Construction Of Nano-Fluorescent Biosensors And Antibacterial Application Of Nanozyme

Fluorescent biosensors with high sensitivity and selectivity,simple and convenient operation have been widely used in the detection of biological small molecules,metal ions,biological enzyme activities and cancer markers.With the rapid development of nanotechnology and the emergence of the concept of nanozymes,nanozymes with the catalytic function of natural enzymes and good stability play an important role in biosensing and nano-therapeutic systems.Up to now,the sensors based on nanozymes mainly focused on the application of the colorimetric determination of the analyte(such as:OPD,TMB,ABTS substrates).However,most of them are mainly single-signal output causing the low sensitivity.Therefore,in view of the above problem,this thesis focuses on the construction of new fluorescent sensors(single-signal and dual-signal output modes)and the expansion of the biological application of nanomaterials with enzymatic activity.The main research contents are described as follows:1.Biocompatible boron nitride quantum dots(BN QDs)are prepared via a facile onespot hydrothermal synthesis.A "turn off-on" nanosensor is designed based on glutathione(GSH)regulating the inner filter effect(IFE)of MnO2 nanosheets(MnO2 NS)on boron nitride quantum dots(BNQDs).The fluorescence of BNQDs is quenched efficiently in the presence of redoxable MnO2 NS due to its superior light absorption capability.The introduction of GSH can trigger the decomposition of MnO2 to Mn2+and weaken the IFE,causing the partial fluorescence recovery.And the recovered fluorescence depends on the concentration of GSH.The linear detection range of the sensing platform for GSH is 0.5 μM～250μM with the detection limit of 160 nM under optimal conditions.The practical application of the proposed system is demonstrated by detecting GSH in human plasma samples with satisfactory results.2.A ratiometric fluorescence sensor based on the signal amplification of twodimensional manganese-iron layered double hydroxide(Mn-Fe LDH)in the presence of ascorbic acid(AA)and its catalytic function is investigated.Two-dimensional(2D)Mn-Fe LDH nanosheets are firstly examined to mediate O-phenylenediamine(OPD)based fluorescent switch in the presence of ascorbic acid(AA).On one hand,Mn-Fe LDH mimicks the functions of oxidase to catalyze the oxidation of OPD to OPDox,emitting the fluorescence at 565 nm.On the other hand,Mn-Fe LDH acts as the superior catalyst for the reaction between AA and OPD to generate the 3-(1,2dihydroxyethyl)furo[3,4-b]quinoxalin-1(3H)-one(named N-heterocyclic compounds)with an emergence of the maximum emission at 425 nm(ca.6 folds).The presence of AA not only induces the enhanced emission at 425 nm from N-heterocyclic compound,but also leads to the fluorescence at 565 nm decreasing due to the decomposition of Mn-Fe LDH nanosheets.On the basis of the reversed fluorescent response at 425 and 565 nm,ratiometric fluorescent sensing methods(ΔF425nm/ΔF565 nm)are developed for the determination of AA.With the assistance of alkaline phosphatase(ALP),the activity of ALP can be monitored with the detection limit of 0.16 mU mL-1 using the ratiometric platform based on the hydrolyzing ascorbic acid 2-phosphate(AAP)to yield AA.Unlike the traditional ratiometric sensing platform with two fluorescent probes,this work is based on in situ generation of fluorescent signals without the introduction of additional nanomaterials,greatly simplifying the experimental process with promising applications in the biological field.3.The fluorescent sensor based on the single-atom nanozyme signal amplification is successfully constructed by introducing Cu2+via the coordination with 2,2’-bipyridine ligands of Zr-MOF just like "Midas touch".More details on the coordination environment of Cu active sites in the Zr-MOF-Cu are disclosed via electron paramagnetic resonance and synchrotron-radiation-based X-ray absorption fine structure.The as-prepared Zr-MOF-Cu exhibits unparalleled catalytic ability,which can catalyze the AA to dehydroascorbic acid and further intrigue the reaction with ophenylenediamine to produce fluorescent signal probes with 8-fold signal amplification.On the basis of the catalyzing dephosphorylation process of AAP to yield AA via ALP and AA-dependent signal response,a universal fluorescent system has been successfully constructed for quantitative measurement of the activity of ALP and ALPrelated enzyme-linked immunosorbent assay with carcinoembryonic antigen as a model showing good linear response in the range of 0～100 ng/mL.Moreover,the stableloading of Cu active sites endows the sensing platform with anti-inference capacity and reuse without loss in catalytic activity after six months.4.In this study,we find that CuFe LDH with oxidase and peroxidase activities for the first time can generate H2O2 by itself.And ROS(hydroxyl radicals,superoxide anions,and singlet oxygen)is proved by electron spin resonance spectroscopy.CuFe LDH not only has the ability to oxidate GSH,which further inhibits the consumption of ROS,achieving the effect of synergistic treatment.Under the premise of no need to introduce additional energy,it can produce more than 99%antibacterial efficiency against Escherichia coli(E.coli)and Methicillin-resistant Staphylococcus aureus(MRSA).In vivo experiments show that MRSA-infected wounds are accelerated wounds heal faster when CuFe LDH sterilization is applied.More importantly,CuFe LDH has good biocompatibility and low toxicity.This work illustrates the synergistic antibacterial mechanism of LDH for the first time and expands its application in antibacterial field.