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

A compact microelectrode array chip with multiple measuring sites for electrochemical applications

In this paper we demonstrate the fabrication and electrochemical characterization of a microchip with 12 identical but individually addressable electrochemical measuring sites, each consisting of a set of interdigitated electrodes acting as a working electrode as well as two circular electrodes functioning as a counter and reference electrode in close proximity. The electrodes are made of gold on a silicon oxide substrate and are passivated by a silicon nitride membrane. A method for avoiding the creation of high edges at the electrodes (known as lift-off ears) is presented. The microchip design is highly symmetric to accommodate easy electronic integration and provides space for microfluidic inlets and outlets for integrated custom-made microfluidic systems on top

Development of Dual-Electrode Amperometric Detectors for Liquid Chromatography and Capillary Electrophoresis

Abstract The body of this research was focused on the use and development dual-electrode detection schemes for liquid chromatography and capillary electrophoresis. These detection schemes were developed to investigate redox chemistries for endogenous and exogenous antioxidants that play key roles in maintaining tissue redox homeostasis under oxidative stress conditions. A parallel adjacent dual electrode detector was first proposed for liquid chromatography in which redox cycling was hypothesized to occur between the electrodes resulting in signal enhancement. Flow rates for these systems were too high (≥ 1.0 mL) to obtain redox cycling and subsequently no signal enhancement was observed for these systems. Flow rates in capillary electrophoresis are significantly lower compared to liquid chromatography. Therefore, a parallel dual–electrode was developed for capillary electrophoresis in this work. The dual–electrode was investigated using reduced phenolic acids, which were chemically reversible, semi–reversible and non-reversible compounds allowing all potential electrochemistry’s to be investigated. Redox cycling and signal enhancement was observed with the developed dual–electrode. Furthermore, the parallel dual–electrode could be operated in either a redox cycling mode or dual–potential mode, where either chemical reversibility or voltammetry could be used as a means to confirm migration based peak identification, respectively. The same design was then applied for a dual Au/Hg electrode for capillary electrophoresis, in which thiols and disulfides were investigated in vivo. With the developed dual Au/Hg electrode redox changes were observed as a result of chemically induced oxidative stress

Lab-on-PCB Devices

Lab-on-PCB devices can be considered an emerging technology. In fact, most of the contributions have been published during the last 5 years. It is mainly focussed on both biomedical and electronic applications. The book includes an interesting guide for using the different layers of the Printed Circuit Boards for developing new devices; guidelines for fabricating PCB-based electrochemical biosensors, and an overview of fluid manipulation devices fabricated using Printed Circuit Boards. In addition, current PCB-based devices are reported, and studies for several aspects of research and development of lab-on-PCB devices are described

Development of Microfluidic Electrophoresis Separation Methods for Calmodulin Binding Proteins

Calmodulin (CaM) is a Ca2+ signaling protein that regulates more than 100 different enzymes in many intracellular pathways. Investigation of this complex CaM-binding "interactome" requires a sensitive and rapid screening mechanism. The objective of this research work is to develop a highly sensitive fluorescence-based detection method coupled with microfluidic electrophoresis separation assay for CBPs. A functional microfluidic separation platform with red laser-induced fluorescent detection was developed. It is a semi-automated system with integrated functional modules, a separation module, an optical module, a detection module and a control module. AF647-labeled CaM, BSA and concanavalin A were separated to test the microchip platform. Different microfluidic devices, separation modes, and separation conditions were used to optimize the separation of a mixture of the standard proteins. Additionally, the three standards were separated in 100 s by capillary zone electrophoresis-based methods using glass chips. Si-nanoparticle colloidal array chips provided better resolution and separation efficiency in comparison with the glass chips. A photochemical bi-functional cross-linker was used to make a covalent link between AF647-labeled CaM and CBP to allow separation under denaturing conditions. Two CBPs, calcineurin (CN) and eNOS, were used as model proteins and photo cross-linked with CaM using different photochemical cross-linkers (BPM and NHS-diazirine). Mass spectrometric analysis of the in-gel digested sample revealed the presence of both CaM and CBPs in the sample, meaning that CaM and CBPs were successfully cross-linked. NHS-LC-SDA was used as the photo chemical cross-linker; and CaM-CN and CaM-eNOS photoproducts were separated on a PDMS/glass, PDMS/PMMA, glass and Si-nanoparticle colloidal array microfluidic device. CaMAF647 and the individual photoproducts were identified by different separation devices and modes. Overall, this work demonstrated the separation of CaM binding model proteins using different microfluidic devices operated under electrophoresis

Master of Science

thesisDNA extraction automation is a major concern in molecular diagnostics where processing of numerous and daily samples of blood represent a labor-intensive task and are difficult to automate. With the rapid growth in the area of DNA diagnostics, there is an urgent need for the development of a microsized total analysis system which can perform all three of the primary tasks of nucleic acid-based diagnostics on a single chip: sample preparation, extraction of the DNA, detection and quantification. This thesis work presents design and fabrication of an integrated system that can extract and electrochemically quantify DNA simultaneously from any unknown sample on a single chip. The system is fabricated using aluminum oxide membranes as substrates for the extraction and quantification of DNA, bonded with the PDMS (Polydimethylsiloxane), whose layer provides the microfluidic inlet and outlet. Cost effective fabrication tools, such as Xurography (a knife plotter) and soft lithography, are used to obtain integrated hybrid fluidic and detector prototypes. Characterization of the DNA quantification system is performed based on several important operational parameters such as different concentrations of gDNA (sample), voltage applied to the electrochemical detector, flow rate of the sample and carrier buffer and channel gap size between the detector electrodes. Three experiments with different experimental setup are applied to quantify the binding of DNA with the surface of aluminum oxide membranes. The change in the current through the detector wires is found to be linear with different concentrations of gDNA. Using these experimental data, a calibration curve is obtained through which concentration and mass of gDNA that is extracted from an unknown sample can be determined successfully. This system thus provides us with several advantages, such as simultaneous extraction and quantification of gDNA, low detection limit of DNA (3.3ng/^L), low sample volume (200^L), high sensitivity and selectivity, low cost, small size, easy fabrication, portability, and disposability, when compared to other quantification systems