Construction Of Fluorescent Nanosensors Based On Metal Nanoclusters And Its Applications In Bio-enzyme Detection

Bio-enzymes play a key role in maintaining normal life activities by regulating the rate of intracellular biochemical reactions and controlling the processes of various physiological processes.Researches have shown that abnormal activity of some enzymes is closely related to the occurrence of some major diseases,such as cancer.Thus,enzymes are usually regarded as biomarkers for the diagnosis of related diseases and drug discovery.At present,a variety of methods have been reported for bio-enzymes detection,including enzyme-linked immunosorbent assay,high performance liquid chromatography,surface-enhanced Raman,optical detection and so on.Each analysis method has its unique advantages,but they are also limited by the expensive precision instruments and equipment,tedious detection steps and long detection time.Therefore,it is of great significance to establish a new analytical method for the simple,rapid and accurate determination of enzymes in complex biological samples for the diagnosis and treatment of diseases and the protection of human health.Owing to its high sensitivity,good selectivity,simple operation,low cost and real-time monitoring,fluorescence analysis has attracted wide attention and rapid development.As a new type of nanomaterials,metal nanoclusters possess the advantages of low toxicity,good biocompatibility and light stability.Based on the excellent physical and chemical properties of metal nanoclusters combined with the advantages of sensors,a series of fluorescent nanosensors have been constructed for the simple,sensitive and selective detection of various enzymes in complex biological samples,improving the early prevention and precise treatment of major diseases and contributing to human health.The main research is briefly summarized as follows:1.A new fluorescent biosensing method for the detection of PKA activity was developed by using 11-mercaptoundecanoic acid-capped gold nanoclusters?Au NCs?and graphene oxide?GO?with low background noise.In this strategy,the special designed peptide could be anchored on the surface of Au NCs by the Au-S bond and also adsorbed on the surface of GO owing to the electrostatic interaction.As a result,the fluorescence of Au NCs was quenched leading to low background fluorescence due to the forster resonance energy transfer?FRET?between Au NCs and GO via peptide as a bridge.However,when the substrate peptide was phosphorylated by PKA,the FRET between GO and Au NCs was disrupted because of the weakened interaction between the phosphorylated peptide and the GO,resulting in recovery of the fluorescence intensity.The developed label-free fluorescence“turn-off-on”method can detect PKA activity in the range of 0.6-2.0 U/mL with a detection limit of 0.17 U/mL.The feasibility of this present method for kinase inhibitor screening was also studied by assessment of H-89 with an IC₅₀ value of 0.049?mol/L.2.A sensitive fluorometric assay is reported for label-free detection of the activity of protein kinase?PKA?and its inhibitor.It is based on the Eu³⁺-modulated fluorescence of peptide-stabilized gold nanoclusters?Au NCs?.Both adenosine-5?-triphosphate?ATP?and adenosine-5?-diphosphate?ADP??formed from ATP by PKA-catalyzed hydrolysis?can enhance the fluorescence of Au NCs.Due to the strong binding ability between ATP and Eu³⁺,the addition of Eu³⁺can quench the fluorescence of the ATP/Au NCs system to,while addition of Eu³⁺had no effect on the fluorescence of the ATP/PKA/Au NCs system.Based on this“turn-on”method,the activity of PKA can be detected with high sensitivity in 0.05-1.6 U/mL activity range,with a detection limit of 0.02 U/mL?3??.The feasibility of this method for screening of inhibitors was also studied.The IC₅₀value for the commercially available inhibitor H-89 was found to be 0.043?mol/L.Moreover,the detection method was also applied to monitoring the drug-stimulated activation of PKA in HepG-2 cell lysates.3.In this work,a novel fluorescence nanosensor for the determination of pyrophosphatase?PPase?activity based on tannic acid capped copper nanoclusters?Cu NCs?was established.The fluorescence of Cu NCs can be quenched by Fe³⁺due to electron transfer from electron-rich tannic acid to electron-deficient Fe³⁺.Subsquently,pyrophosphate?PPi?can restore the Fe³⁺-quenched Cu NCs fluorescence owing to the strong binding affinity between PPi and Fe³⁺.However,when PPi was hydrolysed by PPase into two phosphate ions?Pi?,free Fe³⁺was released from PPi,and leading to quench the fluorescence of Cu NCs again.Under optimized conditions,fluorescence intensity linearly correlated with the activity of PPase in the range from 0.50 to 18.0U/L with a detection limit of 0.19 U/L.Additionally,the proposed fluorescence strategy was employed for the determination of PPase in human serum samples with satisfactory results,indicating the great potential application for biological analysis.4.Herein,a fluorescent bi-enzymes sensing platform for ultrasensitive detection of alkaline phosphatase activity was developed based on 3-aminophenylboronic acid functionalized gold nanoclusters?APBA-Au NCs?.Alkaline phosphatase?ALP?can catalyze hydrolysis of phenyl phosphate to phenol,which can be subsequently hydroxylated to generate catechol in the presence of tyrosinase?TYR?.Due to the special covalent combination between the catechol and boric acid group,the five-membered cyclic esters can be formed on the ligands of APBA-Au NCs,leading to the fluorescence quenching of the Au NCs.Thus,according to the change of fluorescence signal,the proposed biosensor can be utilized to ultrasensitive detection ALP.Under the optimal condition,there exhibited good linear relationships between fluorescence intensity change of Au NCs and the concentration of ALP in the range of 0.02-2.0 U/L.The detection limits reached 0.005 U/L.The developed strategy was employed to monitor enzyme activity in real samples with satisfactory results obtained,illustrating the potential application of this biosensor.5.Bimetallic nanoclusters,compared to monometallic nanoclusters,possess better chemical performance.Herein,with the assistance of chondroitin sulfate,a novel fluorescent gold/silver nanoclusters?Au/Ag NCs?have been developed and applied to establish a sensing platform for sensitive detection of hyaluronidase?HAase?.The Au/Ag NCs fluorescence intensity can be distinctly quenched by gold nanoparticles?Au NPs?due to the inner-filter effect?IFE?.With the introduction of protamine?PRO?,the positively charged PRO can bind to negatively charged Au NPs via electrostatic attraction resulting in the aggregation of gold nanoparticles.When PRO was preferential interaction with the negatively charged hyaluronic acid?HA?,it would lead to the disaggregation of Au NPs and quench the fluorescence again.However,when HAase hydrolyzed HA into small fragments,the interaction between HA and protamine was blocked,which led to the aggregation of Au NPs again,thus causing the change of fluorescence signal in the system.Therefore,this sensing system can be utilized to detect HAase level in the range of 0.5-37.5 U/mL.The developed strategy not only expands the application scope of bimetallic nanoclusters,but also provides a new approach for the determination of HAase.