Development Of Enzyme-catalyzed Biosensors And Their Analytical Applications

As catalysts, natural enzymes are proteins which catalyze the reactions of living creature. Since it has strong specificity and high catalytic efficiency, they’re widely used in biomedicine, analysis, testing and other fields. But at the same time they also exposed some defects: the catalytic activity is susceptible to environmental impact and easy to inactivation, the high cost of purification and storage. Therefore, scientific researchers work for the development of new types of enzyme-like catalysis method. On the one hand, a large number of nanomaterials enzymes-like were developed, which not only synthetic method is simple, easy to store, and can be modified to apply in different systems. On the other hand, researchers develop some nanomaterials which can activate natural enzymes. This way can not only to provide a wide range of reaction conditions for the enzyme catalysis, but also improve the catalytic efficiency. These two kinds of enzyme catalytic way made up for the shortcoming of pure natural enzymes, thus has high research value and broad market prospects. Based on this, in order to solve the faults of natural enzymes, this paper develop two kinds of enzyme catalysis method, and applied them to the detection of food and biological. The concrete research content is as follows:(1) The synthetic gold clusters with HRP as the template synthesis showed superior simulation oxidase activity under the condition of visible light. It can still maintain a high catalytic efficiency in more severe reaction conditions, but also has stronger affinity of TMB than the pure natural enzyme HRP. We found that the addition of bivalent mercury ions can significantly inhibit enzyme activity because of the bivalent mercury ions and gold ions form Au- Hg alloy on the surface of the gold clusters, and change the surface state of gold clusters, thus loss of catalytic activity. And melamine and bivalent mercury has a stronger binding force, so it can relieve the inhibition, making gold clusters regain simulated enzyme activity. Based on these, we set up a detection method for melamine, which is successfully applied in the actual samples with adding method.(2) We found that the in situ synthesis of Cd S QDs show optical simulation enzyme activity, and the kinds of modifier and the concentration of sulfur ions directly affects the Cd S QDs simulated enzyme activity. Then we find the highest catalytic activity PO43- @ Cd S QDs and combined the phenomenon of decompose the glucose oxidase 1-β-D-glucose can produce sulfur ions, we set up a colorimetric method to detect the activity of glucose oxidase. Due to the in situ synthesis, the background signal is weak, and the amount of required enzyme in catalytic reaction is rare, thus greatly improving the detection sensitivity(3) Catechol(CA) and titanium dioxide(Ti O2) compounds can produce active intermediates h+ and O2?- under visible light irradiation, and h+ and O2?- can activate natural enzyme HRP, make it catalytic oxidation of substrates TMB and ABTS without the presence of hydrogen peroxide. The activity of Ti O2-CA activation HRP is closely related with the the concentration of CA, and CA can be produced by alkaline phosphatase(ALP) catalytic substrate pyrocatechol phosphate OPP. Based on this, we set up three times signal amplification method to detect the ALP strategy.(4) We use the improved synthesis Hummers method to get GO, which can produce reactive intermediates h+ and O2?- under visible light irradiation and they can also activate natural enzyme HRP, make it catalytic oxidized substrate TMB and ABTS without hydrogen peroxide. The amount of HRP is directly related to catalytic oxidation effect in the system. Due to GO has good biological compatibility, high activation efficiency, the activation of HRP enzyme catalytic method is applied to the immune analysis experiment. By the mean of DNA amplification technology, DNA connect on a large number of HRP, we set up an ultrasensitive detection antigen of AFP. The detection limit is 0.0001 pg/m L of AFP, this is far less than the traditional methods of detecting AFP results.