Development of advanced analytical chemistry approaches for small and large molecule detection

Abstract

In this dissertation, the first chapter is about the Chapter One summarize the basis of building biosensors including the introduction of biorecognition elements and binding affinity characterization techniques; the working principle of electrochemical and electronic detection techniques including cyclic voltammetry (CV), amperometry, and field-effect transistor. Then the recent five years sensor design improvement development for the neurotransmitter detections. In Chapter Two, a polymer-modified electrolyte-gated transistor (EGT) is used for the detection of soybean agglutinin (SBA), a common food allergen protein. The polymer affinity agent, poly(N-acetyl galactosamine ethyl methacrylamide) or P(MAGalNAc), was chemically synthesized, and the affinity between SBA and the P(MAGalNAc) polymer of varied molecular weight was determined through isothermal titration calorimetry (ITC) to identify the optimal affinity agent. This platform has been used to detect different concentrations of aqueous soybean agglutinin with a simple electronic readout and achieved a limit of detection of 5.7 nM. Furthermore, excellent selectivity was maintained even in the presence of common interfering proteins. In Chapter Three, reusable electrolyte-gated transistors (EGT) was fabricated with a structure shifting aptamer as a binding affinity agent to achieve sensitive and selective detection of serotonin. The device was designed such that the sensing happens away from the transistor portion of the device, which facilitates stable and convenient affinity agent modification and detection. The aptamer functionalization was characterized with a range of techniques. The binding between the aptamer and serotonin was characterized for the first time using surface plasmon resonance (SPR). Finally, the reusability of the device following treatment with base suggests that the regeneration mechanism is related to pH-driven changes to the negatively charged aptamer structure. In Chapter Four, we explored the impacts of two antimalaria drugs, chloroquine and quinine, on the chemical messenger secretion by blood platelets. The secreted neurotransmitters were explored at bulk cell and single cell level respectively through high performance liquid chromatography (HPLC) and carbon-fiber microelectrode amperometry. The results showed that the two drugs reduce the number of platelet exocytosis events and delay fusion pore opening and closing. This work promotes understanding of how the two antimalaria drugs quantitatively and qualitatively influence exocytosis, which informs future therapeutic malarial treatment development. The second part of my thesis will be about the co-op project I performed in Merck. Chapter Five and Chapter Six evaluated the influence of polysorbate 80 hydrolysis byproducts (oleic acid) on mAb stability through the mixing study of oleic acid and mAbs solutions. Polysorbate(PS) has been widely used in pharmaceutical formulations as the surfactant to stabilize the monoclonal antibody (mAb). The correlation between polysorbate degradation and the increased subvisible particles in the protein drug products is not well understood. In these two studies, we revealed that the oleic acid is related to the proteinaceous particle formation and proposed the mechanism of the mAb aggregation through the electrostatic interactions by tuning the ionic strength, pH, and isoelectric points. The formation of the particles are also reversible upon the addition of additional salt or human serums protein. All these findings help with the understanding of the root cause of particle formation in the pharmaceutical industry.