Mountain Scholar :: Browsing by Author "Henry, Charles S., advisor"

Browsing by Author "Henry, Charles S., advisor"

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Open Access
Advancing point-of-need bacteria detection using microfluidic paper-based analytical devices
(Colorado State University. Libraries, 2018) Boehle, Katherine Elizabeth, author; Henry, Charles S., advisor; Krummel, Amber T., committee member; Geiss, Brian J., committee member; Ackerson, Christopher J., committee member
Bacteria are responsible for more hospitalizations and deaths than any other foodborne contaminant, making the detection of these pathogens of utmost importance. To further complicate bacteria detection, the overuse of antibiotics and genetic plasticity of bacteria has caused antimicrobial resistant (AMR) bacteria to become a more prevalent issue that threatens to be the number one cause of death worldwide by 2050 unless significant innovations are made. Although bacteria detection in the field is ideal, the current gold standards for detection require trained personnel and a central laboratory. The primary work in this dissertation acts to improve upon current bacteria detection methods by designing, developing, and optimizing inexpensive user-friendly tests that detect bacteria at the point-of-need without trained personnel or expensive equipment. These goals are accomplished using microfluidic paper-based analytical devices (μPADs), a growing field for point-of-need detection that have been used for a variety of analytes and applications. Using paper as a platform has allowed for the simple development of user-friendly devices because of their easily designed and modifiable material that typically costs <$0.01 USD per device and allows for multiple tests to be completed from one sample addition. Devices that will be described include colorimetric spot tests that detect common fecal indicator bacteria (FIB) species Escherichia coli and Enterococci spp. based on enzymes that are naturally produced by the bacteria. Utilizing these enzymes, a test was developed that turns from clear to yellow as an indication of live bacteria. These tests were successfully used in the detection of bacteria in food and water samples to demonstrate its efficacy in food safety applications. To improve specificity and sensitivity of bacteria detection, a second spot test was developed that utilizes immunomagnetic separation (IMS) and an enzymatic sandwich immunoassay in the detection of another common foodborne pathogen, Salmonella typhimurium. This assay was developed specifically for detecting pathogens in complex matrices, such as one of the most common causes of pathogen contamination: animal feces. Because AMR bacteria are becoming a more prevalent problem, devices were developed to specifically detect bacteria resistant to β-lactam antibiotics, the most common case of antimicrobial resistance observed in bacteria. The first generation of devices were developed to detect β-lactamase activity, an enzyme that facilitates resistance against β-lactam antibiotics. These devices were successful in detecting AMR in different species of bacteria isolated from environmental samples, and in the detection of AMR in sewage water. The second generation of devices enables detection of resistance against specific antibiotics through hydrolysis of the antibiotic and detecting a change in pH. Although not yet demonstrated, these devices will eventually be used to determine if bacteria are resistant against specific classes of β-lactam antibiotics, including a commonly used class of last resort antibiotics, carbapenems. Beyond bacteria detection, this dissertation also explores developing a field-ready device to identify falsified and substandard antibiotics. Because antibiotics are most commonly counterfeited in resource-limited settings, it is imperative to develop user-friendly point-of-need devices that can quantify the amount of active pharmaceutical ingredient in antibiotics. This was accomplished using enzyme competition, a method that had not been demonstrated paper-based devices. Finally, all devices that have been developed and optimized in this dissertation utilized colorimetric detection. While a user-friendly and easily implemented method of detection, it does suffer from drawbacks such as sensitivity and user subjectivity when using the devices. To eliminate subjectivity, a portable system using a Raspberry Pi computer and 3D-printed light box and device holder have been optimized. Although the system has been demonstrated by automatically analyzing images and calculating Michaelis-Menten enzyme kinetic values, this system has limitless possibilities in automatically analyzing colorimetric paper-based devices for truly objective colorimetric readouts and quantitative infield detection of pathogens or other analytes.
Open Access
Assay development for pathogen detection at the point-of-need
(Colorado State University. Libraries, 2020) Carrell, Cody S., author; Henry, Charles S., advisor; Farmer, Delphine K., committee member; McNally, Andrew, committee member; Reardon, Kenneth F., committee member
Infectious diseases are responsible for roughly one third of worldwide deaths, which disproportionately occur in low- and middle-income countries. Government health agencies recognize high quality diagnostics as a key tool to slow the spread and reduce the burden of disease in these countries. The same diagnostics that have minimized deaths from infectious disease in developed nations, however, cannot simply be implemented in all locations. Low- and middle-income countries lack the financial resources and infrastructure required to use the sophisticated instruments found in modern hospital laboratories. Instead of relying on current diagnostic technologies to reduce the burden of infectious disease, there is an urgent need to develop new technologies suited for the resource-limited settings they will be used in. The work described in this thesis aims to advance the capabilities of diagnostic sensors for use at the point-of-need. Microfluidic devices have been used for decades to perform complex analysis using compact devices with small sample and reagent volumes. Their portability and low-cost make them ideal candidates for analysis in resource limited settings, but their fabrication is tedious and expensive. To improve the fabrication process, Chapter Two of this thesis describes two methods for simplified 3D-printing of microfluidic devices. The 3D printer and resin used are inexpensive and commercially available and the fabrication process is not limited by the need to remove uncured resin from enclosed channels. Instead, open-faced channels in 3D-printed pieces were silanized and sealed to a secondary substrate. Common microfluidic devices including a droplet generator and herringbone mixer were created with the new fabrication method to demonstrate the strength of the seal and ability for the printer to create microfluidic channels. We envision this method being used for rapid prototyping and increased innovation in the field of microfluidic sensors. Traditional polymer microfluidics are limited in their usefulness in point-of-need situations because they require a pump to drive flow. Paper-based microfluidics use capillary action to drive flow instead of a pump and have emerged as an easy-to-use and inexpensive alternative to traditional microfluidics in situations where a power source is not available. However, paper-based microfluidics often suffer from poor analytical performance, and efforts to improve result in increased complexity. Chapter Three of this thesis describes a paper-based device that increases the sensitivity of a Salmonella assay while retaining ease-of-use. The device combines paper-pads for reagent storage with a 3D-printed rotational manifold to perform an enzyme-linked immunosorbent assay (ELISA). Typically, this assay requires dozens of complex pipetting steps, but the rotary device simplifies this process into four semi-automated steps. A detection limit of 440 colony forming units/mL was found using the paper-based device. As demonstrated in Chapter Three, common issues with paper-based microfluidics can be solved by integrating paper with other inexpensive components like 3D-printed polymer. In the final study in Chapter Four, we created a device to further simplify the steps of an ELISA using a combination of paper, polyester transparency film, and double-sided adhesive. The device, termed a disposable ELISA (dELISA), automatically performed the sequential reagent delivery and washing steps required for a traditional ELISA and require only two end user steps. The dELISA was then used to perform a serology assay for SARS-CoV-2 antibodies from whole-blood. The detection limit of the assay was 2.8 ng/mL for the dELISA, which was nearly identical to the detection limit found using a tradition well-plate assay (1.2 ng/mL).
Open Access
Bioanalytical applications of capillary electrophoresis and microfluidics: from metabolomics to biofuels
(Colorado State University. Libraries, 2010) Holcomb, Ryan E., author; Henry, Charles S., advisor
Capillary electrophoresis (CE) and related microfluidic technologies are increasingly being utilized as state of the art analysis tools in the field of bioanalytical chemistry. The following dissertation highlights selected applications of CE and microfluidics for metabolomics and microalgal-based biofuels research. Metabolomics research focused on targeted metabolic profiling and fingerprinting of biofluids using both conventional and microchip CE. Metabolite analysis in biofluids was of interest as this can be a useful clinical tool for monitoring disease states and treatment efficacy. Initial work in this area focused on targeted metabolic analysis of the cardiovascular disease biomarker homocysteine (Hcys). In this work, serum Hcys was analyzed using microchip CE (MCE) coupled with pulsed amperometric detection. Using this system, Hcys could be resolved from other electrochemically active serum components in under a minute by employing appropriate separation conditions. Following this targeted metabolic analysis, research shifted to a more comprehensive metabolic fingerprinting study of dogs undergoing chemotherapy for diffuse large B cell lymphoma. Urine samples from diseased and non-diseased dogs were obtained at various clinical time points and analyzed using CE with UV detection. The resulting fingerprints were compared for differences in metabolite make-up using multivariate statistical techniques. In an attempt to conduct this type of research at the microscale, a MCE device was developed with an integrated electrode array detector for resolving the multiple components present in biological samples. Selective detection and electrochemical resolution of co-migrating analytes could be facilitated with this device via judicious choice of detection potential at the multiple working electrodes. Improvement in detection capability of this system compared to single electrode MCE systems should allow for its use in rapid metabolic fingerprinting and profiling analyses. The final area of research presented in this dissertation involved use of microfluidics for culturing and screening cellular lipid accumulation in microalgae exposed to various environmental stressors. A microfluidic device was developed which contained integrated valves for facilitating cell culture and conducting imaging assays on-chip. Lipid accumulation in stressed microalgae was determined using fluorescence microscopy techniques. Additional experiments were conducted using gas chromatography to determine the types of lipids being accumulated in these stressed microalgae.
Open Access
Characterization and modification of carbon composite electrodes towards more affordable biosensing applications and integration into fluidic devices
(Colorado State University. Libraries, 2022) Clark, Kaylee M., author; Henry, Charles S., advisor; Van Orden, Alan, committee member; Prieto, Amy L., committee member; Volckens, John, committee member
Fast, accurate, and low-cost medical tests and platforms for biomolecule monitoring are essential to the diagnosis, management, and treatment of many diseases. Electrochemical detection allows for highly sensitive measurements with fast response times. Carbon composite electrodes are an attractive option for electrochemical detection due to their low cost, resistance to biological fouling, large electrochemical solvent windows, and ability to be patterned. However, they often suffer from poor electrocatalytic activity, inability to be molded, and need for complex modifications to effectively detect certain analytes. Combining electrochemistry with fluidics is attractive for a wide array of applications including multiplexing, automation, and high-throughput screening. However, fabrication of electrochemical fluidic devices with integrated carbon electrodes remains a challenge. Thermoplastic electrodes (TPEs) are a new class of composite electrodes discussed in this dissertation that exhibit superior electrochemical properties to typical carbon composite electrodes and can be easily molded into intricate structures. Overall, this dissertation aims to improve carbon composite materials for biosensing applications and integration of electrochemical sensors into fluidic devices. Chapter 2 introduces polycaprolactone (PCL) as a new binder material for TPEs and focuses on the electrochemical characterization of the new material. The PCL-based TPEs have excellent electrochemical activity towards a wide range of analytes as well as high electrical conductivity. Chapter 2 also introduces a simple technique for integrating PCL and carbon composite electrodes into microfluidics. The presented electrode-integrated microfluidic devices are quickly fabricated with a laser cutter using PCL as a bonding layer. As a proof-of-concept application, water-in-oil droplets are electrochemically analyzed. Chapter 3 focuses on use of PCL-based TPEs for enzymatic sensors. The simple fabrication of TPEs also allows catalysts and enzymes to be mixed directly into the material to enhance detection. In Chapter 3, the TPE material is bulk-modified with cobalt phthalocyanine, an electrocatalyst, and glucose oxidase, resulting in a robust glucose sensor that demonstrates long-term current response stability. These sensors can be molded into intricate shapes and sanded for surface renewal (without requiring additional steps to maintain the modification), allowing the sensors to be continuously reused even if damaged or fouled. Chapter 4 investigates the properties of TPEs using two different binders – polycaprolactone (PCL) and polystyrene (PS) – with sanded and heat-pressed surface treatment. XPS and SEM analysis suggested that sanded TPEs have a higher density of graphitic edge planes and improved electrochemistry as a result. Electrochemical detection of O2 and H2O2, which are typically difficult to detect on carbon composites without complex modification, was demonstrated on sanded PS-based TPEs. Additionally, Chapter 4 introduces a new 3D-printed TPE sensor module that is reversibly sealed with magnets. A proof-of-concept sensor for detecting H2O2 in flow with the sensor module is presented. Chapter 5 presents a low-cost flow device, made of inexpensive polyethylene terephthalate (PET) and adhesive films, developed to detect SARS-CoV-2 nucleocapsid (N) protein. Upon addition of a sample in the device, reagents and washes are sequentially delivered to an integrated screen-printed carbon electrode for detection thus automating a full sandwich immunoassay with a single end-user step. The modified electrodes are sensitive and selective for COVID-19 N protein and stable for over seven weeks. The flow device was also successfully applied to detect nine different SARS-CoV-2 variants, including Omicron. In summary, this dissertation presents work to improve carbon composite electrodes, their modification, and integration into fluidic devices for applications as biosensors and beyond. The TPEs presented show improved electrochemical and physical properties, that allow for simple modifications. This work also demonstrates simple electrode integration strategies in several types of fluidic devices for easier and more sensitive detection of biologically relevant analytes. Moreover, the platforms established in this dissertation can be easily adapted for a wide variety of analytes and applications. This work provides materials, methods, and platforms to create more affordable biosensors for medical and other biological sensing.
Open Access
Coupling electrochemistry and microfluidics for biosensor development
(Colorado State University. Libraries, 2016) Feeny, Rachel M., author; Henry, Charles S., advisor; Van Orden, Alan, committee member; Sambur, Justin B., committee member; Kipper, Matthew J., committee member
Biosensors are valuable analytical tools across both scientific and medical fields. Improving and miniaturizing biosensors is an area of great interest in academic, medical, and diagnostic settings. There is a constant need to improve these systems by increasing accessibility through lower costs, greater portability, and enhanced ease-of-use. Interfacing microfluidics and electrochemical methods shows great potential to address these needs. The work described in this thesis aims to address gaps in biosensor development, including increasing accessibility and improving sensing capabilities such as sensitivity, selectivity, and resolution, by combining electrochemistry and microfluidics to develop new tools for use in a range of biosensor systems. The primary focus of this research was to couple electrochemical detection methods with microfluidic devices for bioanalytical applications. Two main topics are reported. The first is a fluid transport mechanism employing the gas permeability of poly(dimethylsiloxane) (PDMS). Degassed PDMS pumps provide a simple, portable, inexpensive method to generate controlled fluid flow in a microfluidic device to transport a sample to electrodes for electrochemical detection. The second topic reported does not aim to address cost or portability of the system, but rather focuses on improving the capabilities of electrode arrays as chemical imaging platforms. In this work, a platinum microelectrode system was developed for biomarker detection, primarily nitric oxide and norepinephrine. Microfluidic devices interfaced directly with the electrodes provided precise control of fluid delivery to the sensors enabling both localized control of chemical concentrations as well as selective chemical stimulation of living tissue. The microelectrodes, when arranged in a high-density array, provided a platform capable of achieving electrochemical biomarker detection and imaging from live tissue slices with high spatiotemporal resolution. Both technologies described required the effective interfacing of microfluidic devices with electrochemical sensors to generate biomarker detection platforms. Custom microfluidic systems were developed to directly integrate biological samples into the platforms, including dried serum spots on a filter paper matrix and live ex vivo murine adrenal slices embedded in agarose. To achieve reproducible biomarker detection in complex biological matrices, electrochemical cleaning methods were developed and utilized for electrode maintenance. All of the tools described in this thesis were designed to address specific applications, but were also intended to be translatable to other systems. The degassed PDMS pump could be used as a fluid transport mechanism for other microfluidic devices, improving the simplicity and portability of systems that could otherwise be limited by external pumping equipment. Similarly, the strategies described for interfacing microfluidic devices with the reported electrode arrays and platinum microelectrodes could be applied to other silicon microchips to accomplish precise control of fluid delivery to the electrodes. The technology developed to generate an electrochemical imaging platform could be further pursued to achieve a high level of chemical selectivity by employing alternative waveforms, such as fast scan cyclic voltammetry, or electrode modifications to better elucidate the role of chemical gradients in biological systems. All of these tools, when applied to other bioanalytical platforms, could continue to advance the field of biomarker detection using microfluidic systems.
Open Access
Detection of disease biomarkers using a novel multi-analyte immunoassay
(Colorado State University. Libraries, 2007) Caulum, Meghan M., author; Henry, Charles S., advisor
Biomarkers provide clinicians with an important tool for disease assessment. Many different biomarkers have been discovered making it readily apparent that no single biomarker can be relied upon for accurate disease detection which has led towards the push for new multi-analyte screening methods. There are, however, currently few inexpensive multi-analyte methods that can use these biomarkers for disease detection in a point-of-care setting. This dissertation details the development of a novel immunoassay which bridges the gap between traditional immunoassays and high density microarrays by utilizing microfluidics, immunoassays and micellar electrokinetic chromatography (MEKC). This chemistry, the Cleavable Tag Immunoassay (CTI), is a low- to medium-density heterogeneous immunoassay designed to detect up to 20 analytes simultaneously. The multi-analyte CTI is shown to be a useful tool for the detection and quantification of cardiac biomarkers in serum samples. Limit of detection and linear range are reported for all of the biomarkers with LODs on the order of low ng/mL to low pg/mL. The linear range for each of the biomarkers spans the boundary between normal and elevated levels. In addition, the use of a combination of two surfactants in the run buffer which form mixed micelles is shown to improve resolution of CTI tags. Preliminary studies of cleavage kinetics using single particles for an integrated CTI analyzer are also presented including demonstration of the ability to trap and release magnetic particles in a microchip device. Two methods for improving separation efficiency of poly(dimethylsiloxane) including the use of polyelectrolyte multilayers and a simple and effective way to generate a stable hydrophilic glass-like surface for use with microchip CE-EC and CE-fluorescence are also presented. The outcome of this dissertation demonstrates the marriage of immunoassays and microchip MEKC in the CTI shows promise as a new medium-density chemistry for rapid biomarker detection. This work is the first step towards the development of an integrated assay for rapid point-of-care analysis of AMI.
Open Access
Developing high-performance microfluidic paper-based analytical devices
(Colorado State University. Libraries, 2018) Nguyen, Michael Paul, author; Henry, Charles S., advisor; Van Orden, Alan, committee member; Strauss, Steven H., committee member; Marchese, Anthony J., committee member
Small-scale systems to manipulate fluids, also referred to as microfluidics, have proven effective at reducing analytical costs by increasing the portability of diagnostic devices. Microfluidic paper-based analytical devices (μPADs) have also proven to be cost-effective while remaining disposable, possessing the capacity to store reagents, and producing quantitative diagnostic results. These benefits have lead the field to increase exponentially since the seminal publication, with 63 review articles currently published on the subject. Most articles in the field focus on three topics: 1) new applications, 2) new methods of analysis with broad applicability, and 3) new ways to manipulate fluids in devices. A host of new analytes and clever architectures are being developed for a variety of applications, including environmental analysis and diagnostics. However, several critical obstacles remain for μPADs including improving detection limits, reducing analysis time, increasing selectivity, and increasing the range of measurable analytes. The work described in this dissertation presents three studies that address these issues. The first study examines simple factors to improve sample delivery through a cellulose channel that directly and significantly impact detection limits. Here I show how common μPAD designs lose roughly 50% of sample prior to quantification. This major challenge has been solved through geometry changes that led to a 94% increase in signal when compared to standard designs. While Ni(II) detection was used to study the system, the methods are translated to Mn(II) detection, antibiotic purity tests and determination of nitrite in saliva suggesting the broad applicability of the methods. The second study aimed at decreasing analysis time by utilizing multiple layers of paper in μPADs. I present the ability to tune speed, distance, and time at which the fluid travels with the formation of a microchannel between the layers. By increasing both the number of paper layers and the distance between them, the solution flux is dramatically increased in agreement with theoretical predictions. However, experimental flow rates deviate from predictions at large spacings. The detailed characterization and current understanding of the fast flow properties allow us to design assays that take seconds to complete instead of minutes along with improved analytical performance.Developing a selective test for Al(III) in food, mining and water samples is the goal of the last study. To address this need, a fluorescent ligand selective for Al(III) was synthesized and characterized on a μPAD for the first time. A distance-based μPAD for Al(III) exhibited a linear response from 2–55 ppm and a limit of detection of 2 ppm. This chemistry was also further developed with a radial uPAD that measures diameter of a color response as opposed to distance. Despite a smaller linear range with this radial device, the limit of detection is 0.9 ppm, which is below the concentration relevant to plant health. All three of these studies highlight improving the analytical performance of μPADs with carefully selected assays and deliberate device design.
Open Access
Developing paper-based devices for mapping agricultural pesticides and environmental contaminants
(Colorado State University. Libraries, 2021) Menger, Ruth F., author; Henry, Charles S., advisor; Borch, Thomas, advisor; Ravishankara, A. R., committee member; Neilson, James R., committee member; Trivedi, Pankaj, committee member
The detection of environmental contaminants is important to ensure the health of both humans and the environment. Currently, detection is done by instrumentation like liquid or gas chromatography coupled with mass spectrometry. While sensitive and selective for multiple analytes, these instruments suffer from disadvantages like large size, high sample cost, and the need for a trained analyst to run the samples. As an alternative, microfluidic paper-based analytical devices (µPADs) are becoming more common as inexpensive, fast, easy to use devices to detect and quantify a variety of analytes. My research has been focused on developing µPADs for three different analytes: pesticides, PFAS, and heavy metals. In order to ensure proper crop protection and pest management, it is important to manage and optimize pesticide application. Currently, this is done by water-sensitive papers, which often inaccurately portray the presence of pesticide due to humidity and extraneous water droplets that are not pesticide. In Chapter 2, I have developed a method that uses filter paper to capture a fluorescent tracer dye that has been mixed with the pesticide and then sprayed over the crop. The filter papers are imaged with a lightbox and Raspberry Pi camera system and then analyzed to determine percent coverage. After optimization and validation of the method to WSP, the filter paper method was used to evaluate pesticide distribution in a citrus grove in Florida (Chapter 3). The data from these field studies was used to make recommendations for which application method is best for the different types of pesticides. Paper-based devices are inherently limited by the inability to control fluid properties like mixing. In order to incorporate mixing but also retain a small device that does not require external power to initial flow, a microfluidic device was fabricated out of two glass slides. A staggered herringbone pattern is laser ablated into the slides, and a channel is formed by double-sided adhesive (Chapter 4). Mixing was quantified using blue and yellow dyes. A reaction between horseradish peroxidase and hydrogen peroxide was used as a representative enzymatic reaction and also to determine enzyme kinetics. Since the microfluidic device is made of glass, it is also compatible with non-aqueous solvents. Paper-based devices do not work well with organic solvents because the hydrophobic wax on the paper is dissolved by the solvent. In Chapter 5, the dissertation returns to traditional µPADs for environmental contaminants. Per- and polyfluoroalkyl substances (PFAS) are class of compounds that are highly persistent, toxic, bioaccumulative, and ubiquitous. While multiple instrument-based methods exist for sensitive and selective detection in a variety of matrices, there is a huge need for a fast, inexpensive, and easy-to-use sensor for PFAS detection. This would enable widespread testing of drinking water supplies, ensuring human health. A µPAD was developed for the detection of perfluorooctane sulfonate (PFOS) where the ion-pairing of PFOS and methylene green forms a purple circle. The diameter of the purple circle can be measured by the naked eye with a ruler or with the help of a smartphone to correlate the diameter back to PFOS concentration. At a cost of cents per sample, this µPAD enables fast and inexpensive detection of PFOS to ensure safe drinking water. A common issue with environmental µPADs is the relatively high limits of detection compared to what is needed for regulatory purposes. It can be challenging to lower the limits of detection without incorporating an external pretreatment and/or preconcentration step. As µPADs are small and handle only a small volume of sample (<120 µL), there is the possibility of increasing the sample capacity of the device but without significantly increasing the device size or analysis time. By adding multiple layers of absorbent filter paper underneath radial device for heavy metal detection, the sample volume increased to 1 mL, decreasing the limit of detection for a radial copper detection card from 100 ppb to 5 ppb (Chapter 6). The research presented here achieves the goal of developing µPADs for environmental contaminants. They can be used in different ways to visualize the presence of the contaminant for monitoring and management purposes, ultimately ensuring human and environmental health.
Open Access
Development of a microchip electrophoresis environmental monitoring system: from surface to separation chemistry
(Colorado State University. Libraries, 2008) Dressen, Brian M., author; Henry, Charles S., advisor
Environmental contaminants are an increasing problem in today's modern world. Methods of incorporation into the human body are numerous, but perhaps the most common is ingestion with food or drinking water. Of more recent concern is perchlorate in drinking water. Human exposure to perchlorate is of concern because of the potential for impaired thyroid function, leading to a number of developmental delays and other medical problems. Its prevalence in the environment only gained interest in the late 1990's, once a method was developed for its detection at the 4 ppb level. In 2005 after being added to the Environmental Protection Agency's (EPA) Unregulated Contaminant Monitoring Rule (UCMR1) list, a sampling of 2800 large water systems and 800 smaller systems, representing less than 10% of all US water systems, revealed contamination in 153 sites over 25 states. Numerous methods of analysis exist relying on expensive bench-top analysis devices that require long analysis times. This dissertation details the development of a perchlorate sensor capable of sub ppb detection limits and 4 min analysis times, with a total cost of less than $10K. The size of the sensor device and supporting equipment lends itself to portability for near-real-time in-field monitoring. This device combines microchip capillary electrophoresis with sensitive detection technology to reliably separate and detect perchlorate in surface and waste water samples. Here I describe the comprehensive development of the microchip system including control of surface chemistry, flow magnitude and direction, bulk material selection, and chemical selectivity to allow for the separation of perchlorate in water. Optimization of the microchip system, including the use of a selective zwitterionic surfactant to enhance on-chip separations is also presented. The final outcome of this dissertation work is a near-portable environmental sensor for perchlorate capable of meeting EPA regulations for sensitivity to perchlorate as well as several advances in controlling the chemistry within microchip electrophoresis devices through modification or bulk material selection.
Open Access
Development of electrochemical imaging methods using micro-electrode arrays and microfluidic networks
(Colorado State University. Libraries, 2016) Wydallis, John B., author; Henry, Charles S., advisor; Van Orden, Alan, committee member; Barisas, B. George, committee member; McNaughton, Brian R., committee member; Dandy, David S., committee member
Distribution of molecules over space and time drive a multitude of macroscopic and microscopic biological processes. There is a need to design novel imaging techniques that can map molecular distributions with spatiotemporal resolution. In this thesis, new electrochemical approaches to provide spatiotemporal imaging are presented. The bulk of this work utilizes high-density platinum micro-electrode arrays fabricated using complementary metal oxide semiconductor (CMOS) fabrication techniques as well as microfluidics and carbon-based electrodes fabricated using soft lithography fabrication techniques. The systems described in this dissertation focus on quantification of biologically relevant neurotransmitters, mainly catecholamines and nitric oxide with concentration ranges from nM to mM. The pitch, or resolution between two "pixels" of electrochemical data, was 250 µm for microfluidic based sampling methods and 12.5 µm for the CMOS based sensors. Descriptions of fabrication methods for the carbon based electrodes and CMOS electrodes are described in this work. Finally, potential future directions of this technology is discussed in the final chapter.
Open Access
Development of fluidic devices to facilitate more accessible monitoring of human health
(Colorado State University. Libraries, 2024) Cherwin, Amanda E., author; Henry, Charles S., advisor; Tobet, Stuart A., advisor; Snow, Christopher, committee member; Abdo, Zaid, committee member
In December of 2023, the World Health Organization (WHO) Director-General Tedros Adhanom Ghebreyesus outlined the 'Five P's' of global health priorities: Promoting health, Providing health, Protecting health, Powering health, and Performing for health. Despite the mantra of 'prevention is better than cure,' many countries still prioritize treating the sick over proactive health promotion, leading to inadequate prevention of non-communicable diseases (NCDs). Access to healthcare services poses a significant barrier to early recognition and treatment of health issues, particularly in low-income communities. To address these challenges, harnessing the power of science and technology becomes imperative. Powering health involves leveraging scientific research and collaboration to understand disease mechanisms better. Physiologically relevant models, such as microfluidic systems, offer insights into disease progression. Microfluidics, especially when combined with 2D and 3D culture systems, enhances functionality by mimicking physiological conditions. These devices provide cost-effective solutions for diagnostic challenges, bridging the gap between in vitro and in vivo studies. Protecting health requires a deeper understanding of organ systems. Chapter 2 examines a microfluidic model of the gut, an organ that plays a critical role in maintaining overall health. Two devices are discussed, an organotypic device for maintaining ex vivo gut tissue explants, and an electrochemical sensor module for monitoring relevant molecules such as oxygen or hydrogen peroxide within the tissue media. Dysbiosis in the gut microbiome has been linked to various pathologies, emphasizing the need for accurate models for studying gut barrier integrity. Ex vivo models using microfluidic devices offer promising avenues for studying disease mechanisms. The devices described in Chapter 2 serve as an effective model of the intestinal barrier that can be closely monitored in real-time. Providing health involves making effective healthcare solutions universally accessible. Point-of-care (POC) diagnostics, facilitated by microfluidics, enable rapid and cost-effective disease detection. Capillary-driven flow microfluidic devices enhance accessibility by eliminating the need for bulky external pumps, making POC testing feasible even in resource-limited settings. Combining the concepts of Powering and Providing health leads to the development of innovative diagnostic devices. Capillary-driven flow microfluidics enables the development of portable devices for diagnosing conditions from viscous sample matrices like blood and saliva. These devices offer less invasive and more accessible alternatives to traditional diagnostic methods, potentially revolutionizing healthcare delivery. Chapter 3 describes a capillary flow device used to quantify levels of two salivary biomarkers (Galectin-3 and S100A7) correlated to Heart Failure (HF) outcomes. This rapid, noninvasive, accessible POC test can drastically improve the quality of life for HF patients, particularly in rural and resource-limited areas. Using an electrochemical detection method, we demonstrate successful multiplexed detection of both biomarkers in spiked buffer solutions. Chapter 4 focuses on microfluidic devices probing rheological properties of whole blood related to Sickle Cell Disease (SCD) and clotting using capillary flow. For the SCD device, our goal was to develop a low-cost Point-of-Care (POC) multiplexed device for rapid and accurate identification of SCD phenotypes using three key reagents tied to altered sickle cell blood rheology: calcium chloride, sodium metabisulfite, and adenosine diphosphate. We developed an integrated device where whole blood reacts with reagent pads, enabling rapid assessment of a patient's SCD phenotype to inform appropriate treatment. We also introduced the Paper-based Clotting Analysis Test (PCAT) for efficient, low-cost analysis of primary hemostasis. Current methods for monitoring hemostasis are expensive and slow. Our capillary flow device uses whole blood moving at high flow rates for sustained durations to induce thrombus formation. This dissertation bridges the gap between effective health monitoring and accessibility through fluidic devices using either pump-driven or capillary-driven flow. Chapters detail the development of microfluidic systems for monitoring intestinal barrier function, detecting biomarkers in saliva for Heart Failure prognosis, and processing blood samples for Sickle Cell Disease phenotyping and clotting analysis. Ultimately, these devices hold the potential to transform healthcare management, particularly in underserved communities.
Embargo
Development of low-cost capillary driven immunoassays for improved medical diagnostics
(Colorado State University. Libraries, 2023) Link, Jeremy S., author; Henry, Charles S., advisor; Van Orden, Alan, committee member; Ackerson, Christopher J., committee member; Kipper, Matt J., committee member
Rapid medical diagnostics are a crucial part of an effective healthcare system. While traditional laboratory diagnostic methods are well established and sensitive, they are also time consuming and expensive. Point of care (POC) diagnostics offer an attractive alternative to traditional testing for more affordable, fast results. Their simplicity allows for POC devices to be run quickly by untrained personnel, but the simplicity often limits their detection range and sensitivity. In this dissertation I discuss affordable, capillary-driven immunoassay devices that are capable of passively delivering reagents associated with a traditional well-plate enzyme linked immunosorbent assay (ELISA) to test strips. These devices are made of patterned and laser cut double-sided adhesive. When stacked and laminated together, the patterns cut from the layers form hollow microfluidic channels that can passively transport fluids through capillary action. The devices in this dissertation require only a single end-user step to perform a sandwich immunoassay, and signal from the enzyme/substrate reaction is detectable in under 30 min. Chapter 2 discusses the first application for visual detection of SARS-CoV-2 in these affordable capillary-driven immunoassay devices. The device in this chapter uses the enzyme horseradish peroxidase (HRP) and the substrate 3,3',5,5'-tetramethylbenzydine (TMB) to produce signal at the test line. Upon sample addition, the device channels fill, rehydrating the detection antibody and substrate dried on conjugate release pads that are stored in the channels of the device. Within 20 min, target, reagents, and washing steps are passively delivered to a nitrocellulose test strip containing a capture antibody test line. The device performance was compared to a well-plate ELISA, and the detection limits for inactivated SASR-CoV-2 were 86 PFU/mL and 8 PFU/mL for the device and ELISA respectively. A dose response curve was also generated for spiked nasal swab samples with a detection limit of 222 PFU/mL, demonstrating the device's use with complex biological samples. Chapter 3 expands on the work in Chapter 2 by demonstrating an alternative detection method. Chemiluminescent immunoassays are highly sensitive assays that rely on the energy provided by a chemical reaction to excite electrons. When the electrons move back to the ground state, they produce light that can be detected with an imager. In Chapter 3, I demonstrate the first example of a one-step, capillary driven immunoassay for chemiluminescent detection. The device is similar to that in Chapter 2, but the detection system relies on the reaction between HRP and a luminol based substrate to detect SARS-CoV-2 antigen. This work was done in collaboration with Burst Diagnostics Inc. and will be published when the appropriate patents and protections are in place. Chapter 4 introduces the first capillary driven enzyme-linked immunoassay for the simultaneous detection of multiple biomarkers. This multiplexed device is made of the same inexpensive materials as the previous chapters, but the microfluidic channels are designed in such a way that reagents are delivered to two, spatially separated test strips. This separation allows for simultaneous detection of two targets without cross-reactivity between reagents, reducing the chance of false positives. To demonstrate the purpose of this device, they were used to detect SARS-CoV-2 antigen on one test strip, and influenza antigen on the other. The illnesses caused by these two viruses lead to very similar symptoms, so distinguishing between the two illnesses from a single device would be beneficial. Dose response curves were gathered for both antigens, and the device was able to detect both diseases visually without false positives on the other test strip. Another form of multiplexed detection is simultaneous detection of two targets. To demonstrate this, SARS-CoV-2 and influenza antigen were detected simultaneously. Additionally, SARS-CoV-2 virus and c-reactive protein (CRP), a biomarker that can be used to determine the severity of COVID-19 cases, were detected simultaneously. This multiplexed assay has the potential to tell a healthcare provider 1) if an infection is or is not SARS-CoV-2, and 2) what level of care might be needed. This dissertation introduces three capillary driven immunoassay devices primarily for the use of detecting communicable diseases. The devices all run from a single end-user step, and fully automate the steps required for a more time consuming and expensive ELISA. Although the focus of this dissertation was on detecting communicable diseases, these devices can (and are) being further developed for chronic illnesses. In the future, by swapping the antibodies used in the immunoassay, the applications of these devices are innumerable. Additionally, different detection methods, such as fluorescent, electrochemical, and further chemiluminescent work could continue to push the detection limit down, widening the application of these devices even further.
Open Access
Development of methods for assessing oxidative stress caused by atmospheric aerosols
(Colorado State University. Libraries, 2012) Sameenoi, Yupaporn, author; Henry, Charles S., advisor; Rovis, Tomislav, committee member; Farmer, Delphine K., committee member; Van Orden, Alan K., committee member; Kipper, Matthew J., committee member
Extensive epidemiological studies show strong associations between the exposure to atmospheric aerosol particulate matter (PM) in the size range of 0.1- 10 µm and health problems, including respiratory, atherosclerosis and cardiovascular diseases. However, the mechanisms of PM-induced toxicity are poorly understood. A leading hypothesis states that airborne PM induces harm by generating reactive oxygen species in and around human tissues, leading to oxidative stress. To improve understanding of this effect, methods including biological assays and chemical assays for assessing oxidative stress caused by atmospheric aerosols have been developed and are described in this dissertation. For biological assays, a cleavable tag immunoassay (CTI) was developed with an ultimate goal of measuring multiple oxidative stress biomarkers in a single run. As a proof-of-concept, the multianalyte analysis system CTI was performed in competitive, non-competitive, and mixed formats for detection of small molecules and protein biomarkers simultaneously. For chemical assays, a microfluidic electrochemical sensor and a microfluidic paper-based analytical device (µPAD) have been developed for assessing aerosol oxidative stress in an area-based exposure study and a personal exposure study, respectively. The microfluidic electrochemical sensor was used for assessing aerosol oxidative stress by measuring the oxidative activity. The sensor was coupled directly to a Particle-into-Liquid-Sampler (PILS) to create an on-line aerosol sampling/analysis system. The system offers analysis with 3 minute temporal resolution, making it the best available temporal resolution for aerosol oxidative activity. The sensor was also used to analyze the ability of aerosols to generate hydroxyl radicals as another parameter for assessing aerosol oxidative stress. The ultimate goal of this system is to create an on-line monitoring system using a similar approach for oxidative activity analysis. As a first step toward this goal, assay optimization and system characterization in an off-line format employing flow injection analysis and amperometric detection, were carried out and presented in this dissertation. A microfluidic paper-based analytical device (µPAD) was developed for measuring oxidative activity of aerosol collected by a personal sampler. The system allows analysis with minimal sample preparation and requires 100-fold less particulate matter mass than existing analysis methods.
Open Access
Development of paper-based analytical devices for particulate metals in welding fume
(Colorado State University. Libraries, 2015) Cate, David M., author; Henry, Charles S., advisor; Volckens, John, advisor; Dandy, David, committee member; Peel, Jennifer, committee member; Lear, Kevin, committee member
Exposure to metal-containing particulate matter places a tremendous burden on human health. Studies show that exposures lead to cardiovascular disease, asthma, flu-like illnesses, other respiratory disorders, and to increased morbidity. Individuals who work in occupations such as metalworking, construction, transportation, and mining are especially susceptible to unsafe exposures because of their proximity to the source of particle generation. Despite the risk to worker health, relatively few are routinely monitored for their exposure due to the time-intensive and cost-prohibitive analytical methods currently employed. The current paradigm for chemical speciation of workplace pollution is outdated and inefficient. Paper-based microfluidic devices, a new type of sensor technology, are poised to overcome issues associated with chemical analysis of particulate matter, specifically the cost and timeliness of exposure assessment. Paper sensors are designed to manipulate microliter liquid volumes and because flow is passively driven by capillary action, analysis costs are very low. The objective of this work was to develop new technology for rapidly measuring Ni, Cu, Fe, and Cr in welding fume using easy-to-use paper devices. This dissertation covers the development of two techniques for quantifying metal concentration: spot integration and distance-based detection. Metal concentrations as low as 0.02 ppm are reported. A method for controlling reagent deposition as well as a new interface for multiplexed detection of metals, is discussed.
Open Access
Development of paper-based devices for point-of-need, bioanalytical applications
(Colorado State University. Libraries, 2020) Noviana, Eka, author; Henry, Charles S., advisor; Reynolds, Melissa M., committee member; Chung, Jean, committee member; Geiss, Brian J., committee member
The growing demand for reliable analytical tools to perform testing at the point-of-need has necessitated the development of novel sensors that are low cost (USD 1-10), portable, sensitive, selective, easy to use, and rapid (i.e. provide results within minutes or a few hours). Miniaturization of the sensors into microfluidic platforms has become a promising approach to achieve these sensors. However, traditional microfluidics often require relatively expensive and complicated pumping mechanisms that increase the cost and limit the portability of the sensors. From a material perspective, cellulosic paper is an attractive substrate for constructing point-of-need sensors due to its affordability, vast availability, self-pumping ability via capillary action, and easy fabrication using various printing and patterning techniques. My dissertation research has been focused on developing paper-based devices to address several key gaps that exist between the current technologies and the desired properties of point-of-need sensors. Chapter 2 describes the development of a steady flow paper device that enabled a function similar to conventional flow injection analysis (FIA) without external pumps. Two-layer paper devices increased the attainable flow rate and reduced the analysis time to only a minute, compared with 10-20 min analysis time reported in previous paper-based FIA. Disposable Pt microwire electrodes were used as a detector in the electrochemical paper-based device (ePAD) and the proposed sensor has been used to detect the activity of β-galactosidase (a bacterial indicator for coliform detection and a common detection label in enzyme-linked immunosorbent assay). Similar enzyme kinetics to those reported in the literature was obtained using the proposed sensor, showing a great promise for semi-automation in bioanalysis. Implementing a similar flow ePAD, the goal has now expanded toward improving the detection sensitivity as well as reducing the cost of the sensors. In Chapter 3, low-cost (~1 USD) and reusable thermoplastic electrodes (TPEs) were fabricated by mixing carbon and a plastic binder and pressing the material into an acrylic mold. These TPEs showed an improved electrochemical activity over conventional carbon paste electrodes typically used in ePADs. In addition, electrode arrays can also be fabricated using the technique to improve detection sensitivity via a generation-collection experiment, where the first electrode in the array oxidizes the analyte, the second reduces it, and the process is repeated across the entire array to provide an enhanced cumulative signal. Nanomolar detection limits were achieved using TPEs in both single detector and detector arrays configurations. A 5× improved sensitivity was obtained by employing electrode arrays over the single detector. In Chapter 4, the dissertation shifts focus to a more specific application, detecting nucleic acid, an important biological analyte that has been largely targeted to diagnose various diseases including genetic disorders, cancer, neurodegenerative, and infectious diseases. This chapter describes the integration of nuclease protection assay (NPA), a highly specific hybridization-based technique, with a reader-free colorimetric detection via lateral flow assay (LFA). In NPA, the hybridization of an antisense probe to the target sequence is followed by single-strand nuclease digestion. The protected double-stranded target-probe hybrids are then captured on the LFA device, followed by the addition of a colorimetric enzyme-substrate pair for signal visualization. The proposed paper-based NPA can detect sub-femtomole (~108 copies) of target DNA with high specificity. While the paper-based NPA can serve a good screening tool for several types of chronic infection in which large copies of pathogen DNA is present in the samples, the high detection limit hinders the application of this method for early disease diagnosis and detecting pathogens in environmental samples. In Chapter 5, polymerase chain reaction (PCR), a nucleic acid amplification technique, was coupled to the colorimetric LFA to improve the detection limit and enable the detection of antimicrobial-resistant (AMR) genes and bacteria in environmental samples. Six orders of magnitude lower detection limit (i.e. 102 plasmid DNA copies) was achieved by the PCR-LFA. The proposed method can be applied for rapid detection (less than 3 h) of AMR bacteria in environmental samples. Several works presented in this dissertation provided different approaches to achieve viable paper-based sensors for point-of-need applications. Progress has been made in improving both analytical figures of merit (i.e. sensitivity and detection limit) and practical specifications of the paper sensors (i.e. reduced sensor cost, semi-automation via an external pump-free flow-based system, instrument-free colorimetric readout, and improved assay time).
Open Access
Microchip capillary electrophoresis: improvements using detection geometry, on-line preconcentration and surface modification
(Colorado State University. Libraries, 2012) Guan, Qian, author; Henry, Charles S., advisor; Strauss, Steven H., committee member; Van Orden, Alan K., committee member; Krummel, Amber, committee member; Hanneman, William H., committee member
Capillary electrophoresis and related microfluidic technologies have been utilized with great success for a variety of bioanalytical applications. Microchip capillary electrophoresis (MCE) has the advantages of decreased analysis time, integrated sample processing, high portability, high throughput, minimal reagent consumption, and low analysis cost. This thesis will focus on the optimization of our previous microchip capillary electrophoresis coupled electrochemical detection (MCE-ECD) design for improved separation and detection performance using detection geometry, on-line preconcentration and surface modification. The first effort to improve detection sensitivity and limits of detection (LODs) of our previous MCE-ECD system is established by an implementation of a capillary expansion (bubble cell) at the detection zone. Bubble cell widths were varied from 1× to 10× the separation channel width (50 μm) to investigate the effects of electrode surface area on detection sensitivity, LOD, and separation efficiency. Improved detection sensitivity and decreased LODs were obtained with increased bubble cell width, and LODs of dopamine and catechol detected in a 5× bubble cell were 25 nM and 50 nM respectively. In addition, fluorescent imaging results demonstrate ~8% to ~12% loss in separation efficiency in 4× and 5× bubble cell, respectively. Another effort for enhancing detection sensitivity and reducing LODs involves using field amplified sample injection and field amplified sample stacking. Stacking effects were shown for both methods using DC amperometric and pulsed amperometric detections. Decreased LODs of dopamine were achieved using both on-line sample preconcentration methods. The use of mixed surfactants to affect electroosmotic flow (EOF) and alter separation selectivity for electrophoretic separations in poly(dimethylsiloxane) (PDMS) is also presented in this thesis. First the effect of surfactant concentration on EOF was studied using the current monitoring method for a single anionic surfactant (sodium dodecyl sulfate, SDS), a single zwitterionic surfactant (N-tetradecylammonium-N,N-dimethyl-3-ammonio-1-propane sulfonate, TDAPS), and a mixed ionic/zwitterionic surfactant system (SDS/TDAPS). SDS increases the EOF as reported previously while TDAPS shows an initial increase in EOF followed by a reduction in EOF at higher concentrations. The addition of TDAPS to a solution containing SDS makes the EOF decrease in a concentration dependent manner. The mixed SDS/TDAPS surfactant system allows tuning of the EOF across a range of pH and concentration conditions. After establishing EOF behavior, the adsorption/desorption rates were measured and show a slower adsorption/desorption rate for TDAPS than SDS. Next, capacitively coupled contactless conductivity detection (C4D) is introduced for EOF measurements on PDMS microchips as an alternative to the current monitoring method to improve measurement reproducibility. EOF measurements as a function of the surfactant concentration were performed simultaneously using both methods for three nonionic surfactants, (polyoxyethylene (20) sorbitan monolaurate (Tween 20), polyoxyethylene octyl phenyl ether (Triton X-100), polyethylene glycol, (PEG 400)), mixed ionic/nonionic surfactant systems (SDS/Tween 20, SDS/Triton X-100, and SDS/PEG 400) and mixed zwitterionic/nonionic surfactant systems (TDAPS/Tween 20, TDAPS/Triton X-100, and TDAPS/PEG 400). EOF for the nonionic surfactants decreases with increasing surfactant concentration. The addition of SDS or TDAPS to a nonionic surfactant increases EOF relative to the pure nonionic surfactant. Next, separation and electrochemical detection of two groups of model analytes were explored using mixed surfactant systems. Similar analyte resolution with greater peak heights was achieved with mixed surfactant systems relative to the single surfactant system. Finally, the utility of mixed surfactant systems to achieve improved separation chemistry of biologically relevant compounds in complex sample matrixes was demonstrated in two applications, which include the detection of catecholamine release from rat pheochromocytoma (PC12) cells by stimulation with 80 mM K+ and the detection of reduced glutathione (GSH) in red blood cells (RBCs) exposed to fly ash suspension as a model environmental oxidant.
Open Access
Microfluidics for environmental analysis
(Colorado State University. Libraries, 2018) Gerold, Chase T., author; Henry, Charles S., advisor; Krummel, Amber, committee member; Levinger, Nancy, committee member; Finke, Richard, committee member; Dandy, David, committee member
During my graduate dissertation work I designed and utilized microfluidic devices to study, model, and assess environmental systems. Investigation of environmental systems is important for areas of industry, agriculture, and human health. While effective and well-established, traditional methods to perform environmental assessment typically involve instrumentation that is expensive and has limited portability. Because of this, analysis of environmental systems can have considerable financial burden and be limited to laboratory settings. To overcome the limitations of traditional methods researchers have turned to microfluidic devices to perform environmental analyses. Microfluidics function as a versatile, inexpensive, and rapidly prototyped analytical tool that can achieve analysis in field setting with limited infrastructure; furthermore, microfluidic devices can also be used to study fundamental chemistry or model complex environmental systems. Given the advantages of microfluidic devices, the research presented herein was accomplished using this alternative to traditional instrumentation. The research projects described in this dissertation involve: 1) the study of fundamental chemistry associated with surfactant surface fouling facilitated by divalent metal cations; 2) the creation of a microfluidic device to study fluid interactions within an oil reservoir; and 3) the fabrication of a paper-based microfluidic to selectively quantify K+ in complex samples. The first research topic discussed involves observation of dynamic evidence that supports the hypothesized cation bridging phenomenon. Experimental results were acquired by pairing traditional microfluidics with the current monitoring method to observe relative changes to a charged surface's zeta potential. Divalent metal cations were found to increase surfactant adsorption, and cations of increasing charge density were found to have a greater effect on surface charge. Analysis of the experimental data further supports theoretical cation bridging models and expands on knowledge relating to the mechanism by which surfactant adsorption occurs. This work was published in the ACS journal Langmuir (2018, 34 (4), pp 1550–1556). The second project discussed herein focuses on the development of the microfluidic Flow On Rock Device (FORD) that was designed to study fluid interactions within complex media. The FORD was designed to be an alternative to existing fluid modeling methods and microfluidic devices that test oil recovery strategies. Fabrication of the FORD was accomplished by incorporating real reservoir rock core samples into the device. The novelty of this device is due to the simplicity and accuracy by which the physical and chemical characteristics are represented. This project has been accepted for publication pending minor revisions in Microfluidics and Nanofluidics. The final project discussed the creation of the first non-electrochemical microfluidic paper-based analytical device (µPAD) capable of quantitatively measuring alkali or alkaline earth metals using K+ as a model analyte. This device was fabricated by combining distance-based analytical quantification in µPADs with optode nanosensors. Experimental results were obtained using the naked eye without the requirement of a power source or external hardware. The resulting distance-based µPAD showed high selectivity and the capacity to quantify K+ in real undiluted human serum samples. This work has been published in the ACS journal Analytical Chemistry (2018, 90 (7), pp 4894–4900). The research projects briefly described above and thoroughly discussed later within this dissertation were made possible by the utilization of microfluidic devices. These projects investigated various aspects of environmental chemistry without the use of traditional instrumentation or methods. The experimental results that were obtained further the fundamental understanding of surfactant adsorption, provide an inexpensive and accurate model to observe fluid interactions within reservoir rock material, and allow for the selective quantification of K+ in a paper-based device without the use of a power source. The funding for each of these projects was supplied by BP plc and Global Good, as is mentioned accordingly within this dissertation.
Open Access
Molecular diagnostic platforms for point-of-need pathogen detection
(Colorado State University. Libraries, 2021) Jain, Sidhartha, author; Henry, Charles S., advisor; Geiss, Brian J., advisor; Dandy, David S., committee member; Magzamen, Sheryl L., committee member
Rapid, accurate, reliable nucleic acid testing (NAT) platforms are essential in the diagnosis and management of diseases. The inherent complexity associated with NAT requires that such testing be performed in centralized laboratories by highly trained personnel. Modified molecular technologies that can be used at the point-of-care (POC) are needed to improve the turnaround times of results and lower the global burden of infectious diseases. To help address this urgent need, we have developed a nucleic acid sensor platform utilizing nuclease protection and lateral flow detection for rapid, point-of-need nucleic acid analysis. We have also improved the analytical performance of the assay by pairing it with isothermal padlock rolling circle amplification (RCA). RCA is one of the simplest and most versatile isothermal amplification techniques as it only requires one primer and a strand-displacing polymerase. Utilizing our rolling circle amplification lateral flow platform, we have developed assays for beta-lactamase resistance genes for antimicrobial resistance monitoring and severe acute respiratory virus coronavirus 2 (SARS-CoV-2). We have also explored the use of exponential isothermal amplification to further improve the assay limit of detection. We also propose a microfluidic device to rapidly detect the RCA amplicons. The device allows programmable sequential delivery of reagents to a detection region, reducing the number of user steps. With further development, such microfluidic devices can be used to develop fully integrated sample-to-result molecular diagnostic platforms that integrate sample pretreatment, amplification, and detection in an easy-to-use, point-of-need nucleic acid sensor platform. Chapter 1 presents a brief review of the nucleic acid testing landscape, the challenges associated with the development of point-of-need nucleic acid sensors and recent successes utilizing paper-based devices for fully integrated sample-to-result sensors. Chapters 2 and 3 discuss the development of the nuclease protection lateral flow assay and padlock probe-based rolling circle amplification lateral flow assay. Chapter 4 describes our work on the use of exponential RCA to improve the limit of detection of the SARS-CoV-2 assay. In Chapter 5, we present our work on a paper-plastic microfluidic device for the rapid detection of the RCA amplicon. We believe that such devices can be used for the development of integrated molecular diagnostic sensor platforms that can be used at the point-of-need in resource-limited settings.
Open Access
Predicting the physical stability of biopolymers by self-interaction chromatography
(Colorado State University. Libraries, 2008) Payne, Robert W., author; Henry, Charles S., advisor
Biopolymers, including proteins and peptides, are increasingly taking the place of small molecules in manufacturing, health and home applications. The advantage of using biopolymers over small molecules for therapeutic applications include high activity, high specificity and low toxicity. The disadvantages of using biopolymers include large scale production, chemical instability and physical degradation. A biopolymer that shows promise for a specific application will not advance past early development if the protein lacks either chemical or physical stability. Of these two parameters, physical stability is often harder to predict and/or measure. The physical stability of a biopolymer can be improved by site-directed mutagenesis, glycosylation, post-translational modification and adjusting the solvent/co-solvent system. Quantitatively measuring the change in the physical stability of a protein in different solvent/co-solvent systems can increase the number of early developmental stage proteins that advance to commercial scale. Physical stability of proteins can be described by protein-protein interactions and measured by the osmotic second virial coefficient (B). However, current methods used to measure B such as static light scattering are not practical for large screening studies because of large protein consumption, low throughput, method variability and analyte size limitations. An alternative method to measure B is Self-Interaction Chromatography (SIC), which requires less protein, shorter analysis time, allows for miniaturization and capable of measuring B for small size biopolymer. The ability of SIC to measure B for a therapeutic protein, a peptide and membrane proteins in different solvent/co-solvent systems has been demonstrated in this dissertation.
Open Access
Pump-free magnetophoresis for improved point-of-care diagnostics
(Colorado State University. Libraries, 2022) Call, Zachary D., author; Henry, Charles S., advisor; Reynolds, Melissa M., committee member; Dandy, David S., committee member; Snow, Christopher D., committee member
Infectious diseases are one of the largest health burdens for low-income countries and claim millions of lives every year. The loss of life in low-income countries is largely due to the lack of access to preventative healthcare and appropriate diagnostic testing. Several health agencies have recognized the need for improved diagnostics to reduce the burden of infectious diseases. The following works described in this thesis are focused on improving the capabilities of point-of-care (POC) testing to improve patient healthcare. Microfluidic devices are a popular approach for diagnostics because they offer reduced assay times, reduced sample volume, and are small (<10 cm). Additionally, microfluidic devices can be used with magnetophoresis to improve sensitivity and specificity. However, traditional microfluidic devices have difficulty translating to the POC because of tedious and expensive fabrication. Microfluidic paper-based analytical devices (µPADs) are a popular alternative to traditional microfluidics due to the natural capillary action through cellulose fibers and simple fabrication. µPADs are portable, low-cost, and do not require external instrumentation, making them ideal for POC settings. However, µPADs often suffer from poor analytical performance resulting in failing to translate to POC testing. In Chapters 2, 3 and 4 of this thesis, I described combining µPADs with magnetophoresis to improve the analytical performance without sacrificing the advantages of µPADs. Coupling magnetophoresis with µPADs is a novel approach and was not reported until the publication of chapter two. Chapter 2 of this thesis describes the first reported example of paper-based magnetophoresis. Magnetophoresis has always needed external pumps to drive flow, however we demonstrate the ability to perform magnetophoresis completely pump-free in a µPAD. We demonstrated the ability to detect E.coli at 105 colony forming units (CFU/mL) with a fluorescent label in a pooled human urine sample. Chapter 3, describes improvements to the device described in chapter two. The limit of detection was improved by three orders of magnitude and calculated at 4.67 x 102 CFU/mL in pooled human urine, which is below detection limits for commercial urinary tract infection tests. Colorimetric detection was used instead of fluorescence detection to eliminate any instrumentation needed and create an easy read-by-eye assay. Additionally, the device design was modified to incorporate a burst valve to generate more consistent laminar flow and simplify user-end steps. We envision this technology to be used a platform for future paper-based devices incorporating magnetophoresis for improved POC devices. In Chapter 4 of this thesis, we describe a new platform for microfluidic magnetophoresis that simplifies user-end steps further through a simple magnet sliding operation. Here we introduce a MagnEtophoresis Slider Assay (MeSA) for sequential binding and washing steps without the need for any external instrumentation. A competitive biotin assay and a sandwich immunoassay are demonstrated to display the functionality of this new platform. The limit of detection was calculated at 1.62 x 103 CFU/mL using colorimetric detection. The MeSA is extremely user-friendly, provides sensitive and rapid results (<15 min), and can be applied to a wide range of applications.