Electrochemiluminescent microemulsion, generation method, generation device and application thereof

Abstract

The present invention discloses an electrochemiluminescent microemulsion and a generation method, a generation device and an application, including an external phase solution; the external phase solution encapsulates an intermediate phase solution; the intermediate phase solution encapsulates an internal phase solution; the volume ratio of the external phase solution, the intermediate phase solution and the internal phase solution is 4-20:1.25:1; the external phase solution is an aqueous solution containing 5wt% PVA, mineral oil or an aqueous solution containing 2wt% tween 20; the intermediate phase solution is FC 40 containing 2wt% fluorine surfactant, HFE 7500 containing 2wt% fluorine surfactant or an aqueous solution containing 15wt% glycerol; the internal phase solution is phycocyanin, biliverdin, mesobililiverdin or urobilin. The generation device of the present invention can adjust the flow rate to generate electrochemiluminescent microemulsions of different diameters, the diameter is 80μm-200μm adjustable, and the electrochemiluminescent microemulsion detects a strong current signal in the electrochemiluminescent platform; and the detection range of quantitative detection of alpha-fetoprotein is 0.05-500ng / ml.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis, and in particular relates to an electrochemiluminescent microemulsion and a generation method, a generation device and application thereof. Background Art

[0002] Double emulsion droplets are composed of a droplet and another immiscible droplet contained inside. The core-shell, pore-shell, and multi-chamber functional microparticles generated using double emulsion droplets as templates are widely used in drug delivery and controlled release, active substance encapsulation, chemical catalysis, and biochemical separation. The performance of the prepared material mainly depends on the structure of the double emulsion droplets, so the stable and high-speed generation of monodisperse double emulsion droplets is the key to its application. The double emulsion droplets generated by microfluidics have good monodispersity, uniformity, and sphericity, and are highly controllable and have high raw material utilization.

[0003] The microfluidic method for preparing microdroplets mainly uses two incompatible liquids as the continuous phase and the dispersed phase, respectively, and controls the generation of droplets by controlling the microtubule structure and the flow rate ratio of the two phases. Driven by a driving pump with a fixed volume flow rate, the continuous phase and the dispersed phase enter different microchannels respectively. When the two fluids meet at the intersection, the dispersed phase fluid continues to extend to form a "plug-like" or "jet-like" liquid column, which is then pinched off by the shear and extrusion of the continuous phase fluid and dispersed in the continuous phase in the form of microdroplets. Generally, the generation of microdroplets requires a sufficiently large force to disturb the interfacial tension between the continuous phase and the dispersed phase. When the force applied to a certain point in the dispersed phase is greater than its interfacial tension, the trace amount of liquid at that point will break through the interfacial tension and enter the continuous phase to form droplets. At the microscale, interfacial tension and liquid viscosity both play a very important role, and the most commonly used means of regulation is to add different surfactants.

[0004] Uniformly sized and stable microemulsions collected by droplet microfluidics are used for electrochemical bioassays, where droplet collisions, also known as nanoimpact, are studied. Nanoimpact is an electrochemical measurement method involving the random collision of single particles on a microelectrode and the subsequent Faradaic current transient. The method originated from the collision of insoluble particles on a calomel electrode. So far, a variety of biological species including DNA, RNA, enzymes, bacteria, vesicles, and cells have been studied using nanoimpact electrochemistry.

[0005] The nano-impact method is mainly that when a droplet collides with an electrode, its components are oxidized under the applied potential, thereby generating a current spike signal. If the components in the droplet are electrochemiluminescent bodies and co-reactants, the subsequent reaction will produce light that conforms to the reported ECL mechanism, allowing the simultaneous observation of nano- and micron-sized droplet collisions using electrochemistry and ECL. When the microelectrode under potential control is immersed in the solution, the freely diffusing analyte particles in the solution will collide randomly with the electrode surface by virtue of their Brownian motion. The current signal can be identified by sensing various electrochemical processes. This method does not require complex instruments and operations, and can obtain rich information quickly and conveniently.

[0006] Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer and one of the leading causes of cancer-related deaths worldwide. Early and accurate HCC detection is urgently needed to achieve effective treatment and improve cancer survival. Proteins are particularly useful molecules that can serve as serum biomarkers because they are often effectors of the disease and can be diagnosed by convenient non-invasive tests. Alpha-fetoprotein (AFP) is the most widely used serum biomarker for clinical diagnosis of HCC and has been shown to be useful for early screening, recurrence monitoring, and prognosis estimation. Currently, there are a variety of analytical techniques, such as enzyme-linked immunosorbent assay (ELISA), blotting, and liquid chromatography-mass spectrometry. Nanoparticle collision electrochemistry is an electroanalytical method that has developed rapidly in recent years, which is based on the random collision of single nanoparticles with inert ultramicroelectrodes caused by Brownian motion. Summary of the invention

[0007] The purpose of the present invention is to provide an electrochemiluminescent microemulsion and a generation method, a generation device and an application thereof in view of the deficiencies of the prior art. The purpose of the present invention is achieved through the following technical scheme: an electrochemiluminescent microemulsion, comprising an external phase solution; the external phase solution envelops an intermediate phase solution; the intermediate phase solution envelops an internal phase solution; the volume ratio of the external phase solution, the intermediate phase solution and the internal phase solution is 4-20:1.25:1; the external phase solution is an aqueous solution containing 5wt% PVA, mineral oil or an aqueous solution containing 2wt% tween 20; the intermediate phase solution is FC 40 containing 2wt% fluorine surfactant, HFE 7500 containing 2wt% fluorine surfactant or an aqueous solution containing 15wt% glycerol; the internal phase solution is phycocyanin, biliverdin, mesobililiverdin or urobilin.

[0008] Furthermore, the volume ratio of the external phase solution, the intermediate phase solution and the internal phase solution is 8:1.25:1.

[0009] Furthermore, the inner phase solution is phycocyanin.

[0010] A method for generating the electrochemiluminescent microemulsion comprises the following steps:

[0011] (1) preparing an external phase solution, an intermediate phase solution and an internal phase solution; the external phase solution is an aqueous solution containing 5 wt.% PVA, mineral oil or an aqueous solution containing 2 wt.% tween 20; the intermediate phase solution is FC40 containing 2 wt.% fluorine surfactant, HFE 7500 containing 2 wt.% fluorine surfactant or an aqueous solution containing 15 wt.% glycerol;

[0012] (2) filtering and ultrasonically degassing the external phase solution, the intermediate phase solution, and the internal phase solution;

[0013] (3) mixing the intermediate phase solution and the internal phase solution to form a water-in-oil monolayer emulsion;

[0014] (4) The external phase solution is introduced into the water-in-oil single-layer emulsion formed in step (3) to form a water-in-oil-in-water double-layer emulsion, i.e., an electrochemiluminescent microemulsion.

[0015] An electrochemiluminescent microemulsion generating device for realizing the above-mentioned generating method comprises a fluid injection pump module, a microfluidic chip, a droplet collecting device and a cover glass; the cover glass is provided with a microfluidic chip; the microfluidic chip is provided with an external phase channel inlet, an intermediate phase channel inlet, an internal phase channel inlet, a first cross aggregation structure, a second cross aggregation structure, and a channel outlet; the fluid injection pump module comprises an external phase injection pump, an intermediate phase injection pump and an internal phase injection pump, which are sequentially connected to the external phase channel inlet, the intermediate phase channel inlet and the internal phase channel inlet; the channel outlet is connected to the droplet collecting device;

[0016] The first cross-aggregation structure is provided with an inner phase shearing opening, a single emulsion generating opening, a middle phase left shearing opening and a middle phase right shearing opening; the inner phase shearing opening and the single emulsion generating opening are subjected to a narrowing treatment;

[0017] The second cross-aggregation structure is provided with a single emulsion shearing opening, a double emulsion generating opening, an external phase left shearing opening and an external phase right shearing opening; the single emulsion shearing opening is subjected to a narrowing treatment.

[0018] Furthermore, the connection between the external phase injection pump and the external phase channel inlet, the connection between the intermediate phase injection pump and the intermediate phase channel inlet, and the connection between the internal phase injection pump and the internal phase channel inlet are all connected through polytetrafluoroethylene microtubes.

[0019] Furthermore, the channel outlet and the droplet collecting device are connected via a polytetrafluoroethylene microtube.

[0020] The application of the electrochemiluminescent microemulsion in the quantitative detection of alpha-fetoprotein is as follows:

[0021] (1) The microemulsion generation platform constructed by the present invention can quickly and stably generate water-in-oil-in-water (W / O / W) double-layer emulsions of uniform size. The platform can not only efficiently generate double emulsions of phycocyanin and related water-soluble materials, but also can observe the double emulsion generation state in real time and adjust the flow rate in time.

[0022] (2) The present invention uses phycocyanin (PCB) with photoluminescent properties as the research object and as the inner phase of the double emulsion, which can detect a strong current signal in the electrochemiluminescence platform;

[0023] (3) The microfluidic optical platform of the present invention can observe the encapsulation morphology of the double-layer emulsion in real time, and has high feasibility;

[0024] (4) Based on the collision ECL system of phycocyanin double emulsion, an immunosensor was constructed to detect the AFP content in the sample, and a linear range of 0.05 ng / mL to 500 ng / mL was obtained with a linear correlation coefficient of 0.99, which has a lower detection limit compared with the detection limit of the existing AFP detection kit.

[0025] (5) The electrochemiluminescent microemulsion generation platform constructed by the present invention provides a new research direction for the electrochemical collision of soft nanoparticles. BRIEF DESCRIPTION OF THE DRAWINGS In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for the description of the embodiments are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Design drawings for microfluidic chips;

[0027] Figure 2 Schematic diagram of the microfluidic channel device;

[0028] Figure 3 Generate system diagrams for microemulsions;

[0029] Figure 4 Double emulsion bright field microscopy image (left), double emulsion fluorescence microscopy image (right), scale bar: 100 μm;

[0030] Figure 5 is the particle size distribution histogram of double emulsion;

[0031] Figure 6 This is the electrochemical cyclic voltammogram of PCB double emulsion;

[0032] Figure 7 is the collision electrochemical intensity diagram of microemulsion;

[0033] Figure 8 It is the UV spectra of phycocyanin (PCB) and phycocyanin (CPC);

[0034] Fig. 9 The fluorescence spectra of phycocyanin (PCB) and phycocyanin (CPC);

[0035] Fig.10 This is a curve diagram showing the relationship between alpha-fetoprotein (AFP) concentration and electrochemiluminescence intensity. DETAILED DESCRIPTION

[0036] Exemplary embodiments will be described in detail herein, examples of which are shown in the accompanying drawings. When the following description refers to the drawings, the same numbers in different drawings represent the same or similar elements unless otherwise indicated. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present invention. Instead, they are merely examples of devices and methods consistent with some aspects of the present invention as detailed in the appended claims.

[0037] The terms used in the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The singular forms "a", "the" and "the" used in the present invention and the appended claims are also intended to include plural forms unless the context clearly indicates other meanings. It should also be understood that the term "and / or" used herein refers to and includes any or all possible combinations of one or more associated listed items.

[0038] It should be understood that although the terms first, second, third, etc. may be used in the present invention to describe various information, these information should not be limited to these terms. These terms are only used to distinguish the same type of information from each other. For example, without departing from the scope of the present invention, the first information may also be referred to as the second information, and similarly, the second information may also be referred to as the first information. Depending on the context, the word "if" as used herein may be interpreted as "at the time of" or "when" or "in response to determining".

[0039] The present invention is described in detail below in conjunction with the accompanying drawings. In the absence of conflict, the features of the following embodiments and implementations can be combined with each other.

[0040] Embodiment 1:

[0041] An electrochemiluminescent microemulsion comprises an external phase solution of 5wt% PVA, an intermediate phase solution of FC 40 containing 2wt% fluorine surfactant, and an internal phase solution of 0.1mM phycocyanin. The intermediate phase solution and the internal phase solution are mixed at a volume ratio of 1.25:1 to form an oil-in-water monolayer emulsion, and the external phase solution is then added to form the electrochemiluminescent microemulsion; wherein the volume ratio of the external phase solution to the internal phase solution is 8:1.

[0042] In some embodiments, the concentration of phycocyanin is 0.1-1 mM.

[0043] In some embodiments, the internal phase solution is 0.1-1 Mm biliverdin, 0.1-1 Mm mesobilidin or 0.1-1 Mm urobilin Example 2:

[0044] Different from Example 1, the external phase solution is mineral oil, the middle phase solution is HFE7500 containing 2wt% fluorine surfactant, the internal phase solution is 0.1-1Mm biliverdin, and the volume ratio of the external phase solution, the middle phase solution and the internal phase solution is 4:1.25:1.

[0045] Embodiment 3:

[0046] Different from Example 1, the external phase solution contains 2% tween 20, the middle phase solution is an aqueous solution containing 15wt% glycerol, the internal phase solution is 0.1-1Mm mesobililiverdin, and the volume ratio of the external phase solution, the middle phase solution and the internal phase solution is 20:1.25:1.

[0047] Embodiment 4:

[0048] The difference from Example 1 is that the internal phase solution is urobilin.

[0049] Embodiment 5:

[0050] like Figure 1 As shown, an electrochemiluminescent microemulsion generating device of the present invention comprises a fluid injection pump module 1, a microfluidic chip 2, a droplet collecting device 3 and a cover glass 4; the cover glass 4 is provided with a microfluidic chip 2; the microfluidic chip 2 is provided with an external phase channel inlet 21, an intermediate phase channel inlet 22, an internal phase channel inlet 23, a first cross aggregation structure 24, a second cross aggregation structure 25, and a channel outlet 26; the fluid injection pump module 1 comprises an external phase injection pump, an intermediate phase injection pump and an internal phase injection pump, which are connected to the external phase channel inlet 21, the intermediate phase channel inlet 22, and the internal phase channel inlet 23 in sequence; the channel outlet 26 is connected to the droplet collecting device 3;

[0051] The first cross-aggregation structure 24 is provided with an inner phase shearing opening 24-1, a single emulsion generating opening 24-2, a middle phase left shearing opening 24-3 and a middle phase right shearing opening 24-4; the inner phase shearing opening 24-1 and the single emulsion generating opening 24-2 are subjected to a narrowing treatment;

[0052] The second cross-aggregation structure 25 is provided with a single emulsion shearing opening 25-1, a double emulsion generating opening 25-2, an external phase left shearing opening 25-3 and an external phase right shearing opening 25-4; the single emulsion shearing opening 25-1 is subjected to a narrowing treatment.

[0053] The present invention designs a microfluidic chip 2 for the needs of generating electrochemiluminescent microemulsions, such as Figure 2 As shown in the figure, a cross-shaped chip is used, and a narrow necking treatment is performed on this structure. The main purpose is to use capillary instability to emulsify droplets. The fluid velocity will increase sharply at the narrow necking, resulting in an increase in the symmetrical shear force of the external phase flow on the internal phase flow. The fluid produces a focusing effect under the extrusion of the narrow necking to complete the emulsification.

[0054] The material of the microfluidic chip 2 is polydimethylsiloxane (PDMS), and the PDMS chip is bonded to the cover glass by electric spark treatment to form a closed channel. The fluid injection pump module 1 and the polytetrafluoroethylene microtube can both tolerate weakly polar non-water-soluble reagents. The external phase injection pump, the intermediate phase injection pump and the internal phase injection pump of the fluid injection pump module 1 can control the flow rate individually, or any two or three of them can be controlled simultaneously. After the reagent is introduced into the channel, an oil-in-water (W / O) single emulsion is formed at the first cross aggregation structure 24, and a water-in-oil-in-water (W / O / W) double emulsion is formed at the second cross aggregation structure 25, and finally the microemulsion is collected by a collecting bottle at the channel outlet. In this embodiment, the collecting bottle is the droplet collection device 3.

[0055] The electrochemiluminescent microemulsion generating device of the present invention can also generate double-layer emulsions of different diameters by controlling the flow rates of the external phase, the intermediate phase and the internal phase, and the diameter is adjustable from 80 μm to 200 μm; the flow rate control of the internal phase at 1.7 μl / min, the intermediate phase at 3.4 μl / min and the external phase at 13 μl / min can generate a double-layer emulsion with a diameter of 80 μm; when the flow rates of the internal phase and the intermediate phase remain unchanged and the flow rate of the external phase is 10 μl / min, a double-layer emulsion of 100 μm can be generated; when the flow rate of the internal phase remains unchanged, the flow rate of the intermediate phase is 2 μl / min and the flow rate of the external phase is 6 μl / min, a double-layer emulsion of 200 μm can be generated.

[0056] Embodiment 6:

[0057] The method for generating an electrochemiluminescent microemulsion of the present invention comprises the following steps:

[0058] S1: preparing an external phase solution, an intermediate phase solution and an internal phase solution; the external phase solution is 5wt% PVA, the intermediate phase solution is FC 40 containing 2wt% fluorine surfactant, and the internal phase solution is 0.1mM phycocyanin;

[0059] S2: filtering the external phase solution, the intermediate phase solution and the internal phase solution and subjecting them to ultrasonic degassing treatment; and loading them into the external phase injection pump, the intermediate phase injection pump and the internal phase injection pump respectively;

[0060] S3: introducing the external phase solution, the intermediate phase solution and the internal phase solution in sequence, and exhausting the gas in the microfluidic chip 2;

[0061] S4: adjusting the flow rates of the internal phase and intermediate phase injection pumps; wherein the flow rate of the internal phase injection pump is 1.7 μl / min, and the flow rate of the intermediate phase injection pump is 3.4 μl / min; forming an oil-in-water monolayer emulsion at the first cross aggregation structure 24;

[0062] S5: adjusting the flow rate of the external phase injection pump again; wherein the flow rate of the external phase injection pump is 13 μl / min; forming a water-in-oil-in-water (W / O / W) double-layer emulsion, i.e., an electrochemiluminescent microemulsion, at the second cross aggregation structure 25;

[0063] S6: Collect the electrochemiluminescent microemulsion formed in step S5 through the droplet collection device 3.

[0064] Embodiment 7:

[0065] like Figure 3 As shown, through the microemulsion generation platform, the flow rates of the inner phase, the middle phase and the outer phase are adjusted to stably generate a double-layer emulsion of uniform size, such as Figure 4 As shown in the bright field microscopy and fluorescence microscopy images of the double-layer emulsion, the generated double-layer emulsion has uniform size and stable morphology, with a diameter range of about 86.5 μm. Figure 5 As shown. The collected phycocyanin microemulsion and phycocyanin microemulsion were characterized by UV and fluorescence spectra, as shown Figure 8 and Fig. 9 As shown, the black dotted line represents the UV and fluorescence curves of phycocyanin (PCB), and the black solid line represents the UV and fluorescence curves of C-phycocyanin (CPC).

[0066] The prepared PCB double-layer emulsion was used for electrochemical collision, and the droplets were added to the electrolytic cell for electrochemical analysis. The current change of the microemulsion was detected by cyclic voltammetry (CV). First, 1 mL of the generated double-layer emulsion was added to 2-3 mL of PBS buffer solution, and a 10 mM TOAB solution was used to modify the glassy carbon electrode as the working electrode, the silver / silver chloride electrode as the reference electrode, and the platinum sheet electrode as the counter electrode. The electrochemical parameters were set, the high potential was set to 0 V, the low potential was set to -2 V, and the forward scan was performed at a scan rate of 0.1 V / s. Figure 6 The electrochemical cyclic voltammogram of double emulsion collision is shown. An obvious oxygen reduction peak can be observed at -0.6V, and a characteristic reduction peak at -1.7V. And when the droplet collides with the electrode, a sharp change in the current signal will occur. It can be seen that there are a large number of collisions between the microemulsion and the electrode between -1.5V-2V, and the amplitude of the signal change is closely related to the strength of the collision. It can be seen from the figure that the probability of collision increases at high potentials, which may be due to the excessively high voltage causing gas to precipitate on the electrode surface, resulting in instability of the electrode interface. Figure 7 This is the time-intensity diagram of microemulsion collision electrochemistry. It can be seen that the ECL intensity of microemulsion electrochemical collision is stable. Using three buffer solutions (Hepes, Tris, PBS) with a pH of 7.4 as electrolytes, the electrochemiluminescence (ECL) intensity of the double emulsion structure was tested by an electrochemiluminescence instrument, and the optimal buffer was PBS. The ECL intensity was tested at different scanning speeds, and the optimal scanning speed was 0.2V / s. The amount of the co-reactant H2O2 was then selected and optimized, and 7 H2O2 solutions of different concentrations (0.1mM, 5mM, 10mM, 15mM, 20mM, 25mM, 30mM) were added to the electrolyte. The electrochemiluminescence (ECL) intensity of the microemulsion collision was tested by an electrochemiluminescence instrument, and the optimal amount of H2O2 was 20mM.

[0067] Embodiment 8:

[0068] Based on the above-prepared microemulsion, AFP was detected; an ECL immunosensor was designed: first, p-aminobenzoic acid (ABA) was modified on the electrode surface, then EDC-NHS was modified to activate the carboxyl group to form an amide bond and assembled on the electrode surface, and AFP antibody was added and incubated at 37°C for 30 to 60 minutes, and then bovine serum albumin (BSA) was added for blocking, and then AFP antigen was modified. Six different concentrations of AFP antigens (500ng / mL, 50ng / mL, 5ng / mL, 0.5ng / mL, 0.05ng / mL, and 0.005ng / mL) were prepared. The modified glassy carbon electrode was used as the working electrode, the silver / silver chloride electrode was used as the reference electrode, and the platinum sheet electrode was used as the counter electrode. The double emulsion droplets were added to the electrolytic cell, and the electrochemical parameters were set. The high potential was set to 0V, the low potential was set to -2V, and the forward scan rate was 0.1V / s. The relationship between the AFP concentration and the electrochemiluminescence intensity was detected by cyclic voltammetry, as shown in Figure 2. Fig.10 As shown, it can be concluded that when the AFP concentration is in the range of 0.05 to 500 ng / mL, its logarithmic function is linearly related to the electrochemiluminescence intensity, and the relationship is y=3495.08x+5585.63, where x is the logarithm of the AFP concentration and y is the electrochemiluminescence intensity.

[0069] The detection range of the present invention is 0.05-500ng / ml, while the detection range of the existing AFP detection kit is 2-480ng / ml, which is lower than the detection limit of the existing AFP detection kit. In addition, the normal reference value of human serum alpha-fetoprotein content is less than 25ng / ml, that is, it can be used to detect human serum alpha-fetoprotein content.

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0071] The above embodiments are only used to illustrate the design ideas and features of the present invention, and their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. The protection scope of the present invention is not limited to the above embodiments. Therefore, any equivalent changes or modifications made based on the principles and design ideas disclosed by the present invention are within the protection scope of the present invention.

[0072] Those skilled in the art will readily appreciate other embodiments of the present application after considering the description and practicing the contents disclosed herein. The present application is intended to cover any variations, uses or adaptations of the present application, which follow the general principles of the present application and include common knowledge or customary technical means in the art that are not disclosed in the present application. The description and examples are intended to be exemplary only.

[0073] It should be understood that the present application is not limited to the exact construction that has been described above and shown in the drawings, and that various modifications and changes may be made without departing from the scope thereof.

Claims

1. An electrochemiluminescent microemulsion, characterized in that: It comprises an external phase solution; the external phase solution encapsulates an intermediate phase solution; the intermediate phase solution encapsulates an internal phase solution; the volume ratio of the external phase solution, the intermediate phase solution and the internal phase solution is 4-20:1.25:1; the external phase solution is an aqueous solution containing 5 wt% PVA or an aqueous solution containing 2 wt% tween 20; the intermediate phase solution is FC 40 containing 2 wt% fluorine surfactant or HFE 7500 containing 2 wt% fluorine surfactant; the internal phase solution is phycocyanin.

2. An electrochemiluminescent microemulsion according to claim 1, characterized in that: The volume ratio of the external phase solution, the intermediate phase solution and the internal phase solution is 8:1.25:

1.

3. A method for producing an electrochemiluminescent microemulsion according to any one of claims 1 to 2, characterized in that: The following steps are involved: (1) preparing an external phase solution, an intermediate phase solution and an internal phase solution; the external phase solution is an aqueous solution containing 5 wt% PVA or an aqueous solution containing 2 wt% tween 20; the intermediate phase solution is FC 40 containing 2 wt% fluorinated surfactant or HFE 7500 containing 2 wt% fluorinated surfactant; (2) filtering and ultrasonically degassing the external phase solution, the intermediate phase solution and the internal phase solution; (3) mixing the intermediate phase solution and the internal phase solution to form an oil-in-water monolayer emulsion; (4) The external phase solution is introduced into the oil-in-water single-layer emulsion formed in step (3) to form a water-in-oil-in-water double-layer emulsion, i.e., an electrochemiluminescent microemulsion.

4. The generation method according to claim 3, characterized in that: The electrochemiluminescent microemulsion is generated based on an electrochemiluminescent microemulsion generating device, the device comprising a fluid injection pump module (1), a microfluidic chip (2), a droplet collecting device (3) and a cover glass (4); the cover glass (4) is provided with a microfluidic chip (2); the microfluidic chip (2) is provided with an external phase channel inlet (21), an intermediate phase channel inlet (22), an internal phase channel inlet (23), a first cross aggregation structure (24), a second cross aggregation structure (25), and a channel outlet (26); the fluid injection pump module (1) comprises an external phase injection pump, an intermediate phase injection pump and an internal phase injection pump, which are connected to the external phase channel inlet (21), the intermediate phase channel inlet (22), and the internal phase channel inlet (23) in sequence; the channel outlet (26) is connected to the droplet collecting device (3); The first cross-aggregation structure (24) is provided with an inner phase shearing opening (24-1), a single emulsion generating opening (24-2), an intermediate phase left shearing opening (24-3) and an intermediate phase right shearing opening (24-4); the inner phase shearing opening (24-1) and the single emulsion generating opening (24-2) are subjected to a narrowing treatment; The second cross-aggregation structure (25) is provided with a single emulsion shearing opening (25-1), a double emulsion generating opening (25-2), an external phase left shearing opening (25-3) and an external phase right shearing opening (25-4); the single emulsion shearing opening (25-1) is subjected to a narrowing treatment; Prepare an external phase solution, an intermediate phase solution and an internal phase solution; and load them into the external phase injection pump, the intermediate phase injection pump and the internal phase injection pump respectively; The flow rates of the external phase injection pump, the intermediate phase injection pump and the internal phase injection pump can be controlled individually; after the injection, an oil-in-water single emulsion is formed at the first cross aggregation structure (24), a water-in-oil-in-water double emulsion is formed at the second cross aggregation structure (25), and finally a microemulsion is collected at the channel outlet (26) through a droplet collection device (3); The device can generate double-layer emulsions with different diameters by controlling the flow rates of the outer phase, the middle phase and the inner phase.

5. The generation method according to claim 4, characterized in that: The connection between the external phase injection pump and the external phase channel inlet (21), the connection between the intermediate phase injection pump and the intermediate phase channel inlet (22), and the connection between the internal phase injection pump and the internal phase channel inlet (23) are all connected via polytetrafluoroethylene microtubes.

6. The generation method according to claim 4, characterized in that: The channel outlet (26) and the droplet collecting device (3) are connected via a polytetrafluoroethylene microtube.

7. Use of the electrochemiluminescent microemulsion according to any one of claims 1 to 2 in the preparation of an immunosensor for quantitative detection of alpha-fetoprotein.

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