Immunodetection liquid for preventing fluorescence leakage, kit and application of immunodetection liquid

Abstract

The invention provides an immunodetection solution for preventing fluorescence leakage, a kit and application of the immunodetection solution, and the immunodetection solution is characterized by comprising 5-100mM of a buffer solution; 0.1%-10% (W / V) of cyclodextrin or a derivative thereof; and 0.05% to 0.2% (W / V) of a surfactant. The invention also provides an application of the immunodetection liquid for preventing fluorescence leakage in droplet immunodetection. The immunodetection liquid has the beneficial effects that the cyclodextrin or the derivative thereof is added into the immunodetection liquid, so that the leakage of resorufin is effectively controlled, and the accuracy of a single-molecule droplet photographing immunodetection result is guaranteed; by controlling the concentration of magnesium ions in the immunodetection liquid, the positive rate is remarkably increased, and then the sensitivity of a liquid drop system is improved; the immunodetection liquid is stable and can be stored at room temperature, and related temperature control components on an instrument are prevented from being used; the immunodetection solution is simple in preparation process and controllable in cost.

Description

Technical Field

[0001] The invention belongs to the technical field of immunoassay, and in particular relates to an immunoassay liquid and a reagent kit capable of preventing fluorescence leakage, and applications thereof. Background Art

[0002] Droplet immunoassay is an immunoassay method based on microdroplet technology. This method uses microfluidics technology to generate droplets with nanoliter to picoliter volumes, and an independent immune reaction is performed in each droplet. Droplet immunoassay has broad application prospects in clinical diagnosis, food safety, environmental monitoring and other fields. In droplet immunoassays based on galactosidase conjugates as reagent components, the most commonly used fluorescent substrates are fluorescein di-β-D-galactoside (FDG) and resorufin-β-D-galactoside (RGP). Under the catalysis of galactosidase, the fluorescent product of FDG is fluorescein, which is highly hydrophilic and stable in the "oil-in-water" structure formed by the microfluidic chip, without obvious leakage of fluorescein. However, FDG exhibits a slow reaction in the galactosidase-catalyzed reaction, and has obvious disadvantages in the development of instrument reagents that pursue detection speed. Under the catalysis of galactosidase, the fluorescent product of RGP is resorufin, which has both certain hydrophilicity and hydrophobicity. In the "oil-in-water" structure formed by the microfluidic chip, the fluorescent product is prone to leakage, thus affecting the accuracy of the immunoassay results. However, RGP exhibits the characteristics of rapid response in the galactosidase catalytic reaction, which meets the speed measurement requirements of instrument reagents.

[0003] Regarding the leakage of fluorescent products, some research results are given in the literature. The research of Philipp Gurner et al. shows that different fluorescent substances show different leakage rates in the same droplet oil, and the droplet oil containing different concentrations of surfactants also affects the leakage rate. Research in this direction is time-consuming and costly, and according to the author's experimental results, its effect in preventing the leakage of resorufin is very limited. Yin Kun et al. used Pickering emulsification to solve the problem of resorufin leakage. This method has a certain effect, but there are disadvantages such as the complex and time-consuming preparation process of Pickering emulsion, and the unstable finished product, which is difficult to use in commercialization. The research of Yousr Skhiri et al. shows that adding bovine serum albumin (BSA) to the buffer solution can control the leakage of resorufin to a certain extent. In the author's experiment, it was found that this method has a certain effect, but the droplets generated after using the buffer containing BSA have a phenomenon of fusion and enlargement, which has an adverse effect on the analysis of experimental data.

[0004] Therefore, there is an urgent need for a reagent that can effectively control fluorescence leakage and is suitable for commercialization to solve the problem of fluorescence leakage in droplet immunoassay. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide an immunoassay liquid, a kit and its application that prevent fluorescence leakage, so as to solve the problems of fluorescence leakage, poor droplet stability and inaccurate test results in current droplet immunoassay.

[0006] In order to achieve the above-mentioned object and other related objects, the present invention provides an immunoassay solution for preventing fluorescence leakage, characterized in that it comprises: 5mM~100mM buffer; Cyclodextrin or its derivatives with a mass volume fraction of 0.1% to 10%; Surfactant with a mass volume fraction of 0.05%~0.2% The mass volume fraction of the preservative is 0.05%~0.2%.

[0007] As a preferred embodiment of the present application, the immune detection solution comprises: 10mM~100mM buffer; Cyclodextrin or its derivatives with a mass volume fraction of 0.1% to 10%; 1mM~20mM soluble magnesium salt; Surfactant with a mass volume fraction of 0.05% to 0.2%; The mass volume fraction of the preservative is 0.05%~0.2%.

[0008] As a preferred embodiment of the present application, the cyclodextrin or its derivative is one or more of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxypropyl-β-cyclodextrin, carboxymethyl-β-cyclodextrin, and sulfobutyl-β-cyclodextrin sodium. More preferably, it is hydroxypropyl-β-cyclodextrin.

[0009] As a preferred embodiment of the present application, the buffer is one or more of PBS buffer, Tri-HCl buffer, and HEPES buffer, and more preferably PBS buffer.

[0010] As a preferred embodiment of the present application, the pH value of the buffer solution is 7.0-8.0, and more preferably 7.4.

[0011] As a preference of the present application, the surfactant is one or more of anionic surfactant, cationic surfactant, amphoteric surfactant or nonionic surfactant.

[0012] As a preferred embodiment of the present application, the surfactant is one or more of Triton X-100, Tween 20, Tween 80, Pluronic F-127, and sodium dodecyl sulfate. More preferably, the surfactant is Tween 20.

[0013] As a preferred embodiment of the present application, the soluble magnesium salt is one or more of magnesium chloride, magnesium nitrate and magnesium sulfate, and more preferably magnesium chloride.

[0014] As a preferred embodiment of the present application, the preservative is Proclin 950.

[0015] As a preferred embodiment of the present application, the immune detection solution includes: 10 mM PBS buffer; 1% (W / V) hydroxypropyl β-cyclodextrin; 5 mM magnesium chloride; 0.05% (W / V) Tween 20; 0.1% (W / V) Proclin950.

[0016] The present invention also provides a fluorescence immunoassay quantitative assay kit, characterized in that it comprises the immunoassay liquid for preventing fluorescence leakage.

[0017] The present invention also provides an application of the fluorescent immunoassay quantitative assay kit in droplet immunoassay.

[0018] The present invention also provides an application of the immunoassay liquid capable of preventing fluorescence leakage in droplet immunoassay.

[0019] As a preferred embodiment of the present application, the application method includes some or all of the following steps: Step 1, preparing a dilution solution of the sample to be tested: diluting the sample to be tested by a known multiple with a buffer solution to obtain a dilution solution of the sample to be tested; Step 2, preparing magnetic bead immune complexes: using immunomagnetic beads coated with specific antibodies to capture antigens in the dilution of the sample to be tested, then incubating with biotin-coupled specific antibodies, washing, incubating with streptavidin-galactosidase complexes, and washing again to obtain magnetic bead immune complexes; Step 3, resuspending the magnetic bead immune complex with the immune detection solution, oscillating and mixing, and obtaining a resuspended magnetic bead solution; Step 4, using the resuspended magnetic bead solution as the magnetic bead phase, the substrate solution as the substrate phase, and the droplet generation oil as the oil phase, and generating water-in-oil droplets after passing through a microfluidic chip; the substrate solution is a resorufin-β-D-galactoside solution; Step 5, incubate the water-in-oil droplets in the droplet storage channel of the microfluidic chip at 37° C.-60° C. for 2-5 minutes; Step 6. After the incubation is completed, the microfluidic chip is placed in an inverted microscope to take bright field and dark field photos, and bright field photos and dark field fluorescence photos of sample dilutions of different concentrations are obtained. According to the results of the bright field photos and dark field photos of the sample dilutions, the total number of magnetic beads, the number of positive magnetic beads and the number of fluorescent leakage magnetic beads corresponding to each group are determined, and then the positive rate and the fluorescent leakage rate are calculated according to formula (1) and formula (2); Positive rate = number of positive magnetic beads / total number of magnetic beads (1); Fluorescence leakage rate = number of fluorescent leakage beads / number of positive beads (2); Step 7: Find the corresponding antigen concentration value according to the antigen concentration-positive rate standard curve and multiply it by the dilution factor of the sample to be tested to obtain the concentration and content of the antigen in the sample to be tested.

[0020] As a preferred embodiment of the present application, the volume ratio of the magnetic bead phase to the substrate phase is 1:1.

[0021] As a preferred embodiment of the present application, in step 3, the droplet generating oil is HFE7500.

[0022] As a preferred embodiment of the present application, the substrate solution is RGP reagent (Cat. No.: 103159) from quanterix.

[0023] As a preferred embodiment of the present application, the material of the microfluidic chip is PDMS.

[0024] As a preferred embodiment of the present application, the height of the droplet storage channel of the microfluidic chip is 10 μm to 25 μm, more preferably 15 μm.

[0025] As a preferred embodiment of the present application, the connecting medium between the microfluidic chip and the sample is an elastic quartz capillary, and the inner diameter of the capillary is 100 μm.

[0026] As a preferred embodiment of the present application, in step 3, the diameter of the water-in-oil droplet is 7 μm to 15 μm, and more preferably 10 μm.

[0027] As a preferred embodiment of the present application, in step 4, the incubation temperature of the water-in-oil droplets is 50° C., and the incubation time is 3 min.

[0028] As a preferred embodiment of the present application, the antigen concentration-positive rate standard curve is established as follows: The antigen with known purity and concentration is gradiently diluted with a buffer to obtain an antigen dilution solution; Preparation of magnetic bead immune complexes: using immunomagnetic beads coated with specific antibodies to capture antigens in the antigen dilution solution, then incubating with biotin-coupled specific antibodies, washing, then incubating with streptavidin-galactosidase complexes, and washing again to obtain magnetic bead immune complexes; Resuspending the magnetic bead immune complex with the immune detection solution, oscillating and mixing, and obtaining a resuspended magnetic bead solution; The resuspended magnetic bead solution is used as the magnetic bead phase, the substrate solution is used as the substrate phase, and the droplet generation oil is used as the oil phase, and water-in-oil droplets are generated after passing through a microfluidic chip; the substrate solution is a resorufin-β-D-galactoside solution; The water-in-oil droplets collected in the droplet storage channel of the microfluidic chip were incubated at 37°C-60°C for 2-5 min; After the incubation is completed, the microfluidic chip is placed in an inverted microscope for bright field and dark field photography, and bright field photos and dark field fluorescence photos of sample dilutions of different concentrations are obtained. The bright field photos and the corresponding dark field photos are automatically fused and analyzed. According to the results of the bright field photos and dark field photos of the sample dilutions, the total number of magnetic beads, the number of positive magnetic beads and the number of fluorescent leakage magnetic beads corresponding to each group are determined. Then, the positive rate and the fluorescent leakage rate are calculated according to formula (1) and formula (2), and the antigen concentration-positive rate standard curve is established; Positive rate = number of positive magnetic beads / total number of magnetic beads (1); Fluorescence leakage rate = number of fluorescent leakage beads / number of positive beads (2).

[0029] As a preferred embodiment of the present application, in the step of establishing the antigen concentration-positive rate standard curve, the antigen is gradiently diluted with a 1% BSA PBST solution to obtain an antigen dilution solution.

[0030] The present invention also provides a microfluidic chip, including a chip body, characterized in that: a liquid inlet and a droplet storage channel connected to the liquid inlet are provided on the chip body, the liquid inlet includes an oil phase inlet, a magnetic bead phase inlet, a substrate phase inlet, and a droplet generation port that are interconnected, the droplet generation port is connected to the liquid inlet of the droplet storage channel, and the liquid outlet of the droplet storage channel is located at an end away from the liquid inlet.

[0031] As a preferred embodiment of the present application, the microfluidic chip also includes a temperature control module, which includes a microscope stage and a heating module, a temperature sensing probe and a heat conductive block arranged on the microscope stage. A chip droplet generation observation window is provided on the microscope stage for observing a single droplet flowing through the chip droplet generation observation window; the temperature sensing probe is arranged on the heat conductive block for detecting the temperature of the heat conductive block; the heat conductive block is in contact with the heating module for realizing heat conduction.

[0032] Compared with the prior art, the present invention has the following beneficial effects: 1. Cyclodextrin or its derivatives are added to the immunoassay solution to effectively control the leakage of resorufin; 2. It overcomes the problem of droplet fusion caused by bovine serum albumin in the solution, ensuring the accuracy of the single-molecule droplet photo-immunoassay results; 3. By controlling the magnesium ion concentration in the immunoassay solution, the positive rate is significantly increased, thereby improving the sensitivity of the droplet system; 4. The immunoassay solution is stable and can be stored at room temperature, avoiding the use of related temperature control components on the instrument; 5. The preparation process of the immunoassay solution is simple and the cost is controllable. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 and Figure 2 Schematic diagram of fluorescence leakage of the immune detection solution of Example 1 ( Figure 1 For bright field images, Figure 2 is a dark-field fluorescence image).

[0034] Figure 3 and Figure 4 Schematic diagram of fluorescence leakage of the detection solution 1 of comparative example 1 ( Figure 3 For bright field images, Figure 4 is a dark-field fluorescence image).

[0035] Figure 5 and Figure 6 Schematic diagram of fluorescence leakage of the detection solution 2 of comparative example 2 ( Figure 5 For bright field images, Figure 6 is a dark-field fluorescence image).

[0036] Figure 7 This is the antigen concentration-positive rate standard curve of the immune detection solution in Example 5 in human IL-6 antigen detection.

[0037] Figure 8 Schematic diagram of the structure of the microfluidic chip of this application.

[0038] Fig. 9 This is a schematic diagram of the back side of the temperature control module of the microfluidic chip of this application. DETAILED DESCRIPTION

[0039] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0040] It should be noted that the process equipment or devices not specifically specified in the following embodiments are all conventional equipment or devices in the art.

[0041] In addition, it should be understood that one or more method steps mentioned in the present invention do not exclude the existence of other method steps before or after the combination step or the insertion of other method steps between these explicitly mentioned steps, unless otherwise specified; it should also be understood that the combination connection relationship between one or more devices / apparatuses mentioned in the present invention does not exclude the existence of other devices / apparatuses before or after the combination device / apparatus or the insertion of other devices / apparatuses between these explicitly mentioned two devices / apparatuses, unless otherwise specified. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or the scope of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the present invention without substantially changing the technical content.

[0042] Example 1 Step 1. Prepare antigen diluent: Use 1% (W / V) BSA (MERCK, catalog number: B2064-100g) in PBST as antigen diluent; Step 2, prepare magnetic bead immune complex: 500,000 / test of immunomagnetic beads coated with human IL-6 antibody (GenScript Biotech, catalog number: V06401) as reagent 1, 50 ng / mL of biotin-coupled human IL-6 antibody (GenScript Biotech, catalog number: V06403) as reagent 2, 50 ng / mL of streptavidin-galactosidase complex (Thermo Fisher Scientific, catalog number: S931) as reagent 3, and NIBSC / WHO (89 / 548) INTERLEUKIN-6 dissolved solution as antigen. Dilute to 30 pg / mL with the antigen diluent prepared in step 1, perform a three-step immune reaction with an incubation procedure of 30 minutes + 15 minutes + 15 minutes, then place the obtained product on a magnetic stand for magnetic absorption, discard the supernatant solution, and obtain immune complex magnetic beads; Step 3, resuspend the magnetic bead immune complex with an immunoassay solution, oscillate and mix, and obtain a resuspended magnetic bead solution for subsequent droplet fluorescence leakage test; the immunoassay solution includes: 10mM PBS solution (pH7.4), 1% (W / V) hydroxypropyl-β-cyclodextrin, 0.05% (W / V) Tween 20, and 0.1% Proclin950; Step 4: The resuspended magnetic bead solution is used as the magnetic bead phase, the RGP reagent (Cat. No.: 103159) of quanterix is ​​used as the substrate phase, and HFE7500 is used as the oil phase. The microfluidic chip is connected to the sample tube through a quartz capillary with an inner diameter of 100 μm. The sample tube is then connected to the air pump through a hose. The volume ratio of the magnetic bead phase and the substrate phase is controlled to be 1:1 by air pressure. The air pump applies an air pressure of 115 kPa to the substrate phase and the magnetic bead phase in the microfluidic chip, and an air pressure of 140 kPa to the oil phase. Under this condition, oil-in-water droplets are generated, and the size of the droplets is about 10 μm. Step 5: When the generated droplets completely fill the droplet storage channel of the microfluidic chip for storing droplets, turn off the air pump and incubate at 50° C. for 3 min in the temperature control module of the microfluidic chip; Step 6: After the incubation is completed, the microfluidic chip is placed in an inverted microscope to take bright field and dark field photos; bright field photos and dark field fluorescence photos of sample dilutions of different concentrations are obtained; Specifically, the inverted fluorescence microscope was adjusted to 10x, and the location where the droplets were densely concentrated in the chip was first found through the bright field, and a bright field photo was taken; then the bright field light source was turned off, switched to the green laser channel, the exposure time was set to 1000 milliseconds, and a dark field fluorescence photo was taken; it should be noted that multiple groups of photos were taken for each sample to obtain sufficient analysis data; Processing the photos: automatically fusion analyzing a group of pictures (including bright field and dark field), identifying the droplets containing only a single magnetic bead in the picture by setting the magnetic bead particle size parameter, identifying the fluorescence positive droplets in the picture by setting the fluorescence intensity parameter, and analyzing the average fluorescence intensity of the surrounding droplets within the center diameter of the magnetic bead droplet to determine the droplet leakage situation, and finally outputting the total number of magnetic beads, the number of positive magnetic beads and the number of fluorescent leakage magnetic beads for each group of pictures, and then calculating the positive rate and fluorescence leakage rate according to formula (1) and formula (2); Positive rate = number of positive magnetic beads / total number of magnetic beads (1); Fluorescence leakage rate = number of fluorescent leakage beads / number of positive beads (2).

[0043] Test results such as Figure 1 and Figure 2 .

[0044] Embodiments 2 to 14 The difference between Example 1 and Example 1 is that the immunoassay solution is different (the immunoassay solution ratio is shown in Table 1), the test antigen concentration is 30 pg / mL and 0.37 pg / mL, and the other process conditions are exactly the same. The test results are shown in Table 2.

[0045] Table 1 Test solution ratio table Buffer Soluble salt Cyclodextrin or its derivatives ((W / V) Surfactant (W / V) Preservatives (W / V) Example 1 10mM PBS pH7.4 - 1% Hydroxypropyl β-cyclodextrin 0.05% Tween 20 0.1%Proclin950 Example 2 10 mM PBS pH 7.4 - 1% α-cyclodextrin 0.05% Tween 20 0.1%Proclin950 Example 3 10 mM PBS pH 7.4 - 0.1% Hydroxypropyl β-cyclodextrin 0.05% Tween 20 0.1%Proclin950 Example 4 10 mM PBS pH 7.4 - 10% Hydroxypropyl-β-cyclodextrin 0.05% Tween 20 0.1%Proclin950 Example 5 10 mM PBS pH 7.4 Magnesium chloride, 5 mM 1% Hydroxypropyl-β-cyclodextrin 0.05% Tween 20 0.1%Proclin950 Example 6 10 mM PBS pH 7.4 Magnesium chloride, 20 mM 1% Hydroxypropyl-β-cyclodextrin 0.05% Tween 20 0.1%Proclin950 Example 7 10 mM PBS pH 7.4 Magnesium chloride, 5 mM 1% Hydroxypropyl-β-cyclodextrin 0.1%Pluronic F-127 0.1%Proclin950 Example 8 20 mM HEPES, pH 7.4 Magnesium chloride, 5 mM 1% Hydroxypropyl-β-cyclodextrin 0.05% Tween 20 0.1%Proclin950 Example 9 20 mM Tris, pH 8.0 Magnesium chloride, 5 mM 1% Hydroxypropyl-β-cyclodextrin 0.05% Tween 20 0.1%Proclin950 Example 10 5mM PBS pH7.4 Magnesium chloride, 5 mM 1% Hydroxypropyl-β-cyclodextrin 0.05% Tween 20 0.1%Proclin950 Embodiment 11 100 mM PBS pH 7.4 Magnesium chloride, 5 mM 1% Hydroxypropyl-β-cyclodextrin 0.05% Tween 20 0.1%Proclin950 Example 12 10 mM PBS pH 7.4 Magnesium sulfate, 5 mM 1% Hydroxypropyl-β-cyclodextrin 0.05% Tween 20 0.1%Proclin950 Embodiment 13 10 mM PBS pH 7.4 Magnesium chloride, 1 mM 1% Hydroxypropyl-β-cyclodextrin 0.05% Tween 20 0.1%Proclin950 Embodiment 14 10 mM PBS pH 7.4 - 1% β-cyclodextrin 0.05% Tween 20 0.1%Proclin950 Comparative Example 1 The difference between this comparative example and Example 1 is that the immune detection solution is replaced by detection solution 1, which includes: 10mM PBS solution, 0.05% Tween 20, 0.1% (W / V) Proclin 950, and the pH of the PBS solution is 7.4. The rest of the process is exactly the same. The test results are as follows Figure 3 and Figure 4 .

[0046] Comparative Example 2 The difference between this comparative example and Example 1 is that the immune detection solution is replaced by the detection solution 2, which includes: 10mM PBS solution, 5% (W / V) BSA, 0.05% Tween 20, 0.1% (W / V) Proclin 950, and the pH of the PBS solution is 7.4. The rest of the process is exactly the same. The test results are as follows Figure 5 and Figure 6 .

[0047] Comparative Example 3 The difference between this comparative example and Example 1 is that 1% (W / V) PVP K30 is used instead of 1% (W / V) cyclodextrin. The rest of the processes are exactly the same.

[0048] Comparative Example 4 The difference between this comparative example and Example 1 is that 1% (W / V) PEG6000 is used instead of 1% (W / V) cyclodextrin. The rest of the processes are exactly the same.

[0049] Comparative Example 5 The difference between this comparative example and Example 1 is that 1% (W / V) urea is used instead of 1% (W / V) cyclodextrin. The rest of the processes are exactly the same.

[0050] Comparative Example 6 The difference between this comparative example and Example 1 is that 1% (W / V) DMSO is used instead of 1% (W / V) cyclodextrin. The rest of the processes are exactly the same.

[0051] Result analysis: From Figure 1 to Figure 6 As shown, in the detection solution 1 of Comparative Example 1 ( Figure 3 and Figure 4 ), the fluorescent product resorufin leaked out obviously (the positive droplets and the adjacent droplets formed a "flower" shape); in the detection solution 2 of comparative example 2 ( Figure 5 and Figure 6 ), there was no obvious leakage of the fluorescent product, but the droplet size was uneven (further research found that the droplets would fuse during the incubation process); in the immunoassay solution of Example 1, there was no obvious leakage of the fluorescent substance, and the droplet size was uniform (such as Figure 1 and Figure 2). The above experimental results show that the immunoassay solution of the present application has the advantage of solving the fluorescence leakage in droplet immunoassay, and can overcome the droplet fusion caused by the presence of bovine serum albumin in the solution.

[0052] Table 2 Leakage rate of different ratios of test solution at antigen dilution concentrations of 30 pg / mL and 0.37 pg / mL From the positive rate and / or leakage rate data of Examples 1 to 14 and Comparative Examples 1 to 6 statistically shown in Table 2, the addition of cyclodextrin or its derivatives significantly controls the fluorescence leakage rate of the 30pg / mL antigen system, which can be reduced from the original 89.42% to less than 40.78%. The anti-fluorescence leakage rate of Examples 1, 4 to 13 is controlled below 1%, and there is almost no fluorescence leakage problem. It can be judged that cyclodextrin or its derivatives play a key role in the detection solution, and the detection solution to which cyclodextrin or its derivatives are added can effectively control the leakage of the fluorescent product (resorufin). According to the positive rate and leakage rate data of Example 2 and Example 1, even when the concentration of hydroxypropyl β-cyclodextrin is 0.1%, the leakage rate is better than that of Example 2, from which it can be seen that hydroxypropyl β-cyclodextrin is better than cyclodextrin. As can be seen from Examples 1, 3 to 4, with the increase in the concentration of hydroxypropyl β-cyclodextrin, the 30pg / mL leakage rate is significantly reduced, but when the concentration of hydroxypropyl-β-cyclodextrin reaches 10%, the 0.37pg / mL positive rate is significantly suppressed. Considering comprehensively, the concentration of hydroxypropyl β-cyclodextrin is 1%, which is the optimal concentration. According to the changes in the types of buffer solutions of Examples 5, 7 to 9 as shown in Table 2, the changes in pH and the replacement of surfactants have little effect on the changes in leakage rate and positive rate. As can be seen from Examples 1, 5, 6 and 12 as shown in Table 2, the addition of magnesium chloride or magnesium sulfate has increased the positive rate of the 0.37pg / mL antigen system to a certain extent and the effect is comparable, but there is no obvious effect on the leakage rate. Analysis believes that it may be caused by the promotion of magnesium ions in the activity of galactosidase. Considering comprehensively according to the results of Examples 2 to 13, it is determined that the immunoassay solution for preventing fluorescence leakage is best selected from Example 5.

[0053] Embodiment 15 According to Example 5, a test solution was re-formulated for use in a human IL-6 antigen detection kit to determine the effect of the formula in actual use. The sample preparation refers to the step of obtaining the immune complex magnetic beads in Example 1, and the specific antigen concentration is set to 30pg / mL, 10pg / mL, 3.333pg / mL, 1.111pg / mL, 0.37pg / mL, 0.123pg / mL, 0.041pg / mL, 0pg / mL. The subsequent steps refer to the other contents of Example 1.

[0054] Table 3 Verification of the effect of formula 6 detection solution in human IL-6 antigen detection Antigen concentration (pg / mL) Total number of beads Number of positive magnetic beads Fluorescence leakage magnetic beads Positive rate Fluorescence leakage rate 0.000 6482 90 0 1.39% 0.00% 0.041 6031 122 0 2.02% 0.00% 0.123 5975 245 0 4.10% 0.00% 0.370 6981 641 0 9.18% 0.00% 1.111 6589 1439 0 21.84% 0.00% 3.333 7210 3824 2 53.04% 0.03% 10.000 5844 5256 6 89.94% 0.10% 30.000 6571 6525 11 99.30% 0.17% From the statistical data in Table 3, it can be seen that under the conditions of various antigen concentrations of human IL-6 standard curve, the leakage rate of droplets is extremely low, indicating that the solution can effectively control the leakage of droplet fluorescence and ensure the accuracy of the positive rate statistics of the experimental results. Figure 4 From the plotted standard curve, each concentration point has good discrimination and the sensitivity can reach fg / mL level, which indicates that this group of immunoassay solutions can meet product requirements.

[0055] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limitations of the present invention. In addition, the various modifications listed herein and the variations of methods and compositions in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications obvious to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. An immunoassay solution for preventing fluorescence leakage, characterized in that: include: 5mM~100mM buffer; Cyclodextrin or its derivatives with a mass volume fraction of 0.1% to 10%; Surfactant with a mass volume fraction of 0.05% to 0.2%; The mass volume fraction of the preservative is 0.05%~0.2%.

2. The immunoassay solution for preventing fluorescence leakage according to claim 1, characterized in that: include: 5mM~100mM buffer; Cyclodextrin or its derivatives with a mass volume fraction of 0.1% to 10%; 1mM~20mM soluble magnesium salt; Surfactant with a mass volume fraction of 0.05% to 0.2%; The mass volume fraction of the preservative is 0.05%~0.2%.

3. The immunoassay solution for preventing fluorescence leakage according to claim 1, characterized in that: The cyclodextrin or its derivative is one or more of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxypropyl-β-cyclodextrin, carboxymethyl-β-cyclodextrin, and sulfobutyl-β-cyclodextrin sodium.

4. The immunoassay solution for preventing fluorescence leakage according to claim 1, characterized in that: The buffer is one or more of PBS buffer, Tri-HCl buffer and HEPES buffer.

5. The immunoassay solution for preventing fluorescence leakage according to claim 3, characterized in that: The pH value of the buffer solution is 7.0-8.

0.

6. The immunoassay solution for preventing fluorescence leakage according to claim 2, characterized in that: The soluble magnesium salt is one or more of magnesium chloride, magnesium sulfate and magnesium nitrate.

7. The immunoassay solution for preventing fluorescence leakage according to claim 6, characterized in that: include: 10 mM PBS buffer; Hydroxypropyl β-cyclodextrin with a mass volume fraction of 1%; 5 mM magnesium chloride; Tween 20 with a mass volume fraction of 0.05%; Proclin950 with a mass volume fraction of 0.1%.

8. A fluorescent immunoassay kit, characterized in that: The invention comprises the immunoassay liquid for preventing fluorescence leakage as described in any one of claims 1 to 7.

9. Use of the immunoassay solution for preventing fluorescence leakage as claimed in any one of claims 1 to 7 in immunoassay.

10. The use according to claim 9, characterized in that: The application method comprises some or all of the following steps: Step 1, preparing a dilution solution of the sample to be tested: diluting the sample to be tested by a known multiple with a buffer solution to obtain a dilution solution of the sample to be tested; Step 2, preparing magnetic bead immune complexes: using immunomagnetic beads coated with specific antibodies to capture antigens in the dilution of the sample to be tested, then incubating with biotin-coupled specific antibodies, washing, incubating with streptavidin-galactosidase complexes, and washing again to obtain magnetic bead immune complexes; Step 3, resuspending the magnetic bead immune complex with the immune detection solution, oscillating and mixing, and obtaining a resuspended magnetic bead solution; Step 4, using the resuspended magnetic bead solution as the magnetic bead phase, the substrate solution as the substrate phase, and the droplet generation oil as the oil phase to generate water-in-oil droplets through a microfluidic chip; The substrate solution is a resorufin-β-D-galactoside solution; Step 5, incubate the water-in-oil droplets in the droplet storage channel of the microfluidic chip at 37° C.-60° C. for 2-5 minutes; Step 6. After the incubation is completed, the microfluidic chip is placed in an inverted microscope for photographing to obtain bright field photographs and dark field fluorescence photographs of samples of different concentrations. The bright field photographs and dark field fluorescence photographs of each group are combined for analysis to determine the total number of magnetic beads, the number of positive magnetic beads, and the number of fluorescent leakage magnetic beads corresponding to each group, and then the positive rate and the fluorescent leakage rate are calculated according to formula (1) and formula (2); Positive rate = number of positive magnetic beads / total number of magnetic beads (1); Fluorescence leakage rate = number of fluorescent leakage beads / number of positive beads (2); Step 7: Substitute the positive rate of the sample to be tested into the standard curve of antigen concentration-positive rate fitting for calculation to obtain the antigen concentration in the sample to be tested.

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