Study On Preparation And Application Of Amperometric Enzyme Biosensor For Lactose Detection

The preparation of lactose biosensor can provide us a beneficial means for the present rapid detection. Lactose biosensor has the characteristics of high selectivity, simplicity, fast speed, and tending to be monitoring on line.Amperometric enzyme biosensor is the most important and sensitive biosensor in the biosensor area and it has also been paid more attention and commercialized successfully. The core component of such biosensor is namely the immobilized enzyme membrane. Based on the commercialized SBA-40C biosensor, the immobilized enzyme membrane of satisfied capability has been developed in this paper. The capability of lactose enzyme membrane mainly depends on the selection of immobilized materials and the methods of immobilization. The capability characteristics of immobilized enzyme membranes prepared from some materials such as yeast cell wall particulates,β-cyclodextrin particulates (β-CDP), starch adhesive and Calcium alginate and their mixture and capability differences of lactose enzyme membranes prepared by adsorption, cross link, entrapment and mixture were studied and compared. The effect of glutaraldehyde concentration and acting time on enzyme membrane activities was also studied. The results indicated that the enzyme membranes made from the former three materials performed high sensitivity. The lowest lactose detection concentration was below 0.0001% for lactose and 0.00001% for glucose, while their service lives were not satisfied. When calcium alginate was mixed with the other three materials, enzyme membrane with high sensitivity and long service life can be obtained and the sensitivity of the enzyme membrane wouldn't decrease evidently. The optimum condition was: 0.2% calcium alginate, 0.5g starch adhesive (or 0.5gβ-CD)and 20ml 1% glutaraldehyde solution are heated and dissolved as colloid; Then, the colloid are mixed with enzyme solution for 10min, made into sandwich-like membrane sheet; And finally the membrane was immerged into calcium chloride solution for 15 seconds and washed. The lowest lactose detection concentration was below 0.0005%, with an acting time of 20 seconds. With a linear relation in the range of 0 and 2% that R2 is above 0.995, the activity of enzyme membrane was detected 40 times a day and it wouldn't obviously decline after a week. The rule of effect of glutaraldehyde onβ-galactosidase specificity was also studied in this study. It was found that glutaraldehyde caused the changes ofβ-galactosidase specificity .And the results suggested that the special substrate ofβ-galactosidase transformed from lactose to sucrose, with the increase of glutaraldehyde concentration and acting time. Hence, the sucrose enzyme membrane of high capability was also prepared. The lowest lactose detection concentration was below 0.0005%, with an acting time of 20 seconds. Also with a linear relation in the range of 0%-3% that R2 was above 0.999, the activity of enzyme membrane was detected 40 times a day and but it wouldn't obviously decline after a week.Mathematical models of lactose biosensor were constructed by Matlab software (edition: 2007a) and simulated by computer. Meanwhile, the effect of every parameter on the capability of biosensor studied in this model was corresponding to our experimental results.