The Fabrication Of Cytochrome P450 Biosensors And Its Application In The Detection Of Signaling Biomolecules

Biosensors are widely used in fields such biomedical analysis,environmental monitoring and food qulity and safty.In this thesis,cytochrome P450 55B1 and cytochrome P450 707A3 are prepared by prokaryotic expression and used to construct two electrochemical biosensors.And new methods are established for the detection of signaling biomeloculars including nitric oxide and abscisic acid.The thesis mainly includes the following three parts:1.Cytochrome P450 55B1(CYP55B1)from Chlamydomonas reinhardtii was expressed in E.coli and purified by Ni column.The prepared CYP55B1was immobilized onto the pyrolytic graphite electrode by glutaraldehyde with its strong crosslinking ability.CYP55B1 was realized the direct electrochmeistry with the anodic and cathodic peak potential of-0.35 V and-0.38V under the scan rate of 0.5V/s,respectively.CYP55B1 can catalyze the reduction of nitric oxide to nitrous oxide with the peak potential of-0.85V.The reduction peak current was used to the electrochemical detection of nitric oxide.The linear range of nitric oxide is 14.4?108?mol�L^-1with the detection limit(LOD)10.47?mol�L^-1(S/N=3)and the linear regression equation y=0.01696x 0.0344(R2=0.9766).The biosensor has high specificity for NO and can avoid interference from other nitrogen-containing analogues.2.Cytochrome P450 707A3(CYP707A3)from Arabidopsis was prokaryotic expressed and purified by Ni column.Dihexadecylphosphate(DHP)can form bilayer lipid membrane.CYP707A3 was immobilized onto the pyrolytic graphite electrode by DHP.CYP707A3 was realized the direct electrochmeistry with the anodic and cathodic peak potential of-0.50V and-0.58V under the scan rate of 0.5 V/s,respectively.CYP707A3 can catalyze the oxidation of abscisic acid(ABA)with the peak potential of-0.58 V.The peak current was used to the electrochemical detection of ABA.The linear range of ABA detection by the biosensor is 0-200nmol�L^-1with the detection limit(LOD)4.85nmol�L^-1 and the linear regression equation is:y=0.1041x-0.4860(R²=0.9757).The biosensor has good specificity and does not respond to common plant hormones such as auxin and cytokinin.3.The interaction between abscisic acid and CYP707A3 was studied by fluorescence spectrometry.Studies show that abscisic acid quenches the fluorescence signal of iron porphyrin in CYP707A3 through a static quenching method.The bingding constant was calculated to be 6.26�10⁴L/mol at 25?and the bingding sites was to be one.The binding forces between ABA and CYP707A3 are mainly hydrogen bond and Van der Waals force.The interaction between ABA and iron porphyrin in CYP707A3 results in the dynamic quenching of fluorescence intensity of Trp and Tyr amino acid residues and the conformation change of CYP707A3.Synchronous fluorescence spectroscopy showed that the interaction sites between ABA and CYP707A3 were close to tryptophan residues and led to the polarity increase in the microenvironment of tryptophan residues.