Construction Of Fluorescent Nanosensor And Its Application In Detection Of Biological Enzymes And Small Molecules

Biological enzymes and small molecules play a vital role in maintaining the normal life activities of organisms.Therefore,the development of sensitive and efficient methods for the detection of biological enzymes and biological small molecules is of great significance in the field of biomedicine.Fluorescent nanosensor has the advantages of high sensitivity,good selectivity,quick analysis,simple operation and low cost,which has attracted the attention of researchers.Fluorescent nanosensor can detect analyte through the change of the generated fluorescent signal.In this paper,a series of fluorescent nanosensors have been constructed and applied to the detection of biological enzyme activity and biological small molecules.The specific content is as follows:1.A fluorescent nanosensor based on nitrogen-doped graphene quantum dots（N-GQDs）was constructed for tyrosinase（TYR）and acid phosphatase（ACP）activity detection.Firstly,the activity of TYR was detected by using the catalytic property of TYR for tyrosine（Tyr）to produce dopaquinone,and the generated dopaquinone effectively quenched the fluorescence of N-GQDs.In addition,the ascorbic acid（AA）produced by the reaction of ACP and L-Ascorbic acid-2-phosphate（AAP）can reduce dopaquinone and inhibit the quenching effect of dopaquinone on N-GQDs,leading to the recovery of the fluorescence of N-GQDs,so as to realize the detection of ACP activity.The fluorescence quenching and recovery reaction mechanism of N-GQDs can simultaneously detect the activities of TYR and ACP,with the corresponding detection limits of 0.15 U m L^-1 and 0.014 m U m L^-1,respectively.2.A fluorescent nanosensor based on glutathione（GSH）functionalized graphene quantum dots（GQDs@GSH）was designed and successfully fabricated for the detection of phytic acid（PA）and hydrogen peroxide（H₂O₂）.Firstly,we synthesized GQDs@GSH using citric acid and GSH as raw materials.Fe³⁺ions can interact with the carboxyl and hydroxyl groups on the surface of GQDs@GSH to effectively quench the fluorescence of GQDs@GSH.When PA was added into the above system,due to its strong reducibility,it can reduce Fe³⁺to Fe²⁺and form PA/Fe²⁺complex,resulting in the fluorescence recovery of GQDs@GSH.Then adding strong oxidizing H₂O₂ into the system,it can destroy the complex structure of PA/Fe²⁺,release Fe²⁺and oxidize Fe²⁺to Fe³⁺,resulting in the fluorescence quenching of GQDs@GSH again.According to the linear relationship between the recovery and quenching degree of GQDs@GSH fluorescence intensity and the concentration of PA and H₂O₂,the simultaneous detection of PA and H₂O₂can be achieved in a certain concentration range,and the detection limits were 14 nmol L^-1 and 0.134μmol L^-1,respectively.In addition,this method has been successfully used for the determination of PA and H₂O₂in real samples.3.A dual-wavelength ratiometric fluorescence sensor was constructed using blue-emitting N-GQDs and red-emitting dopamine（DA）-functionalized Cd Te QDs（DA-Cd Te QDs）,and was applied to the detection of TYR activity.TYR specifically oxidized DA on the surface of Cd Te QDs to dopaquinone.Dopaquinone,as an electron receptor,quenched the red fluorescence of DA-Cd Te QDs by electron transfer effect,while the blue fluorescence of N-GQDs was not affected.Different concentrations of TYR can cause the corresponding fluorescence intensity ratio of red and blue quantum dots to change,and the fluorescence color of the whole system can change from red to blue.The detection limit of this method for TYR reached 0.0045U m L^-1.The ratiometric fluorescent sensor can not only detect TYR activity sensitively,but also realize the visualization of TYR detection.4.Based on the combination of oxidase activity of manganese dioxide（Mn O₂）nanosheet and the luminescent properties of thiamine（TH）,a fluorescent nanosensor was developed for the detection of butylcholinesterase（BCh E）activity.Mn O₂nanosheets can catalyze the oxidation of TH to generate blue fluorescent product thiochrome（TC）.The thiocholine generated by the reaction between BCh E and S-butyrylthiocholine iodide（BTCh）reduced Mn O₂ nanosheets to Mn²⁺ions.As a result,the amount of TC produced by catalytic oxidation of Mn O₂ nanosheets decreased and the fluorescence intensity decreased.The fluorescence reduction of TC was used as a signal to quantitatively detect BCh E activity.The detection range of this sensor for BCh E was 0.036 U L^-1.The method was applied to the determination of BCh E in human serum samples with satisfactory results,which indicates that the fluorescent nanosensor has a great application prospect in biological analysis.