Sample preparation method for single nanoparticle fluorescence imaging of low-magnification objective lens

Abstract

The invention provides a sample preparation method for single nanoparticle fluorescence imaging of a low-magnification objective lens, which comprises the following steps of: processing a layer of metal film on the surface of glass or a silicon wafer by adopting a film coating technology to form a metal substrate; mixing an aqueous solution of the nanoparticles with a water-soluble polymer to form a mixed solution, dropwise adding the mixed solution to the surface of the substrate, and evaporating water; butting the metal substrate with the substrate on which the nanoparticles are fixed, controlling the distance between the metal substrate and the nanoparticles through the concentration of the water-soluble polymer, and sealing by adopting an adhesive tape; a prepared nanoparticle sample is placed on a microscope objective table, and a low-magnification objective lens is combined with an imaging element to carry out fluorescence imaging of a single nanoparticle. According to the invention, the low-magnification objective lens is adopted to carry out fluorescence imaging on a single nanoparticle, so that the detection flux of single imaging is effectively increased, the operation is simple, the application range is wide, the problems of automatic scanning, focusing, imaging and counting of single-particle fluorescence imaging are expected to be solved, and the technical conversion of digital immunoassay is facilitated.

Description

Technical Field

[0001] The invention relates to a sample preparation method for single nanoparticle fluorescence imaging under a low-magnification objective lens, and belongs to the technical field of single particle imaging and digital immunoassay. Background Art

[0002] Digital immunoassay is a protein analysis method that quantifies the immune complexes produced by a single target protein. It has shown great application prospects in early disease screening and prognosis detection. Fluorescence imaging is a commonly used detection method for digital immunoassay. Generally, fluorescent nanoparticles (such as quantum dots and upconversion nanoparticles) are used as labeling signals for immune complexes. The technology of immune complexes is converted into the counting of nanoparticles. Nanoparticles or dimers are transferred to the surface of the substrate by means of charge attraction, and high-power objective lenses are combined with imaging elements to perform single-particle fluorescence imaging of nanoparticles or dimers. Counting and protein quantification are performed based on fluorescent photos. Due to the low fluorescence intensity of a single nanoparticle, an oil immersion lens with a high numerical aperture (≥0.6) and a high magnification (≥60 times) is required to collect enough fluorescence to achieve fluorescence imaging of a single nanoparticle. High-magnification oil immersion lenses have the following problems: (1) The field of view is small, which limits the detection throughput of a single imaging; (2) The immersion oil is inconvenient to use and is not suitable for autofocus and automatic scanning imaging. Therefore, the use of low-magnification non-oil objective lenses (≤40x) has become an urgent need for automated, high-throughput imaging detection. However, low-magnification objective lenses have a weak ability to collect fluorescence, and there are challenges in single-particle imaging of extremely small nanoparticles such as quantum dots and upconversion particles. Although some fluorescence enhancement technologies have been developed, such as microsphere lens enhancement, photonic crystal structure enhancement, and noble metal enhancement, there are still problems such as nanoparticle omission (microsphere lens enhancement and photonic crystal structure enhancement), difficulty in controlling the distance between nanoparticles and noble metal layers (noble metal enhancement), and incompatibility with digital immunoassay processes. Therefore, the development of sample preparation methods suitable for single nanoparticle fluorescence imaging with low-magnification objectives is of great significance to the automation and commercialization of digital immunoassays. Summary of the invention

[0003] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a sample preparation method for single nanoparticle fluorescence imaging with a low-magnification objective lens, so as to solve the problem caused by the need to use a high-magnification oil objective lens for single-particle fluorescence imaging. At the same time, it also solves the problems existing in the application of low-magnification objective lenses in the field of digital immunoassay, such as omission of nanoparticles, difficulty in controlling the distance between nanoparticles and the precious metal layer, and incompatibility with the digital immunoassay process.

[0004] The present invention provides a sample preparation method for single nanoparticle fluorescence imaging under a low-magnification objective lens, comprising the following steps: Step 1: Metal substrate production: Use coating technology to process a metal film on the surface of glass or silicon wafer to form a metal substrate; Step 2, fixing nanoparticles on the substrate surface: mixing an aqueous solution of nanoparticles with a water-soluble polymer to form a mixed solution, and dropping the mixed solution onto the substrate surface to evaporate water; Step 3, metal substrate and substrate composition - docking the metal substrate with the substrate on which the nanoparticles are fixed, controlling the distance between the metal substrate and the nanoparticles by the concentration of the water-soluble polymer, and then sealing with an adhesive tape to obtain a nanoparticle sample; Step 4: Fluorescence imaging: Place the prepared nanoparticle sample on the microscope stage, and use a low-magnification objective lens combined with an imaging element to perform fluorescence imaging of a single nanoparticle.

[0005] The present invention provides a sample preparation method for single nanoparticle fluorescence imaging under a low-power objective lens, comprising the processes of metal substrate production, substrate surface nanoparticle fixation, controllable assembly of metal substrate and substrate, fluorescence imaging, etc.; a thin metal layer is plated on the surface of a glass slide or silicon wafer by using a coating technology to form a metal substrate; a nanoparticle aqueous solution and a water-soluble polymer are evenly mixed in a certain proportion to form a mixed solution, a certain volume of the mixed solution is dripped on the substrate surface, water is evaporated, the polymer is precipitated on the substrate surface, and an edge ring-shaped film is formed on the substrate surface, and the nanoparticles are dispersed and fixed in the film; the metal substrate is docked with a substrate for fixing nanoparticles, the distance between the metal substrate and the nanoparticles is controlled, and the tape is sealed. The packaged metal substrate and substrate are placed on a microscope stage, and the low-power objective lens combined with an imaging element can realize the fluorescence imaging of a single nanoparticle.

[0006] The technical solution further optimized as the present invention is as follows: In the step 1, the coating technology is one of thermal evaporation coating, magnetron sputtering, and chemical vapor deposition technology, and the material of the metal film is at least one of gold, silver, chromium, nickel, titanium, and platinum.

[0007] Preferably, the metal film is a single-layer structure or a multi-layer structure.

[0008] In step 2, the nanoparticles are fluorescent materials with a size of less than 500 nm, including at least one of quantum dots, carbon dots, gold clusters, silver clusters, upconversion particles, fluorescent polystyrene balls, and fluorescent silica balls; the water-soluble polymer is at least one of polyvinyl alcohol, dextran, polyacrylic acid, hydroxypropyl methylcellulose, gelatin, alginate, and sucrose; and the material of the substrate is one of glass, silicon wafer, polymethyl methacrylate, polydimethylsiloxane, polycarbonate, polystyrene, poly-4-methyl-1-pentene, and polysulfone.

[0009] Preferably, the concentration of the nanoparticles in the mixed solution is 0.1aM to 1mM, and the mass percentage concentration of the water-soluble polymer is 0.005% to 1%.

[0010] In step 3, the distance between the metal substrate and the nanoparticles is controlled by the concentration of the water-soluble polymer. The thickness of the edge of the annular film formed by water-soluble polymers of different concentrations is different. The thickness of the annular edge directly determines the distance between the nanoparticles and the metal substrate in the film.

[0011] Preferably, the distance between the nanoparticles and the metal substrate is 5 nm to 200 nm.

[0012] In step 4, the low-magnification objective lens is a dry lens with a magnification of less than 60 times and air as the medium, and the imaging element is one of CMOS and CCD.

[0013] Compared with the prior art, the present invention uses a low-magnification objective lens to achieve fluorescent imaging of a single nanoparticle. The low-magnification objective lens has a large field of view, which effectively increases the detection flux of a single imaging. The method does not involve any expensive and complex instruments, is easy to operate, and has a wide range of applications. It is expected to solve the problems of automatic scanning, focusing, imaging, and counting of single-particle fluorescent imaging, and is conducive to the technical transformation of digital immunoassay. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram of the principle of the method of the present invention.

[0015] Figure 2 This is a scanning electron microscope image of the metal layer in the present invention.

[0016] Figure 3 This is a diagram showing the combination of the cover glass and the metal substrate in the present invention.

[0017] Figure 4 These are single particle images of quantum dots of different concentrations using the sample preparation method of the present invention. (ad) The concentrations of the quantum dots are 500fM, 1pM, 2pM and 5pM, respectively.

[0018] Figure 5 Schematic diagram of the effect of dextran concentration on single quantum dot particle imaging in the present invention, (af) dextran concentrations are 1%, 0.5%, 0.1%, 0.05%, 0.01% and 0.005%, respectively.

[0019] Figure 6 This is a single particle imaging diagram of polyvinyl alcohol on quantum dots in the present invention. Figure 7 This is a 100x oil-immersed objective lens image of quantum dots using the conventional sample preparation method of the present invention.

[0020] Figure 8 This is a fluorescence image formed by directly dropping a mixed solution of quantum dots and polymers on a metal substrate.

[0021] Fig. 9 This is a fluorescence imaging diagram of upconversion nanoparticles using the sample preparation method of the present invention. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings: This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and specific operation process are given, but the protection authority of the present invention is not limited to the following embodiments. Example 1

[0023] The sample preparation method for low-magnification objective lens single nanoparticle fluorescence imaging and 40x objective lens imaging of single quantum dots is as follows: Figure 1 As shown, a metal layer is processed on the glass surface by magnetron sputtering technology to form a metal substrate (see Figure 2 ), the material composition of the metal layer is 10 nanometers of chromium, 10 nanometers of nickel and 30 nanometers of silver. Take a certain mass of dextran (DEX, 40kDa) and dissolve it in deionized water to form a 0.05% (w / v) DEX solution. Take a certain volume of quantum dot solution (using Qdot 655ITK carboxyl quantum dots, 1nM, purchased from Thermo Fisher Scientific) and mix it with 0.05% (w / v) DEX solution to form a quantum dot mixture (the final concentration of quantum dots in the mixture is 1aM~1mM). Take 2.5 microliters of the quantum dot mixture and drop it on a clean coverslip. Evaporate the water, and phase separation causes DEX to precipitate, forming a ring-shaped film. The quantum dots are evenly dispersed in the DEX film. The DEX film on the surface of the coverslip contacts the surface of the metal substrate, and the coverslip and the metal substrate are sealed with tape so that the annular edge of the film is in close contact with the surface of the metal substrate (see Figure 3 ).

[0024] The combination of the cover glass and the metal substrate was placed on the microscope stage, and fluorescence imaging was performed using a 40x objective lens (UPLFLN40X) combined with sCMOS (photometrics95B). Fluorescence photos of quantum dots with different concentrations were obtained as shown below: Figure 4 shown. Figure 4 The quantum dot concentrations corresponding to ad are 500fM, 1pM, 2pM and 5pM respectively.

[0025] Changing the concentration of DEX, repeating the above steps, the fluorescence photos of quantum dots obtained are as follows: Figure 5 shown. Figure 5The DEX concentrations corresponding to af in the figure are 1%, 0.5%, 0.1%, 0.05%, 0.01% and 0.005% respectively. The edge thickness of the film formed by DEX solutions of different concentrations is different. The thickness of the annular edge directly determines the distance between the quantum dots and the metal substrate in the film, affecting the fluorescence intensity of the quantum dots.

[0026] The DEX solution was replaced with a polyvinyl alcohol (PEG) solution and mixed with a certain volume of quantum dot solution. The concentration of quantum dots in the mixed solution was 1 pM and the concentration of PEG was 0.1% (w / v). The rest of the steps remained unchanged. The combination of the cover glass and the glass substrate was placed on the microscope stage, and fluorescence imaging was performed using a 40x objective lens (UPLFLN40X) combined with sCMOS (photometrics95B). The obtained quantum dot fluorescence photos are shown in Figure 1. Figure 6 shown.

[0027] Comparative Example 1 Conventional sample preparation method for 100x oil-immersed imaging of a single quantum dot In this example, the metal substrate in Example 1 is replaced with a glass substrate, and the remaining steps are the same as in Example 1. The combination of the cover glass and the glass substrate is placed on the microscope stage, and fluorescence imaging is performed using a 100 oil objective (UPlanSApo100X) combined with sCMOS (photometrics95B). The obtained quantum dot fluorescence photos are shown in FIG. Figure 7 The results show that the single imaging flux under the sample preparation method provided by the present invention is significantly greater than the imaging flux of the 100 oil objective lens combined with sCMOS under the conventional method, and there is no significant difference in the imaging quality between the two.

[0028] Comparative Example 2 A mixed solution of quantum dots and water-soluble polymers was directly dripped onto a metal substrate for imaging Take 2.5 microliters of the quantum dot mixture of Example 1 and drip it directly on the surface of the metal substrate. The water evaporates and the phase separation causes DEX to precipitate, forming a ring-shaped film. The quantum dots are evenly dispersed in the DEX film. The metal substrate is placed on the microscope stage, and the 40x objective lens (UPLFLN40X) is combined with sCMOS (photometrics95B) for fluorescence imaging. The obtained quantum dot fluorescence photo is as follows: Figure 8 Compared with the sample preparation method provided by the present invention, at the same concentration, the quantum dot image obtained by directly dropping the mixed solution of quantum dots and polymer on the metal substrate for imaging is significantly reduced. Example 2

[0029] The upconversion nanoparticles were mixed with 0.05% (w / v) DEX solution according to the steps of Example 1 to form an upconversion nanoparticle mixed solution. 2.5 μl of the mixed solution was dropped on a clean coverslip to evaporate the water. Phase separation caused DEX to precipitate and form an annular film. The upconversion nanoparticles were dispersed in the DEX film. The DEX film on the surface of the coverslip was in contact with the surface of the metal substrate. The coverslip and the metal substrate were sealed with tape so that the annular edge of the film was in close contact with the surface of the metal substrate. The combination of the coverslip and the metal substrate was placed on a microscope stage, and fluorescence imaging was performed using a 40x objective lens (UPLFLN40X) combined with sCMOS (photometrics95B). The obtained fluorescence photograph is shown as follows. Fig. 9 shown.

[0030] The above description is only a specific implementation of the present invention, but the protection scope of the present invention is not limited thereto. Any person familiar with the technology can understand and think of any changes or substitutions within the technical scope disclosed by the present invention, which should be included in the scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A sample preparation method for single nanoparticle fluorescence imaging under a low-magnification objective lens, characterized in that: The following steps are involved: Step 1: Metal substrate production: Use coating technology to process a metal film on the surface of glass or silicon wafer to form a metal substrate; Step 2, fixing nanoparticles on the substrate surface: mixing an aqueous solution of nanoparticles with a water-soluble polymer to form a mixed solution, and dropping the mixed solution onto the substrate surface to evaporate water; Step 3, metal substrate and substrate composition - docking the metal substrate with the substrate on which the nanoparticles are fixed, controlling the distance between the metal substrate and the nanoparticles by the concentration of the water-soluble polymer, and then sealing with an adhesive tape to obtain a nanoparticle sample; Step 4: Fluorescence imaging: Place the prepared nanoparticle sample on the microscope stage, and use a low-magnification objective lens combined with an imaging element to perform fluorescence imaging of a single nanoparticle.

2. The sample preparation method for single nanoparticle fluorescence imaging under a low-magnification objective lens according to claim 1, characterized in that: In the step 1, the coating technology is one of thermal evaporation coating, magnetron sputtering, and chemical vapor deposition technology, and the material of the metal film is at least one of gold, silver, chromium, nickel, titanium, and platinum.

3. The sample preparation method for single nanoparticle fluorescence imaging under a low-magnification objective lens according to claim 2, characterized in that: The metal film is a single-layer structure or a multi-layer structure.

4. The sample preparation method for single nanoparticle fluorescence imaging under a low-magnification objective lens according to claim 1, characterized in that: In step 2, the nanoparticles are fluorescent materials with a size of less than 500 nm, including at least one of quantum dots, carbon dots, gold clusters, silver clusters, upconversion particles, fluorescent polystyrene balls, and fluorescent silica balls; the water-soluble polymer is at least one of polyvinyl alcohol, dextran, polyacrylic acid, hydroxypropyl methylcellulose, gelatin, alginate, and sucrose; and the material of the substrate is one of glass, silicon wafer, polymethyl methacrylate, polydimethylsiloxane, polycarbonate, polystyrene, poly-4-methyl-1-pentene, and polysulfone.

5. The sample preparation method for single nanoparticle fluorescence imaging under a low-magnification objective lens according to claim 4, characterized in that: The concentration of the nanoparticles in the mixed solution is 0.1aM-1mM, and the mass percentage concentration of the water-soluble polymer is 0.005%-1%.

6. The sample preparation method for single nanoparticle fluorescence imaging under a low-magnification objective lens according to claim 1, characterized in that: In step 3, the distance between the metal substrate and the nanoparticles is controlled by the concentration of the water-soluble polymer. The thickness of the edge of the annular film formed by water-soluble polymers of different concentrations is different. The thickness of the annular edge directly determines the distance between the nanoparticles and the metal substrate in the film.

7. The sample preparation method for single nanoparticle fluorescence imaging under a low-magnification objective lens according to claim 6, characterized in that: The distance between the nanoparticles and the metal substrate is 5nm to 200nm.

8. The sample preparation method for single nanoparticle fluorescence imaging under a low-magnification objective lens according to claim 1, characterized in that: In the step 4, the low-magnification objective lens is a dry lens with a magnification of less than 60 times and air as the medium, and the imaging element is one of CMOS and CCD.

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