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Doctor of Philosophy / Department of Chemistry / Jun Li / Reduction in electrode size down to nanometers dramatically enhances the detection sensitivity and temporal resolution. Here we explore nanoelectrode arrays (NEAs) and nanoparticles in building high performance biosensors. Vertically aligned carbon nanofibers (VACNFs) of diameter ~100 nm were grown on a Si substrate using plasma enhanced chemical vapor deposition. SiO[subscript]2 embedded CNF NEAs were then fabricated using techniques like chemical vapor deposition, mechanical polishing, and reactive ion etching, with CNF tips exposed at the final step. The effect of the interior structure of CNFs on electron transfer rate (ETR) was investigated by covalently attaching ferrocene molecules to the exposed end of CNFs. Anomalous differences in the ETR were observed between DC voltammetry (DCV) and AC voltammetry (ACV). The findings from this study are currently being extended to develop an electrochemical biosensor for the detection of cancerous protease (legumain). Preliminary results with standard macro glassy carbon electrodes show a significant decrease in ACV signal, which is encouraging. In another study, NEA was employed to capture and detect pathogenic bacteria using AC dielectrophoresis (DEP) and electrochemical impedance spectroscopy (EIS). A nano-DEP device was fabricated using photolithography processes to define a micro patterned exposed active region on NEA and a microfluidic channel on macro-indium tin oxide electrode. Enhanced electric field gradient at the exposed CNF tips was achieved due to the nanometer size of the electrodes, because of which each individual exposed tip can act as a potential DEP trap to capture the pathogen. Significant decrease in the absolute impedance at the NEA was also observed by EIS experiments. In a final study, we modified gold nanoparticles (GNPs) with luminol to develop chemiluminescence (CL) based blood biosensor. Modified GNPs were characterized by UV-Vis, IR spectroscopy and TEM. We have applied this CL method for the detection of highly diluted blood samples, in both intact and lysed forms, which releases Fe[superscipt]3[superscript]+ containing hemoglobin to catalyze the luminol CL. Particularly, the lysed blood sample can be detected even after 10[superscript]8 dilution (corresponding to ~0.18 cells/well). This method can be readily developed as a portable biosensing technique for rapid and ultrasensitive point-of-care applications.

Rapid and sensitive detection of biomolecules is an important issue in nanomedicine. Many disorders are manifested by changes in protein levels of serum and other biofluids. Rapid and effective differentiation between normal and cancerous cells is an important challenge for the diagnosis and treatment of tumor. Likewise, rapid and effective identification of pathogens is a key target in both biomedical and environmental monitoring. Most biological recognition processes occur via specific interactions. Gold nanoparticles (AuNPs) feature sizes commensurate with biomacromolecules, coupled with useful physical and optical properties. A key issue in the use of nanomaterials is controlling the interfacial interactions of these complex systems. Modulation of these physicochemical properties can be readily achieved by engineering nanoparticles surface. Inspired by the idea of mimicking nature, a convenient, precise and rapid method for sensing proteins, cancerous cells and bacteria has been developed by overtaking the superb performance of biological olfactory systems in odor detection, identification, tracking, and location. On the fundamental side, an array-based/`chemical nose' sensor composed of cationic functionalized AuNPs as receptors and anionic fluorescent conjugated polymers or green fluorescent proteins or enzyme/substrates as transducers that can properly detect and identify proteins, bacteria, and cancerous cells has been successfully fabricated.

Yes / 2-Amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) is a central dietary mutagen, produced when proteinaceous food is heated at very high temperatures potentially causing DNA strand breaks. This study investigates the protective potential of a well-researched flavonoid, myricetin in its bulk and nano-forms against oxidative stress induced ex vivo/in vitro by PhIP in lymphocytes from pre-cancerous monoclonal gammopathy of undetermined significance (MGUS) patients and those from healthy individuals. The results from the Comet assay revealed that in the presence of myricetin bulk (10 µM) and myricetin nano (20 µM), the DNA damage caused by a high dose of PhIP (100 µM) was significantly (P < 0.001) reduced in both groups. However, nano has shown better protection in lymphocytes from pre-cancerous patients. Consistent results were obtained from the micronucleus assay where micronuclei frequency in binucleated cells significantly decreased upon supplementing PhIP with myricetin bulk (P < 0.01) and myricetin nano (P < 0.001), compared to the PhIP treatment alone. To briefly determine the cellular pathways involved in the protective role of myricetin against PhIP, we studied gene expression of P53 and ATR kinase (ATM- and Rad3-related), using the real-time PCR technique.

Yes / The present study investigated the genoprotective and genotoxic effects of myricetin bulk (10 μM) and nano forms (20 μM) in the lymphocytes from pre-cancerous, monoclonal gammopathy of unknown significance (MGUS) patients and healthy individuals using the Comet and micronucleus assays. The study also evaluated the effect of myricetin on P53 expression levels, using the Western blot technique. Results showed that throughout the in-vitro treatment, lymphocytes from the patients group had higher levels of baseline DNA damage compared to the healthy group. Myricetin in both forms induced significant DNA damage, only at higher concentrations (>40 μM). The micronucleus assay showed a significant reduction (P < 0.01) in the frequency of micronuclei in mono-nucleated cells in the patient group treated with the nano form of myricetin at the non-toxic dose of 20 μM. There was a significant increase in both gene and protein P53 levels in lymphocytes isolated from healthy individuals and pre-cancerous patients. These results suggested a protective effect of myricetin and indicated its nutritional supplement potential for protection against cancer development among patients suffering from MGUS. / This study was kindly funded by Mr Nasir Qayyum, Bradford, UK.

Cancer is one of the leading causes of death across the globe which needs appropriate and cost-effective treatment. Several recent studies have suggested that dietary intake of various flavonoids such as myricetin have a protective effect against different types of cancers and cardiovascular diseases. The present study was conducted to investigate the genoprotective and genotoxic effects of myricetin nano and bulk forms on the lymphocytes from pre-cancerous and multiple myeloma cancer patients compared to those from healthy individuals. Also, to investigate the protective potential of myricetin bulk and nano against the oxidative stress produced in vitro by 2- amino-1-methyl-6 phenylimidazo [4, 5-b] pyridine and reactive oxygen species- induced DNA damage using the Comet assay, micronucleus assay, cellular reactive oxygen species and glutathione detection assay, Western blotting, real-time polymerase chain reaction and immunofluorescence. Lymphocytes from the patient groups showed significantly higher levels of basal DNA damage compared to the lymphocytes from healthy individuals which was observed throughout the in vitro treatment. Myricetin in both forms has not induced any significant DNA damage in all of the investigative groups at selective lower concentrations; in fact, the results demonstrate a reduction in DNA damage upon treating with myricetin nano in lymphocytes from pre-cancerous patients demonstrated by significant reduction in micronuclei formation in mononucleated cells. DNA repair capacity of myricetin bulk and nano was determined by co-treating the drugs with hydrogen peroxide. Myricetin significantly reduced the oxidative stress related damage caused by hydrogen peroxide, where myricetin nano seemed to be more effective employing the Comet assay. In the presence of myricetin bulk and nano, the damaging effects of 2- amino-1-methyl-6 phenylimidazo [4,5-b] pyridine were considerably decreased, where myricetin nano was more effective. This could be because nanoparticles have a larger surface area which could improve their reactivity and also the reduction in size of the particles could improve the anti-cancer properties of this compound. Myricetin has shown genoprotective and anti-oxidant effects by demonstrating the potential to reduce DNA damage caused by over-production of reactive oxygen species and oxidative stress. It has also shown anti-cancer potential in the lymphocytes from multiple myeloma patients by regulating the apoptosis related proteins, dependent on oxidative stress. Therefore, this study suggests that myricetin supplementation in our regular diet with enhanced bioavailability could have potential health beneficial effects and possibly protect against various diseases including cancer.

This research presents a framework for enhancing and analyzing time-sequenced mammographic images for detection of cancerous tissue, specifically designed to assist radiologists and physicians with the detection of breast cancer. By using computer aided diagnosis (CAD) systems as a tool to help in the detection of breast cancer in computed tomography (CT) mammography images, previous CT mammography images will enhance the interpretation of the next series of images. The first stage of this dissertation applies image subtraction to images from the same patient over time. Image types are defined as temporal subtraction, dual-energy subtraction, and Digital Database for Screening Mammography (DDSM). Image enhancement begins by applying image registration and subtraction using Matlab 2012a registration for temporal images and dual-energy subtraction for dual-energy images. DDSM images require no registration or subtraction as they are used for baseline analysis. The image data are from three different sources and all images had been annotated by radiologists for each image type using an image mask to identify malignant and benign. The second stage involved the examination of four different thresholding techniques. The amplitude thresholding method manipulates objects and backgrounds in such a way that object and background pixels have grey levels grouped into two dominant and different modes. In these cases, it was possible to extract the objects from the background using a threshold that separates the modes. The local thresholding introduced posed no restrictions on region shape or size, because it maximized edge features by thresholding local regions separately. The overall histogram analysis showed minima and maxima of the image and provided four feature types--mean, variance, skewness, and kurtosis. K-means clustering provided sequential splitting, initially performing dynamic splits. These dynamic splits were then further split into smaller, more variant regions until the regions of interest were isolated. Regional-growing methods used recursive splitting to partition the image top-down by using the average brightness of a region. Each thresholding method was applied to each of the three image types. In the final stage, the training set and test set were derived by applying the four thresholding methods on each of the three image types. This was accomplished by running Matlab 2012a grey-level, co-occurrence matrix (GLCM) and utilizing 21 target feature types, which were obtained from the Matlab function texture features. An additional four feature types were obtained from the state of the histogram-based features types. These 25 feature types were applied to each of the two classifications malignant and benign. WEKA 3.6.10 was used along with classifier J48 and cross-validation 10 fold to find the precision, recall, and f-measure values. Best results were obtained from these two combinations: temporal subtraction with amplitude thresholding, and temporal subtraction with regional-growing thresholding. To summarize, the researcher's contribution was to assess the effectiveness of various thresholding methods in the context of a three-stage approach, to help radiologists find cancerous tissue lesions in CT and MRI mammography images.

L’exérèse des tumeurs cancéreuses est une procédure courante pour le traitement de nombreux cancers. L’enjeu est de réaliser une excision la plus complète possible pour éviter les récidives tout en épargnant le plus possible les tissus sains bordant la tumeur. La détection de positons est une modalité d’imagerie particulièrement adaptée au repérage de résidus tumoraux lors de l'éxerèse car sa forte sélectivité spatiale permet de s'affranchir du bruit provenant de la fixation non spécifique du radiotraceur dans les tissus situés en profondeur, offrant ainsi une meilleur sensibilité et un meilleur rapport signal sur bruit que la détection de photons gamma. L’utilisation pour le contrôle d’exérèse impose cependant une contrainte forte sur les dimensions du détecteur qui doit être manipulable facilement par le chirurgien et pouvoir être inséré dans des plaies opératoires potentiellement étroites. Une nouvelle génération de photodétecteurs appelés photomultiplicateurs silicium (SiPM) est particulièrement adaptée à cette application car ceux-ci allient la compacité et la robustesse des technologies silicium avec d'excellentes performances de détection. Mon travail de thèse porte sur le développement et la caractérisation de nouvelles sondes positon basées sur ces photodétecteurs. Dans un premier temps, un travail de caractérisation des SiPMs a été réalisé pour évaluer leurs performances pour la détection de positons. Deux prototypes de prototypes de détecteurs aux rôles complémentaires ont ensuite été réalisé: le premier est un imageur, basé sur l’assemblage de deux scintillateurs avec une ou deux matrices de SiPMs, qui permet de réaliser rapidement l'image de la distribution de traceur sur une large surface de tissus. Le second détecteur est une sonde de comptage, constituée de fibres scintillantes couplées à des SiPMs individuels via des fibres claires et capable d'être couplée à l'outil d'exérèse. Elle permet de guider l'outil du chirurgien vers les tissus repérés préalablement avec l'imageur. La caractérisation de l’imageur a montré sa capacité à détecter des résidus tumoraux de petite taille (15mg) avec une résolution submillimétrique. La sonde de comptage présente quant à elle, une efficacité de détection de 80%. / Excision of cancerous tumors is a common procedure for the treatment of numerous cancers. The stake is to perform the most complete excision to prevent recurrences while preserving as much as possible surrounding healthy tissues. Positron detection is a well suited imaging modality for detection of tumor remains during excision because its strong spatial selectivity makes it insensitive to the noise coming from the non-specific accumulation of the radiotracer in healthy tissues located far from the detector, leading to a better sensitivity and a better signal-to-noise ratio than gamma photon detection. Its use for the control of excision implies however strong constraints on detector dimensions which must be easy to handle by the surgeon and easy to insert in tight surgical wound. A new generation of photodetectors called Silicon Photmultipliers (SiPMs) is particularly suited for this application because they present the compactness and robustness of silicon technologies and very good detection performances. My thesis aims to develop and characterize a new generation of new positron probes based on these photodetectors. Two prototypes of detectors with complementary roles were realized: the first one is an imaging device based on the assembly of two scintillators with one or two SiPMs arrays which allows to quickly make an image of tracer distribution along a wide surface of tissues. The second detector is a counting probe made of scintillating fibers associated with individual SiPMs through clear optical fibers and can be associated to the excision tool. It guides the surgeon tool to the tissues previously localized with the imaging probe. Characterization of the imaging probe showed its ability to detect small tumor remains (15mg) with a submillimetric resolution. The counting probe showed a detection efficiency of 80%.