Construction Of Activity Evaluation And Sensing Detection Methods Based On Enzyme Catalysis

Enzymes are a kind of high-performance biocatalysts with high substrate specificity,which participate in various reactions in the organism and are closely related to life activities such as homeostasis,growth and development,as well as individual reproduction.Therefore,enzymes are an important class of drug targets,and screening of their inhibitors can provide references for the new drug development.In reality,developing safe and effective enzyme inhibitory active ingredients from natural products is one of the important directions of current new drug research and development.However,the traditional free enzyme-activity determination method has the disadvantages of long operation cycle,time-consuming process and labor-intensive operation.Therefore,the development of simple,effective and high-throughput screening methods based on immobilized enzyme using a variety of immobilization materials and methods could provide a method reference for the discovering effective enzyme inhibitory active ingredients from natural products.On the other hand,due to the high efficiency,specificity,adjustability and simplicity of enzyme catalysis,enzyme has important applications in the field of analysis and detection,especially in sensing detection.Portable personal glucose meters and paper-based sensors are widely used in the point-of-care testing(POCT)field because of their low price,portability,small sample usage and simple operation.At present,the traditional portable personal glucose meters is mostly used for the measurement of blood glucose;while the paper-based detection platform mainly relies on light and electrochemical signals for detection.The quantitative analysis of paper-based detection often involves complex instruments and equipment,which limits its wide application in remote areas.Therefore,based on the catalytic reaction of enzymes,this thesis has developed portable personal glucose meters and paper-based quantitative detection method readable by naked eyes,which has important practical application for expanding the application of portable personal glucose meters and the rapid and effective identification and determination of target analytes in remote areas and emergency public health events.This thesis mainly includes the following eleven chapters,including nine chapters of experimental research.The first chapter is the introduction of this thesis.Firstly,the classification of enzyme,the basic theoretical knowledge of enzyme catalysis and reaction kinetics,as well as the advantages and disadvantages of different calculation models to estimate the kinetic parameters of enzyme catalytic reaction(K_m and V_max)were briefly introduced.Secondly,the research progress of screening enzyme inhibitors from traditional Chinese medicine and natural products by combining the current analysis techniques with free enzyme and immobilized enzyme as targets was reviewed,and the enzyme immobilized technology was also introduced.On the other hand,the application progress of POCT(portable personal glucose meter,portable paper-based sensor,microfluidic chip,electrochemical paper-based chip,pH meter,thermometer and pressure meter)in analysis and detection was reviewed.In the second chapter,tyrosinase(TYR)-mediated dopamine polymerization and internal release of Ca²⁺were used to realize the encapsulation of TYR and?-glucosidase(?-Glu)double enzymes by polydopamine-sodium alginate magnetic beads.The effects of temperature and pH on the catalytic activity of free enzyme and immobilized enzyme were compared.Under the optimum conditions,using levodopa and p-nitrophenyl-?-D-glucopyranoside(p NPG)as the substrates of TYR and?-Glu,the measured K_m values of immobilized TYR and?-Glu were 2.72 m M and 3.45 m M,respectively.The IC₅₀ values of the positive drugs kojic acid and castosperamine on immobilized TYR and?-Glu were 13.04?M and 56.23?M,respectively.Finally,the immobilized double enzyme was used to evaluate the enzyme inhibitory activity of tea polyphenol extracts.The results showed that the polyphenols extracted from black tea and white tea had good inhibitory effect on TYR,while the polyphenols extracted from black tea and black tea showed good inhibitory activity on?-Glu.The study of this chapter provides a reference for the development of support materials and methods for multiple enzymes'immobilization.In the third chapter,magnetic silica material combined with Con A affinity immobilization technology was used to directionally immobilize coagulation factor Xa(FXa),and the effects of temperature and pH on the catalytic activity of free and immobilized enzymes were investigated and compared.The measured K_m values of free and targeted immobilized FXa under optimization conditions were 0.17 m M and 0.10m M,respectively.The IC₅₀ value of the positive drug rivaroxaban on immobilized FXa was 0.35?M.The method was further used to screen potential direct FXa inhibitors from natural small-molecule compounds.The results showed that epigallocatechin gallate(EGCG),gallocatechin gallate(GCG)and epicatechin gallate(ECG)had good inhibitory activities.Cryptochlorogenic acid had promotion potency on FXa.Finally,molecular docking study showed that the interactions between EGCG,GCG,ECG,cryptochlorogenic acid and FXa were mainly hydrophobic,electrostatic and hydrogen bonding.The study of this chapter provides a reference for the development of support materials and methods for directional immobilization of enzymes.In addition,the established method for evaluating inhibitor activity can provide an effective way for screening FXa inhibitors in natural products.In the fourth chapter,a simple,rapid and automated method was used to develop an immobilized trypsin microreactor(IMER)based on capillary electrophoresis(CE)by immobilizing trypsin onto the inner surface of the capillary through physical adsorption.Using N?-benzoyl-L-arginine ethyl ester(BAEE)as the substrate,the measured K_mvalue of immobilized trypsin under optimum conditions was 0.99 m M.Then,the commercially available trypsin inhibitor(benzamidine)was used as a positive drug to verify the feasibility of CE-based trypsin-IMER for enzyme inhibitor screening,and the IC₅₀ and K_i values were determined as 3.39 m M and 1.68 m M,respectively.Meanwhile,the established method was used to evaluate the inhibitory effects of six phenolic acids(sinapic acid,ferulic acid,gallic acid,caffeic acid,cinnamic acid and vanillic acid)on trypsin.It was found that among the six phenolic acids,ferulic acid,gallic acid and caffeic acid showed better enzyme inhibitory activity at the concentration of 0.15 m M.Finally,the results of molecular docking study showed that phenolic acid could inhibit the activity of the enzyme by changing the structure of the enzyme in a non-competitive mode.The study of this chapter provides an efficient and rapid method for on-line evaluating the trypsin inhibitory activity of phenolic acids from functional foods,which is helpful to promote the rational applications of phenolic acids in functional foods.In the fifth chapter,an effective and simple immobilized trypsin CE-IMER was established using the strong adhesion and good biocompatibility of the polydopamine coating.The S-2765(molecular formula:Z-D-Arg-Gly-Arg-p NA·2HCl)was used for the first time as a chromogenic substrate to determine the enzymatic properties of immobilized trypsin.The measured K_m of immobilized trypsin under optimization conditions was(0.47±0.08)m M.The commercially available trypsin inhibitor(benzamidine)was used as the positive compound and its IC₅₀ value and K_i value were determined as 3.34 m M and 3.00 m M,respectively.The established method was used to evaluate the enzyme inhibitory activities of four small-molecule compounds of catechins and three kinds of tea polyphenol extracts.The results showed that EGCG and ECG,as well as green tea,white tea and black tea extracts had good enzyme inhibitory activities.Finally,molecular docking study showed that EGCG,ECG,EC and EGC were all located not only in the catalytic cavity,but also in the substrate-binding pocket of trypsin.The study of this chapter establishes an effective method for evaluating the inhibitory activity of catechins on trypsin,which is helpful to explain the effect of tea drinking on the enzyme activity of the digestive system and promote their application in functional foods and beverages.In the sixth chapter,a simple and portable enzyme activity assay and inhibitor screening method was developed based on the?-Glu catalyzes hydrolysis of D(-)-salicin to produce glucose,which reacted with glucose dehydrogenase on the glucose strips to produce a signal that can be detected by the personal glucose meter.Under the optimum experimental conditions,quantitative detection of?-Glu was achieved within the linear ranges from 1.0–9.0 U/m L.The detection limit was 0.45 U/m L,and the recovery rates of?-Glu in bitter almond extract were 96.2%and 84.3%.A commercially available?-Glu inhibitor(castanospermine)was used as the positive compound and its IC₅₀ value was determined to be 4.81?M.Furthermore,the established personal glucose meter method was used to evaluate the enzyme inhibitory activity of fourteen small-molecule compounds and six tea polyphenol extracts.The results showed that gallic acid,protocatechualdehyde,cryptochlorogenic acid,EC,EGC and vanillic acid had good inhibitory effect on?-Glu(all higher than 40%).Finally,molecular docking study was used to predict the binding sites and modes of small-molecule compounds to?-Glu.It was found that gallic acid,protocatechualdehyde,cryptochlorogenic acid,EC,EGC and vanillic acid were all located in the active site pocket of?-Glu.This study is based on the double-enzyme cascade reaction of the personal glucose meter to detect non-glucose targets,which not only expands the application of personal glucose meters,but also provides a simple and efficient method for the determination of?-Glu and its inhibitors'screening.In the seventh chapter,a rapid and portable enzyme activity assay and inhibitor screening method was developed based on alkaline phosphatase(ALP)catalyzes hydrolysis of amifostine to produce electrochemically active product WR-1065,and then the WR-1065 reacted with the medium K₃[Fe(CN)₆]on the glucose strips to produce a signal that can be detected by the personal glucose meter.Under the optimum experimental conditions,quantitative detection of ALP was achieved within the linear range from 0.33–3.33 U/?L.The detection limit was 0.13 U/?L,and the recovery of ALP in three milk samples were range from 87.7%–116.9%.The ALP inhibitor(Na₂FPO₃)was used as the positive compound and its IC₅₀ value was determined to be15.4 m M.The developed method was applied to evaluate inhibitory activity of ten small-molecule compounds and six Cordyceps sinensis(CS)extracts on ALP.The results showed that adenosine-5'-monophosphate(AMP)and theophylline had good enzyme inhibitory activity.The CS02 and CS05 extracts had promotion potency on ALP.In addition,the presence of a phosphate group(ionic or compound form)on AMP and AMP-2Na may influence the inhibitory effect against ALP.Finally,molecular docking study was used to predict the binding sites and modes of small-molecule compounds to ALP,and a new compound was designed by scaffold hopping study.The results showed that hydrogen bonds and metal-receptor interactions were important forces in the process of binding between small-molecule ligands and ALP.The study of this chapter establishes a method for the rapid determination of ALP and its inhibitor screening.The screening results can provide a reference for the development of ALP inhibitors and this study further expands the application of personal glucose meters.In the eighth chapter,the principles of rapid chelation between sodium alginate and Ca²⁺to form a hydrogel and resulted in the change of solution viscosity were used to encapsulate the glucose oxidase into the sodium alginate gel,the glucose oxidase catalyzes oxidation of glucose to generate gluconic acid,and then gluconic acid reacts with calcium carbonate to release Ca²⁺.Thereafter,the calcium alginate hydrogel is quickly formed and resulted in the change of solution viscosity.Glucose can be detected by a vernier caliper to measure the diffusion diameter of the solution spot on the microporous membrane.Under the optimum experimental conditions,quantitative detection of glucose was achieved within the linear range from 1.4–7.0 m M,and the limit of quantification was 1.4 m M.Finally,the established method was used in the detection of glucose in pear,apple and longan samples.The recovery of the fruit samples spiked with 2.0 m M glucose were 91.8%–99.1%.The paper-based sensor combined with visual distance-readout method in this study is a convenient,low-cost and accurate method,which can provide a method reference for the on-site determination of glucose in the fruit samples.In the ninth chapter,the principle of pH response of the viscosity and solubility of chitosan was used to encapsulate urease into the chitosan-acetate sol,and then the urease catalyzes hydrolysis of urea to produce ammonia,which increases the pH value of the chitosan-acetic acid sol and changed the viscosity of the sol solution.Urea could be detected by a vernier caliper to measure the diffusion diameter of the solution spot on the microporous membrane.Under the optimum experimental conditions,quantitative detection of urea was achieved within the linear range from 3.8–15.1 m M,and the limit of quantification was 3.8 m M.Finally,the established method was applied to detect urea in diesel exhaust fluid,and the recovery were 91.4%and 109.9%,respectively.The method established in this chapter integrates paper-based sensor and visual distance-readout technique,which provides a simple method for POCT of urea in remote areas.At present,the synthesis process of nanozyme is complex and its biocompatibility is poor,especially the disposable use of heavy mental compounds or precious metal nanozyme will cause the waste of resource and environmental pollution.Therefore,in the tenth chapter,a simple and environmentally friendly co-precipitation method was used to prepare hydrophilic phytic acid/copper phosphate(PA/Cu₃(PO₄)₂·3H₂O)for studying its enzyme activity and the detection of H₂O₂.The peroxidase-like activities of Cu₃(PO₄)₂·3H₂O and PA/Cu₃(PO₄)₂·3H₂O were investigated.Then,the effects of buffer pH,temperature and incubation time on the activity of nanozyme and the kinetic parameter K_m value of nanozyme were investigated.Under the optimum experimental conditions,this method was used for the quantitative detection of H₂O₂.The absorbance value and H₂O₂ concentration had a good linear relationship in the two ranges of0.1–1.0 m M and 1.0–5.0 m M.The detection limit of H₂O₂ was 79.0?M.The recovery of H₂O₂ in the milk samples were from 92.4%–101.7%.The method developed in this chapter provides a method reference for improving the peroxidase-like activity of metal phosphate,and proves that the inorganic material Cu₃(PO₄)₂·3H₂O has a catalytic activity similar to horseradish peroxidase,which provides a reference for subsequent study using Cu₃(PO₄)₂·3H₂O as an enzyme immobilization carrier.The eleventh chapter is the summary and prospect of this thesis.In this paper,several new methods for activity evaluation and sensing detection were constructed based on the catalytic action of enzymes.On the one hand,physical,covalent and affinity immobilized enzyme technology was used to establish offline magnetic immobilized single/double enzyme reactors and on-line CE-IMER,and then the enzymatic kinetics study and inhibition activity evaluation were carried out.On the other hand,POCT methods were constructed to apply the portable personal glucose meter to the detection of non-glucose targets based on the?-Glu hydrolyze the substrate D(-)-salicin to produce glucose and ALP hydrolyze substrate amifostine to produce the electrochemically active product WR-1065,and then the enzyme activity determination and inhibitory activity evaluation of small-molecule compounds or extracts on enzyme were achieved.Thirdly,GOx and urease were used to catalyze the substrate to produce corresponding acid and alkaline products,thereby changing the viscosity of the sodium alginate-Ca CO₃ and chitosan-acetic acid sol solution.Glucose and urea could be quickly detected using a vernier caliper to measure the diffusion diameter of the solution spot on the microporous membrane.Fourthly,based on the enzyme-like properties of PA/Cu₃(PO₄)₂·3H₂O nanonzyme,the colorimetric detection of H₂O₂ was achieved.In conclusion,a variety of materials and methods for enzymes immobilization were used for developing a simple,effective and high-throughput screening method,which can provide a method reference for discovering effective enzyme inhibitory active ingredients from natural products.Secondly,the construction of portable personal glucose meters and paper-based quantitative detection method with naked eyes has an important application value for the rapid and effective identification and determination of target analytes in remote areas and emergency public health events.Meanwhile,it also expands the application of personal glucose meter.In addition,a universal,simple,green and fast method to enhance the peroxidase-like activity of nanozymes was developed,which enriched the research of nanozymes.Finally,based on the studies of this thesis,the suggestions for future research in relevant filed were prospected.