CN116735501A - Optical imaging and biological detection method based on zero-dimensional nanostructure - Google Patents
Optical imaging and biological detection method based on zero-dimensional nanostructure Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/255—Details, e.g. use of specially adapted sources, lighting or optical systems
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/47—Scattering, i.e. diffuse reflection
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Abstract
The invention relates to the technical field of optical nano imaging, and discloses an optical imaging and biological detection method based on a zero-dimensional nano structure. The method comprises the following steps: (1) The white light incident light irradiates the zero-dimensional nano-structure array in an oblique incidence mode, and a common optical microscope is used as a receiving end to collect scattered light. (2) The introduction of nanoparticles around the zero-dimensional nanostructures changes the overall scattering cross section, causing a color change in the scattered light. (3) In the visible region, variations in the scattering cross section cause variations in the spectral intensity and peak position. (4) The carboxyl modified nano particles are combined with amino groups in the antibody to enable the surface of the nano particles to be connected with a specific antibody, so that the specific substance to be detected is combined. The introduction of the substance to be detected changes the overall scattered light behavior and thus causes a change in the color of the scattered light. The detection method is convenient and fast, and can realize the specific detection of pathogens. The detection method is simple to operate, low in cost, portable and visual.
Description
Technical Field
The invention relates to the technical field of optical nano imaging, in particular to a photon imaging and biological detection method based on a zero-dimensional nano structure.
Background
It is important to develop a method for direct, ultrasensitive, rapid detection of pathogens. Due to the limits of abbe diffraction limits, conventional optical microscopy imaging limits in the visible range are about 200 nanometers. This indicates that conventional optical microscopes are unable to image sub-wavelength scale objects in the visible range. Therefore, virus particles or other nanoscale objects cannot be visualized by conventional optical microscopy imaging. The surface plasmon technology and the surface enhanced raman spectroscopy method are currently commonly used optical detection methods based on interaction between light and substances, but detection instruments of the two methods are complex and have relatively high detection cost. Fluorescence imaging methods are also commonly used for biological detection, but require fluorescent labeling of the substance to be detected, limited by photobleaching and a light source that excites fluorescence. In addition, the common optical detection method is a colorimetric method, but the colorimetric method has low sensitivity and relatively large error. The current optical detection method has a great challenge to the practical application of the nano-scale substance characterization due to the technical limitation.
Disclosure of Invention
In order to improve the above technical problems, the present invention provides an optical detection method, which includes the following steps: placing carboxyl modified nanoparticle suspension on a substrate, obtaining a zero-dimensional nanoparticle array by a self-assembly method, adding an activating agent for activation, and then modifying the activated zero-dimensional nanoparticle array by an antibody to obtain a zero-dimensional nanoparticle array structure capable of detecting corresponding antigens; and mixing the sample to be detected containing the nanoscale antigen substance with the antibody modified zero-dimensional nanoparticle array structure, incubating, imaging by an optical microscope, and determining the antigen in the sample to be detected based on the scattered light color change.
According to the embodiment of the invention, the method for obtaining the zero-dimensional nanoparticle array by the self-assembly method comprises the steps of placing carboxyl modified nanoparticle suspension on a substrate, then covering a hydrophobic silicon column template with the surface treated by fluorosilane on the substrate, and obtaining the zero-dimensional nanoparticle array by self-assembly of the nanoparticles in the suspension under the induction of a template structure.
According to an embodiment of the present invention, the nanoparticle material may be metal or nonmetal. For example, polystyrene nanoparticles. Preferably, the nanoparticle has a particle size of 300 to 320nm, and exemplary 310nm.
According to an embodiment of the invention, the carboxyl-modified nanoparticle employs a carboxylated material ratio of 100-150ueq/g.
According to an embodiment of the present invention, the method for synthesizing the carboxyl-modified nanoparticle may employ a modification method of the nanoparticle known in the art, such as a copolymerization method.
According to embodiments of the present invention, the substrate may be a rigid substrate or a flexible polymer substrate. Such as a silicon substrate or a flexible PET film substrate.
According to an embodiment of the invention, the optical detection method further comprises sintering the zero-dimensional nanoparticle array to enhance connectivity of the carboxyl-modified nanoparticle to the substrate. Preferably, the sintering temperature is 100-120 ℃, and is exemplified by 100 ℃, 110 ℃ and 120 ℃; the sintering time is 20-40 min, and exemplary is 20min, 30min, 40min.
According to an embodiment of the present invention, the activator may be at least one of carbodiimide (EDC), N-hydroxysuccinimide (NHS) and Dimethylacetamide (DMAC); preferably, the activator is both EDC and NHS. Illustratively, the activator is a mixed solution of EDC and NHS. For example, the activator is a mixed solution configured from solid powders of EDC and NHS.
According to an exemplary embodiment of the invention, the activator is a mixed solution of 0.05mol/L carbodiimide (EDC) and 0.2mol/L N-hydroxysuccinimide (NHS). 200 μl of activation solution is required for each piece of zero-dimensional nanoarray structure.
According to the present invention, the antibody may be a plurality of types of antibodies, which can specifically bind to a nanoscale antigen substance, and for example, may be an anti-IgM antibody, an anti-IgG antibody, an anti-SIg antibody, or the like.
In the present invention, the antibody may be a fluorescent-labeled antibody or a non-fluorescent-labeled antibody.
According to an embodiment of the present invention, the method of antibody modification may employ various antibody modification methods known in the art, such as electrostatic adsorption method, chemical coordination method and chemical bonding method; according to an exemplary embodiment of the invention, the method is a chemical bonding method. For example, the activated product is placed in an antibody solution for incubation, thereby obtaining the nano detection probe.
According to the present invention, the antigen is an antigen having specific affinity such as an anti-IgM antibody, an anti-IgG antibody, an anti-SIg antibody, etc., and may be a virus, or may be other nanoscale biological particles, for example, may be exosome vesicles secreted by various cells, etc.
According to an exemplary embodiment of the present invention, the sample to be tested is blood or sputum.
According to an embodiment of the present invention, 100 to 500. Mu.L (200. Mu.L, for example) of sample solution to be tested is required per zero-dimensional nanoparticle array.
According to the embodiment of the invention, the sample to be detected containing the nanoscale antigen substance is mixed with the antibody-modified zero-dimensional nanoparticle array structure (namely, the sample to be detected containing the nanoscale antigen substance is dripped on the antibody-modified zero-dimensional nanoparticle array structure) to enable the antigen to perform a specific recognition reaction with the antibody on the surface of the antibody-modified zero-dimensional nanoparticle array structure, so that the antigen is adsorbed on the surface of the zero-dimensional nanoparticle structure through the antibody.
According to an embodiment of the invention, the temperature of the incubation may be 25-37 ℃, exemplary 25 ℃; the incubation time may be 15-60min, and is exemplified by 15min.
According to an embodiment of the invention, the optical microscope comprises an objective lens that can receive light and a camera that can image.
According to an embodiment of the present invention, in the optical microscope imaging, oblique incident light is further included.
In the present invention, oblique incident light means that the angle of incident light is 70 ° or more, and exemplary angles are 70 °, 75 °, 80 °, and preferably 70 °.
According to an embodiment of the present invention, the wavelength range of the incident light is 350nm to 1100nm.
According to an embodiment of the present invention, the scattered light color change may be compared by obtaining gray values of pixels of the optical image by image processing software.
The method comprises the steps of firstly, activating carboxyl on the surface of a carboxyl modified nanoparticle by an activating agent, and connecting the carboxyl of the activated carboxyl modified nanoparticle with amino in an antibody to obtain a zero-dimensional nanoparticle array with a specific antibody modified on the surface; and then, combining the sample to be detected containing the nanoscale antigen substances onto a zero-dimensional nano particle array structure with the surface modified with the specific antibodies, imaging by an optical microscope, and determining the antigen in the sample to be detected based on the color change of scattered light.
According to an embodiment of the present invention, the optical detection method includes the steps of:
(1) Placing carboxyl modified nanoparticle suspension on a substrate, and obtaining a zero-dimensional nanoparticle array by a self-assembly method;
(2) Sintering the zero-dimensional nano particle array structure in the step (1) to enhance connectivity of particles and a substrate;
(3) Immersing the sintered zero-dimensional nano structure in a mixed solution of NHS (N-hydroxyysulfosuccinimide) and EDC (N-methyl-N' - (dimethylmineopyl) carbodiimide) to activate the carboxyl on the surface of the nano particle, and then placing the nano particle in an antibody solution to connect the carboxyl with the amino in the antibody so that the surface of the nano particle can be connected with a specific antibody;
(4) Dripping a sample to be detected containing nanoscale antigen substances onto the zero-dimensional nanoparticle array structure of the surface modification specific antibody obtained in the step (3), so that the antigen and the antibody on the surface of the zero-dimensional nanoparticle array structure generate specific recognition reaction;
(5) Placing the sample obtained in the step (4) on an objective table of an optical microscope, obliquely irradiating incident light on the surface of a zero-dimensional particle array structure, receiving scattered light by an objective lens, and performing optical imaging by a camera;
(6) And (5) acquiring gray values of the pixel points from the optical image obtained in the step (5) through image processing software.
The invention also provides application of the optical detection method in pathological detection, in-vitro diagnosis, immune recognition and the like. For example for the detection of nanoscale objects.
The invention also provides a kit comprising a zero-dimensional nanoparticle array.
The invention also provides a biosensor comprising a zero-dimensional nanoparticle array.
The invention also provides application of the kit and/or the biosensor in pathological detection, in-vitro diagnosis, immune recognition and the like. For example for the detection of nanoscale objects.
The invention has the beneficial effects that:
in the dark field imaging method, the change of the scattering cross section can influence the whole scattering behavior of the object, and the imaging method for analyzing the change of the scattering behavior to represent the nano-scale object is gradually developed by enhancing the collection of the scattering light of the substance. Based on the above, the invention prepares a photon structure of a single nanoparticle array, the structure can be prepared in a large quantity, the surface can be modified with various biological substances, and the photon structure has important application prospect in the aspect of label-free detection of nanoscale substances. Specifically:
the invention provides an optical imaging and biological detection method based on a zero-dimensional nano structure, which specifically detects antigens by connecting an antibody to a functional group on the surface of a zero-dimensional single nano particle array. Wherein: the virus particles are combined on the surface of the zero-dimensional single nano particles to change the whole scattering cross section, so that the whole scattering light behavior is changed, and the change of the scattering light can be captured by a common optical microscope. Compared with the traditional method, the method is a non-marking detection method without complex marking, so that the sample to be detected is not destructive. Meanwhile, the method of the invention makes the detection method simpler, more convenient, faster and more accurate through the specific combination of the antibody and the antigen, and has important significance in aspects of pathological detection, in-vitro diagnosis, immune recognition and the like.
Drawings
FIG. 1 is a schematic diagram of the application of the single particle nanostructure of example 1 of the present invention to virus detection.
FIG. 2 is a schematic diagram of optical detection of virus particles adsorbed on the surface of a single nanoparticle in example 1 of the present invention.
FIG. 3 is an optical photograph of the surface of a single nanoparticle of example 1 of the present invention before and after detection of virus particles adsorbed thereon.
In fig. 4, a1 to a4, b1 to b4, and c1 to c4 are respectively a scanning electron microscope photograph, a dark field microscopic photomicrograph, and a dark field microscopic photomicrograph of a lattice structure of different numbers of silica particles surrounding polystyrene nanoparticles obtained by the solution self-assembly method in example 2 of the present invention.
Fig. 5 is a line graph of the red, green and blue three-channel gray scale value analysis of the optical image of the single particle nanostructure of example 2 of the present invention applied to nanoparticle detection using ImageJ software.
Fig. 6 is a comparison of the fluorescent photograph after the fluorescent antibody was modified on the surface of the zero-dimensional nanostructure in example 3 of the present invention, the optical photograph before the antibody was modified with a single nanoparticle (single nanoparticle), and the optical photograph after the antibody was modified (antibody incubated), and the scanning electron microscope photograph.
FIGS. 7 (a) and (b) are respectively schematic diagrams of the detection process and the results of virus detection at different concentrations in example 4 of the present invention.
Detailed Description
The technical scheme of the invention will be further described in detail below with reference to specific embodiments. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.
Unless otherwise indicated, the starting materials and reagents used in the following examples are commercially available or may be prepared by known methods.
Example 1
10 mu L of a carboxyl-modified polystyrene nanoparticle suspension (available from Hui Biotech Co., ltd.) was applied dropwise to an area of 1cm 2 After being covered on a clean and smooth hydrophilic silicon substrate, a hydrophobic silicon column template with the upper surface treated by fluorosilane is covered, and a zero-dimensional single nanoparticle lattice structure is obtained by a solution self-assembly method induced by the template structure, wherein the particle size of nanoparticles is 310nm, and the refractive index is 1.59.
As shown in FIG. 1, the obtained zero-dimensional single nanoparticle lattice structure was immersed in a mixed solution of 200. Mu.L of 0.05mol/L N-hydroxythiosuccinimide (N-Hydroxysulfosuccinimide sodium salt abbreviated as NHS) and 0.2mol/L of 1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride abbreviated as EDC) to activate the polystyrene surface carboxyl groups, and then the zero-dimensional single nanoparticle lattice structure after the carboxyl groups were activated was placed in 10. Mu.g/mL influenza virus antibody solution, and the antibody was incubated at 25℃for one hour. The zero-dimensional single nanoparticle lattice structure modified with the antibody was placed in a matrix containing the corresponding influenza virus (antigen concentration: 10) 6 PFU mL -1 ) The serum solution of (or put the single nanoparticle lattice structure of zero dimension into the sputum diluted 10 times with 0.01mol/L PBS buffer solution (pH 7.2-7.4) extracted from the patient to finish the specific recognition reaction of antibody and antigen), place the structure on the objective table of the optical microscope after the recognition reaction is finished, use xenon lamp as the external incident light source, the incident angle is 70 degrees oblique incidence. The objective lens receives incident light and scattered light, the scattered light is integral scattered light of interaction of the zero-dimensional nano particles and the virus particles, the working distance is 11mm, and the numerical aperture is 0.4.
Fig. 2 and 3 are schematic diagrams of an apparatus of the zero-dimensional single nanoparticle lattice structure in example 1 of the present invention when applied to influenza virus detection, and optical photographs before and after the surface of a single nanoparticle adsorbs influenza virus particles. As shown in fig. 3, the color change of the scattered light is closely related to the overall scattering cross section of the nanoparticle. The virus particles are adsorbed on the surfaces of the nano particles, so that the scattering cross section of the zero-dimensional nano particles is increased, and the color of scattered light is gradually changed from yellow to red to purple. And under the condition of oblique incidence of an external light source, the common optical microscope is utilized to directly detect whether viruses exist and the virus content in the real sample by comparing the structural color difference.
Example 2
10. Mu.L of carboxyl-modified polystyrene nanoparticle suspension (available from Hui Biotechnology Co., ltd., diameter: 310nm, concentration: 0.01 mg/mL) was applied dropwise to a clean and smooth area of 1cm according to example 1 2 After the hydrophilic silicon substrate is covered, a hydrophobic silicon column template with the upper surface treated by fluorosilane is covered, a zero-dimensional single nanoparticle lattice structure is obtained by a solution self-assembly method induced by the template structure, the particle size of the nanoparticles is 310nm, and the refractive index is 1.59.
And then 20 mu L of silicon dioxide nanoparticle suspensions with different concentrations (purchased from Nanjing color Natechnology Co., ltd., particle size: 120nm, concentration: 0mg/mL,0.05mg/mL,0.1mg/mL,0.2 mg/mL) are respectively dripped on the zero-dimensional single nanoparticle lattice structure, and different numbers of silicon dioxide particles encircle the polystyrene nanoparticle lattice structure through a solution self-assembly method.
Fig. 4 a1 to a4, b1 to b4, and c1 to c4 are respectively a scanning electron microscope photograph, a dark field microscope photograph, and an optical photograph under oblique incidence of incident light of the lattice structure of different numbers of silica particles surrounding polystyrene nanoparticles obtained by the solution self-assembly method in example 2. As can be seen from the figures: the number of the silica particles surrounding the polystyrene particles is different, the whole scattering cross section is also different, and along with the gradual increase of the number of the silica particles, the color of scattered light gradually changes from yellow to red to purple in an oblique incidence mode. In the traditional dark field mode, the color change of scattered light is not very different before and after the small-particle silicon dioxide nano particles are introduced around the polystyrene particles. Thus, under oblique incidence of incident light, the polystyrene nanoparticle lattice can detect the incorporated silica nanoparticles.
The image j software was used to analyze the gray values of the images acquired in the oblique incidence mode in fig. 4 after red, green and blue three-channel segmentation, and the result is shown in fig. 5. As can be seen from the figures: the scattering behavior of the whole nano particles can be effectively changed along with the increase of the scattering cross section caused by the increase of the number of the silicon dioxide nano particles, so that the color of the whole scattered light is changed.
Example 3
According to example 1, 10. Mu.L of a carboxyl-modified polystyrene nanoparticle suspension (available from Hui Biotechnology Co., ltd., diameter: 310nm, concentration: 0.01 mg/mL) was dropped onto a clean and smooth area of 1cm 2 After the hydrophilic silicon substrate is covered, a hydrophobic silicon column template with the upper surface treated by fluorosilane is covered, and a zero-dimensional single nanoparticle lattice structure is obtained by a solution self-assembly method induced by the template structure, wherein the diameter of the nanoparticle is 310nm, and the refractive index is 1.59.
The obtained zero-dimensional single particle array structure is placed in a mixed solution of 0.05mol/L N-hydroxy thiosuccinimide (N-Hydroxysulfosuccinimide sodium salt is abbreviated as NHS) and 0.2 mol/L1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1- (3-dimethylaminoopyl) -3-ethylcarbodiimide hydrochloride is abbreviated as EDC) to activate the carboxyl on the surface of the nano particle.
The zero-dimensional single particle array structure after carboxyl activation is placed in a solution of fluorescence-labeled rabbit anti-goat Cy3 fluorescent antibody (purchased from Boschner biotechnology Co., ltd., 10. Mu.g/mL) and incubated for 1 hour at 25 ℃. After antibody modification, the surface unbound antibody was removed by elution with PBST (0.01% Tween-20 in PBS). The zero-dimensional single particle array structure modified with the antibody was placed on a fluorescent microscope stage, and a mercury lamp was used as an incident light source, and the result is shown in fig. 6. As can be seen from i) in fig. 6: the chip structure before the fluorescent antibody is modified (zero-dimensional single-particle array structure) has no fluorescent signal under a fluorescent microscope, and the chip structure after the fluorescent antibody is modified shows a remarkable fluorescent signal under the fluorescent microscope, and the fluorescent signals are uniform and consistent. Thus, the method that the carboxyl on the surface of the carboxyl modified polystyrene nanoparticle is connected with the amino on the surface of the modified fluorescent antibody after activation proves that the antibody can be successfully modified on the surface of the carboxyl modified nanoparticle. Ii) in FIG. 6 is an optical photograph and a scanning electron microscope photograph of a single 310nm carboxyl-modified polystyrene particle under incident light oblique incidence conditions. Iii) in FIG. 6 is an optical photograph and a scanning electron microscope photograph under the same experimental conditions (1 hour incubation at 25 ℃) as in i) in 6 under oblique incidence of incident light after the surface of the 310nm carboxyl group-modified polystyrene microsphere was modified with an influenza virus antibody. Comparing the oblique incidence optical photograph and the scanning electron microscope photograph of the zero-dimensional single particle array structure before and after the antibody modification in ii) and 6) to obtain the antibody modified nano-structure, wherein the antibody modification has no influence on the zero-dimensional nano-structure surface.
In summary, it can be seen by comparing the scanning electron microscope photograph and the optical photograph before and after the surface modification of the antibody with the zero-dimensional single particle array structure: the antibody modification has no influence on the surface of the zero-dimensional single particle array structure, and the color of scattered light has no change basically.
Example 4
According to example 1, 10. Mu.L of a carboxyl-modified polystyrene nanoparticle suspension (available from Hui Biotech Co., ltd., diameter: 310nm, concentration: 0.01 mg/mL) was dropped onto a clean and smooth area of 1cm 2 After the hydrophilic silicon substrate is covered, a hydrophobic silicon column template with the upper surface treated by fluorosilane is covered, and a zero-dimensional single nanoparticle lattice structure is obtained by a solution self-assembly method induced by the template structure, wherein the diameter of the nanoparticle is 310nm, and the refractive index is 1.59.
The obtained zero-dimensional single particle array structure is placed in a mixed solution of 0.05M N-hydroxy thiosuccinimide (N-Hydroxysulfosuccinimide sodium salt is abbreviated as NHS) and 0.2M 1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is abbreviated as EDC) to activate the surface carboxyl groups of the nano particles.
The zero-dimensional single particle array structure after carboxyl activation is placed in an influenza virus antibody (H1N 1 influenza A virus hemagglutinin antibody, 10 mug/mL, 200 mug) solution, and incubated for 1 hour at 25 ℃ (the antibody can be more efficiently and uniformly combined on the surface of polystyrene microspheres after carboxyl activation). After blocking the antibody-modified zero-dimensional single particle array structure with 1% BSA buffer solution at 25℃for 30 minutes, excess BSA was removed by elution with PBST (0.01% Tween-20 in PBS). The zero-dimensional single particle array structure modified with antibodies is placed in a matrix containing the corresponding influenza virus (antigenConcentration: 0PFU mL -1 ，10PFU mL -1 ，10 3 PFU mL -1 ，10 6 PFU mL -1 200 μl) of serum solution (or placing the array structure in sputum 10 times diluted with PBS buffer solution extracted from influenza patient to complete specific recognition reaction of antibody and antigen, virus sample is supplied by 301 hospital), recognizing for 15min, placing the antigen-combined zero-dimensional single-particle array structure on microscope carrier, and xenon lamp as incident light source. As shown in FIG. 7 (b), the scanning electron microscope photograph and the optical photograph before and after virus binding are compared with the surface of the zero-dimensional single particle array structure (the antigen concentration from top to bottom is 0PFU mL in sequence) -1 ，10PFU mL -1 ，10 3 PFU mL -1 ，10 6 PFU mL -1 ) After the virus is combined on the surface of the zero-dimensional single-particle nano structure, the color of scattered light can be effectively changed, and the gray value change reflects the intensity of the color change of the scattered light.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. An optical detection method, characterized by comprising the steps of: placing carboxyl modified nanoparticle suspension on a substrate, obtaining a zero-dimensional nanoparticle array through a self-assembly method, adding an activating agent for activation, and then modifying the activated zero-dimensional nanoparticle array by using an antibody to obtain a zero-dimensional nanoparticle array structure capable of detecting corresponding antigens; and mixing the sample to be detected containing the nanoscale antigen substances with the antibody modified zero-dimensional nanoparticle array structure, incubating, imaging by an optical microscope, and determining the antigen in the sample to be detected based on the color change of scattered light.
2. The optical detection method according to claim 1, wherein the method for obtaining the zero-dimensional nanoparticle array by the self-assembly method comprises the steps of placing a carboxyl-modified nanoparticle suspension on a substrate, and then covering the substrate with a hydrophobic silicon column template with the surface treated by fluorosilane, wherein the nanoparticles in the suspension are self-assembled under the induction of a template structure to obtain the zero-dimensional nanoparticle array.
3. The optical detection method according to claim 1 or 2, wherein the nanoparticle material may be metal or nonmetal.
4. The optical detection method of claim 3, wherein the nanoparticle is a polystyrene nanoparticle.
Preferably, the particle size of the nanoparticle is 300-320 nm.
5. The optical detection method of any one of claims 1-4, further comprising sintering the zero-dimensional nanoparticle array to enhance connectivity of the carboxyl-modified nanoparticles to the substrate.
Preferably, the sintering temperature is 100-120 ℃; the sintering time is 20-40 min.
6. The optical detection method according to any one of claims 1 to 5, wherein the incubation temperature is 25 to 37 ℃; the incubation time may be 15-60min.
Preferably, the optical microscope comprises an objective lens capable of receiving light and a camera capable of imaging.
Preferably, the optical microscope imaging further comprises oblique incident light.
Preferably, the scattered light color change may be compared by image processing software to obtain gray values of pixels of the optical image.
7. The optical detection method according to any one of claims 1 to 6, comprising the steps of:
(1) Placing carboxyl modified nanoparticle suspension on a substrate, and obtaining a zero-dimensional nanoparticle array by a self-assembly method;
(2) Sintering the zero-dimensional nano particle array structure in the step (1) to enhance connectivity of particles and a substrate;
(3) Immersing the sintered zero-dimensional nano structure in a mixed solution of NHS (N-hydroxyysulfosuccinimide) and EDC (N-methyl-N' - (dimethylmineopyl) carbodiimide) to activate the carboxyl on the surface of the nano particle, and then placing the nano particle in an antibody solution to enable the carboxyl to be connected with amino in the antibody so as to enable the surface of the nano particle to be connected with a specific antibody;
(4) Dripping a sample to be detected containing nanoscale antigen substances onto the zero-dimensional nano-particle array structure of the surface modification specific antibody obtained in the step (3), so that the antigen and the antibody on the surface of the zero-dimensional nano-particle array structure generate specific recognition reaction;
(5) Placing the sample obtained in the step (4) on an objective table of an optical microscope, obliquely irradiating incident light on the surface of a zero-dimensional particle array structure, receiving scattered light by an objective lens, and performing optical imaging by a camera;
(6) And (5) acquiring gray values of the pixel points from the optical image obtained in the step (5) through image processing software.
8. Use of the optical detection method according to any one of claims 1-7 for pathological detection, in vitro diagnosis, immunological recognition, etc. Preferably for the detection of nanoscale objects.
9. Kit, characterized in that it comprises a zero-dimensional nanoparticle array according to any one of claims 1 to 7.
10. A biosensor comprising the zero-dimensional nanoparticle array of any one of claims 1-7.
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