Two-color quantum dot fluorescent ball two-joint detection immunochromatographic test strip and preparation method thereof

Abstract

The invention discloses a two-color quantum dot fluorescent ball combined detection immunochromatography test strip and a preparation method thereof. A test strip combination pad is coated with a first antibody marked by a green quantum dot fluorescent ball and a second antibody marked by a red quantum dot fluorescent ball; a T1 line, a T2 line, a C line and a calibration line are sequentially arranged on the reaction pad, the T1 line is coated with a first antibody, the T2 line is coated with a second antibody, the C line is coated with a second antibody, and the calibration line is coated with yellow water-soluble quantum dots with the emission wavelength of lambda 3. According to the invention, two kinds of quantum dot fluorescent balls with different particle sizes and different colors are respectively used as fluorescent markers of two detection indexes, so that the influence of cross reaction during multi-index detection in immunochromatography detection is reduced, and the problems of interference among the fluorescent markers during multi-index simultaneous detection and the like are avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of immunochromatography, and in particular to a two-color quantum dot fluorescent ball dual detection immunochromatography test strip and a preparation method thereof. Background Art

[0002] Fluorescence immunochromatography is a new type of membrane detection technology based on antigen-antibody specific immune reactions. This technology uses a strip of fiber chromatography material with fixed detection lines and quality control lines as the stationary phase, the test liquid as the mobile phase, and the fluorescent labeled antibody fixed to the binding pad. The analyte moves on the chromatography membrane as the reaction pad through capillary action.

[0003] Quantum dots are fluorescent nanoparticles. In quantum dot immunochromatographic test strips that use quantum dots as fluorescent markers, the combination of quantum dots and antibody proteins needs to be coated on the binding pad. Existing immunochromatographic tests will have cross-reactions when used for multi-indicator testing, which will lead to interference between fluorescent markers in the simultaneous detection of multiple indicators.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a two-color quantum dot fluorescent ball dual-detection immunochromatographic test strip and a preparation method thereof, so as to improve the sensitivity of the immunochromatographic test strip and reduce the proportion of false positive and false negative results in the test process.

[0006] The present invention is achieved in that:

[0007] In a first aspect, the present invention provides a two-color quantum dot fluorescent ball dual-test immunochromatographic test strip, comprising a bottom plate, on which a sample pad, a binding pad, a reaction pad and a water absorbent pad are sequentially arranged;

[0008] The conjugate pad is coated with a first antibody labeled with a green quantum dot fluorescent ball and a second antibody labeled with a red quantum dot fluorescent ball; wherein the average particle size of the green quantum dot fluorescent ball is 50-70nm, and the average particle size of the red quantum dot fluorescent ball is 300-330nm; the emission wavelength of the quantum dots in the green quantum dot fluorescent ball is λ1, and the emission wavelength of the quantum dots in the red quantum dot fluorescent ball is λ2, wherein λ1 is 500-550nm, and λ2 is 600-650nm;

[0009] The reaction pad is provided with a T1 line, a T2 line, a C line and a calibration line in sequence, wherein the T1 line is coated with a first antibody, the T2 line is coated with a second antibody, the C line is coated with a secondary antibody, and the calibration line is coated with yellow water-soluble quantum dots with an emission wavelength of λ3, wherein λ3 is 565-585nm.

[0010] In an optional embodiment, the yellow water-soluble quantum dot surface ligand is an amphiphilic polymer;

[0011] And / or, the quantum dot surface ligands in the green quantum dot fluorescent ball and the red quantum dot fluorescent ball are both oleic acid.

[0012] In an optional embodiment, the CV of the green quantum dot fluorescent ball is less than 10%; and / or the CV of the red quantum dot fluorescent ball is less than 10%.

[0013] In an optional embodiment, the method for preparing the green quantum dot fluorescent ball comprises: adding an initiator to an emulsion including styrene monomer and green quantum dots under mechanical stirring conditions to carry out an emulsion polymerization reaction to obtain a green quantum dot fluorescent ball;

[0014] And / or, the method for preparing the red quantum dot fluorescent ball comprises: adding an initiator to an emulsion including styrene monomer and red quantum dots under mechanical stirring conditions to carry out emulsion polymerization reaction to obtain the red quantum dot fluorescent ball.

[0015] In an optional embodiment, during the preparation of the green quantum dot fluorescent ball, the initiator is potassium persulfate, the amount of styrene monomer used is 3-5 ml, and the amount of potassium persulfate used is 20-30 mg.

[0016] In an optional embodiment, during the preparation of the red quantum dot fluorescent ball, the initiator is potassium persulfate, the amount of styrene monomer used is 14-18 ml, and the amount of potassium persulfate used is 20-30 mg.

[0017] In an optional embodiment, in the emulsion polymerization reaction, the rotation speed of the mechanical stirrer is 14000-16000 r / min, and the emulsification time is 4-6 min.

[0018] In an optional embodiment, the mass ratio of the green quantum dots to the styrene monomer is 1:5-10; and / or the mass ratio of the red quantum dots to the styrene monomer is 1:15-30.

[0019] In an optional embodiment, the first antibody is a mouse anti-human CRP antibody, the second antibody is a mouse anti-human PCT antibody, and the secondary antibody is a goat anti-mouse secondary antibody.

[0020] In a second aspect, the present invention provides a method for preparing the two-color quantum dot fluorescent ball dual-detection immunochromatographic test strip described in the aforementioned embodiment, comprising:

[0021] S1. Preparation of green quantum dot fluorescent balls and preparation of red quantum dot fluorescent balls;

[0022] S2. Green quantum dot fluorescent ball labeled specific antibody and red quantum dot fluorescent ball labeled specific antibody;

[0023] S3. Preparation of test strips.

[0024] The present invention has the following beneficial effects:

[0025] The present invention designs two quantum dot fluorescent balls with different particle sizes and different colors as fluorescent markers for two detection indicators respectively, and utilizes the fluorescence property of quantum dots with single excitation and multiple emission to reduce the influence of cross-reaction during multi-indicator detection in immunochromatographic detection, and avoids the interference between fluorescent markers when multiple indicators are detected simultaneously.

[0026] For immunochromatographic readers, interference from factors such as the intensity of the excitation light sometimes causes deviations in the test signal. Adding a calibration line allows the instrument error to be judged while testing. Calibrating the test signal with the calibration line can reduce the test error. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0028] Figure 1 This is a detection principle diagram of the two-color quantum dot fluorescent ball dual-detection immunochromatographic test strip in Example 1. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0030] The present invention provides a two-color quantum dot fluorescent ball dual-test immunochromatographic test paper strip, comprising a bottom plate, on which a sample pad, a binding pad, a reaction pad and a water absorption pad are sequentially arranged;

[0031] The conjugate pad is coated with a first antibody labeled with a green quantum dot fluorescent ball and a second antibody labeled with a red quantum dot fluorescent ball; wherein the average particle size of the green quantum dot fluorescent ball is 50-70nm, and the average particle size of the red quantum dot fluorescent ball is 300-330nm; the emission wavelength of the quantum dots in the green quantum dot fluorescent ball is λ1, and the emission wavelength of the quantum dots in the red quantum dot fluorescent ball is λ2, wherein λ1 is 500-550nm, and λ2 is 600-650nm;

[0032] The reaction pad is provided with a T1 line, a T2 line, a C line and a calibration line in sequence, wherein the T1 line is coated with a first antibody, the T2 line is coated with a second antibody, the C line is coated with a secondary antibody, and the calibration line is coated with yellow water-soluble quantum dots with an emission wavelength of λ3, wherein λ3 is 565-585nm.

[0033] In order to make the quantum dot fluorescent ball achieve better immunochromatography effect, two fluorescent balls, one large and one small, are designed respectively, which can effectively avoid the steric effect in the chromatography process. The small-particle fluorescent ball can shuttle in the laminar flow system of the large-particle fluorescent ball. The enrichment of the T1 line will not hinder the chromatographic flow and signal enrichment of the small-particle T2 line. When the 50-70nm green fluorescent ball is selected as a fluorescent marker and the 300-330nm red fluorescent ball is used as the second marker, the markers of the dual-index detection can be non-interfering with each other. The green quantum dot fluorescent ball with a small particle size and the red quantum dot fluorescent ball with a large particle size are designed on the binding pad respectively. The T1 line that appears first is designed as a red fluorescent signal detection indicator, and the T2 line that comes out later is designed as a green fluorescent signal indicator. The C line can detect both green and red signal indicators, and the calibration line is directly sprayed with a fixed concentration of yellow water-soluble quantum dots.

[0034] The present invention designs two different particle sizes and quantum dot fluorescent balls of different colors as fluorescent markers of two detection indicators, respectively, and utilizes the fluorescence properties of quantum dots with one-element excitation and multiple emission to reduce the influence of cross-reactions during multi-index detection in immunochromatographic detection, and avoids the interference between fluorescent markers that occur when multiple indicators are detected simultaneously. The problem of cross-reactions between indicators can be effectively avoided by performing dual-index detection with green and red quantum dots. In the projects of detecting two indicators with the same fluorescent signal available on the market, there will be factors of mutual interference between indicators. The fluorescent marker of the A indicator may be enriched in the detection line of the B indicator to produce a fluorescent signal. Since a fluorescent marker is used, this cross-interference will be misjudged as the fluorescent signal of the B indicator itself, thereby resulting in a false positive result of the B indicator. If the A and B indicators are detected using fluorescent signals of different colors, the above problems will not exist, thereby avoiding false positive results.

[0035] In order to avoid drying between quantum dot fluorescent balls of different colors, the emission wavelengths of the quantum dots contained in each fluorescent ball need to be different. In the present invention, the emission wavelengths are used to distinguish the green quantum dot fluorescent balls and the red quantum dot fluorescent balls: the emission wavelengths are λ1500-550nm; λ2600-650nm, and in some embodiments, λ 1、 λ 2、 λ3 is 525nm, 575nm and 625nm respectively.

[0036] For immunochromatographic readers, due to interference from factors such as the intensity of the excitation light, deviations in the test signal may sometimes occur. By adding a calibration line, the instrument error can be judged while testing, and the test error can be reduced by calibrating the test signal through the calibration line. The calibration line uses yellow water-soluble quantum dots with a stable emission wavelength of 575nm for fluorescent signals, and shares the excitation light source with the red and green quantum dots of the test line, which plays a role in calibrating the intensity of the excitation light source. Under this system, a calibration line is added to each test strip, which greatly improves the accuracy of the test, reduces the test deviation from all aspects, and accurately reflects a test result. In an embodiment of the present invention, yellow water-soluble quantum dots are added to the nitrocellulose membrane (NC membrane) as a calibration line. The stability of the instrument excitation light and detection can be judged while testing, and the test signal can be calibrated in time. The C line and the calibration line can be used together as the basis for quality control of multiple indicators.

[0037] The purpose of designing the C line as the quality control line of the immunochromatographic test strip is to determine whether the activity and fluorescence properties of the marker on the test strip binding pad have failed, thereby avoiding false negative results caused by the failure of the fluorescent marker. Quantum dots have the optical properties of univariate excitation and multivariate emission. Green and red quantum dots are used for dual-index detection at the same time. Only when the C line detects both green and red fluorescence signals can the test be judged to be qualified. Such a C line is a true quality control line. According to the quality control line, it can be judged whether the test is valid. For dual-index detection, false negative results of a single indicator are avoided. The dual-index combined detection uses green and red quantum dot fluorescent balls respectively, which can achieve the detection sensitivity required for clinical use, while effectively avoiding false positive and false negative results during the test process, and accurately reflecting the test results.

[0038] The embodiment of the present invention adopts green and red quantum dot fluorescent balls respectively through dual-index combined detection, which can achieve the detection sensitivity required by clinical practice, effectively avoid false positive and false negative results in the test process, and accurately reflect the test results.

[0039] In an optional embodiment, the yellow water-soluble quantum dot surface ligand is an amphiphilic polymer;

[0040] In an optional embodiment, the quantum dot surface ligands in the green quantum dot fluorescent ball and the red quantum dot fluorescent ball are both oleic acid, which is oil-soluble.

[0041] The surface of the fluorescent ball is carboxylated and sealed, which can label any protein and effectively avoid interference between different fluorescent balls. While retaining the optical properties of quantum dots, the quantum dot fluorescent ball also reduces the difficulty of separating the product after labeling. The present invention designs two fluorescent balls that can be coupled with proteins by carboxyl activation, and purified, precipitated and enriched by centrifugation, and the labeling process is simple.

[0042] In an optional embodiment, the CV of the green quantum dot fluorescent ball is less than 10%; and / or the CV of the red quantum dot fluorescent ball is less than 10%.

[0043] In an optional embodiment, the method for preparing the green quantum dot fluorescent ball comprises: adding an initiator to an emulsion including styrene monomer and green quantum dots under mechanical stirring conditions to carry out an emulsion polymerization reaction to obtain a green quantum dot fluorescent ball;

[0044] And / or, the method for preparing the red quantum dot fluorescent ball comprises: adding an initiator to an emulsion including styrene monomer and red quantum dots under mechanical stirring conditions to carry out emulsion polymerization reaction to obtain the red quantum dot fluorescent ball.

[0045] By encapsulating quantum dots with different emission wavelengths in polystyrene nanospheres, fluorescent spheres of various colors can be obtained. At the same time, multiple quantum dots can be encapsulated in one nanosphere to amplify the fluorescent signal and improve the sensitivity of biological detection. The size of the fluorescent sphere can be controlled by adjusting the amount of monomer and initiator and the parameters of the emulsification process.

[0046] In an optional embodiment, during the preparation of the green quantum dot fluorescent ball, the initiator is potassium persulfate, the amount of styrene monomer used is 3-5 ml, and the amount of potassium persulfate used is 20-30 mg.

[0047] In an optional embodiment, during the preparation of the red quantum dot fluorescent ball, the initiator is potassium persulfate, the amount of styrene monomer used is 14-18 ml, and the amount of potassium persulfate used is 20-30 mg.

[0048] If the amount of initiator used is too large, the PDI of the particle size test will be larger and the distribution will be wider. Therefore, whether it is the preparation of red quantum dot fluorescent balls or green quantum dot fluorescent balls, the amount of initiator should not be too much.

[0049] In this scheme, the particle size of the fluorescent sphere can be controlled by adjusting various parameters in the emulsion polymerization process, but the amount of styrene monomer has a greater influence on the particle size of the fluorescent sphere.

[0050] In an optional embodiment, in the emulsion polymerization reaction, the rotation speed of the mechanical stirrer is 14000-16000 r / min, and the emulsification time is 4-6 min.

[0051] In an optional embodiment, the mass ratio of the green quantum dots to the styrene monomer is 1:5-10; and / or the mass ratio of the red quantum dots to the styrene monomer is 1:15-30.

[0052] In an optional embodiment, the first antibody is a mouse anti-human CRP antibody, the second antibody is a mouse anti-human PCT antibody, and the secondary antibody is a goat anti-mouse secondary antibody.

[0053] The present invention provides a method for preparing the two-color quantum dot fluorescent ball dual-detection immunochromatographic test strip described in the aforementioned embodiment, comprising:

[0054] S1. Preparation of green quantum dot fluorescent balls and preparation of red quantum dot fluorescent balls;

[0055] S2. Green quantum dot fluorescent ball labeled specific antibody and red quantum dot fluorescent ball labeled specific antibody;

[0056] S3. Preparation of test strips.

[0057] Taking the combined detection index of C-reactive protein (CRP) and procalcitonin (PCT) in human blood as an example, the characteristics and performance of the present invention are further described in detail in combination with the embodiments. It should be noted that in the following embodiments, the green quantum dots are oil-soluble cadmium selenide quantum dots-525 products of Jiayuan Company, the red quantum dots are oil-soluble cadmium selenide quantum dots-625 products, and the yellow quantum dots are carboxyl water-soluble quantum dots-575 products of Jiayuan Company. In some other embodiments, each quantum dot can also select other existing products.

[0058] Embodiment 1:

[0059] This embodiment provides a method for preparing a two-color quantum dot fluorescent ball dual-detection immunochromatographic test strip, comprising:

[0060] S1. Preparation of green quantum dot fluorescent balls: 4 ml of styrene monomer and 0.6 g of green quantum dots with an emission wavelength of 525 nm were subjected to emulsion polymerization by adding 25 mg of potassium persulfate as an initiator under mechanical stirring at 15000 r / min. The emulsification time was 5 min to obtain green quantum dot fluorescent balls;

[0061] Preparation of red quantum dot fluorescent balls: Add 25 mg of initiator potassium persulfate to an emulsion including 16 ml of styrene monomer and 0.6 g of red quantum dots with an emission wavelength of 625 nm under mechanical stirring at 15,000 r / min for emulsion polymerization. The emulsification time is 5 min to obtain red quantum dot fluorescent balls.

[0062] S2. Green quantum dot fluorescent balls label specific antibodies, and red quantum dot fluorescent balls label specific antibodies.

[0063] S3. Prepare the test strip: coat the mouse anti-human CRP antibody labeled with green quantum dot fluorescent balls and the mouse anti-human PCT antibody labeled with red quantum dot fluorescent balls on the binding pad of the test strip, the T1 line is coated with the mouse anti-human CRP antibody, the T2 line is coated with the mouse anti-human PCT antibody, the C line is coated with the goat anti-mouse secondary antibody, and the calibration line is coated with the yellow water-soluble quantum dots with an emission wavelength of 575nm. The rest of the design is the same as the procalcitonin assay reagent (immunofluorescence chromatography) model test strip of Guangzhou Weimi Biotechnology Co., Ltd. The detection principle of the test strip obtained in this embodiment is as follows Figure 1 shown.

[0064] Comparative Example 1:

[0065] This embodiment provides a method for preparing a two-color quantum dot fluorescent ball dual-detection immunochromatographic test strip, which is different from Embodiment 1 only in that in step S3:

[0066] Mouse anti-human CRP antibody labeled with red quantum dot fluorescent balls and mouse anti-human PCT antibody labeled with red quantum dot fluorescent balls were coated on the binding pad of the test strip, the T1 line was coated with mouse anti-human CRP antibody 2, the T2 line was coated with mouse anti-human PCT antibody 2, the C line was coated with goat anti-mouse secondary antibody, and the calibration line was coated with yellow water-soluble quantum dots with an emission wavelength of 575 nm.

[0067] Comparative Example 2-Simulation of the failure of PCT markers in actual applications:

[0068] This embodiment provides a method for preparing a two-color quantum dot fluorescent ball dual-detection immunochromatographic test strip, which is different from Embodiment 1 only in that in step S3:

[0069] The mouse anti-human CRP antibody 1 labeled with red quantum dot fluorescent balls was coated on the binding pad of the test strip, the T1 line was coated with mouse anti-human CRP antibody 2, the T2 line was coated with mouse anti-human PCT antibody 2, the C line was coated with goat anti-mouse secondary antibody, and the calibration line was coated with yellow water-soluble quantum dots with an emission wavelength of 575 nm.

[0070] For samples with different CPR concentrations and PCT concentrations detected by the test strips in the above embodiments and comparative examples, the signal intensities of the corresponding lines detected by the corresponding immunochromatographic analyzer are shown in Table 1.

[0071] Table 1

[0072]

[0073] According to Table 1, the test strips in the embodiment can meet the sensitivity requirements of clinical detection for the two detection indicators of CRP and PCT.

[0074] For comparative example 1, when all substances in the test strip are not invalid: both indicators use red quantum dot fluorescent balls as detection signals, and when the CRP concentration in the sample is 0, a small red fluorescent signal is measured on the T1 detection line, which is the interference of the PCT indicator. It may be the interference between the antibodies themselves that leads to the misjudgment of the result. For the embodiment, the CRP indicator and the PCT indicator use signals with independent fluorescence wavelengths of 530nm and 625nm, respectively. Even if there is a cross-reaction between the antibodies, the CRP indicator reads the signal value at the green 530nm, and the PCT indicator reads the signal value at 625nm, which does not affect the accuracy of the judgment.

[0075] For comparative example 2, when simulating the failure of the PCT marker, its C line still shows a red signal, so it is not possible to effectively judge whether a single marker has failed. Only when both markers fail at the same time will there be no signal in the C line; when there is 0.1ng / mL of PCT to be tested in the sample, its T2 line does not show a detection signal, and the C line still shows a fluorescent signal. The result shows that there is no PCT in the sample, resulting in a false negative result of PCT detection. In the embodiment, CRP is detected using green quantum dot fluorescent balls, and PCT is detected using red quantum dot fluorescent balls. Only when the C line can detect both green and red signals, the markers of the two indicators are valid and the detection is valid. If any indicator marker fails, its C line will not show both green and red signals at the same time, so there is no false negative result.

[0076] Study Example 1

[0077] This test example provides a method for preparing quantum dot fluorescent balls, in which an emulsion including styrene monomer and quantum dots is added with an initiator potassium persulfate under mechanical stirring conditions to carry out an emulsion polymerization reaction to obtain quantum dot fluorescent balls. The specific parameters and the particle size of the obtained quantum dot fluorescent balls are shown in Table 2: Taking oil-soluble cadmium selenide quantum dots-625 as an example.

[0078] Table 2

[0079]

[0080]

[0081] S3: Screening of the particle sizes of two fluorescent balls. The fluorescent ball marker is sprayed on the binding pad for chromatography by combining the fluorescent ball-labeled secondary antibody with its detection line antibody. The steric effect of fluorescent balls of different particle sizes is observed and the interference between the two balls is analyzed. The effects of different particle size combinations are shown in Table 3.

[0082] Table 3

[0083]

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A two-color quantum dot fluorescent ball dual-detection immunochromatographic test strip, characterized in that: It comprises a bottom plate, on which a sample pad, a binding pad, a reaction pad and a water absorption pad are arranged in sequence; The conjugate pad is coated with a first antibody labeled with a green quantum dot fluorescent ball and a second antibody labeled with a red quantum dot fluorescent ball; wherein the average particle size of the green quantum dot fluorescent ball is 50-70nm, and the average particle size of the red quantum dot fluorescent ball is 300-330nm; the emission wavelength of the quantum dots in the green quantum dot fluorescent ball is λ1, and the emission wavelength of the quantum dots in the red quantum dot fluorescent ball is λ2, wherein λ1 is 500-550nm, and λ2 is 600-650nm; The reaction pad is provided with a T1 line, a T2 line, a C line and a calibration line in sequence, wherein the T1 line is coated with a first antibody, The T2 line is coated with the secondary antibody, the C line is coated with the secondary antibody, and the calibration line is coated with yellow water-soluble quantum dots with an emission wavelength of λ3, where λ3 is 565-585nm.

2. The two-color quantum dot fluorescent ball dual-detection immunochromatographic test strip according to claim 1, characterized in that: The yellow water-soluble quantum dot surface ligand is an amphiphilic polymer; and / or, the quantum dot surface ligands in the green quantum dot fluorescent ball and the red quantum dot fluorescent ball are both oleic acid.

3. The two-color quantum dot fluorescent ball dual-detection immunochromatographic test strip according to claim 1, characterized in that: The CV of the green quantum dot fluorescent ball is less than 10%; and / or the CV of the red quantum dot fluorescent ball is less than 10%.

4. The two-color quantum dot fluorescent ball dual-detection immunochromatographic test strip according to claim 1, characterized in that: The preparation method of the green quantum dot fluorescent ball comprises: adding an initiator to an emulsion including styrene monomer and green quantum dots under mechanical stirring conditions to carry out an emulsion polymerization reaction to obtain a green quantum dot fluorescent ball; And / or, the method for preparing the red quantum dot fluorescent ball comprises: adding an initiator to an emulsion including styrene monomer and red quantum dots under mechanical stirring conditions to carry out emulsion polymerization reaction to obtain the red quantum dot fluorescent ball.

5. The two-color quantum dot fluorescent ball dual-detection immunochromatographic test strip according to claim 4, characterized in that: In the preparation process of the green quantum dot fluorescent ball, the initiator is potassium persulfate, the amount of styrene monomer used is 3-5 ml, and the amount of potassium persulfate used is 20-30 mg.

6. The two-color quantum dot fluorescent ball dual-detection immunochromatographic test strip according to claim 4, characterized in that: In the preparation process of the red quantum dot fluorescent ball, the initiator is potassium persulfate, the amount of styrene monomer used is 14-18 ml, and the amount of potassium persulfate used is 20-30 mg.

7. The two-color quantum dot fluorescent ball dual-detection immunochromatographic test strip according to claim 4, characterized in that: In the emulsion polymerization reaction, the rotation speed of the mechanical stirrer is 14000-16000 r / min, and the emulsification time is 4-6 min.

8. The two-color quantum dot fluorescent ball dual-detection immunochromatographic test strip according to claim 4, characterized in that: The mass ratio of the green quantum dots to the styrene monomer is 1:5-10; and / or the mass ratio of the red quantum dots to the styrene monomer is 1:15-30.

9. The two-color quantum dot fluorescent ball dual-detection immunochromatographic test strip according to claim 1, characterized in that: The first antibody is mouse anti-human CRP antibody, the second antibody is mouse anti-human PCT antibody, and the secondary antibody is goat anti-mouse secondary antibody.

10. A method for preparing the two-color quantum dot fluorescent ball dual-detection immunochromatographic test strip according to any one of claims 1 to 9, characterized in that: include: S1. Preparation of green quantum dot fluorescent balls and preparation of red quantum dot fluorescent balls; S2. Green quantum dot fluorescent ball labeled specific antibody and red quantum dot fluorescent ball labeled specific antibody; S3. Preparation of test strips.

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