Novel Electrochemcial Biosensors Based On Nucleic Acid-modified Electrodes

The quick, low-cost, highly sensitive and selective detection of biologically active proteins and small molecules, pathogenically associated sequence-specific nucleic acids and highly toxic heavy metal ions is one of the most important and attractive topics in modern biochemical analysis, which has activated the development of biosensors. An electrochemical nucleic acid-based biosensor is a biosensor that integrates an nucleic acid as the biological recognition element and an electrode as the physicochemical transducer. Electrochemical nucleic acid-based biosensor combines the high specificity of nucleic acid probes and the excellent sensitivity of electrochemical detection techniques; it has become the most important branch of biosensors. Typically, the design of an electrochemical nucleic acid-based biosensor includes three key steps:immobilization of the nucleic acid probe, interaction with the target molecule and electrochemical signal transduction of the molecular recognition event. Optimization of each step is required to improve the overall performance of the devices. For the development of novel electrochemical nucleic acid-based biosensors that meet the challenges of modern biochemical analysis, it is essential to explore new properties and application of the nucleic acid-modified electrodes, to construct simple and effective methods for immobilizing of the nucleic acid probes and highly sensitive signal transduction of the molecular recognition event. Focused on these basic items in the development of high-performance electrochemical nucleic acid-based biosensors, the extending application of nucleic acid-modified electrodes to new bioanalysis, the design of novel nucleic acid probes and the novel strategies for immobilizing nucleic acid probes and amplifying sensing signals have been investigated in the present dissertation and described as follows:In chapter2, A DNA-modified glass carbon electrode (DNA-GCE) obtained by one-step electrodeposition was developed for electrochemical detection of dopamine (DA). The negatively charged DNA film electrodeposited on the GCE could electrostatically attract the positively charged DA in neutral buffer solution (pH:7.0), which greatly increased the electrochemical oxidation current of DA. The common overlapped oxidation peaks of DA and ascorbic acid were separated completely on the DNA-GCE. Based on this selective electrocatalysis of the DNA-GCE toward the oxidation of DA, a highly sensitive and selective electrochemical platform has been successfully established for DA detection.In chapter3, a novel electrochemical sensor for Hg2+detection was developed using two mercury-specific oligonucleotide probes and streptavidin-horseradish peroxidase (HRP) enzymatic signal amplification. The two mercury-specific oligonucleotide probes comprised a thiolated capture probe and a biotinated signal probe. The thiolated capture probe was immobilized on a gold electrode. In the presence of Hg2+, the thymine-Hg2+-thymine (T-Hg2+-T) interaction between the mismatched T-T base pairs directed the biotinated signal probe hybridizing to the capture probe and yielded a biotin-functioned electrode surface. HRP was then immobilized on the biotin-modified substrate via biotin-streptavidin interaction. The immobilized HRP catalyzed the oxidation of hydroquinone (H2Q) to benzoquinone (BQ) by hydrogen peroxide and the generated BQ was further electrochemically reduced at the modified gold electrode, producing a readout signal for quantitative detection of Hg2+. The results showed that the enzyme-amplified electrochemical sensor system was highly sensitive to Hg2+in the concentration of0.5nM to1μM with a detection limit of0.3nM, and it also demonstrated excellent selectivity against other interferential metal ions.In chapter4, a novel electrochemical catalytic biosensor for sensitive and selective detecting of Hg2+was developed. To construct the electrochemical catalytic Hg2+sensing system, a thiolated, mercury-specific oligonucleotide capture probe was first immobilized on gold electrode surface. In the presence of Hg2+, a oligonucleotide signal probe integrating a mercury-specific oligonucleotide sequence and a G-quadruplex (G4) sequence was then captured on the gold electrode surface through the thymine-mercury(Ⅱ)-thymine interaction-mediated surface hybridization. The further interaction of the immobilized G4sequence with hemin generated a G4-Hemin complex monolayer. The G4-Hemin units exhibited good electrochemical catalytic activity toward the electrochemical reduction of hydrogen peroxide, producing amplified readout signals for Hg2+sensing events. The electrochemical catalytic Hg2+sensor system was highly sensitive and selective to Hg2+in the concentration of1.0nM to1μM with a detection limit of0.5nM. This simple and effective electrochemical catalytic sensor system provides a potential alternative for Hg2+assay.In chapter5, the electrochemical redox behavior of ferricyanide at the dodecanethiol modified electrode (H25C12S-Au), the single-walled carbon nanotube-dodecanethiol modified electrode (SWCNT-H25C12S-Au) and the single-stranded DNA-single-walled carbon nanotube-dodecanethiol modified electrode (ssDNA-SWCNT-H25C12S-Au) was thoroughly investigated. The results showed that SWCNT adsorbed on the surface of the H25C12S-Au could restore the restrained redox current of ferricyanide on the H25C12S-Au modified electrode, and this so-called "tunneling current effect" could be flexibly mediated by C-Ag+-C interaction between Ag+and the Ag+-specific oligonucleotide probe adsorbed on the sidewall of SWCNT. Based on this unique phenomenon, a sensitive, selective and simple electrochemical platform for Ag+detection was successfully developed.In chapter6, a novel scheme for scanning electrochemical microscopy (SECM) assay of DNA based on molecular beacon (MB) and enzymatic amplification biosensor was described. In this method, streptavidin-horseradish peroxidase (HRP) was captured by double-stranded DNA (ds-DNA) modified gold substrate via biotin-streptavidin interaction after hybridization of target DNA to the immobilized MB probe functioned with a biotin at its3’-end. In the presence of H2O2, hydroquinone (H2Q) was oxidized to benzoquinone (BQ) at the modified substrate surface through the HRP catalytic reaction, and then the generated BQ corresponding to the amount of target DNA was electrochemically reduced by a SECM tip. The resulting reduction current allowed sensitive concentration determination of target DNA and SECM imaging of hybridization between the target DNA and the immobilized MB probe. The detection limit of this method was as low as17pM for the complementary target DNA and it has good selectivity to discriminate between the complementary target and the sequence containing mismatched bases.