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Cell membranes are integral for many biological processes. In addition to containing and protecting cellular contents and maintaining the chemical integrity of the cell, these interfaces host a variety of ligand-receptor interactions. These ligand-receptor interactions are important for cell signaling and transport and the ability to monitor them is key to understanding these processes. In addition, therapeutics and drug discovery is also aided by membrane-specific study, as the majority of drugs target receptors associated with the cell surface. The cell membrane can be effectively mimicked by the use of supported lipid bilayers, which provide a robust platform exhibiting the lateral fluidity and composition associated with cell membranes. The ability to study both ligand-receptor interactions as well as small molecule-membrane interactions on these model membranes is aided by the fact that these assays can be multiplexed and are amenable to use with low sample volumes with high throughput. Our laboratory has recently developed a strategy for fluorescent microscopy studies of ligand-receptor interactions on supported lipid bilayers without the use of fluorescently-labeled analytes. This technique involves the incorporation of pH-sensitive fluorophores into the composition of the supported lipid bilayer as embedded reporter dyes. It was determined that this assay can operate as either a “turn-on” or a “turn-off” sensor depending on the analyte to be detected. It was additionally found that modulating the ionic strength of the operating buffer allows for tuning the operating pH and sensitivity of the assay. This label-free technique can be utilized to monitor small peptide interactions with bilayers containing specific phospholipids. Basic amino acid sequences which are associated with transporting contents across membranes or anti-microbial activity can be monitored binding to negatively charged bilayers without the use of labels. Not only is this a sensitive technique for detecting small peptides, but thermodynamic data can be extracted as well. In a final set of experiments, the interaction of proteins with phosphatidylserine (PS) in supported lipid bilayers is observed by utilizing PS-Cu2+-induced quenching of fluorophores. Disruption of this metal-phospholipid, specifically by Ca2+-dependent protein kinases, results in a turn-on fluorescent assay, which can be used to monitor the binding of the protein to PS and the effects of other metal interference.

The brightness and photostability of semiconductor quantum dots (QDs) has prompted the exploration of their use in a wide variety of fields. Several examples of QD-based biosensors have been reported but none have actually replaced their preexisting technologies. This work reveals the barriers hindering widespread use of QD based biosensors and examines how QDs can be engineered for improved utility in bioassay designs. The first portion of this project aims to improve Förster Resonance Energy Transfer (FRET) that use QDs as both the donor and acceptor. FRET-based sensors often use fluorescent dyes (FD) or proteins (FPs), but their photo- and chemical instability can be problematic. Contemporary QD-QD FRET systems suffer from unacceptably high background signal due to direct acceptor excitation. Materials engineering is used to create QD donors that are brighter than their QD acceptors to mitigate this effect. First, CdSe/xCdS/xZnS QDs of increasing shell thickness were synthesized and tested in a QD-fluorescent dye system to elucidate the effect of increased donor size on the performance of a FRET sensor. The optimal donors were medium-sized and 8 times brighter than commercially available QDs while retaining ~60% FRET efficiency. When used in a sensor, changes in sensor brightness were visible by eye. Moving towards QD-QD systems, a pH-based aggregation assay was used to test how QD heterostructures comprised of different semiconductor materials perform as FRET donors or acceptors. The fundamental principles uncovered are used to improve contemporary QD-QD FRET sensing and show that sensors can be designed to use color change as a visible, easy-to-decipher readout. Color change-based sensor output is further explored in an allosteric transcription factor-based small-molecule sensor that employs QDs as the sole fluorescent label. A highly modular design is presented that achieves a nanomolar concentration visual limit of detection. The ease of use, and fast, instrument-free readout of the sensor shows promise for its development into a fully integrated point-of-care device, endorsing the value of further developing QD-based in vitro biosensors for clinical or commercial translation. / 2020-06-04T00:00:00Z

by Tsang Kee Kung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1978. / Includes bibliographical references (leaves 99-100).

Carbon nanoparticles constitute a class of important materials that have uses in many different fields. This thesis focuses on the synthesis and surface modification of different carbon nanoparticles and each novel nanomaterial is demonstrated to have a specific sensing application. Carbon blacks play a significant role in the research that is presented herein. Emperor 2000, a commercial bulk-produced carbon black available from Cabot Corporation, is the starting material for many of the investigations. The surface of Emperor 2000 is shown to be susceptible to physisorption, through π-π stacking. These interactions are exploited to append pyrene-based compounds onto the surface of the carbon nanoparticles. This methodology results in carbon nanoparticles with surface boronic acid functionality that is demonstrated to be affective in the electrochemical detection of catecholic caffeic acid. Emperor 2000 carbon nanoparticles are commercially produced with phenylsulphonic acid functional groups on the surface. This functionality is subjected to synthetic methods to obtain carbon nanoparticles with extremely hydrohphobic character, which are demonstrated as important substrates for probing lipophilic redox systems and lipid character under different experimental conditions. Fluorescent carbon nanodots (C-dots) are another important form of carbon nanoparticle. Herein, the facile synthesis of C-dots that possess intrinsic pyridine functionality is described. These nanodots exhibit two-photon fluorescence that is exhibited both in solution and in HeLa cells. The nanodots are demonstrated to have the potential to be developed into nanomedicines and biocompatible scaffolds for new drug delivery mechanisms. These straightforward synthesis, modification, and application methods demonstrate the effectiveness and the versatility of carbon nanoparticles. This class of nanomaterial is generally outclassed by modern and more fashionable carbon nanotubes and graphene-based systems. However, carbon nanoparticles are more cost effective and readily available carbon-based nanomaterials that can be used for a wide range of applications.

Cette thèse concerne la détection des métaux lourds dans l’environnement et en particulier dans les eaux de surface et les sous-produits de désinfection de l’eau potable. Les deux catégories de contaminations ont des propriétés différentes de sorte que deux méthodes correspondantes ont été dévéloppées : l’une est basée sur des capteurs moléculaires fluorescents mis en oeuvre dans un micro-dispositif, l’autre est basée sur une détection électrochimique. Deux capteurs moléculaires fluorescents, Rhod-5N et DPPS-PEG, et plusieurs dispositifs microfluidiques ont été fabriqués et appliqués pour la détection des ions de métaux lourds, Cd (II) et Hg (II),dans les eaux de surface. Une nouveau circuit en PMMA est fabriqué par ablation laser femtoseconde et testé pour la détection de Cd2+ avec le Rhod-5N. De plus, des améliorations de la performance des circuits microfluidiques ont été faites. Une nouvelle méthode de détermination sensible de cinq acides haloacétiques (AHAs) dans les d'eaux a été développée. Elle est basée sur l'extraction électromembranaire (EME) avant électrophorèse capillaire avec détection de conductivité sans contact à couplage capacitif (CE-C4D). / This thesis is aimed at environmental contaminations detection, mainly heavy metal ions in surface water and disinfection by-products (DBPs) in drinking water. The two categories of contaminations have different properties so that two correspondent methods were developed: one is based on fluorescent molecular sensors in a microfabricated device, the other one is based on conductive detection. Two fluorescent molecular sensors, Rhod-5N and DPPS-PEG, and several microfluidic devices were developed and applied for heavy metal ions Cd (II) and Hg (II) detection in surface water. A new microchip made of PMMA was fabricated by femtosecond laser ablation and tested for Cd (II) sensing based on a fluorescent molecular sensor Rhod-5N. Further more, some improvements of the performance of microfluidic chips were made. A novel method for sensitive determination of five priority haloacetic acids (HAAs) in water systems has been developed based on electromembrane extraction (EME) prior to capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D).

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