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- Member of: ASU Electronic Theses and Dissertations
Description
Detection of molecular interactions is critical for understanding many biological processes, for detecting disease biomarkers, and for screening drug candidates. Fluorescence-based approach can be problematic, especially when applied to the detection of small molecules. Various label-free techniques, such as surface plasmon resonance technique are sensitive to mass, making it extremely challenging to detect small molecules. In this thesis, novel detection methods for molecular interactions are described.
First, a simple detection paradigm based on reflectance interferometry is developed. This method is simple, low cost and can be easily applied for protein array detection.
Second, a label-free charge sensitive optical detection (CSOD) technique is developed for detecting of both large and small molecules. The technique is based on that most molecules relevant to biomedical research and applications are charged or partially charged. An optical fiber is dipped into the well of a microplate. It detects the surface charge of the fiber, which does not decrease with the size (mass) of the molecule, making it particularly attractive for studying small molecules.
Third, a method for mechanically amplification detection of molecular interactions (MADMI) is developed. It provides quantitative analysis of small molecules interaction with membrane proteins in intact cells. The interactions are monitored by detecting a mechanical deformation in the membrane induced by the molecular interactions. With this novel method small molecules and membrane proteins interaction in the intact cells can be detected. This new paradigm provides mechanical amplification of small interaction signals, allowing us to measure the binding kinetics of both large and small molecules with membrane proteins, and to analyze heterogeneous nature of the binding kinetics between different cells, and different regions of a single cell.
Last, by tracking the cell membrane edge deformation, binding caused downstream event – granule secretory has been measured. This method focuses on the plasma membrane change when granules fuse with the cell. The fusion of granules increases the plasma membrane area and thus the cell edge expands. The expansion is localized at the vesicle release location. Granule size was calculated based on measured edge expansion. The membrane deformation due to the granule release is real-time monitored by this method.
First, a simple detection paradigm based on reflectance interferometry is developed. This method is simple, low cost and can be easily applied for protein array detection.
Second, a label-free charge sensitive optical detection (CSOD) technique is developed for detecting of both large and small molecules. The technique is based on that most molecules relevant to biomedical research and applications are charged or partially charged. An optical fiber is dipped into the well of a microplate. It detects the surface charge of the fiber, which does not decrease with the size (mass) of the molecule, making it particularly attractive for studying small molecules.
Third, a method for mechanically amplification detection of molecular interactions (MADMI) is developed. It provides quantitative analysis of small molecules interaction with membrane proteins in intact cells. The interactions are monitored by detecting a mechanical deformation in the membrane induced by the molecular interactions. With this novel method small molecules and membrane proteins interaction in the intact cells can be detected. This new paradigm provides mechanical amplification of small interaction signals, allowing us to measure the binding kinetics of both large and small molecules with membrane proteins, and to analyze heterogeneous nature of the binding kinetics between different cells, and different regions of a single cell.
Last, by tracking the cell membrane edge deformation, binding caused downstream event – granule secretory has been measured. This method focuses on the plasma membrane change when granules fuse with the cell. The fusion of granules increases the plasma membrane area and thus the cell edge expands. The expansion is localized at the vesicle release location. Granule size was calculated based on measured edge expansion. The membrane deformation due to the granule release is real-time monitored by this method.
ContributorsGuan, Yan (Author) / Tao, Nongjian (Thesis advisor) / LaBaer, Joshua (Committee member) / Goryll, Michael (Committee member) / Wang, Shaopeng (Committee member) / Arizona State University (Publisher)
Created2015
Description
Surface plasmon resonance (SPR) has emerged as a popular technique for elucidating subtle signals from biological events in a label-free, high throughput environment. The efficacy of conventional SPR sensors, whose signals are mass-sensitive, diminishes rapidly with the size of the observed target molecules. The following work advances the current SPR sensor paradigm for the purpose of small molecule detection. The detection limits of two orthogonal components of SPR measurement are targeted: speed and sensitivity. In the context of this report, speed refers to the dynamic range of measured kinetic rate constants, while sensitivity refers to the target molecule mass limitation of conventional SPR measurement. A simple device for high-speed microfluidic delivery of liquid samples to a sensor surface is presented to address the temporal limitations of conventional SPR measurement. The time scale of buffer/sample switching is on the order of milliseconds, thereby minimizing the opportunity for sample plug dispersion. The high rates of mass transport to and from the central microfluidic sensing region allow for SPR-based kinetic analysis of binding events with dissociation rate constants (kd) up to 130 s-1. The required sample volume is only 1 μL, allowing for minimal sample consumption during high-speed kinetic binding measurement. Charge-based detection of small molecules is demonstrated by plasmonic-based electrochemical impedance microscopy (P-EIM). The dependence of surface plasmon resonance (SPR) on surface charge density is used to detect small molecules (60-120 Da) printed on a dextran-modified sensor surface. The SPR response to an applied ac potential is a function of the surface charge density. This optical signal is comprised of a dc and an ac component, and is measured with high spatial resolution. The amplitude and phase of local surface impedance is provided by the ac component. The phase signal of the small molecules is a function of their charge status, which is manipulated by the pH of a solution. This technique is used to detect and distinguish small molecules based on their charge status, thereby circumventing the mass limitation (~100 Da) of conventional SPR measurement.
ContributorsMacGriff, Christopher Assiff (Author) / Tao, Nongjian (Thesis advisor) / Wang, Shaopeng (Committee member) / LaBaer, Joshua (Committee member) / Chae, Junseok (Committee member) / Arizona State University (Publisher)
Created2013
Description
An imaging measurement technique is developed using surface plasmon resonance. Plasmonic-based electrochemical current imaging (P-ECi) method has been developed to image the local electrochemical current optically, it allows us to measure the current density quickly and non-invasively [1, 2]. In this thesis, we solve the problems when we extand the P-ECi technique to the field of thin film system. The P-ECi signal in thin film structure was found to be directly proportional to the electrochemical current. The upper-limit of thin film thickness to use the proportional relationship between P-ECi signal and EC current was discussed by experiment and simulation. Furthermore, a new algorithm which can calculate the current density from P-ECi signal without any thickness limitation is developed and tested. Besides, surface plasmon resonance is useful phenomenon which can be used to detect the changes in the refractive index near the gold sensing surface. With the assistance of pH indicator, by applied EC potential on the gold film as the working electrode, the detection of H2 evolution reaction can be enhanced. This measurement technique is useful in analyzing local EC information and H2 evolution. References [1] S. Wang, et al., "Electrochemical Surface Plasmon Resonance: Basic Formalism and Experimental Validation," Analytical Chemistry, vol. 82, pp. 935-941, 2010/02/01 2010. [2] X. Shan, et al., "Imaging Local Electrochemical Current via Surface Plasmon Resonance," Science, vol. 327, pp. 1363-1366, March 12, 2010 2010.
ContributorsZhao, Yanjun (Author) / Tao, Nongjian (Thesis advisor) / Wang, Shaopeng (Committee member) / Tsow, Tsing (Committee member) / Arizona State University (Publisher)
Created2013
Description
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.
ContributorsWang, Yuan (Author) / Qing, Quan (Thesis advisor) / Lindsay, Stuart (Committee member) / Wang, Shaopeng (Committee member) / Ros, Robert (Committee member) / Arizona State University (Publisher)
Created2021
Description
Endothelial cells lining the blood vessels are a critical component of the Blood-brain barrier (BBB), that supplies oxygen and nutrients to the brain. These cells in the brain are constantly under shear stress caused by blood flow, which gives them their distinctive features separating from other endothelial cells. The primary aim of the thesis is to study the effect of shear induced changes in endothelial cell morphology using a microfluidic platform. Incorporating primary brain endothelial cells (bEC) in the model, the aim was to develop a more physiologically relevant BBB-on-chip model, since studies often use Human Umbilical Vein Endothelial Cells (HUVEC) in their model, which do not represent the characteristics of endothelial cells specific to the brain. The parallel channel microfluidic system has; (i) central hydrogel channel which is injected with fibrin, a component of the extra cellular matrix (ECM) and (ii) two media channels where endothelial cells were seeded to form a monolayer. After two days of culture, the endothelial cells were subjected to various shear conditions for 24 hours. Significant changes in aspect ratio of cells was observed and a trend in directionally in aligning to the flow, when cultured under linearly increasing shear stress from 1-5 dyne/cm². Additionally, reducing the height of the microfluidic platform helped in modeling higher shear with less flow rate. These findings highlight the effects of shear induced morphological changes in endothelial cells.
ContributorsSatyanayaranan, Ritvik Srivant (Author) / Reynoso, Roberto (Thesis advisor) / Nikkhah, Mehdi (Committee member) / Migrino, Raymond (Committee member) / Wang, Shaopeng (Committee member) / Arizona State University (Publisher)
Created2024
Description
Quantifying molecular interactions is pivotal for understanding biological processes at molecular scale and for screening drugs. Although various detection technologies have been developed, it is still challenging to quantify the binding kinetics of small molecules because the sensitivities of the mainstream technologies scale down with the size of the molecule. To address this problem, two different optical detection methods, charge sensitive optical detection (CSOD) and virion
ano-oscillators, are developed to measure the binding-induced charge change instead of the mass change, which enables quantification of the binding kinetics for both large and small molecules.
In particular, the nano-oscillator approach provides a unique capability to image individual nanoparticles and measure the size and charge of each nanoparticle simultaneously. This approach is applied to measure one of the smallest biological particles - single protein molecules. By tracking the oscillation of each protein molecule, the size, charge, and mobility are measured in real-time with high precision. This capability also allows to monitor the conformation and charge changes of single protein molecules upon ligand binding. Measuring the size and charge of single proteins opens a new revenue to protein analysis and disease biomarker detection at the single molecule level.
The virion
ano-oscillators and the single protein approach employ a scheme where a particle is tethered to the surface with a polymer molecule. The dynamics of the particle is governed by two important forces: One is entropic force arising from the conformational change of the molecular tether, and the other is solvent damping on the particle and the molecule. The dynamics is studied by varying the type of the tether molecule, size of the particle, and viscosity of the solvent. The findings provide insights into single molecule studies using not only tethered particles, but also other approaches, including force spectroscopy using atomic force microscopy and nanopores.
ano-oscillators, are developed to measure the binding-induced charge change instead of the mass change, which enables quantification of the binding kinetics for both large and small molecules.
In particular, the nano-oscillator approach provides a unique capability to image individual nanoparticles and measure the size and charge of each nanoparticle simultaneously. This approach is applied to measure one of the smallest biological particles - single protein molecules. By tracking the oscillation of each protein molecule, the size, charge, and mobility are measured in real-time with high precision. This capability also allows to monitor the conformation and charge changes of single protein molecules upon ligand binding. Measuring the size and charge of single proteins opens a new revenue to protein analysis and disease biomarker detection at the single molecule level.
The virion
ano-oscillators and the single protein approach employ a scheme where a particle is tethered to the surface with a polymer molecule. The dynamics of the particle is governed by two important forces: One is entropic force arising from the conformational change of the molecular tether, and the other is solvent damping on the particle and the molecule. The dynamics is studied by varying the type of the tether molecule, size of the particle, and viscosity of the solvent. The findings provide insights into single molecule studies using not only tethered particles, but also other approaches, including force spectroscopy using atomic force microscopy and nanopores.
ContributorsMa, Guangzhong, Ph.D (Author) / Tao, Nongjian (Thesis advisor) / Wang, Shaopeng (Thesis advisor) / Ros, Alexandra (Committee member) / Guo, Jia (Committee member) / Arizona State University (Publisher)
Created2019
Description
Measuring molecular interaction with membrane proteins is critical for understanding cellular functions, validating biomarkers and screening drugs. Despite the importance, developing such a capability has been a difficult challenge, especially for small molecules binding to membrane proteins in their native cellular environment. The current mainstream practice is to isolate membrane proteins from the cell membranes, which is difficult and often lead to the loss of their native structures and functions. In this thesis, novel detection methods for in situ quantification of molecular interactions with membrane proteins are described.
First, a label-free surface plasmon resonance imaging (SPRi) platform is developed for the in situ detection of the molecular interactions between membrane protein drug target and its specific antibody drug molecule on cell surface. With this method, the binding kinetics of the drug-target interaction is quantified for drug evaluation and the receptor density on the cell surface is also determined.
Second, a label-free mechanically amplification detection method coupled with a microfluidic device is developed for the detection of both large and small molecules on single cells. Using this method, four major types of transmembrane proteins, including glycoproteins, ion channels, G-protein coupled receptors (GPCRs) and tyrosine kinase receptors on single whole cells are studied with their specific drug molecules. The basic principle of this method is established by developing a thermodynamic model to express the binding-induced nanometer-scale cellular deformation in terms of membrane protein density and cellular mechanical properties. Experiments are carried out to validate the model.
Last, by tracking the cell membrane edge deformation, molecular binding induced downstream event – granule exocytosis is measured with a dual-optical imaging system. Using this method, the single granule exocytosis events in single cells are monitored and the temporal-spatial distribution of the granule fusion-induced cell membrane deformation are mapped. Different patterns of granule release are resolved, including multiple release events occurring close in time and position. The label-free cell membrane deformation tracking method was validated with the simultaneous fluorescence recording. And the simultaneous cell membrane deformation detection and fluorescence recording allow the study of the propagation of the granule release-induced membrane deformation along cell surfaces.
First, a label-free surface plasmon resonance imaging (SPRi) platform is developed for the in situ detection of the molecular interactions between membrane protein drug target and its specific antibody drug molecule on cell surface. With this method, the binding kinetics of the drug-target interaction is quantified for drug evaluation and the receptor density on the cell surface is also determined.
Second, a label-free mechanically amplification detection method coupled with a microfluidic device is developed for the detection of both large and small molecules on single cells. Using this method, four major types of transmembrane proteins, including glycoproteins, ion channels, G-protein coupled receptors (GPCRs) and tyrosine kinase receptors on single whole cells are studied with their specific drug molecules. The basic principle of this method is established by developing a thermodynamic model to express the binding-induced nanometer-scale cellular deformation in terms of membrane protein density and cellular mechanical properties. Experiments are carried out to validate the model.
Last, by tracking the cell membrane edge deformation, molecular binding induced downstream event – granule exocytosis is measured with a dual-optical imaging system. Using this method, the single granule exocytosis events in single cells are monitored and the temporal-spatial distribution of the granule fusion-induced cell membrane deformation are mapped. Different patterns of granule release are resolved, including multiple release events occurring close in time and position. The label-free cell membrane deformation tracking method was validated with the simultaneous fluorescence recording. And the simultaneous cell membrane deformation detection and fluorescence recording allow the study of the propagation of the granule release-induced membrane deformation along cell surfaces.
ContributorsZhang, Fenni (Author) / Tao, Nongjian (Thesis advisor) / Chae, Junseok (Committee member) / Borges, Chad (Committee member) / Jing, Tianwei (Committee member) / Wang, Shaopeng (Committee member) / Arizona State University (Publisher)
Created2018
Description
Proteins play a central role to human body and biological activities. As powerful tools for protein detections, many surface plasmon resonance based techniques have been developed to enhance the sensitivity. However, sensitivity is not the only final goal. As a biosensor, four things really matter: sensitivity, specificity, resolution (temporal/spatial) and throughput.
This dissertation presents several works on developing novel plasmonic based techniques for protein detections on the last two aspects to extend the application field. A fast electrochemically controlled plasmonic detection technique is first developed with the capability of monitoring electrochemical signal with nanosecond response time. The study reveals that the conformational gating of electron transfer in a redox protein (cytochrome c) takes place over a broad range of time scales (sub-µs to ms). The second platform integrates ultra-low volume piezoelectric liquid dispensing and plasmonic imaging detection to monitor different protein binding processes simultaneously with low sample cost. Experiment demonstrates the system can observe binding kinetics in 10×10 microarray of 6 nL droplet, with variations of kinetic rate constants among spots less than ±5%. A focused plasmonic imaging system with bi-cell algorithm is also proposed for spatial resolution enhancement. The two operation modes, scanning mode and focus mode, can be applied for different purposes. Measurement of bacterial aggregation demonstrates the higher spatial resolution. Detections of polystyrene beads binding and 50 nm gold nanoparticles oscillation show a high signal to noise ratio of the system.
The real properties of protein rely on its dynamic personalities. The above works shed light upon fast and high throughput detection of protein kinetics, and enable more applications for plasmonic imaging techniques. It is anticipated that such methods will help to invoke a new surge to unveil the mysteries of biological activities and chemical process.
This dissertation presents several works on developing novel plasmonic based techniques for protein detections on the last two aspects to extend the application field. A fast electrochemically controlled plasmonic detection technique is first developed with the capability of monitoring electrochemical signal with nanosecond response time. The study reveals that the conformational gating of electron transfer in a redox protein (cytochrome c) takes place over a broad range of time scales (sub-µs to ms). The second platform integrates ultra-low volume piezoelectric liquid dispensing and plasmonic imaging detection to monitor different protein binding processes simultaneously with low sample cost. Experiment demonstrates the system can observe binding kinetics in 10×10 microarray of 6 nL droplet, with variations of kinetic rate constants among spots less than ±5%. A focused plasmonic imaging system with bi-cell algorithm is also proposed for spatial resolution enhancement. The two operation modes, scanning mode and focus mode, can be applied for different purposes. Measurement of bacterial aggregation demonstrates the higher spatial resolution. Detections of polystyrene beads binding and 50 nm gold nanoparticles oscillation show a high signal to noise ratio of the system.
The real properties of protein rely on its dynamic personalities. The above works shed light upon fast and high throughput detection of protein kinetics, and enable more applications for plasmonic imaging techniques. It is anticipated that such methods will help to invoke a new surge to unveil the mysteries of biological activities and chemical process.
ContributorsWang, Yan (Author) / Tao, Nongjian (Thesis advisor) / Chae, Junseok (Committee member) / Goryll, Michael (Committee member) / Wang, Shaopeng (Committee member) / Arizona State University (Publisher)
Created2018
Description
Signal transduction networks comprising protein-protein interactions (PPIs) mediate homeostatic, diseased, and therapeutic cellular responses. Mapping these networks has primarily focused on identifying interactors, but less is known about the interaction affinity, rates of interaction or their regulation. To better understand the extent of the annotated human interactome, I first examined > 2500 protein interactions within the B cell receptor (BCR) signaling pathway using a current, cutting-edge bioluminescence-based platform called “NanoBRET” that is capable of analyzing transient and stable interactions in high throughput. Eighty-three percent (83%) of the detected interactions have not been previously reported, indicating that much of the BCR pathway is still unexplored. Unfortunately, NanoBRET, as with all other high throughput methods, cannot determine binding kinetics or affinities. To address this shortcoming, I developed a hybrid platform that characterizes > 400 PPIs quantitatively and simultaneously in < 1 hour by combining the high throughput and flexible nature of nucleic programmable protein arrays (NAPPA) with the quantitative abilities of surface plasmon resonance imaging (SPRi). NAPPA-SPRi was then used to study the kinetics and affinities of > 12,000 PPIs in the BCR signaling pathway, revealing unique kinetic mechanisms that are employed by proteins, phosphorylation and activation states to regulate PPIs. In one example, activation of the GTPase RAC1 with nonhydrolyzable GTP-γS minimally affected its binding affinities with phosphorylated proteins but increased, on average, its on- and off-rates by 4 orders of magnitude for one-third of its interactions. In contrast, this phenomenon occurred with virtually all unphosphorylated proteins. The majority of the interactions (85%) were novel, sharing 40% of the same interactions as NanoBRET as well as detecting 55% more interactions than NanoBRET. In addition, I further validated four novel interactions identified by NAPPA-SPRi using SDS-PAGE migration and Western blot analyses. In one case, we have the first evidence of a direct enzyme-substrate interaction between two well-known proto-oncogenes that are abnormally regulated in > 30% of cancers, PI3K and MYC. Herein, PI3K is demonstrated to phosphorylate MYC at serine 62, a phosphosite that increases the stability of MYC. This study provides valuable insight into how PPIs, phosphorylation, and GTPase activation regulate the BCR signal transduction pathway. In addition, these methods could be applied toward understanding other signaling pathways, pathogen-host interactions, and the effect of protein mutations on protein interactions.
ContributorsPetritis, Brianne Ogata (Author) / LaBaer, Joshua (Thesis advisor) / Lake, Douglas (Committee member) / Wang, Shaopeng (Committee member) / Arizona State University (Publisher)
Created2018
Description
Chimeric antigen receptor (CAR)-T cell therapy is a type of cancer immunotherapy has shown promising results in engineering the T cells which targets a specific antigen. Despite their success rate, there are certain limitations to the use of CAR-T therapies that includes cytokine release syndrome (CRS), neurologic toxicity, lack of response in approximately 50% of treated patients, monitoring of patients treated with CAR-T therapy. However, rapid point- of- care testing helps in quantifying the circulating CAR T cells and can enhance the safety of patients, minimize the cost of CAR-T cell therapy, and ease the management process. Currently, the standard method to quantify CAR-T cell in patient blood samples are flow cytometry and quantitative polymerase chain reaction (qPCR). But these techniques are expensive and are not easily accessible and suitable for point- of- care testing to assist real- time clinical decisions. To overcome these hurdles, here I propose a solution to these problems by rapid optical imaging (ROI)- based principle to monitor and detect CAR-T cells. In this project, a microfluidic device is developed and integrated with two functions: (1) Centrifuge free, filter- based separation of white blood cells and plasma; (2) Optical imaging- based technique for digital counting of CAR T- cells. Here, I carried out proof- of- concept test on the laser cut prototype microfluidic chips as well as the surface chemistry for specific capture of CAR-T cells. These data show that the microfluidic chip can specifically capture CAR-T positive cells with concentration dependent counts of captured cells. Further development of the technology could lead to a new tool to monitor the CAR-T cells and help the clinicians to effectively measure the efficacy of CAR-T therapy treatment in a faster and safer manner.
ContributorsElanghovan, Praveena (Author) / Wang, Shaopeng (Thesis advisor) / Forzani, Erica (Committee member) / Nikkhah, Mehdi (Committee member) / Arizona State University (Publisher)
Created2023