Coumaphos is an organophosphate insecticide used predominantly by the US Department of Agriculture, Animal and Plant Health Inspection Services for its tick eradication program. Bioremediation of the hydrolysis products of coumaphos, chlorferon and diethylthiophosphate (DETP), using Ca-alginate immobilized cells was the focus of this research. Consortia of indigenous microorganisms capable of degrading chlorferon and DETP were isolated separately. Since chlorferon inhibited both chlorferon-degrading and DETP-degrading organisms, it was not possible to enrich a consortium of organisms for simultaneous degradation of chlorferon and DETP. A two-step growth procedure was developed for degradation studies to provide biomass acclimated to the target compound and reaction medium since cells lost their degradation activity during the growth in a rich medium. Without acclimation, approximately a week-long lag period was required before degradation was initiated. Optimum reaction conditions were found for the degradation of chlorferon and DETP using free cells. Reaction kinetics of chlorferon and DETP were determined using enzyme kinetics because cell growth was not observed during the degradation. Chlorferon degradation followed substrate inhibition kinetics and DETP degradation followed simple Michaelis-Menten kinetics. A calcium-alginate immobilized cell system was developed, and the optimum bead loadings in the reactor were determined. Degradation rates for immobilized cells were enhanced up to five times that for free cells in untreated cattle dip (UCD) solution. The enhanced degradation of immobilized cells was due to protection of the cells from inhibitory substances present in the UCD solution. In addition, physiological changes of cells caused by Ca-alginate immobilization may have contributed to a slightly increased reaction rate in pure solution. Diffusion coefficients of chlorferon and DETP into Ca-alginate gel beads were studied to assist in designing and operating bioreactor systems. Diffusion coefficients of chlorferon and DETP increased with increasing agitation speed and decreasing substrate concentration. Increased cell concentration in gel beads caused lower diffusivity. Calcium-alginate gel beads used in this study were not subject to diffusional limitations. Both external and internal mass transfer resistances were negligible, and the degradation rate inside Ca-alginate gel beads was reaction-limited.

Coumaphos is an organophosphate insecticide used predominantly by the US Department of Agriculture, Animal and Plant Health Inspection Services for its tick eradication program. Bioremediation of the hydrolysis products of coumaphos, chlorferon and diethylthiophosphate (DETP), using Ca-alginate immobilized cells was the focus of this research. Consortia of indigenous microorganisms capable of degrading chlorferon and DETP were isolated separately. Since chlorferon inhibited both chlorferon-degrading and DETP-degrading organisms, it was not possible to enrich a consortium of organisms for simultaneous degradation of chlorferon and DETP. A two-step growth procedure was developed for degradation studies to provide biomass acclimated to the target compound and reaction medium since cells lost their degradation activity during the growth in a rich medium. Without acclimation, approximately a week-long lag period was required before degradation was initiated. Optimum reaction conditions were found for the degradation of chlorferon and DETP using free cells. Reaction kinetics of chlorferon and DETP were determined using enzyme kinetics because cell growth was not observed during the degradation. Chlorferon degradation followed substrate inhibition kinetics and DETP degradation followed simple Michaelis-Menten kinetics. A calcium-alginate immobilized cell system was developed, and the optimum bead loadings in the reactor were determined. Degradation rates for immobilized cells were enhanced up to five times that for free cells in untreated cattle dip (UCD) solution. The enhanced degradation of immobilized cells was due to protection of the cells from inhibitory substances present in the UCD solution. In addition, physiological changes of cells caused by Ca-alginate immobilization may have contributed to a slightly increased reaction rate in pure solution. Diffusion coefficients of chlorferon and DETP into Ca-alginate gel beads were studied to assist in designing and operating bioreactor systems. Diffusion coefficients of chlorferon and DETP increased with increasing agitation speed and decreasing substrate concentration. Increased cell concentration in gel beads caused lower diffusivity. Calcium-alginate gel beads used in this study were not subject to diffusional limitations. Both external and internal mass transfer resistances were negligible, and the degradation rate inside Ca-alginate gel beads was reaction-limited.

Ideally, every person in the world should have access to a safe and clean water supply; if not all sources of water are clean and safe, at the very least, an effective method to detect water contamination should be readily available. An effective detection method should not only be sensitive, rapid, robust, and affordable, but, ideally, it should also be equipment-free and easy to transport and deliver to the end-users. The main goal of this project is to develop a variety of bits and pieces of bioassay systems, with a particular focus on paper-based bioactive devices in order to provide portable and ready-to-use biosensors which can be useable by anyone anywhere around the world without requiring formal training. According to the World Health Organization (WHO), 76,000 people each year die in India alone because of pesticide poisoning. Long term exposure to organophosphate pesticides is known to have adverse effects on neurological function and can lead to Alzheimer's Disease, Attention Deficit Hyperactivity Disorder (ADHD), and reduced Intelligence Quotient (IQ). The likelihood of long term exposure to pesticides is heightened in developing countries, so a reliable and inexpensive pesticide sensor is a much-needed device in the developing world. To address this need, this project reports on the development of a fully-automated bioactive paper-based sensor for the detection of organophosphate pesticides. In the proposed biosensor, two innovations were implemented to achieve a full-automated format for the pesticide sensor: (I) First is a PUMP ON A PAPER (Jahanshahi-Anbuhi et al., LOC, 2012) that increases the flow rate of fluids within paper-based microfluidic analytical devices and sequentially brings two separate liquid streams to the enzyme test zone on the paper sensor, and (II) the second innovation is a PIPETTE ON A PAPER (Jahanshahi-Anbuhi et al., LOC, 2014) that involved the creation of a pullulan (a natural non-ionic polysaccharide) temporary bridge-system to transfer a known amount of solution to the sensing zone that, gives the enzyme zone a chance to dry and accept the substrate solution from the slow channel after a fixed period of time. This proposed format results in a simplified assay that detects the presence of pesticides automatically without any further manipulation from the user. However, the shelf life of this assay kit is challenging due to instability of both enzyme (AChE) and substrate (IDA) at room temperature. AChE loses its enzymatic activity when stored at room temperature and IDA becomes oxidized quickly. This problem is not unique to these two bio reagents, however; almost all bioassays which use bio-reagents (such as enzymes and small-molecular substrates) are unstable to varying degrees and require special shipping and storage. The instability of these molecules can arise from either thermal denaturation or chemical modification, such as oxidation or hydrolysis. Because of these issues, they often have to be shipped on dry ice with special packaging, which is costly. The cost of maintaining a cold chain for distributing bio-reagents accounts for up to 80% of the cost. Aside from the cost, these reagents also have to be stored in bulk in refrigerators or freezers to minimize the loss of activity, but they must be thawed and aliquoted for their intended tests. Repeated freezing and thawing can result in a significant loss of activity, which often leads to less reliable test results. These issues make running such assays in resource-limited settings a significant challenge. There is, therefore, an urgent need for an assay system with stable reagents that is easy to use, simple to read, inexpensive, and that includes a method for the long-term stabilization of enzymes and other unstable reagents in pre-measured quantities. To overcome to all these issues, pullulan is utilized for the development of pill-based-biosensors. Pullulan dissolves quickly in aqueous solutions and shows very high oxygen barrier properties in its film form. Considering the unique properties of pullulan, it is hypothesized that pullulan may be suitable for producing assay pills with encapsulated enzymes or other unstable molecules and may provide a simplified platform for carrying out bioassays in resource-limited settings. The application of these pill-based-biosensors is shown via the entrapment of AChE and IDA for the creation of an assay kit that can detect organophosphate pesticides (Jahanshahi-Anbuhi et al., Angew. Chem., 2014). Moreover, this thesis reports on the stabilization of highly unstable firefly luciferase for the detection of microorganisms and, more particularly, ATP. Through the use of pullulan, this thesis demonstrates that both the enzyme and the substrate can be protected, immobilized, and stabilized at room temperature, instead of the existing storage methods, which require temperatures <-20˚C. This innovation allows for a more convenient method of shipping the bioassay kits around the world without any extra care. Furthermore, pullulan-based films are utilized for the development of a method for controlled multidirectional flow within paper-based biosensors. This method provides the possibility of trapping labile and volatile reagents and stabilizing them by forming thin films with pullulan. The trapped reagents within pullulan films can be strategically stacked and assembled on a paper strip in different directions. Furthermore, should the need arise, these reagents can be released and delivered sequentially or simultaneously in both vertical and lateral directions through the paper. The application of this method is shown for: (I) creation of "ready-to-use" assay kit for the detection of Escherichia coli (E. Coli). This assay kit has the step of cell lysing and proceeds automatically to the step in which enzymes react. The second application (II) shows the trapping of Simon’s reagents, which is widely used for methamphetamine detection. Overall, these unique fabrication techniques can be widely used for the preparation of highly stable, ready-to-use, and user-friendly biosensors. We are currently working on the detection of other contaminants such as heavy metals, and we are starting on vaccine stabilization and delivery, which would have a tremendous impact for society. / Dissertation / Doctor of Engineering (DEng)