Rapid and sensitive fluorescence enzyme-linked immunosorbent assay based on optical microspheres

Abstract

The present invention belongs to the field of immunoassay technology, specifically a rapid and sensitive fluorescent enzyme-linked immunosorbent assay method based on optical microspheres. The present invention uses optical microspheres made of transparent materials with a refractive index higher than the environment as detection carriers; the particle size of the microspheres is sub-millimeter level; the surface of the microspheres is treated with capture antibodies; the asymmetric emission effect of fluorescence at the interface of the microspheres with a higher refractive index is utilized to make most of the light emitted by the fluorescence be in a medium with a higher refractive index, and multiple reflections are achieved in the microspheres to amplify the fluorescence emission, thereby achieving rapid and sensitive detection of ELISA. The method of the present invention has fast detection speed and high sensitivity; and the microspheres are small in size, and can also be developed into on-site, portable ELISA detection equipment.

Description

Technical Field

[0001] The invention belongs to the technical field of immune detection, and in particular relates to a fluorescent enzyme-linked immunosorbent assay method. Background Art

[0002] Immunoassay has evolved from radioimmunoassay (RIA), chemiluminescence immunoassay (CLIA), to enzyme-linked immunosorbent assay (ELISA) and lateral flow immunochromatography (LFIA). ELISA is an important tool for clinical testing, health monitoring and disease diagnosis because it is based on the specific binding between antigen and antibody, and uses enzyme-labeled antibodies to participate in the catalysis of the production of detectable products to achieve specific quantitative detection of antigens, pathogens, hormones and bacteria. It is widely used in a wide range of fields such as clinical diagnosis, disease monitoring, food safety and environmental monitoring due to its high specificity and good detection sensitivity. It is known as the "gold standard" of immunoassay reactions due to its excellent specificity. The current mainstream ELISA is achieved by combining standard microplates and microplate readers to achieve ELISA detection. The general detection time is 3-6 hours, and the detection limit is about ng / mL. Therefore, the traditional ELISA detection method has the disadvantages of insufficient sensitivity and long time consumption, which directly leads to the shortcomings of ELISA detection in early detection and diagnosis as well as rapid response strategies.

[0003] Of course, lateral flow immunochromatography (LFIA) represented by colloidal gold method can achieve rapid detection of target antigens, pathogens, hormones, etc. within minutes. However, this method is a semi-quantitative immunoassay method, and its sensitivity is far from comparable to ELISA detection. Therefore, it is of great practical value to establish a rapid and highly sensitive ELISA detection method. Summary of the invention

[0004] The purpose of the present invention is to provide a fluorescent enzyme chain immunosorbent assay (ELISA) method with short detection time and high detection sensitivity based on the principle of microsphere fluorescent amplification.

[0005] The rapid and sensitive ELISA detection method provided by the present invention adopts optical microspheres as detection carriers, that is, microspheres made of transparent materials with a refractive index higher than that of the environment, to achieve rapid and sensitive ELISA detection.

[0006] The particle size of the microspheres is sub-millimeter level; the surface of the microspheres is treated with capture antibodies; the asymmetric fluorescence emission effect of the microsphere interface with a higher refractive index is utilized to make most of the fluorescence emission light in the medium with a higher refractive index, and multiple reflections are achieved in the microspheres to amplify the fluorescence emission, thereby achieving rapid and sensitive detection of ELISA.

[0007] The surface of the microsphere is treated with capture antibodies, including pretreatment, 3-aminopropyltriethoxysilane (APTES), p-phenylene diisothiocyanate (PDITC) immersion treatment, capture antibody and non-specific blocking treatment.

[0008] In the present invention, the microsphere material includes but is not limited to inorganic glass, organic polymer materials, etc.; its shape is spherical or other cavities that can achieve internal reflection, and the particle size is from several microns to several millimeters (that is, the particle size is at the submillimeter level); the surface can be a smooth surface, a rough surface, a porous surface, a wrinkled surface, etc.

[0009] The surface of the microspheres is treated with capture antibodies, and the specific steps are:

[0010] (1) Microsphere pretreatment, including removal of surface organic matter and surface hydroxylation;

[0011] The specific operation is as follows: soak the microspheres in piranha solution (H2O2 / H2SO4 = 1: (2.5-3.5) (v / v)) for 35-45 minutes to remove organic matter on the surface of the microspheres; then soak the microspheres in 0.1-0.3M sodium hydroxide (NaOH) solution for 25-40 minutes for hydroxylation; then wash the microspheres with a large amount of deionized water and dry them in an oven or fume hood;

[0012] (2) The microspheres are soaked in APTES and PDITC to modify the surface of the microspheres to bond with thiocyanate groups of the capture antibody;

[0013] The specific operation is as follows: the dried microspheres are immersed in a 1.5-2.5% (v / v) 3-aminopropyltriethoxysilane (APTES) solution (prepared with a 95% acetone solution), placed on a shaker for oscillation reaction at room temperature for 1.5-2.5 hours; washed with anhydrous acetone five times and dried; then the microspheres are immersed in a 0.1-0.3% (v / v) PDITC solution (prepared with a 10% pyridine / N,N-dimethylformamide (DMF) solution), placed on a shaker for reaction at 37°C for 1.5-2.5 hours; then washed with methanol five times, acetone twice, and double distilled water once and dried; microspheres with thiocyanate groups on the surface are obtained;

[0014] (3) Capture antibodies and block nonspecific binding sites on the microsphere surface;

[0015] The specific operation is: place the microspheres in a 1.2μg / mL -12μg / mL capture antibody solution (prepared with PBS buffer at pH=7.4), and oscillate the reaction at room temperature for 25-35 minutes, then wash 5 times with TBST buffer; then place the microspheres in a 1%-5% BSA solution (prepared with PBS buffer at pH=7.4) at 3-5°C for 8-14 hours, and wash five times with TBST to obtain rapid and sensitive ELISA microspheres for specific detection of target antigens / antibodies.

[0016] In the rapid and sensitive ELISA detection method provided by the present invention, light emission detection and enzyme-linked immunosorbent assay reaction are carried out on the same carrier, that is, the detection medium and the detection reaction site are superimposed on a transparent microsphere.

[0017] The rapid and sensitive ELISA detection method provided by the present invention comprises the following specific steps:

[0018] (1) Place the optical microspheres in 20-100 μL of the target detection antigen solution and incubate at 37°C for 1-10 minutes; then rinse with TBST buffer 3-5 times;

[0019] (2) Then place the optical microspheres in 20-100 μL of enzyme-labeled antibody solution, incubate at 37°C for 1-10 minutes, and then rinse with TBST buffer 3-5 times;

[0020] (3) Finally, add 20-100 μL of luminescent substrate solution, incubate for 1-10 minutes, and place in a laser induced fluorescence detector, fluorescence microscope or other fluorescence imaging equipment to detect the fluorescence intensity of the optical microspheres.

[0021] The present invention provides a rapid and sensitive ELISA detection method, which is suitable for laser induced fluorescence detection, fluorescence microscope imaging and other fluorescence imaging equipment.

[0022] The advantages of the present invention are as follows:

[0023] The present invention can achieve ultra-high sensitivity on sub-millimeter transparent microspheres, and can rapidly detect target antigens or pathogens at or below the fM level, and the entire detection process is completed in about 10 minutes. It has great potential for rapid and highly sensitive immune detection. Due to the small size of the microspheres used, it can also be developed into an on-site, portable ELISA detection device. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The figure is a flow chart of microsphere preparation and ELISA detection in the microsphere ELISA detection method of the present invention, wherein the upper dotted line frame part is the preparation of microspheres, and the lower dotted line frame part is the rapid and sensitive ELISA detection.

[0025] Figure 2 The microscopic fluorescence images and standard working curves of human IgA measured by microsphere ELISA detection method. Among them, A is the microscopic fluorescence image of human IgA of different concentrations measured by microsphere ELISA detection method; B is the standard working curve for quantifying IgA in the tested sample using microsphere fluorescence intensity. DETAILED DESCRIPTION

[0026] Example 1, Detection of human IgA

[0027] Preparation of microspheres: Soak the microspheres in piranha solution (H2O2 / H2SO4 = 1:3 (v / v)) for 40 min to remove organic matter on the surface of the microspheres. Then soak the microspheres in 0.1 M sodium hydroxide (NaOH) solution for 30 min for hydroxylation. Then wash the microspheres with a large amount of deionized water and dry them in an oven or fume hood. Then immerse the dried soaked microspheres in 2% (v / v) 3-aminopropyltriethoxysilane (APTES) solution (prepared with 95% acetone solution) and place them on a shaker for 2 hours at room temperature. Then wash them five times with anhydrous acetone and dry them. Then immerse the microspheres in 0.2% (v / v) PDITC solution (prepared with 10% pyridine / N,N-dimethylformamide (DMF) solution) and place them on a shaker for 2 hours at 37°C. Then wash them 5 times with methanol, 2 times with acetone, and 1 time with double distilled water and dry them. The microspheres were placed in a 1.2μg / mL -3.0μg / mL rabbit anti-human IgA antibody solution (prepared with PBS buffer at pH=7.4) and oscillated at room temperature for about 30 minutes, then washed five times with TBST buffer. The microspheres were then placed in a 5% BSA solution (prepared with PBS buffer at pH=7.4) and blocked at 4°C for 14 hours, then washed five times with TBST to obtain microspheres for rapid and sensitive ELISA detection.

[0028] Rapid ELISA test:

[0029] Place the microspheres in 20-100 μL human IgA sample and incubate at 37°C for 3-5 minutes, then rinse with TBST buffer 3-5 times. Then place the microspheres in 20-100 μL HRP-labeled rabbit anti-human IgA solution and incubate at 37°C for 3-5 minutes. Then rinse with TBST buffer 3-5 times. Finally, add 20-100 μL of 0.1 mM Amplex@Red and 0.5 mM H2O2 substrate solution and incubate for 3-5 minutes. Then place in a laser induced fluorescence detector, fluorescence microscope or other fluorescence imaging equipment to detect the fluorescence intensity of the microspheres. Fluorescence microscopy imaging can be used to obtain the attached Figure 2(A) Fluorescence image, the fluorescence intensity of the microspheres in the fluorescence image was analyzed using graphic analysis software, and the fluorescence intensity of the microspheres in the fluorescence image was compared with the attached Figure 2 The standard curve shown in (B) can be used to obtain the concentration of IgA in the sample. The minimum detection limit is 3.25fM, and the standard curve covers the range from 0pM to 100pM.

[0030] Example 2, Detection of Measles IgG ELISA

[0031] Preparation of microspheres: Soak the microspheres in piranha solution (H2O2 / H2SO4 = 1:3 (v / v)) for 40 min to remove organic matter on the surface of the microspheres. Then soak the microspheres in 0.1 M sodium hydroxide (NaOH) solution for 30 min for hydroxylation. Then wash the microspheres with a large amount of deionized water and dry them in an oven or fume hood. Then immerse the dried soaked microspheres in 2% (v / v) 3-aminopropyltriethoxysilane (APTES) solution (prepared with 95% acetone solution) and place them on a shaker for 2 hours at room temperature. Then wash them five times with anhydrous acetone and dry them. Then immerse the microspheres in 0.2% (v / v) PDITC solution (prepared with 10% pyridine / N,N-dimethylformamide (DMF) solution) and place them on a shaker for 2 hours at 37°C. Then wash them 5 times with methanol, 2 times with acetone, and 1 time with double distilled water and dry them. The microspheres were placed in a 1.2μg / mL -3.0μg / mL goat anti-IgG solution (prepared with PBS buffer at pH=7.4) and oscillated at room temperature for about 30 minutes, then washed five times with TBST buffer. The microspheres were then placed in a 5% BSA solution (prepared with PBS buffer at pH=7.4) and blocked at 4°C for 14 hours, then washed five times with TBST to obtain microspheres for rapid and sensitive ELISA detection.

[0032] Rapid ELISA test:

[0033] Place the microspheres in 20-100 μL of IgG sample and incubate at 37 °C for 3-5 minutes, then rinse with TBST buffer 3-5 times. Then place the microspheres in 20-100 μL of HRP-labeled goat anti-IgG solution and incubate at 37 °C for 3-5 minutes. Then rinse with TBST buffer 3-5 times. Finally, add 20-100 μL of 0.1mM Amplex@Red and 0.5mM H2O2 substrate solution and incubate for 3-5 minutes, then place in a laser induced fluorescence detector, fluorescence microscope or other fluorescence imaging equipment to detect the fluorescence intensity of the microspheres. The same quantitative method as in Example 1 is used, that is, the fluorescence intensity of the microspheres of the sample is obtained by using a fluorescent image, and the concentration of IgG in the sample is obtained by comparing with the standard curve.

[0034] Example 3, Detection of tumor marker CEA

[0035] Preparation of microspheres: Soak the microspheres in piranha solution (H2O2 / H2SO4 = 1:3 (v / v)) for 40 min to remove organic matter on the surface of the microspheres. Then soak the microspheres in 0.1 M sodium hydroxide (NaOH) solution for 30 min for hydroxylation. Then wash the microspheres with a large amount of deionized water and dry them in an oven or fume hood. Then immerse the dried soaked microspheres in 2% (v / v) 3-aminopropyltriethoxysilane (APTES) solution (prepared with 95% acetone solution) and place them on a shaker for 2 hours at room temperature. Then wash them five times with anhydrous acetone and dry them. Then immerse the microspheres in 0.2% (v / v) PDITC solution (prepared with 10% pyridine / N,N-dimethylformamide (DMF) solution) and place them on a shaker for 2 hours at 37°C. Then wash them 5 times with methanol, 2 times with acetone, and 1 time with double distilled water and dry them. The microspheres were placed in 1.2μg / mL -3.0μg / mL human anti-CEA solution (prepared with PBS buffer at pH=7.4) and shaken at room temperature for about 30 minutes, and then washed five times with TBST buffer. The microspheres were then placed in a 5% BSA solution (prepared with PBS buffer at pH=7.4) and blocked at 4°C for 14 hours, and then washed five times with TBST to obtain microspheres for rapid and sensitive ELISA detection.

[0036] Rapid ELISA test:

[0037] The microspheres were placed in 20-100 μL CEA sample and incubated at 37 ° C for 3-5 minutes, then rinsed 3-5 times with TBST buffer. The microspheres were then placed in 20-100 μL HRP-labeled human anti-CEA solution and incubated at 37 ° C for 3-5 minutes. Then rinsed 3-5 times with TBST buffer. Finally, 20-100 μL of 0.1 mM AmplexRed and 0.5 mM H2O2 substrate solution were added and incubated for 3-5 minutes, and then placed in a laser induced fluorescence detector, fluorescence microscope or other fluorescence imaging equipment to detect the fluorescence intensity of the microspheres. The same quantitative method as in Example 1 was used, that is, the fluorescence intensity of the microspheres of the sample was obtained by using a fluorescent image, and the concentration of CEA in the sample was obtained by comparing it with the standard curve.

Claims

1. A rapid and sensitive fluorescent enzyme-linked immunosorbent assay based on optical microspheres, characterized in that: Optical microspheres made of transparent materials with a higher refractive index than the environment are used as detection carriers; the particle size of the microspheres is sub-millimeter level; the surface of the microspheres is treated with capture antibodies; the asymmetric fluorescence emission effect of the microsphere interface with a higher refractive index is utilized to make most of the fluorescent emission light in the medium with a higher refractive index, and multiple reflections are achieved in the microspheres to amplify the fluorescent emission, thereby achieving rapid and sensitive detection of ELISA.

2. The rapid and sensitive fluorescent enzyme-linked immunosorbent assay method according to claim 1, characterized in that: The microsphere material is inorganic glass or organic polymer material; its shape is spherical or other cavity that can realize internal reflection.

3. The rapid and sensitive fluorescent enzyme-linked immunosorbent assay method according to claim 2, characterized in that: The microsphere surface is a smooth surface, a rough surface, a porous surface or a wrinkled surface.

4. The rapid and sensitive fluorescent enzyme-linked immunosorbent assay method according to claim 3, characterized in that: The microsphere surface is treated with a capture antibody, including: (1) Microsphere pretreatment, including removal of surface organic matter and surface hydroxylation; (2) The microspheres are soaked in APTES and PDITC to modify the surface of the microspheres to bond with thiocyanate groups of the capture antibody; (3) Capture antibodies and block nonspecific binding sites on the microsphere surface.

5. The rapid and sensitive fluorescent enzyme-linked immunosorbent assay method according to claim 4, characterized in that: The light emission detection and ELISA reactions are carried out on the same carrier, that is, the detection medium and the detection reaction site are superimposed on the transparent microspheres; the specific steps are as follows: (1) Place the microspheres treated with capture antibodies in 20-100 μL of the target detection antigen solution and incubate at 37°C for 1-10 minutes; then rinse with TBST buffer 3-5 times; (2) Then place the microspheres in 20-100 μL of enzyme-labeled antibody solution, incubate at 37°C for 1-10 minutes, and then rinse with TBST buffer 3-5 times; (3) Finally, add 20-100 μL of luminescent substrate solution, incubate for 1-10 minutes, and place in a laser induced fluorescence detector, fluorescence microscope or other fluorescence imaging equipment to detect the fluorescence intensity of the microspheres.

Images