Miniaturization of electrodes has played a key role in the advancement of electrochemical measurements. Ultramicroelectrodes (UME) have a critical dimension that is of the same order as the diffusion layer thickness at the electrode, thus reducing the time scale of an electrochemical measurement. In the case of macroelectrodes, the electroactive species diffuses linearly normal to the electrode surface; whereas in UMEs diffusion is radial thereby increasing the current density. This phenomenon makes UMEs extremely advantageous for the following reasons: 1) the steady state is reached much faster, 2) electrochemical kinetic measurements are possible and 3) highly accurate measurements can be done even in nonpolar solvents or in resistive solutions. One such arrangement is that of interdigitated electrode arrays (IDEAs). In IDEAs two working electrodes are held in close proximity such that their diffusion profiles overlap. When specific potential are applied to the two working electrodes the electroactive redox species will cycle (oxidize and reduce) multiple times before diffusing out into the bulk solution of the sample. This redox cycling effect has been exploited in last couple of decades for enhancing current signal and lowering the limit of detection. However, most of the work involves use of noble metal IDEAs, which are expensive, require sophisticated instrumentation, and are difficult to fabricate. In this contribution, we report on simpler fabrication technique that involves patterning of SU-8, a negative photoresist, using conventional UV photolithography followed by pyrolysis in an inert environment to create conductive carbon IDEAs with sub-micron resolution. The sensor performance was electrochemically characterized under different geometries under stationary and flow conditions. The capability of the carbon IDEA sensor was demonstrated by applying them in selective detection of dopamine against ascorbic acid interference, selective amplification of neurotransmitters such as dopamine and epinephrine in their mixture, and finally as an electrochemical immunoassay.

For the last few decades, the field of carbon Microelectromechanical systems (CMEMS) has taken inspiration from the patterning and microfabrication techniques from microelectronics industry to create unique and intricate three dimensional carbon materials with a variety of properties and applications. Carbon has excellent electrochemical stability, chemical inertness, biocompatibility, and is able to be fabricated from a number of easily machinable polymer precursors. These features have allowed researchers to fabricate high performance electrochemical sensors, scaffolds for tissue engineering, batteries with excellent lithium intercalation capacity, and other microelectronic platforms.

In this work, microfabrication techniques are used to create a fragmented and graphitic carbon material that is used as a biosensor to detect iodide and Dopamine in complex fluids. The excellent microstructural properties of the material were able to detect both iodide and Dopamine at clinically relevant concentration levels in the presence of interfering ions in complex body fluids, making it an ideal biosensor.

Since the emergence of the coronavirus SARS-CoV-2 in 2019, unprecedented rates of transmission coupled with alarmingly high mortality rates have remained the focus of the current healthcare climate. Despite the development of vaccines designed to combat virulence and the severity of the symptoms, the global healthcare crisis has continued through to 2022. Additionally, existing golden standard RNA-based diagnostics such as RT-PCR require expensive instrumentation, careful handling and extensive training, services that may not be immediately available to some vulnerable communities and limit their response during an epidemic. These issues have become increasingly evident with continued transmission stemming from mutation of the virus, resulting in a multitude of different lineages with varying degrees of infectivity, endangering the efficacy of existing solutions. To address these shortcomings in the global response, an RNA-based diagnostic tool has been developed that provides an isothermal and easy-to-handle option for screening samples for the presence of the Wuhan strain of the SARS-CoV-2 virus (Wuhan-Hu-1) and differentiation from the UK (B.1.1.7) variant. In this work, two key steps have been incorporated for detection of regions of interest: 1) upstream isothermal amplification of key regions using Reverse Transcription-Recombinase Polymerase Amplification (RT-RPA), and 2) a proof-read step using duplex-specific nuclease that mediates further signal amplification only in the presence of a perfect DNA:RNA duplex. We further demonstrate compatibility with alternative low-resource signal enhancement strategies with a signal concentration method utilizing electro-osmotic concentration of bead-based fluorescence and detection on lateral flow nitrocellulose strips. The potential of this novel nuclease-based fluorescent assay is demonstrated as a user-friendly alternative for screening between relevant SARS-CoV-2 strains through detection of key regions within the ORF1a and S genes necessary to distinguish between the aforementioned strains (Wuhan-Hu-1, B.1.1.7).

Carbon-MicroElectroMechanical systems (C-MEMS) use patterning and nanofabrication techniques developed for integrated circuits (ICs) in combination with traditional carbon pyrolysis to create intricate 3-D carbon materials and electrodes from organic precursors. The fabrication of specialized, pyrolytic carbon microfeatures and geometries, such as carbon micro-post arrays or interdigitated electrode arrays (IDEAs), significantly enhances carbon’s electrical, chemical and mechanical properties to create high performance sensors and energy storage devices.

However, C-MEMS is generally limited to using amorphous or glassy carbons since most inexpensive carbon microfabrication processes require cross-linking a non-graphitizing polymer precursor. The inability to change the microstructure of a polymer precursor to a configuration that yields graphitic carbon inhibits the performance of the resulting material. Furthermore, traditional processes for functionalizing carbon using surface modifications are typically too harsh for its small microfeatures. The ideal solution is to develop a fabrication process that allows for the structural and surface modification of pyrolytic carbons without sacrificing cost or scalability. The results of this work present a novel method for electromechanically modifying the microstructure and surface properties of carbon to significantly enhance its electrochemical activity. Specifically, the results of a comprehensive materials and electrochemical characterization of the structurally modified carbon reveals a new class of stress-activated pyrolytic carbon (SAPC). The SAPC showed a remarkable improvement in graphitization and demonstrated an inherently high and enduring electrocatalysis that has yet to be seen with any other pyrolytic carbon to date. Furthermore, the SAPCs simple and scalable synthesis process is compatible with common C-MEMS fabrication processes, creating a new tool for C-MEMS operators to tune the properties of their carbon electrodes.

Fabrication of a microfluidic ELISA assay can be a very time-consuming method, due to the curing process required for molded parts. This thesis examines Fused Deposition Modeling and Stereolithography as candidates for rapid prototyping microfluidic devices. Individual components of the device were designed on SolidWorks, and underwent several generations of revisions to address problems of air and fluid leakage. We present an automated ELISA assay device created using a combination of Fused Deposition Modeling and Stereolithography as a comprehensive demonstration of additive manufacturing capabilities, as well as the methodology used to create such a device. A detailed explanation on how to troubleshoot the fabrication process and machines is also discussed.

Carbon is one of the most adaptable element due to its unique flexibility in atomic composition to form sp, sp3, and sp3 hybridize covalent bonds with various elements [4,19,18]. Depending on its atomic conformation, carbon can greatly vary, ranging from a fully sp3-bonded crystalline network like a diamond to a fully-sp3 bonded 2-D structure like graphite. Each of the allotropes also have vastly different electrical, mechanical, thermal and chemical properties which have led to its adoption in the realms of biotechnology, electronics and energy storage [5,34,30]. Particularly, in the fields of materials sciences, carbon nanomaterials have become an excellent model system to study the impact of nanoscale defects on electrochemistry that can potentially be use as biosensors or wastewater filtration. However, fabrication of such materials is still arduous due to its tendency to break easily or inflexibility to mold. C-MEMS fabrication technology is, therefore, to address these challenges to engineer 3-D structures of carbon. However, chemically-inert surface of these carbons still makes its implementation for various electrochemical application problematic. Although various methods have been introduced to functionalize carbons, these processes can be unstable, detrimental to microfeatures of carbon and time-consuming due to additional step of activation. Herein, we show a new class of carbon, stress-activated pyrolytic carbons (SAPCs), that has a highly-graphitic structure with abundance in edge planes and nitrogen heteroatoms without external activation steps. In this study, we reveal an outstanding capacity of SAPCs as a promising material for improving laccase-driven electrocatalysis in biofuel cell application.

Centrifugal microfluidics is a widely known research tool for biological sample and water quality analysis. Currently, the standard equipment used for such diagnostic applications include slow, bulky machines controlled by multiple operators. These machines can be condensed into a smaller, faster benchtop sample-to-answer system.

Sample processing is an important step taken to extract, isolate, and convert biological factors, such as nucleic acids or proteins, from a raw sample to an analyzable solution. Volume definition is one such step. The focus of this thesis is the development of a model predicting monodispersed droplet formation and the application of droplets as a technique for volume definition.

First, a background of droplet microfluidic platforms is presented, along with current biological analysis technologies and the advantages of integrating such technologies onto microfluidic platforms. Second, background and theories of centrifugal microfluidics is given, followed by theories relevant to droplet emulsions. Third, fabrication techniques for centrifugal microfluidic designs are discussed. Finally, the development of a model for predicting droplet formation on the centrifugal microfluidic platform are presented for the rest of the thesis.

Predicting droplet formation analytically based on the volumetric flow rates of the continuous and dispersed phases, the ratios of these two flow rates, and the interfacial tension between the continuous and dispersed phases presented many challenges, which will be discussed in this work. Experimental validation was completed using continuous phase solutions of different interfacial tensions. To conclude, prospective applications are discussed with expected challenges.

Centrifugal microfluidics, or compact disc (CD) microfluidics, has been gaining popularity in the lab-on-a-chip field as an advanced diagnostic platform over the last fifteen years. The lab-on-a-disc (LoD) platform embodies advantages of the lab-on-a-chip platform, including small volumes, fast reaction times, low power consumption, and portability; it also has other unique advantages, including embedded fluid pump operation and ease of automation and multiplexing. These advantages make the lab-on-a-disc diagnostic system attractive due to its capability for rapid disease diagnosis. In clinical diagnostics, rapid nucleic acid biomarker detection generally has been a challenge especially due to the rigorous thermocycling required to bring the biomarker quantity up to a delectable level. This work follows the development of a diagnostic microfluidic disc that utilizes polymerase chain reaction for DNA amplification and a DNA microarray that allows for visual detection of numerous target biomarkers. The assay begins with a sample volume that undergoes preparation, thermocycling, post-processing, and detection, so a number of challenges must be addressed; these challenges include precise fluid manipulation, application of heat, and storage and release of reagents. Along the way, modular fluid-handling techniques, including the thermo-pneumatic pump (TPP) and the multifunctional wax valves (MWV), were implemented to reduce the complexity and cost of the overall hardware system. This system would not only be capable of nucleic acid amplification but also specialized multiplexed biomarker amplification and detection. It is anticipated that this platform will provide a foundation for the development of other fully-integrated LoD systems for rapid disease diagnosis in the near future.

Waterborne diseases cause millions of deaths worldwide, especially in developing communities. The detection of waterborne pathogens is a critical step for the selection of water treatment processes that will lead to the prevention of disease transmission. Unfortunately, analyzing water samples is a lengthy and laborious process that requires 1) collecting and transporting large volumes of samples (>1 L) to a centralized lab, 2) sample preparation for downstream analysis, 3) using appropriate detection methods to identify pathogens and 4) estimating the proportion of viable pathogens that pose a risk to public health. This lengthy “sample-to-answer” process significantly delays the risk mitigation actions, and subsequently exposes those using the water for daily necessities to pathogen infection. This dissertation aims to streamline the water sample analysis timeline by incorporating the major laboratory steps onto centrifugal microfluidic platforms for implementation at the point-of-sample collection, mitigating many of the aforementioned limitations. We first investigate pathogen detection methods by describing the integration of a droplet digital loop mediated isothermal DNA amplification assay onto a centrifugal microfluidic disc that coupled with a portable analysis instrument, can detect E. faecalis, a common waterborne pathogen. We highlight the fluidic and functional integration of the major steps of the assay onto the disc as well as the rapid, quantitative, hands-free aspects of the system. Next, to process samples in our detection system, we tackle upstream sample preparation by focusing on adopting simple and accessible water sample concentration mechanisms onto a centrifugal microfluidic platform. We integrate super absorbent polymer beads with the disc system to concentrate E.coli, another common waterborne pathogen, and observe how the concentration is affected by various relevant parameters. We conclude by discussing strategies for downstream assessment of pathogen viability and relevant infectivity in water samples. We describe the method and initial feasibility of a RNA-based, enzyme-mediated signal amplification strategy to detect E.coli and highlight its integration into modified culture-based assays for rapid, viable pathogen detection and risk assessment. Together, this work highlights the critical challenges in water analysis and innovations in centrifugal microfluidics for pathogen detection at the point-of-sample collection.

Life-saving treatment starts with proper diagnosis. This thesis therefore discusses two central aspects: detection methods and integration of fluidic steps onto fully automated diagnostic platforms. To investigate performance of different common commercial detection methods with regard to detection characteristics, such as limit of detection (LOD), sensitivity, and dynamic range (DR), horseradish peroxidase (HRP) was chosen as label and detection was conducted via absorbance, chemiluminescence, and amperometry. While these performance parameters vary for different substrates and depend on specific procedure for each technique, the importance of choosing the proper detection method for a given application is conspicuous. Integration of a colorimetric, HRP-based enzyme-linked immunosorbent assay (ELISA) for detection of malaria is demonstrated on a rapid prototyped platform (3D printed microfluidic structure) and electrochemical detection is integrated on a compact disc (CD) microfluidic device. The latter is most commonly produced via injection molding and thus compatible with mass manufacturing. While CD microfluidics excels in simplicity of pumping principle based on centrifugal force from a single motor and elegance of instrumentation-free, passive valving based on changing capillary forces at cross-section changes (burst valves), this type of valving often shows unreliable. Burst valves are therefore analytically modelled and experimentally analyzed and evaluated. While instrumentation-free principle incites their application, control of burst pressure underlies effects of surface imperfections and dimensional control is critical. While pumping on CD microfluidic platforms inherently enables fluid motion towards the outer perimeter of the disc, reversed flow is demonstrated on material jetted CD-inserts.

Integration of electrochemical detection on the CD, allows for signal amplification via enhanced mass transport. The effect of flow onto electrochemical measurements is investigated in cyclic voltammetry experiments on Interdigitated Electrode Arrays (IDAs) on the CD. While flow generally increases mass transport to the electrode surface, redox amplification suffers. Nevertheless, dual mode cyclic voltammetry during flow results in lowest LOD.