Induced Formaldehyde Dehydrogenase In Escherichia Coli For Biosensor Research

As formaldehyde is the substance that can lead to deformlty and cancer, the world pays more and more attention to it. People get muted with long-term contact to it. So it is important to find a way for rapid and sensitive detection. Formaldehyde dehydrogenase (FADH) can catalyze formaldehyde with high specificity, while the electrochemical test can convert weak biological signal to strong one. So it can be a new way to detect formaldehyde fast and accurately if we combine formaldehyde dehydrogenase and electrochemical test method together.In order to fix formaldehyde dehydrogenase to the surface of the glassy carbon electrode more effectively, we optimized conditions for inducing E.coli and the methods for purifying formaldehyde dehydrogenase preliminary. We also attempt to chose different materials to modify electrode, as well as test the way to fix enzyme to the surface of the glassy carbon.In this study, we chose three kinds of E.coli (DH5a, JM109, BL21) as the source of formaldehyde dehydrogenase. The results show that DH5a, compare to the others, has a higher efficiency for producing enzyme. After cultured 12 hours, DH5a can produce more enzyme at 6ppm formaldehyde concentration in 1hour inducing. The enzyme activity can reach to 58.926U/ml with 1ml bacterial suspension after treated by crushing. In the process of purification of formaldehyde dehydrogenase, ultrasonication method is more effective than enzymatic hydrolysis and combination of the two. As to the process of purifying the crude product, we used salting-out method and isoelectric point precipitation method. By comparison, when the concentration of ammonium sulfate is 45%, the increments of the purified enzyme at 340nm is 3.165; while at the isoelectric point the increments is 3.425, indicating that the isoelectric point method is better than salting-out method.When testing enzyme electrodes, compared the CNT/PPY/GCE electrode with PPY/GCE electrode in the aspact of electrochemical test response and enzyme-fixed of electrochemical test response,the CNT/PPY/GCE modified electrode is better. In the aspect of fixing enzymes, we chose electrostatic self-assembly method and embedding method, the former one is better in electrochemical response, and its stability is good.In this study, we chose polypyrrole for modifying glassy carbon electrodes, with electrostatic self-assembly method used single-walled carbon nanotubes, the final layers for modifying is three. The standard curve equation of the biosensor is:Y=0.3717X+4.5169, the correlation coefficient R2 is 0.9983. The optimal pH value is 7.5. The relative error is weak. The detection range is between 0.1991ppm and 300ppm,with better anti-jamming performance and stability The deadline for an accurate test results is 15days.