CN113049549B

CN113049549B - Electrochemical luminescent agent, biological detection reagent and electrochemical immunoassay method

Info

Publication number: CN113049549B
Application number: CN201911380593.8A
Authority: CN
Inventors: 苏彬; 彭笑刚; 曹芷源; 舒宇飞; 殷俊
Original assignee: Zhejiang University ZJU; Najing Technology Corp Ltd
Current assignee: Zhejiang University ZJU; Najing Technology Corp Ltd
Priority date: 2019-12-27
Filing date: 2019-12-27
Publication date: 2022-12-02
Anticipated expiration: 2039-12-27
Also published as: CN113049549A

Abstract

The invention provides an electrochemical luminescent agent, a biological detection reagent and an electrochemical immunoassay method. The electrochemical luminescent agent comprises quantum dots, wherein the quantum dots are CdSe/CdS/ZnS core-shell quantum dots, cdSe is used as a core, cdS is used as a first shell layer, znS is used as a second shell layer, and the surfaces of the quantum dots comprise water-soluble ligands. The quantum dot has excellent electrochemical luminescence performance and stability.

Description

Electrochemical luminescent agent, biological detection reagent and electrochemical immunoassay method

Technical Field

The invention relates to the field of electrochemical luminescence, in particular to an electrochemical luminescence agent, a biological detection reagent and an electrochemical immunoassay method.

Background

Electrochemiluminescence (ECL) is a light emission phenomenon that is generated by an electrochemical reaction to generate an excited state and returning from the excited state to a ground state. The electrochemical luminescence does not need an excitation light source, has the advantages of low background, high sensitivity, wide linear range and space-time controllability of a luminescence process, and is one of the most advanced immunoassay technologies at present. Currently, ruthenium terpyridyl (Ru (bpy) ₃ ²⁺ ) Is the most widely used electrochemical luminescence molecule and is widely applied to the commercialized electrochemical luminescence analysis and detection system. However, ru (bpy) ₃ ²⁺ The fluorescence quantum yield (PLQY) is only 4.2%, and the optical properties (80 nm half-peak width, hundred nanosecond radiation life and difficult adjustment of emission wavelength) of the fluorescence quantum yield (PLQY) seriously limit the sensitivity of electrochemical luminescence analysis and detectionAnd (4) degree. Therefore, although ECL is considered to be the most advanced immunoassay technology at present, the development of efficient electrochemiluminescence is a core problem to be solved in the current ECL field.

Quantum Dots (QDs) are a class of semiconductor nanocrystals with dimensions smaller than the exciton bohr radius. Under the influence of quantum confinement effect, the quantum dots have the advantages of wide absorption, narrow emission and continuous and adjustable luminescence wavelength along with size. With the continuous development of synthesis technology, it has become possible to prepare quantum dots with ideal optical properties (PLQY-100%, single exponential decay of fluorescence lifetime, stability and no flicker). Contrast QDs with Ru (bpy) ₃ ²⁺ The QDs has great advantages and application prospects in the field of ECL. Bard and colleagues report electrochemiluminescence of Si quantum dots for the first time. Subsequently, the ECL properties of group II-VI quantum dots (e.g., cdSe/ZnSe, cdTe, etc.) have been extensively studied. However, these quantum dots are often only dispersible in organic solvents, which is not conducive to biochemical analysis applications. Zou Guizheng teaches the development of aqueous phase synthesis strategies for quantum dots and the construction of multicolor electrochemiluminescent systems based on CdSe and CdTe QDs. However, all quantum dots used in the ECL field have poor electrochemical luminescence performance and poor stability, and cannot be practically used. Therefore, the development of the water-dispersed quantum dots with high performance for the field of electrochemical luminescence is urgently needed, and the great advantage of the quantum dots as an ideal luminescent material is really realized.

Disclosure of Invention

The invention aims to provide an electrochemical luminescent agent, a biological detection reagent and an electrochemical immunoassay method, which are used for solving the problems of poor electrochemical luminescence performance and poor stability of quantum dots in the prior art.

According to a first aspect of the present application, there is provided an electrochemiluminescent agent comprising quantum dots, wherein the quantum dots are core-shell quantum dots of CdSe/CdS/ZnS, the CdSe is the core, the CdS is the first shell, the ZnS is the second shell, and the surface of the quantum dots comprises a water-soluble ligand.

Further, the thickness of the first shell layer is 5 to 8 monolayers.

Further, the thickness of the second shell layer is 2 to 3 monolayers.

Furthermore, the electrochemical luminescence peak value of the quantum dot is adjustable within the range of 549-643 nm, and the half-peak width of the electrochemical luminescence of the quantum dot is preferably less than 40nm.

Further, the water-soluble ligand is a mercaptocarboxylic acid compound.

Furthermore, the quantum dots are prepared by performing water-soluble ligand exchange reaction on oil-soluble quantum dot raw materials.

Further, the relative electrochemiluminescence efficiency of the quantum dot is more than 100 times of that of the terpyridyl ruthenium compound.

Furthermore, the relative standard deviation of the electrochemiluminescence intensity of the electrochemiluminescence agent in continuous cyclic voltammetry scanning in a potential range of-1.2-0V is less than 1%.

According to a second aspect of the present application, there is provided a biological detection reagent comprising at least one electrochemiluminescent agent as described above, an antigen or an antibody coupled to the electrochemiluminescent agent as described above.

According to a third aspect of the present application there is provided an electrochemical immunoassay in which one or more electrochemiluminescent agents as defined above are used.

By applying the technical scheme of the invention, the surface of the quantum dot is coated with the ZnS shell layer, so that the electrochemiluminescence with high performance and high stability can be realized. Furthermore, by changing the structure of the quantum dots, the emission of various light-emitting wavelengths can be realized, so that the quantity of the detected substances can be detected simultaneously. Finally, the narrow electrochemical emission peak of the quantum dot can improve the detection sensitivity. The advantageous effects of the present application include, but are not limited to, the above-described effects.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

fig. 1 shows a fluorescence spectrum change graph a and a fluorescence decay kinetic curve change graph b of the quantum dot in comparative example 1 before and after ligand exchange.

Fig. 2 shows a fluorescence spectrum change graph a and a fluorescence decay kinetic curve change graph b of the quantum dot in comparative example 2 before and after ligand exchange.

Fig. 3 shows a fluorescence spectrum change diagram a and a fluorescence decay kinetic curve change diagram b of the quantum dots before and after ligand exchange in the quantum dots in example 1.

FIG. 4 shows ECL spectra at different potentials for the ECL system of comparative example 3.

FIG. 5 shows ECL spectra and ECL, PL spectra comparisons for different potentials for the ECL system of comparative example 4.

FIG. 6 shows ECL spectra and ECL, PL spectra in comparison of different potentials for the ECL system of example 2.

FIG. 7 shows the current and ECL intensity as a function of potential for the ECL system of comparative example 5.

FIG. 8 shows the current and ECL intensity as a function of potential for example 2.

Figure 9 shows the results of the ECL system stability test of example 2.

Fig. 10 shows a graph a of a change in fluorescence spectrum of the quantum dot and a graph b of a change in fluorescence decay kinetics curve of the quantum dot before and after ligand exchange in comparative example 6.

FIG. 11 shows ECL spectra at different potentials for the ECL system of comparative example 7.

Fig. 12 shows the current and ECL intensity versus potential for comparative example 8.

Figure 13 shows the ECL spectra at different potentials for the ECL system of example 3.

Figure 14 shows the ECL spectra for the ECL system of example 4 at different potentials.

FIG. 15 shows ECL spectra at different potentials for the ECL system of example 5.

FIG. 16 is a graph showing the current and ECL intensity as a function of potential for the ECL system of example 3.

FIG. 17 is a graph showing the current and ECL intensity as a function of potential for the ECL system of example 4.

FIG. 18 is a graph showing the current and ECL intensity as a function of potential for the ECL system of example 5.

FIG. 19 shows a histogram of the relative ECL efficiencies of examples 3-5.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

In conjunction with the background, the inventors believe that the best quantum dots to generate ECL for biomedical applications should have their own criteria, rather than the quantum dot design principle established based on ideal fluorescence, both ECL and fluorescence have differences and associations.

According to a first aspect of the present application, an electrochemical luminescent agent is provided, the electrochemical luminescent agent comprises quantum dots, the quantum dots are core-shell quantum dots of CdSe/CdS/ZnS, the CdSe is a core, the CdS is a first shell, the ZnS is a second shell, and the surfaces of the quantum dots comprise water-soluble ligands. The inventor finds that a large number of surface defects are introduced on the surface of the quantum dot in the ligand exchange process of oil-to-water conversion, the injection efficiency of electrons and holes is seriously reduced due to the existence of the surface defects, so that electrochemiluminescence is quenched, meanwhile, the quantum dot shows an ECL spectrum which is red-shifted and broadened compared with a fluorescence spectrum due to the existence of the surface defects, and the advantages of the quantum dot in the ECL field are difficult to embody. For example, it was found experimentally that CdSe/CdS core/shell quantum dots with perfect fluorescence emission in non-aqueous solution decreased the fluorescence quantum yield (PLQY) to 31% and increased the short-component fluorescence lifetime after conversion to water-soluble quantum dots. The ZnS has wider forbidden bandwidth, and the ZnS shell layer epitaxially grown on the CdSe/CdS core/shell quantum dot can confine excitons in the quantum dot more effectively, so that the optical property of the quantum dot after ligand exchange is not affected, and the quantum dot has no surface defect or nearly perfect optical property. Thereby ensuring the electrochemical luminescence property and stability of the electrochemical luminescence agent.

In some embodiments, it is desirable that the shell thickness be selected so as not to interfere with carrier injection into the quantum dot, or to interfere with a significant reduction in the quantum dot's electrochemical luminescence properties.

In some embodiments, the first shell layer has a thickness of 5 to 8 monolayers.

In some embodiments, the second shell layer has a thickness of 2 to 3 monolayers.

In some embodiments, the peak wavelength of the electrochemiluminescence of the quantum dots is adjustable within a range of 549-643 nm. The electrochemical luminescence peak wavelength of the quantum dot can be adjusted by controlling the size of the quantum dot, and the quantum dot based on the characteristic structure can realize the fluorescent labeling of multiple luminescence wavelengths, thereby realizing the simultaneous detection of multiple disease markers. Preferably, the half-width of the electrochemiluminescence of the quantum dots is less than 40nm, which is beneficial for providing more distinguishable electrochemiluminescence signals in a limited spectral range.

In some embodiments, the water-soluble ligand is a mercaptocarboxylic acid. The mercapto carboxylic acid compound is preferably one or more of mercaptopropionic acid, mercaptoacetic acid, mercaptobutyric acid, mercaptopentanoic acid and mercaptohexanoic acid. In some embodiments, the surface ligands of the quantum dots have other specific groups to allow attachment of nucleic acids, antibodies, and like biomolecules.

In some embodiments, the quantum dots are prepared by performing a water-soluble ligand exchange reaction on an oil-soluble quantum dot raw material. Compared with the water-soluble quantum dot prepared by a direct aqueous phase synthesis method, the quantum dot obtained by the method has more excellent electrochemical luminescence property.

In some embodiments, the electrochemiluminescence efficiency of the quantum dots is more than 100 times that of ruthenium terpyridyl. It should be noted that there is no method for measuring the absolute luminous efficiency in the electrochemical luminescence field, and therefore the relative efficiency is used for comparison. The present application uses a classical and widely used commercial electrochemiluminescent molecule, a terpyridyl ruthenium compound (Ru (bpy) ₃ ²⁺ ) And (4) comparing. The inventors found that when the same experimental conditions were applied to bothWhen the quantum dots and the terpyridyl ruthenium compound are all in reduction luminescence, the relative luminescence efficiency difference is 6 orders of magnitude; when the quantum dots are in reduction luminescence and the terpyridyl ruthenium compound is in oxidation luminescence (respective optimal conditions), the relative luminous efficiency difference is two orders of magnitude, and the electrochemical luminous efficiency of the quantum dots is more than 100 times of that of the terpyridyl ruthenium.

In some embodiments, the relative standard deviation of the electrochemiluminescence intensity of the electrochemiluminescence agent in the cyclic voltammetry scan between-1.2 and 0V is less than 1%.

According to a second aspect of the present application, there is provided a biological detection reagent comprising at least one of the above-mentioned electrochemiluminescent agents, and an antigen or antibody coupled to the above-mentioned electrochemiluminescent agent. The biological detection reagent can realize the high-efficiency detection of one or more detected objects.

According to a third aspect of the present application, there is provided an electrochemical immunoassay method in which any of the above-described electrochemiluminescent agents is used. The electrochemical immunoassay method has higher reproducibility, namely test stability.

In some embodiments, the testing method comprises: preparing an electrochemical cell comprising a working electrode (fluorine-doped tin oxide glass electrode), a counter electrode (e.g., pt wire) and a reference electrode (e.g., ag/AgCl), and adding an electrochemiluminescent agent (QDs), a co-reactant (e.g., K) ₂ S ₂ O ₈ ) And a supporting electrolyte solution (e.g., PBS buffer) is added to the electrochemical cell. An electrochemical signal is applied at an electrochemical workstation and an ECL signal is detected with a photomultiplier tube (PMT) or spectrometer. The combination of the above components and reagents is abbreviated as ECL system.

Hereinafter, the above-mentioned electrochemical luminescence agent and its application will be further described by the following specific examples.

Chemicals and materials. All chemicals above analytical grade were used directly without further purification. All aqueous solutions were prepared with ultrapure water (18.2M. Omega. Cm). Na (Na) ₂ HPO ₄ ·12H ₂ O(99％)，NaH ₂ PO ₄ ·2H ₂ O(99％)，K ₂ S ₂ O ₈ (99.99%), sodium hydroxide (96%) and tri-n-propylamine (TPrA, 99%) were purchased from alatin. Ru (bpy) ₃ Cl ₂ ，NH ₃ ·H ₂ O (25% by weight), cadmium oxide (CdO, 99.99%), cadmium acetate dihydrate (Cd (Ac) ₂ ·2H ₂ O，>98%), zinc acetate dihydrate (Zn (Ac) ₂ ·2H ₂ O，>99%), selenium powder (Se, 200 mesh, 99.999%), sulfur powder (S, 200 mesh, 99.999%), 1-octadecene (ODE, 90%), decanoic acid (HCa, 98%), oleic acid (OA, 90%), mercaptopropionic acid (MPA,>99%) and tetramethylammonium hydroxide (TMAH, 25% w/w in methanol), purchased from Sigma-Aldrich. Cetyl trimethyl ammonium bromide (CTAB,>99%) from Acros Organics. The stearic acid (HSt,>90%) by tokyo chemical industries, inc. Ethyl acetate, toluene, hexane, methanol, ethanol and chloroform were purchased from the ministry of china medicine. Fluorine doped tin oxide glass electrodes (FTO,<15 ohm/sq) from Kyoki, photoelectric technologies, inc.

Comparative example 1

And preparing CdSe nuclear quantum dots. Selenium powder (1 mmol) was dispersed in ODE (10 mL) to prepare a selenium suspension (Se-SUS). In a typical synthesis procedure, cdO (0.2 mmol) and stearic acid (0.5 mmol) were added to a 50mL three-necked flask containing 4mL ODE to give a mixture. The mixture was heated to 280 ℃ to obtain a colorless cadmium stearate solution. The reaction temperature was lowered to 250 ℃, and then 1mL of selenium powder suspension was rapidly injected into the solution in the three-necked flask, and the reaction was carried out at 250 ℃. After 5 minutes, se-SUS was dropped into the solution at a rate of 0.2mL/min to obtain a quantum dot stock solution of CdSe core quantum dots having a target size. The reaction process was monitored by uv-vis absorption spectroscopy.

Purifying CdSe nuclear quantum dots and exchanging ligands. In the first step, 2mL of a stock solution of CdSe nuclear quantum dots was mixed with 2mL of ethyl acetate. After centrifugation at 4000rpm, the supernatant was removed. Further, 2mL of ethyl acetate was added thereto, and the above operation was repeated once more. In the second step, the precipitate was dissolved using 1mL of toluene and the resulting mixture was heated at 110 ℃ until a clear solution was formed. Then, 1mL of methanol was added, and the mixture was centrifuged at 4000rpm to remove the supernatant. The resulting precipitate was redissolved in 1mL of toluene and the procedure repeated once more to give purified CdSe quantum dots (3.1 nm in average size by TEM observation) for ligand exchange of the quantum dots. The ligand exchange method is described below.

Comparative example 2

And preparing CdSe/CdS core/shell quantum dots. Mixing Cd (Ac) ₂ ·2H ₂ O (0.7 mmol), decanoic acid (0.7 mmol), oleic acid (2.1 mmol), the oil-soluble CdSe core quantum dots (0.1. Mu. Mol) obtained after purification in comparative example 1, and ODE (4 mL) were charged in a 50mL three-necked flask to obtain a mixture. The mixture was then heated to 260 ℃ and sulfur dissolved in ODE (0.1M) was injected into the solution in a three-necked flask at a rate of 2mL/h until a quantum dot stock solution of CdSe/CdS core/shell quantum dots with the target size was obtained. The reaction process was monitored by uv-vis absorption spectroscopy.

And (3) purifying the CdSe/CdS core/shell quantum dots and exchanging ligands. First, 3mL of quantum dot stock solution was mixed with 9mL of ethanol. After centrifugation at 4000rpm, the supernatant was removed. The precipitate was dissolved in 2mL of toluene, then 2mL of methanol was added and the mixture was held at 110 ℃ for 1 minute. After that, the mixture was centrifuged at 4000rpm for 3 minutes to obtain a supernatant. And repeating the steps again to obtain the purified CdSe/CdS core/shell quantum dots (the average size is 5.6nm measured by TEM observation, wherein the CdS shell is 5 CdS single layers) for ligand exchange of the quantum dots. The ligand exchange method is described below.

Example 1

And (3) preparing CdSe/CdS/ZnS core/shell quantum dots. Zn (Ac) ₂ ·2H ₂ O (0.5 mmol), decanoic acid (0.4 mmol), oleic acid (1.2 mmol), cdSe/CdS core/shell quantum dots purified in comparative example 2 (0.05. Mu. Mol), and ODE (4 mL) were charged into a 50mL three-necked flask. The mixture was then heated to 290 ℃ and sulfur (0.1M concentration) dissolved in ODE was injected into the reaction solution at a rate of 2mL/h until a quantum dot stock solution of CdSe/CdS/ZnS core/shell quantum dots with a target size was obtained. The reaction was monitored by UV-Vis spectroscopy.

And (3) purifying and ligand exchanging the CdSe/CdS/ZnS core/shell quantum dots. 2mL of the quantum dot stock solution was mixed with 6mL of ethyl acetate and heated at 50 ℃ for 3 minutes. After centrifugation at 4000rpm, the supernatant was removed and 0.5mL of toluene was added to dissolve the precipitate. The above steps are repeated again. Obtaining CdSe/CdS/ZnS core/shell quantum dots (the average size measured by TEM observation is 7.1nm, and the ZnS shell layer is 3 single layers) for ligand exchange of the quantum dots. The ligand exchange method is described below.

In comparative examples 1 to 2 and example 1, the ligand on the surface of the quantum dot before ligand exchange was carboxylate, and the ligand was dispersed only in the organic solvent, and it was necessary to disperse the quantum dot in the aqueous solution by ligand exchange with mercaptopropionic acid (MPA). A typical process for ligand exchange of quantum dots with MPA is as follows: about 0.5. Mu. Mol of the purified oil-soluble quantum dots were dissolved in 1mL of chloroform, and then 0.2mL of MPA was added to the solution. After centrifugation at 4000rpm for 5 minutes, the supernatant was removed and the precipitate was washed twice with n-hexane. The precipitate was dried under vacuum at room temperature to completely remove the organic solvent. Finally, the precipitate was dissolved in 1mL of water and 200. Mu.L of TMAH was added. It should be noted that the concentration of CdSe nuclei in the present application is calculated by Lambert beer' S law, where the extinction coefficient of CdSe is calculated by the method developed in the teaching of Peng Xiaogang (Li, J.; chen, J.; shen, Y.; peng, X.extraction coefficient useful CdE (E = Se or S) unit for zinc-blank CdSe nanocrystals. Nano Res.2018,11, 3991-4004.). The concentration of the quantum dots is not changed in the process of epitaxially growing the CdS and ZnS shells.

Fig. 1, 2 and 3 show a fluorescence spectrum change graph a and a fluorescence decay kinetic curve change graph b of the quantum dots before and after ligand exchange in the quantum dots in comparative example 1, comparative example 2 and example 1 respectively. In comparative examples 1-2 and example 1, the fluorescence quantum yield corresponding to each quantum dot before ligand exchange was greater than 90% and the fluorescence decay kinetic curve was single exponential; however, after MPA ligand exchange, the quantum dots in comparative example 1 had a fluorescence quantum yield of almost 0; the quantum dots in comparative example 2 showed a decrease in fluorescence quantum yield to 31% and an increase in the short-component fluorescence lifetime of the fluorescence decay kinetic curve (see. Tau. In FIG. 2) ₁ A dotted dispersion portion corresponding to =13.0 ns), a hole defect corresponding to a surface of the quantum dot; the quantum yield of the fluorescence quantum of the quantum dots in the embodiment 1 is kept above 90% and is still single exponential fluorescence decay, and the necessity and the effectiveness of the ZnS shell epitaxial growth are proved.

Comparative example 3

The aqueous phase quantum dots (concentration of 0.3. Mu. Mol/L) in comparative example 1 were added to the solution containing 10mmol/L K ₂ S ₂ O ₈ 0.1mol/L PBS buffer (pH = 7.4); fluorine-doped tin oxide (FTO) conductive glass is used as a working electrode, a platinum wire is used as a counter electrode, and a silver/silver chloride electrode (filled with saturated potassium chloride) is used as a reference electrode to test the electrochemical luminescence property of the quantum dot. FIG. 4 shows the ECL spectra at different potentials for the ECL system of comparative example 3, which can be seen to have no ECL signal in the potential range of-0.6V to-1.2V.

Comparative example 4

The quantum dot that completed ligand exchange in comparative example 2 was used, which was different from comparative example 3 in the kind of quantum dot. FIG. 5 shows the ECL spectra at different potentials for the ECL system of comparative example 4. The light emission potential of the quantum dot is-0.83V, the maximum luminous intensity is reached at-1.06V, and the ECL spectrum is coincident with the fluorescence spectrum.

Comparative example 5

The difference from comparative example 3 was that, in terms of the kind of luminescent material, 0.3. Mu. Mol/L Ru (bpy) was used ₃ ²⁺ And (3) solution. And ECL intensity was recorded using a photomultiplier tube (PMT) biased at 500V, otherwise identical to comparative example 3. FIG. 7 shows the current and ECL versus potential for the ECL system of comparative example 5.

Example 2

Different from comparative example 3 in that the ligand-exchanged water-soluble quantum dot obtained in example 1 was used, and FIG. 6 shows an ECL spectrum at different potentials of example 2, the quantum dot having a light emission potential of-0.87V and reaching a maximum light emission intensity at-1.19V, the ECL spectrum coinciding with a fluorescence spectrum; FIG. 8 is a graph showing the change of current and ECL intensity with potential of example 2, which is different from comparative example 5 in that a neutral filter (ND = 4) is disposed before a photomultiplier tube (PMT) to prevent light saturation when ECL is measured, and the electrochemiluminescence intensity of example 2 is 4.7X 10 of comparative example 5 ⁵ Doubling; FIG. 9 shows the ECL stability of the quantum dots in example 2, which is different from comparative example 5 only in that the standard deviation of the maximum luminescence intensity of the ECL is less than the standard deviation under continuous cyclic voltammetric scanning at a bias of 300V from PMT1% (fig. 9 a) and the optical properties did not change after ECL testing (fig. 9 b). The ECL system still has stable ECL luminescence intensity after multiple potential scans, see fig. 9a; and still has stable fluorescence absorption and single exponential fluorescence decay after 125 th round of potential scanning, see fig. 9b.

Comparative example 6

Preparation of CdSe/CdS core/shell quantum dots (shell with 8 monolayers of CdS). The difference from comparative example 2 is that the CdS epitaxial growth time is prolonged until CdSe core epitaxially grows 8 single-layer CdS, which is measured to have an average size of 8.1nm by TEM observation. FIG. 10 shows the fluorescence spectrum change diagram a and fluorescence decay kinetic curve change diagram b of the quantum dots before and after ligand exchange, and PLQY is reduced to 38% after ligand exchange. The necessity of epitaxially growing a ZnS layer was demonstrated.

Comparative example 7

The water-soluble CdSe/CdS quantum dots with 8 CdS layers prepared in comparative example 6 were used, differing from comparative example 2 only in the kind of quantum dots. FIG. 11 shows ECL spectra at different potentials for the ECL system of comparative example 7. From fig. 11 it can be seen that the ECL properties are similar to those of comparative example 4, demonstrating that the high ECL performance of example 2 does not result from an increase in the size of the quantum dots, but rather the outer shell layer.

Comparative example 8

The difference from example 2 is in the ECL luminophores, the co-reactants, the working electrode and the potential interval. At 0.3. Mu. Mol/L Ru (bpy) ₃ ²⁺ In place of the core/shell quantum dots, K was replaced with 10mM tripropylamine (TPrA) ₂ S ₂ O ₈ And replacing an FTO electrode with a glassy carbon electrode, wherein the potential scanning rate is 100mV/s, and the scanning interval is 0-1.2V. The current and ECL intensity versus potential for comparative example 8 is shown in figure 12. Quantum dot relative Ru (bpy) ₃ ²⁺ The ECL efficiency calculation formula of (a) is as follows:

ECL represents ECL intensity, I represents voltammetric current, QD represents quantum dot, ru represents Ru (bpy) ₃ ²⁺ A luminescent material.

The ECL efficiency of example 2 was calculated to be 6.9X 10 of comparative example 5 ⁵ The magnification was 165 times that of comparative example 7.

Example 3

Water-soluble green CdSe/CdS/ZnS quantum dots were prepared according to the method of example 1, differing only in that the first exciton absorption peak with the CdSe core was 450nm. The average size of the quantum dots was measured by TEM to be 5.9nm. ECL system set-up and ECL testing was performed as in example 2. FIG. 13 shows ECL spectra at different potentials for the ECL system of example 3. FIG. 16 is a graph of ECL current and ECL intensity as a function of potential for the ECL system of example 3.

Example 4

Water-soluble yellow CdSe/CdS/ZnS quantum dots were prepared according to the method of example 1, differing only in that the first exciton absorption peak with the CdSe core was 500nm. The average size of the quantum dots was measured by TEM to be 6.6nm. ECL system set-up and ECL testing was performed as in example 2. FIG. 14 shows ECL spectra at different potentials for the ECL system of example 4. FIG. 17 is a graph of ECL current and ECL intensity as a function of potential for the ECL system of example 4.

Example 5

Water-soluble red CdSe/CdS/ZnS quantum dots were prepared according to the method of example 1, differing only in that the first exciton absorption peak with the CdSe core was 590nm. The average size of the quantum dots measured by TEM was 9.0nm. ECL system set-up and ECL testing was performed as in example 2. FIG. 15 shows ECL spectra at different potentials for the ECL system of example 5. FIG. 18 is a graph of ECL current and ECL intensity as a function of potential for the ECL system of example 5. FIG. 19 shows a histogram of the relative ECL efficiencies of examples 3-5.

The test method and the apparatus are as follows:

TEM (Transmission Electron microscope lens) images were obtained at 100kV on a transmission electron microscope (SU-8020, hitachi, japan). Absolute PL quantum yield was measured on an integrating sphere (Ocean Optics FOIS-1) combined with a spectrometer (QE 65000) and the solvent for the quantum dots was toluene. Fluorescence decay kinetics curves were measured on a time-dependent single photon counting (TCSPC) fluorescence spectrometer (FLS 920, edinburg instruments, uk). The sample was diluted with toluene and excited with a 405nm laser at 2MHz.

All ECL measurements were performed using a classical three-electrode system. For ECL intensity measurements, an MPI-E ECL analysis system (Remex analyzer, west ampere, china) consisting of a photomultiplier tube (PMT) and an electrochemical workstation was used, which simultaneously recorded the ECL intensity and current as a function of voltage. ECL spectrograms at different potentials were measured by a CHI440A electrochemical workstation (Shanghai, china) in combination with a spectrometer (QE-Pro, ocean Optics). The potential sweep rate was 100mV/s and the integration time for each ECL spectrum was 100ms.

The inventors have found that CdSe/CdS/ZnS core/shell quantum dots synthesized in non-aqueous solution with a ZnS shell, after conversion to aqueous solubility, can produce very bright and stable ECLs with ECL efficiencies approximately proportional to their PL quantum yields. The ECL efficiency of the CdSe/CdS core/shell quantum dot system is much lower than that of the CdSe/CdS/ZnS core/shell system. Therefore, the quantum dot electrochemical luminescence with high efficiency can be realized by designing the energy band and the lattice structure of the quantum dot.

The present application develops a fluorescent quantum yield of nearly 100: (>90%), a narrow fluorescence emission peak and a single exponential fluorescence decay kinetic curve. The quantum dot has ECL properties of high efficiency, stability and narrow bandwidth. It is worth noting that the ECL efficiency of CdSe/CdS/ZnS core/shell quantum dots with perfect fluorescence luminescence property in water is higher than that of traditional Ru (bpy) under the condition of not increasing the barrier of charge injection ₃ ²⁺ 2 to 6 orders of magnitude higher. In addition, by adjusting the size of the CdSe core, the ECL emission wavelength of the CdSe/CdS/ZnS core/shell quantum dot can be adjusted along with the size of the CdSe core, and an ECL system for wavelength resolution of red, yellow and green is constructed according to the ECL emission wavelength.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A biological detection reagent, wherein the biological detection reagent comprises at least one electrochemiluminescent agent, an antigen or an antibody coupled to the electrochemiluminescent agent; the electrochemical luminescent agent comprises quantum dots, the quantum dots are CdSe/CdS/ZnS core-shell quantum dots, the CdSe is a core, the CdS is a first shell layer, the ZnS is a second shell layer, and the surfaces of the quantum dots comprise water-soluble ligands; the electrochemical luminescence peak value of the quantum dot is adjustable within the range of 549-643 nm; the half-peak width of the electrochemiluminescence of the quantum dots is less than 40nm; the water-soluble ligand is a mercapto carboxylic acid compound; the thickness of the first shell layer is 5-8 single layers; the thickness of the second shell layer is 2-3 single layers.

2. The biological detection reagent of claim 1, wherein the quantum dot is prepared from an oil-soluble quantum dot raw material by a water-soluble ligand exchange reaction.

3. The bioassay reagent as set forth in claim 1, wherein the relative standard deviation of the electrochemiluminescence intensity of the electrochemiluminescence reagent in the continuous cyclic voltammetry scan at a potential interval of-1.2 to 0V is less than 1%.

4. An electrochemical immunoassay method, wherein the bioassay reagent according to any one of claims 1 to 3 is used in the immunoassay method.