Made available in DSpace on 2016-06-02T19:19:56Z (GMT). No. of bitstreams: 1 BUENO_Carolina_2013.pdf: 2152454 bytes, checksum: fde5d928317281f1a9960dccbc2768a7 (MD5) Previous issue date: 2013-02-26 / Financiadora de Estudos e Projetos / The elaboration of highly-sensitive and selective functional nanobiosensors has significant applications for purposes of resilience and conservation of natural resources, to contribute on projects aimed to pointing out degraded and contaminated areas (soil and water), as well as being a quality indicator. In the present work, a nanobiosensor has been developed based on the biomimicry of the action mechanism of the herbicides in plants coupled with the Atomic Force Microscopy (AFM) tools. To sense the herbicide molecule at very low concentrations, the technique of the sensor construction was based on chemical functionalization of the surfaces of the AFM probes and substrate in order to prioritize covalent bonds and to improve the molecules flexibility, as well as to achieve reproducibility and accurate results. The architecture and molecular design of the nanobiosensor were based on the molecular spatial arrangement, binding efficiency and localization, host-guest specificity, and its binding energies which were analyzed by Molecular Docking and Molecular Dynamics Simulation. The results were based on the adhesion force (carried out by force curves data) between the AFM probe functionalized with ACCase enzyme and the substrate functionalized with herbicides. The results indicate that the specific target molecule of agrochemical was efficient, when compared with others nonspecific agrochemicals. The difference between the values of specific recognition (diclofop) and nonspecific (imazaquin, metsulfuron and glyphosate) is, on average, 90%. This evidence validates the selectivity and specificity of the nanobiosensor. This work presents the first evidence of nanolevel detection of diclofop by AFM probes sensors. / A elaboração de nanobiossensores altamente sensíveis e com seletividade funcional tem aplicações importantes para fins de resiliência e conservação dos recursos naturais que podem contribuir em projetos que visam a apontar áreas degradadas e contaminadas (solo e água), além de ser um indicador de qualidade. No presente trabalho, um nanobiossensor foi desenvolvido com base na biomimética do mecanismo de ação dos herbicidas nas plantas, juntamente com as ferramentas de Microscopia de Força Atômica (AFM). Para detectar a molécula do herbicida diclofop, a técnica da construção do biossensor se baseou na funcionalização química das superfícies de cantilevers de AFM e do substrato, onde a molécula alvo foi fixada. Através da funcionalização química dos cantilevers e substratos priorizam-se as ligações químicas, aumentando a especificidade do nanossensor. O design e arquitetura molecular do nanobiossensor foram baseados no arranjo molecular espacial, na eficiência e localização da ligação, na especificidade de interações enzima-substrato e suas energias de ligação, as quais foram analisadas por Docking Molecular e Dinâmica Molecular. Os resultados foram baseados na força de adesão (obtidos através de curvas de força) entre a ponta do AFM funcionalizada com a molécula sensora (enzima ACCase) e do substrato funcionalizado com herbicidas. Os resultados indicam que a molécula alvo do agroquímico que inibiu especificamente essa enzima foi eficiente, quando comparado com outros herbicidas não inibidores. A diferença entre os valores de reconhecimento específico (diclofop) e não específico (imazaquin, metsulfuron e glifosato) foi, em média, 90%. Esta evidência comprova a seletividade e especificidade do nanobiossensor. Este trabalho apresenta a primeira evidência da detecção ao nível molecular do diclofop por sensores de ponta de AFM.

Italian ryegrass (Lolium multiflorum Lam.) is a competitive weed in small grain production areas throughout the northwestern and southeastern US. In small grains, Italian ryegrass has generally been controlled with postemergence treatments of diclofop, or diclofop-methyl, a member of the subfamily of the aromatic carboxylic acid family, the aryloxyphenoxypropionates. The first incidence of diclofop resistance in Italian ryegrass was reported in Virginia in 1995. Experiments to characterize diclofop resistance in several Virginia biotypes of Italian ryegrass included the following objectives: (1) evaluation of the presence of diclofop resistance in several Italian ryegrass biotypes collected across Virginia, (2) evaluation of alternative herbicide efficacy for diclofop resistant Italian ryegrass control, and (3) characterization of the aryloxyphenoxypropionate (APP) resistance mechanism in resistant Italian ryegrass biotypes. The response of 32 biotypes to diclofop collected from various locations statewide with varying histories of diclofop applications confirmed diclofop resistance in Virginian Italian ryegrass populations. At 4-times the label-recommended application rate, only 50% of biotypes previously exposed to diclofop in a cropping situation were adequately controlled versus 94% of the biotypes not previously treated with diclofop. Tralkoxydim provided the most effective control of four of the biotypes. No postemergence treatment effectively controlled one biotype previously exposed to diclofop applications. Effective preemergence herbicide treatments for Italian ryegrass control in the greenhouse included acetochlor (two formulations) and flufenacet plus metribuzin. In the field, flufenacet plus metribuzin resulted in excellent Italian ryegrass control, little crop injury, and acceptable barley yields. Acetyl-coenzyme A carboxylase (ACCase) assays and herbicide absorption, translocation, and metabolism studies were conducted to investigate resistant mechanism(s) to two APP herbicides, diclofop and quizalofop. ACCase assays indicated no differences in enzyme activity between the two biotypes of Italian ryegrass evaluated. Furthermore, no significant differences in the specific activity of ACCase were detected between the two biotypes in the absence of diclofop. [14C]Quizalofop-P absorption, translocation, and metabolism did not differ between resistant and susceptible Italian ryegrass biotypes. Lack of a significant biotype effect suggests that differential metabolism does not explain the differential response to diclofop treatments observed in the herbicide dose-plant response experiment. / Ph. D.

Made available in DSpace on 2016-06-02T19:19:55Z (GMT). No. of bitstreams: 1 AMARANTE_Adriano_2013.pdf: 11925080 bytes, checksum: 6de5e4ba7ae233d30b78c7f1d927740a (MD5) Previous issue date: 2013-03-19 / Universidade Federal de Sao Carlos / In this work was developed a prototype of a new nanobiosensor with molecular specificity through a study of theoretical models of Chemical Force Microscope. For the sensing were used molecular modeling techniques as well as experimental models of the functionalized Atomic Force Microscope tip with the Acetil co-A Carboxylase (ACC) attached. Specific and non-specific inhibitors were used to evaluate substrate-enzyme interactions. The nanobiosensor investigates specific enzymatic inhibition characteristics of the ACC enzyme through the herbicide Diclofop by reversing this process applying a force in a determined direction. The force is theoretically calculated by using molecular dynamic techniques associated to the adhesion force experimentally obtained. Theoretical and experimental questions involving nanobiosensors of AFM tips still obscure until now, such as, the number of functional enzymes attached on the AFM tip, the number of the active sites available to interact after immobilization process, the consequences of the enzyme immobilization as well as the substrate and theoretical adhesion between AFM tip and substrate were analyzed here. / Este trabalho teve como objetivo principal desenvolver o protótipo de um novo nanobiossensor de alta especificidade por intermédio do estudo e desenvolvimento de modelos teóricos específicos para a Microscopia de Força Química (MFQ). Para o sensoriamento foram utilizadas técnicas de Modelagem Molecular Computacional (MMC) e resultados experimentais de MFQ, do qual a ponta do Microscópio de Força Atômica (AFM, do inglês Atomic Force Microscopy) foi funcionalizada com enzimas Acetil-coA Carboxilase (ACC). O nanobiossensor foi utilizado para detectar especificamente substratos de herbicidas específicos e não-específicos. O nanobiossensor explora as características de inibição enzimática específica da enzima ACC pelo herbicida Diclofop revertendo esse processo aplicando-se uma força numa determinada direção. Essa força foi calculada teoricamente por intermédio de cálculos de técnicas de Dinâmica Molecular e associada à força de adesão experimental. Os resultados experimentais validaram os modelos teóricos de forma inequívoca. Questões teóricas e experimentais envolvendo nanobiossensores de ponta de AFM não respondidas até o momento (número de enzimas úteis na ponta do AFM que podem interagir com o substrato, o número de sítios ativos disponíveis, consequências da imobilização das enzimas e do substrato, força de adesão teórica entre a ponta do AFM e o substrato de herbicidas, etc.) foram solucionadas neste trabalho.