Novel Label-free Fluorescent Biosensing Methods Based On Poly(T)DNA And BSA Templated Copper Nanoclusters

Fluorescent metal nanoclusters are a new type of luminescent nanomaterials with the advantages of small size,easy synthesis,strong anti-photobleaching,good biocompatibility and so on.In recent years,due to the characteristics of low cost,good water solubility,stable optical properties and adjustable fluorescence emission,fluorescent copper nanoclusters have attracted a wide range of research interests in bioanalysis,biological imaging,medical applications and environmental monitoring.In this paper,poly（T）DNA and bovine serum albumin（BSA）were used as synthesis templates to synthesize fluorescent copper nanoclusters with different fluorescence emission wavelengths as a fluorescent signal probe to detect biological small molecules and mercapto compounds.In addition,the fluorescent nanoclusters with protein as template can form complexes with MnO₂ nanosheets,and a new,sensitive and unlabeled new sensing method has been developed for the detection of proteins The main research contents are as follows:（1）a simple,rapid and label-free fluorescent sensor has been developed for the detection of kojic acid based on the kojic acid-induced blocking of the formation of Poly（thymine）（Poly T）-templated copper nanoparticles.Fluorescent copper nanoclusters were synthesized with Poly（T）DNA as a template and ascorbic acid as a reducing agent within 5 minutes at room temperature.When in the presence of kojic acid,since Cu²⁺ and kojic acid form stable complexes,the formation of fluorescent copper nanoparticles of Poly（T）DNA template is effectively prevented,which leads to the weakening of the fluorescence signal of the system.Under the optimized conditions,the linear range of kojic acid was from 0.1 μmol/L to 300 μmol/L,and the detection limit was 10 nmol/L.At the same time,the method can be used for the determination of kojic acid content in food samples.（2）A rapid,sensitive and label-free fluorescence sensor has been developed for Hg（II）ion detection on the basis of blocking of cysteine-quenching of fluorescent Poly T-templated copper nanoparticles.In the presence of Cys,a stable Cu-S bond is formed between Cys and copper nanoclusters,thereby effectively quenching the fluorescent signal of copper nanoclusters.When the system was added to Hg²⁺,Cys and Hg²⁺ formeda more stable Hg-S bond,leading to Cys away from the surface of fluorescent copper nanoclusters,the fluorescence intensity of the system was restored.This method realizes the sensitive detection of target Hg²⁺,the linear range is from 0.5 nmol/L to 100 nmol/L,the detection limit is 0.1 nmol/L.（3）a novel fluorescent sensing strategy has been developed for sensitive detection of glutathione on the basis of MnO₂ nanosheets–copper nanoclusters composites.Fluorescent copper nanoclusters were prepared by simple step-by-step chemical reduction using bovine serum albumin as a template.When the MnO₂nanosheets-fluorescent copper nanoclusters are formed,the fluorescence signals of the copper nanoclusters can be effectively quenched by the fluorescence energy resonance transfer by the MnO₂ nanosheets.When the target glutathione was added,MnO₂ nanosheets could be reduced to produce Mn2+,which destroyed the MnO₂ nanosheets and inhibited the fluorescence quenching effect caused by MnO₂ nanosheets.The fluorescence signal of the final system was restored.The linear range of this method is from 0 μmol/L to 300 μmol/L,the detection limit is 100 nmol/L,and it has a good selectivity.The results show that the method is simple,rapid,low cost,easy to prepare and so on.（4）A novel method for fluorescence biosensor was developed for the determination of glucose based on H₂O₂-induced destruction of quencher MnO₂ nanosheets by using MnO₂ nanorods-fluorescent copper nanoclusters as fluorescence probes.When in the presence of H₂O₂,the copper nanoclusters are released from the surface of MnO₂ nanosheets,and the fluorescence signal of the system is recovered.On the other hand,glucose can be oxidized by dissolved oxygen（O2）in the presence of glucose oxidase（GOx）to produce glucuronic acid and H₂O₂.Consequently,the concentration of glucose can be determined indirectly by the amount of enzymatically generated H₂O₂ according to the fluorescence recovery.The linear range of this method is from 1 μmol/L to 200μmol/L,and the detection limit is 100 nmol/L.At the same time,this method can be used for the determination of glucose in human serum samples.