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Thesis (Ph. D.)--University of Texas at Austin, 2002. / Vita. Includes bibliographical references. Available also from UMI Company.

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Hemimicelles and admicelles are well-investigated wonders in modern science; they are surfactant monolayers and surface adsorbed micelles, respectively. Capacitance measurements for monitoring the formation of dodecyl sulfate (DS) surfactant monolayer on positively charged gold substrates (planar gold) and the adsorbance of 2-naphthol onto DS surfactant monolayer were performed. The investigation of the electrical control of DS at various concentrations (4, 6, 16, and 32 mM) below and above the critical micelle concentration (CMC= 8 mM) on gold surfaces for analyte preconcentration, prior to chromatographic analysis, is presented. Charged ionic surfactants, such as DS, drawn to a surface of opposite charge (porous nickel substrates coated with gold) serve as a stationary phase to trap organic analytes. It is believed that these DS assemblies gain stability through surfactant chain–chain interactions. The attachment and the removal of the surfactant are controlled using an electric field. Due to the fact that the surfactantanalyte association is released by electrical control, organic solvents, which are used in conventional solid phase extraction, are not required, making this procedure environmentally friendly. Electrical Impedance Spectroscopy was used to investigate the formation of the DS layer and the preconcentration of 2-naphthol in the presence of an applied electric field. High performance liquid chromatography was used to determine 2- naphthol concentrations. Anthracene and 9-anthracenecarboxylic acid were substituted as additional test molecules as well. Presented are the results of the preconcentration of 2-naphthol, anthracene and 9-anthracenecarboxylic acid using the DS layer with various concentrations of sodium dodecyl sulfate on a gold electrode surface.

E-commerce and retail companies are seeking ways to cut delivery time and cost by exploring opportunities to use drones for making last-mile deliveries. In recent years, drone routing and scheduling have become a highly active area of research. This research addresses the concept of a truck-drone combined delivery by allowing autonomous drones to fly from delivery trucks, make deliveries, and fly to delivery trucks nearby. The first part of the research considers the synchronized truck drone routing model by allowing multiple drones to fly from any truck, serve customers and immediately return to any available truck or depot in the system. The goal is to find the optimal routes of both trucks and drones which minimize the arrival time of both trucks and drones at the depot after completing the deliveries. The problem can be solved by the formulated Mixed Integer Programming (MIP) for the small-size problems and our proposed heuristic called Adaptive Insertion Heuristics (ADI) which is based on the insertion technique for the medium/large-size problems. The second part of the research extends the first studied problem by allowing drones to serve multiple customers before merging with trucks as well as considering the capacity requirement for both vehicles. The problem is mathematically formulated and two efficient heuristic algorithms are designed to solve the large-size problems: Drone Truck Route Construction (DTRC) and Large Neighborhood Search (LNS). In the third study, the goal is to study the potential benefits of combining different types of fleet vehicles to deliver packages to the customers. Three types of vehicles are considered in this study including large drones, traditional trucks and hybrid trucks. The problem can be optimally solved by a mathematical formulation on a small scale. Two efficient metaheuristics based on Variable Neighborhood Search (VNS) and Large Neighborhood Search (LNS) are proposed to solve for approximate solutions of the large-size problems. A case study and numerical analysis demonstrate the better delivery time of the proposed model when compared with the delivery time of other delivery models with a single fleet type.

Addition of a small amount of polar solvent (i.e. modifier) which contains an ionic component (i.e. additive) to a CO2 mobile phase has shown major improvement in the elution of ionic analytes via packed column supercritical fluid chromatography (SFC). Firstly, we focused on the elution of sodium arylsulfonate analytes by using various ionic additives, such as lithium acetate, ammonium acetate, tetramethylammonium acetate, tetrabutylammonium acetate, and ammonium chloride. The analytes were successfully eluted with all additives with good peak shape under isocratic/isobaric/isothermal conditions. Three stationary phases with different degrees of deactivation were considered. They were conventional Cyanopropyl, Deltabond Cyanopropyl, and non-chemically bonded silica. The effect of additive concentration and additive functionality on retention was also investigated. Secondly, solid state NMR of the silica packing material before and after being flushed with supercritical CO2 modified by methanol containing the ionic additives was performed to gain some insight into the retention mechanism(s). A fraction of silanol protons were undetected after being treated with the mobile phase which suggested replacement by the cationic component of the additive. CaChe calculations were carried out on several of the additives in an attempt to explain why different ionic additives produce different effects on chromatographic retention. Modification of the stationary phase and ion pairing with the analyte are two possible retention mechanisms being considered. As ion-pair formation was considered to be one of the retention mechanisms, the use of sodium sulfonates as mobile phase additives to elute secondary and quaternary ammonium salts was then studied. Propranolol HCl, benzyltrimethylammonium chloride, and cetylpyridium chloride were chosen as the probe analytes. Sodium ethansulfonate, sodium 1-heptanesulfonate, and sodium 1-decanesulfonate were studied as mobile phase additives. The analytes were successfully eluted from Deltabond Cyano phase within 5 minutes, but were retained strongly without additive or with ammonium acetate as the additive. An Ethylpyridine column showed dramatic advantages on the elution of these ammonium analytes. No additive was required to elute these ionic compounds. Protonation of some fraction of the pyridine functional groups and the deactivation of active silanol sites were believed to be the major mechanisms responsible for this behavior. Lastly, we successfully eluted large peptides (up to 40 mers) containing a variety of acidic and basic residues in SFC. We used trifluoroacetic acid as additive in a CO2/methanol mobile phase to suppress deprotonation of peptide carboxylic acid groups and to protonate peptide amino groups. The Ethylpyridine column was used for the majority of this work. The relatively simple mobile phase was compatible with mass spectrometric (MS) detection. To our knowledge, this is the first report of the elution of peptides of this size with a simple, MS-compatible mobile phase. Fast analysis speed, the possibility of coupling multiple columns to achieve desired resolution, a normal-phase retention mechanism, and less use of organic solvents are the advantages of SFC approach for peptide separation. / Ph. D.

Diabetes mellitus is one of the metabolic disorders prevailing all the over world. About 381.8 million people are affected by diabetes mellitus (DM) during 2013 and it is estimated to increase to 80% by 2035. Nephropathy, retinopathy, neuropathy and cardiovascular diseases are common complications arise in the patients suffering from diabetes Type I and Type II. Continuous monitoring of glucose will give greater clinical acumen on glucose metabolism of patients than conventional intensified glucose monitoring. It benefits patients to plan their meals and insulin dosages to prevent hypo-and hyperglycemia. Diabetes is a major cause of chronic kidney disease (CKD). Chronic kidney disease increases the risk of cardiovascular diseases (CVD). CVD and CKD are strongly intertwined. Urea and potassium are the major markers used in the diagnosis of chronic kidney disease and cardiovascular disease, respectively. Continuous monitoring of urea and potassium will help to initiate appropriate medical intervention to decelerate the progression of chronic kidney disease and cardiovascular disease. Conventional invasive blood withdrawal procedure has potential risks like infection, pain and discomfort to the patients. Moreover, invasive techniques deter periodic blood sampling as it requires for frequent vascular puncturing. At present, no medical device is available for continuous monitoring of blood analytes non-invasively. Present investigations aim at developing a non-invasive technique for monitoring blood analytes (glucose, urea and potassium) which have great potential to use as a point of care diagnostics. Interstitial fluid bathes the cells of the body and it is ultrafiltrate of plasma. It contains ions like potassium, sodium, etc., and neutral molecules like glucose, urea, etc. Analytes (glucose, urea and potassium) level in interstitial fluid equilibrates with blood with lag time of 0 – 15 minutes. Reverse iontophoresis is a process in which a small current is applied through the skin to enable the transdermal extraction of interstitial fluid. Reverse iontophoresis is a non-invasive method and it is suitable for developing an integrated system to extract and analyze the extracted analyte. It enables frequent analyte sampling in high risk patients like elderly and paediatric with more comfort than conventional methods. In the present work, investigations are conducted on non-invasive monitoring of blood glucose, urea and potassium using reverse iontophoresis (RI). As part of experimental investigations, in vitro models are developed. In vitro investigations are conducted to optimize the reverse iontophoresis parameters current density and time of extraction. With these optimized parameters, the in vivo investigations are conducted on human subjects. A dedicated instrumentation suitable for extraction of analytes is developed. Screen printed electrochemical glucose sensors suitable for revere iontophoresis applications are developed using mediated carbon ink. Glucose oxidase is immobilized on screen printed sensor using cross linking method. Electrochemical and material characterization studies are conducted on the developed sensors. The obtained results confirm that the suitability of developed sensor can be used for serum glucose measurement as well as for reverse iontophoresis. Screen printed potentiometric urea biosensors are also developed to monitor the blood urea level non-invasively using reverse iontophoresis. The extraction and sensing system consists of a reverse iontophoresis electrodes, a working electrode, and a reference electrode. Unease enzyme is immobilized in the polypyrrole matrix on the working electrode using cyclic voltammetry. The electrochemical and material characterizations are conducted on screen printed sensors. The sensitivity, selectivity and sensing range of sensors show that they have a potential application in reverse iontophoresis applications. The in vitro models are used to evaluate the developed (urea and glucose) sensors. They are further validated by this conducting the clinical investigations on 15 human subjects. A correlation between blood analyte (glucose and urea) level and transdermally extracted analytes (glucose and urea) is established. It is attempted to integrate both the sensors (glucose and urea) and evaluated their performance on human subjects. The effect of potassium present in the stratum corneum of skin during reverse iontophoresis is investigated by conducting in vivo studies on human subjects. Tape stripping technique is used to detect the presence of potassium in stratum corneum. Reverse iontophoresis investigations with and without passive diffusion are also conducted to analyze the effect of potassium in stratum corneum. Skin impedance is measured during reverse iontophoresis in order to study the effect of reverse iontophoreteic current on skin properties. The clinical investigations are conducted on human subjects to validate the performance of the developed sensors (glucose and urea) with the approval of Institute Human Ethical Committee (IHEC), IISc, Bangalore. Non-invasive monitoring of blood analytes (glucose and urea) on human subjects is successfully demonstrated with the indigenously developed sensors through reverse iontophoresis.

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The SCP-RICM assay employs the measurable surface energy (or adhesive work W_adh) of a micrometer-sized polymeric sphere (soft colloidal probe, SCP) interacting with a glass chip using reflection interfer-ence contrast microscopy (RICM). Depending on those two interacting surfaces' nature and functional-ization, the SCP will deform, creating a contact area with the hard glass chip. This contact area is clearly distinguishable from the sphere’s interference ring pattern and can be measured. The adhesive surface energy W_adh can be calculated from the size of the contact area. An immobilization can be overcome by choosing a two-component analyte-dependent interaction, here presented for the copper (Cu) detection. The detection of Cu was chosen as a proof-of-concept system. However, detecting metal ions is an essential endeavor because, in excessive amounts, they present a severe threat to health and the environment. The copper-dependent interaction of the yeast chaperones yCox17 (also Cox17) and ySco1 (also Sco1) were chosen as the two-component analyte-dependent interaction. The chaperones partic-ipate in vivo in the formation of the electron transport chain of S. cerevisiae and interact in the mito-chondrial inner membrane to transfer one Cu(I) ion from Cox17 to Sco1. It was necessary to immobilize one protein to the SCPs and one to the chip surface, to transfer the copper chaperones' interaction into the SCP-RICM assay core detection components. The unique self-assembling characteristics of the class I hydrophobin Ccg-2 from N. crassa were used to immobilize one interaction partner to the chip surface. Class I hydrophobins are known for the formation of re-sistant and uniform layers at hydrophilic/hydrophobic interfaces. Initial SCP-RICM assay measurements with Sco1Δ95_a-SCPs and the Cox17_c-chips indicate that copper detection using the proposed mechanism is possible (Figure 39-3). Measurements can be differentiated between 0 and 0.1 mM Cu(I) concentration in solution. Further screening of concentrations be-low 0.1 mM is still necessary. The presented proof-of-principle system for the indirect detection of copper shows copper-dependent behavior. These positive results give rise to many more options to use the SCP-RICM assay as an indirect detection system. The application range of the SCP-RICM assay could be enlarged for different analytes such as other heavy metals, bacteriophages, biomarkers, et cetera, and is relevant for fields from medicine to environmental monitoring.:TABLE OF CONTENT Table of Content I List of Figures VII List of Tables IX List of Abbreviations XI 1 Introduction 1 1.1 Biosensors 1 1.2 Analytical Detection Methods: Copper 2 1.3 SCP-RICM Assay 3 1.3.1 Sensor Chip Surface 4 1.3.2 Soft Colloidal Probes 5 1.3.3 Reflection Interference Contrast Microscopy 6 1.4 Hydrophobins 9 1.4.1 Structure and Functions of Hydrophobins 9 1.4.2 Ex vivo Applications of Hydrophobins 11 1.4.3 Class I Hydrophobin: Ccg-2 12 1.5 Mitochondrial Respiratory Chain 14 1.5.1 Copper Transport in Yeast 14 1.5.2 S. cerevisiae Sco1 protein 18 1.5.3 S. cerevisiae Cox17 protein 21 1.6 SCP-RICM Assay for Copper Detection 23 1.7 Aim of the Study 24 2 Materials and Methods 25 2.1 Laboratory Equipment 25 2.1.1 Devices 25 2.1.2 Chemicals 26 2.1.3 Consumables 28 2.1.4 Antibodies 29 2.1.5 Enzymes 30 2.1.6 Molecular Weight Standards 30 2.1.7 DNA Oligonucleotides 31 2.1.8 Plasmids and Vectors 32 2.2 Microorganisms 33 2.2.1 Strains 33 2.2.2 Cultivation of Microorganisms 34 2.2.3 Preparation of Electrocompetent E. coli Cells 36 2.2.4 Preparation of E. coli Glycerol Stocks 36 2.3 Protein Design 37 2.4 Molecular Cloning Methods 38 2.4.1 Vector Template Preparation 38 2.4.2 Agarose Gel Electrophoresis 40 2.4.3 DNA Extraction from Agarose Gels 41 2.4.4 Polymerase Chain Reaction 41 2.4.5 DNA Restriction Digest 42 2.4.6 DNA Dialysis 43 2.4.7 Ligation of DNA Fragments 43 2.4.8 Isolation of DNA from E. coli 44 2.4.9 DNA Sequencing 45 2.4.10 Transformation of E. coli via Electroporation 45 2.5 Protein Detection and Quantification 46 2.5.1 SDS PAGE 46 2.5.2 Coomassie Staining 50 2.5.3 Western Blot Analysis 51 2.5.4 Immunological Detection 51 2.5.5 Protein Quantification: Lowry Assay 52 2.5.6 Protein Quantification: Bradford Assay 53 2.5.7 Protein Quantification: NanoDrop Measurement 53 2.6 Protein Purification and Storage 54 2.6.1 Expression Analysis of Recombinant Proteins 54 2.6.2 Solubility Analysis 54 2.6.3 Protein Purification by Ni2+ Affinity Chromatography 55 2.6.4 Quantification of Purified Proteins 64 2.6.5 Dialysis of Purified Proteins 65 2.7 Glass Surface Functionalization 65 2.7.1 Glass Surface Preparation 66 2.7.2 Hydrophobin and Fusion Protein-Based Coating 66 2.7.3 Contact Angle Measurement 67 2.7.4 DRoPS Test 67 2.7.5 Atomic Force Microscopy 67 2.8 SCP Functionalization 68 2.8.1 Functionalization of SCPs with Proteins 68 2.8.2 Validation of SCP Functionalization with FITC Staining 69 2.9 SCP-RICM Assay and Its Analysis 69 3 Results 73 3.1 Generation of Recombinant Fusion Proteins 73 3.1.1 Sco1 and Sco1∆95 73 3.1.2 Cox17 84 3.1.3 Ccg-2 88 3.1.4 Overview: Optimization of Expression and Purification of Recombinant Proteins 90 3.2 His-Tag Cleavage 92 3.3 Chip Surface Functionalization 94 3.3.1 Optimization of the Glass Chip Preparation 94 3.3.2 Macroscopic Properties of the Functionalized Chip Surface 95 3.3.3 AFM Measurements 102 3.3.4 Theoretical Package of Hydrophobin Ccg-2 on the Chip Surface 103 3.4 SCP Functionalization 104 3.4.1 SCP Functionalization and FITC Staining 104 3.4.2 Theoretical Package of Proteins on SCPs 106 3.5 SCP-RICM Assay 107 4 Discussion and Further Prospectives 113 4.1 Discussion: SCP-RICM Assay and Protein-Protein Interaction 113 4.2 Outlook and Further Prospects 119 4.2.1 Heterologous Protein Expression and Purification: Methods, Cleavage and Refolding 119 4.2.2 Further Analysis of Chip Surface Functionalization 124 4.2.3 Alternative Chip Surface Functionalization Methods 126 4.2.4 SCP-RICM Assay: Data Acquisition and Evaluation 128 4.2.5 SCP-RICM Assay: Copper Detection 130 4.2.6 Exploiting the SCP-RICM Assay using Protein-Protein Interactions 131 4.2.7 Exploiting the SCP-RICM Assay with Alternative Interactions 133 5 Summary 137 6 Bibliography 141 7 Appendix 165 7.1 Sequences of Protein Constructs 165 7.1.1 Sequences of the Protein Construct Cox17_a 165 7.1.2 Sequences of the Hydrophobin-Cox17 Fusion Protein Cox17_b 165 7.1.3 Sequences of the Hydrophobin-Cox17 Fusion Protein Construct Cox17_c 166 7.1.4 Sequences of the Protein Construct Sco1_a and Sco1Δ95_a 167 7.1.5 Sequences of the Hydrophobin-Sco1 Fusion Protein Constructs Sco1_b and Sco1Δ95_b 169 7.1.6 Sequences of the Hydrophobin-Sco1 Fusion Protein Constructs Sco1_c and Sco1Δ95_c 171 7.1.7 Sequences of the Hydrophobin Ccg-2 173 7.2 pET-28b(+): Plasmid Map 173 7.3 Nickel Removal During Dialysis 175 7.4 DGR Assay 176 7.5 SCP diameter 179 Acknowledgements 181 Declaration of Authorship 183