| Global prevalence of foodborne diseases has increased over the years, resulting in major public health problems. Foodborne pathogens such as Escherichia coli (E. coli) can originate from different sources, hence there is a need for rapid detection methods to ensure safe food and consequently avert foodborne diseases. Conventional methods for bacterial detection have been challenged on several fronts including the speed with which they produce results. As such, this has necessitated the call for research in fast methods that are also cost effective, selective and sensitive.This thesis describes the synthesis of a novel sugar coated poly(p-phenylene ethynylene) (PPE) and its subsequent functional properties. Sugar coated fluorescent polymers are an emerging technology that is fast attracting a lot of interest as a promising tool for screening and detecting bacterial targets because of their unique ability to act as both recognition and communication elements. In addition, PPEs simulate several interactions by exhibiting a number of ligands on a single polymer chain. Although the last decade has seen the emergence of PPEs, their application has largely been limited to substrate scope and the high cost of synthesis. However, amidst all these concerns, the design and production of such intermediates needs to be as simple as possible and economically viable if the use of PPEs in biosensing is to be a meaningful option. Therefore, efforts were made to design and synthesize a polymer that is not only highly emissive but also cost effective. The key concept developed in this study was the design principle that facilitates the combination of biological receptor molecules (recognition elements) for specific detection of target molecules and PPEs as signal transduction and amplification units.The work was carried out in three phases, i.e synthesis, photo-physical property measurements and performance evaluation. Synthetic work was initiated by the preparation of aminoethyl mannoside and amino galactose to introduce the amine functional group without affecting the alcohol functionality. The sugars were designed to act as recognition elements once conjugated with the main polymer chain because of their high affinity for cell surface lectins. N-Cbz-aminoethanol was employed as the glycosyl acceptor because it is commercially available, crystalline and its deprotection can easily be achieved in a single step as described in chapter 2. The techniques used were carefully selected to make sure they were easy enough to be used by less experienced organic chemists because of excellent overall yields without the need for chromatographic purifications.Preparation of the PPE is outlined in chapter 3 and started with the synthesis of basic intermediate compounds based on 1,4-dimethoxybenzene as starting material. The synthetic route followed a multi-step process to produce diethynyldimethoxybenzene and a diidodipropanoic acid as principle monomers, with a number of intermediate products in-between. These monomers were polymerized via Pd-catalyzed Sonogashira reaction and further coated with mannose units to mitigate the effect of nonspecific interactions. UV-Vis and fluorescence spectra were employed to measure the fluorescence properties and investigate the response of the polymer towards concanavalin A (Con A, a carbohydrate-binding protein). Confocal microscopy was engaged for fluorescent cell imaging and sensitivity studies. Optical measurement results showed that the polymer exhibited a green fluorescence whose intensity can be reduced by mannose binding lectin. The protocols reported herein provide intermediate products that can be converted into useful sensing elements from cheap and commercially available starting materials, in a few number of steps.Chapter 4 details the photo-physical properties of the polymer. These are some of the key indicators for the quality of fluorescent units and determine fitness for biosensing studies. The polymer exhibited absorbance and emission wavelengths of 393 and 466.5nm respectively. Although the quantum yield of the polymer is not as high (10.8%), it has a high Stokes shift making it ideal for sensing studies. In studying the effect of fluorescence quenchers for this sugar coated PPE, it was demonstrated that the polymer’s fluorescence is quenched by Con A due to complex formation at ground state. Con A is a lectin which specifically binds sugars (mannose) and plays a key role in cell signaling, cell surface recognition and pathogen docking. In this study, Con A was chosen because it is less toxic and usually the obvious choice as proof of principle for novel methods which detect ligand-protein interactions.A description of the performance of the polymer as a detection mechanism is detailed in chapter 5. Incubation of E.coli (BL21), a normal flora of the gut, with the polymer, yielded fluorescent bacterial aggregates through polyvalent interactions. Subsequently, there was a reduction in fluorescence intensity of the solution. To determine the optimum binding time, fluorescence intensity of the polymer-bacteria suspension was measured at intervals. These measurements showed that binding, in this system, will reach an optimum level within 30 minutes of incubation. The polymer’s affinity for bacteria has been demonstrated with a detection limit of 103cfu/ml. One major advantage for this system is that detection can be achieved within a minimum period of 30 minutes, unlike in conventional methods which can take up to several days to report a result.The sugar coated PPE produced herein has revealed great promise in biosensing applications. Having a carboxylic acid group on the terminal end of the spacer allowed for expediency for further bioconjugation with various other target elements. The utility of the mannose functionalized polymer to detect E. coli expressing functional FimH mannose-specific lectin on their surface was also demonstrated. The sugar units displayed on the surface of the polymer retained their functional ability to interact with mannose binding lectin. |
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