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Browsing by Subject "Biosensors"

Now showing 1 - 5 of 5
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    Dielectrophoresis on nanostructured substrates for enhanced plasmonic biosensing
    (2017-02) Barik, Avijit
    Performance of surface-based plasmonic biosensors is often plagued by diffusion-limited transport, which complicates detection from low-concentration analytes. By harnessing gradient forces available from the sharp metallic edges, tips or gaps that are often found in the plasmonic sensors, it is possible to combine a dielectrophoretic concentration approach to overcome the mass transport limitations. A transparent electrode is integrated with the plasmonic substrates that allow dielectrophoresis without interfering with the label-free sensing schemes such as surface plasmon resonance or Raman spectroscopy. Furthermore, by shrinking the gap between gold electrodes to sub-10 nm, we show ultralow-power trapping of nanoparticles and biomolecules. Reducing the operating voltages diminishes Joule heating, bubble formation and electrochemical surface reactions - hurdles associated with traditional electrodes for dielectrophoresis. The ultralow power electronic operation combined with plasmonic detection has potential in high-density on-chip integration and portable biosensing.
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    Investigating Novel Hetero-Fret Biosensors for Environmental Ionic Strength Using Time-Resolved Fluorescence And Anisotropy
    (2020-08) Aplin, Cody
    One aspect of the biocomplexity of eukaryotic cells is the compartmentalized ionic strength, which impacts a myriad of biological functions. In this thesis, we investigate a class of environmental ionic strength sensors (mCerulean3–linker–mCitrine) that can be genetically encoded in site-specific compartments in living cells. In these sensors, mCerulean3–mCitrine acts as a Förster resonance energy transfer (FRET) pair that is tethered by two oppositely charged α-helices in the linker region. We hypothesize that as the ionic strength is increased, the attractive force between these charged alpha helices will be screened by the dissolved ions, resulting in an increase in the donor–acceptor distance (i.e., reduced FRET efficiency). To test this hypothesis, we investigated the rotational dynamics of these sensors using a newly developed time-dependent polarization anisotropy approach of the acceptor (mCitrine) emission, following pulsed excitation of the mCerulean3 (donor) in cleaved and intact mCerulean3–linker–mCitrine sensors (KE, RD, and RE) in different Hofmeister salt solutions (namely, KCl, NaCl, NaI, and Na2SO4). Our results show that the rotational dynamics of the intact and cleaved sensors are distinct under the excitation-detection conditions. Importantly, the FRET efficiency decreases and the donor-acceptor distance increases as the environmental ionic strength increases, with slight sensitivity to the Hofmeister salt type. In contrast, FRET efficiency of E6G2 with electrostatically neutral amino acids in the linker region exhibits salt-independent rotational and FRET dynamics. Our time-resolved anisotropy data were also used to test existing theorical models concerning the steady-state anisotropy of these hetero-FRET sensors, while revealing the ionic strength effects on the angle between the dipole moments of the donor and acceptor in these sensors. Using our complementary, traditional time-resolved fluorescence method, we optimized our time-resolved anisotropy approach for FRET analysis and developed a theoretical model using Debye ionic screening of the two charged alpha helices in the linker region. These results help establishing time-resolved anisotropy of donor-acceptor pairs as a quantitative means for FRET analysis, which complement other traditional methods such as time-resolved fluorescence.
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    Label-Free, Microfluidic Biosensors with Printed, Floating-Gate Transistors
    (2017-12) White, Scott
    Printed electronics and microfluidics are two emerging and developing technologies with the common attractive feature of scalability. Advancements in fabrication capabilities have evolved research questions from, “What can we build?” to, “What should we build?”. This work focuses on the combination of these two technologies and their application to biosensing. The motivating theme is to understand how integrated, functional materials interact, elucidate the underlying molecular phenomena, then utilize the emergent advantages to address the outstanding limitations of conventional biosensing strategies. Printed electronics have recently been applied to biological detection with a variety of techniques1 while microfluidics, since their inception, have been used to handle biological fluids.2 The work presented here outlines a patented sensing strategy based off Floating-Gate Transistors (FGTs). The FGT design physically separates the electronic materials and biological fluids and thus bypasses various compatibility obstacles limiting other next-generation sensor technologies.3 The specific changes in interfacial properties that lead to robust signal transduction are derived empirically.4 This is followed by a mechanistic investigation into the molecular origin of sensor operation when FGTs are used in biomolecular detection. Finally, the versatility and scalability engendered by facile prototyping of FGTs is exemplified by successful iterations to DNA,3 ricin,5 and gluten proteins. The first proof-of-principle experiments incorporated printed electronics with an elementary biological system of DNA oligonucleotides. The results successfully demonstrated the potential of FGTs but failed to solidify their concrete value. Systematic investigation into the complex dynamics at the interface of chemically functionalized electrodes and electrolytes uncovered the most attractive features of the FGT technology. The chemistry was tuned with molecules that range in complexity from simple, short-chain alkyl-thiols to reversible protein-protein interactions. The observed responses with well-controlled systems were generalized to real systems like protein capture in food matrices (e.g. ricin in milk, orange juice). The resulting versatility originated from the label-free, electronic sensing mechanism and opened a range of possibilities for FGTs’ impact. The fundamental insights into interfacial dynamics, device operation, and biomolecular interactions were made possible by the advancements in the materials science and fabrication techniques underlying the presented results. Future avenues of development are hypothesized along with the most promising strategies. The continued elucidation of the physical mechanism and engineering upgrades justify the proposed strategies and inspire the continued effort to fully realize the potential of FGT biosensors.
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    Magnetic Biosensing Technologies and Applications
    (2021-01) Su, Diqing
    Magnetoresistance (MR) biosensors have been widely employed in the detection of small molecules, proteins, and nucleic acids. The major contribution of this thesis is the development of novel giant magnetoresistance (GMR) biosensing technologies to improve the portability, sensitivity, and flexibility of the detection process through both modeling and experimental studies. The work in this thesis can be divided into two major parts, i.e., the development of novel GMR sensors and the development of novel magnetic nanolabels as the labels for the target analytes. The possibility of employing spintronic structures for neuron stimulation is also explored.The optimization of the GMR sensors are from three different aspects. Firstly, GMR biosensors are successfully integrated with a handheld biosensing system, which is capable of fast, accurate, and onsite disease diagnosis. The detection of influenza A virus in swine nasal swab samples is demonstrated with comparable sensitivity to the enzyme-linked immunosorbent assay (ELISA), which is a lab-based golden standard technology for protein detection. Secondly, flexible GMR sensors with a bending radius lower than 1 mm and similar magnetic properties to their rigid counterparts are fabricated with a two-step thinning process. A lab-on-a-needle detection platform is used for cell detection with a LOD of 200 cells in the testing sample, exhibiting great potential in the onsite biopsy at the tumor site, as well as in drug delivery efficacy monitoring as an implanted device. Thirdly, a large-area GMR biosensing scheme based on the reverse nucleation mechanism is proposed, modeled, and demonstrated, which leads to a sensitivity 20 times higher than traditional GMR biosensors. In addition, magnetic nanowires (MNWs) are employed as the magnetic labels for the first time in the cell detection process. The MNWs can be readily internalized into cells without inducing much cytotoxicity. The distance, angular, and concentration dependence of sensor signal generated by the MNWs are studied. Single-cell detection has been successfully realized in the GMR biosensors when the cells are in direct contact with the sensor surface.
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    Self-assembling multienzyme systems at oil-water interface for biphasic biotransformations
    (2008-11) Narayanan, Ravindrabharathi
    Living cells are highly organized with many functional units or organelles separated by membranes. The membrane is comprised of specific proteins and lipid components that enable it to perform its unique roles for that cell or organelle. At cellular membranes, lipid bilayers are stabilized laterally with the help of integral proteins. This stability is provided through a clustering of the hydrophobic core of both the lipid bilayer and the integral protein. Surface and interfacial phenomenon involving the activities of enzymes are wide spread in cellular systems and occur within the interfacial constraints of substrate accessibility, distribution and partitioning. Similar mechanisms can be used to enhance productivity of industrial biotransformations at oilwater interface. Detailed study and manipulation of interfacial enzyme catalysis is of great interest for biotechnology, chemical technology, biology, and offers new opportunities in protein and polymer chemistry, separation science, bio-renewable products, environmental science and waste minimization. Herein, novel self-assembling enzyme systems were developed by manipulation of microenvironment of the enzymes for interfacial biotransformations at oil-water interface. The enzyme molecules were modified to self-assemble at oil-water interface by conjugation with hydrophobic moieties like polymers. The present work focused on (i) characterization of enzyme assembly morphology, (ii) stabilizing the enzymes at the interface, (iii) broadening the scope of interfacial biocatalysis with multienzyme-cofactor system and developing method to assemble cofactors at the interface, (iv) investigating the kinetic parameters of the interfacial reaction, (v) improving the activity of interfacial iii enzyme by interfacial mobility enhancement and (vi) extending the hydrophobic manipulation of enzyme’s microenvironment for development of biosensors based on nanofibers containing organic soluble enzymes. Four sets of model reactions system, a single enzyme system and three multiple enzyme system was employed for interfacial biocatalysis study and oxidation of glucose by glucose oxidase was chosen as a model system for biosensor development. In a previous study, it was demonstrated that interface-assembled enzymes improved the reaction rate by two orders of magnitude. As a part of the present work, the important role of mobility and the assembly morphology of the interface-assembled enzyme on regulating the enzymatic liquid membrane fluidity at the interface were investigated. To characterize the surface assembly morphology of the interfaceassembled enzyme by surface pressure analysis, Langmuir film balance was used. The resulting surface pressure isotherm exhibited monolayer assembly, with intramolecular rearrangement of the interface-assembled enzymes. The mobility of the novel interfaceassembled enzymes was evaluated by using fluorescence recovery after photobleaching technique that gave the diffusion coefficient of 6.7×10-10 cm2/sec, three orders of magnitude less than that of native enzymes in aqueous solution, due to localization of the modified enzyme at the interface. Though modification of enzymes with polymer for interfacial assembly reduced its mobility, the conjugation of polymer to enzyme stabilized the enzymes against interfacial denaturation. The polymer stabilizes the three dimensional structure of enzymes and prevent it from unfolding at hydrophobic interfaces. Apart from the iv interfacial stabilization of interface-assembled enzyme by polymer, the localization of enzymes at the interface offered a unique opportunity to enhance the stability of the enzymes against the deactivation effect of compounds in bulk phase. Chloroperoxidase (CPO) was chosen as a model enzyme to explore the factors that determine the stability of interface-assembled enzymes. Although the interface-assembled CPO showed improved stability as compared to native CPO, enzyme deactivation by peroxide reactants like hydrogen peroxide (H2O2) in the bulk phase, still limited the overall productivity of the enzyme. Two approaches to further improve the stability of interfaceassembled CPO were examined in this work. In one approach, several chemical stabilizers were used to prevent highly reactive intermediates from oxidizing the porphyrin ring active site of CPO; polyethylene glycol (PEG) was found exceptional in that it increased both the operational and storage stability of CPO with a productivity increase of 57%, an operational stability improvement by almost 2 folds and a storage stability of 60% activity retention after 24 hours incubation in 1 mM H2O2. On the other hand, glucose enhanced the operational stability by 2 folds, but exhibited no significant effect on storage stability. While in a second approach, in situ generation of hydrogen peroxide (H2O2) by using glucose oxidase (GOx) to keep H2O2 concentration low was applied. It was found that the combined effect of presence of glucose and lowered concentration of H2O2, extended operational lifetime to 60 minutes for CPO with in situ generation of H2O2 by GOx. To expand the scope of interfacial enzyme catalysis, multienzyme oxidoreductases-cofactor systems were employed. The structure of cofactors involves unique combination of functional groups that are required by oxidoreductases enzymes to carry out biotransformations and any modification to cofactor for interfacial assembly should not affect the enzyme-cofactor interaction. The challenge of modifying cofactors to assemble at the interface was overcome by structural manipulation of the adenine group of nicotinamide cofactor. The synthesis of interface-assembled cofactor gave a process yield of 67%, the modified cofactor was highly stable with a continuous operation of 2150 hours and turnover number of 2617 for a biphasic reaction involving reduction of acetophenone in organic phase and oxidation of glucose in aqueous phase. The Damkohler number that gives the ratio between reaction rate and mass transfer rate was obtained to be 0.12 with interface assembled cofactor, compared to 87.5 with native enzymes and free cofactor, indicating mass transfer limitations with interface assembled cofactor. The kinetic analysis of interface-assembled cofactor gave the binding resistance of enzyme to cofactor at the interface, Kc, as 0.18 mM compared to 0.03 mM of native enzyme and free cofactor, which indicated that limited interfacial interaction between molecules and two-dimensional mobility of the enzymes contributed significant resistance towards interfacial reaction. A novel mechanism of nanostirring was developed to improve the twodimensional mobility of interface-assembled enzymes. Iron oxide (Fe3O4) superparamagnetic nanoparticles were coupled with polymer conjugated enzymes for interfacial assembling and applied to improve the mobility of the interface-assembled enzyme under external electromagnetic field. The enhanced mobility of the interfaceassembled enzymes was quantified through fluorescent microscopic visualization, and enabled over 600% of improvement in the observed reaction rate for both single enzyme and multienzyme systems as compared to reactions in the absence of the magnetic field. The combination of slow reactions and denaturation of dehydrogenase enzymes due to stirring posed a major constraint for realizing reactions with configuration of both cofactor and enzymes assembled at the interface. This limitation was overcome by development of a unique interfacial biotransformation with interface assembled cofactor and interface-assembled multiple enzymes was realized by employing relatively shear resistant dehydrogenase, ADH RS1, coupled with GluDH for faster NADH turnover. A maximum NADH turnover of 13 was achieved by optimizing the reaction conditions enzyme ratio, organic phase and aqueous phase substrates concentrations, and polymer modifier concentration added during modification of enzymes. In another effort, the manipulation of microenvironment of enzymes for enhanced hydrophobicity was extended to develop completely organic-soluble enzymes. The organic soluble enzymes were utilized in the development of polymer-enzyme composite nanofibers for biosensing applications. Polyurethane nanofibers of diameters of 100-140 nm containing up to 20% (w/w) protein were prepared via electrospinning. The enzyme, glucose oxidase (GOx), was complexed with an ionic surfactant and was thus transformed into organic soluble prior to electrospinning. When examined for biosensor applications, such prepared nanofibers showed a sensitivity of up to 66 A M-1 mgenzyme- 1 (or 0.39 A M-1 cm-2), 100 times improvement from previous studies. The high enzyme loading coupled with the high specific surface area of the nanofibers enhanced the reaction kinetics and thus enabled strong responses for small changes in glucose concentration. The confinement of the enzyme within the body of nanofibers also stabilized the enzyme, such that the biosensor retained 80% of its sensitivity after 70 days. The interface-assembled enzymes with their improved interfacial stability can substitute soluble enzymes that are presently used for many industrial applications with biphasic systems. Also, Interface-assembled enzymes offer simultaneous access to reactants in both the bulk phases across the interface and thus improve the overall efficiency for the biotransformations between immiscible chemicals. The novel polymerenzyme conjugates and functional materials that were developed through this research with their unique structural, magnetic and mechanical properties can be used in broad range of applications like sensors, membrane technology, generate alternative strategies for encapsulation and delivery of therapeutic agents, and will enable minimum downstream processing for specialty chemical synthesis. The present work is of great interest in the search for production of different important industrial chemicals including bio-renewable products and for sustainable environmental quality.