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Designing and Researching the Biosensor Using Scanning Electrochemical Microscopy

Author: FanHuaJun
Tutor: HePinGang; FangYuZhi
School: East China Normal University
Course: Analytical Chemistry
Keywords: Scanning Microscope Nanoparticles Gold substrate Sensor platform On the substrate Array Fixed probe Glass substrate Dodecanethiol Micron Electrochemical detection Electrolyte solution Biosensor Technology Immune Sensors Biosensor Alkylmercaptans DNA Scanning tunneling microscopy Coulomb force Self-assembled monolayer
CLC: TP212
Type: PhD thesis
Year: 2014
Downloads: 73
Quote: 0
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Abstract


Since the discovery of DNA double helix structure by Watson and Crick in1953, a hot wave of DNA research raised between each subject. As molecular biology subject gradually been established, the boom continue rising until now. Plotted of the draft of human genome marked the development of life science entered the post genome era, which make us recognize and understand the origin and evolution of human disease from a new angle.And how to study so many genes’role in the process of life became a global joint project of the life science. So, to establish a kind of miniaturization, rapid, high sensitivity, high throughput detection technology is especially important. These demands promoted the birth and development of DNA chips.In this paper, we combined the electrochemical scanning microscope micro processing technology, the array chip production techniques and nanoparticles signal amplification technology, and developed the new DNA micro biosensor, immune sensor and DNA chip with high sensitivity and high selectivity. This thesis was divided into six chapters, and the concrete content is as follows:Scanning electrochemical microscopy (SECM), which presented by the Bard in1989, is a new type of scanning probe microscope with high spatial resolution.Since it has "chemical sensitivity", SECM can not only characterize the surface topography of conductor and insulator, but also distinguish electrochemical activity on the substrate surface. It also could research the biological molecules and cells, which generated electroactive substances or were charged themselves. It is different from other scanning techniques, such as scanning tunneling microscope (STM) and atomic force microscope (AFM), SECM could directly provide information of electrochemical activity. Moreover micro surface processing could be achieved using the interaction between SECM probe and substrate, and this can extend to other aspects of applied research.Chapter1IntroductionIn this chapter, we systematically introduced the work principle and characteristics of scanning electrochemical microscope (SECM), and observed the current application of SECM. Then we emphatically summarized the SECM using in DNA detection and immune detection. Finally we expounded the design thinking and research significance of this paper.Chapter2Scanning electrochemical microscope building micro biosensor platformIn this chapter, we successfully built a micro biosensor platform on the1-dodecanethiolmodified gold substrate surface using SECM probe as a tool.1-dodecanethiol can be modified on the gold substrate and orderly formed a hydrophobic monolayer. As there is sufficiently long carbon chain in this monolayer, the modified substrate is insulating. In this experiment, we applied a negative potential pulse between the SECM probe and modified substrate, broken the SH bond of1-dodecanethiol monolayer beneath the SECM probe, thus complete the etching. By adjusting the pulse voltage, pulse cycle and the distance between the probe and substrate, we can control the size of the range of etching. In order to investigate the stability of etching, E, C, N, U pattern of letters were etched out.Chapter3Detection of DNA sequences modified on the gold substrate using SECM feedback modeIn this chapter, thiol-sequenced DNA was assembled on the prepared micro biosensor platform and detected by SECM using feedback mode, which confirmed the feasibility of construct DNA sensors in this mode. In feedback mode, the detection of DNA was achieved by the interaction between the different charged mediator and negative charged DNA phosphate backbone. Positive charged mediator could pass though the DNA molecule and recycled at the gold substrate, thus the current of the approach curve was increased. While the electrostatic repulsion between negative charged mediator and DNA phosphate backbone prevent the mediator diffusing to the gold substrate, and the current of the approach curve was decreased.Chapter4Qualitative and quantitative detection of DNA amplified withHRP-modified SiO2nanoparticles using scanningelectrochemical microscopyIn this chapter, a"sandwich" DNA structure was formed by the hybridization of thiol-tethered oligodeoxynucleotide probes (capture probe), assembled on the prepared micro biosensor platform, with target DNA and biotinylated indicator probe. HRP (horseradish peroxidase)-wrapped SiO2nanoparticles were linked to the sandwich structure through biotin-streptavidin interaction. Hydroquinone (H2Q) was oxidized to benzoquinone (BQ) at the modified substrate surface where sequence-specific hybridization had occurred through the HRP-catalyzed reaction in the presence of H2O2. The detection was based on the reduction of BQ generated on the modified substrate by SECM tip. The detection limit of complementary DNAwas as low as0.8pM. This technique is promising for the application on electrochemical DNA chip.Chapter5Scanning Electrochemical Microscopy of DNA Hybridization on DNA Microarrays Enhanced by HRP-Modified SiO2NanoparticlesIn this chapter, based on the sandwich structured DNA sensor, we reported a DNA microarray method. With this DNA microarray, a number of genes could be detected simultaneously and selectively enough to discriminate between complementary sequences and those containing base mismatches. DNA arrays were fabricated by robotic printing of capture DNA probes onto a glass substrate. The capture probe (CP) sequences were amine-tethered oligodeoxynucleotides that could be immobilized on the aldehyde-covered glass substrate through the amine-aldehyde interaction. Passivation of the tip during large scale scanning was avoided by adding surfactant to the solution, which keeps the tip clean and the background of the tip current was stable during scanning. The DNA targets at prepared spots could be imaged in SECM GC mode over a wide concentration range (10-7-10-12M). This technique may find applications in genomic sequencing.Chapter6Detection of Human IgG using GC mode of SECMIn this chapter, sheep-anti-human IgG was directly dropped on the glass substrate, then reacted with human IgG and Indicating element, which formed by rabbit-anti-human IgG-HRP wrapped SiO2nanoparticles, forming a sandwich-typed immunocomplex. In this SECM immune sensor, detecting mode is still generation/collection. Hydroquinone (H2Q) was oxidized to benzoquinone (BQ) through the HRP-catalyzed reaction in the presence of H2O2. The detection was based on the reduction of BQ generated on the modified substrate by SECM tip. The detection limit of human IgG was as low as10pg/L.

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