Microchip electrophoresis bioanalytical applications

Microchip electrophoresis (MCE) is a novel analytical technique resulting from miniaturization of capillary electrophoresis (CE) to a planar microfabricated separation device. The consequences of the transfer of CE to MCE in terms of benefits and drawbacks have been identified and commented. The strategies developed to overcome the unfavourable features of the chip with respect to the capillary are briefly described. A method for simultaneous separation of catecholamines and their cationic metabolites has been developed on the microchip. The addition of three modifiers was required to resolve all analytes. The sensitivity of on-chip amperometric detection has been improved by employing an enzyme-catalyzed reaction on the amperometric electrode, as well as by using a carbon nanotube-modified electrode. The developed analytical methodology has been successfully applied for a direct on-chip determination of catecholamines and their metabolites in a mouse brain homogenate. The feasibility of performing affinity measurements as well as isoelectric focusing on the microchip has been demonstrated and available applications of these two electrophoretic modes on a chip have been reviewed. A commercial Shimadzu microchip station has for the first time been applied for high-throughput microchip isoelectric focusing of therapeutic proteins and obtained results have been compared to conventional capillary isoelectric focusing

Development of a Separation-Based Sensor using Microdialysis Coupled to Microchip Electrophoresis with Electrochemical Detection for Monitoring Catecholamines

Microdialysis is a powerful separation technique capable of simultaneously monitoring multiple analytes in the extracellular fluid of the brain. This technique generates small sample volumes in a continuous flow stream. Traditional methods used for sample analysis forfeit temporal information regarding dynamic neurochemical processes due to the larger volumes necessary for analysis. Additionally, sample acquisition methods traditionally involve some form of tethering or anesthetizing the animal under study, greatly reducing the available behavioral information. In order to preserve both temporal resolution and behavioral information, the ideal analysis system is one that can be employed on-line, has fast analysis times of small sample volumes, and can be placed on a freely-roaming animal. Microdialysis sampling coupled on-line to microchip electrophoresis with electrochemical detection creates a separation-based sensor that fulfills these constraints. The ability to place the device directly on-animal, without tethering, allows for the neurochemical information to be correlated with the animal’s behavior, allowing for further understanding of the neurochemical basis behind each behavior. Additionally, neuroactive drug metabolism can be monitored alongside behavior when employing an on-animal separation-based sensor, potentially aiding in drug development. The goal of this thesis is therefore to develop a separation-based sensor that is capable of monitoring neurochemicals in vivo. Towards this aim, the separation and detection of analytes in the dopamine metabolic pathway was accomplished using microchip electrophoresis with electrochemical detection at a carbon electrode. The substrate material in this separation was also optimized. In order to integrate this separation and detection with microdialysis sampling, a novel fabrication procedure was developed. This procedure creates a PDMS/glass hybrid device capable of integrating hydrodynamic microdialysis flow with electrophoretic flow and detection at a carbon electrode using a flow-gated interface. Lastly, the developed method was used to monitor the dopamine metabolic pathway in vivo in rat after the administration of L-DOPA. In the future, the complete device and associated instrumentation can be used remotely and on-animal, for near-real time in vivo monitoring

Development of Analytical Methodology for Neurochemical Investigations

David J. Fischer Department of Pharmaceutical Chemistry University of Kansas Neurochemical Applications of Microchip Electrophoresis The development of sensitive and selective analytical tools has facilitated the investigation of complex neurological pathways and enhanced our understanding of neurodegenerative diseases. The development of sensitive analytical methodology for the determination of neurotransmitters and proteins related to neurodegenerative disease is described. The goal of the work performed in the first part of this dissertation was to develop analytical methodology for the analysis of catecholamine neurotransmitters (NTs) by microchip electrophoresis with electrochemical (EC) detection. Much of this work focused on the fabrication and characterization of the novel carbon-based electrode material, pyrolyzed photoresist. The fabrication of pyrolyzed photoresist film (PPF) electrodes was optimized for use in microchip electrophoresis and analytical performance was characterized using catecholamine NTs. In addition, an extensive comparison of the analytical performance of several commonly used electrode materials and electrode alignment schemes and the PPF electrode material was performed. Aspects such as sensitivity, limit of detection (LOD), resolution, reproducibility, and ease of fabrication were examined. In addition to the development of EC detection methods for catecholamine NTs, analytical methods for the determination of myc-tagged proteins were developed. The development of an electrophoretic immunoaffinity assay for the detection of a myc-tagged protein expressed in cell culture is described. While this is a general assay that can be applied to a variety of myc-tagged proteins, mutant huntingtin protein (mHtt) was used as a specific example. The development and optimization of capillary and microchip electrophoresis assays were performed for this purpose. In addition, the results obtained using these methods were directly compared to traditional analysis by Western blotting. The long term goal of this project is integrate both of these assays into a single lab-on-a-chip device capable of detecting NT release and mHtt protein in single cells

Dispositivos electroanalíticos miniaturizados: estrategías para la mejora de la separación y la detección

Tesis doctoral por el sistema de Compendio de PublicacionesActualmente, la miniaturización es una de las principales tendencias en todos los campos científicos y particularmente en la Química Analítica. En este contexto, las técnicas de separación y especialmente la electroforesis, han sido pioneras en el desarrollo de dispositivos analíticos miniaturizados (Microchips de Electroforesis, MEs). Estos se incluyen dentro de los dispositivos denominados ¿lab-on-a-chip¿ (LOC), capaces de integrar todas las etapas de análisis en un dispositivo portátil. En lo que se refiere a su aplicación clínica, también pueden considerarse dispositivos ¿point-of-care¿ (POC), desarrollados con el objetivo de realizar análisis descentralizados en el punto de diagnóstico o tratamiento. Con el fin de obtener dispositivos con altas prestaciones analíticas, el trabajo realizado en la presente memoria puede ser estructurado en tres partes: la modificación de los microcanales (para mejorar la selectividad), la obtención de un sistema de detección electroquímico miniaturizado y competitivo (para mejorar la sensibilidad), y la introducción de nuevos materiales (con el fin de obtener ¿zero-cost¿ análisis). Los microchips de electroforesis son dispositivos analíticos miniaturizados que pretenden ser una potente herramienta de análisis. La separación de los analitos va a depender de dos factores: (i) los fenómenos electrocinéticos, que vienen determinados en gran medida por la acumulación de cargas en la pared del microcanal y (ii) de la interacción de los analitos con el material inmovilizado. Por ello es de gran importancia la modificación superficial de los microcanales, especialmente cuando se trata de analitos con una relación masa/carga parecida. Las estrategias propuestas en esta memoria para la consecución de este objetivo pueden ser clasificadas en dos grandes grupos: modificaciones estáticas y modificaciones dinámicas, dependiendo de si son irreversibles o si se producen por adición del modificador a la disolución reguladora (fase pseudoestacionaria). A lo largo de los estudios aquí realizados se han evaluado polímeros comerciales [poli(ácido acrílico)], polímeros de síntesis propia derivados del poli(glicidil metacrilato) y modificadores orgánicos de reciente introducción (líquidos iónicos). Todos ellos han demostrado ser necesarios para conseguir la separación de especies estructuralmente muy parecidas, como son las catecolaminas (moléculas además de gran interés desde el punto de vista clínico dada su relación con enfermedades como por ejemplo el Alzheimer). Debido a los pequeños volúmenes de muestra empleados, estos dispositivos miniaturizados requieren un sistema de detección muy sensible. En los estudios recogidos en esta Tesis los nanotubos de carbono crecidos directamente sobre la superficie han demostrado ser un material electródico apropiado para la detección de las catecolaminas. Dado que la medida que se realiza es una medida electroquímica interfacial, el estado de la superficie es muy importante. Por ello, se evaluó la influencia de distintos parámetros como el empleo de catalizadores y capas intermedias, pretratamientos, o la densidad e influencia de la orientación de los nanotubos en la señal electroquímica. Por otro lado, la detección electroquímica ofrece muchas posibilidades, entre ellas el empleo de electrodos miniaturizados y de series de electrodos o ¿arrays¿ para obtener la señal analítica. Esta estrategia, permite además la obtención de mejores sensibilidades. Así, se describen los primeros pasos y pruebas hacia la consecución de este objetivo mediante el diseño de máscaras por métodos fotolitográficos. La parte final de esta Tesis está basada en la integración de la detección electroquímica en dispositivos analíticos miniaturizados realizados en materiales de bajo coste, los denominados ¿paper-based analytical devices¿. Así, se han evaluado tanto papel de filtro como transparencias, demostrando su potencial en la detección de metales pesados como el plomo o el cadmio

Development of online microdialysis-microchip system for in vivo monitoring

Microdialysis is a sampling technique that can be employed to monitor biological events in vivo and chemical reactions in vitro. When it is coupled to an analytical system, microdialysis can provide near real time information on the time dependent concentration changes of analytes in the extracellular space or other aqueous environments. Online systems for the analysis of microdialysis samples enable fast, selective and sensitive analysis while preserving the temporal information. Analytical methods employed for on-line analysis include liquid chromatography (LC), capillary (CE) and microchip electrophoresis and flow-through biosensor devices. This dissertation is focused on the development of microchip systems coupled to microdialysis sampling for online near-real time monitoring with high temporal resolution for the analysis of amino acid neurotransmitters in vivo. Fluorescence detection was utilized for all the studies. Also, naphthalene-2,3-dicarboxaldehyde (NDA) and b-mercaptoethanol (b-ME) or CN- are used as the derivatization reagents for rendering the amino acids fluorescent. Initial studies were performed to evaluate a twin-t design with a 3 cm separation channel that incorporated on-column derivatization with NDA and b-ME. Biogenic amines and peptides were separated on chip demonstrating the feasibility of online microdialysis sampling with online derivatization and analysis in vitro. Subsequently, the chip was further modified by incorporating a 20 cm serpentine separation channel and excitatory amino acid neurotransmitters (glutamate, aspartate) were separated from other analytes in rat brain microdialysis samples. Next, precolumn derivatization was incorporated into the chip and online in vivo experiments were performed for the continuous analysis of glutamate from rat brain microdialysis sample. Also, fluorescein was included in the experiment for the possibility to simultaneously monitor the permeability of the blood brain barrier (BBB) along with changes in levels of excitatory neurotransmitters during online analysis applied to the rat stroke model. The chip was further modified and optimized for experiments that require fast injections and separations thereby attaining high temporal resolution. This work described an online microdialysis-microchip system that can be used for monitoring neurochemical events that involve fast changes in analyte concentration in the brain

NOVI ELEKTROKEMIJSKI SENZOR TEMELJEN NA METALNIM NANOČESTICAMA I MOLEKULARNO UTISNUTOM NANOKOMPOZITNOM POLIMERU S PRIMJENOM U BIOLOGIJI

Metal nanoparticles trapped in a biopolymer composite due to electrical conductivity properties improve electrochemical sensors with biomedical and environmental applications. The study aims are to design a novel molecularly imprinted polymer (MIP) composite based on magnetic graphene oxide (Fe3O4@GO) modified silica (SiO2) and gold nanoparticles (AuNPs) to electrochemical detect serotonin (5-hydroxytryptamine, 5-HT). A suitable amount of 5-HT is effective on motivational functions and the environment because it is a serotonergic neurotransmitter. But the desired nanocomposite may have a relatively low recognition, therefore must be in choosing the type of functional monomer be careful. In this regard, the design of the electrochemical sensor began by synthesis of Fe3O4@GO-SiO2@AuNPs nanocomposite. Then, MIP electropolymerization was carried out by using p-aminothiophenol (PATP)-functionalized Fe3O4@GO-SiO2@AuNPs nanocomposite in the presence of 5HT as a template molecule. Electrochemical polymerization of MIP nanocomposite was developed using cyclic voltammetry (CV) and the electrochemical properties of 5-HT were studied use differential pulse voltammetry (DPV) technology in the 5HT solution. After optimization of preparation and measurement conditions on the designed sensor, the 5HT concentration range is 0.1 μM to 10 μM linearly, and the detection limit was 1 × 10-5 μM (S / N = 3). The wide concentration range and low detection limit were presented metal nanoparticles functionalized MIP with appropriate functional monomer have a great effect on the performance of the sensor. Furthermore, PATP-functionalized metal nanoparticles increase the conductivity and recognition of the prepared MIP electrochemical sensor to the quantification of 5-HT in biological samples with high selectivity and recovery.Metalne nanočestice zadržane u biopolimernome kompozitu zbog električne vodljivosti poboljšavaju svojstva elektrokemijskih senzora u biomedicini i izučavanju okoliša. Ovdje je prikazan novi molekularni utisnuti kompozitni polimer temeljen na grafenskom oksidu (Fe3O4→GO), modificiranome silicijevim dioksidom (SiO2) i nanočesticama zlata (AuNPs), a s ciljem elektrokemijskoga opažanja serotonina (5-hidroksitriptamin, 5-HT). Određeni iznos 5-HT-a utječe na motivaciju i doživljaj okoliša jer se radi o neurotransmiteru središnjega živčanog sustava. Kod odabira nanokompozita, a zbog učinkovitosti, funkcionalni monomer mora biti pažljivo odabran. Stoga je molekularno utisnuti polimer (MUP) generiran uporabom p-aminotiofenola (PATP), funkcionalnoga nanokompozita Fe3O4→GO-SiO2→AuNPs, u nazočnosti 5HT-a kao templatne molekule. Elektrokemijska polimerizacija nanokompozitnoga MIP-a načinjena je uporabom cikličke voltametrije (CV). Elektrokemijska svojstva 5-HT-a izučena su uporabom različitih tehnologija pulsne voltametrije (PV) u njegovoj otopini. Nakon optimizacije mjernih uvjeta i senzora, koncentracija 5-HT-a linearno je mijenjana u rasponu 0,1 – 10 μM, uz granicu opažanja 1 × 10-5 μM (S / N = 3). Širok raspon koncentracije i nizak prag opažanja te korištene metalne nanočestice MUP-a uz odabrani monomer imali su velik utjecaj na svojstva senzora. Nadalje, metalne čestice funkcionalizirane PATP-om povećale su vodljivost i mogućnost opažanja elektrokemijskih senzora na MIP te kvantificiranje sadržaja 5-HT-a u biološkim uzorcima s velikom točnošću

Development of low-cost sensing and separation devices based on macro, micro and nano technology for health applications

The work presented in this thesis described the development of low-cost sensing and separation devices with electrochemical detections for health applications. This research employs macro, micro and nano technology. The first sensing device developed was a tonerbased micro-device. The initial development of microfluidic devices was based on glass or quartz devices that are often expensive to fabricate; however, the introduction of new types of materials, such as plastics, offered a new way for fast prototyping and the development of disposable devices. One such microfluidic device is based on the lamination of laser-printed polyester films using a computer, printer and laminator. The resulting toner-based microchips demonstrated a potential viability for chemical assays, coupled with several detection methods, particularly Chip-Electrophoresis-Chemiluminescence (CE-CL) detection which has never been reported in the literature. Following on from the toner-based microchip, a three-electrode micro-configuration was developed on acetate substrate. This is the first time that a micro-electrode configuration made from gold; silver and platinum have been fabricated onto acetate by means of patterning and deposition techniques using the central fabrication facilities in Tyndall National Institute. These electrodes have been designed to facilitate the integration of a 3- electrode configuration as part of the fabrication process. Since the electrodes are on acetate the dicing step can automatically be eliminated. The stability of these sensors has been investigated using electrochemical techniques with excellent outcomes. Following on from the generalised testing of the electrodes these sensors were then coupled with capillary electrophoresis. The final sensing devices were on a macro scale and involved the modifications of screenprinted electrodes. Screen-printed electrodes (SPE) are generally seen to be far less sensitive than the more expensive electrodes including the gold, boron-doped diamond and glassy carbon electrodes. To enhance the sensitivity of these electrodes they were treated with metal nano-particles, gold and palladium. Following on from this, another modification was introduced. The carbonaceous material carbon monolith was drop-cast onto the SPE and then the metal nano-particles were electrodeposited onto the monolith materia

Nuevas estrategias electrocinéticas y nuevas aportaciones a la detección electroquímica en microchips de electroforesis capilar basadas en el empleo de nanotubos de carbono y otras nanoestructuras

Premio Extraordinario de Doctorado 2012El trabajo se ha centrado en la exploración de las posibilidades analíticas de los microchips de electroforesis capilar con detección electroquímica (microEC-DE) en el campo alimentario y, de una manera más concreta, en el análisis de alimentos funcionales que tanto interés suscita en la industria alimentaría. Antioxidantes naturales (polifenoles), vitaminas e isoflavonas han sido determinados en una gran variedad de alimentos y suplementos dietéticos debido a sus beneficios para nuestra salud (propiedades anticarcenogénicas y protectoras contra distintas enfermedades). El trabajo desarrollado estos cuatro años se puede dividir en dos grandes bloques; el primero, que corresponde al desarrollo de metodologías que mejoraran el análisis alimentario mediante el uso del microEC-DE proponiéndose un nuevo concepto denominado "calibración integrada" con el cual se mejoró tanto el rendimiento analítico como la precisión y exactitud del método y, un segundo bloque, que consistió en la exploración del uso de nanomateriales (nanotubos de carbono y nanohilos) como detectores electroquímicos en el sistema microfluídico dando lugar a enormes mejoras en los límites de detección y en la eficacia de la separación electroforética

Synthesis & retention mechanisms of novel mixed mode stationary phase for detection of specific polar low molecular weight biological samples

The thesis primarily reports the synthesis, characterization and application of novel mixed mode stationary phases for Hydrophilic Interaction Liquid Chromatography (HILIC). HILIC is a rapidly emerging chromatographic mode that is finding great applicability in the analysis of polar organic molecules. In addition, there is a chapter on the analysis of Bisphenol A and related species using capillary electrophoresis (CE) coupled with boron-doped diamond electrodes for electrochemical detection. The synthesis and characterization of the novel mixed mode stationary phases prepared in this work is an important contribution to the field as the materials prepared exhibited better performance than similar materials obtained commercially. In addition a more thorough characterization of the materials (e.g.,thermogravimetric analysis, various NMR modes, elemental analysis, etc.) and resulting columns (e.g., H) than is typically encountered. The application of these new materials to the analysis of sugars using evaporative light scattering is also novel. In CE studies, electrochemical detection is sufficiently rare that the work is also novel