Study And Application Of Immobilized Enzyme Reactor Based On Alginate Gel Encapsulated Method

As in the 1960's,immobilized enzyme technology has been widely used in the study of enzyme analysis.The attachment of an enzyme to an insoluble support is termed as immobilization.Compared with the free enzyme,immobilized enzyme offer several unique advantages including(1)the separation of the enzyme and reaction mixture was easy;(2)the cost can be reduced because the studied enzymes can be continuous reused or recycled;(3)the enzyme stability can be also promoted,etc.Immobilized enzyme micro-reactors(IMERs)based on enzyme immobilization technology are increasingly being used in the filed of fermentation industry,biological medicine and biochemical research in recent years.To greatly reduce the consumption of samples and reagents(i.e.?L to nL),reduce costs,short analysis time with efficient analysis,the present paper mainly focused on the research of miniaturized IMER.The contents were listed below:1.A facile,cost-effective and high-throughput screening method was developed for enzyme-based assays based on Robolid/Microplate(RLMP)platform.The RLMP platform is constructed by immobilizing enzyme on commercial robolids and combining it with a standard 96-well microplate to achieve high-throughput analysis.The initiation and quenching of enzymatic reaction can be performed by simply sandwiching or unsealing the enzyme-immobilized robolids and the sample-containing microplate.This platform enables measurements of multiple target analytes simultaneously based on immobilized enzymatic reactions,with analysis time independent of the number of wells in the microplate.By using urea as the model analyte,we have shown that the RLMP platform exhibits large linear detection range of up to 10 mM,fast analysis time of 30 min/96 samples,as well as good reproducibility and stability.Measurements of urea in human urine and serum samples were performed using the RLMP platform and were compared with the commercial urea test kit.Good correlation was found between the two methods.This study shows that the present RLMP platform has promising prospects for detection of clinical markers and application in disease diagnosis and biochemical analysis.2.This work reports a simple,rapid,and enzyme-selective method to induce gelation for in-situ encapsulated enzyme into capillary inlet to fabricated about 2 cm long microreactor.The method utilizes acetylcholinesterase(AChE)to selectively identify and catalyze the conversion of thioacetyl chloride to thiochromine and acetic acid,and the produced proton acts to CaCO3 to release Ca2+,which triggers gelation of alginate sol to form a calcium-alginate hydrogel.AChE was in-situ encapsulated to form AChE-IMER.The preparation of the enzyme reactor is simple and controllable,and the enzyme loading is high.In this experiment,we systematically studied the feasibility and accuracy of the enzymereactor for on-line enzyme analysis and organophosphorus pesticide detection.The Km values of the immobilized AChE were 1.01 ± 0.04 mM.The inhibitory constants of the inhibitors Tacrine and Huperzine-A were 0.31 ± 0.08 ?M and 1.47 ± 0.14 ?M,respectively,which were similar to those obtained by the free enzyme in off-line.The immobilization method did not significantly alter the structure and activity of the enzyme.After 40 consecutive runs,the RSD of the product migration time and peak height was 3.2% and 4.3%,respectively.In addition,the enzyme activity was 80% of the initial enzyme activity after 10 times of continuous inhibition and activation of organophosphorus pesticide-solution.The results show that the enzyme reactor has good reproducibility and stability,and it has a good prospect in the field analysis of enzyme and the detection of organophosphorus pesticide in environmental pollutants.