Study And Application With Horseradish Peroxidase,Laccase Biosensores

Objective: Using enzyme systems of electrochemical biosensor to detect organic pollutants in the analytic environment is easily and quickly. In this study,sequential injection analysis (SIA) was combined with the screen-printed enzyme sensor technology to detect catalytic current of horseradish peroxidase(HRP) and laccase,which can reflect the concentration of phenol and other organic pollutants.It provides an accurate,sensitive and automated detection technology for rapidly detect environmental pollutants.Methods: This study based on the screen-printed electrode of Single enzyme sites HRP,laccase and four enzyme point(HRP,laccase),using bovine serum albumin(BSA) and glutaraldehyde to immobilize HRP,laccase or mix liquid of two enzymes with osmium polymer([Os(bipyridine)2Cl2]poly(4-vinylpyridine),Os(bpy)2(PVP)10Cl2,Os-PVP) on the screen-printed electrodes and the surface of four point electrode. And then utilizing the technology of SIA coupled with screen-printed biosensor to process detect and analysis on each kind of Enzyme electrode,and optimize the relevant test conditions such as the buffer pH and concentration, pump flow rate, sample volume and etc.Results:(1) Analyzed the detection system of single enzyme sites,no Os-PVP modified HRP electrode,got the optimum conditions of the experiment via analysis on detection of free ferricyanide modified laccase electrode.The optimum conditions of the experiment are as follows:①HRP electrode:153.9μL/min flow rate,100μL sample volume and pH 6.5,0.1 mol/L buffer.The reaction of HRP catalyzing o-phenylenediamine(OPD)-H₂O₂,which the current response linear equationIn of H₂O₂ concentration is y=14.59+114.61x,R²=0.9845,and Current response sensitivity is 110.11nA/(mmol/L).The current response linear equationIn of OPD concentration is y=27.595x+17.925,R²=0.9941, and Current response sensitivity is 26.88 nA/(mmol/L).In addition, the biosenser had advantages of repeatability,RSD was 3.8%.The recovery ration for OPD in the real sample was more than 60%.②free ferricyanide modified laccase electrodes:0.1 mol/L,pH 4.5 PBS buffer,the current response linear equationIn of laccase catalyzed catechol and ferricyanide is y=49.7x+17.582,R²=0.9827,and Current response sensitivity is 50 nA/(mmol/L),they Showed a good linear relationship.(2)Analyzed the detection system of single enzyme sites of HRP electrode and laccase electrode, which modified by Os-PVP. Under the respectively optimum PBS buffer conditions of 0.1 mol/L, pH 7.5 and 4.5, the current response linear equation ,which HRP catalyzed OPD-H₂O₂, is y=300.39x-6.7031,R²=0.9875,and Current response sensitivity is 97.46 nA/ (mmol/L).The current response linear equations, which laccase catalyzed catechol, is y=1.166x+0.078,R²=0.9801,and Current response sensitivity is 1.13μA/ (mmol/L). The recycle rate of HRP-(Os-PVP) electrode used for detecting real water samples varies from 70% to 93%. And the laccase-(Os-PVP) electrode used for detecting the landfill leachate,the average concentration of catechol is 0.028 mmol/L,and the standard deviation(RSD) is 0.34%.(3) To analyzed the Four-point enzyme electrode, respectively mixed HRP,laccase,HRP,laccase with Os-PVP in sequence on the surface of screen-printed electrodes.The optimum conditons are as follows: 0.15 mmol/L,pH 6.0 of PBS buffer.The first enzyme point(HRP) current response linear equations of a common reaction of H₂O₂ and catechol is y=1.572x-0.165,R²=0.9559,and current response sensitivity is 1.44μA/(mmol/L).The seconde enzyme point(laccase) current response linear equations of a common reaction of H₂O₂ and catechol is y=0.5519x-0.023,R²=0.949,and current response sensitivity is 0.54μA/(mmol/L).The third enzyme point(HRP) current response linear equations of a common reaction of H₂O₂ and catechol is y=1.022x+0.01, R²=0.9604,and current response sensitivity is 0.91μA/(mmol/L).The forth enzyme point(laccase) current response linear equations of a common reaction of H₂O₂ and catechol is y=0.349x-0.004,R²=0.9641,and current response sensitivity is 0.33μA/(mmol/L).In addition,to detect the respectively catechol concentrition of landfill leachate is 1.09 mmol/L,1.02 mmol/L,1.04 mmol/L,1.09 mmol/L,and average value is 1.05 mmol/L. The relative deviation (RSD) was 3.4%.Conclusion: Here established a technology of screen-printed enzyme sensor coupled with sequential injection analysis(SIA) and studied on four point enzyme electrode. This technology improved the sensor analytical accuracy and sensitivity, and it will use to testing environment of long-term,real-time online. Enzyme electrode of the four points provided a method for the complex environment detection,which widened the scope of testing and selective.