Microscopic analysis method using microsphere reflector as probe

Abstract

The present application provides a kind of microanalysis method with microsphere reflector as probe, comprising the following steps: 1) carboxylation modification of SiO2 microsphere, including carboxylation modification;Activation of carboxylated silica microsphere surface carboxyl;2) antibody coupling on probe microsphere and capture microsphere;3) reaction with target object;4) acquisition of microscopic image;5) quantitative analysis, the obtained image is analyzed using image analysis software.The present application realizes the detection of probe microsphere with particle size less than 300nm using optical microscope equipped with digital camera, which is low in cost;After reaction of sample with capture and probe microsphere, it can be directly detected without separation;Using capture microsphere and corresponding antibody encoded by different colors and particle sizes can realize simultaneous detection of multiple target objects;Sample consumption is low, only ul level finger blood is needed;It has high sensitivity, and theoretically can realize single molecule detection.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay technology, specifically relating to a microscopic analysis method using a microsphere reflector as a probe. Background Technology

[0002] Immunoassay is currently one of the most widely used biological detection methods, characterized by high accuracy, sensitivity, and specificity. It is a technique that uses solid-phase labeled immunoprobes to qualitatively, semi-quantitatively, or quantitatively detect the content of analytes in samples. The development of immunoassay technology is closely related to immunoprobes. Commonly used immunoprobes include colloidal gold, colored microspheres, fluorescent microspheres, paramagnetic nanoparticles, upconversion phosphorescence, quantum dots, etc. These immunoprobes share the common feature of having a particle size mostly below 200 nm, aiming to achieve high sensitivity in immunoassays.

[0003] In addition, the surface of silica microspheres is easily functionalized, making them suitable for immunoassays. Silica microspheres are non-toxic, pollution-free, high-strength, high-toughness, stable, with a large specific surface area and high mechanical strength, making them suitable for use in ceramics, rubber modification, plastics, coatings, sunscreens, and pigments. Due to their excellent optical and mechanical properties, good shape uniformity, controllable size, and simple composition, silica microspheres have significant potential applications in photonic crystal self-assembly, the construction of ordered materials, chromatographic packing materials, particle size standards, and flat panel displays. Furthermore, the surface of silica microspheres is easily functionalized, readily modified with carboxyl, amino, and streptavidin groups. Their low fluorescence and extremely low non-specific adsorption of biomolecules also make them widely applicable in biomedical fields such as nucleic acid separation, cell separation, and immunoassays.

[0004] In the prior art, microspheres encoded by multicolor upconversion fluorescent nanoparticles (UCNMs) of 30-50 nm are used as encoding signals and combined with portable fluorescence image processing to develop a platform for the simultaneous quantitative detection of different mycotoxins (Yang M, Zhang Y, Cui M, et al. A smartphone-based quantitative detection platform of mycotoxins based on multiple-color upconversion nanoparticles[J].Nanoscale,2018,10(33):15865-15874.). Upconversion fluorescent nanoparticles emitting red / green / blue light were doped into mesoporous polystyrene microspheres as labels. Antigens were conjugated to the surface of the labels to serve as immunoprobes. The probes were incubated with the target analyte, washed with PBST, and then incubated with phycoerythrin-labeled secondary antibody. After washing again with PBST and resuspending in ultrapure water, the microspheres were dried. Excitation with laser diodes at 980 nm and 488 nm, respectively, resulted in the microspheres emitting different colored barcode signals, representing three different mycotoxins. The fluorescently labeled secondary antibody bound to the microspheres served as the detection signal. Images were observed and captured using a microscope with magnification of 200×–240× mounted on a mobile phone lens. Subsequent calculations were performed by comparing the images obtained under the two excitation conditions. However, this technology also has the following problems:

[0005] 1. The preparation of upconversion luminescent microspheres involves filling upconversion luminescent substances into polyethylene microspheres, which is a complicated and costly process.

[0006] 2. When observing upconversion luminescent microspheres under a microscope, it is necessary to use two different special light sources, such as excitation light, to excite the microspheres and make them emit light.

[0007] 3. The reaction probe needs to be separated from the sample and the unreacted probe before detection, which is a complicated process.

[0008] Another quantitative fluorescence image analysis method based on multicolor upconversion nanoparticles (UCN) encoded microspheres (Yang M, Cui M, Wang W, et al. Background-free upconversion-encoded microspheres for mycotoxin detection based on a rapid visualization method[J]. Analytical and Bioanalytical Chemistry, 2020, 412(1):81-91.) is used to detect ochratoxin A and zearalenone. Red and blue upconversion fluorescent nanomaterials were doped into mesoporous polystyrene microspheres as prepared labels. The red and blue labels were then bound to the target antigen, respectively, to serve as detection probes. The probes were then mixed with the target antigen, incubated with antibody, washed to remove unbound antibody, and then phycoerythrin-labeled secondary antibody was added. After incubation and washing three times to remove unbound phycoerythrin-labeled secondary antibody, the microspheres were centrifuged, resuspended in deionized water, and dried on a glass slide. Background-free encoded images of the UCN-doped microspheres were captured using a fluorescence microscope under excitation at 980 nm and 480 nm, respectively. Encoding signals of the two target antigen microspheres (red and blue fluorescence) were captured under 980 nm excitation, while detection images of the phycoerythrin-labeled secondary antibody coupled to the microspheres were captured under 480 nm blue light excitation. After image processing, the addition of the target antigen to the sample was calculated. However, this technology also has the following problems:

[0009] 1. The preparation of upconversion luminescent microspheres involves filling upconversion luminescent substances into polyethylene microspheres, which is costly.

[0010] 2. When observing upconversion luminescent microspheres under a microscope, special light sources such as different excitation lights are needed to excite the microspheres and make them emit light.

[0011] 3. The reaction probe needs to be separated from the sample and the unreacted probe before detection, which is a complicated process.

[0012] In addition, a smartphone-based immune detection system based on hydrogel microspheres was constructed (Zhang L, Zhang Z, Tian Y, et al. Rapid, simultaneous detection of mycotoxins with smartphone recognition-based immune microspheres[J]. Analytical and Bioanalytical Chemistry, 2021, 413(14):3683-3693.), which can simultaneously and rapidly detect ochratoxin A (OTA) and zearalenone (ZEN). After preparing the hydrogel prepolymer, red / blue / green upconversion fluorescent substances were added, and the mixture solidified into solid particles after ultraviolet irradiation. The hydrogel particles were irradiated with a 980 nm laser and showed three colors: red, green, and blue. Two types of hydrogel particles with diameters of 0.6 mm and 0.35 mm, respectively, were conjugated with monoclonal antibodies against two fungal toxins as detection probes. In another test tube, standards for the two fungal toxins were added, followed by the addition of OTA urease and ZEN urease, and incubation at 37°C. Excess enzymes were then washed away to obtain artificial antigens. The probes, target compounds, and prepared artificial antigens were then mixed and incubated. Unbound artificial antigens and target compounds were removed by centrifugation. An aqueous solution containing urea was then added to the system, and incubation was performed at 37°C. Bromocresol purple (BCP) was added and the mixture was stored in the dark for color development. Finally, the microspheres were placed on a clean glass slide, and a photograph was taken under natural light using a mobile phone equipped with a portable microscope. The resulting image was then analyzed and calculated. However, this technique also has the following problems:

[0013] 1. Preparation of hydrogel microspheres: First, a hydrogel prepolymer is synthesized. Then, a microfluidic injection pump is combined with a heavy-duty plastic tubing to form a microdroplet generating device. Under this device, an upconversion luminescent material is filled into the hydrogel prepolymer and cured by ultraviolet irradiation to form hydrogel microspheres. The preparation steps are complicated and costly.

[0014] 2. Using urease and urea as chromogenic substrates for color development of the reaction presents challenges in the transportation and storage of urease, as well as the short shelf life of the product.

[0015] 3. The probe needs to be separated from the sample and unreacted urease after the reaction before detection. The presence of urease can lead to false positives and incorrect results. Separation of the probe and unreacted urease makes the detection process cumbersome. Summary of the Invention

[0016] To address the aforementioned technical problems, this invention provides a microscopic analysis method using a microsphere mirror as a probe, thereby solving the following technical issues:

[0017] 1) Solving the problem of observing probe microspheres smaller than 200 nm without relying on high-resolution optical microscopy. The detection signal is obtained by using the reflected light spot from the microsphere. For over a century, the "Abbe limit" of 200 nm has been considered the theoretical resolution limit of optical microscopy; objects smaller than this size must be observed using electron microscopy or scanning tunneling microscopy. However, commonly used immunoassay labeled probes typically have particle sizes below 200 nm. This invention utilizes the property that SiO2 microspheres with particle sizes of 150-350 nm can reflect visible light, and that the diameter of the reflected light spot when observed under an electron microscope is several times the particle size itself. This achieves the goal of detecting microspheres without the need for a sophisticated high-resolution optical microscope. Other types of probe microspheres with particle sizes below 200 nm are difficult to observe with an optical microscope.

[0018] 2) Solving the problem of adjusting the probe signal intensity. The diameter of the reflected light spot of the microsphere can be adjusted by adjusting the light intensity of the light source on the microscope, which brings more flexibility to signal detection. This is something that other microspheres used as probes do not have.

[0019] 3) Solving the problem of special excitation light sources. Existing technologies such as microscopic observation of fluorescent microspheres and upconversion luminescent microspheres actually utilize the emission spot of the microspheres, but these technologies require the use of other special light sources to excite the microspheres and make them emit light. This invention only requires the light source of a common microscope. The equipment is simpler and the cost is lower.

[0020] 4) Probe cost issue. Existing fluorescent microspheres and upconversion luminescent microspheres are usually prepared by filling SiO2 microspheres with fluorescent or upconversion luminescent materials, while this invention uses SiO2 microspheres directly, which is obviously cheaper.

[0021] 5) Convenience of sample detection. This invention combines color-coded microspheres (or other carriers with coding functions) with probe microspheres. It quantifies the target analyte by simultaneously identifying the code and detecting the number of probe microspheres bound to the color-coded microspheres (or other carriers with coding functions). This allows for direct detection without separating and cleaning unbound probes. Existing technologies sometimes fail to achieve simultaneous identification and detection, and sometimes require separating and cleaning unbound probes before detection.

[0022] To solve the above-mentioned technical problems, the specific technical solution of the present invention is as follows:

[0023] The microscopic analysis method using a microsphere reflector as a probe includes the following steps:

[0024] 1) Carboxylation modification of SiO2 microspheres

[0025] Carboxylation modification: Monodisperse silica microspheres of different colors and particle sizes were ultrasonically dispersed for 10-20 min, and a certain amount of microspheres were added to an equimolar amount of silane coupling agent KH-550 (γ-aminopropyltriethoxysilane) and DMF (N,N-dimethylformamide) solution after mixing succinic anhydride. The reaction was carried out for 1-3 h. After the reaction was completed, the microspheres were centrifuged and washed to obtain carboxylated silica microspheres, which were then resuspended in PBS solution.

[0026] Activation of carboxyl groups on the surface of carboxylated silica microspheres: Place the carboxylated silica microspheres in a 2ml imported centrifuge tube, add N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) to the centrifuge tube, react at room temperature for 30-90 min to activate the carboxyl groups on the surface of the carboxylated silica microspheres, centrifuge to remove excess activator, and resuspend the activated carboxylated silica microspheres in PBS solution.

[0027] 2) Antibody conjugation on probe microspheres and capture microspheres

[0028] The activated colored carboxylated microspheres with a particle size of 1-5 μm were used as the target analyte capture microspheres. After being mixed with the target analyte capture antibody, they were incubated at room temperature for 1 h to obtain the prepared capture microspheres.

[0029] The activated white carboxylated silica microspheres with a particle size of 150-350 nm were used as probe microspheres and incubated with the detection antibody at room temperature for 1 h to obtain the prepared probe microspheres.

[0030] 3) Reaction with the target substance

[0031] Mix the sample, capture microspheres and probe microspheres in a certain proportion in a centrifuge tube, react in the centrifuge tube for 3-10 minutes, take 2 microliters of the reaction solution, spread it evenly on a glass slide, dry it and observe it.

[0032] 4) Acquisition of microscopic images

[0033] After placing the slide under the microscope, the image is directly obtained using the light source on the microscope with constant parameters. During microscopic imaging, the probe microspheres with a particle size of 200nm become a light source themselves due to the reflection of the light source on the mirror surface. Due to the overexposure of the photosensitive element, a halo larger than the light source itself is formed, creating a bright spot in the field of view, which is then used as a detection signal.

[0034] 5) Quantitative analysis

[0035] The acquired images were analyzed using image analysis software (such as ImageJ). First, the centroid coordinates of the two different sizes of silica microspheres were statistically analyzed. Then, the number of centroids where the coordinate radii intersect was calculated. From the number of intersecting centroids, the number of binding sites where the capture microsphere and probe microsphere bind to the target analyte can be obtained. The number of binding sites is proportional to the concentration of the target analyte in the sample, thus enabling quantitative detection of the target analyte.

[0036] The present invention has the following technical effects:

[0037] 1) The detection of probe microspheres with a particle size of less than 300 nm was achieved using an optical microscope equipped with a digital camera, which was cost-effective;

[0038] 2) The sample can be directly detected after reacting with the capture and probe microspheres, without the need for separation;

[0039] 3) Simultaneous detection of multiple targets can be achieved by using capture microspheres encoded with different colors and particle sizes and corresponding antibodies;

[0040] 4) Small sample volume is required; only ul-level fingertip blood is needed.

[0041] 5) It has high sensitivity and can theoretically achieve single-molecule detection. Attached Figure Description

[0042] Figure 1 This is a schematic diagram of the present invention;

[0043] Figure 2 This is a schematic diagram of the present invention. Detailed Implementation

[0044] The specific technical solutions of the present invention will be described with reference to the embodiments.

[0045] This invention requires the use of an optical microscope equipped with a digital camera. Under illumination from a light source on the microscope, SiO2 microspheres of a specific size reflect the light through a mirror surface, thus becoming a light source themselves and forming a bright area in the field of view. Because the CMOS or CCD sensor of a digital camera overexposes this bright area, it creates a halo much larger than the diameter of the light source. This drawback, which is detrimental to capturing realistic images, is utilized in this invention as a signal amplification method to achieve imaging detection of probes that are difficult to detect at ordinary resolutions.

[0046] Antibodies against the target analyte were labeled on SiO2 probe microspheres, and capture antibodies were labeled on capture microspheres. After mixing the two types of microspheres with the sample and reacting for a period of time, images were taken under a microscope to observe the binding patterns. Figure 1As shown in the diagram: a) capture microspheres and probe microspheres are bound together by the target; b) capture microspheres are not bound to the target or probe microspheres; c) probe microspheres are not bound to the target; and d) aggregated probe microspheres are bound to capture microspheres by the target.

[0047] Its image is as follows Figure 2 As shown, the number of binding points between the light spot and the captured microspheres is counted. Figure 2 The content of the target substance is quantified by using three methods (three in total).

[0048] Example 1

[0049] Protein detection using a double-antibody sandwich method: determination of gastrin 17 (G-17) by multicolor coded microsphere chemiluminescence imaging immunoassay.

[0050] 1) Carboxylation modification of SiO2 microspheres

[0051] Carboxylation modification: Monodisperse silica microspheres (5% solid content) of different colors and particle sizes (red 2μm, white 200nm) were ultrasonically dispersed for 10 min. Then, 1 ml of microspheres were added to equimolar (0.01 mol) DMF solutions of silane coupling agent KH-550 and succinic anhydride. The reaction was carried out for 3 h. After the reaction was completed, the microspheres were centrifuged and washed to obtain carboxylated silica microspheres, which were then resuspended in PBS solution.

[0052] Activation of carboxyl groups on the surface of carboxylated silica microspheres: 100 μL of carboxylated silica microspheres were placed in a 2 ml imported centrifuge tube. 50 μL of 20 mg / ml NHS and 50 μL of 20 mg / ml EDC were added to the centrifuge tube. The mixture was reacted at room temperature for 30 min to activate the carboxyl groups on the surface of the carboxylated silica microspheres. Excess activator was removed by centrifugation. The activated carboxylated silica microspheres were then resuspended in PBS solution.

[0053] 2) Antibody conjugation on probe microspheres and capture microspheres

[0054] The activated red carboxylated silica microspheres with a particle size of 2 μm were used as G-17 capture microspheres. They were mixed with 20 μg of G-17 capture antibody and incubated at room temperature for 1 h to obtain the prepared capture microspheres.

[0055] The activated white carboxylated silica microspheres with a particle size of 200 nm were used as probe microspheres and incubated with 20 μg of G-17 detection antibody at room temperature for 1 h to obtain the prepared probe microspheres.

[0056] 3) Reaction with the target substance

[0057] Take 2 μL of fingertip blood and mix it with 14 μL of diluent (40 mmol / L phosphate buffer containing 0.15 mol / L EDTA, 0.26 mol / L NaCl, 10 mg / mL Tween-20, 0.1 mg / mL γ-globulin, and 10 mg / mL casein). Add 2 μL of the prepared capture microspheres and 2 μL of the prepared probe microspheres in sequence. React in a centrifuge tube for 3 min. Take 2 μL of the reaction solution and spread it evenly on a glass slide. After drying, observe the solution.

[0058] 4) Acquisition of microscopic images

[0059] After the slide is placed under the microscope, the image is directly obtained using the light source on the microscope with constant parameters. During microscopic imaging, the probe microspheres with a particle size of 200nm become a light source themselves because they reflect the illumination light from the light source through the mirror surface. Due to the overexposure of the photosensitive element, a halo larger than the light source itself is formed, creating a bright spot in the field of view, which is then used as a detection signal.

[0060] 5) Quantitative analysis

[0061] ImageJ was used to analyze the obtained images. First, the centroid coordinates of the two different sizes of silica microspheres were statistically analyzed. Then, the number of centroids where the coordinate radii intersected was calculated. From the number of intersecting centroids, the number of binding sites where the capture microsphere and probe microsphere intersect through G-17 binding can be obtained. The number of binding sites is proportional to the G-17 concentration in the sample.

[0062] Example 2

[0063] Competitive method for detecting small molecules: Determination of aflatoxin B1 (AFB1) by multicolor coded microsphere chemiluminescence imaging immunoassay.

[0064] 1) Carboxylation modification of SiO2 microspheres

[0065] Carboxylation modification: Monodisperse silica microspheres (5% solid content) of different colors and particle sizes (5 μm for blue, 300 nm for white) were ultrasonically dispersed for 10 min. Then, 1 ml of microspheres were added to equimolar (0.01 mol) DMF solutions of silane coupling agent KH-550 and succinic anhydride. The reaction was carried out for 3 h. After the reaction was completed, the microspheres were centrifuged and washed to obtain carboxylated silica microspheres, which were then resuspended in PBS solution.

[0066] Activation of carboxyl groups on the surface of carboxylated silica microspheres: Take 100 μL of carboxylated silica microspheres and put them into 2 ml imported centrifuge tubes. Add 50 μL of 20 mg / ml NHS and 50 μL of 20 mg / ml EDC to the centrifuge tubes. React at room temperature for 30 min to activate the carboxyl groups on the surface of carboxylated silica microspheres. Centrifuge to remove excess activator and resuspend the activated carboxylated silica microspheres in PBS solution.

[0067] 2) Coupling of antibody / antigen on probe microspheres and capture microspheres

[0068] Activated blue carboxylated silica microspheres with a particle size of 5 μm were used as AFB1 capture microspheres. They were mixed with 200 μL of PBS solution containing 10 μg of AFB1-BSA and incubated at room temperature for 3 h. Then, 1% BSA was added to a final concentration of 1% for blocking to obtain the prepared capture microspheres.

[0069] The activated white carboxylated silica microspheres with a particle size of 300 nm were used as probe microspheres and incubated with 20 μg of AFB1 monoclonal antibody at room temperature for 1 h to obtain the prepared probe microspheres.

[0070] 3) Fixation of microspheres on a glass slide

[0071] Immunofluorescence in situ hybridization slides (commercial product, each slide with 6 circular reaction wells of 8 mm diameter, separated by a superhydrophobic material) were used. The reaction wells were washed three times with anhydrous ethanol and then three times with ultrapure water. A mixture of 1 mmol of silane coupling agent KH-550 and 1 mmol of succinic anhydride was uniformly coated onto the reaction wells and reacted for 3 hours. After the reaction, the slides were washed three times with anhydrous ethanol and then three times with ultrapure water to obtain carboxylated slides.

[0072] Add 20 μL of a mixture of 20 mg / ml NHS and 20 mg / ml EDC to the reaction wells and react at room temperature for 30 min. After the reaction is complete, absorb the excess liquid with absorbent paper. Add 20 μL of the trapping microspheres prepared in the previous step and react at room temperature for 30 min. After the reaction is complete, absorb the excess liquid with absorbent paper and wash the reaction wells three times with ultrapure water to obtain a glass slide with the trapping microspheres fixed.

[0073] 4) Reaction with the target substance

[0074] Take 20 μL of sample solution into a centrifuge tube, add 2 μL of probe microspheres, react in the centrifuge tube for 3 min, then transfer these solutions to the reaction wells of a glass slide, react for 10 min, absorb excess liquid with absorbent paper, dry and observe.

[0075] 5) Acquisition of microscopic images

[0076] After the slide is placed under the microscope, the image is directly obtained using the light source on the microscope with constant parameters. During microscopic imaging, the probe microsphere with a particle size of 300nm becomes a light source itself because it reflects the light from the light source through the mirror. Due to the overexposure of the photosensitive element, a halo larger than the light source itself is formed, creating a bright spot in the field of view, which is then used as a detection signal.

[0077] 6) Quantitative analysis

[0078] The acquired images were analyzed using the developed software. First, the centroid coordinates of two different sized silica microspheres were statistically analyzed. Then, the number of centroids where the coordinate radii intersect was calculated. From the number of intersecting centroids, the number of binding sites between the capture microsphere and the probe microsphere can be obtained. The number of binding sites is inversely proportional to the AFB1 concentration in the sample. Experiments were conducted using AFB1 standard substances at different concentrations, and a standard curve was established based on this. The corresponding number of binding sites was used as the target concentration of AFB1.

[0079] Example 3

[0080] Competitive assay for simultaneous detection of multiple small molecules: Multicolor coded microsphere chemiluminescence imaging immunoassay for simultaneous determination of aflatoxin B1 (AFB1), zearalenone (ZEN), and vomitoxin (DON).

[0081] 1) Carboxylation modification of SiO2 microspheres

[0082] Carboxylation modification: Purchased white 200nm monodisperse silica microspheres (5% solid content) were ultrasonically dispersed for 10 min. Then, 1 ml of microspheres were added to an equimolar (0.01 mol) solution of silane coupling agent KH2550 and succinic anhydride mixed in DMF. The reaction was carried out for 3 h. After the reaction was completed, the microspheres were centrifuged and washed to obtain carboxylated silica microspheres, which were then resuspended in PBS solution.

[0083] 2) Activation of silica microspheres and carboxylated latex microspheres

[0084] Activation of carboxyl groups on the surface of carboxylated silica microspheres: 100 μL of carboxylated white 200 nm silica microspheres were placed in a 2 ml imported centrifuge tube. 50 μL of 20 mg / ml NHS and 50 μL of 20 mg / ml EDC were added to the centrifuge tube. The mixture was reacted at room temperature for 30 min to activate the carboxyl groups on the surface of the carboxylated silica microspheres. Excess activator was removed by centrifugation. The activated carboxylated silica microspheres were then resuspended in PBS solution.

[0085] Activation of carboxyl groups on the surface of carboxylated latex microspheres: Take 100 μL of each of the purchased 3 μm carboxylated latex microspheres (red, green, and blue) into 2 ml imported centrifuge tubes. Add 50 μL of 20 mg / ml NHS and 50 μL of 20 mg / ml EDC to the centrifuge tubes and react at room temperature for 30 min to activate the carboxyl groups on the surface of the carboxylated latex microspheres. Centrifuge to remove excess activator and resuspend the activated carboxylated latex microspheres in PBS solution.

[0086] 2) Coupling of antibody / antigen on probe microspheres and capture microspheres

[0087] a) Blue carboxylated latex microspheres with an activated particle size of 3 μm were used as AFB1 capturing microspheres. 20 μg of AFB1-BSA was added to 200 μL of PBS solution and incubated with the activated 3 μm blue carboxylated latex microspheres at room temperature for 3 h. Then, 1% BSA was added to a final concentration of 1% for blocking to obtain the prepared AFB1 capturing microspheres.

[0088] b) The activated red carboxylated latex microspheres with a particle size of 3 μm were used as ZEN capture microspheres. 20 μg of ZEN-BSA was added to 200 μL of PBS solution and incubated with the activated 3 μm red carboxylated latex microspheres at room temperature for 3 h. Then, 1% BSA was added to a final concentration of 1% for blocking to obtain the prepared ZEN capture microspheres.

[0089] c) Green carboxylated latex microspheres with an activated particle size of 3 μm were used as DON capture microspheres. 20 μg of DON-BSA was added to 200 μL of PBS solution and incubated with the activated 3 μm green carboxylated latex microspheres at room temperature for 3 h. Then, 1% BSA was added to a final concentration of 1% for blocking to obtain the prepared DON capture microspheres.

[0090] d) The activated white carboxylated silica microspheres with a particle size of 200 nm were used as probe microspheres for three target substances (AFB1, ZEN and DON). 10 μg of AFB1 monoclonal antibody / ZEN monoclonal antibody / DON monoclonal antibody were added to 200 μL of PBS solution and incubated with the activated 200 nm white carboxylated silica microspheres at room temperature for 1 h. Then, 1% BSA was added to a final concentration of 1% for blocking, and the three probe microspheres were obtained.

[0091] 3) Fixation of microspheres on a glass slide

[0092] Immunofluorescence in situ hybridization slides (commercial product, each slide with 6 circular reaction wells of 8 mm diameter, separated by a superhydrophobic material) were used. The reaction wells were washed three times with anhydrous ethanol and then three times with ultrapure water. A mixture of 1 mmol of silane coupling agent KH-550 and 1 mmol of succinic anhydride was uniformly coated onto the reaction wells and reacted for 3 hours. After the reaction, the slides were washed three times with anhydrous ethanol and then three times with ultrapure water to obtain carboxylated slides.

[0093] Add 20 μL of a mixture of 20 mg / ml NHS and 20 mg / ml EDC to the reaction wells and react at room temperature for 30 min. After the reaction is complete, absorb the excess liquid with absorbent paper. Add 6 μL of each of the three types of trapping microspheres prepared in the previous step to the same reaction well and react at room temperature for 30 min. After the reaction is complete, absorb the excess liquid with absorbent paper and wash the reaction wells three times with ultrapure water to obtain a glass slide with the three types of trapping microspheres fixed.

[0094] 4) Reaction with the target substance

[0095] Take 20 μL of sample solution into a centrifuge tube, add 2 μL of each of the three probe microspheres, react in the centrifuge tube for 3 min, then transfer these solutions to the reaction wells of a glass slide, react for 10 min, absorb excess liquid with absorbent paper, dry and observe.

[0096] 5) Acquisition of microscopic images

[0097] After the slide is placed under the microscope, the image is directly obtained using the light source on the microscope with constant parameters. During microscopic imaging, the probe microspheres with a particle size of 200nm become a light source themselves because they reflect the illumination light from the light source through the mirror surface. Due to the overexposure of the photosensitive element, a halo larger than the light source itself is formed, creating a bright spot in the field of view, which is then used as a detection signal.

[0098] 6) Quantitative analysis

[0099] The acquired images were analyzed using the developed software. First, the centroid coordinates of all silica microspheres were statistically analyzed. Then, the number of centroids intersecting the coordinate radii of the target probe microspheres and the capturing microspheres was calculated. The number of intersecting centroids yielded the number of binding sites between the capturing and probe microspheres. The number of binding sites was inversely proportional to the target concentration in the sample. Experiments were conducted using AFB1, ZEN, and DON standards at different concentrations to establish standard curves. The corresponding number of binding sites was used as the target concentration for AFB1, ZEN, and DON.

Claims

1. A microscopic analysis method using a microsphere mirror as a probe, characterized in that, Includes the following steps: 1) Carboxylation modification of SiO2 microspheres Carboxylation modification: Monodisperse silica microspheres of different colors and sizes were ultrasonically dispersed for 10-20 min, and a certain amount of microspheres were added to a solution of silane coupling agent γ-aminopropyltriethoxysilane and succinic anhydride in N,N-dimethylformamide. After the reaction was completed, the microspheres were centrifuged and washed to obtain carboxylated silica microspheres, which were then resuspended in PBS solution. Activation of carboxyl groups on the surface of carboxylated silica microspheres: Place carboxylated silica microspheres in a 2ml imported centrifuge tube, add N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to the centrifuge tube, react at room temperature to activate the carboxyl groups on the surface of the carboxylated silica microspheres, centrifuge to remove excess activator, and resuspend the activated carboxylated silica microspheres in PBS solution; 2) Antibody conjugation on probe microspheres and capture microspheres Activated colored carboxylated microspheres with a particle size of 1-5 μm were used as target microspheres. After being mixed with the target antibody, they were incubated at room temperature to obtain the prepared target microspheres. The activated white carboxylated silica microspheres with a particle size of 150-200 nm were used as probe microspheres and incubated with the detection antibody at room temperature to obtain the prepared probe microspheres. 3) Reaction with the target substance Mix the sample, capture microspheres and probe microspheres in a centrifuge tube and react for 3-10 minutes. Take 2 μL of the reaction solution, spread it evenly on a glass slide, dry it and observe it. 4) Acquisition of microscopic images After placing the slide under the microscope, the image is directly obtained using the light source on the microscope with constant parameters. During microscopic imaging, the probe microspheres with a particle size of 150-200nm become a light source themselves because the light is reflected by the mirror. Due to the overexposure of the photosensitive element, a halo larger than the light source itself is formed, creating a bright spot in the field of view, which is then used as a detection signal. 5) Quantitative analysis The obtained images were analyzed using the image analysis software ImageJ. First, the centroid coordinates of the two microspheres of different sizes were statistically analyzed. Then, the number of centroids where the coordinate radii intersected was calculated. The number of intersecting centroids was used to obtain the number of binding sites where the captured microsphere and the probe microsphere bound to the target substance. The number of binding sites is proportional to the concentration of the target substance in the sample, thus enabling the quantitative detection of the target substance.

2. The microscopic analysis method using a microsphere mirror as a probe according to claim 1, characterized in that, In step 1), the silane coupling agent γ-aminopropyltriethoxysilane and succinic anhydride are mixed in equal molar amounts in N,N-dimethylformamide to form a solution.

3. The microscopic analysis method using a microsphere mirror as a probe according to claim 1, characterized in that, In step 1), the carboxylation modification process takes 1-3 hours. The carboxyl groups on the surface of activated carboxylated silica microspheres were reacted at room temperature for 30-90 min.

4. The microscopic analysis method using a microsphere mirror as a probe according to claim 1, characterized in that, In step 2), the room temperature incubation was 1 hour.

Images