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Item Electrochmical impedance spectroscopy biosensor platform for evaluation of biofilm(Montana State University - Bozeman, College of Engineering, 2023) McGlennen, Matthew Connor Dusenbery; Co-chairs, Graduate Committee: Christine Foreman and Stephan Warnat; This is a manuscript style paper that includes co-authored chapters.Microbial biofilms are organized communities of surface-attached microorganisms encased in a self-produced extracellular matrix that pose significant challenges in medicine, the environment, and industry. Biofilms can cause chronic infections, biofouling, and equipment failure, while existing methods for biofilm detection are slow, costly, and labor-intensive. Recently, the use of microfabricated electrochemical impedance spectroscopy (EIS) biosensors has emerged as a promising technique for evaluating biofilm growth in real-time with advantages of small-size, adaptability, low-cost, and high-sensitivity. In this work, EIS biosensors featuring gold micro-interdigitated electrodes were produced using standard microfabrication techniques. Sensors were integrated into a custom 3D-printable flow cell system, enabling EIS measurements and confocal laser scanning microscopy (CLSM) imaging simultaneously. Green fluorescently labeled Pseudomonas aeruginosa PA01, a model biofilm forming bacteria, was introduced into flow chambers and subsequent growth was monitored by EIS, CLSM, and biomass enumeration. Using the system, biofilm growth, dispersal, and the effects of cell-signaling suppression were evaluated. The sensors were also tested in an oil-water emulsion and field-tested on an alpine snow-patch and pond. Improved stability of EIS measurements was achieved by coating the sensors' counter and reference electrodes with an electrically conductive polymer. Biofilm growth was successfully detected using EIS biosensors at an optimized single-frequency, with average decreases in impedance of ~22% by 24 hours. Likewise, biofilm dispersal via chemical treatments were successfully detected with average increases in impedance of ~14% over the ensuing 12 hours. When cells were exposed to a quorum sensing inhibition agent, impedance did not decrease for 18 hours. Impedance changes due to biofilm growth, dispersal, and effects of quorum sensing inhibition were validated by CLSM images and biofilm enumeration. Similarly, in an oil-water emulsion the biosensors successfully detected biofilm growth, dispersal, and effects of quorum sensing inhibition. In an alpine field-test, samples containing varying concentrations of microbes could be detected using the EIS biosensors. This work demonstrates that EIS biosensors are a promising tool for real-time monitoring of biofilm dynamics in a variety of aqueous environments. Overall, EIS biosensing holds great potential for in situ and real-time data regarding biofilm colonization that is not possible with existing techniques.Item Techniques for improving activity based biosensors: a Kuhl platform for engineering(Montana State University - Bozeman, College of Agriculture, 2020) Thomas, Merrilee Anne; Chairperson, Graduate Committee: Thomas Hughes and Susy C. Kohout (co-chair); Thomas E. Hughes was a co-author of the article, 'Optically activated, customizable, excitable cells Kuhl platform for evolving next gen biosensors' submitted to the journal 'PLOS One' which is contained within this dissertation.According to Kuhn, ''there are three classes of problems - determination of significant facts, matching of facts with theory, and articulation of that theory (Kuhn 2012).'' The current paradigm in molecular neuroscience is that there is a need for revolutionary tool development in neuroscience. Interestingly, the need for better tools in neuroscience is to answer neuroscience theories and provide the determination and articulation of those theories. Currently, the neuroscientist's toolbox is growing and the ways in which those tools are used is rapidly changing. Neuroscience underwent a revolution when we were able to take single-cell recording in vivo and then assign field properties to individual neurons based upon those responses (O'Keefe and Bouma 1969; O'Keefe and Dostrovsky 1971; Moser et al. 1995). Scientists became adept at imaging increasingly smaller regions of the functioning human brain (Price 2012). We have since been able to genetically encode and manipulate proteins and pathways while recording from them using fluorescence (Southern and Berg 1982; Chalfie 2009). In vitro and in vivo we have harnessed the use of light to stimulate or inhibit specific neurons or ligands (Boyden 2011; Adamantidis et al. 2007). These tools are just the beginning and by no means is this an exhaustive list. We introduce the Kuhl synthetic cell system that provides a customizable de-novo excitable cell. The Kuhl system is activated using a blue light photo activated cyclase bPAC. It can be used to create better tools to image the brain and can be used to screen multi-color fluorescent sensors. Interestingly, sensors that are within bPACs activation spectrum can be used in these synthetic cells. We show that both red and green Ca 2+ sensors can be imaged simultaneously, and both Ca 2+ and Voltage sensors can be screened in the Kuhl system. The Kuhl system has the potential to be used to screen for drug compounds and in theory, they could be used in studying pathways that are less understood, such as the mTOR pathway.Item Improving the two-photon absorption properties of fluorescent proteins for neuroscience(Montana State University - Bozeman, College of Letters & Science, 2020) Molina, Rosana Sophia; Chairperson, Graduate Committee: Thomas Hughes; Yong Qian, Jiahui Wu, Yi Shen, Robert E. Campbell, Mikhail Drobizhev and Thomas E. Hughes were co-authors of the article, 'Understanding the fluorescence change in red genetically encoded calcium ion indicators' in the journal 'Biophysical Journal' which is contained within this dissertation.; Tam M. Tran, Robert E. Campbell, Gerard G. Lambert, Anya Salih, Nathan C. Shaner, Thomas E. Hughes and Mikhail Drobizhev were co-authors of the article, 'Blue-shifted green fluorescent protein homologues are brighter than enhanced green fluorescent protein under two-photon excitation' in the journal 'The Journal of physical chemistry letters' which is contained within this dissertation.; Jonathan King, Jacob Franklin, Nathan Clack, Christopher McRaven, Vasily Goncharov, Daniel Flickinger, Karel Svoboda, Mikhail Drobizhev, Thomas E. Hughes were co-authors of the article, 'An instrument to optimize fluorescent proteins for two-photon excitation' which is contained within this dissertation.Untangling the intricacies of the brain requires innovative tools that power basic research. Fluorescent proteins, first discovered in jellyfish, provide a genetically encodable way to light up the brains of animal models such as mice and fruit flies. They have been made into biosensors that change fluorescence in response to markers of neural activity such as calcium ions (Ca 2+). To visualize them, neuroscientists take advantage of two-photon excitation microscopy, a specialized type of imaging that can reveal crisp fluorescence images deep in the brain. Fluorescent proteins behave differently under twophoton excitation compared to one-photon excitation. Their inherent two-photon properties, namely brightness and peak absorption wavelength, limit the scope of possible experiments to investigate the brain. This work aims to understand and improve these properties through three projects: characterizing a set of red fluorescent protein-based Ca 2+ indicators; finding two-photon brighter green fluorescent proteins; and developing an instrument to screen for improved fluorescent proteins for two-photon microscopy. Analyzing nine red Ca 2+ indicators shows that they can be separated into three classes based on how their properties change in a Ca 2+-dependent manner. In one of these classes, the relative changes in one-photon properties are different from the changes in two-photon properties. In addition to characterizing, identifying and directly improving fluorescent proteins for enhanced two-photon properties is important. Presented here is a physical model of the light-absorbing molecule within the green fluorescent protein (the chromophore). The model predicts that green fluorescent proteins absorbing at higher energy wavelengths will be brighter under two-photon excitation. This proves to be the case for 12 blueshifted green fluorescent proteins, which are up to 2.5 times brighter than the commonly used Enhanced Green Fluorescent Protein. A way to directly improve fluorescent proteins is through directed evolution, but screening under two-photon excitation is a challenge. An instrument, called the GIZMO, solves this challenge and can evolve fluorescent proteins expressed in E. coli colonies under two-photon excitation. These results pave the way for better two-photon fluorescent protein-based tools for neuroscience.Item If you build it, they will come: engineering the next generation of optical tools to image neural activity deep within the living brain(Montana State University - Bozeman, College of Letters & Science, 2017) Barnett, Lauren Marie; Chairperson, Graduate Committee: Thomas Hughes; Thomas E. Hughes and Mikhail Drobizhev were co-authors of the article, 'Deciphering the molecular mechanism responsible for GCAMP6M's Ca 2+ dependent change in fluorescence' in the journal 'PLoSONE' which is contained within this thesis.; Mikhail Drobizhev and Thomas E. Hughes were co-authors of the article, 'Making pKa-altering mutations in GCAMP6M changes the Ca 2+-dependent fluorescence response' submitted to the journal 'PLoSONE' which is contained within this thesis.; Jelena Platisa, Marko Popovic, Vincent A. Pieribone and Thomas Hughes were co-authors of the article, 'A fluorescent, genetically-encoded voltage probe capable of resolving action potentials' in the journal 'PLoSONE' which is contained within this thesis.; Lauren M. Barnett, Mikhail Drobizhev, Geoffrey Wicks, Alexander Mikhaylov, Thomas E. Hughes and Aleksander Rebane were co-authors of the article, 'Two-photon directed evolution of green fluorescent proteins' in the journal 'Nature Scientific Reports' which is contained within this thesis.To see the activity of large, integrated neural circuits functioning in real-time inside of a living brain, neuroscientists will need multiple genetically-encoded fluorescent activity sensors that can be individually targeted to specific cell types, are fast enough to resolve multiple action potentials, can be distinguished from one another and imaged deep within the brain. The goal of this work is to better understand and improve upon the most recent generations of genetically-encoded Ca 2+ and voltage sensors, and to expand biosensor utility in two-photon excitation, which will be necessary to image neural activity deep within the brain. Genetically-encoded Ca 2+ sensors measure the intracellular Ca 2+ release that occurs downstream of an action potential. The GCaMP6 series are the best Ca 2+ sensors available, however little is known about how they work. Measurements of four different states in GCaMP6m reveal that its large Ca 2+-dependent change in 470 nm excited fluorescence is due to a redistribution of the chromophore protonation state, from a neutral form excited at ~400 nm to an anionic form excited at ~470 nm, via a change in pK a. Making pK a-altering mutations in GCaMP6m changes the Ca 2+-dependent fluorescence response. This highlights the importance of Delta pK a and identifies key amino acid positions that will be important for improving GCaMP6m and GCaMP-like biosensors. A direct readout of an action potential would be ideal for capturing complex signal transduction in the brain. This will require a bright, fast voltage sensor. ElectricPk is the first genetically-encoded voltage sensor with a fluorescence response fast enough to resolve multiple action potentials in mammalian neurons. This design indicates it is possible to couple a fluorescence change with a very fast (~1 ms) voltage-dependent movement in the Ciona intestinalis voltage-sensitive phosphatase protein. Whether imaging a downstream Ca 2+ signal or a direct change in membrane potential, to image neuronal activity in deep brain tissue biosensors will need to be brightly fluorescent in two-photon excitation. The two-photon directed evolution of green fluorescent proteins presented here is a proof-of-principle design that shows a high-throughput screen focused on improving the two-photon properties of a fluorescent protein is possible.Item Glycosylation of Anthrax Protective Antigen : engineering of a promiscuous O-glycosyltransferase(Montana State University - Bozeman, College of Letters & Science, 2003) Hyman, Deborah Anne; Chairperson, Graduate Committee: Charles SpanglerItem Synthesis and characterization of anti-body self-assembling monolayers on the surface of a quartz crystal microbalance for use as a biosensor(Montana State University - Bozeman, College of Engineering, 2000) Kositruangchai, NapawanItem Substrate integrated waveguide resonant cavity sensor(Montana State University - Bozeman, College of Engineering, 2013) Revia, Richard Aaron; Chairperson, Graduate Committee: James P. BeckerA current area of active research is the development of biosensors. Biosensors have been constructed to examine a large range of target analytes such as enzymes, antibodies, DNA, and cells. The majority of currently developed biosensors require the use of labels which attach to an analyte to enhance the sensitivity of the sensor to a significant level. Labeling targets introduces many drawbacks: added complexity to the detection process, increased preparation time, and most importantly, the possible modification of analyte properties due to the attachment of the label. For these reasons, there has been much attention on electronic means of label-free biological agent detection. One such electronic biosensing method is the use of a resonant circuit operating at microwave frequencies for impedance and dielectric spectroscopy. In these spectroscopic measurements, changes in the resonant frequency of the circuit are detected and correlated to the presence of a specific analyte. Various resonator circuits have been utilized in dielectric spectroscopy and biomolecule detection; however, these biosensors, although label-free, possess their own idiosyncratic complications such as imprecise and convoluted test sample deposition schemes. To address some of the challenges associated with existing biosensors, a device is presented demonstrating the potential to be used for label-free biosensing and promises a convenient sample deposition procedure. The instrument is based on the construction of a substrate integrated waveguide analog of an enclosed section of rectangular waveguide. Classical microwave engineering principles were used to give an outline of key electrical characteristics and dimensions, and full-wave finite element analysis software was utilized to further refine and optimize the device. A fabricated prototype was tested through measurement of scattering parameters using a network analyzer. The archetypal resonant circuit discussed herein can be used to extract the complex permittivity from test materials. Discussions of the cardinal design parameters, sensitivity analysis, and permittivity extraction techniques are provided. Suggestions for continued development are presented based on experience gained from the design of the prototype sensor. Proposed future work includes a scaled-down version of the substrate integrated waveguide resonator and testing with biological agents such as biofilms and single cells.