Construction and characterization of novel amperometric plant tissue-based biosensors

Thesis (Ph. D.)--University of Hawaii at Manoa, 1992.Includes bibliographical references (leaves 134-141)Microfiche.xiv, 141 leaves, bound ill. (some col.) 29 cmA biosensor is an analytical device in which a biological material, capable of specific chemical recognition, is in intimate contact with a physicochemical transducer to give an electrical signal. In this research, plant tissue materials (as the molecular recognition element) are coupled to electrochemical transducers to construct selective analytical biosensors for the detection of important analytes. The use of eggplant fruit as a source of polyphenol oxidase to construct an amperometric biosensor for catechol detection is demonstrated. Amperometric measurements are based on the detection of catechol quinone produced at the electrode surface. The characterization of the novel sensor with respect to biocatalytic loading, linear dynamic range, sensitivity, selectivity and lifetime is reported. A use of a novel thin layer electrode design, with a plant tissue reactor and a sensing tin oxide collector electrode, is reported for amperometric detection in flow injection analysis and compared with previous carbon paste electrode designs. Eggplant fruit containing the enzyme, polyphenol oxidase is used as the selective biocatalyst. The new system, evaluated for the case of catechol measurements, is shown to offer superior sensitivity, dynamic range and detection limits. In vitro cultured tobacco callus tissue, which contains high activity of peroxidase, is used to construct an amperometric biosensor for the detection of hydrogen peroxide. The tissue is incorporated into a carbon paste matrix along with ferrocene, an electron mediator. The detection of hydrogen peroxide was accomplished through its enzymatic reduction mediated by ferrocene. Comparison studies conducted with normally grown types of tobacco tissues indicate that the callus tissue has the highest biocatalytic activity and the least variance between tissue preparations with respect to sensor response toward hydrogen peroxide. Additionally, the sensor exhibits a remarkably long lifetime of about five months. Other important characteristics of the sensor include fast response times, wide dynamic range, and very low detection limits. The possibility of using genetically transformed (transgenic) plants; specifically, transgenic potato (Solanum tuberosum) and transgenic tobacco (Nicotiana tabacum), in the construction of an amperometric biosensor for the detection of phenyl-B-glucuronide was attempted as an application of recombinant DNA technology in biosensor construction

Effect of Exposure of Ultraviolet B Radiation on Two Anuran Species, Polypedates cruciger and Duttaphrynus melanostictus under Empirical Conditions

Increase in ultraviolet (UV)-B radiation has been identified as a suspect in amphibian deformities and decline. Eggs of two amphibian species, Polypedates cruciger and Duttaphrynus melanostictus were exposed to UV-B radiation (312 nm) to receive erythemal dose (0.08 relative response) of the action spectra under laboratory conditions. Biologically estimated doses were calculated to match 50 % (high), 25% (medium) and 10 % (low) of the average daily irradiance given for the Asian region (17.92 × 10-3 Wm-2). Hatching success of the eggs and survival, growth of tadpoles and development of malformations were recorded until metamorphosis. Although the exposure had no effect on the hatching success of the eggs of the two species, the survival of tadpoles of both species was significantly reduced compared to the tadpoles coming from unexposed eggs in the control. Tadpoles of both species experienced growth retardations due to UV-B exposure. Exposed individuals were smaller at metamorphosis and they took longer to metamorphose. In addition, malformations such as ulcers, edemas, abnormally pigmented skin were common among the exposed tadpoles. Species-specific differences in the growth were observed between D. melanostictus and P. cruciger but not in the other measured parameters. This study shows that the two anuran species are highly sensitive to levels of UV-B radiation that is present in their natural environment and suffered both lethal and sub-lethal effects

Effects of agrochemicals on disease severity of Acanthostomum burminis infections (Digenea: Trematoda) in the Asian common toad, Duttaphrynus melanostictus

Abstract Background Agrochemicals are widely used in many parts of the world posing direct and indirect threats to organisms. Xenobiotic-related disease susceptibility is a common phenomenon and a proposed cause of amphibian declines and malformations. For example, parasitic infections combined with pesticides generally pose greater risk to both tadpoles and adult frogs than either factor alone. Here, we report on experimental effects of lone and combined exposures to cercariae of the digenetic trematode Acanthostomum burminis and ecologically relevant concentrations of (0.5 ppm) four pesticides (insecticides: chlorpyrifos, dimethoate; herbicides: glyphosate, propanil) on the tadpoles and metamorphs of the Asian common toad, Duttaphrynus melanostictus. Results All 48 cercaraie successfully penetrated each host suggesting that the pesticides had no short-term detrimental effect on cercarial penetration abilities. When the two treatments were provided separately, both cercariae and pesticides significantly decreased the survival of tadpoles and metamorphs and induced developmental malformations, such as scoliosis, kyphosis, and skin ulcers. Exposure to cercariae and the two insecticides additively reduced host survival. In contrast, mortality associated with the combination of cercariae and herbicides was less than additive. The effect of cercariae on malformation incidence depended on the pesticide treatment; dimethoate, glyphosate, and propanil reduced the number of cercarial-induced malformations relative to both the control and chlorpyrifos treatments. Conclusions These results show that ecologically relevant concentrations of the tested agrochemicals had minimal effects on trematode infections, in contrast to others studies which showed that these same treatments increased the adverse effects of these infections on tadpoles and metamorphs of the Asian common toad. These findings reinforce the importance of elucidating the complex interactions among xenobiotics and pathogens on sentinel organisms that may be indicators of risk to other biota

Combined Effects of Pesticides and Trematode Infections on Hourglass Tree Frog Polypedates cruciger.

The impact of widespread and common environmental factors, such as chemical contaminants, on infectious disease risk in amphibians is particularly important because both chemical contaminants and infectious disease have been implicated in worldwide amphibian declines. Here we report on the lone and combined effects of exposure to parasitic cercariae (larval stage) of the digenetic trematode, Acanthostomum burminis, and four commonly used pesticides (insecticides: chlorpyrifos, dimethoate; herbicides: glyphosate, propanil) at ecologically relevant concentrations on the survival, growth, and development of the common hourglass tree frog, Polypedates cruciger Blyth 1852. There was no evidence of any pesticide-induced mortality on cercariae because all the cercariae successfully penetrated each tadpole host regardless of pesticide treatment. In isolation, both cercarial and pesticide exposure significantly decreased frog survival, development, and growth, and increased developmental malformations, such as scoliosis, kyphosis, and also edema and skin ulcers. The combination of cercariae and pesticides generally posed greater risk to frogs than either factor alone by decreasing survival or growth or increasing time to metamorphosis or malformations. The exception was that lone exposure to chlorpyrifos had higher mortality without than with cercariae. Consistent with mathematical models that suggest that stress should increase the impact of generalist parasites, the weight of the evidence from the field and laboratory suggests that ecologically relevant concentrations of agrochemicals generally increase the threat that trematodes pose to amphibians, highlighting the importance of elucidating interactions between anthropogenic activities and infectious disease in taxa of conservation concern