Investigation Of Biomarkers Of Neurodegenerative Diseases And Cancers By Surface Plasmon Resonance And Electrochemisty

Abstract:Alzheimer’s disease (AD) is one of the neurodegenerative diseases with high morbidity, and no significant cognitive or functional impairment was detectable at the earliest stages. Amyloid-β (Aβ) and Aβ-binding proteins may provide possible diagnostic and therapeutic information for AD. Cancer is another leading cause to human death. Human fluids (e.g., urine, blood, saliva, and cerebrospinal fluids) in these two diseases could provide useful chemical and biological information which reflects the progress of disease, especially the expression level of biomarkers. Investigation of the biomarkers is of great importance to early diagnosis and treatment of these diseases.Surface plasmon resonance (SPR) is a sensitive optical technique, being capable of measuring the refractive index or thickness change occurring at the sensor chip. Electrochemistry is a technique that studies the charge transfer process between the redox moiety (or tag) on the biomarkers and the electrode. SPR is label-free, while electrochemistry is a representative of the label-based techniques. Both SPR and electroanalytical biosensors possess promising characteristics for early clinical diagnosis of AD and cancers in their sensitivity and selectivity.In this thesis, SPR and electroanalytical methods have been proposed for the detection of AD and glioma biomarkers.1. Two major biomarkers of AD, Aβ(1-42) and transtherithin (TTR) in cerebrospinal fluids (CSFs) were quantified simutaniously by a dual-channel SPR. First, capture antibodies specific to Aβ(1-42) and TTR were immobilized onto the two fluidic channels. Via injection of TTR, and the mixed solution of Aβ or CSF with the pre-added detection conjugate (streptavidin conjugated to biotinylated Aβ(1-16) antibodies), levels of Aβ(1-42) and TTR in CSFs were determined. The lowest detection level of Aβ(1-42) and TTR were4.7pM and1.1nM respectively. The concentrations of both proteins in CSFs of AD patients are lower than that in healthy donors. The assay of two biomarkers could provide strong evidence for the early diagnosis of AD.2. An SPR method for the continuous screening of β-site amyloid precursor protein-(APP-) cleaving enzyme1(β-secretase, BACE1) inhibitors at a single SPR chip has been developed. In the presence of a non-inhibitor, BACE1clips the peptide substrate at the cleavage site, detaching a fragment that is homologous to the N-terminus of the A(3peptide. Consequently, a subsequent injection of the Aβ antibody does not lead to any molecular recognition or SPR signal change at the chip. In contrast, abolishment of the BACE1activity by a strong inhibitor leaves the peptide substrate intact, and the subsequent antibody attachment produces an easily detectable SPR signal. The method reported here is cost-effective, as the unlabeled peptide is used as the BACE1substrate. Two inhibitors were screened and their half maximal inhibitory concentrations (IC50) determined by the SPR method are in excellent agreements with the values deduced from enzyme linked immunosorbent assay (ELISA) and mass spectrometry.3. A voltammetric method for the monitoring of BACE1activity and screening of BACE1inhibitors has been proposed.BACE1in the absence or presence of BACE1inhibitors was exposed to the biotinylated peptide substrate-modified electrode, followed by the attachment of ferrocene (Fc)-capped gold nanoparticle/streptavidin conjugates. Due to the blockage of the BACE1activity by select inhibitors, well-defined voltammetric peaks of high signal intensity were obtained. However, featureless voltammogram was obtained upon initiating the cleavage reaction. The proposed method is simple, sensitive, and suitable for monitoring of BACE1activity and screening of BACE1inhibitors.4. Amplified voltammetric detection of miRNA sequences in glioma patients was carried out. A biotinylated miRNA (biotin-miRNA) whose sequence is the same as that of a miRNA target is introduced into samples of interest 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 Fc-capped gold nanoparticle /streptavidin conjugates. The Fc oxidation current was found to be inversely proportional to the concentration of target miRNA between10fM and2.0pM. The low detection levels allow the direct quantification of miRNA-182to be performed in serum samples without sample pretreatment and RNA extraction and enrichment. The obviations of the requirement of an internal reference in qPCR, simplicity, and cost-effectiveness are other additional advantages of this method for the detection of nucleic acids in clinical samples.