Metal enhanced detection of salivary proteins, Bacillus globigii and novel reagents for bioimaging and sensing

Recent developments in proteomics and emerging technologies have resulted in better understanding and characterization of saliva biomarkers. Consequently, the major objective of this research is to develop novel oral biosensors for detection of major salivary proteins including amylase and histatin. Herein, we make the first report on the development of a label-free electrochemical biosensor for the detection of amylase and histatin. The biosensor is based on a new Ab-Ag sensor platform which utilizes metal-enhanced electrochemical detection (MED). The MED concept exploits our recent discovery that an immobilized metal layer, either as continuous film, particle, colloid or monolayer significantly amplifies electrochemical signals, while reducing reorganization energy and distance dependence electron transfer kinetics following the formation of the Ab-Ag complex at the electrode surface. The MED immunosensor exhibits remarkable sensitivity with detection limits in the picogram range. The analytical characteristics of the MED immunosensor are shown to be superior to those of the standard ELISA and the capillary fluorescence ELISA. The successful detection of the salivary biomarkers amylase and histatin can therefore serve as initial steps for monitoring oral health status and may lead to the universal acceptance of saliva as a diagnostic tool for oral diseases and systemic diseases.; The concept of the MED biosensor has been extended to the detection of the biological warfare agent simulant (BWA) Bacillus atrophaeus (formerly B. globigii). A modified signaling strategy, the M-NED, which relies on oleate stabilized silver nanoparticles adsorbed on the surface of the gold quartz electrode, is presented. This is the first report on the application of silver nanoparticles as a redox probe in the detection of BWA in a label-less detection system. A detection limit of 760 spores/ml, well below the infectious dose of Bacillus pathogens (2 x 10 5 spores/ml) was achieved. The M-NED immunosensor overcomes some of the challenges that affect electrochemical biosensors developed for BWA detection (such as poor sensitivity and lack of specificity) and could therefore serve as a benchmark for future designs in the development of electrochemical biosensors for biodefense and first responder applications.; The second part of this study is dedicated to the development of a new class of fluorescence imaging and contrasting agents. The simple synthesis of eight new monoazo-derivatives based on diazotized 3,2-aminobenzimidazole and 3-aminoquinoline that were coupled to either resorcinol, 2-naphthol, 3-hydroxybenzoic acid or 3-aminophenol is presented. The new compounds are 1-(2-benzimidazolylazo)-2-naphthol (BAN), 4-(2-benzimidazolylazo)-2-resorcinol (BAR), 2-(2-benzimidazolylazo)-3-hydroxybenzoicacid (BABA), 2-(2-benzimidazolylazo)-3-phenylaniline (BAA), 1-(3-Quinolinoazo)-2-napthol (QAN), 4-(3-Quinolinoazo)resorcinol (QAR), 4-(3-Quinolinoazo)-3-hydroxybenzoic acid (QABA) and 2,3-(Quinolinoazo)-3-hydroxyphenylaniline (QAA). These have been extensively characterized by IR, UV-Vis, 1HNMR, 13CNMR and fluorescence spectroscopy. The potential application of these new compounds in environmental remediation of uranium and as DNA stains is also demonstrated.; Finally the development of a HPLC method and a proposed electrochemical sensor for risedronate (Actonel(TM)) are presented.