A problem driven approach to the miniaturizsation and automation of enzyme-based assays and an investigation of the dissipation of cyanazine and bromide in wetland mesocosms

Dissertation (Ph.D.)--University of Kansas, Chemistry, 1997.Analytical chemistry, as a discipline, has something of an identity crisis. Its role in the development of instrumental methods frequently relegates it into the category of a technology, while the curiosity-driven fundamental studies of the technology drive it into the category of a science. Fortunately, it is possible to do both science and technology simultaneously. Fundamental studies and innovation in technology lead to the same result, better analyses. For this reason, regardless of the strategy of the study, analytical chemistry at its best is problem driven research. This work reflects the approach that it is of paramount importance to answer fundamental and technological questions in the context of real world problems. Biological recognition elements have enjoyed popularity recently in analytical chemistry. Receptors such as antibodies have been demonstrated to achieve low limits of detection in diverse complex matrices from serum to ground water. Enzymes are an integral part of such bioassays, providing increased sensitivity as a result of the time dependence of their product production. The power of enzyme based immunoassays has been demonstrated since their introduction in 1971. However, enzymes alone have been shown to be valuable bioanalytical tools as exemplified by the plethora of research in the area of implantable amperometric glucose oxidase (GOx) based biosensors for glucose. Biosensors or biologically based analytical methods must continue to evolve to prove useful in the changing landscape. Recent trends in new drug development have made different demands on the analytical chemist. No longer are simply good limits of detection, sensitivities, or high theoretical plates satisfactory for pharmaceutical companies. As thousands of combinatorial libraries queue up for analysis, speed and sample throughput have become increasingly important. Joining drug companies are hundreds of superfund sites, with millions of complex, toxic samples to be analyzed in a limited time frame. New demands brought about by the political climate in hospitals are forcing clinical labs to consider innovations to improve the speed and cost effectiveness of routine screening. Other considerations, such as minimizing contact with biological fluids brought about by concerns over hepatitis and HIV, renew interest in fundamental research in sample handling and preparation. This work reflects the approach that it is of paramount importance to answer fundamental and technological questions in the context of real world problems. In Part One two related problems are described. Chapter One demonstrates that the enzyme linked immunosorbent assay (ELISA) may be used to assess the immunogenicity of the immobilized GOx on an implantable glucose sensor. Chapter Two addresses the problem of how enzyme based analytical methods such as those used in Chapter One are automated and miniaturized. Part Two presents in Chapter Three the results of the environmental analysis of pond water drawn from wetland mesocosms treated with a herbicide and a volume tracer. The significance of this work is two-fold. First, inherent in a field study of this scope is the analytical challenges associated with sampling, sample handling and analysis specifically with respect to throughput. Explicitly, and more importantly, are the valuable discoveries related to the non-conservative behavior of both compounds applied

An insulator-based (electrodeless) dielectrophoretic concentrator for microbes in water

Dielectrophoresis (DEP), the motion of a particle caused by an applied electric field gradient, can concentrate microorganisms non-destructively. In insulator-based dielectrophoresis (iDEP) insulating microstructures produce non-uniform electric fields to drive DEP in microsystems. This article describes the performance of an iDEP device in removing and concentrating bacterial cells, spores and viruses while operated with a DC applied electric field and pressure gradient. Such a device can selectively trap particles when dielectrophoresis overcomes electrokinesis or advection. The dielectrophoretic trapping behavior of labeled microorganisms in a glass-etched iDEP device was observed over a wide range of DC applied electric fields. When fields higher than a particle-specific threshold are applied, particles are reversibly trapped in the device. Experiments with Bacillus subtilis spores and the Tobacco Mosaic Virus (TMV) exhibited higher trapping thresholds than those of bacterial cells. The iDEP device was characterized in terms of concentration factor and removal efficiency. Under the experimental conditions used in this study with an initial dilution of 1 × 105 cells/ml, concentration factors of the order of 3000× and removal efficiencies approaching 100% were observed with Escherichia coli cells. These results are the first characterization of an iDEP device for the concentration and removal of microbes in water

The zeta potential of cyclo-olefin polymer microchannels and its effects on insulative (electrodeless) dielectrophoresis particle trapping devices

While cyclo-olefin polymer microchannels have the potential to improve both the optical detection sensitivity and the chemical resistance of polymer microanalytical systems, their surface properties are to date not thoroughly characterized. These surface properties dictate, among other things, electrokinetic effects when electric fields are present. Here, we report the measurement of the zeta potential of cyclo-olefin polymers (injection-molded and hot-embossed Zeonor® 1060R and 1020R) microchannels as a function of pH, counter-ion concentration, storage conditions, and chemical treatment in aqueous solutions both with and without EOF-suppressing additives. In contrast with previous reports, significant surface charge is measured, consistent with titration of charged sites with pKa = 4.8. Storage in air, acetonitrile, or aqueous solutions has relatively minor effects. While the source of the surface charge is unclear, chemical functionalization has shown that carboxylic acid groups are not present at the surface, consistent with the chemical structure of Zeonor®. EOF-suppressing additives (hydroxypropylmethylcellulose) and conditioning in perchloric acid allow the surface charge to be suppressed. We demonstrate dielectrophoretic particle trapping devices in Zeonor® 1060R substrates that show reduced trapping voltage thresholds as compared to previous implementations in glas

The development of polymeric devices as dielectrophoretic separators and concentrators

Efficient and reliable particle separators and concentrators are needed to support a wide range of analytical functions including pathogen detection, sample preparation, high-throughput particle sorting, and biomedical diagnostics. The advent of lab-on-a-chip devices based on the phenomenon of dielectrophoresis offers advantages that can meet several of the challenges associated with cell sorting and detection. The majority of the devices presented in the scientific literature have used glass-based devices for these applications, but there has been recent activity that indicates that polymer-based devices can operate as effectively as their glass progenitors. Processing and operational advantages motivate the transition from glass and silicon to polymer microdevices: mechanical robustness, economy of scale, ease of thermoforming and mass manufacturing, and the availability of numerous innate chemical polymer compositions for tailoring performance. We present here a summary of the developments toward, and results obtained from, these polymeric dielectrophoretic devices in the selective trapping, concentration, and gated release of a range of biological organisms and particles

Effect of surfactants on electroosmotic flow and trapping behavior in a polymeric insulator-based dielectrophoretic (idep) device

We have previously reported on the use of insulator-based dielectrophoresis (iDEP) for the separation and concentration of biological particles in water. We have found that the applied DC field required to trap these particles depends on particle size, shape, and the zeta potential of the material utilized to form the device. In order to improve device performance, and decrease the power required for optimal performance, it is necessary to adjust one (or several) of these parameters. Surfactants are known to adsorb onto polymeric surfaces in a dynamic fashion, and have been utilized extensively to modify device performance in such related fields as capillary electrophoresis and micellar electrokinetic chromatography. We present here the effect of the anionic surfactant, sodium dodecyl sulfate, on the applied field strengths required to achieve effective isolation and trapping of polystyrene beads

Performance impact of dynamic surface coatings on polymeric insulator-based dielectrophoretic particle separators

Efficient and robust particle separation and enrichment techniques are critical for a diverse range of lab-on-a-chip analytical devices including pathogen detection, sample preparation, high-throughput particle sorting, and biomedical diagnostics. Previously, using insulator-based dielectrophoresis (iDEP) in microfluidic glass devices, we demonstrated simultaneous particle separation and concentration of various biological organisms, polymer microbeads, and viruses. As an alternative to glass, we evaluate the performance of similar iDEP structures produced in polymer-based microfluidic devices. There are numerous processing and operational advantages that motivate our transition to polymers such as the availability of numerous innate chemical compositions for tailoring performance, mechanical robustness, economy of scale, and ease of thermoforming and mass manufacturing. The polymer chips we have evaluated are fabricated through an injection molding process of the commercially available cyclic olefin copolymer Zeonor 1060R. This publication is the first to demonstrate insulator-based dielectrophoretic biological particle differentiation in a polymeric device injection molded from a silicon master. The results demonstrate that the polymer devices achieve the same performance metrics as glass devices. We also demonstrate an effective means of enhancing performance of these microsystems in terms of system power demand through the use of a dynamic surface coating. We demonstrate that the commercially available nonionic block copolymer surfactant, Pluronic F127, has a strong interaction with the cyclic olefin copolymer at very low concentrations, positively impacting performance by decreasing the electric field necessary to achieve particle trapping by an order of magnitude. The presence of this dynamic surface coating, therefore, lowers the power required to operate such devices and minimizes Joule heating. The results of this study demonstrate that iDEP polymeric microfluidic devices with surfactant coatings provide an affordable engineering strategy for selective particle enrichment and sorting. [Figure not available: see fulltext.

Polymeric microfluidic devices for the monitoring and separation of water-borne pathogens utilizing insulative dielectrophoresis

We have successfully demonstrated selective trapping, concentration, and release of various biological organisms and inert beads by insulator-based dielectrophoresis within a polymeric microfluidic device. The microfluidic channels and internal features, in this case arrays of insulating posts, were initially created through standard wet-etch techniques in glass. This glass chip was then transformed into a nickel stamp through the process of electroplating. The resultant nickel stamp was then used as the replication tool to produce the polymeric devices through injection molding. The polymeric devices were made of Zeonor® 1060R, a polyolefin copolymer resin selected for its superior chemical resistance and optical properties. These devices were then optically aligned with another polymeric substrate that had been machined to form fluidic vias. These two polymeric substrates were then bonded together through thermal diffusion bonding. The sealed devices were utilized to selectively separate and concentrate a variety of biological pathogen simulants and organisms. These organisms include bacteria and spores that were selectively concentrated and released by simply applying D.C. voltages across the plastic replicates via platinum electrodes in inlet and outlet reservoirs. The dielectrophoretic response of the organisms is observed to be a function of the applied electric field and post size, geometry and spacing. Cells were selectively trapped against a background of labeled polystyrene beads and spores to demonstrate that samples of interest can be separated from a diverse background. We have implemented a methodology to determine the concentration factors obtained in these devices