Concanavalin Electrochemical Impedance Sensor

Three kinds of most important biological molecules include saccharides, protein and nucleic acid, concerning the essence of living activities. Glycomics has also increasingly attracted our attention besides the development of genomics and proteomics. Saccharides is not only simple energy substance, but important informational molecules. It participates in many pathological and physiological processes. Serious diseases and their treatment have close connection with saccharides which plays a significant role in the research fields such as life science, biochemistry and medical science. The sugar of the cell surface play key roles in recognizing normal cells, cell adhesion, signal transmission among cells and in the research of cytopathy pathogen infection. The studies on saccharides will be a major concern of future bioscience. It can be said that test of protein and saccharides can provide new tech and major studies method of life sciences such as early diagnosis of diseases, drug screening and glycomics. The testing plays significant roles in establishing high sensitive and high selective methods for the detection of the saccharides or proteins. Electrochemical biosensors have many advantages such as high sensitivity, fast response, simplicity, cheaper apparatus, therefore, they were widely used in the research of the biomolecules. The research of the biosensor of sugar has been a focus after electrochemical immunosensor and DNA electrochemical sensor.The research of sugar biosensors centers on the immobilization methods which can increase detective sensitivity and develop new testing tech. At present, most literatures of sugar biosensors deal with chemical modification of sugar chains (animation or sulphur) and purification before the sugar coupled to solid substrate, this methods is complicated with a high cost but difficult to practice. Moreover, many problems exist in the process of chemical modification such as stereo configuration, this weakness became the restrictive factors of the development of sugar biosensors. Thus it is a potential preparation method by covalent bonding without modification sugar molecules to the substrate. So a challenge arises when we try directionally fixing sugar on condition that the maintenance of the function and natural construction of sugar.The aim of this thesis is to develop a simple and practical sugar immobilization method. The article doesn't chemically modify the natural sugar substance which directly to immobilize on substrate surface and establishes a method which has highly sensitive and selective testing of ConA. It is promised in the application between bioanalysis and clinical test. It is of great significance in the development of life sciences and biosensors.This thesis consists of two parts. First part, it involves general introduction the concepts of saccharide and lectin, biological significance, the specificity recognition principle of sugar-lectin and immobilization method. The research developments of electrochemical sugar biosensor were reviewed, and finally the purpose and significance of this research work is presented, while the second part including two chapters presents research work.In the first part of research work, we investigated the detection of Con A based on carboxyl links D-mannose. We designed label-free electrochemical impedance spectroscopy (EIS) biosensor for the determination of Con A, based the change of the electron transfer resistance of the interactions of monosaccharide (D-mannose) and Con A. The Con A biosensor was fabricated by covalently immobilizing D-mannose on the surface of the gold electrode modified with 11-Mercapto-undecanoic acid, researching the interactions of D-mannose and Con A. The results of experiment shows that the change of the electron transfer resistance of the biosensor is linear with the concentration logarithm of Con A in the range from 2.0×10-10 to 2.0×10-7 mol·L-1. The linear regression equation wasΔRet (kΩ)=119.3 1g c+121.8 (c is concentration of Con A, the unit is mol·L-1, r=0.9938). The detection limit for ConA was 6.7×10-11 mol·L-1(S/N=3).The sensor has many advantageous such as simply, rapid, label-free and high sensitivity.In the second part of research work, we designed the detection of Con A based on amino links D-mannose. The Con A biosensor was fabricated by covalently binding D-mannose to cysteamine self-assembled on the surface of the gold electrode, using [Fe(CN)6]3-/4- as a redox probe to detect Con A. Optimized self-assembled time, the immobilization time and interaction time was 4.5 h,12 h and 1 h. Under optimum conditions, according to the increasing of electron transfer resistance of [Fe(CN)6]3-/4- after D-mannose binding with Con A realized the quantitative detection of ConcanavalinA.The results demonstrated that the change of the electron transfer resistance of the biosensor linearly increased with the concentration logarithm of ConA in the range from 2.0×10-9 to 2.0×10-6 mol·L-1.The linear regression equation wasΔRet (kΩ)=32.04 1g c+312.5(c is concentration of Con A, the unit is mol·L-1, r=0.9931). The detection limit for Con A was 6.7×10-10 mol·L-1(S/N=3). It has some reference value to the research on the detection method of Concanavalin A, applicable to the detection of the sample, the diagnosis and treatment of diseases, and significant to clinical and pharmacological studies.