Research And Application Of Biosensor Based On Clark Oxygen Electrode

The biosensor is a special chemical sensor.It uses the biologically sensitive components as active units(such as enzyme,antibody,nucleic acid,cell, tissue) and promises highly selectively to analyte.The analyte was sensed through various kinds of physical-chemical signal transducer.The analytical methods based on biosensor are characterized by high selectivity,easy realization for on-line continuous monitoring,fast response and low cost. This paper mainly concerns the application of enzyme or cell-based Clark oxygen electrode biosensors in the field of bio-chemical analysis.The paper consists of six chapters.Chapter 1:Biosensors are analytical devices composed of a recognition element of biological origin and a physico-chemical transducer.The biological element is capable of sensing the presence,activity or concentration of a chemical analyte in solution.The conception,basal constitution,the types,work principle and the merit of the biosensor and the development of application of the biosensor in the fields of environment monitoring,ferment,food analysis,bio-medicine and the military field.Chapter 2:A galactose biosensor using an enzyme-immobilized eggshell and the Clark oxygen electrode for galactose determination has been fabricated. Galactose oxidase was covalently immobilized on an eggshell membrane with glutaraldehyde as a cross-linking agent.The galactose biosensor was fabricated by positioning the enzyme-immobilized eggshell membrane on the surface of a Clark oxygen electrode.When galactose passed through sensor, galactose was oxidized by dissoloving oxygen under catalysis of galactose oxidase present in the eggshell membrane,then produced hydrogen peroxide and the depleted dissolved oxygen.The decrease in the oxygen level was monitored by Clark oxygen electrode.The effect of pH,phosphate buffer concentration and temperature on the response of the biosensor has been studied in detail.Common matrix interferents did not give significant interference.The results showed response time of biosensor is 100 s,the linear range of is 1.0×10^-4—8.5×10^-3mol/L galactose(r²=0.9984) with a detection limit of 50μM(S/N =3),RSD=3.74%for 5.0 mM galactose(n=7). It can retain 83.6%of initial enzyme activity after 3 months when stored in a pH 7.0 phosphate.In brief,the biosensor has displayed a faster response to the galactose,good reproducibility,selectivity,and stability for long-term use. The results showed that the eggshell membrane technology holds a great promise for the fabrication of a biosensor as well as practical.Chapter 3:A biosensor for determination of ethanol has been fabricated by employing an alcohol oxidase immobilized onto an eggshell membrane with chitosan as a cross-linking agent and a Clark oxygen electrode as the transducer.The detection scheme was based on the enzymatic reactions of ethane leading to the depletion of the dissolved oxygen level in the base solution and then the decrease in the oxygen level was monitored and related to the ethanol concentration.The effects of enzyme loading,chitosan concentration,pH,and temperature on the response of the biosensor were investigated.Storage and repeatability of the biosensor were also studied in detail.The analytical range of the biosensor was from 6.0×10^-5-8.0×10^-4 mol/L ethanol with a detection limit of 30μM(S/N=3) and 1 min response time.In the optimization studies of the biosensor the most suitable enzyme and chitosan amounts were found to be 1.0 mg and 0.30%(w/v), respectively.The phosphate buffer(pH 7.4,25 mM) and room temperature (20-25℃) were chosen as the optimum working conditions.In the characterization studies of the biosensor some parameters such as interference effects,operational and storage stability were studied in detail. The biosensor was also tested withvarious wine samples.There were no significant differences between the values obtained from the proposed biosensor and gas chromatographic methods.Furthermore,the recovery tests for ethanol were performed by adding various known amounts of ethanol to the beer and liquor samples.The amounts of added ethanol were then evaluated by the proposed ethanol biosensor.The recovery tests demonstrate that the proposed biosensor offers an excellent,accurate and precise method for the determination of ethanol in real samples.Chapter 4:A microbial biosensing system for the measurement of methane has been developed using an immobilized mixed culture of Pseudomonas aeruginosa and Klebsiella sp.together with Clark oxygen(O₂) electrode. When methane was applied to the biosensing system,methane was oxidized by dissoloving oxygen under catalysis of methane-oxidizing bacteria present in the immobilized small ball,then the depleted dissolved oxygen.The dissolved O₂ content decreased until a steady-state was reached and was monitored by Clark oxygen electrode.The biosensing system response depends linearly on methane concentration between 1.0 and 5.0%v/v with a detection limit of 0.3%v/v(S/N=3) and a 100-s response time.Phosphate buffer(pH 7.0,25 mM) and room temperature(20-25℃) were chosen as the optimum working conditions.Some parameters including pH,temperature, operational and storage stability were studied in detail for characterization of the biosenisng system.Satisfactory repeatability was confirmed with a relative standard deviation of 3.1%obtained by subjecting the microbial biosensing system to 3.0%v/v methane in pH 7.0 phosphate buffer(25 mM) eight times.Our proposed microbial biosensing system exhibits fast response to methane with good reproducibility.The most promising features of our system are cost-effective,simple sensing design and ease of operation.This microbial biosensing system using co-immobilized Pseudomonas aeruginosa and Klebsiella sp appears very attractive for rapid determination of methane.Chapter 5:On the base of previous experiments,the mechanisms of immobilization of enzyme andbacteria were brought forward. Chapter 6:A novel material of sensing membrane-synthesis and characterization of Cryptophane ACryptophane-A was synthesized from vanillin using a three-step method,it was purified by elution through a silica gel column and characterized by nuclear magnetic resonance(NMR) spectroscopy.Cryptophanes consist of two rigid cone-shaped units(cyclotriveratrylene) linked together by three bridges.It can complex with neutral molecules and plays an important part in molecular probe and gas sensor field,so the fluorescence and UV spectra of it were studied in the article.