Recent breakthroughs in optical scattering-based imaging have enabledvisualization of entities as small as single proteins. Leveraging our innovation, Surface Enhanced Scattering Microscopy (SESM), detection of single protein binding kinetics and single DNA conformational changes have been achieved, showcasing the feasibility of single molecule imaging. In this dissertation, I aim to harness the potential of SESM and extend its relevance in the biomedical realm. My first goal is to conduct multiplexed protein detection and parallel binding kinetics analysis with label-free digital single- molecule counting. My second goal is focused on accurate quantification of cell force. An elastic model has been developed to quantify the cell-substrate interactions and have continuously tracked cell force evolutions upon small-molecule drugs (for example, acetylcholine) stimulation, achieving a temporal resolution of approximately 60 ms over the course of 30 min without attenuating the signals. The third goal is to achieve real- time tracking of DNA self-assembly dynamics. I have demonstrated SESM's capability to image individual DNA origami monomers and established an on-chip temperature annealing system to monitor the real-time progression of DNA self-assembly. The applications of the imaging method, spanning single proteins, single DNA origami, and single cells, are poised to impact the field of biology

Emerging pathogens present several challenges to medical diagnostics. Primarily, the exponential spread of a novel pathogen through naïve populations require a rapid and overwhelming diagnostic response at the site of outbreak. While point-of-care (PoC) platforms have been developed for detection of antigens, serologic responses, and pathogenic genomes, only nucleic acid diagnostics currently have the potential to be developed and manufactured within weeks of an outbreak owing to the speed of next-generation sequencing and custom DNA synthesis. Among nucleic acid diagnostics, isothermal amplification strategies are uniquely suited for PoC implementation due to their simple instrumentation and lack of thermocycling requirement. Unfortunately, isothermal strategies are currently prone to spurious nonspecific amplification, hindering their specificity and necessitating extensive empirical design pipelines that are both time and resource intensive. In this work, isothermal amplification strategies are extensively compared for their feasibility of implementation in outbreak response scenarios. One such technology, Loop-mediated Amplification (LAMP), is identified as having high-potential for rapid development and PoC deployment. Various approaches to abrogating nonspecific amplification are described including a novel in silico design tool based on coarse-grained simulation of interactions between thermophilic DNA polymerase and DNA strands in isothermal reaction conditions. Nonspecific amplification is shown to be due to stabilization of primer secondary structures by high concentrations of Bst DNA polymerase and a mechanism of micro-complement-mediated cross-priming is demonstrated as causal via nanopore sequencing of nonspecific reaction products. The resulting computational model predicts primer set background in 64% of 67 test assays and its usefulness is illustrated further by determining problematic primers in a West Nile Virus-specific LAMP primer set and optimizing primer 3’ nucleotides to eliminate micro-complements within the reaction, resulting in inhibition of background accumulation. Finally, the emergence of Orthopox monkeypox (MPXV) as a recurring threat is discussed and SimCycle is utilized to develop a novel technique for clade-specific discrimination of MPXV based on bridging viral genomic rearrangements (Bridging LAMP). Bridging LAMP is implemented in a 4-plex microfluidic format and demonstrates 100% sensitivity in detection of 100 copies of viral lysates and 45 crude MPXV-positive patient samples collected during the 2022 Clade IIb outbreak.

The response of living cells to electric field (EF) has been observed for more than a hundred years, but the mechanism of how cells interact with EF is not entirely ascertained. Although many efforts have been devoted to the application of EF stimulation in tissue engineering and regeneration, the fundamental scientific principle of such practice remains unveiled and keeps drawing attention during the pursuit of consistent outcomes. In this regard, my research focuses on the underlying mechanism by which EF stimulation evokes cellular responses and the EF modulation of cell signaling pathways to physiological behaviors. The first part of my research focuses on developing the platform for controlled EF stimulation and real-time imaging/analysis. High-k dielectric passivated microelectrodes are fabricated to send capacitively coupled alternating current electric field (AC EF) stimulation to cells. I have developed two generations of EF stimulation devices with environmental control chambers: the first one is used to study cell signaling pathway dynamics; the second one is upgraded with long-term culture capability to study cell physiological behaviors. The second part of my research focuses on the quantification and mechanistic study of AC EF perturbation of the extracellular signal-related kinase (ERK) signaling pathway. I demonstrate that AC EF stimulation can induce both inhibition and activation of the ERK pathway, with different AC EF amplitude thresholds and time and magnitude scales. The mechanistic study shows that the ERK activation is initiated by AC EF-induced epidermal growth factor receptor (EGFR) phosphorylation, and the ERK inhibition is related to AC EF-induced change of Ras activities. In addition, these ERK responses show high sensitivity to AC EF waveform and timing, indicating electrostatic coupling mechanism and providing new parameter spaces for further investigation on the modulation of the ERK signaling pathway via AC EF stimulation. The last part of my research steers to cell physiological behaviors under prolonged AC EF stimulation. I report that AC EF stimulation can clearly inhibit cell proliferation and migration, and the inhibition in cell proliferation is sensitive to AC EF amplitude, stimulation pattern, and pulse rising time. These findings can benefit the AC EF application in medical treatment.

Exploration of long-range conductance in non-redox-active proteins at the single molecule scale is aided by the development of innovative, tailor-made quantitative data analysis techniques. This thesis details the rationale behind the proposed approaches, the steps taken to design and implement every method, and the validation of the methodologies using appropriate experiments, benchmarks, and rigorous statistical data analysis. The first chapter conducts a thorough literature review, sets the stage for the subsequent investigation, and underscores the importance of the research questions addressed in this thesis. The second chapter describes the solvent effects on the electronic conductance of a series of Consensus Tetratricopeptide Repeat proteins (CTPR) measured with Scanning Tunneling Microscopy (STM). The study reveals a reversible reduction in electronic conductance when water (H2O) is replaced with heavy water (D2O) due to a ~6-fold decrease in the carrier diffusion constant as proteins become solvated by D2O. Similar observations are made in a ~7 nm long tryptophan zipper protein, while a phenylalanine zipper protein of comparable length remains unchanged in D2O, highlighting the critical role of aromatic residues in proteins lacking redox cofactors. As an extension to this finding, the third chapter describes the development of a machine-learning model to detect the presence of a protein and identify essential features helping in the detection. For this purpose, a solid-state device was engineered to measure the conductance of CTPR-16 protein wires. This approach addresses the limitations in characterizing the STM gap, enables the collection of stable current vs. time data, and provides a statistical understanding of the electronic transport through a protein. The final chapter investigates real-time changes in conductance in response to protein conformation alterations. A deoxyribonucleic acid (DNA) polymerase Φ29 was chosen for its potential utility as a single-molecule DNA sequencing device. The modified enzyme was bound to electrodes functionalized with streptavidin. Φ29 connected by one biotinylated contact and a second nonspecific contact showed rapid small fluctuations in current when activated. Signals were greatly enhanced with two specific contacts. Features in the distributions of conductance increased by a factor of 2 or more over the open-to-closed conformational transition of the polymerase.

Cubic boron nitride (c-BN), hexagonal boron nitride (h-BN), and semiconducting diamond all have physical properties that make them ideal materials for applications in high power and high frequency electronics, as well as radiation detectors. However, there is limited research on the unique properties and growth of c-BN or h-BN thin films. This dissertation addresses the deposition of c-BN via plasma enhanced chemical vapor deposition (PECVD) on boron doped diamond substrates. In-Situ X-ray photoelectron spectroscopy (XPS) is used to characterize the thickness and hexagonal to cubic ratio of boron nitride thin films. The effects of hydrogen concentration during the deposition of boron nitride are investigated. The boron nitride deposition rate is found to be dependent on the hydrogen gas flow. The sp2 to sp3 bonding is also found to be dependent on the hydrogen gas flow. Preferential growth of h-BN is observed when an excess of hydrogen is supplied to the reaction, while h-BN growth is suppressed when hydrogen flow is reduced to be the limiting reactant. Reduced hydrogen flow is also observed to promote preferential growth of c-BN. The hydrogen limited reaction is used to deposit c-BN on single crystal (100) boron-doped diamond substrates. In-situ ultra-violet photoelectron spectroscopy (UPS) and XPS are used to deduce the valence band offset of the diamond/c-BN interface. A valence band offset of -0.3 eV is measured with the diamond VBM above the VBM of c-BN. This value is then discussed in context of previous experimental results and theoretical calculations. Finally, UPS and XPS are used to characterize the surface states of phosphorus-doped diamond. Variations within the processing parameters for surface preparation and the effects on the electronic surface states are presented and discussed.

My research focuses on studying the interaction between spatiotemporally encoded electric field (EF) and living cells and biomolecules. In this thesis, I report two projects that I have been working on to address these questions. My first project studies the EF modulation of the extracellular-signal-regulated kinase (ERK) pathway. I demonstrated modulation of ERK activities using alternative current (AC) EFs in a new frequency range applied through high-k dielectric passivated microelectrodes with single-cell resolution without electrochemical process induced by the EF stimulation. Further experiments pinpointed a mechanism of phosphorylation site of epidermal growth factor (EGF) receptor to activate the EGFR-ERK pathway that is independent of EGF. AC EFs provide a new strategy to precisely control the dynamics of ERK activation, which may serve as a powerful platform for control of cell behaviors with implications in wide range of biomedical applications. In the second project, I used solid-state nanopore system as the base platform for single molecule experiments, and developed a scalable bottom-up process to construct planar nanopore devices with self-aligned transverse tunneling junctions, all embedded on a nanofluidic chip, based on feedback-controlled reversible electrochemical deposition in a confined nanoscale space. I demonstrated the first simultaneous detection of translocating DNA molecules from both the ionic channel and the tunneling junction with very high yield. Meanwhile, the signal amplitudes from the tunneling junction are unexpectedly high, indicating that these signals are probably dominated by transient currents associated with the fast motion of charged molecules between the transverse electrodes. This new platform provides the flexibility and reproducibility required to study quantum-tunneling-based DNA detection and sequencing. In summary, I have developed two platforms that engineer heterogenous EF at different length scales to modulate live cells and single biomolecules. My results suggest that the charges and dipoles of biomolecules can be electrostatically manipulated to regulate physiological responses and to push detection resolution to single molecule level. Nevertheless, there are still many interesting questions remain, such as the molecular mechanism of EF-protein interaction and tunneling signal extraction. These will be the topics for future investigations.

My research centers on the design and fabrication of biomolecule-sensing devices that combine top-down and bottom-up fabrication processes and leverage the unique advantages of each approach. This allows for the scalable creation of devices with critical dimensions and surface properties that are tailored to target molecules at the nanoscale.

My first project focuses on a new strategy for preparing solid-state nanopore sensors for DNA sequencing. Challenges for existing nanopore approaches include specificity of detection, controllability of translocation, and scalability of fabrication. In a new solid-state pore architecture, top-down fabrication of an initial electrode gap embedded in a sealed nanochannel is followed by feedback-controlled electrochemical deposition of metal to shrink the gap and define the nanopore size. The resulting structure allows for the use of an electric field to control the motion of DNA through the pore and the direct detection of a tunnel current through a DNA molecule.

My second project focuses on top-down fabrication strategies for a fixed nanogap device to explore the electronic conductance of proteins. Here, a metal-insulator-metal junction can be fabricated with top-down fabrication techniques, and the subsequent electrode surfaces can be chemically modified with molecules that bind strongly to a target protein. When proteins bind to molecules on either side of the dielectric gap, a molecular junction is formed with observed conductances on the order of nanosiemens. These devices can be used in applications such as DNA sequencing or to gain insight into fundamental questions such as the mechanism of electron transport in proteins.

Mechanical properties, in particular elasticity, of cancer cells and their microenvironment are important in governing cancer cell fate, for example function, mobility, adhesion, and invasion. Among all tools to measure the mechanical properties, the precision and ease of atomic force microscopy (AFM) to directly apply force—in the range of Pico to micronewtons—onto samples—with length scales from nanometers to tens of micrometers—has made it a powerful tool to investigate the mechanics of materials. AFM is widely used to measure deformability and stiffness of soft biological samples. Principally, these samples are indented by the AFM probe and the forces and indentation depths are recorded. The generated force-indentation curves are fitted with an elastic contact model to quantify the elasticity (e.g. stiffness). AFM is a precise tool; however, the results are as accurate as the contact model used to analyze them. A new contact model was introduced to analyze force-indentation curves generated by spherical AFM probes for deep indentations. The experimental and finite element analysis results demonstrated that the new contact model provides more accurate mechanical properties throughout the indentation depth up to radius of the indenter, while the Hertz model underestimates the mechanical properties. In the classical contact models, it is assumed that the sample is vertically homogenous; however, many biological samples—for example cells—are heterogeneous. A novel two-layer model was utilized to probe Polydimethylsiloxane hydrogel (PDMS) layers on PDMS substrates with stiffness mismatch. In this experiment the stiffness of the substrate was deconvoluted from the AFM measurements to obtain the stiffness of the layer. AFM and confocal reflectance microscopy were utilized along with a novel 3D microengineered breast cancer tumor model to study the crosstalk between cancer tumor and the stromal cells (CAFs) and the ECM remodeling caused by their interplay. The results showed that as the cancer cells invade into the extracellular matrix (ECM), they release PDGF ligands which enable Cafes to remodel the ECM and this remodeling increased the invasion rate of the cancer cells. Next, the effect of the ECM remodeling on anti-cancer drug resistant was investigated within the 3D microengineered cancer model. It was demonstrated that the combinatory treatment by anti-cancer and-anti-fibrotic drugs enhance the efficiency of the cancer treatment. A novel DNA-based 3D hydrogel model with tunable stiffness was investigated by AFM. The results showed the hydrogel stiffness can be enhanced by adding DNA crosslinkers. In addition, the stiffness was reduced to the control sample level by introducing the displacement DNA. Biophysical quantifications along with the in vitro microengineered tumor models provide a unique frame work to study cancer in more detail.

Atomic force microscopy (AFM) was used to study structural differences in the chromatin of cancerous (CP-D) and non-cancerous (EPC2) cell lines. Chromatin samples were extracted using a salt fractionation protocol and subject to Mnase digestion for 2, 4, 8, and 16 minutes. Samples were then immobilized on APTES-functionalized mica sheets. Images were produced using the tapping mode capabilities of the AFM and structural differences between cell lines were quantified using image processing software. Vast differences in chromatin structure were observed between cancerous and non-cancerous cell lines and it was discovered that CP-D chromatin is present as scattered nucleosomes and nucleosome aggregates while EPC2 chromatin is present in intricate arrays. It was also observed that in both the CP-D and EPC2 cell lines, nucleosomes were more isolated and less apparent at longer Mnase digestion times. These findings lead to the conclusion that as the DNA becomes sufficiently digested, chromatin and nucleosomal arrays begin to deteriorate and lose their complex and elaborate structure.

Measurements of the response of superconducting nanowire single photon detector (SNSPD) devices to changes in various forms of input power can be used for characterization of the devices and for probing device-level physics. Two niobium nitride (NbN) superconducting nanowires developed for use as SNSPD devices are embedded as the inductive (L) component in resonant inductor/capacitor (LC) circuits coupled to a microwave transmission line. The capacitors are low loss commercial chip capacitors which limit the internal quality factor of the resonators to approximately $Qi = 170$. The resonator quality factor, approximately $Qr = 23$, is dominated by the coupling to the feedline and limits the detection bandwidth to on the order of 1MHz. In our experiments with this first generation device, we measure the response of the SNSPD devices to changes in thermal and optical power in both the time domain and the frequency domain. Additionally, we explore the non-linear response of the devices to an applied bias current. For these nanowires, we find that the band-gap energy is $\Delta_0 \approx 1.1$meV and that the density of states at the Fermi energy is $N_0 \sim 10^{10}$/eV/$\mu$m$^3$.

We present the results of experimentation with a superconducting nanowire that can be operated in two detection modes: i) as a kinetic inductance detector (KID) or ii) as a single photon detector (SPD). When operated as a KID mode in linear mode, the detectors are AC-biased with tones at their resonant frequencies of 45.85 and 91.81MHz. When operated as an SPD in Geiger mode, the resonators are DC biased through cryogenic bias tees and each photon produces a sharp voltage step followed by a ringdown signal at the resonant frequency of the detector. We show that a high AC bias in KID mode is inferior for photon counting experiments compared to operation in a DC-biased SPD mode due to the small fraction of time spent near the critical current with an AC bias. We find a photon count rate of $\Gamma_{KID} = 150~$photons/s/mA in a critically biased KID mode and a photon count rate of $\Gamma_{SPD} = 10^6~$photons/s/mA in SPD mode.

This dissertation additionally presents simulations of a DC-biased, frequency-multiplexed readout of SNSPD devices in Advanced Design System (ADS), LTspice, and Sonnet. A multiplexing factor of 100 is achievable with a total count rate of $>5$MHz. This readout could enable a 10000-pixel array for astronomy or quantum communications. Finally, we present a prototype array design based on lumped element components. An early implementation of the array is presented with 16 pixels in the frequency range of 74.9 to 161MHz. We find good agreement between simulation and experimental data in both the time domain and the frequency domain and present modifications for future versions of the array.