Colorimetric Detection Of Cholesterol And Organophosphorus Pesticides Based On Metal-organic Framework Materials Immobilized Enzymes

Enzymes have the advantages of high substrate specificity,catalytic efficiency and green chemistry.However,the use of enzymes is often hindered by their low stability,recycling difficulties and the lack of reusability in industrial applications.Immobilization of the enzymes is one of the most attractive ways to overcome these defects.Compared with free enzymes,immobilized enzymes can prevent the aggregation,autolysis or hydrolysis of enzyme molecules,rigidification of the enzyme structure through multi-point covalent attachment,and form a favorable microenvironment.Immobilized enzyme has the advantages of high operational stability,strong enantioselectivity,simple reactor operation and convenient product separation.In addition,the immobilized enzyme can be recycled to improve the enzyme recovery,reduce the contamination of the enzyme in the product and the cost of the production process.Metal-organic frameworks(MOFs)are porous coordination polymers composed of metal nodes and organic ligands connected by coordination bonds.Generally speaking,MOFs has the advantages of ultra-high porosity,large specific surface area(beyond 6000 m2/g),diverse functions,high thermal/mechanical stability and photoelectric properties.Due to the extraordinary degree of variability of organic and inorganic components in the structure,MOFs has become a research hotspot of enzyme immobilized materials.In this paper,cholesterol and organophosphorus pesticides were detected in colorimetry by using the composites of MOF-enzyme.In the introduction chapter.Firstly,the research progress of immobilized enzyme was summarized,and then the synthesis method of MOFs was introduced.Finally,the application and advantages of MOFs in immobilized enzyme were introduced.In the second chapter,high levels of cholesterol lead to atherosclerosis,which is one of the main causes of coronary heart disease,myocardial infarction and stroke.It is very important for human health to test cholesterol content.The co-immobilization of two enzymes onto single support commonly exhibits low efficiency due to the competition against limited sites.Water-stable MOFs[i.e.,PCN-333(Al)]with a high surface area and ultra-large cavities were employed to efficiently adsorb cholesterol oxidase(ChOx)and encapsulate horseradish peroxidase(HRP),respectively.The prepared PCN-333/ChOx&HRP was characterized through SEM,XRD,confocal microscopy,N2 adsorption isotherms,and thermal gravity analysis(TGA).The high surface area and high concentration of mesoporous cages resulted in the high loadings of both ChOx and HRP.The absorbed ChOx and the encapsulated HRP presented excellent activities without additional chemical modification.The immobilized enzymes were stable against protease digestion,organic solvents,temperature changes,and pH variation.Thus,a colorimetric biosensor was fabricated depending on cascade catalytic reactions of the immobilized bi-enzymes for cholesterol detection.An extended linear range from 0.0 ?M to 40.0 ?M with a low detection limit of 0.6 ?M was obtained using the biosensor.The co-immobilization of the enzymes onto the surface and into the mesopores of MOFs provided a new and excellent platform for the development of highly stable and sensitive colorimetric biosensor.In the third chapter,organophosphorus pesticides(OPs)have been widely used as pesticides in the agricultural field causing great harm to human health due to the OPs residues in agricultural products.Both organophosphorus and carbamate pesticides can inhibit the activity of AChE.We immobilized AChE with the help of MOFs ZIF-8 as the carrier,and used the reactivator pralidoxime compounds to reactivate the activity of AChE inhibited by OPs to exclude the influence of other inhibitors.The ZIF-8/AChE was characterized by SEM,XRD,TGA,N2 adsorption isotherms,and CLSM.The experimental principle is as follows:ZIF-8/AChE catalyzes the hydrolysis of substrate thioacetylcholine to thiocholine,5,5'-dithiobis(2-nitrobenzoic acid)(DTNB)acted as a chromogenic reagent further reacted with thiocholine to generat yellow product 5-thio-2-nitrobenzoic acid(TNB).We measured the absorbance value change of TNB at 412 nm to determine organophosphorus compounds and used the reactivator to confirm the inhibitory effect of organophosphorus compounds with the reactivation of reduced activity of immobilized AChE.The linear response range of detecting paraoxon was 0.1-5.0 ?M with the detection limit of 0.08 ?M.This study provided the basis for the application of biosensor with MOFs as the enzyme immobilized carrier,and offered a simple and fast method for the detection of organophosphorus.The established method also had fantastic sensitivity,selectivity and stability.