Browsing by Subject "Biosensor"
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Item Embargo A Vertically Oriented Passive Microfluidic Device for Automated Point-Of-Care Testing Directly from Complex Samples(2023) Kinnamon, David StanleyDetection and quantification of biomarkers directly from complex clinical specimens is desired and often required by healthcare professionals for the effective diagnosis and screening of disease, and for general patient care. Current methodologies to accomplish this task have critical shortcomings. Laboratory immunoassays, most notably enzyme-linked immunosorbent assay (ELISA) require extensive clinical infrastructure and complex user intervention steps to generate results and often are accompanied by a lengthy time-to-result. Conversely, available point-of-care (POC) diagnostic solutions, most notably available lateral flow immunoassays (LFIAs), often struggle with sensitivity and specificity in complex fluids, lack quantitative output and are not easily multiplexed. In this dissertation I will discuss the design, fabrication, testing, and refinement of an all-in-one fluorescence microarray integrated into a passive microfluidic fluid handling system to create a versatile and automated POC platform that can detect biomarkers from complex samples for disease management with the relative ease-of-use of an LFIA and the performance of a laboratory-grade test. The platform is driven by capillary and gravitational forces and automates all intervention steps after the addition of the sample and running buffer at the start of testing. The microfluidic cassette is built on a (poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) polymer brush which imparts two key functionalities, (1) it eliminates cellular and protein binding, and when combined with the vertical orientation of the microfluidic cassette prevents settling of debris during all assay steps. This allows for impressive sensitivities and specificities to be obtained from samples as complex as undiluted whole blood even when relying on gentle capillary and hydrostatic pressures for cassette operation. (2) Paradoxically, printed biorecognition elements can be stably and non-covalently immobilized into the POEGMA allowing for all reagents needed to conduct a sandwich immunoassay in a single step to be easily inkjet printed as spatially discrete spots into the POEGMA brush, which also stabilizes them at room temperature. Additionally, the microfluidic cassette is compatible with the “D4Scope” a handheld fluorescence detector that can quantify the output of the microfluidic cassette in seconds at the POC and is the only piece of auxiliary equipment required to operate the test.
This dissertation discusses early cassette prototypes and characterizes the performance of major device iterations (Chapter 2) before moving into three clinical applications of the cassette. First, a multiplexed serological test to detect antibodies against different proteins of the SARS-CoV-2 virus was developed (Chapter 3). Second, a multiplexed COVID-19 diagnostic test that simultaneously differentiates which variant you are infected with was developed (Chapter 4). Third, a sensitive fungal infection test for the diagnosis of talaromycosis was developed (Chapter 5). Finally, a rapidly iterative yet highly scalable injection molding fabrication process flow was created and characterized to improve performance and translatability of the cassette (Chapter 6).
Item Open Access Characterization of porous, dexamethasone-releasing polyurethane coatings for glucose sensors.(Acta Biomaterialia, 2014-11) Vallejo-Heligon, Suzana G; Klitzman, Bruce; Reichert, William MCommercially available implantable needle-type glucose sensors for diabetes management are robust analytically but can be unreliable clinically primarily due to tissue-sensor interactions. Here, we present the physical, drug release and bioactivity characterization of tubular, porous dexamethasone (Dex)-releasing polyurethane coatings designed to attenuate local inflammation at the tissue-sensor interface. Porous polyurethane coatings were produced by the salt-leaching/gas-foaming method. Scanning electron microscopy and micro-computed tomography (micro-CT) showed controlled porosity and coating thickness. In vitro drug release from coatings monitored over 2 weeks presented an initial fast release followed by a slower release. Total release from coatings was highly dependent on initial drug loading amount. Functional in vitro testing of glucose sensors deployed with porous coatings against glucose standards demonstrated that highly porous coatings minimally affected signal strength and response rate. Bioactivity of the released drug was determined by monitoring Dex-mediated, dose-dependent apoptosis of human peripheral blood derived monocytes in culture. Acute animal studies were used to determine the appropriate Dex payload for the implanted porous coatings. Pilot short-term animal studies showed that Dex released from porous coatings implanted in rat subcutis attenuated the initial inflammatory response to sensor implantation. These results suggest that deploying sensors with the porous, Dex-releasing coatings is a promising strategy to improve glucose sensor performance.Item Open Access Development of Tunable Molecular Tension Sensors to Visualize Vinculin Loading and Detect Mechanosensitive Protein Recruitment to Focal Adhesions(2018) LaCroix, Andrew ScottMechanical forces are potent drivers of many biological processes. The form and function of many tissues depends on cells receiving the proper mechanical signals either from neighboring cells or from the underlying matrix. During development, dynamic tissue movements are driven by cell contractility and stem cell fate depends in large part to the mechanical forces they feel in their local surroundings. Conversely, aberrant mechanosensitive signaling is associated with the pathological progression of several disease states, such as cancer and atherosclerosis, for which effective treatments are scarce. As such, understanding how cells physically interact with and detect mechanical aspects of their microenvironment is critical to both understanding developmental processes and developing new treatments for disease.
Mechanical information from the microenvironment is converted into biochemical signals inside cells through molecular scale processes, collectively referred to as mechanotransduction. Many of the events associated with mechanosensitive signaling and mechanotransduction are mediated by force-dependent changes in protein structure and function. However, the lack of available tools to study these molecular scale processes in cells is currently preventing further progress. To address this need, the goals of this dissertation were to (1) improve upon and expand the capabilities of existing tools to visualize molecular forces and (2) develop novel methodologies to detect force-sensitive signaling events inside cells.
We began by focusing on the further development and improvement of one of the most critical tools to mechanobiological investigations: Förster Resonance Energy Transfer (FRET)-based molecular tension sensors. While these sensors have contributed greatly to our understanding of mechanobiology, the limited dynamic range and inability to specify the mechanical sensitivity of existing sensors has hindered their use in diverse cellular contexts. Through both experiments and modeling efforts, we developed a comprehensive biophysical understanding of molecular tension sensor function that enabled the creation of new sensors with predictable and tunable mechanical sensitivities. We used this knowledge to create a sensor optimized to study the ~1-6pN loads experienced by vinculin, a critical linker protein that plays an integral role in connecting cells, via focal adhesions (FAs), to the extracellular matrix (ECM). Using this optimized sensor enabled sensitive detection of changes in molecular loads across single cells and even within individual FA structures. We also expanded the capabilities of tension sensors to investigate the potentially distinct roles of protein force and protein extension in activating mechanosensitive signaling. Specifically, a trio of these new biosensors with distinct force- and extension-sensitivities revealed that an extension-based control paradigm underlies cellular control of vinculin loading.
Since these sensors uniquely provide insight into which molecules are physically engaged and could be participating in mechanically-based signaling, we chose to investigate which cytoskeletal structures mediate patterns of vinculin loading at multiple length scales within the cell. Specifically, we focused on two active, but distinct force generating machineries inside cells: stress fibers (SFs) and lamellipodial protrusions (LPs). By measuring vinculin tension in various mechanical and biochemical contexts, we found significant evidence for vinculin’s involvement in force transmission from both LP and SF structures. However, the distribution of loads across vinculin at the level of a single FA was dramatically different between these two distinct actin structures. Specifically, asymmetric distribution of vinculin load along individual FAs was an exclusive feature of SF-associated FAs. Subsequent experiments showed that formation and maintenance of these gradient loading profiles also depends on vinculin’s interactions with key binding partners, suggesting that both the magnitude as well as the pattern of vinculin loading within FAs are independently regulated by cells, and thus might serve distinct purposes in cellular mechanosensing.
Towards understanding the biochemical consequences of protein load at the molecular level, we developed an imaging-based technique to detect one of the events most often implicated in mechanically-based signal transduction: the formation of force-sensitive protein-protein interactions (PPIs). While these force sensitive interactions have been extensively documented in vitro, the extent to which they occur inside living cells is debated. This imaging-based technique, which we refer to as fluorescence-force co-localization (FFC), involves simultaneous FRET imaging of a FRET-based tension sensor to visualize protein loading and correlate this with the recruitment of other species to areas of high molecular loads. With vinculin as a prototypical example and a screen-based approach in mind, we used immunofluorescence to measure the relative enrichment of 20 other key FA proteins in areas of high vinculin tension. Factoring in what we previously learned about (1) the importance of actin architecture and (2) the well-established role of vinculin alone in controlling FA composition, we provide a multiparametric perspective on a potential mechanotransduction node associated with high vinculin loads. Focusing on the top five hits from this FFC screen, subsequent experiments revealed a genuine vinculin tension-dependent interaction with migfilin. While the involvement of both vinculin and migfilin in cardiac settings is a tempting line of future work, the work presented in this dissertation even more powerfully provides a proof-of-principle for the detection of force-sensitive PPIs in cells.
In total, the techniques developed in this dissertation enable detection of multiple molecular events associated with mechanotransduction inside cells. The improvement of FRET-based tension sensors as well as the ability to define their mechanical properties a priori should expedite investigations of molecular forces in diverse biological contexts. Additionally, the realization of force-dependent PPIs inside cells provided by the FFC screen constitutes a significant step towards uncovering mechanically-based signaling mechanisms inside cells. The more widespread application of these tools will undoubtedly fuel our understanding of mechanotransduction and could enable better control of cell behaviors in engineered tissues as well as the development of treatments for mechanosensitive diseases.
Item Open Access Label-free Biodetection with Individual Plasmonic Nanoparticles(2010) Nusz, GregoryThe refractive index sensitivity of plasmonic nanoparticles is utilized in the development of real-time, label-free biodetection. Analyte molecules that bind to receptor-conjugated nanoparticles cause an increase in local refractive index that in turn induces an energy shift in the optical resonance of the particle. Biomolecular binding is quantified by quantitatively measuring these resonance shifts. This work describes the application and optimization of a biomolecular detection system based on gold nanorods as an optical transducer.
A microspectroscopy system was developed to collect scattering spectra of single nanoparticles, and measure shifts of the spectra as a function of biomolecular binding. The measurement uncertainty of LSPR peak shifts of the system was demonstrated to be 0.3 nm. An analytical model was also developed that provides the optimal gold nanorod geometry for detection with specified receptor-analyte pair. The model was applied to the model biotin-streptavidin system, which resulted in sensing system with a detection limit of 130 pM - an improvement by four orders of magnitude over any other single-particle biodetection previously presented in the literature.
Alternative optical detection schemes were also investigated that could facilitate mulitplexed biosensing. A theoretical model was built to investigate the efficacy of using a multi-channel detector analogous to a conventional RGB camera. The results of the model indicated that even in the best case, the detection capabilities of such a system did not provide advantages over the microspectroscopic approach.
We presented a novel hyperspectral detection scheme we term Dual-Order Spectral Imaging (DOSI) which is capable of simultaneously measuring spectra of up to 160 individual regions within a microscope's field of view. This technique was applied to measuring shifts of individual nanoparticles and was found to have a peak measurement uncertainty of 1.29 nm, at a measurement rate of 2-5 Hz.
Item Open Access Mapping Sensitivity of Nanomaterial Field-Effect Transistors(2020) Noyce, Steven GaryAs society becomes increasingly data-driven, the appetite of individuals, corporations, and algorithms for data sources swells, strengthening the demand for sensors. Chemical sensors are of particular interest as they provide highly human-relevant information, such as DNA sequences, cancer biomarker concentrations, blood glucose levels, antibody detection, and viral testing, to name a few. Among the most promising transduction elements for chemical sensors are nanomaterial field-effect transistors (FETs). The nanoscale size of these devices allows them to operate using very small sample sizes (an extremely small volume of patient blood, for instance), be strongly influenced by low concentrations of the target chemical, and be produced at low-cost, potentially using the same methods developed for consumer electronics (which have achieved a cost of less than 0.000001 cents per device). Nanomaterial FET-based chemical sensors also have the advantage of directly transducing a chemical presence or change to an electrical output signal. This avoids components such as lasers, optics, fluorophores, and more, that are frequently used as a part of the transduction chain in other types of chemical sensors, adding size, complexity, and cost. Much work has focused on demonstrating one-off nanomaterial FET-based sensors, but less work has been done to determine the underlying mechanisms that lead to sensitivity by mapping sensitivity against other variables in experimental devices. With challenges of consistency and reproducible operation stifling progress in this field, there is a significant need to improve understanding of nanomaterial-based FET sensitivity and operation mechanisms.
The work contained in this dissertation maps the sensitivity of nanomaterial FETs across a range of parameters, including space, time, device operating point, and analyte charge. This mapping is performed in an effort to yield insight into the underlying mechanisms that govern the sensitivity of these devices to nearby charges. In order to both draw comparisons between different device types and to make the results of this work broadly applicable to the field as a whole, four types of devices were studied that span a broad range of characteristics. The device types spanned from channels of one-dimensional nanotubes to three-dimensional nanostructures, and from partially printed fabrication to cleanroom-based nanofabrication. Specifically, the devices explored herein are carbon nanotube (CNT) FETs, molybdenum disulfide (MoS2) FETs, silicon nanowire FETs, and carbon nanotube thin-film transistors (CNT-TFTs). Fabrication processes were developed to build devices of each of these types that are capable of undergoing long-term electronic testing with reliable contact strategies. Passivation schemes were also developed for each device type to enable testing in solution and formation of solution-based sensors so that results could be extended to the case of biosensors. An automated experimentation platform was developed to enable tight synchronization between characterization instruments so that each variable impacting device sensitivity could be controlled and measured in tandem, in some cases for months on end.
Many of the obtained results showed similar trends in sensitivity between device types, while some findings were unique to a given channel material. All tested devices showed stability after a period of drain current settling caused by the occupation equilibration of charge trap states – an effect that was found to severely reduce sensitivity and dynamic range. For CNTs specifically, two new decay modes were discovered (intermediate between device stability and breakdown) along with respective onset voltages that can be used to avoid them. For CNT-TFTs, it was found that the relationship between signal-to-noise ratio (SNR) and device operating point remained consistent between ambient air and solution environments, indicating that this relationship is governed primarily by properties of the device. A simple chemical sensor made from the same devices showed a clear peak in the SNR near the device threshold voltage – a result that became increasingly meaningful when combined with similar observations in other device types obtained via separate experimental methods.
For both silicon nanowire and MoS2 FETs, sensitivity was mapped in space with sub-nanometer precise control over analyte position. Both device types manifested distinct sensitivity hotspots spread across the geometry of the channel. These hotspots were found to be stable in time, but their prominence depended heavily on the device operating point. When SNR was mapped across a range of operating points for these devices, a clear peak was discovered, with the hotspot intensity culminating at the peak. Ideal operating points were identified to be near the threshold voltage for both device types, with findings (and a developed numerical model) in MoS2 indicating that the operating point where SNR is maximized may depend upon the extent of the channel that is influenced by the analyte. Observations from multiple devices and approaches revealed that SNR peaks below the point of maximal transconductance, offering increased resolution to a matter that has previously been of some debate in the literature. In MoS2 FETs, a significant asymmetry was discovered in the response of devices to analytes of opposing polarity, with analytes that modulate devices toward their off-state eliciting a much larger response (and, correspondingly, SNR). This asymmetry was confirmed by a numerical model that suggested it to be a general result applicable to all FET-based charge detection sensors, leading to the recommendation that sensor designers select FETs that will be turned off by the target analyte.
Each finding contributed by this dissertation provides insight into future sensor designs and increases clarity of the underlying mechanisms leading to sensitivity in nanomaterial FET-based sensors. The discovery of decay modes, hotspots, ideal operating points, asymmetries, and other trends comprise substantial scientific advancements and propel the field closer to the goal of providing ubiquitous access to critical information, diagnoses, and measurements that promptly and correctly inform decisions.
Item Open Access Molecular Imaging and Sensing Using Plasmonic Nanoparticles(2010) Crow, Matthew JamesNoble metal nanoparticles exhibit unique optical properties that are beneficial to a variety of applications, including molecular imaging. The large scattering cross sections of nanoparticles provide high contrast necessary for biomarkers. Unlike alternative contrast agents, nanoparticles provide refractive index sensitivity revealing information regarding the local cellular environment. Altering the shape and composition of the nanoparticle shifts the peak resonant wavelength of scattered light, allowing for implementation of multiple spectrally distinct tags. In this project, nanoparticles that scatter in different spectral windows are functionalized with various antibodies recognizing extra-cellular receptors integral to cancer progression. A hyperspectral imaging system is developed, allowing for visualization and spectral characterization of cells labeled with these conjugates. Various molecular imaging and microspectroscopy applications of plasmonic nanoparticles are then investigated. First, anti-EGFR gold nanospheres are shown to quantitatively measure receptor expression with similar performance to fluorescence assays. Second, anti-EGFR gold nanorods and novel anti-IGF-1R silver nanospheres are implemented to indicate local cellular refractive indices. Third, because biosensing capabilities of nanoparticle tags may be limited by plasmonic coupling, polarization mapping is investigated as a method to discern these effects. Fourth, plasmonic coupling is tested to monitor HER-2 dimerization. Experiments reveal the interparticle conformation of proximal HER-2 bound labels, required for plasmonic coupling-enhanced dielectric sensing. Fifth, all three functionalized plasmonic tags are implemented simultaneously to indicate clinically relevant cell immunophenotype information and changes in the cellular dielectric environment. Finally, flow cytometry experiments are conducted utilizing the anti-EGFR nanorod tag to demonstrate profiling of receptor expression distribution and potential increased multiplexing capability.
Item Open Access Plasmonic Nanoplatforms for Sensing, Diagnostics, and Therapy(2020) Crawford, BridgetRecent advances in nanotechnology have led to the application of nanoparticles in a wide variety of fields. In particular, anisotropic nanoparticles have shown great potential for surface-enhanced Raman scattering (SERS) detection due to their unique optical properties. Gold nanostars are a type of anisotropic nanoparticle with one of the highest SERS enhancement factors in a non-aggregated state. By utilizing the distinct characteristics of gold nanostars, new plasmonic materials for sensing, diagnostics, and therapy can be synthesized. The work described herein is divided into two main themes. The first half demonstrates the development and application of a novel label-free inverse molecular sentinel (iMS) nanoprobe for detection of microRNA biomarkers related to cancer progression as well as those related to gene expression in plants. This work also describes the initial proof-of-concept for a SERS-based electrowetting-on-dielectric (EWD) digital microfluidic platform as a diagnostic platform requiring samples of nanoliter volume. The second half demonstrates the utility of plasmonic nanoparticles for SERS imaging as well as photothermal therapy (PTT) and photodynamic therapy (PDT).
Development of accessible strategies for efficient detection of nucleic acid biomarkers is a major unmet need for applications ranging from cancer screening to agricultural biotechnology and biofuel development. MicroRNAs (miRNAs) have great promise as a new important class of biomarkers for early detection of various cancers; however, these small molecules have not been adopted into early diagnostics for clinical practice because of challenges adapting complex laboratory techniques into accessible clinical tests. In a blinded study, the surface-enhanced Raman scattering (SERS)-based plasmonics-active nanoprobes described herein, referred to as inverse molecular sentinels (iMS), demonstrated diagnostic accuracy for in vitro identification of endoscopic biopsy samples as tumor, Barrett’s esophagus or normal tissue via miRNA detection. The iMS nanoprobe technology can be designed to detect a wide range of nucleic acids for a variety of applications. In addition to medical applications, the knowledge over gene expression dynamics and location in plants is crucial for applications ranging from basic biological research to agricultural biotechnology. However, current methods are unable to provide in vivo dynamic detection of genomic targets in plants, due to the complex sample preparation needed by current methods for nucleic acids detection, which disrupt spatial and temporal resolution. We have developed a multimodal technique utilizing iMS nanoprobes for in vivo imaging and biosensing of microRNA biotargets within whole plants. This work lays the foundations for in vivo functional imaging of RNA biotargets in plants with previously unmet spatial and temporal resolution.
The prevalence of cancer has increasingly become a significant threat to human health and as such, there exists a strong need for developing novel methods for early detection and effective therapy. Gold nanostars (AuNS) with tip-enhanced plasmonics have become one of the most promising platforms in photothermal therapy (PTT) as they exhibit superior photon-to-heat conversion efficiency and can be delivered specifically to tumors. We have demonstrated that AuNS are endocytosed into multiple cancer cell lines irrespective of receptor status or drug resistance and allow for the effective photothermal ablation of tumor cells. Additionally, we demonstrate a unique in vitro preclinical model that mimics the tumor structures assumed by inflammatory breast cancer (IBC) in vivo. IBC has a unique presentation of diffuse tumor cell clusters called tumor emboli. AuNS are able to penetrate the tumor embolic core in 3D culture, allowing effective photothermal ablation of the IBC tumor emboli.
Additionally, we have furthered the development of the gold nanostar treatment platform by developing a theranostic nanoconstruct that consist of Raman-labeled gold nanostars coated with a silica shell that is loaded with photosensitizer molecules for PDT. The outer surface of the nanoconstruct was functionalized for targeting to allow for specific treatment of folate positive breast cancer. SERS detection and PDT are performed at different wavelengths, so there is no interference between the diagnostic and therapeutic modalities. Singlet oxygen generation (a measure of PDT effectiveness) was demonstrated from the drug-loaded nanocomposites. In vitro testing demonstrated the effectiveness of the nanoconstruct for targeted PDT.
Item Open Access The metabolic regulation of anchor cell invasion through basement membrane in C. elegans(2022) Garde, AasthaBasement membranes (BM) are dense, highly crosslinked sheets of extracellular matrix proteins that surround and constrain cells in animal tissues. Specialized cells acquire the ability to invade through BM barriers during development and homeostasis, and aberrant BM invasion underlies many diseases. Invading cells use transient and specialized cellular protrusions to breach the BM, and the membrane dynamics and cytoskeletal rearrangements necessary to build and fuel these structures are both energy intensive and metabolically complex. Thus, it is crucial to understand how invasive cells regulate their catabolic and anabolic metabolism to drive BM invasion, but experimentally dissecting stochastic cell invasion events that occur deep within optically inaccessible tissues in vivo is challenging. Here I use the C. elegans anchor cell (AC) as an experimentally tractable and visually accessible in vivo model for cell invasion through the BM, and use 4D live cell imaging , metabolic biosensors, and RNAi-mediated screening to investigate how invading cells regulate their ATP production and lipid metabolism to drive invasion through the BM. In Chapter 1, I review the mechanisms used by cells to fuel invasion through matrix and identify gaps in our understanding of localized energy production during invasion. In Chapter 2, I discover that localized glucose import, and glycolytic processing support rapid and transient ATP production by mitochondria in the AC to fuel the invasive protrusions for BM invasion. In Chapter 3, I identify that sphingolipid biogenesis and protein prenylation support the formation of the invasive protrusion and the actin-based invasion machinery within in to breach the BM barrier. In Chapter 4, I discuss the implications of these findings on our understanding of the metabolism of cells invading through the BM.