Studies On The Detection Of Small Molecule And Protein Using Electrochemical Biosensors

Electrochemical biosensors have wide applications in chemistry, biomedicine,environmental mornitoring, food industry, medicine and military because of theirexcellent selectivity, high sensitivity, rapid response, low cost, simplicity ofinstrumentation, continuous detection in complex system, easy to be miniaturized.Thisdissertation focuses on developing a series of electrochemical biosensing techniquesfor the detection of ochratoxin A, β-indole acetic acid, TATA binding protein. Thedetailed materials are shown as follows:1. A novel, highly sensitive electrochemical immunosensor has been proposed forthe determination of ochratoxin A (OTA), a common toxic contaminant in agriculturalproducts (in chapter2). A gold colloid layer was firstly formed on the surface of theelectrode via a self-assembled monolayer of HDT. So the OTA-OVA conjugate couldbe adsorbed directly on the gold colloid layer. Then, with the addition of a fixedconcentration of mouse anti-OTA antibody together with the OTA samples,competition for the binding sites of limited antibody molecules was realized betweenthe immobilized OTA on the surface and the free OTA in the solution, therefore, theamount of antibody bound on the interface through antigen-antibody interaction woulddecrease with increasing OTA concentration in the solution. Electrochemical responsearising from the oxidation of enzymatic product of1-naphthyl phosphate was observedto be inversely proportional to OTA concentration in the range from10pg/mL to100ng/mL with a detection limit as low as8.2pg/mL.2. An electrochemical immunosensor based on an indirect competitive assayformat was developed for the determination ofβ-indole acetic acid (in chapter3).phytohormone indole-3-acetic acid (IAA) is one of the most important native auxinsthat can induce cell elongation and division with all subsequent results for plantgrowth and development. Therefore, phytohormone research is significant toagriculture, horticulture and other related fields. The traditional detection methodsincluding HPLC, GC, and CE, possess some shortcomings, such as requiringexpensive apparatus, complicated processing systems and time-consuming. In thisthesis, the immobilized hapten and IAA analyte in sample solution competed for thelimited anti-IAA mouse monoclonal antibody, therefore, the amount of antibody bound on the interface through antigen-antibody interaction would decrease with increasingIAA concentration in the solution. At the optimal conditions, the working range of theelectrochemical immunosensor was from300ng/mL to3mg/mL.3. A simple, sensitive biosensors for the detection of human TATA binding protein(TBP) was developed using electrochemical impedance spectroscopy (EIS)measurement (in chapter4). In this thesis, a designed double strand DNA specificallybound to the hybrid DNA (TBP-DNA) was firstly immobilized on the gold electrodeand then TBP solution was added to the modified interface of the electrode for thespecific combination with TBP-DNA after blocking and washing. Finally, anelectrochemical biosensor for TBP was prepared based on the observation that electrontransfer resistance (Ret) correlated with the changing concentration of TBP due to thealtered surface charge of the electrode caused by the specific binding. A low detectionlimit of4.2ng/ml was achieved under optimal conditions.