Development Of New Chemiluminescent And Electrochemiluminescent Coreactants And Their Bioanalytical Applications

Electrochemiluminescence(ECL)and chemiluminescence provide the highly demanded sensitivity in analytical method developments.Moreover,the discovery that some molecules known as coreactants could facilitate the fast electron transfer reactions to populate the excited states of luminophores further extended the applications of these sensitive analytical techniques to the detection of biomolecules,food adulteration,environmental monitoring,forensic examination of crime scenes,cell imaging,and pharmaceutical drugs analysis.Motivated by the excellent and unique properties of both ECL and CL,we designed a unique sensing strategy to determine the activity of a biologically significant enzyme,superoxide dismutase(SOD),using lucigenin chemiluminescence based on its efficient coreaction with a 1,4-dithiothreitol(DDT).Secondly,an electrochemiluminescent method was developed based on the ECL of tris(2,2-bipyridyl)ruthenium(?)((Ru(bpy)32+)to determine isatin,a biomolecule with a wide range of biological activities and therapeutic roles against several diseases including neuroblastoma and apoptosis.Lastly,we also employed the ECL of Ru(bpy)32+and rongalite as a model food adulterant to detect potential adulteration of Tofu,a popular type of food,particularly in South East Asia.This thesis includes the results of our experimental findings as follows:1.1,4-dithiothreitol(DTT),a highly water-soluble and well-known reducing agent in biomedical applications responsible for preservation and regeneration of sulfhydryl groups,has been developed as an efficient and stable chemiluminescent coreactant for the first time.DTT reacts with lucigenin efficiently to generate chemiluminescence(CL),eliminating the need for catalysts to facilitate the CL generation in the classical lucigenin-hydrogen peroxide(H2O2)system.The intensity of the lucigenin/DTT CL system surpasses the classical lucigenin-H2O2 several orders of magnitude,even without an external catalyst.The newly-developed lucigenin/DTT system achieves sensitive detection of DTT(LOD=65 nM).Owing to the reaction between DTT and dissolved molecular oxygen(02)mediated by lucigenin,reactive oxygen species were generated as confirmed by radical scavengers.Based on the excellent CL quenching facilitated by catalytic disproportionation of U2·-by superoxide dismutase(SOD),a sensitive and facile strategy to assay SOD was developed with LOD of 2.2 ng/mL.Our proposed method herein is not only advantageous in terms of rapidity and sensitivity but eliminated the need for a complicated synthesis of sensing probes and facilitates the efficient detection of both DTT and SOD in human serum with excellent recoveries in the range of 98.6-104.1%.This lucigenin/DTT CL system can serve as a unique sensing probe for applications in clinical and biomedical analysis.2.Isatin is an endogenous indole derivative that has been reported to possess various pharmacological,therapeutic,and clinical importance.To date,isatin has been detected mainly by mass spectrometry.Here,we report for the first time the intense anodic electrochemiluminescence(ECL)of tris(2,2'-bipyridyl)ruthenium(?)with isatin as an efficient co-reactant.Interestingly,the ECL response of the Ru(bpy)32+/isatin system in borate buffer solution is about 12 times more intense than in phosphate buffer of the same pH;and approximately 114 times greater than the ECL intensity of the luminophore.Under the optimal conditions,the ECL of Ru(bpy)32+increases linearly with the concentration of the isatin in the range of 1.0 ?M to 1.0 mM(R2=0.998)with a limit of detection(LOD)of 0.28?M(3?/m).Moreover,the method has been successfully applied to the detection of isatin in human urine.The developed method has shown excellent recoveries in the range of 99.7-101.3%.The anodic ECL assay reported in this work is superior in terms of linear range and selectivity to the previously reported methods and can be applied to detect isatin in real samples.3.The persistent reports on the indiscriminate contamination of foodstuffs with inedible chemicals for economic gains and the high cost of the existing analytical methods for their detection make it necessary to develop simple and cheaper methods for the immediate detection of those toxic substances.Here,we report a fast and highly sensitive electrochemiluminescence(ECL)method to detect foodstuffs adulteration with an industrial bleaching agent-rongalite(RGL)for the first time.Under the optimal conditions,the ECL intensity of the tris(2,2'-bipyridine)ruthenium(?)(Ru(bpy)32+)increases linearly with the concentration of RGL in the range of 1 ?M to 1000 ?M with a limit of detection of 0.57 ?M(S/N=3).The developed method has shown excellent selectivity in the presence of co-existing interfering species.The application of the ECL method in this contribution can be extended to other foodstuffs that might be adulterated with RGL other than the one(Tofu)investigated here.