Affinity Electrochemcial Biosensor For Peptide And Short DNA Strand

Biosensor is a kind of equipment consisting of the bioactive materials,the transducer and the signal analysis device. According to thebiorecognition between the recognition molecules and the target molecules,the biosensor is devided into catalytic biosensor and affinity-basedbiosensor. Owing to its advantages of fast response, easy to use, cheapnessand miniaturization, the affinity electrochemical biosensor is well widelyaccepted. In recent years, with the development of the molecular biology,materials science and analytical chemistry, the electrochemical sensor hasbeen developed rapidly in various areas of research. It can rapidly andaccurately detect a large number of substances, including cells, bacteria,nucleic acids, polypeptide, et al. And the affinity-based biosensor is anactive study in the clinical laboratory diagnostics. In our work, a rapid,simple, and sensitive method for detection of peptides and short DNAsequences based on electrochemical sensor was developed, which wouldbecome a powerful tool for clinical diagnostics, and environmentalmonitoring. The study has two parts as follow:1. A Novel Electrochemical Immunosensor for Angiotensin II Detection based on the Glass Carbon Electrode Modified by CarbonNanotubes/Chitosan FilmAng II is the important peptide of the RAS and plays important roles inthe control of body fluid balance, blood pressure regulation and electrolytehomeostasis. Ang II can bind the type1angiotensin receptor (AT1R), andclosely involve in the regulation of angiogenesis, cell proliferation, cancerand inflammation. So it is a need to detect Ang II for studying the differentactions of Ang II in various systems. Now, a lot of methods are developedfor Ang II detection, for example epitope-immunoassay, high performanceliquid chromatography and radio immunoassay. These assays are widelyused, but they are needing complex and long-time work, requiring high-endinstrument and costliness. Therefore, developing a simple, cheap and fastmethod is a challenge for the direct detection of Ang II.In the assay, we successfully developed a new electrochemicalcompetitive immunosensor for Ang II detection based on the glass carbonelectrode (GCE), which was modified with the single-walled carbonnanotubes/chitosan (SWNTs/CS) nanocomposite film. In the constructionof electrochemical sensor, Ang II was firstly covalently immobilized onSWNTs/CS modified GCE. Then, the mixture solution of target Ang II andBio-Anti-Ang II antibody was dropped on the modified GCE and incubated.After the reaction, ST-AP, which can catalyze the hydrolysis of α-NP in thedetection solution, was captured on the biosensor surface. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) wereused to characterize the every assemble step of the sensor. Under theoptimal assay conditions, the electrochemical biosensor had a specificresponse to the target Ang II. The immunosensor had a wide linear rangefrom0.05to10μg/mL, and the detection limit was0.03μg/mL. Theimmnunosensor presented good specificity and reproducibility, and it wasused to detect Ang II in sample serum. This novel immunosensor methodwas firstly applied to detect Ang II, which might be a potential method forassay of Ang II for clinical diagnosis and treatment in the future.2. Ultrasensitive electrochemical DNA biosensor based onmolecular beacon-mediated circular strand displacement and rollingcircle amplificationHighly sensitive and selective detection of sequence-specific DNAplays essential roles in clinical diagnosis and therapy, pathogen detectionand environmental monitoring. A novel electrochemical biosensing strategywas developed for ultrasensitive target DNA detection using a cascade signalamplification based on molecular beacon (MB) mediated circular isothermalstrand displacement amplification (CISDA), rolling circle amplification(RCA), biotin-strepavidin system and enzymatic amplification. The targetDNA hybridized with the loop portion of MB probe immobilized on the goldelectrode and triggered the CISDA, leading to multiple biotin-tagged DNAduplex. Furthermore, via biotin-streptavidin interaction, the RCA was implemented, producing a lot of long massive tandem-repeat DNAsequences for binding numerous biotinylated detection probes, whichenabled an ultrasensitive electrochemical readout by further employing thestreptavidin-alkaline phosphatase. The proposed biosensor showed veryhigh sensitivity and selectivity with a dynamic linear range from1fM to100pM, and a detect limit as low as0.4fM. The established strategy could havethe potential for applying in clinical molecular diagnostics, pathogendetection and environmental monitoring.