Preparation Of Novel Fluorescent Nanomaterials And Their Application In Alkaline Phosphatase Detection

Biosensor is an interdisciplinary subject that combines bioactive materials（enzyme,protein,DNA,antibody,antigen,biofilm,etc.）with physicochemical transducers.It is an advanced detection method essential for the development of biotechnology.Because of its advantages such as good selectivity,high sensitivity,fast analysis speed and low cost,it has been widely used in food,pharmaceutical,chemical,biomedical,and environmental monitoring.In recent years,nanomaterials have attracted a lot of interest due to their good biocompatibility,excellent electrochemical performance and catalytic performance.The combination of biosensors and new nanomaterials promotes the development of signal conversion technology.Fluorescent biosensors have been widely studied due to their advantages such as high sensitivity,simple operation,low cost,and good selectivity.However,with the continuous advancement of modern analysis technology,the detection requirements of the objects to be detected have also increased,so it is urgent to construct a new type of fluorescent sensors to improve the detection accuracy and sensitivity of detection.Improving the accuracy of the sensor can be achieved by constructing a ratio fluorescence sensor with a reference signal,while the sensitivity of the sensors can be achieved by fabricating new nanomaterials to amplify the detection signal and selecting the quenching agent with high quenching efficiency to reduce the background signal.Focusing on the application of new functional nanomaterials in fluorescent biosensors,this paper uses two strategies of background reduction and ratio signal to build a highly sensitive and accurate new fluorescent biosensor for the detection of alkaline phosphatase（ALP）.The main contents are as follows:（1）The high quenching efficiency of 2,6-dichlorophenolindophenol（DCIP）on gold nanoclusters（AuNCs）was used to reduce the background signal and increase the sensitivity of the sensor,thereby achieving sensitive detection of ALP.In this study,the positively charged polyallylamine hydrochloride（PAH）cross-linked AuNCs（PAH-AuNCs）with aggregation-induced enhancement properties（AIE）were synthesized.It can electrostatically adsorb the negatively charged DCIP.Due to the fluorescence resonance energy transfer（FRET）from PAH-AuNCs to DCIP,the fluorescence of PAH-AuNCs was significantly quenched.However,the reduction reaction of DCIP from blue to colourless by L-ascorbic acid（AA）which is generated by the ALP catalyse hydrolysis of 2-Phospho-L-ascorbic acid（AAP）disturbs the FRET between PAH-AuNCs to DCIP.The quenched PAH-AuNCs fluorescence can be recovered effectively.The innovation of this paper is the synthesis of AIE-enhanced PAH-AuNCs nanomaterials,and then applied for constructing an effective FRET strategy to achieve highly sensitive detection of ALP.More importantly,the distinct colorimetric signal change can be used to visually distinguish the presence of ALP.Good linear relationships of fluorescence and colorimetric sensing towards ALP were obtained in the range from 0.5 to 100 U/L,and the detection limits were 0.2 U/L and 0.5 U/L,respectively.In addition,the proposed FRET sensing system was applied to the detection of ALP in human serum samples with satisfactory results.（2）A novel and convenient ratio fluorescence sensor for detecting ALP activity was designed to improve the detection accuracy.Amino-functionalized mesoporous silica nanoparticle-gold nanoclusters（MSN-AuNCs）nanocomposites were integrated with o-phenylenediamine（OPD）to form a ratiometric fluorescence nanoplatform.MSN-AuNCs are used as reference signals,and OPD is used as identification elements and response signals.The presence of ALP induced the generation of quinoxaline（QX）derivative which called 3-（dihydroxyethyl）furo[3,4-b]quinoxaline-1-one（DFQ）with strong fluorescence emission at 450nm,while the orange-red fluorescence of MSN-Au NCs at 580 nm was slightly quenched by Fe²⁺.Meanwhile,an obvious fluorescence color change from orange-red to purple and fnally to blue can be observed by naked eyes with the increasing of ALP concentration.Therefore,employing the fluorescence emission of DFQ at 450 nm as the reporter signal and the fluorescence emission of MSN-AuNCs at 580 nm as a reference signal,a sensitive ratiometric detection method for ALP was developed.Quantitative detection of ALP activity in the linear range from 0.2 to 80U/L with a detection limit of 0.1 U/L can be realized in this way,which endows the assay with high sensitivity enough for practical detection of ALP in human serum samples,indicating that it has broad application prospects in clinical diagnosis.（3）Based on the stimulation responsive lanthanide coordination polymer,a simple and novel ratio fluorescence sensing strategy for the detection of ALP activity was proposed.A supramolecular lanthanide coordination polymer（Tb-GMP）was constructed with Tb³⁺as the metal ion and guanine single nucleotide（GMP）as the ligand.To construct the ratiometric fluorescence biosensor,the lanthanide coordination polymer Tb-GMP was used as a response signal,and CDs were used as a reference signal due to its good stability.When excited at 290 nm,the polymer network emits green characteristic fluorescence of Tb³⁺at 545 nm,while the CDs encapsulated in the polymer network emit blue fluorescence at 370 nm.After adding ALP to the system,the substrate GMP can be hydrolyzed by the ALP thus induced the destruction of the polymer network,resulting in the significant quenching of the fluorescence of Tb³⁺,while slight increasing of the fluorescence of CDs due to the release of CDs from the polymer network.Using the proposed fluoresence biosensor,the ALP can be detected sensitively by comparing the relationship between the fluorescence intensity ratio of the two signals and the concentration of ALP.The detection linear range is 0.5-80 U/L,and the detection limit is 0.13 U/L.The innovation of this work is that using natural substrate GMP instead of artificial substrate to detect ALP may provide the actual activity level of ALP under physiological conditions,so that it can more accurately evaluate the function of ALP in biological systems.In addition,the proposed ratiometric sensing system was applied to the detection of ALP in human serum samples with satisfactory results,indicating the potentical application in clinical diagnosis.