Electrochemical Sensing Of Biomolecules Associated With Oxidative Stress

Oxidative stress has attracted increasing attention because it has been implicated in several human diseases such as aging and cancer. The related biomoleculars of oxidative stress such as malondialdehyde (MDA), superoxide anion (O2’), p53and oral cancer gene have been detected by many methods. Electrochemical method is an effective method due to its easy operation, high sensitivity and good selectivity. Nanomaterials are widely used in electrochemical biosensing because of their large specific surface area and good biocompatibility. In this thesis, multiwalled carbon nanotubes (MWNTs), Pd-Au alloy nanocrystals and nicking endonuclease were used to amplify signal. Mimic enzymes were used to enhance stability. Based on the principles above, several electrochemical biosensors were constructed to detect the related biomoleculars of oxidative stress.1. A label-free electrochemical biosensor based on multiwalled carbon nanotubes (MWNTs) for detecting malondialdehyde (MDA) was constructed for the first time. The biosensing interface was easily fabricated by human complement factor H (CFH) assembly on MWNTs-CS through amide dehydration reaction. MDA could bind CFH with acceptable selectivity and block the electron transfer of [Fe(CN)6]3/4-, leading to an obvious current signal changes. The biosensor exhibited good analytical performance, such as low detection limit and wide linear range. The biosensor was successfully applied to the assay of MDA content in human serum samples, which had a promising prospect for diagnosis of cancers.2.(E)-4-(4-formylstyryl) pyridine (Formylstyrylpyridine) was prepared through Heck reaction and used as a photopolymer material to immobilize biomimetic superoxide dismutase under ultraviolet irradiation (UV) irradiation in a short time. Mn2P2C>7multilayer sheet, a novel superoxide dismutase mimic, was synthesized. The formed photopolymer could immobilize Mn2?2O7firmly under UV irradiation. Based on fast interfacial electron transfer of Mn2P2O7-Formylstyrylpyridine film and the high efficient catalysis of Mn2P2O? which was used as a SOD mimic, a novel electrochemical biosensing platform called MWCNTs/Mn2P2O7-Formylstyrylpyridine for superoxide anion (O2") detection was constructed. The electrochemical biosensor provided a reliable platform to adhere living cells directly on the modified electrode surface. The biosensor was successfully applied to vitro determination of O2·released from living cells, which had a promising prospect for living cells monitoring and diagnosis of oxidative stress-related diseases. 3. Pd-Au alloy nanocrystals were used to immobilize probe DNA onto the electrode. Nicking endonuclease N.BstNB I could recognize the specific sequence in the double-stranded DNA (dsDNA). Hemin/G-quadruplex was acted as the mimic enzymes of reduced nicotinamide adenine dinucleotide (NADH) oxidase and horseradish peroxidase (HRP). Based on the principles above, the electrochemical biosensor was constructed to detect p53and oral cancer gene. Firstly, probe DNA hybridized with helper DNA partly, and then tagert DNA hybridized with helper DNA mostly to form a sequence of dsDNA and departed from the electrode. At the same time, a part of probe DNA became single-stranded. N.BstNB I recognized the specific sequence in the dsDNA and cleave the fragment of helper DNA. After that, tagert DNA became single-stranded and hybridized with helper DNA on the electrode surface to make more probe DNA become single-stranded. At last, the single-stranded probe DNA reacted with hemin and formed hemin/G-quadruplex. When thionine was functioned as the electron mediator, hemin/G-quadruplex catalyzed the reduction of H2O2which was generated from NADH in the presence of O2to produce electrochemical signal. If the helper DNA was changed, different target DNA could be detected, developing a universal determination method. The designed electrochemical biosensor showed high selectivity against single-base mismatch sequences and wide linear range. These satisfactory performances made a new avenue for potential diagnosis and prevention of cancer.