Dna Identification Based On Thiol Protein Biosensor

Fluorescence spectroscopy and voltammetry have been utilized to the determination of ultra-trace levels of biomolecules such as peptides, proteins and DNA preimmobilized onto the electrode surface.The introduction of the functional thiol-specific reagents in these methods increases the sensitivity,selectivity,and the ability to recognize the species analyzed.The main contents of the thesis are as follows:1.To develop a method for the detection of surface-confined peptides containing cysteine residues or oligodeoxynucleotides(ODNs)whose 3' ends modified with thiol groups,a thiol-specific fluorescent cross-linker, N-(9-acridinyl)maleimide(NAM)was used.The peptides studied herein include both the oxidized and reduced forms of glutathione,and a hexapeptide(FT).Peptides are first attached onto the activated 11-mercaptoundecanoic acid(MUA)-terminated alkanethiol self-assembled monolayers(SAMs)and then derivatized with NAM.The cysteine residues was determined via combination of electrochemical desorption with fluorescence detection.GSH concentration as low as 40 pmol·L^-1can be measured.The fluorescence intensity in the case of FT is about 3 times as high as that for GSH,which is consistent with the molar ratio of cysteine residues in these two molecules.The analytical performance of gene analysis was also evaluated through the analyses of a complementary target and targets with varying numbers of mismatching bases.The method described here is simple,sensitive,reproducible,and does not require sophisticated analytical instrumentation and separation procedures.2.Development of sensitive and selective methods to detect protein at trace levels is of great biological importance.Consensus double-stranded oligonucleotide(ds-DNA)was first formed onto gold electrodes via hybridization of thiolated single-stranded DNA (ss-DNA)/hexanethiol(HT)mixed self-assembled monolayer with the complementary DNA to capture wild-type p53 in solution.The cysteine residues on the exterior of the p53 molecules were derivatized with a thiol-specific reagent,N-(2-ethyl-ferrocene)maleimide(Fc-Mi). Well-defined voltammetric peaks that report on the interaction of p53 with consensus ds-DNA were obtained.The level of p53 binding was found to be dependent on the surface density of the consensus ds-DNA, the level of p53 binding was found to be dependent on the sequence of the double-stranded(ds-DNA).The present method can measure p53 concentration as low as 1.33 nmol·L^-1.