| DNA research is a vitally important aspect in life science. In which, simple, quick and accurate determination of special sequence DNA has taken a very important role in the areas of clinical diagnosis, forensic identification, epidemic prevention, environmental monitoring and bioengineering, espacialy for the early diagnosis and treatment of disease. Among the kinds of developing gene analysis technologies, DNA sensor, which is based on base-pair rule, has been attached much more importance. As one of the DNA biosensor, electrochemical DNA detection is a novel and developing technique that combining electrochemical, biochemical, medical and electronic techniques. With the advantages of being cheap, rapid, simple, reliable, convenient, sensitive and selective for genetic detection, electrochemical DNA detection, which was considered the most promising DNA analysis, has received considerable attentions and has been a hot topic in the field of biochemistry and medicine.Carbon nanotubes (CNTs) are a new type of carbon nanostructure material that possess outstanding properties, such as high electrical conductivity, large surface area, good chemical stability and significant mechanical strength. Owing to their unique structure and physical properties, CNTs have been extensively studied and are expected to attract more attention in the future for numerous potential applications. A special class of CNTs, aligned carbon nanotubes (ACNTs), offering many important additional advantages over conventional electrode materials and normal tangled CNTs, including a larger surface area and high electrical conductivity. Therefore, ACNTs have been developed as one ideal electrode material for biosensors. Because of the special large surface area, more DNA chains and protein molecular could be immobilized onto ACNTs. Moreover, modification provided ACNTs the ability of chemical attachment of some biological molecules, which could improve the stability. Therefore, it possesses promoting potential in electrochemical analysis, especially in life science.Supramolecular chemistry is one of chemistry science focuses on supramolecular which aggregate through supramolecular noncovalent interaction between the molecules. Combing with the biological science, material science, information technology and nanometer technology, it has developed to be supramolecular science with wide range of application. The molecular recognition technology, also called host-guest recognition technology, defined as the supramolecular noncovalent interaction between the "host" and "guest" molecules, has played an important role in the chemical sensing field. Host-guest recognition is the selective inclusion complexation between host and guest, in which some special functions can be realized. It is the base of biological recognition, such as enzyme catalysis, nucleic acid duplicate, antigen and antibody, drug and target molecule interaction. Cyclodextrin (CDs) and cucurbit, as two typical hosts, have received considerable attention because of their particular characterization, and the studies on the host-guest interaction based on cyclodextrin and cucurbit had been transferred from the processes and mechanism of inclusion complex between a pair of host and guest to the. application in the fields such as analysis, medicine, environment protection and sensors.In this paper, we have fabricated some electrochemistry and electrochemical luminescence (ECL) biosensors using ACNTs which was prepared by chemical vapor deposition (CVD) in our laboratory, and developed some non-immobilizing electrochemical DNA biosensors with homogenous hybridization based on the host-guest recognition technology. These biosensors were as follows. A nonenzyme H2O2 sensor immobilized DNA-Cu2+ composite onto ACNTs electrode by electrodeposition; An ACNTs electrochemistry DNA biosensors based on amplification of enzyme using double modification of ACNTs by immobilized DNA molecular and absorbed electronic media; An lable-free ECL aptamer biosensors based on CdS QDs/ACNTs electrode for detection of thrombin; Two types of non-immobilizing electrochemical DNA biosensors employed CDs and ACNTs to detect DNA hybridization in homogeneous solution based on host-guest recognition; An non-immobilizing DNA biosensors using cucurbit as host molecular to detect DNA hybridization in homogeneous solution based on host-guest recognition. These sensors have characteristics of good specificity, high sensitivity, good stability and easy preparation, have been successfully applied to detect the specific DNA sequences and sensing target protein with specificity. The study provided a new idea with simple, convenient and cheap for determination of gene and protein.The thesis consists of seven chapters.Chapter 1:PrefaceIn the first part, we introduce the aligned carbon nanotubes array and its application, including the classification and properties of carbon nanotubes, the preparation methods of ACNTs and the application of ACNTs in biosensors field. Then, we introduce the general situation and research status of electrochemical sensors, in which the principle, the classification and the research status of electrochemical DNA sensors are highlighted. After that, were introduc the principle and application of electrochemiluminescence (ECL) biosensors, including the research status of ECL luminophores, ECL mechanisms and ECL aptamer biosensors. In the last part, we illustrate the technology of host-guest recognition and its application in biosensors. Based on the above introduction, we point out the purpose and signification of the thesis.Chapter 2:A Sensitive Nonenzymatic H2O2 Sensor Based on DNA-Cu2+ Complex Electrodeposition onto Aligned Carbon Nanotubes ElectrodeWe prepared aligned carbon nanotubes (ACNTs) electrode, then fabricated a new nonenzyme H2O2 sensor. ACNTs were fabricated using a thermal chemical vapor deposition system (CVD) and then were prepared to be ACNTs electrode. After that, DNA-Cu2+ complex was electrodeposited onto the surface of ACNTs electrode, which fabricated a DNA-Cu2+/ACNTs electrode sensor to detect H2O2 with nonenzyme. Cyclic voltammogram of DNA-Cu2+/ACNTs electrode showed a pair of well-defined redox peaks for Cu2+/Cu+. Moreover, the electrodeposited DNA-Cu2+ complex exhibited excellent electrocatalytic behavior and good stability for the detection of H2O2. The effects of Cu2+ concentration, electrodeposition time and determination conditions such as pH value, applied potential on the current response of the DNA-Cu2+/ACNTs electrode toward H2O2 were optimized to obtain the maximal sensitivity. The linear range for the detection of H2O2 is 2.0×10-7 M to 2.0×10-3 M with a high sensitivity of -46.46μA mM-1, a low detection limit of 8.0×10-8 M and a fast response time of within 4 s. In addition, the sensor has good reproducibility and long-term stability and is interference free.Chapter 3:Aligned Carbon Nanotubes Electrochemistry DNA Biosensors Based on Amplification of EnzymeAligned carbon nanotubes biosensors based on a horseradish peroxidase enzyme label and a thionine mediator were fully characterized and further used for amplified electrochemical detection of specific DNA sequences of human SARS virus (SARS DNA). For this purpose, aligned carbon nanotubes array was prepared by thermal chemical vapor deposition system and a thin layer of Au was then sputtered coating onto the ACNTs array. First of all, the 'capture probe' was immobilized on the ACNTs electrode with a 5'-end thiol group by self-assembly. Then, after hybridization with target DNA and a complementary HPR-labeled detection probe, the HRP-labeled oligonucleotide hybrids were immobilized onto the sensing surface. After that, thionin, as a redox mediator of horseradish peroxidase enzyme was absorbed to the ACNTs electrode through strongπ-πstocking force. In the last place, the proposed enzyme-based ACNTs biosensor shows catalytic activity toward H2O2 reduction and applied to the detection of target DNA. Cyclic voltammetric and amperometric were implored to study the characterization of the enzyme-based ACNTs biosensor. Under the optimal conditions, a wide linear range of propose biosensor for the detection of DNA was observed from 1.0×10-12 M to 1.0×10-9 M, with a detection limit of 1.0×10-13 M. Furthermore, the proposed biosensor displayed rapid response, high stability, very good specificity and high sensitivity for the detection of DNA. Chapter 4:Electrochemiluminescence Aptamer Biosensor for Detection of Thrombin Based on CdS QDs/ACNTs ElectrodeA label-free ECL aptamer biosensor based on CdS QDs/ACNTs electrode for the sensitive detection of thrombin was fabricated. CdS QDs and Chitosan (CTS) complex films were coated and filled the tubes of ACNTs by carrying out electrodeposition reaction of CTS-CdS QDs on ACNTs electrode, the resulting electrode showed high ECL intensity and good biocompatibility. After aptamer was bound to the film via glutaric dialdehyde, the modified electrode could be used as an ECL aptamer sensor for the thrombin detection. The specific reaction between thrombin and aptamer resulted in the decrease in ECL intensity. The change of the ECL intensity was found to be linear with the logarithm of thrombin concentration in the range from 1.0×10-13 M to 1.0×10-9 M. The ECL aptamer sensor had the advantages of speed, high sensitivity, specificity and stability, which could be a promising technique for protein detection.Chapter 5:A Non-immobilizing Electrochemical DNA Biosensor with Homogenous Hybridization Based on Host-guest Recognition TechnologyWe electrochemically modified ACNTs withβ-cyclodextrin ((3-CD). Subsequently, we employed the polyβ-CD/ACNTs as an electrochemical DNA sensor to detect DNA hybridization in homogeneous solution based on host-guest recognition. For fulfilling the homogeneous phase-based hybridization,4-((4-(Dimethylamino) phenyl) azo) benzoic acid (dabcyl) labeled DNA probe was mixed with the target DNA to form dsDNA hybridizer. When the polyβ-CD/ACNTs electrode was immerged in the dabcyl-labeled DNA probe or the dsDNA hybridizer, the ssDNA or the dsDNA hybridizer was captured onto the polyβ-CD/ACNTs electrode by the force of the host-guest recognition between CD and dabcyl. After that, an intercalator was used as an indicator to detect DNA hybridization. At the same time, several capture electrodes and intercalators were selected in this study. We employed polyβ-CD/ACNTs electrode as capture electrode and the daunomycin as intercalator, under optimized detection conditions, the proposed method gives a high sensitivity with detection limit of 6.0×10-13 M, and also a high selectivity for sequence-specific DNA analysis.Chapter 6:The Application ofβ-cyclodextrin Derivative Functionalized Aligned Carbon Nanotubes for Electrochemically DNA Sensing via Host-guest RecognitionWe functionalized ACNTs with a new kind ofβ-CD derivative through diazotization reaction. The resultingβ-CD/ACNTs electrode was used to detect DNA hybridization in homogeneous solution based on host-guest recognition technology. In the sensing protocol, one special DNA probe was designed with a stem-loop structure and both ends modified, which we called dually-labeled DNA probe (DLP). One end of the DLP was labeled with dabcyl as guest molecule forβ-CD/ACNTs electrode capture, and the other end was labeled with a CdS nanoparticle as an electrochemical tag to indicate the occurrence of DNA hybridization. In the absence of the target DNA sequence, the DLP maintains its hairpin structure in solution phase and would not be captured and detected by theβ-CD/ACNTs electrode. In the presence of the complementary target sequence, the conformational structure of the DLP was altered and a double-stranded DNA (dsDNA) molecule was formed by the hybridization of DLP and complementary DNA sequence. Consequently, the dsDNA was captured by theβ-CD/ACNTs electrode owing to guest-host recognition betweenβ-CD and dabcyl. The electrochemical signal from the CdS nanoparticle-dsDNA/β-CD/ACNTs was then measured. Under optimized detection conditions, the proposed method showed high sensitivity and selectivity with a detection limit of 5.0×10-13 M for complementary DNA sequence.Chapter 7:Study on DNA Biosensor Used Cucurbit as Host Molecular Based on Host-guest Recognition TechnologyWe prepared a new cucurbit [7] modified multiwalled carbon nanotube (Q7/MWNTs) electrode. Subsequently, we employed the Q7/MWNTs as an electrochemical DNA sensor to detect DNA hybridization in homogeneous solution based on host-guest recognition. DLP was employed, which was designed with dabcyl labeled at one end as a guest molecule, and with CdTe QDs labeled at the other end as electrochemical tag to indicate the hybridization occurrence. Before the hybridization, the DLP remained in the stem-loop structure, which forced the dabcyl molecular to be closed to the CdTe QDs. Due to the steric effect of the CdTe QDs, the dabcyl was prevented from conjugating with the Q7 on the Q7/MWNTs electrode and resulting in that the DLP could not be captured by the Q7/MWNTs electrode. After hybridized with the target DNA, the target-binding DLP caused the DLP's loop-stem structure opened and then the dabcyl molecule was easily entering the cavity of the Q7/MWNTs electrode and resulting in that the DLP could be captured by the Q7/MWNTs electrode. Therefore, the target hybridization event can be sensitively transduced via detecting the electrochemical current signal of CdTe QDs. Under optimized detection conditions, the proposed method showed high sensitivity and selectivity with a detection limit of 7.0×10-13 M for complementary DNA sequence.
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