Novel Fluorescence Biosensing Based On The Regulation Of Cu(Ⅱ) Ions Mediated Oxidation Reaction

Fluorescence analysis has the advantages of simple operation,fast response,high sensitivity,excellent selectivity and high cost-effectiveness,and it has a broad application prospect in the fields of biosensing and biochemical analysis.Based on the unique oxidation reaction between o-phenylenediamine and Cu²⁺,the oxidation reaction was regulated by complexation reaction and redox reaction.A series of fluorescence biosensing platforms were developed for the sensitive detection of Cu²⁺ions,kojic acid,ascorbic acid（AA）,alkaline phosphatase（ALP）and interleukin-6（IL-6）antigen.A cascade amplification strategy was developed by coupling fenton-like reaction and chemical redox cycle amplification method,a split-type ultrasensitive fluorescence sensing platform was constructed for the ultrasensitive detection of H₂O₂,glucose and IL-6 antigens.The main research contents are as follows:1.Using copper nanoclusters（GSH-Cu NCs）as energy acceptor and Cu²⁺oxidation of OPD to produce 2,3-diaminophenazine（DAP）as energy donor,a novel label free atio fluorescence sensing platform was developed based on F（?）rster resonance energy transfer（FRET）.With an increase in Cu²⁺concentration,DAP generated by oxidized OPD was served as an energy acceptor to inhibit the fluorescence emission of GSH-Cu NCs（energy donor）,thereby reducing the fluorescence of GSH-Cu NCs at 420 nm and enhancing the fluorescence of DAP at 555 nm.In addition,KA could reacted with Cu²⁺through the strong complexation to form stable copper kojate,which prevented Cu²⁺catalyzing OPD to form DAP.As a consequence,FRET between GSH-Cu NCs and DAP was inhibited.This process restoreed the fluorescence of GSH-Cu NCs and weakened the fluorescence of DAP.Using the ratio(F₅₅₅/F₄₂₀)as signal output,the platform realized the sensitive detection of Cu²⁺and KA.The linear range of Cu²⁺was 0.1～50μmol/L with a limit of detection of 20 nmol/L.The recoveries were in the range of 98.7%～100.9%,the relative standard deviation（RSD）of three parallel determination result was 3.2%～4.0%.The linear range of KA was 0.2～300μmol/L,the detection limit was 50 nmol/L,the recovery was 99.5%～101.5%,RSD was no more than 4.3%.As a result,the sensitivity of the strategy were greatly improved due to the dual-emission reverse change ratiometric profile.2.Using OPD as signal precursor,the in situ generated（DAP）and3-（dihydroxyethyl）furan[3,4-b]quinoxaline-1-one（DFQ）as two fluorescence indicators were used as signal outputs,a novel ratio fluorescence assay was developed for the sensitive detection of ascorbic acid,Alkaline phosphatase and interleukin 6（IL-6）antigens.On the one hand,Cu²⁺can be reduced to suppress the generation of DAP in the presence of AA.On the other hand,the oxidation product of AA was dehydroascorbic acid（DHAA）,it can react with OPD to form DFQ with an emission peak at 420 nm.With AA concentration increased,the fluorescence of DAP（555 nm）decreased and that of DFQ（420 nm）increased significantly.ALP catalyzed the hydrolysis of substrate L-ascorbic acid-2-phosphate（AA2P）to produce AA.The ratio of F₅₅₅/F₄₂₀ is linear with the alkaline phosphatase activity.Using the biomarker interleukin 6（IL-6）as the model analyte,the application of the ratio fluorescence method in immunoassay was verified.The linear range of AA was 0.05～20μmol/L,and the detection limit was 150 nmol/L.The linear range of ALP was 0.02～1.0 m U/m L,the correlation coefficient（R²）was0.9994,and the detection limit was 0.006 m U/m L.The linear range of IL-6 was 0.2～10pg/m L,the detection limit was 0.06 pg/m L,the recoveries were 96.4%～110.5%,RSD was no more than 4.8%.This method is simple and feasible,with clear reaction mechanism,high sensitivity,good selectivity and excellent analytical performance.3.An ultrasensitive split-type fluorescence sensing platform was constructed for the detection of H₂O₂,glucose and IL-6 antigens based on a cascade signal amplification strategy by coupling chemical redox-cycling and fenton-like reaction.In this strategy,Cu²⁺could oxidize chemically o-phenylenediamine（OPD）to generate photosensitive 2,3-diaminophenazine（DAP）and Cu⁺ion.On the other hand,H₂O₂ reacted with Cu⁺to produce hydroxyl radicals（·OH）and Cu²⁺ions through a Cu⁺-mediated Fenton-like reaction.The produced·OH and recycled Cu²⁺ions could take turns oxidizing OPD to generate more photoactive DAP,which triggering a chemical redox-cycling reaction and a remarkable improvement of fluorescence signal.In the presence of glucose,glucose oxidase（GOx）catalyzed the oxidation of substrates（glucose）and dissolved oxygen（O₂）to generate gluconic acid and H₂O₂,and the H₂O₂-triggered cascade signal amplification strategy for determination of glucose.Using IL-6 as a model target,a new method of split-type immunoassay was developed.The linear range of H₂O₂ was 100 pmol/L～1μmol/L,and the detection limit was 20 pmol/L.The linear range of glucose was 1 nmol/L～1μmol/L,and the detection limit was 0.3 nmol/L.The linear range of IL-6 was 20fg/m L～10 pg/m L a lower limit of detection of 5 fg/m L.The RSD was less than 4.5%,and the recoveries were range from 96.5%to 105.8%.