A kind of preparation method and application based on copper nano-cluster-Resonance energy transfer system construction immunosensor
阅读说明：本技术 一种基于铜纳米簇-共振能量转移体系构建的免疫传感器的制备方法及应用 (A kind of preparation method and application based on copper nano-cluster-Resonance energy transfer system construction immunosensor ) 是由 魏琴 贾越 杨磊 薛经纬 马洪敏 赵磊 于 2019-09-12 设计创作，主要内容包括：一种基于铜纳米簇-共振能量转移体系构建的免疫传感器的制备方法及应用,该发明属于纳米材料与共振能量转移FRET领域；本发明以牛血清蛋白BSA包裹的铜纳米团簇Cu NCs作为能量供体,并在其表面利用L-抗坏血酸原位生长金纳米粒子Au NPs作为能量受体,得到Cu NCs-Au NPs三维共振结构,供受体对之间通过偶极-偶极的非辐射作用方式传递能量,其传递速率与距离的六次方呈反比,二者之间光谱的高比率重叠以及超近的配对距离,显著猝灭了Cu NCs的ECL发射并控制发光强度在合理范围内,为生物标志物的痕量检测提供了一种新的ECL-FRET能量转移新模型,为猝灭型ECL传感体系的机理探究提供了一种全新的思路。(A kind of preparation method and application based on copper nano-cluster-Resonance energy transfer system construction immunosensor, the invention belong to nano material and the field Resonance energy transfer FRET；The present invention is using the copper nanocluster Cu NCs that bovine serum albumin BSA is wrapped up as energy donor, and on its surface using L-AA growth in situ gold nanoparticle Au NPs as energy acceptor, obtain Cu NCs-Au NPs three-dimensional resonance structure, energy is transmitted for the non-radiative activity mode between receptor pair by dipole-dipole, six powers of its delivery rate and distance are in inverse ratio, the height ratio overlapping of spectrum between the two and super close matching are adjusted the distance, the ECL for significantly having quenched Cu NCs emits and controls luminous intensity in the reasonable scope, a kind of new ECL-FRET energy transfer new model is provided for the trace detection of biomarker, it is probed into for the quenching type ECL mechanism for sensing system and provides a kind of completely new thinking.)
1. a kind of preparation method based on copper nano-cluster-Resonance energy transfer system construction immunosensor, which is characterized in that The following steps are included:
(1) glass-carbon electrode is cleaned by ultrasonic 30 s in ethyl alcohol and deionized water respectively, successively with 1.0 μm, 0.3 μm and 0.05 μm of Al2O3Polishing powder, which polishes it, is allowed to smooth as similar mirror surface, with being dried with nitrogen；
(2) 6 μ L are uniformly dispersed, Cu NCs-Au NPs solution drop coating that concentration is 1 ~ 3 mg/mL is to glass-carbon electrode table Face, at room temperature naturally dry；
(3) the above-mentioned glass-carbon electrode dried is inserted into the recombinant protein A solution of 5 ~ 10 μ g/mL, in 4oHatch 30 under C Min, ultrapure water are cleaned, at room temperature naturally dry；
(4) bovine serum albumen solution that 6 μ L mass fractions are 0.1% is added dropwise, is allowed to close nonspecific activity site, uses pH 7.4 phosphate buffer solution rinses electrode surface, at room temperature naturally dry；
(5) the antibody standard solution that 6 μ L concentration are 1 ~ 2 μ g/mL is added dropwise, in 4oHatch 1 h under C, with the phosphoric acid of pH 7.4 Salt buffer solution flushing electrode surface, 4oC dries；
(6) antigenic solution corresponding to antibody of 6 μ L unknown concentrations is added dropwise, in 4oHatch 1 h under C, with the phosphoric acid of pH 7.4 Salt buffer solution flushing electrode surface, 4oC dries, and sensor building finishes.
2. a kind of system based on copper nano-cluster-Resonance energy transfer system construction immunosensor as described in claim 1 Preparation Method, the Cu NCs-Au NPs solution, prepares according to the following steps:
It at room temperature, is 1 ~ 2 mg/mL's by 1 mL, the copper-bath that concentration is 5 ~ 10 mmol/L and 1 mL, concentration Bovine serum albumen solution is sufficiently mixed, and after being vigorously stirred 2 ~ 5 min, 0.1 ~ 1 mL is added, concentration is 1 ~ 2 mol/L's Then the mixed liquor is placed in 65 by sodium hydroxide solutiono12 h in C water-bath, until solution colour becomes brown from light blue, At this point, copper ion is successfully restored by BSA, the copper nano-cluster Cu NCs of BSA cladding is obtained；
The Cu NCs of preparation is mixed with the L-AA solution that concentration is 10 mmol/L, 5 mL deionized waters are then added It is diluted, then, the chlorauric acid solution of 50 ~ 150 uL, mass fraction 2% is added in Xiang Shangshu solution, persistently stirs 2 h The Cu NCs-Au NPs three-dimensional resonance nanostructure of atropurpureus is obtained afterwards.
3. a kind of system based on copper nano-cluster-Resonance energy transfer system construction immunosensor as described in claim 1 Preparation Method, which is characterized in that the antigen is human body acute bacterial infection and pyemic marker Procalcitonin.
4. a kind of system based on copper nano-cluster-Resonance energy transfer system construction immunosensor as described in claim 1 The sensor of Preparation Method preparation is used to detect the concentration of Procalcitonin.
5. the concentration of detection Procalcitonin as claimed in claim 4, which is characterized in that detecting step is as follows:
(1) use the three-electrode system of electrochemical workstation as excitaton source, Ag/AgCl electrode is as reference electrode, platinum electrode As to electrode, prepared electroluminescent sensor is as working electrode, by electrochemical workstation and ultraweak optical detector Combination, photomultiplier tube high pressure are set as 800 V, and cyclic voltammetry scan current potential is 0 ~ 1.5 V, and sweeping speed is 200 mV/s；
(2) in 50 ~ 80 mmol/L triethylamine phosphate buffers, pass through electroluminescent containing concentration in 10 mL, pH 7.4 System detects the electroluminescence signal intensity in a series of determinand Modified antigen state lower sensor of various concentrations, draws Working curve；
(3) determined antigen is replaced to detect practical blood serum sample.
Technical field
The invention belongs to nano sciences and immunoassay field.
Background technique
Copper nanocluster is that nano science develops a kind of novel nano-material for being risen, good optical stability, Big stoke shift, high quantum yield and good biocompatibility are that its application in bio-sensing field is established Solid foundation.Copper nanocluster can be synthesized in alkaline environment by a variety of micro- reducing agents such as nucleic acid, aglucon, protein, In the electroluminescent optical signal of shorter wavelength is shown under low potential using bovine serum albumin as the copper nanocluster of template, meanwhile, Its surface coated bovine serum albumin exposes a large amount of functional group's active site, provides greatly for the modification of biomolecule It is convenient, there is advantageous advantage in immunoassay compared to electroluminors such as semiconductor-quantum-points.However, it is higher Electroluminescent efficiency also allow sensing platform signal be difficult to control, be based on this it is proposed that using Resonance energy transfer system come The needs of object trace analysis can be met by controlling its luminous intensity, it is intended to the mechanism of system is sensed for quenching type ECL It probes into and provides a kind of completely new thinking.
Summary of the invention
Technical assignment of the invention first is that in order to make up the deficiency of ECL-FRET model evidence at this stage, a kind of Cu is provided The preparation method of NCs-Au NPs three-dimensional resonance structure.This method is to realize that three conditions that Resonance energy transfer to be met are to grab Hand, gets rid of the unilateral behavior that traditional ECL-FRET sensing model emphasizes merely spectra overlapping, and emphasis probes into verifying quenching efficiency The relationship of distance between co-receptor probes into for the quenching type ECL mechanism for sensing system and provides a kind of completely new thinking.
Technical assignment two of the invention is to provide the ECL-FRET model in the purposes in bio-sensing field, is based on the mould The biosensor of type research and development can quickly detect Procalcitonin, and high sensitivity, detection limit is low, favorable reproducibility, in clinical detection In have broad application prospects.
To achieve the above object, The technical solution adopted by the invention is as follows:
1. a kind of preparation method based on copper nano-cluster-Resonance energy transfer system construction immunosensor, feature exist In, comprising the following steps:
(1) glass-carbon electrode is cleaned by ultrasonic 30 s in ethyl alcohol and deionized water respectively, successively with 1.0 μm, 0.3 μm and 0.05 μm of Al2O3Polishing powder, which polishes it, is allowed to smooth as similar mirror surface, with being dried with nitrogen；
(2) 6 μ L are uniformly dispersed, Cu NCs-Au NPs solution drop coating that concentration is 1 ~ 3 mg/mL is to glass-carbon electrode table Face, at room temperature naturally dry；
(3) the above-mentioned glass-carbon electrode dried is inserted into the recombinant protein A solution of 5 ~ 10 μ g/mL, in 4oHatch 30 under C Min, ultrapure water are cleaned, at room temperature naturally dry；
(4) bovine serum albumen solution that 6 μ L mass fractions are 0.1% is added dropwise, is allowed to close nonspecific activity site, uses pH 7.4 phosphate buffer solution rinses electrode surface, at room temperature naturally dry；
(5) the antibody standard solution that 6 μ L concentration are 1 ~ 2 μ g/mL is added dropwise, in 4oHatch 1 h under C, with the phosphorus of pH 7.4 Hydrochlorate buffer solution flushing electrode surface, 4oC dries；
(6) antigenic solution corresponding to antibody of 6 μ L unknown concentrations is added dropwise, in 4oHatch 1 h under C, with the phosphoric acid of pH 7.4 Salt buffer solution flushing electrode surface, 4oC dries, and sensor building finishes.
A kind of preparation method based on copper nano-cluster-Resonance energy transfer system construction immunosensor described in 2., The Cu NCs-Au NPs solution, prepares according to the following steps:
It at room temperature, is 1 ~ 2 mg/mL's by 1 mL, the copper-bath that concentration is 5 ~ 10 mmol/L and 1 mL, concentration Bovine serum albumen solution is sufficiently mixed, and after being vigorously stirred 2 ~ 5 min, 0.1 ~ 1 mL is added, concentration is 1 ~ 2 mol/L's Then the mixed liquor is placed in 65 by sodium hydroxide solutiono12 h in C water-bath, until solution colour becomes brown from light blue, At this point, copper ion is successfully restored by BSA, the copper nano-cluster Cu NCs of BSA cladding is obtained.It is with concentration by the Cu NCs of preparation The L-AA solution of 10 mmol/L mixes, and 5 mL deionized waters are then added and are diluted, then, in Xiang Shangshu solution The chlorauric acid solution of 50 ~ 150 uL, mass fraction 2% is added, obtains the Cu NCs-Au NPs of atropurpureus after persistently stirring 2 h Three-dimensional resonance nanostructure.
A kind of preparation method based on copper nano-cluster-Resonance energy transfer system construction immunosensor described in 3., It is characterized in that, the antigen is human body acute bacterial infection and pyemic marker Procalcitonin.
A kind of preparation method system based on copper nano-cluster-Resonance energy transfer system construction immunosensor described in 4. Standby sensor is used to detect the concentration of Procalcitonin.
The concentration of detection Procalcitonin described in 5., which is characterized in that detecting step is as follows:
(1) use the three-electrode system of electrochemical workstation as excitaton source, Ag/AgCl electrode is as reference electrode, platinum electrode As to electrode, prepared electroluminescent sensor is as working electrode, by electrochemical workstation and ultraweak optical detector Combination, photomultiplier tube high pressure are set as 800 V, and cyclic voltammetry scan current potential is 0 ~ 1.5 V, and sweeping speed is 200 mV/s；
(2) in 50 ~ 80 mmol/L triethylamine phosphate buffers, pass through electroluminescent containing concentration in 10 mL, pH 7.4 System detects the electroluminescence signal intensity in a series of determinand Modified antigen state lower sensor of various concentrations, draws Working curve；
(3) determined antigen is replaced to detect practical blood serum sample.
Beneficial achievement of the invention
(1) three-dimensional resonance ECL sensing model Cu NCs-Au NPs is prepared for based on copper nanocluster for the first time, which passes through confession The non-radiative activity mode of dipole-dipole, which transmits energy, between receptor pair can effectively control the ECL intensity of copper nano-cluster；
(2) present invention is based on three-dimensional resonance structure C u NCs-Au NPs for the first time, constructs a kind of ECL sensing platform for dropping calcium Plain former actual sample detection, the transducer sensitivity is high, and detection range is wide, and detection limit is down to 2.9 fg/mL.
Specific embodiment:
Present invention will be further explained below with reference to specific examples, it should be appreciated that these embodiments be merely to illustrate the present invention without For limiting the scope of the invention.