Build And Electron Transfer Kinetics Sugar Novel Electrochemical Sensor

As one of three important biological molecules in life, carbohydrates involve in some biological and pathological process, such as signal transduction, cell adhesion, cancer metastasis, as well as the main source of metabolic energy required by organisms. The detection of carbohydrates provides a new novel promising method for early diagnosis of diseases, drug screening, and glycomics research. Therefore, it is urgent needed for the development of a high sensitivity, high selectivity method for the detection of saccharides. Electrochemical biosensor is a kind of analysis device based on specific molecular recognition materials, such as enzyme, antigen/antibody, DNA, sugar, and export electrochemical signals for detection signal. Electrochemical biosensor has received much attention in life sciences, environmental analysis and drug analysis areas due to its unique advantages, such as good selectivity, high sensitivity, low detection limit, small analyte volume, simple instrumentation, and minimal manipulation Electrochemical biosensors are divided into labeled and label-free biosensors. Label-free electrochemical biosensor is on baisi of the variation of the electrochemical signal before and after interaction between the biosensor and target substance with the advantage of simplicity. Labeled electrochemical biosensor is based on the change of electrochemical signal from signal probe labled by electro-active substances. Labeled electrochemical biosensors have attracted much attention because of its high sensitivity. The electron transfer of electric-active substances in the electrode surface is the basis of the electrochemistry in labeled electrochemical biosensor.The aim of the present work is to develop simple, sensitive, novel electrochemical glycan sensors. Taking advantages of the sensitivity of electrochemistry and the specificity of biological molecular recognition substances, a label-free electrochemical saccharide sensor and an ampetrometric glycan biosensor array based on dual signal amplification of gold nanoparticles and enzyme was proposed incorparation of phenylboric acid, lectin as molecular recognition element to detect glucose, lactose, mannan, carcinoembryonic antigen. Moreover, the lectin-based electrochemical biosensor array fabricated with different lectins was used to evaluate the glycan expression of CEA N-glycan and discriminate CEA between healthy and cancer patients serum samples. Additionally, the effect of conformation of aptamer ss-DNA on the electron transfer of ferrocence labeled onto aptamer ss-DNA was examined to reveal the effect of biosensing signal transduction, identifying reaction and response mode on the sensor sensitivity and selectivity.This thesis consists of two parts:First part is general introduction, while second part is research report.Chapter1. General introduction. In this chapter, the definition, classification and biological significance of carbohydrates were briefly introduced. And then the glycan biosensor and classification were described. The developments of glycan electrochemical biosensors were reviewed in detail. Theory of electrochemical electron transfer was described and finally the purpose and significance of this research work was presented.Chapter2. A novel electrochemical impedance spectroscopy sensor for the determination of saccharide using phenylboronic acid as molecular recognition element. Phenylboronic acid was immobilized on gold surfaces by means of self-assembly technique using cysteamine as cross-link to fabricate saccharide biosensor. Phenylboronic acid can react with different saccharides, resulting in an increase of the electron transfer resistance of the biosensor. Under the optimized conditions, the electrochemical impedance response was linear with the glucose concentration from5.05×10-9mol·L-1to5.05×10-5mol·L-1with the detection limit of9.7×10-10mol·L-1(S/N=3); the electrochemical impedance response was linear with the lactose concentration from2.78×10-9mol·L-1to2.78×10-5mol·L-1with a detection limit of8.1×10-10mol·L-1(S/N=3); the electrochemical impedance response was linear with the logarithm of the mannan concentration from4.35×10-9mol·L-1to4.35×10-5mol·L-1with the detection limit of3.4×10-10mol·L-1(S/N=3). This biosensor would be used as a simple, sensitive and inexpensive tool for detection of saccharide.Chapter3. A sensitive electrochemical glycan biosensor array for the detection and discrimination of carcinoembryonic antigen based on dual signal amplification of gold nanoparticles and enzyme. The electrochemical biosensor was fabricated by self-assembly and covalent binding of lectin onto the surface of the gold nanoparticle-modified screen-printed carbon electrodes (SPCE). Carcinoembryonic antigen (CEA) was chosen as model target and horseradish peroxidase was used as enzyme label. The immunoassay in sandwich model was performed by specifically binding the targets, HRP labeled antibody to the immunosensor. With high specific surface area of gold nanoparticles, a sensitive sandwich electrochemical immunoassay was developed for the determination of CEA. Under the optimized experimental conditions, the catalytic current response was linear with the CEA concentration from1.0ng/mL to10ng/mL with the detection limit of0.3ng/mL and0.1ng/mL used ConA and WGA as recognition molecular respectively; the catalytic current response was linear with the CEA concentration from0.5ng/mL to10ng/mL with the detection limit of0.05ng/mL used LCA as recognition molecular. Moreover, the lectin-based biosensor array fabricated with different lectins was used to evaluate the glycan expression of CEA N-glycan and discriminate CEA between healthy and cancer patients serum samples. This work demonstrates that the employment of gold nanoparticle as immobilization platform and lectin as molecular recognition element in biosensor array is promising approach for the determination and discrimination of glycoproteins for cancer diagnosis.Chapter4. Effect of conformation of aptamer ss-DNA on the electron transfer of ferrocence labeled onto aptamer ss-DNA. The effect of conformation of aptamer ss-DNA on the electron transfer of ferrocence labeled onto aptamer ss-DNA was examined to reveal the effect of biosensing signal transduction, identifying reaction and response mode on the sensor sensitivity and selectivity. Ferrocene-labled aptamer ss-DNA was modified onto gold electrode via self-assembly. Electron transfer kinetics behavior of ferrocene in different states and different base lengths self-assembled monolayers were examined. It was found that the electron transfer kinetics increased after self-assembly monolayer blocked by DTT. The electron transfer rate also increased after interacted with cocaine and decreased with hybridized with fully complementary DNA strands combined. Aptamer chains consisting of, the ferrocene on the electrode surface exhibited similar electrochemical behavior, the heterogeneous reaction rate constant is similar when the base of aptamer ss-DNA is32and52bases.In this thesis, we constructed one electrochemical label-free sugar sensor and one electrochemical glycan biosensor array by employing phenylboric acid and lectin as moleculer recognition and to AC impedance and amperometric as detection technology. Sensitive electrochemical methods were developed to detect glucose, lactose, mannan and carcinoembryonic antigen. Electrochemical electron transfer behavior of ferrocene on different electrode surfaces was examined using cyclic voltammograms technology. This work provides novel strategy for the fabrication of the electrochemical sugar biosensor, provides promising analytical devices for the early diagnosis of the clinical disease, and also provides a certain understanding of biosensing signal transduction, recognition reaction and response mode. The achievements in this work will promote the development of electrochemical biosensor and their applications in analytical, clinical, material and biologicial field.