Basic Research On Application Of Microfluidic Electrochemical Immunosensor Chip Basing On4-(3-pyrrolyl) Butyric Acid

Conducting polymers, as a new-type high molecular material, have been attracting greatattentions especially in the field of biosensors for their excellent electrical, optical,mechanical properties. Due to their diversity and easy preparation, conducting polymersdramatically enrich the immobilization methods of the biomolecules, which is quite essentialto enhance the detection sensitivity and specificity of biosensors. Currently, the developmenttendency of biosensor is to combine with microfluidic technology (a powerful tool ofcontrolling fluid in micro-/nano-scales), in which way it can benefit the integration ofbiological analysis system so as to achieve on-line detection. Moreover, there exists inherentmatching between microfluidics and electrochemical biosensors. In conclusion, all these canprovide theoretical and practical basis for the construction of microfluidic electrochemicalimmunosensing chip.This study was aimed to construct a microfluidic electrochemical immunosensing chipbased on the functional conducting polymers. Firstly, a conducting polymer composite film of4-(3-pyrrolyl) butyric acid and pyrrole was fabricated by electrochemical oxidation and thenthe properties of polymer composite were evaluated by various electrochemical methods, suchas cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Meanwhile,the Fourier transformed infrared spectroscopy and Raman spectroscopy was used to identifythe functional groups of the composite film. Atomic force microscope (AFM) and the contactangle measurement were conducted to observe the surface structure and the hydrophilicity ofthe polymer composites respectively.Secondly, an immunosensor using the above-mentioned polymer composite film as probeimmobilization matrix was constructed and the fabrication processes were as follows:Streptavidin, which was covalently anchored on the polymer composite via EDC/NHS, wasused to immobilize the protein probe（biotin conjugated first antibody）, and then a “sandwich”immunosensor was constructed through the interaction between the antibody and antigen.With the help of alkaline phosphatase conjugated on second antibody, a substrate was converted to electro-active substance, which can be detected by the electrochemical method.CV and AFM were taken to confirm the immobilization of proteins on the polymer film anddifferential pulsevoltammetry was used for detection of the concentration of protein.Furthermore, polyvinyl alcohol (PVA) was employed to enhance the hydrophilicity of thepolymer surface, leading to decrease in the nonspecific protein adsorption, which wouldimprove the sensitivity of the immunosensor. Theresults show that after PVA treatment, anexcellent linear relationship (R2=0.997) was observed in the concentration range from7ng/mlto20μg/ml with the detection limit of7ng/ml (S/N≥3).Finally, based on the successful construction of immunosensor, a microfluidic chip wasfabricated and used for the detection of protein. Herein, Au film was coated on the glasssurface by the magnetron sputtering and then the microelectrode was obtained by lift-offtechnology. At the same time, we employed soft lithography to prepare PDMS microchanneland the whole chip was sealed via oxygen plasma treatment. Thereafter, the syringe pumpwas used to inject solution into the channel for the detection of mouse IgG. The difference inthe detection signals between the chip and the gold electrode and the influence of pumpingrate for the detection of mouse IgG were discussed.