Development of a Planar Capillary Electrophoresis device coupled with dual refractive index and fluorescence detection

Capillary electrophoresis (CE) is a versatile separation technique applied in many disciplines due to its speed, small sample consumption and high resolution. While current commercial CE devices have made this technique widely available, limitations remain for universal analyte detection and small footprint field deployable instruments. The objective of this thesis is to design a small-scale planar capillary electrophoresis (PCE) platform equipped with universal and fluorescence detection. This platform uses a short 10 cm long capillary with thin walls (15 um) that enables rapid separations of analytes at high field strengths. The PCE device is coupled with backscatter interferometry (BSI) detection. This refractive index (RI) detection method is capable of universal detection of analytes including small inorganic ions, amino acids, and proteins. Due to the rapid separation capabilities, it can also be used to detect binding interaction between biomolecules.Initial studies showed that this platform can perform rapid separations and detection of amino acids and ions which are important biomarkers in metabolic disorders. A useful feature of BSI detection is that it can detect the sample injection and electroosmotic flow, which are used to enhance the repeatability of the migration times. RI detection methods are generally considered as low sensitive detectors. With BSI, however, we show that the signal can be enhanced using high field strengths. Using a mathematical model and computer simulations we systematically explored this “field enhancement” phenomenon which reveals that BSI detectors act as a refractive index as well as a conductivity detector. The analyte peak area increases linearly with applied field strength, which leads to useful strategies to enhance the BSI detection.Small scale electrophoresis devices can suffer from low resolution due to the short separation length. Phenomenon such as “induced electroosmotic” flows caused by radial electric fields arising from the device compact geometry can also impact performance. We study the existence of this phenomenon in the PCE device and explore ways to mitigate its effects using external (radial) electric fields and negative separation voltages. These two methods are shown to have a significant effect in improving resolution, which is demonstrated using the branched chain amino acids (Leu and Ile) which are important biomarkers of metabolic disorders.An advantage of BSI is that it can easily be coupled with fluorescence detection. We have successfully coupled BSI with fluorescence detection to simultaneously detect proteins in the RI channel and measure immunoassays in the fluorescence channel. We have also used this configuration to study the effects of system peaks on fluorescence signals to improve the quality of detection.Finally, we explored methods to combine digital microfluidics with the PCE platform to integrate miniaturized sample and fluid handling. Digital microfluidics manipulate liquids in the form of small droplets (~ 2uL) on a patterned electrode surface using the electrowetting on dielectric phenomenon (EWOD). We show that the PCE device can be successfully coupled with DMF for sample and separations, thus greatly reducing the footprint of the platform and reducing reagent consumption.Throughout this thesis, important developments of the PCE platform are discussed with the goal of improving universal detection, separation resolution, coupling secondary detection methods (fluorescence) with the platform, and miniaturizing the fluid handling approach. These improvements are discussed in the context of applications that will benefit from these developments.

Grantor dc:publisher

University of Kansas

Year dc:date.issued

2024

Author dc:creator

• De Silva, Kaluwahandi Miyuru Thavisha

Advisor dc:contributor.advisor

• Dunn, Robert C

• Analytical chemistry
• Biochemistry
• Optics
• Amino Acids
• Backscatter Interferometry
• Capillary Electrophoresis
• Electroosmotic flow
• Proteins
• Refractive Index Detection

Statement dc:rights

• Copyright held by the author.

Language dc:language.iso

en

Dc Identifier Other

http://dissertations.umi.com/ku:19478

OAI identifier oai:identifier

oai:kuscholarworks.ku.edu:1808/38513