Capillary Electrophoresis-based Methodology Development for Biomolecule Analysis

Capillary electrophoresis (CE) is a separation tool with wide applications in biomolecule analysis. Fast and high-resolution separation requiring minute sample volumes is advantageous to study multiple components in biological samples. Flexible modes and methods can be developed. In this thesis, I focus on developing and applying novel CE methods to study multi-target nucleic acid sensing with high sensitivity (Part I) and interactions between multiple components, i.e. proteins, nanoparticles and drugs (Part II).;In Part I (Chapter 2--4), rolling circle amplification (RCA) was combined with CE-laser induced fluorescence detection (LIF) for sensitive detection of DNA and small RNA. In Chapter 3, development of the RCA-CE method for DNA detection was systematically carried out. The obstacle of injecting long ssDNA to capillary was overcome by digesting them into identical short ssDNA fragments. Under optimized conditions, LOD was as low as 1.6 fmol. Dual-target sensing was also demonstrated with two padlock probes. In Chapter 4, to further improve the sensitivity for small RNA sensing, the stand-alone RCA was studied. Eliminating ligation and using the target small RNA as RCA primer simplified the assay procedure and lowered the detection limit to 200- or 35- amol with good specificity. Highly reproducible detection and accurate quantification of the target small RNA in plant total RNA extracts was achieved with the complete digestion of the background RNA molecules by RNase A. Two polymerases, Phi29 and Bst were compared on their performance.;In Part II (Chapter 5--7), CE was first explored as a flexible platform to quantitatively measure the dissociation constant of the nanoparticle-protein interaction. Then, mechanism studies on driving force and binding sites identification were carried out. Surface ligand pyrolysis was revealed by mass spectrometry. Desolvation was considered as a driving force which could overcome electrostatic repulsion in our systems. Peptides associated with binding site were identified by cross-linking and mass spectrometry. They were surface peptides within drug binding site 2. Ibuprofen and naproxen, drugs sharing exactly the same binding site, would suppress the binding between human serum albumin and the Fe3O4 NPs. Fusidic acid, binding HSA in a site remote to site 2, showed no effect.