Development of Facile Microfabrication Technologies for the Fabrication and Characterization of Multimodal Impedimetric, Plasmonic, and Electrophysiological Biosensors

The objective of this dissertation was to develop novel methods of patterning inorganic and organic materials, develop biocompatibility evaluations, and subsequently apply these methods toward developing biosensors and lab-on-a-chip devices, such as Interdigitated Electrodes (IDEs) and Microelectrode Arrays (MEAs) on non-traditional (such as nanostructured and plasmonic) polymer substrates or deploy these methods to enhance precision cellular placement on traditional (glass) MEA substrates. It was hypothesized that a combination of such facile microfabrication techniques and patterning technologies on traditional and non-traditional substrates would increase the sensitivity and selectivity of such sensor platforms by several orders of magnitude, and potentially introduce new modalities for cell-based biosensing. In order to demonstrate the biological functionality of these new IDEs and MEAs, a variety of cell cultures were used (cardiac, stem cell, and endothelial cells) to study the growth, proliferation, modes of increasing sensitivity and response to various compounds in vitro (outside the body)

Real time measurement of oxygen by integrating a clark sensor with low cost printed circuit board technology and solid electrolyte membrane

A prototype of a miniaturized Clark type electrochemical oxygen sensor integrated with a 3D printed in vitro cell culturing platform is designed and developed for the purpose of monitoring the cellular oxygen consumption by the solution flowing through the cultured cells on the platform. Oxygen respiration indicates a cell's metabolic activity, so by measuring a chemical's oxygen content as it passes through a cell chamber, we can measure that chemical's potential effectiveness. This miniature micro sensor is designed and fabricated on a printed circuit board for the first time and integrated with a solid electrolyte membrane and 3D printed cell culturing platform to ensure robustness, low manufacturing cost and good electrical conductivity for sensing. Hence the sensor is aimed at enabling the pharmaceutical industry to rapidly test chemical products on animal and cancer cells; and has been designed to be low cost and suitable for mass production. The presented oxygen sensor configuration consists of two identical series of working, reference and counter microelectrodes. The solid polymer electrolyte membrane, Nafion (perfluorosulfunic acid membrane, DuPont Company) removed requirement for extra humidification and increased the shelf life of the sensor. The sensitivity of the oxygen sensor was tested in different oxygen concentration in gas and liquid states and was calibrated with measurements from a Portable Multi-Gas analyzer and a dissolved oxygen analyzer. The prototype can detect the small changes in oxygen concentration in the range of 0 to 5 μA current and has a response time of less than 5 seconds

Stimuli-Responsive Hydrogel Microdomes Integrated with Genetically Engineered Proteins for High-Throughput Screening of Pharmaceuticals

A hydrogel microdome that can swell in response to a stimuli or target molecule is formed by polymerizing a mixture comprising a monomer capable of forming a hydrogel with a biopolymer. An array of hydrogel microdomes can be formed on a substrate by microspotting the mixture and polymerizing. The array can be used for high-throughput screening of analytes as well as for use as an actuator and biosensor using the swelling property of the hydrogel

Simulation of an electrically actuated cantilever as a novel biosensor

Recently, detecting biological particles by analyzing their mechanical properties has attracted increasing attention. To detect and identify different bioparticles and estimate their dimensions, a mechanical nanosensor is introduced in this paper. To attract particles, numerous parts of the substrate are coated with different chemicals as probe detectors or receptors. The principal of cell recognition in this sensor is based on applying an electrical excitation and measuring the maximum deflection of the actuated cantilever electrode. Investigating the critical voltage that causes pull-in instability is also important in such highly-sensitive detectors. The governing equation of motion is derived from Hamilton’s principle. A Galerkin approximation is applied to discretize the nonlinear equation, which is solved numerically. Accuracy of the proposed model is validated by comparison studies with available experimental and theoretical data. The coupled effects of geometrical and mechanical properties are included in this model and studied in detail. Moreover, system identification is carried out to distinguish bioparticles by a stability analysis. Due to the absence of a similar concept and device, this research is expected to advance the state-of-the-art biosystems in identifying particles

Micromachined three-dimensional electrode arrays for in-vitro and in-vivo electrogenic cellular networks

This dissertation presents an investigation of micromachined three-dimensional microelectrode arrays (3-D MEAs) targeted toward in-vitro and in-vivo biomedical applications. Current 3-D MEAs are predominantly silicon-based, fabricated in a planar fashion, and are assembled to achieve a true 3-D form: a technique that cannot be extended to micro-manufacturing. The integrated 3-D MEAs developed in this work are polymer-based and thus offer potential for large-scale, high volume manufacturing. Two different techniques are developed for microfabrication of these MEAs - laser micromachining of a conformally deposited polymer on a non-planar surface to create 3-D molds for metal electrodeposition; and metal transfer micromolding, where functional metal layers are transferred from one polymer to another during the process of micromolding thus eliminating the need for complex and non-repeatable 3-D lithography processes. In-vitro and in-vivo 3-D MEAs are microfabricated using these techniques and are packaged utilizing Printed Circuit Boards (PCB) or other low-cost manufacturing techniques. To demonstrate in-vitro applications, growth of 3-D co-cultures of neurons/astrocytes and tissue-slice electrophysiology with brain tissue of rat pups were implemented. To demonstrate in-vivo application, measurements of nerve conduction were implemented. Microelectrode impedance models, noise models and various process models were evaluated. The results confirmed biocompatibility of the polymers involved, acceptable impedance range and noise of the microelectrodes, and potential to improve upon an archaic clinical diagnostic application utilizing these 3-D MEAs.Ph.D.Committee Chair: Mark G. Allen; Committee Member: Elliot L. Chaikof; Committee Member: Ionnis (John) Papapolymerou; Committee Member: Maysam Ghovanloo; Committee Member: Oliver Bran

Micro- and nano-electrode arrays for electroanalytical sensing

A systematic investigation of the electrochemical behaviour of two sets of microelectrode arrays, fabricated by standard photolithographic and reactive-ion etching techniques, is presented. The first set of microelectrode arrays had a constant relative centre-centre spacing of 10r (where r is the electrode radius). As a value of r was decreased, the cyclic voltammograms recorded from the array became increasingly peak-shaped, due to merging of the diffusion fields of the individual electrodes. Furthermore, it was shown that the peak current densities obtained were largest for the arrays with the smallest individual electrodes, as were the signal-to-noise ratios (SNRs). Electroplating the individuals electrodes with platinum black was also shown to increase the peak currents and the SNRs, due to an increase in the effective surface area. Sigmoidal voltammograms, which are indicative of radial diffusion, were obtained for an individual electrode radius of 25 mm but not for arrays with smaller electrodes. To obtain radial diffusion for an array of 2.5 mm electrodes, it was shown (using a second set of microelectrode arrays) that a minimum relative centre-centre spacing of 40r is required. Further enhancement of the peak current densities were obtained by decreasing the size of the individual electrodes. A series of nanoelectrode arrays were fabricated using electron-beam lithography (EBL). The voltammograms obtained from these arrays exhibited a continual increase in the recorded peak current as the individual electrodes radius was decreased to a value of 110 nm. Since EBL is a slow and costly technique, nanoimprint lithography (NIL) was investigated as an alternative method of fabricating nanoelectrode arrays and comparable results were obtained from arrays produced by EBL and NIL. A dissolved oxygen and temperature sensor incorporating a working microelectrode array was also designed and fabricated. The sector comprised a densely packed array of 2.5 mm radius electrodes and a micro-reference electrode, both of which were covered with an agarose electrolyte gel enclosed in an SU8 chamber. A thermal resistor was included for temperature compensation of the dissolved oxygen measurements. The Ag|AgCl micro-reference electrode was found to be stable for approximately 80 hours in 0.1 M KCl, with 100 nA of current passing through it. Linear calibration curves were obtained from both temperature and dissolved oxygen measurement

Ultrafast Microfluidic Immunoassays Towards Real-time Intervention of Cytokine Storms

Biomarker-guided precision medicine holds great promise to provide personalized therapy with a good understanding of the molecular or cellular data of an individual patient. However, implementing this approach in critical care uniquely faces enormous challenges as it requires obtaining “real-time” data with high sensitivity, reliability, and multiplex capacity near the patient’s bedside in the quickly evolving illness. Current immunodiagnostic platforms generally compromise assay sensitivity and specificity for speed or face significantly increased complexity and cost for highly multiplexed detection with low sample volume. This thesis introduces two novel ultrafast immunoassay platforms: one is a machine learning-based digital molecular counting assay, and the other is a label-free nano-plasmonic sensor integrated with an electrokinetic mixer. Both of them incorporate microfluidic approaches to pave the way for near-real-time interventions of cytokine storms. In the first part of the thesis, we present an innovative concept and the theoretical study that enables ultrafast measurement of multiple protein biomarkers (<1 min assay incubation) with comparable sensitivity to the gold standard ELISA method. The approach, which we term “pre-equilibrium digital enzyme-linked immunosorbent assay” (PEdELISA) incorporates the single-molecular counting of proteins at the early, pre-equilibrium state to achieve the combination of high speed and sensitivity. We experimentally demonstrated the assay’s application in near-real-time monitoring of patients receiving chimeric antigen receptor (CAR) T-cell therapy and for longitudinal serum cytokine measurements in a mouse sepsis model. In the second part, we report the further development of a machine learning-based PEdELISA microarray data analysis approach with a significantly extended multiplex capacity using the spatial-spectral microfluidic encoding technique. This unique approach, together with a convolutional neural network-based image analysis algorithm, remarkably reduced errors faced by the highly multiplexed digital immunoassay at low analyte concentrations. As a result, we demonstrated the longitudinal data collection of 14 serum cytokines in human patients receiving CAR-T cell therapy at concentrations < 10pg/mL with a sample volume < 10 µL and 5-min assay incubation. In the third part, we demonstrate the clinical application of a machine learning-based digital protein microarray platform for rapid multiplex quantification of cytokines from critically ill COVID-19 patients admitted to the intensive care unit. The platform comprises two low-cost modules: (i) a semi-automated fluidic dispensing module that can be operated inside a biosafety cabinet to minimize the exposure of technician to the virus infection and (ii) a compact fluorescence optical scanner for the potential near-bedside readout. The automated system has achieved high interassay precision (~10% CV) with high sensitivity (<0.4pg/mL). Our data revealed large subject-to-subject variability in patient responses to anti-inflammatory treatment for COVID-19, reaffirming the need for a personalized strategy guided by rapid cytokine assays. Lastly, an AC electroosmosis-enhanced localized surface plasmon resonance (ACE-LSPR) biosensing device was presented for rapid analysis of cytokine IL-1β among sepsis patients. The ACE-LSPR device is constructed using both bottom-up and top-down sensor fabrication methods, allowing the seamless integration of antibody-conjugated gold nanorod (AuNR) biosensor arrays with microelectrodes on the same microfluidic platform. Applying an AC voltage to microelectrodes while scanning the scattering light intensity variation of the AuNR biosensors results in significantly enhanced biosensing performance. The technologies developed have enabled new capabilities with broad application to advance precision medicine of life-threatening acute illnesses in critical care, which potentially will allow the clinical team to make individualized treatment decisions based on a set of time-resolved biomarker signatures.PHDMechanical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/163129/1/yujing_1.pd