Preparation And Bioenzyme Sensing Applications Of Fluorescent Copper Nanoclusters-based Composite Probes

Fluorescent copper nanoclusters(Cu NCs)have the advantages of low cost,excellent biocompatibility and adjustable fluorescence properties,thus have great application value in biochemical analysis,disease diagnosis and treatment,and environmental monitoring.However,Cu NCs often have low fluorescence quantum yields,poor stability,and weak fluorescence response to the bioactive molecules,which severely restricts the applications of Cu NCs in the field of analysis and detection.In this article,the fluorescent copper nanoclusters were compounded with chitosan,2D matrix materials(boehmite sheets,manganese dioxide nanosheets),and 3D matrix material(mesoporous silica)to effectively regulate the fluorescence intensity,quantum yield,stability and fluorescence response of Cu NCs by using the confinement induced emission effect and bioactive molecular response capacity of matrixs.On this basis,a series of fluorescent copper nanocluster composite probes were constructed for the detection of various enzyme activities and screening of the related inhibitors.Specific research work includes1.An electrostatic confined in situ method was developed to prepare chitosan-glutathione-Cu NCs(CS-GSH-Cu NCs)composite probe by using CS as the confinement matrix and GSH as both ligand and reducing agent.The fluorescence properties of GSH-Cu NCs were significantly improved by the antioxidant capacity and the confinement effect of CS.The quantum yield of GSH-Cu NCs was increased from0.4%to 5.2%,and the stability of GSH-Cu NCs was also significantly improved.Further studies showed that lysozyme(Lys)can quench the fluorescence of CS-GSH-Cu NCs probe by catalyzing the hydrolysis of CS.Based on this,a fluorescence method was established to determine the activity of Lys.The linear detection range was 0-110n M,and the detection limit was 1.6 n M.This method has high selectivity for Lys and can be used for the determination of Lys in actual serum samples.2.The 2D boehmite(Boe)nanosheets was used as the confinement matrix,the combination of Boe and GSH-Cu NCs was realized by the electrostatic interaction between the GSH-Cu NCs and Boe.The Boe@GSH-Cu NCs composite probe showed good fluorescence intensity and stability,and the excitation wavelength was blue shifted from 385 nm to 330 nm.Therefore,4-nitrophenyl-?-D-glucopyranoside(PNPG,?_max=310 nm)can effectively quench the fluorescence of Boe@GSH-Cu NCs by inner filter effect(IFE).However,?-glucosidase(?-Glu)can catalyze the hydrolysis of PNPG to p-nitrophenol(?_max=410 nm),thus restore the fluorescence of Boe@GSH-Cu NCs.A novel method was developed to detect?-Glu activity in serum samples and screen natural?-Glu inhibitors.3.A facile and efficient sensing platform was proposed for butyrylcholinesterase(BCh E)detection by using a Cu NCs composite fluorescent probe which is composed of polyethyleneimine-protected Cu NCs(PEI-Cu NCs)and Mn O2 nanosheets.The emission of PEI-Cu NCs can be obviously quenched by Mn O2 nanosheets through IFE.In the presence of BCh E,the hydrolysis of butyrylthiocholine iodide produces thiocholine which can reduce Mn O2 nanosheets into Mn²⁺,thus leading to the fluorescence recovery of PEI-Cu NCs.On this basis,a fluorescence method for BCh E activity detection was established with a detection limit of 2.26 U/L.This method can be applied to determining BCh E in real serum samples,and can effectively distinguish the serum of normal people and liver cirrhosis patients,which has potential application value in the clinical diagnosis of liver disease.In addition,the method can be used to screen BCh E inhibitors,which are promising to alleviate the symptoms of Alzheimer's disease.4.The 3D mesoporous silica particles(M-Si O₂)was used as the confinement matrix,the N-acetyl-L-cysteine-protected Cu NCs(NAC-Cu NCs)can be assembled into M-Si O₂ through electrostatic interaction.The assembly process only took 2 min.Owing to the electrostatic confinement effect of M-Si O₂,the fluorescence emission and quantum yield of NAC-Cu NCs can be significantly improved.The prepared M-Si O2@NAC-Cu NCs composite was further integrated with manganese dioxide(Mn O2)nanosheets,the emission intensity of M-Si O2@NAC-Cu NCs was significantly decreased with the addition of Mn O2 nanosheets by IFE.While,acid phosphatase(ACP)can hydrolyze L-ascorbic acid-2-phosphate to ascorbic acid,which can further change Mn O2 nanosheets to Mn²⁺,thus recover the fluorescence of Si O2@NAC-Cu NCs.On this basis,a fluorescence analysis method was developed to detect the activity of ACP,a key indicator in the diagnosis of prostate cancer.