Research On Colorimetric Detection Method Based On Biomolecular Regulation Of Nanoenzyme Activity Strategy

In recent years,due to the advantages such as high stability,low cost and easy for activity regulation,the researches on nanomaterials-based enzyme mimics(nanozymes)have become a research hotspot in the field of chemistry.In analytical chemistry,nanozymes are widely used as the alternatives for natural enzymes in developing sensing methods.In addition to the above advantages,nanozymes also have unique advantages in analytical chemistry.Besides the enzyme-like catalytic activity,nanozymes may have various interactions with different biological molecules and ions,which may change the catalytic activity of nanozymes.Thus,compared with directly using nanozymes as alternatives for natural enzymes,the studies for nanozyme activity regulation have brought wide possibilities for the development of nanozyme-based sensing methods.At present,biological macromolecules such as DNA,some ions and small molecules,etc.,have been reported to possess interactions with nanozymes and affect their catalytic activity.Based on the above principles,detection for various targets can be achieved.Colorimetric sensors possess merits such as stable signal,good accuracy,cheap and low equipment requirements as well as direct visual detection,which are very suitable for real-time and on-site detection.Besides,the advantages of nanozymes also match well with the colorimetric sensing methods.Therefore,colorimetric sensors based on nanozymes have attracted high attentions.However,there are still some shortcomings existed in this field.Firstly,with the increasing demands for early diagnosis and environmental monitoring,sensitivity requirements for sensing methods are increasing.However,the activity of nanozymes is still lower than that of natural enzymes.Moreover,the sensitivity of colorimetric sensing is generally considered to be relatively insufficient.Therefore,improving the sensitivity of nanozyme-based colorimetric sensing is important for the research,development and application of this field.Secondly,the development of nanozyme-based colorimetric sensing still needs to be further explored.At present,the researches on detection methods using biological macromolecules to regulate the activity of nanozymes are still mainly focused on the application of DNA molecules,and there are only a small number of other reports.In addition,the combining of unique advantages of nanozymes with more detection methods to solve the shortcomings of existing analytical methods and to expand nanozyme sensing is still needed to be studied.In this thesis,the detection methods based on the activity regulation strategies of nanozymes were studied.For the above problems,studies were carried out based on the research of sensing methods:1.In this research,the nanozyme was firstly applied in target-responsive hydrogel sensing.The strategy of applying the hydrogel in gel-state for target recognition and using the hydrogel in sol-state for regulating nanozyme activity to realize target sensing was proposed.It was firstly reported that alginate could regulate the peroxide-like activity of g-C₃N₄ nanozymes.Taking the alginate-Cu²⁺hydrogel as an example,the addition of histidine could cause the alginate-Cu²⁺hydrogel to transform into the sol state,and then the alginate molecules could be applied to inhibit the peroxidase-like activity of g-C₃N₄ nanozyme for catalyzing oxidation of the chromogenic substrate ABTS.Based on this,a colorimetric histidine detection method was successfully established,which verified the feasibility of the proposed idea.Besides,the feasibility of using DNA hydrogel to regulate nanozyme activity for sensing was preliminary explored,indicating that this strategy possessed the feasibility to be expanded.Compared with classic hydrogel sensing system based on the target recognition function,the concept of using the sol-state hydrogel for regulating nanozyme activity has the advantage of no need to load signal substances in the hydrogel,which can obviously reduce the complexity of the sensor preparation process.Besides,the system also expands the field of nanozyme sensing,which is expected to inspire related research work and promote the research and development of this field.2.The natural enzyme is composed of an active center and a protein scaffold and the realization of catalytic function depends on the combination of them.Similarly,as the mimics for natural enzymes,the catalytic function of some nanozymes also depends on the combination between two components.Inspired by the structure of natural enzymes and the high efficiency of their active center inhibition,in this chapter,the target inhibited combination of the two components of nanozymes to inhibit nanozyme activity was proposed.The Au NPs-Hg²⁺peroxidase mimic system was adopted for a verification experiment and the strong coordination effect of thiols on mercury ions was used.By interacting with its“active center”-Hg²⁺,the functions of target recognition and high sensitivity could be simultaneously realized.Due to the selective binding between Hg²⁺and thiols,the generation and the catalytic activity of Au NPs-Hg²⁺nanozyme were inhibited.Thus,highly sensitive colorimetric sensing for glutathione was successfully realized with a detection limit of 12.5 n M,which was comparable to some recent reports based on fluorescent and electrochemical methods and obviously better compared with the colorimetric detection methods based on the reduction effect of biological thiols.3.To improve the sensitivity of nanozyme-based sensors for analyzing total antioxidant substance concentration,the strategy of target inhibition for nanozyme activity was adopted and the substrate competition strategy was first simultaneously introduced in the detection system.As a negatively charged biological macromolecule,heparin was firstly used as a template and stabilizer to synthesize manganese dioxide-heparin oxidase mimicking nanozyme.The reaction between the bio-antioxidant substance ascorbic acid and manganese dioxide caused the dissolution of the manganese dioxide-heparin nanozyme,which could reduce its catalytic activity on the oxidation of substrate TMB.At the same time,the dissolution of manganese dioxide-heparin nanozyme would also release heparin molecules into the solution.Since heparin was a negatively charged macromolecule,it competed with the remaining manganese dioxide-heparin for the binding of positively charged TMB through electrostatic interaction,which was beneficial for the improvement of sensitivity.Based on this method,highly sensitive colorimetric detection of ascorbic acid based on nanozyme was realized with a very low detection limit of 0.04?M.