(Amplified Voltammetric Detection Of MiRNA And The Cleavage Of Peptide By BACE1)

MiRNA and BACE1play important roles in glioma and Alzhermiz s Disease, respectively. The expression levels of miRNA-182might be used as an indicator for early diagnosis of glioma. And the inhibition of the activity of BACE1could reduce the production of A(3. Therefore, the investigation of the concentration change of miRNA and the cleavage process of AAP by BACE1is of great significance. However, due to the low concentration of miRNA in biological samples and the lack of effective methods to monitor the cleavage process, it is highly desirable to develop a viable method to meet these requirements. In the thesis, the concentration of miRNA and the cleavage of AAP by BACE1were detected via signal amplification by ferrocene-capped gold nanoparticle/streptavidin conjugates.MiRNA belongs to a family of small non-protein coding RNAs (typically17-25nucleotide long) and exists generally in Eukaryotes. The expression level of miRNA-182is associated with glioma. In this work, a biotinylated miRNA with the same sequence as that of a miRNA target is introduced into samples and allowed to compete with the miRNA target for the oligonucleotide (ODN) probe preimmobilized onto an electrode. Voltammetric quantification of the miRNA target was accomplished after complexation of the biotin-miRNA with ferrocene (Fc)-capped gold nanoparticle/streptavidin conjugates. It was found that the current of the Fc oxidation peak current was inversely proportional to the concentration of target miRNA. The concentration of miRNA-182in glioma patients is3times as high as that in healthy persons, a conclusion in excellent agreement with a RT-qPCR measurement of the expression level. The method is highly reproducible (RSD<5%) and senstive with a concentration level of10fM.BACE1is involved in the production of Aβ from the amyloid precursor protein (APP) by sequential proteolytic cleavages by two enzymes,β-secretase and y-secretase. More and more evidence showed that Aβ played an important role in AD. Therefore, the inhibition of the activity of BACE1has become a significant means for AD therapyas well as drug discivery. In this work, we simulated the the hydrolytic cleavage process of APP by BACE1. BACE1was first exposed to the electrode modified with the biotinylated peptides, this was followed by the attachment of ferrocene-capped gold nanoparticle/streptavidin conjugates. Well-defined voltammetric peaks with high signal intensity were obtained in absense of BACE1. However, the peak current was greatly decreased upon BACE1cleavage. The presence of a potent inhibitor suppressed the above cleavage process, leading to a much larger electrochemical signal.