition with enzymatic discrimination and amplification. Upon target recognition in solution, DNA probes are subjected to thousand-fold amplification in solution, followed by selective detection on arrays and another hundred-fold amplification of reporter molecule created from the first amplification reaction. </p><p>Lastly, I describe a novel graphical representation of nucleic acid sequences using TrueType fonts that can be of value for visual inspection of DNA sequences and for teaching purposes</p>

<p>In later years polymer replication techniques have become a frequently employed fabrication method for microfluidic and micro-optical devices. This thesis describes applications and further developments of microstructures replicated in polymer materials. </p><p>A novel method for homogenous amplified single-molecule detection utilizing a microfluidic readout format is presented. The method enables enumeration of single biomolecules by transforming specific molecular recognition events at nanometer dimensions to micrometer-sized DNA macromolecules. This transformation process is mediated by target specific padlock probe ligation, followed by rolling circle amplification (RCA) resulting in the creation of one rolling circle product (RCP) for each recognized target. Throughout this transformation the discrete nature of the molecular population is preserved. By hybridizing a fluorescence-labeled DNA detection oligonucleotide to each repeated sequence of the RCP, a confined cluster of fluorophores is generated, which makes optical detection and quantification possible. Spectral multiplexing is also possible since the spectral profile of each RCP can be analyzed separately. The microfluidic data acquisition process is characterized in detail and conditions that allow for quantification limited only by Poisson sampling statistics is established. The molecular characteristics of RCPs in solution are also investigated.</p><p>Furthermore a novel thermoplastic microfluidic platform is described. The platform allows for observation of the microchannels using high magnification optics and also offers the possibility of on-chip cell culture and the integration of mechanical actuators.</p><p>A novel fabrication process for the integration of polymer micro-optical elements on silicon is presented. The process is used for fabrication of a micro-optical system consisting of a laser and a movable microlens making beam steering possible. Such a micro-scanning system could potentially be used for miniaturized biochemical analysis.</p>

In later years polymer replication techniques have become a frequently employed fabrication method for microfluidic and micro-optical devices. This thesis describes applications and further developments of microstructures replicated in polymer materials. A novel method for homogenous amplified single-molecule detection utilizing a microfluidic readout format is presented. The method enables enumeration of single biomolecules by transforming specific molecular recognition events at nanometer dimensions to micrometer-sized DNA macromolecules. This transformation process is mediated by target specific padlock probe ligation, followed by rolling circle amplification (RCA) resulting in the creation of one rolling circle product (RCP) for each recognized target. Throughout this transformation the discrete nature of the molecular population is preserved. By hybridizing a fluorescence-labeled DNA detection oligonucleotide to each repeated sequence of the RCP, a confined cluster of fluorophores is generated, which makes optical detection and quantification possible. Spectral multiplexing is also possible since the spectral profile of each RCP can be analyzed separately. The microfluidic data acquisition process is characterized in detail and conditions that allow for quantification limited only by Poisson sampling statistics is established. The molecular characteristics of RCPs in solution are also investigated. Furthermore a novel thermoplastic microfluidic platform is described. The platform allows for observation of the microchannels using high magnification optics and also offers the possibility of on-chip cell culture and the integration of mechanical actuators. A novel fabrication process for the integration of polymer micro-optical elements on silicon is presented. The process is used for fabrication of a micro-optical system consisting of a laser and a movable microlens making beam steering possible. Such a micro-scanning system could potentially be used for miniaturized biochemical analysis.

Improved methods are needed to interrogate the genome and the proteome. Methods with high selectivity, wide dynamic range, and excellent precision, capable of simultaneously analyzing many biomolecules are required to decipher cellular function. This thesis describes a molecular and microfluidic toolbox designed with those criteria in mind. It also presents a tool for graphical representation of nucleic acid sequences. Proximity ligation is a novel protein detection method that requires dual and proximate binding of two oligonucleotide-tagged affinity reagents to a protein or protein complex in order to elicit a signal. The responses from such recognition reactions are the formation of specific nucleic acid reporter molecules that are subsequently amplified and quantitatively detected. A scalable microfluidic platform suitable for fluorescence detection, cell culture, and actuation is also described. The platform uses rapid injection molding to produce microstructures in thermoplastic materials. By applying a thin layer of silica to the structures, a lid made of silicone rubber coated onto a thermoplastic support can be covalently bonded to generate enclosed channels. A method is presented for precise biomolecule counting, termed “amplified single-molecule detection”. The method preserves the discrete nature of biomolecules, converting specific molecular recognition events to fluorescence-labeled micrometer-sized objects that are enumerated in microfluidic channels. I also present a novel microarray-based detection method. To attain high selectivity and a wide dynamic range, the method is based on dual recognition with enzymatic discrimination and amplification. Upon target recognition in solution, DNA probes are subjected to thousand-fold amplification in solution, followed by selective detection on arrays and another hundred-fold amplification of reporter molecule created from the first amplification reaction. Lastly, I describe a novel graphical representation of nucleic acid sequences using TrueType fonts that can be of value for visual inspection of DNA sequences and for teaching purposes

The cyclical interaction between the force-generating protein myosin and actin is the mechanism responsible for muscle contraction among all muscle types. Cardiac muscle contraction is tightly controlled to ensure that blood pumps effectively and efficiently from the heart to peripheral organs. Mutations in various cardiac proteins can lead to cardiac dysfunction and a number of cardiomyopathies. The first part of this dissertation studies two disease-linked mutations in the regulatory light chain of the cardiac myosin molecule, D166V and K104E, and assesses the kinetic and mechanochemical effects of the mutations via the in vitro motility assay. The data show that D166V mutant myosin force generation is reduced compared to wild type, and exogenous phosphorylation of the mutant light chain rescues force generation. In contrast, the K104E mutation showed no deficit in force production but exhibited increased calcium sensitivity of activation. These results are consistent with contractile defects associated with cardiomyopathies caused by various mutation-induced changes to protein function and mechanism of interaction. The second part uncovers the actin-binding mechanism of one of the chief muscle regulatory proteins tropomyosin. In cardiac and skeletal muscle, tropomyosin and troponin modulate muscle contraction. Tropomyosin binds along the length of actin filaments and blocks myosin-binding sites. Following an excitatory stimulus, calcium binds troponin and causes tropomyosin to shift its position on actin, allowing myosin to bind. The precise mechanism of how tropomyosin monomers with low actin affinity bind to form a stably bound, high affinity chain is unknown. By directly observing fluorescently labeled tropomyosin binding to actin filaments, it was shown that tropomyosin molecules bind randomly along the actin filament. Subsequent monomer binding, and formation of tropomyosin end-to-end bonds, increases the probability of sustained chain growth by decreasing the probability of detachment prior to additional monomer binding. Tropomyosin molecules added to the growing chain at approximately 100 monomers/(μM*s). Different tropomyosin isoforms segregate to distinct functional and structural regions of cells. The last chapter presents data that show spatial segregation of two different tropomyosin isoforms on actin filaments. This suggests that tropomyosin sorting in cells is, at least partly, an intrinsic property of the binding mechanism.