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The electrochemical enzymatic electrodes dominate the world market for blood glucose monitoring devices for controlling, as well as reducing the detrimental effects of diabetes. However, the enzymatic electrodes exhibit constraints restricting their reliance on the enzyme’s activity which can be influenced by the external, and the environmental factors such as temperature, pH, and humidity etc. However, the greater thickness of the enzyme layer hinders the performance of the glucose biosensors resulting in signal dampening or loss. In addition, the selectivity of the electrodes is affected by the interferents present in blood. Moreover, the invasive nature of the electrodes is a major problem considering the patient’s perspective. In contrast, recent research activities demonstrated that the electrochemical non-enzymatic electrodes possess huge potential for inexpensive and highly sensitive glucose monitoring devices, yet these electrodes are invasive in nature. Therefore, the purpose of this research was to fabricate electrochemical non-enzymatic non-invasive electrodes for sweat glucose monitoring devices. A very simple low-cost fabrication technique has been shown to make the facile, flexible, and inexpensive electrodes to detect sugar in sweat bio-analyte for a non-invasive glucose monitoring system using the native stable Cu oxides (CuNOx), Cu2O, layers grown on 35 µm thin Cu foils keeping under ambient conditions (25℃- and 760-mm Hg) for more than 2 years so that the oxide layers are full-grown, and fully stable. Moreover, the foils also annealed at various temperatures such as 160, 230, and 280℃ with new temperature profile for reducing the required time of growing stable oxides and producing oxides with larger crystallized structures with higher surface – to – volume ratio. The X-ray photoelectron spectroscopy (XPS) and high-resolution transmission electron microscopy (HRTEM) results supported that at 280℃ annealing temperature the surface, mostly, transformed into highly electrocatalytic CuO with larger grain sizes, crystallized structures, and the uniform layer of ~ 140 nm. The electrochemical characterization, and sensing performance of the electrodes have been done by cyclic voltammetry (CV), one of the excellent and well accepted electrochemical methods, with the 3 – electrode configuration of the potentiostat. The CuNOx sensors of having ~10 nm layer of stable Cu2O exhibited a sensitivity of 603.42 μA mM−1 cm−2, a linear range beyond the desired limit of 7.00 mM with excellent linearity (R2 = 0.9983) and a low limit of detection of 94.21 μM. In contrast, the new annealing profile has. the CuNOx sensors annealed at 280 ºC using new temperature profile provided twin calibration curves of linear ranges of 0.05 – 1.00 mM and 1.00 – 7.00 mM, that applicable for sweat and blood glucose sensing, respectively, and exhibited a sensitivity of 1795 μA mM−1 cm−2, a linear range up to the desired limit of 1.00 mM for sweat glucose sensing with excellent linearity (R2 = 0.9844), and a lower limit of detection of 135.39 μM. In addition, it has been shown that the peak electro-oxidation current of glucose sensing is linearly related with the squire root of the annealing temperature, √T. This can help to figure out the required applied annealing temperature for getting desired peak electro-oxidation current of glucose in a human health monitoring system. / Dissertation / Doctor of Philosophy (PhD)

This thesis focuses on the optimazation of the electrochemical method, which characterizes products of untemplated nonenzymatic polymerization of 3',5' -cyclic guanosine monophosphate (cGMP) under conditions modeling prebiotic environment. An adsorptive transfer stripping techniques on carbon electrode and gel electrophoresis were used. The method was optimized on the model system of oligonucleotides located in solution of cGMP on carbon electrode, where DNA and RNA adsorb. This technique allows simple removing of interfering substances such as cGMP, which are not present in the original sample, but they do not adsorb on the surface of electrode or they adsorb weaker than oligonucleotides or polynucleotides. Analyses are based on the selective desorption of cGMP from the surface of the carbon electrode by the chemical and physical methods before the measurement of linear voltammetry itself. Detergents, such as SDS, Tween 20 and Triton x-100 with different concentrations and electrostatic repulsions of cGMP with different negative potentials on the carbon electrode were used for the selective desorption of cGMP. The selective desorption of cGMP was observed for all detergents and inserted negative potentials. Used methods were compared and the most effective detergent for selective desorption of cGMP was SDS. Desorption of oligonucleotides was minimalized by inserted positive potential on washed carbon electrode in 0,01% SDS in basic medium. This optimized method was used on electrochemical analysis of preliminary samples of untemplated nonenzymatic polymerization of 3',5' -cGMP and compared to the analysis of gel electrophoresis.

Utilization of cyanide as a nutritional nitrogen source in P. fluorescens NCIMB 11764 was shown to involve a novel metabolic mechanism involving nonenzymatic neutralization outside of cells prior to further enzymatic oxidation within. Several cyanide degrading enzymes were produced by NCIMB 11764 in response to growth or exposure to cyanide, but only one of these cyanide, oxygenase (CNO), was shown to be physiologically required for assimilation of cyanide as a growth substrate.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Acute Myocardial Infarction (AMI) is one of the major public health problems in the world. Coronary occlusion, which occurs when an atherosclerotic plaque breaks, is the main mechanism that leads to AMI. With the objective of tissue reparation, there is an increase in platelet aggregation and thrombus formation. When ischemia/reperfusion occurs in AMI, Reactive Oxygen Species (ROS) are produced. The imbalance between ROS production and degradation may lead to an increase in oxidative stress. This study aimed to determine the activity of enzymes involved in thromboregulation, such as NTPDase and 5 -nucleotidase, as well as oxidative stress parameters. Evaluation of the oxidant system was carried out by lipid peroxidation and carbonyl protein determination, and the enzymatic and nonenzymatic antioxidant defense measurements were performed in platelets, total blood, plasma, and serum of AMI patients. The results demonstrated that an increase in the activity of NTPDase by ATP (54%) and ADP (45%) hydrolysis occurred in AMI patients when compared to the control group. The same occurred with 5 -nucleotidase activity. The hydrolysis of AMP increased 46% in AMI patients compared to the control group. The increase in ectonucleotidase activities could be related to a compensatory organic response to the pathologic state formed. Regarding oxidant levels, an increase in TBARS and carbonyl protein levels was observed in AMI patients when compared to the control group. The same occurred for the activities of the enzymatic antioxidants, superoxide dismutase (SOD), and catalase (CAT). However, a decrease in nonenzymatic antioxidants, such as vitamin C and vitamin E, was observed in AMI patients when compared to control. These results suggest an increase in oxidative stress in AMI, which was probably a result of the ischemic/reperfusion moment, as well as a decrease of antioxidant defenses. Furthermore, the increased antioxidant defense may act as a compensatory mechanism in consequence of the overproduction of ROS after AMI. No differences in the parameters tested were observed with the drugs utilized in AMI treatment in vitro. In conclusion, AMI results in oxidative damage as well as an increase in the organism s defenses as a compensatory response. / O Infarto Agudo do Miocárdio (IAM) é um dos maiores problemas de saúde pública no mundo. O principal mecanismo que leva ao IAM é a oclusão coronariana que ocorre quando uma placa aterosclerótica sofre fissura. Com o objetivo de reparação tecidual temos um aumento na agregação plaquetária e formação do trombo. Acompanhando o momento de isquemia e reperfusão que ocorre no IAM temos uma produção elevada de Espécies Reativas de Oxigênio (EROs). Um desequilíbrio entre a produção e a degradação de EROs pode levar a geração de estresse oxidativo. Neste trabalho determinaram-se a atividade das enzimas envolvidas na tromboregulação: NTPDase e 5 -nucleotidase, e parâmetros de estresse oxidativo em pacientes que sofreram IAM e em pacientes controles. Foi realizada a avaliação do sistema oxidante através da determinação da peroxidação lipídica e da carbonilação protéica e a medida das defesas antioxidantes enzimáticas e não enzimáticas do organismo, em plaquetas, sangue total, plasma e soro destes pacientes. Os resultados demonstraram um aumento na atividade da NTPDase, revelada através da hidrólise dos nucleotídeos ATP (54%) e ADP (45%), em pacientes com IAM quando comparados com o grupo controle. O mesmo ocorreu com a atividade da enzima 5 -nucleotidase. A hidrólise do AMP aumentou 46% nos pacientes com IAM em relação ao grupo controle. O aumento na atividade das ectonucleotidases pode estar relacionado a uma resposta orgânica compensatória do organismo frente ao estado patológico formado. Em relação aos níveis de oxidantes determinados, observou-se um aumento nos níveis de TBARS e proteína carbonil em soro de pacientes com IAM quando comparados com o grupo controle. Esse aumento também foi observado para as defesas antioxidantes enzimáticas superóxido dismutase (SOD) e catalase (CAT). Entretanto, observou-se um decréscimo das defesas antioxidantes não enzimáticas como a vitamina C e a vitamina E no soro de pacientes com IAM. Estes dados sugerem um aumento do estresse oxidativo como resultado do momento de isquemia/reperfusão e da diminuição das defesas antioxidantes não enzimáticas. Além disso, o aumento das defesas antioxidantes enzimáticas poderiam agir como um mecanismo compensatório como consequência da superprodução de EROS após o IAM. Nenhuma interferência dos medicamentos utilizados no tratamento do IAM foram observadas sobre os parâmetros testados in vitro. Concluí-se então, que o IAM resulta tanto em danos oxidativos como mobilização das defesas do organismo para uma resposta compensatória.