Several Non-electric Detection Of Biologically Active Molecules Biosensor

Several kinds of electrochemical biosensors have been constructed for the determination of ultra-trace levels of redox-inactive peptides, proteins and DNA preimmobilized onto the electrode surface.The introduction of the electrochemical markers in these methods increases the sensitivity,selectivity,and the ability to recognize the species analyzed.The main contents of the thesis are as follows:A thiol-specific electroactive cross-linker,N-(2-ethyl-ferrocene) maleimide(Fc-Mi),has been used to tag surfaceconfined peptides containing cysteine residues or oligodeoxynucleotides(ODNs)whose 3' ends have been modifed with thiol groups.The peptides studied herein include both the oxidized and reduced forms of glutathione and a hexapeptide.Cyclic voltammograms(CVs)of the Fc-Mi groups attached to the surfaces were used to quantify the total number of cysteine residues that are tagged and/or can undergo facile electron transfer reactions with the underlying electrodes.A quartz crystal microbalance was used in conjunction with CV to estimate the total number of cysteine groups labeled by Fc-Mi per peptide molecule.The methodology is further extended to the determination of ODN samples in a sandwich assay wherein the thiol linker on the 3' end can be tagged with Fc-Mi.The analytical performance was evaluated through determinations of a complementary ODN target and targets with varying numbers of mismatching bases.ODN samples as low as 10 fmol can be detected.Meanwhile,via derivatization with a bifunctional cross-linker 4-maleimidobutyric acid N-hydroxysuccinimide ester(GMBS)and an electrochemical marker 11-ferrocenyl-1-undecanethiol(Fc-SH), voltammetric determination of surface-confined proteins electrostatically adsorbed onto the polyelectrolyte of poly(sodium 4-styrenesulfonate) (PSS)or poly(allylamine hydrochloride)(PAH)-covered surfaces could be realized.The utilization of PSS or PAH was anticipated to reduce the nonspecific adsorption of the proteins on the surface.Two kinds of proteins with no redox activity or exhibiting complex or ill-defined voltammetric peak(s),i.e.the positively charged lysozyme and negatively charged metallothionein(MT)were demonstrated.Due to the incorporation of the bifunctional reagent GMBS and the redox active Fc groups onto the protein-modified electrodes,well-defined voltammetric peaks of high signal intensity were obtained.The anodic peak heights were found to be dependent on the surface density of the proteins electrostatically binded to the polyelectrolyte-coated surface.The present method can measure lysozyme concentration as low as 0.1 nM.The protocol described herein is simple,reproducible,selective,and does not require sophisticated analytical instrumentation.