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Biosensores amperométricos basados en la inmovilización de Escherichia coli y acetilcolinesterasa en organoarcillas aplicados a la detección de 3,5-Diclorofenol y Clorpirifos.
| dc.contributor.advisor | Prieto Castañeda, Natalia | |
| dc.contributor.advisor | Taborda-Ocampo, Gonzalo | |
| dc.contributor.author | Ospina Rodríguez, Sergio Andrés | |
| dc.date.accessioned | 2021-10-19T16:32:41Z | |
| dc.date.available | 2022-06-26 | |
| dc.date.available | 2021-10-19T16:32:41Z | |
| dc.date.issued | 2021-10-16 | |
| dc.identifier.uri | https://repositorio.ucaldas.edu.co/handle/ucaldas/17166 | |
| dc.description | Ilustraciones, fotos | spa |
| dc.description.abstract | spa:El avance en el seguimiento de sustancias ambientales de forma “On-Site” ha ido aumentando en los últimos años, sin embargo, no todos los parámetros pueden ser medidos en tiempo real y se requieren largos tiempos para el tratamiento y análisis de las muestras. La gran variedad de sustancias contaminantes del medio ambiente implican exhaustivas etapas de tratamiento acompañado de técnicas instrumentales de altos costos que no permiten la detección en el sitio de estudio ni posibilitan el seguimiento de la normativa ambiental vigente en el país, reduciendo la prevención oportuna de las consecuencias que éstos múltiples contaminantes pueden generar a corto plazo. Generalmente, se llevan a cabo análisis de compuestos halogenados, plaguicidas y metales pesados mediante diversas técnicas analíticas como la cromatografía liquida de alta eficiencia (HPLC) y la cromatografía de gases (GC) asociadas con técnicas de extracción y micro extracción en fase líquida (LPE, LMPE) y fase sólida (SPE, SPME), las técnicas cromatográficas de gases y líquida acoplada a espectrometría de masas (GC-MS) y (HPLC-MS) respectivamente; las técnicas tándem como (GC-MS/MS), que aunque presentan buenas respuestas y bajos límites en la detección de múltiples contaminantes no permiten analizar los efectos tóxicos que estos generan en los organismos vivos. Para contrarrestar estas dificultades se han venido desarrollando dispositivos analíticos que conservan la robustez de las técnicas tradicionales pero que posibilitan el análisis oportuno de diversas sustancias contaminantes en tiempo real y a muy bajos límites de detección. Es así como el desarrollo de biosensores ha presentado un gran avance en los últimos años. Los biosensores son dispositivos de carácter analítico conformados por un elemento de reconocimiento biológico (ERB) acoplado a un mecanismo de detección electrónico que interpreta los cambios fisicoquímicos presentados entre el analito y el ERB en tiempo real. El desarrollo de estos biosensores viene acompañado por modificaciones del elemento transductor con materiales que permitan mejorar la fijación de los ERB sin que se pierda su actividad bioquímica. Diversos son los transductores empleados en el desarrollo de estos dispositivos, sin embargo, los biosensores de base electroquímica, surgen como una gran oportunidad para realizar análisis altamente específicos, con mínima preparación de muestra y con altas posibilidades de miniaturización. La mayoría de trabajos en el desarrollo de biosensores electroquímicos se centran en la utilización de materiales orgánicos o inorgánicos como polímeros sintéticos u óxidos metálicos que aunque le brindan alta sensibilidad de respuesta a estos dispositivos, pueden presentar problemas de estabilidad y biocompatibilidad con los elementos biológicos de reconocimiento (ERB) que se estén empleando además de los altos costos de producción. Por otro lado, los materiales inorgánicos ricos en silicatos se han ido presentando como una alternativa en el desarrollo de estos sistemas dados los bajos costos de producción, la capacidad que tienen estos materiales para ser modificados con otras sustancias, la alta sensibilidad, la estabilidad y la bioafinidad que presentan éstos con los ERB. En la presente tesis de Maestría se intercaló la arcilla Montmorillonita con diferentes compuestos orgánicos (dimetilamina, quitosano, hexadeciltrimetilamonio y líquidos iónicos de metilimidazolio) para generar matrices de inmovilización de células bacterianas de Escherichia Coli y la enzima acetilcolinesterasa aplicadas a la detección de plaguicidas organofosforados (Clorpirifos) y organoclorados (3,5-Diclorofenol). Se realizaron estudios electroquímicos para medir la respuesta de los biosensores a partir de las técnicas voltamperometria cíclica (VC) y cronoamperometría (CA). Con éstas, se analizaron variables como la bioafinidad, temperatura de inmovilización, tiempo de incubación, carga bacteriana, la selectividad y sensibilidad de los dispositivos. Los resultados obtenidos a partir de las técnicas electroquímicas fueron respaldados con técnicas espectroscópicas, microscópicas y pruebas cualitativas de laboratorio encontrando una alta compatibilidad entre los ERB empleados y arcillas modificadas con líquidos iónicos. El enfoque aquí presentado representa una nueva perspectiva hacia la utilización de arcillas modificadas con líquidos iónicos en el desarrollo de biosensores de alta biocompatibilidad, sensibilidad y de bajo costo. | spa |
| dc.description.abstract | eng:Advances in the monitoring of environmental substances in an “On-Site” way have been increasing in recent years, however, not all parameters can be measured in real time and long times are required for the treatment and analysis of the samples. The great variety of polluting substances in the environment involve exhaustive treatment stages accompanied by high-cost instrumental techniques that do not allow detection at the study site or allow the monitoring of current environmental regulations in the country, minimizing the timely prevention of consequences that these multiple pollutants can generate in the short term. Generally, analyzes of halogenated compounds, pesticides and heavy metals are carried out using various analytical techniques such as high performance liquid chromatography (HPLC) and gas chromatography (GC) associated with liquid phase extraction and micro extraction (LPE) techniques. , LMPE) and solid phase (SPE, SPME), gas and liquid chromatographic techniques coupled to mass spectrometry (GC-MS) and (HPLC-MS) respectively; tandem techniques such as (GC-MS / MS), which, although they present good responses and low limits in the detection of multiple pollutants, do not allow analyzing the toxic effects that these generate in living organisms. To counteract these difficulties, analytical devices have been developed that preserve the robustness of traditional techniques but that allow the timely analysis of various polluting substances in real time and at very low detection limits. This is how the development of biosensors has presented a great advance in recent years. Biosensors are analytical devices made up of a biological recognition element (ERB) coupled to an electronic detection mechanism that interprets the physicochemical changes presented between the analyte and the ERB in real time. The development of these biosensors is accompanied by modifications of the transducer element with materials that make it possible to improve the fixation of ERBs without losing their biochemical activity. The transducers used in the development of these devices are diverse, however, electrochemical-based biosensors appear as a great opportunity to perform highly specific analyzes, with minimal sample preparation and with high miniaturization possibilities. Most of the work in the development of electrochemical biosensors focuses on the use of organic or inorganic materials such as synthetic polymers or metal oxides that, although they provide high response sensitivity to these devices, can present stability and biocompatibility problems with the biological elements of recognition (ERB) that are being employed in addition to the high production costs. On the other hand, inorganic materials rich in silicates have been presented as an alternative in the development of these systems given the low production costs, the ability of these materials to be modified with other substances, high sensitivity, stability and the bioaffinity that these present with ERBs. In this Master's thesis, Montmorillonite clay was interspersed with different organic compounds (dimethylamine, chitosan, hexadecyltrimethylammonium and ionic methylimidazolium liquids) to generate immobilization matrices for bacterial cells of Escherichia Coli and the enzyme acetylcholinesterase applied to the detection of pesticides Chlorpyrifos) and organochlorines (3,5-Dichlorophenol). Electrochemical studies were carried out to measure the response of the biosensors using cyclic voltammetry (VC) and chronoamperometry (CA) techniques. With these, variables such as bioaffinity, immobilization temperature, incubation time, bacterial load, selectivity and sensitivity of the devices are analyzed. The results obtained from the electrochemical techniques were supported with spectroscopic and microscopic techniques and qualitative laboratory tests, finding a high compatibility between the ERBs used and clays modified with ionic liquids. The approach presented here represents a new perspective towards the use of clays modified with ionic liquids in the development of biosensors of high biocompatibility, sensitivity and low cost. | eng |
| dc.description.tableofcontents | 1.Resumen / 1.1 Abstract / 2.Agradecimientos / 3.Abreviatura / 6. Planteamiento del problema / 6.1 Justificación / 6.2 Pregunta de investigación / 7.Objetivos / 7.1 Objetivo general / 7.2 Objetivos específicos / Bibliografía / Marco teórico / 8.1 Toxicidad de compuestos utilizados en la agricultura / 8.1.1 Plaguicidas, breve clasificación / 8.1.2 Efecto de los plaguicidas en el medio ambiente / 8.1.3 Efecto de los plaguicidas en la salud / 8.1.4 Intoxicación por organofosforados / 8.1.5 Intoxicación por organoclorados / 8.2 Sensores y biosensores en la detección de compuestos / 8.2.1 Sensores físicos y químicos / 8.2.2 Biosensores / 8.2.3 Electroquímica en la detección de compuestos contaminantes / 8.3 Sistemas de detección electroquímica / 8.3.1 Celdas de análisis electroquímicos / 8.4 Sistemas Amperométricos / 8.4.1 Técnicas de detección comúnmente aplicadas / 8.5 Biosensores basados en enzimas / 8.5.1 Biosensores basados en la inhibición de la enzima acetilcolinesterasa (AchE) / 8.6 Biosensores basados en células microbianas / 8.6.1 Biosensores basados en Escherichia Coli (E.coli) / 8.7 Transductores electroquímicos / 8.7.1 Tipos de electrodos utilizados en la detección de sustancias / 8.7.2 Electrodos serigrafiados / 8.7.3 Modificación de los elementos transductores / 8.8 Materiales arcillosos en los procesos de modificación de electrodos / 8.8.1 Clasificación de las arcillas / 8.8.2 Caracterización del material arcilloso / 8.8.3 Aplicaciones de la Montmorillonita / 8.8.4 Aplicaciones de las matrices arcillosas en el desarrollo de biosensores / Bibliografía / Materiales y métodos / 9.1 Preparación del soporte arcilloso para la inmovilización de los ERB / 9.2 Desarrollo de biosensor enzimático / 9.2.1 Biosensor basado en la enzima acetilcolinesterasa (AchE) / 9.2.2 Prueba de actividad enzimática / 9.2.4 Procesos de inmovilización del biosensor enzimático / 9.2.5 Medidas electroquímicas / 9.2.6 Pruebas de bioafinidad / 9.2.7 Temperatura de inmovilización / 9.2.8 Selectividad / 9.2.9 Repetibilidad de los biosensores enzimáticos / 9.2.10 Sensibilidad del biosensor enzimático / 9.3 Desarrollo de biosensor bacteriano / 9.3.1 Biosensor basado en células bacterianas de Escherichia Coli / 9.3.2 Preparación del cultivo bacteriano / 9.3.3 Procesos de inmovilización del biosensor bacteriano / 9.3.4 Medidas electroquímicas / 9.3.5 Pruebas de bioafinidad / 9.3.6 Optimización del biosensor / 9.3.7 Análisis del pH de adsorción / 9.3.8 Análisis de carga bacteriana / 9.3.9 Análisis microscópico / 9.3.10 Sensibilidad del biosensor bacteriano / Bibliografía / Caracterización de las arcillas empleadas como soporte de inmovilización / 10.1 Resultados de la caracterización / 10.1.1 Espectros infrarrojos de las arcillas modificadas / 10.1.2 Análisis por difracción de rayos X (DRX) / 10.1.3 Punto de carga cero (PZC) / Resultados y discusión del biosensor enzimático / 10.2 Desarrollo del biosensor enzimático / 10.2.1 Actividad enzimática / 10.2.2 Respuestas electroquímicas / 10.2.3 Modificación de los electrodos / 10.2.4 Protocolo de inmovilización / 10.2.5 Pruebas de actividad enzimática / 10.2.6 Estabilidad enzimática en las arcillas / 10.2.7 Temperatura de inmovilización / 10.2.8 Selectividad / 10.2.9 Reproducibilidad entre los biosensores / 10.2.10 Tiempo de incubación / 10.2.11 Sensibilidad / 10.2.12 Porcentaje de inhibición / Bibliografía / Resultados y discusiones del biosensor bacteriano / 10.3 Desarrollo del biosensor bacteriano / 10.3.1 Prueba de protocolos / 10.3.2 Estabilidad microbiana en arcillas / 10.3.3 Pruebas de Carga bacteriana / 10.3.4 Pruebas de pH / 10.3.5 Pruebas de caracterización / 10.3.6 Pruebas de viabilidad celular / 10.3.7 Pruebas de sensibilidad / Bibliografía / 11. Conclusiones / 12. Recomendaciones / 13. Tendencias / Productos de investigación | spa |
| dc.format.mimetype | application/pdf | spa |
| dc.language.iso | eng | spa |
| dc.language.iso | spa | spa |
| dc.title | Biosensores amperométricos basados en la inmovilización de Escherichia coli y acetilcolinesterasa en organoarcillas aplicados a la detección de 3,5-Diclorofenol y Clorpirifos. | spa |
| dc.type | Trabajo de grado - Maestría | spa |
| dc.contributor.researchgroup | Grupo de Investigación en Cromatografía y Técnicas Afines (Categoría A1) | spa |
| dc.description.degreelevel | Maestría | spa |
| dc.identifier.instname | Universidad de Caldas | spa |
| dc.identifier.reponame | Repositorio Institucional Universidad de Caldas | spa |
| dc.identifier.repourl | https://repositorio.ucaldas.edu.co | spa |
| dc.publisher.faculty | Facultad de Ciencias Exactas y Naturales | spa |
| dc.publisher.place | Manizales | spa |
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| dc.rights.accessrights | info:eu-repo/semantics/closedAccess | spa |
| dc.subject.proposal | Biosensores | spa |
| dc.subject.proposal | Montmorillonita | spa |
| dc.subject.proposal | Arcilla | spa |
| dc.subject.proposal | E.coli | spa |
| dc.subject.proposal | Acetilcolinesterasa | spa |
| dc.subject.unesco | Electroquímica | |
| dc.subject.unesco | Compuesto químico | |
| dc.type.coar | http://purl.org/coar/resource_type/c_bdcc | spa |
| dc.type.content | Text | spa |
| dc.type.driver | info:eu-repo/semantics/masterThesis | spa |
| dc.type.version | info:eu-repo/semantics/publishedVersion | spa |
| oaire.version | http://purl.org/coar/version/c_ab4af688f83e57aa | spa |
| oaire.accessrights | http://purl.org/coar/access_right/c_f1cf | spa |
| dc.description.degreename | Magister en Química | spa |
| dc.publisher.program | Maestría en Química | spa |
| dc.description.researchgroup | Química Analítica | spa |
