Method for measuring molar concentration of nanoparticles

Abstract

The invention discloses a method for measuring the molar concentration of nano-particles. The method comprises the following steps: diluting a nano-particle suspension with a high-molecular polymer solution or dispersing nano-particle powder into the high-molecular polymer solution; transferring the suspension liquid to a spacing net on the side of a sample pool in an electrophoresis tank; nano-particles migrate from the sample pool to the buffer pool under the driving of the electric field and are uniformly distributed on the spacing net in a single-particle manner; performing single-particle imaging on the spacing net by using an optical microscope; and counting the number of the nanoparticles in the image so as to measure the molar concentration of the nanoparticles in the sample. The method can be used under the condition that the properties of the nanoparticles are unknown or no standard substance exists, the detection object has no additional condition, and the accuracy is high.

Description

Technical Field

[0001] This invention relates to a method for measuring the molar concentration of nanoparticles based on the combined use of electrophoretic deposition and optical microscopy imaging, which belongs to various application fields related to nanoparticles. Background Technology

[0002] Measuring the molar concentration of nanoparticles often requires prior knowledge such as complex physical parameters or calibration of standard samples. This process is complicated and consumes a lot of manpower and resources. Therefore, it is essential to establish a method that can measure molar concentration without prior knowledge.

[0003] Currently, there are two main methods for measuring the molar concentration of nanoparticles: macroscopic methods based on the overall signal intensity of nanoparticles and single-particle counting methods that count the number of nanoparticles. Macroscopic methods are indirect methods for measuring nanoparticle concentration, often requiring known physical parameters of the nanoparticles or calibration with standards, such as NanoDrop OD600 light scattering and backscattering DLS. While convenient, macroscopic methods are severely affected by particle shape and size heterogeneity, resulting in poor accuracy and significant systematic differences between methods. Measurement results for the same sample can show deviations of several orders of magnitude, making them only suitable for nanoparticles with specific properties. NanoDrop OD600 light scattering is limited to materials responding to specific wavelengths, such as polymer nanoparticles; backscattering DLS requires optical path optimization and is suitable for complex systems such as food and coatings. Single-particle counting methods directly measure the molar concentration of nanoparticles; the number of nanoparticles counted can be converted into molar concentration. These include nanoparticle tracking analysis (NTA) and single-particle inductively coupled plasma mass spectrometry (spICP-MS). Single-particle counting is unaffected by particle heterogeneity and, theoretically, can achieve accurate measurement of nanoparticle molar concentration without prior knowledge. However, existing single-particle counting techniques rely on sampling, which fails to meet the necessary condition for accurate measurement of nanoparticle molar concentration: detecting all particles in the sample without omission, and its applicability is limited. NTA is suitable for nanoparticle samples larger than 10 nm, such as extracellular vesicles; spICP-MS is only used for aqueous samples and is generally used for inorganic nanoparticles. Summary of the Invention

[0004] The technical problem this invention aims to solve is to overcome the shortcomings of existing technologies and establish a precise method for measuring the molar concentration of nanoparticles. This method combines electrophoretic deposition with optical microscopy imaging, which on the one hand efficiently achieves uniform and monodisperse deposition of nanoparticles, and on the other hand directly measures the number of nanoparticles, thereby calculating the molar concentration. This method can be applied to fields related to nanoscience.

[0005] To achieve the above objectives, the present invention includes the following steps: Step 1: Mix the polymer solution with the nanoparticles to prepare a nanoparticle suspension; Step 2: A certain volume of nanoparticle suspension is added to the spacer mesh in the electrophoresis tank to make it evenly dispersed on the mesh. Then, the two ends of the electrophoresis tank are sealed with electrode plates, the electrophoresis buffer is filled with the electrophoresis tank, and the power is turned on. Under the action of the external electric field, the nanoparticles migrate directionally to the spacer mesh and are finally uniformly and monodispersely deposited on the spacer mesh. Step 3: Image the spacer grid using an optical microscope, achieving image resolution down to the level of a single particle. Step 4: Randomly select m (m≥1) imaging regions, acquire images of the imaging regions, count the number of nanoparticles in the images, and calculate the total number of nanoparticles N. 总 , (1) In the formula, S 随机 Let N be the area of ​​m randomly selected imaging regions. 随机 S represents the number of nanoparticles in the image of the imaging region. 总 The area of ​​the spacer mesh inside the electrophoresis tank; Calculate the concentration C of the nanoparticle suspension. (2) Where D is the factor by which the polymer solution dilutes the nanoparticles, and N... A V represents Avogadro's constant. 总 This refers to the volume of the nanoparticle suspension added dropwise onto the spacer mesh.

[0006] This invention has two key advantages: first, it utilizes glue-free sieving electrophoresis technology to synergistically inhibit the diffusion and directional movement of nanoparticles, controlling their uniform deposition on a spacer mesh; second, it eliminates factors that induce particle aggregation common in other deposition methods, ensuring that nanoparticles are deposited as single particles. Combined with microscopy for counting, it allows for accurate extrapolation from localized counting to counting across the entire deposition area.

[0007] The further preferred technical solution of the present invention is as follows: In step 1, the polymer includes one or more of linear polyacrylamide, polyvinylpyrrolidone, polyethylene oxide, cellulose and its derivatives, polyethylene glycol, polyN,N-dimethylacrylamide, agarose, dextran, sucrose, mannitol, sodium alginate, and polyacrylic acid; the nanoparticles include one or more of quantum dots, upconversion nanoparticles, carbon dots, metal nanoparticles, and inorganic material nanoparticles.

[0008] Furthermore, the mass concentration of the polymer buffer solution is between 0.01% and 10%; the molecular weight of the polymer is between 600 and 10,000,000; and the size of the nanoparticles is between 1 and 150 nm.

[0009] In step 1, the nanoparticle suspension is diluted with a polymer solution, and the dilution factor D is recorded, where D is greater than 1; the concentration of the nanoparticle solution is 10. -21 -10 -6 mol•L -1 between.

[0010] In step 1, the nanoparticle powder is dispersed in a polymer solution to form a nanoparticle suspension, and the dilution factor D of the nanoparticle powder to the polymer solution is 1.

[0011] In step 2, the electrophoresis tank consists of a sample cell, a spacer mesh, and a buffer cell; the sample cell and the buffer cell are separated by a spacer mesh, forming a sample cell and a buffer cell with a shared spacer mesh; the ends of the sample cell and the buffer cell are respectively sealed with electrode plates for applying voltage and forming an electric field; the electric field strength is 5 × 10⁻⁶. 3 -22.5×10 3 V•m -1 Between; a suspension of nanoparticles is dropped onto the spacer mesh in the sample cell. After the electrophoresis tank is filled with electrophoresis buffer, an electric field is applied, and the nanoparticles migrate from the sample cell to the buffer cell. Because their size is larger than the pore size of the spacer mesh, they are uniformly and monodispersely trapped on the spacer mesh.

[0012] In step 2, both the sample cell and the buffer cell are equipped with vent holes and liquid inlets.

[0013] In step 2, the spacer includes one or more of the following: polymer composite film, porous hydrogel, modified nanoporous silica gel, and transparent pressure-sensitive tape treated with pores.

[0014] In step 3, the optical imaging technology includes one or more of the following: bright-field imaging, dark-field imaging, fluorescence imaging, Raman imaging, luminescence imaging, phase contrast imaging, and polarization imaging.

[0015] In step 4, the area of ​​any randomly selected imaging region is less than 0.01 μm. 2 -60 μm 2 Between; the statistical methods for counting the number of nanoparticles include one or more of manual counting, software counting, and artificial intelligence counting.

[0016] In summary, this invention utilizes capillary gel-free sieving electrophoresis technology to efficiently, uniformly, and monodisperse nanoparticles onto a spacer mesh, and then accurately counts the number of nanoparticles by single-particle counting. Compared with existing technologies, this invention can be used when the properties of nanoparticles are unknown or when no standards are available, requires no additional conditions for the detection object, and has high accuracy. Attached Figure Description

[0017] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0018] Figure 1 This is a schematic diagram of the nanoparticle deposition process. In the figure, a is a schematic diagram of the deposition process of negatively charged nanoparticles, and b is a schematic diagram of the deposition process of positively charged nanoparticles.

[0019] Figure 2 This is a bright-field image of the cellulose ester membrane (spacer mesh) under a 20x objective lens.

[0020] Figure 3 This is a fluorescence image of negatively charged core-shell quantum dots after deposition, viewed under a 40x objective lens (Example 1).

[0021] Figure 4 This is a fluorescence image of positively charged core-shell quantum dots after deposition, viewed under a 100x objective lens (Example 2).

[0022] Figure 5 This is a dark-field image of negatively charged gold nanoparticles after deposition, viewed under a 100x objective lens (Example 3).

[0023] Figure 6 This is a fluorescence image of negatively charged carbon dots after deposition under a 100x objective lens (Example 4).

[0024] Figure 7 This is a fluorescence image of negatively charged upconversion nanoparticles after deposition, viewed under a 100x objective lens (Example 5). Detailed Implementation

[0025] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings: This embodiment is implemented under the premise of the technical solution of the present invention, and provides detailed implementation methods and specific operation processes, but the protection scope of the present invention is not limited to the following embodiments.

[0026] This invention provides a method for measuring the molar concentration of nanoparticles, comprising the following steps: Step 1: Dilute the nanoparticle suspension with a polymer solution. The concentration of the nanoparticle suspension before dilution is 10. -21 -10 -6 mol•L -1Record the dilution factor D between the two, and D is greater than 1; or disperse the nanoparticle powder into the polymer solution to prepare a nanoparticle suspension, and the dilution factor D of the nanoparticle powder being diluted by the polymer solution is 1.

[0027] The polymer solution has a mass concentration between 0.01% and 10%; the polymer includes, but is not limited to, one or more of linear polyacrylamide, polyvinylpyrrolidone, polyethylene oxide, cellulose and its derivatives, polyethylene glycol, polyN,N-dimethylacrylamide, agarose, dextran, sucrose, mannitol, sodium alginate, and polyacrylic acid; and the molecular weight of the polymer is between 600 and 10,000,000.

[0028] Nanoparticles include, but are not limited to, one or more of quantum dots, upconversion nanoparticles, carbon dots, (noble) metal nanoparticles, and inorganic material nanoparticles. The size of nanoparticles is between 1 and 150 nm.

[0029] Step 2, add a certain volume of nanoparticle suspension (V) dropwise. 总 The nanoparticles are then uniformly dispersed on the spacer mesh located in the electrophoresis tank. Next, the two ends of the electrophoresis tank are sealed with electrode plates, the tank is filled with electrophoresis buffer, and a power source is connected and energized. Under the influence of the applied electric field, the nanoparticles migrate directionally to the spacer mesh and are ultimately deposited uniformly and monodispersely on it.

[0030] The electrophoresis tank consists of a sample cell, a spacer mesh, and a buffer cell. Both the sample cell and the buffer cell are tubular, with vent holes and inlets on their walls. The first port of the sample cell is positioned opposite the first port of the buffer cell, and a spacer mesh is placed between them. Electrode plates are used to seal the second ports of the sample cell and the buffer cell, respectively, to apply voltage and create an electric field. The electric field strength is 5 × 10⁻⁶. 3 -22.5×10 3 V•m -1 Between the sample and buffer cells, a nanoparticle suspension is transferred to a spacer mesh on the sample cell side, and electrophoresis buffer fills both the sample and buffer cells. Upon application of voltage, the nanoparticles migrate directionally from the sample cell to the buffer cell under the drive of the electric field. Because the size of the nanoparticles is larger than the pore size of the spacer mesh, they are uniformly and monodispersely trapped on the spacer mesh.

[0031] The spacer mesh is made of a polymer material with a pore size between 1 and 100 nm. The polymer material includes, but is not limited to, one or more of the following: polymer composite films, porous hydrogels, modified nanoporous silica gel, and transparent pressure-sensitive tape treated with pores.

[0032] Step 3: Perform optical imaging of the spacer grid at the single-particle level using an optical microscope. Optical imaging includes, but is not limited to, one or more of the following: bright-field imaging, dark-field imaging, fluorescence imaging, Raman imaging, luminescence imaging, phase contrast imaging, and polarization imaging.

[0033] Step 4: Randomly select m (m≥1) imaging regions (the total area is denoted as S). 随机 The area of ​​any randomly selected imaging region is 0.01 μm. 2 -60 μm 2 Between. Image acquisition is performed on the imaging area, and the number of nanoparticles (N) in the image is counted. 随机 The total number of nanoparticles (N) is calculated using formula (1). 总 ): (1) S 随机 Let N be the area of ​​m randomly selected imaging regions. 随机 S represents the number of nanoparticles in the image of the imaging region. 总 The area of ​​the spacer mesh inside the electrophoresis tank; Calculate the concentration (C) of the nanoparticle suspension using formula (2): (2) Where D is the factor by which the polymer solution dilutes the nanoparticles, and N... A V represents Avogadro's constant. 总 This refers to the volume of the nanoparticle suspension added dropwise onto the spacer mesh.

[0034] The number of nanoparticles can be counted using one or more of the following methods: manual counting, software counting, and artificial intelligence counting. Example 1: Measurement of the molar concentration of negatively charged core-shell quantum dots

[0035] Take negatively charged core-shell quantum dots as an example (see Figure 1 (a) describes the specific implementation method of the present invention, and the specific steps are as follows: (1) Preparation of electrophoresis samples: The quantum dots used were from Wuhan Jiayuan Quantum Dot Technology Development Co., Ltd., catalog number CAT. Q2625. The size of the quantum dots was 10 nm. The polymer used was (hydroxypropyl) methylcellulose (HPMC, molecular weight approximately 86 kDa), purchased from Merck, catalog number H7509. 0.4%, 0.8%, 1.0%, 1.5%, and 2.0% HPMC solutions were prepared. The 100 pM quantum dot suspension was diluted 100 times with the above HPMC solutions as electrophoresis samples.

[0036] (2) Electrophoresis: Using pH 9.0 50 mM boric acid solution as the electrophoresis buffer, with... Figure 2 The cellulose ester membrane (pore size 1 nM, purchased from Shanghai Chenggong Biotechnology Co., Ltd., item number YY131057-1m) was used as the spacer. Silicone gaskets (Shenzhen Sanyang Fasteners Co., Ltd.) served as the walls of the sample cell and buffer cell, and platinum sheets (31×50×0.1 mm, purchased from Boyan Technology (Jiangsu Taizhou) Co., Ltd.) were used as electrode plates. The height of the sample cell and buffer cell was 2 mm. The spacer and sample cell walls were sealed with adhesive to facilitate subsequent imaging under an optical microscope. 15 μL of the quantum dot suspension was added to the spacer and shaken to ensure uniform distribution. After sealing the sample cell and buffer cell with the platinum electrode plates, electrophoresis buffer was injected to fill the electrophoresis tank. The electrode plates were connected to a power supply (purchased from Beijing Liuyi Instrument Factory, item number DYY-6C), with the voltage set to 90 V (electric field strength 22.5×10⁻⁶). 3 V•m -1 The power is turned on, and electrophoresis begins. After 5 minutes, the power is turned off, ending the electrophoresis process.

[0037] (3) Remove the sample cell and place it under a fluorescence microscope (purchased from Olympus (China) Co., Ltd., model Olympus IX71). Perform single-particle horizontal imaging under a 40x objective lens. Figure 3 A single-particle fluorescence image (one imaging region) of quantum dots after deposition with 2% HPMC as diluent.

[0038] (4) with Figure 3 Taking an example, let's illustrate the concentration calculation process. The camera chip area is 13.2 mm × 13.2 mm. Under a 40x objective lens, the chip imaging area is 13.2 / 40 mm × 13.2 / 40 mm, meaning the area of ​​one imaging region is 0.1089 mm². 2 In this embodiment, only one imaging region was randomly selected, that is, S 随机 It is 0.1089 mm 2 The total area of ​​the sedimentary zone is 200.96 mm. 2 Statistics using ImageJ software Figure 3 N 随机 The value is 5193. The calculation process for the concentration of the quantum dot suspension is as follows:

[0039]

[0040] (5) The table below lists the measurement results when quantum dots are diluted with polymer solutions of different concentrations.

[0041] Example 2: Determination of the molar concentration of positively charged quantum dots

[0042] Taking positively charged core-shell quantum dots as an example (see...) Figure 1 b) describes the specific implementation method of the present invention as follows: (1) Preparation of electrophoresis samples: The quantum dots used were from Thermo Fisher Scientific Inc., catalog number Q21531MP. The quantum dot size was 11 nm. The polymers used were three dextrans (DEX), all purchased from Merck. The molecular weights of the three DEXs were 40 kDa, 100 kDa and 450-650 kDa, respectively, with corresponding catalog numbers 31389, 09184 and 31392. A 1.0% DEX solution was prepared. The 600 pM quantum dot suspension was diluted 100 times with the above DEX solution as the electrophoresis sample.

[0043] (2) Electrophoresis: Use pH 3.0 10 mM phosphate solution as electrophoresis buffer, and the other conditions are the same as step 2 of Example 1.

[0044] (3) Remove the sample cell and place it under a fluorescence microscope (purchased from Olympus (China) Co., Ltd., model Olympus IX71). Perform single-particle horizontal imaging under a 100x objective lens. Figure 4 Single-particle fluorescence images (two imaging regions) after quantum dot deposition with 1% DEX (100 kDa) as dilution.

[0045] (4) Same as step 4 in Example 1. Note: Under a 100x objective lens, the chip imaging area is 13.2 / 100 mm × 13.2 / 100 mm, that is, the area of ​​one imaging region is 0.017424 mm². 2 In this embodiment, two imaging regions were randomly selected, that is, S 随机 It is 0.034848 mm. 2 .

[0046] (5) The table below lists the measurement results when quantum dots are diluted with DEX solutions of different molecular weights.

[0047] Example 3: Determination of the molar concentration of negatively charged gold nanoparticles

[0048] Taking negatively charged gold nanospheres as an example (see...) Figure 1 (a) describes the specific implementation method of the present invention, and the specific steps are as follows: (1) Preparation of electrophoresis samples: The gold nanospheres used were from Nanopartz Inc., catalog number A11-100-CIT-DIH-1-25-CS. The size of the gold nanospheres was 100 nm. The polymers used were polyethylene glycol (PEG) with a molecular weight of 8 kDa and linear polyacrylamide (LPA) with a molecular weight of 40 kDa. Both PEG and LPA were purchased from Merck, catalog numbers 89510 and 738743, respectively. A 2.0% PEG and 2.0% LPA solution was prepared, and the 300 pM gold nanosphere suspension was diluted 500 times with the above PEG and LPA solution as the electrophoresis sample.

[0049] (2) Use pH 7.40 10 mM phosphate solution as electrophoresis buffer, and the other conditions are the same as step 2 of Example 1.

[0050] (3) Remove the sample cell and place it under a dark-field microscope (purchased from Olympus (China) Co., Ltd., model Olympus IX71). Perform single-particle horizontal imaging under a 100x objective lens. Figure 5 Dark-field image of a single gold nanosphere after deposition with 2% PEG (8 kDa) as diluent (one imaging region).

[0051] (4) Same as step 4 in Example 2.

[0052] (5) The table below lists the measurement results when gold nanospheres were diluted with PEG and LPV solutions of the same concentration.

[0053]

[0054] The above three embodiments basically confirm the reliability of the present invention. The present invention will now be extended to samples with unknown molar concentrations. Example 4: Determination of the molar concentration of negatively charged carbon dots

[0055] Take negatively charged carbon dots as an example (see Figure 1 (a) describes the specific implementation method of the present invention, and the specific steps are as follows: (1) Preparation of electrophoresis samples: The carbon dots used were synthesized in our laboratory, and the synthesis method was based on the literature Nano Lett. 2024, 24, 2264−2272. The carbon dot size was 6 nm. The polymer used was 1000 kDa polyethylene oxide (PEO), purchased from Merck, catalog number 372781. A 0.50% PEP solution was prepared and used to dilute the carbon dot suspension by 2 times as the electrophoresis sample.

[0056] (2) Use pH 7.40 10 mM phosphate solution as electrophoresis buffer, and the other conditions are the same as step 2 of Example 1.

[0057] (3) Remove the sample cell and place it under a fluorescence microscope (purchased from Olympus (China) Co., Ltd., model Olympus IX71). Perform single-particle horizontal imaging under a 100x objective lens. Figure 6 Single-particle fluorescence images (four imaging regions) after carbon dot deposition with 0.5% PEO (8 kDa) dilution.

[0058] (4) Except for manually counting carbon dots, the procedure is the same as step 4 in Example 2. In this example, four imaging regions were randomly selected, S 随机 It is 0.069696 mm. 2 N 随机 The value is 341. The measured molar concentration of carbon points is 0.22 pM. Example 5: Determination of the molar concentration of negatively charged upconversion nanoparticles

[0059] Taking negatively charged upconversion nanoparticles as an example (see...) Figure 1 (a) describes the specific implementation method of the present invention, and the specific steps are as follows: (1) Preparation of electrophoresis samples: The upconversion nanoparticles used were synthesized in our laboratory, and the synthesis method was based on the literature Angew. Chem. Int. Ed. 2011, 50, 1-7. The size of the upconversion nanoparticles was 52 nm. The polymer used was HPMC, with a molecular weight of approximately 86 kDa, purchased from Merck, catalog number H7509. A 2.0% HPMC solution was prepared and used to disperse the upconversion nanoparticle powder. The resulting suspension was the electrophoresis sample.

[0060] (2) Use pH 7.40 10 mM phosphate solution as electrophoresis buffer, and the other conditions are the same as step 2 of Example 1.

[0061] (3) Remove the sample cell and place it under a fluorescence microscope (purchased from Nikon Instruments (Shanghai) Co., Ltd., model Nikon Ti-U). Perform single-particle horizontal imaging under a 100x objective lens. Figure 7 Single-particle fluorescence image (one imaging region) after upconversion nanoparticle deposition with 2% HPMC as dilution.

[0062] (4) Except for manually counting the upconversion nanoparticles, the procedure is the same as step 4 in Example 2. The measured molar concentration of the upconversion nanoparticles was 0.13 pM.

[0063] It should be noted that this invention utilizes capillary sieving technology to achieve uniform and monodisperse deposition of nanoparticles on a spacer mesh. Capillary sieving electrophoresis is a relatively mature technology, and the sieving conditions (including the type and concentration of the medium) used in this technology can theoretically be transferred to this invention.

[0064] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any transformations or substitutions that can be conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for measuring the molar concentration of nanoparticles, characterized in that, Includes the following steps: Step 1: Mix the polymer solution with the nanoparticles to prepare a nanoparticle suspension; Step 2: Add the nanoparticle suspension to the spacer mesh in the electrophoresis tank to disperse it evenly. Then, seal both ends of the electrophoresis tank with electrode plates, fill the electrophoresis tank with electrophoresis buffer, connect the power supply and turn on the power. Under the action of the external electric field, the nanoparticles migrate directionally to the spacer mesh and are finally deposited evenly and monodispersely on the spacer mesh. Step 3: Image the spacer grid using an optical microscope, achieving image resolution down to the level of a single particle. Step 4: Randomly select m imaging regions, acquire images of the imaging regions, count the number of nanoparticles in the images, and calculate the total number N of nanoparticles. 总 , （1） In the formula, S 随机 Let N be the area of ​​m randomly selected imaging regions. 随机 S represents the number of nanoparticles in the image of the imaging region. 总 The area of ​​the spacer mesh inside the electrophoresis tank; Calculate the concentration C of the nanoparticle suspension. （2） Where D is the factor by which the polymer solution dilutes the nanoparticles, and N... A V represents Avogadro's constant. 总 This refers to the volume of the nanoparticle suspension added dropwise onto the spacer mesh.

2. The method for measuring the molar concentration of nanoparticles according to claim 1, characterized in that, In step 1, the polymer includes one or more of linear polyacrylamide, polyvinylpyrrolidone, polyethylene oxide, cellulose and its derivatives, polyethylene glycol, polyN,N-dimethylacrylamide, agarose, dextran, sucrose, mannitol, sodium alginate, and polyacrylic acid; the nanoparticles include one or more of quantum dots, upconversion nanoparticles, carbon dots, metal nanoparticles, and inorganic material nanoparticles.

3. The method for measuring the molar concentration of nanoparticles according to claim 2, characterized in that, The mass concentration of the polymer solution is between 0.01% and 10%; the molecular weight of the polymer is between 600 and 10,000,000; and the size of the nanoparticles is between 1 and 150 nm.

4. The method for measuring the molar concentration of nanoparticles according to claim 1, characterized in that, In step 1, the nanoparticle suspension is diluted with a polymer solution, and the dilution factor D is recorded, where D is greater than 1; the concentration of the nanoparticle suspension is 10. -21 -10 -6 mol•L -1 between.

5. The method for measuring the molar concentration of nanoparticles according to claim 1, characterized in that, In step 1, the nanoparticle powder is dispersed in a polymer solution to form a nanoparticle suspension, and the dilution factor D of the nanoparticle powder to the polymer solution is 1.

6. The method for measuring the molar concentration of nanoparticles according to claim 1, characterized in that, In step 2, the electrophoresis tank consists of a sample cell, a spacer mesh, and a buffer cell; the sample cell and the buffer cell are separated by a spacer mesh to form a sample cell and a buffer cell with a shared spacer mesh; the ends of the sample cell and the buffer cell are respectively sealed with electrode plates to apply voltage and form an electric field. The electric field strength is 5×10 3 -22.5×10 3 V•m -1 Between; a nanoparticle suspension is dropped onto the spacer mesh in the sample cell, and after the sample cell and buffer cell are filled with electrophoresis buffer, a voltage is applied; driven by the electric field, the nanoparticles migrate from the sample cell to the buffer cell, and because their size is larger than the pore size of the spacer mesh, they are uniformly and monodispersely trapped on the spacer mesh.

7. The method for measuring the molar concentration of nanoparticles according to claim 6, characterized in that, Both the sample pool and the buffer pool are equipped with vent holes and liquid inlets.

8. The method for measuring the molar concentration of nanoparticles according to claim 6, characterized in that, The spacer mesh includes one or more of the following: polymer composite film, porous hydrogel, modified nanoporous silicone, and transparent pressure-sensitive tape treated with pores.

9. The method for measuring the molar concentration of nanoparticles according to claim 1, characterized in that, In step 3, optical imaging includes one or more of the following: bright-field imaging, dark-field imaging, fluorescence imaging, Raman imaging, luminescence imaging, phase contrast imaging, and polarization imaging.

10. The method for measuring the molar concentration of nanoparticles according to claim 1, characterized in that, In step 4, the area of ​​any randomly selected imaging region is less than 0.01 μm. 2 -60 μm 2 Between; the counting methods for nanoparticles include one or more of manual counting, software counting, and artificial intelligence counting.

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