Engineering bilayer membranes for nano- and microscale sensing

Boyd, Margrethe

doi:https://doi.org/10.21985/n2-xtm0-pb97

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Engineering bilayer membranes for nano- and microscale sensing

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In environments ranging from natural ecosystems to living organisms, small molecule signals and nanoscale forces communicate important information regarding chemical contamination and pollution, pathogenesis, and physical stressors. As these stimuli are often well below detection limits for our own senses, we depend on biosensing technologies to monitorthem. Many biosensors operate through direct molecular interactions with a target analyte, which can be sensitive to off-target molecules or to dilution and matrix effects in environmental or biological samples. Other stimuli, particularly mechanical forces, remain extremely difficult to monitor without highly specialized equipment. As a result, our ability to reliably and accurately detect biologically and technologically important signals in many contexts remains limited. One way to introduce additional sensing features to these systems is to reintroduce a key component of many cellular biosensing mechanisms: the bilayer membrane. As the primary barrier between a cell and its environment, the membrane plays a critical role in signal transduction and information transfer, force response, and protection and containment ofintracellular components. Membranes offer a unique sensing platform in that they can incorporate both hydrophobic, membrane-localized sensors as well as encapsulated aqueous sensors. Similarly, as bio-inspired structures, they can incorporate a range of naturally and synthetically derived components, ranging from amphiphiles to protein transporters, which can introduce additional functionality to the sensor as a whole. In this dissertation, I discuss how synthetic membranes can be engineered to advance approaches to biosensing. I first focus on the development of membrane-based sensors for mechanical force, exploring an optical method to monitor stretch and lipid uptake in a population of nanoscale sensors in response to a changing external environment. I then establish three approaches to lipid vesicle-based sensing with encapsulated aqueous sensors; I design these vesicle sensors with increasing complexity, encapsulating a chemical ion indicator, a transcription-based sensor, and a cell-free protein expression-based sensor. First, I establish a vesicle-based nanosensor for potassium ions. By encapsulating a fluorescent indicator for potassium, I show that dye encapsulation and incorporation of an ion-specific membrane transporter can improve specificity to an ion of interest and allow detection in the presence of live bacteria. Building upon this system, I next incorporate a transcription-based sensor which transcribes an RNA aptamer in the presence of magnesium. I incorporate nonspecific pores into the membrane and demonstrate that both pore concentration and the presence of the targetmolecule modulate sensor output. Finally, I encapsulate a cell-free protein expression-based sensor for a biologically important analyte, fluoride. I demonstrate function of the encapsulated riboswitch sensor and show how membrane composition can be changed to modulate sensitivity to externally added fluoride ions. I then deploy these sensors in real-world samples and demonstrate detection of fluoride in complex environments. Through the incorporation of a variety of sensing strategies, this work highlights the ways in which synthetic membranes can be introduced and engineered to enhance sensing capabilities. These sensing strategies ultimately result in tradeoffs between sensitivity, specificity, and stability and allow the incorporation of a wide variety of sensor types, enabling the detection of both chemical and physical signals in new contexts.

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