A method for measuring concentration of nanoparticles in a liquid for calibration of a particle counter

Abstract

The application discloses a kind of liquid nanoparticle concentration measurement methods for particle counter calibration, belong to nanomaterial characterization technical field.The method is first by adding specific concentration polyvinyl alcohol solution to nanoparticle suspension, and the evaporation of droplet on pretreated silicon substrate is controlled under low temperature, high humidity condition, effectively inhibit the coffee ring effect, obtain the solid sample with high particle monodispersity, uniform distribution.It can be directly used for high-resolution imaging equipment such as scanning electron microscope to clearly observe and count.Further, combined with the particle statistics number of imaging area and sample preparation parameters, the concentration of nanoparticle in original suspension can be calculated back.This application provides an integrated solution from sample preparation, observation to quantitative analysis, with the advantages of simple operation, low cost, wide application range, high accuracy, etc., especially suitable for characterization and quantification of nanoparticle system without standard or unknown properties.

Description

Technical Field

[0001] This invention relates to the field of nanomaterial characterization and analysis technology, specifically to a method for measuring the concentration of nanoparticles in liquids for particle counter calibration. Background Technology

[0002] The physicochemical properties of nanoparticles are closely related to their concentration, size, morphology and dispersion state. Accurate characterization of these parameters is the foundation of nanoscience research and applications (such as biosensoring, drug delivery, catalysis, nanoelectronics, etc.). Among them, preparing nanoparticles from liquid dispersion systems into solid samples for observation by high-resolution microscopes (such as SEM, TEM) is a commonly used characterization method.

[0003] Currently, the simplest sample preparation method is to drop a drop of nanoparticle suspension onto a substrate (such as a silicon wafer, mica, or glass slide) and observe it after it dries naturally. However, this method generally suffers from a severe "coffee ring effect": because the evaporation rate at the edge of the droplet is faster than that at the center, the internal capillary flow will continuously transport the dispersed particles to the edge and accumulate them, resulting in a ring-shaped non-uniform distribution of particles on the substrate after drying, with sparse or even absent particles in the central region.

[0004] This non-uniformity makes: (1) It is difficult to find statistically significant observation areas under a microscope; (2) Particles overlap and accumulate in the edge area, making it impossible to accurately count individual particles; (3) It seriously affected the accuracy of size measurement, morphology observation and concentration calculation.

[0005] To suppress the coffee ring effect, existing technologies have proposed various strategies, such as using surfactants to alter the three-phase pinning, adding binary solvents to regulate Marangoni convection, or using electrowetting to change the contact angle and thus control the evaporation mode. Among these, the principle of using surfactants to alter the three-phase pinning is that surfactants can adsorb at the liquid-solid interface, changing the wetting characteristics of the droplet edge (three-phase contact line). When the surfactant is unevenly distributed or "pinned" on the contact line, it leads to an increase in the variation of the contact angle, making it easier for oil or liquid droplets to detach or remain in a specific position under external force. However, surfactants are prone to residue, making it difficult to completely reverse the "pinning" effect, affecting the reproducibility of subsequent experiments. Furthermore, this method has high requirements for the liquid surface; if the surface is not smooth or chemically homogeneous, the "pinning" effect will be uneven. Adding a binary solvent to modulate Marangoni convection artificially creates a surface tension gradient, disrupting the original Marangoni flow field and thus altering the evaporation pattern. However, this method introduces new variables; the mixing ratio of the binary solvent, the differences in evaporation rates of different components, and solubility all require precise control, increasing experimental complexity. Furthermore, solvent mixing may lead to decreased solubility or the formation of precipitates, affecting droplet purity and causing unintended effects. Electrowetting, which alters the contact angle, applies a voltage between a conductive droplet and an electrode, using an electric field to change the droplet's contact angle, fundamentally altering the droplet's wetting state and enabling real-time control of the evaporation pattern. However, this method requires sophisticated equipment, is only suitable for liquids with a certain degree of conductivity, and may cause electrochemical reactions under high pressure, leading to electrode corrosion or liquid electrolysis.

[0006] Regarding concentration measurement, Chinese patent CN120869908A discloses a method for measuring the molar concentration of nanoparticles. Although uniform deposition can be achieved by using electrophoretic deposition combined with microscopic counting, it requires a specialized electrophoresis device and has certain requirements on the charge properties of the particles and the buffer system.

[0007] Therefore, developing a simpler, more universal, and lower-cost method for processing nanoparticles that can simultaneously obtain uniform samples and accurate concentration information has significant practical application value. Summary of the Invention

[0008] The technical problem this invention aims to solve is to overcome the severe coffee ring effect and uneven particle distribution problems existing in the current droplet drying method for preparing nanoparticle samples, as well as the resulting difficulties in microscopic observation, inaccurate single-particle counting, and difficulty in concentration calculation. This invention aims to provide a simple, low-cost, and highly versatile method for preparing uniform nanoparticle samples, and to achieve reliable concentration measurement based on uniform samples.

[0009] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A method for measuring the concentration of nanoparticles in liquid for particle counter calibration includes the following steps: Step S1, Sample preparation and pretreatment: The flat silicon substrate is thoroughly cleaned, and the nanoparticle suspension to be tested is mixed with a polyvinyl alcohol solution with a specific degree of hydrolysis (98%-99%) in a certain proportion to form a homogeneous mixed suspension. Furthermore, the cleaning process for the silicon substrate is as follows: ultrasonic cleaning with 70% isopropanol (IPA) for 5 minutes, then ultrasonic cleaning with anhydrous ethanol for 5 minutes, and finally ultrasonic cleaning with ultrapure water for 5 minutes. Furthermore, the mixed suspension sample was obtained by mixing a polyvinyl alcohol solution, a nanoparticle suspension, and ultrapure water; the polyvinyl alcohol solution had a degree of hydrolysis of 98%-99% and a mass concentration of 1%; the nanoparticle suspension needed to be diluted, with the dilution factor depending on the concentration of the nanoparticle suspension, and the dilution factor needed to ensure that the particle solids content in the mixed suspension sample was within a certain range. %arrive %between; Step S2, Controlled Droplet Evaporation Deposition: Accurately aspirate a certain volume (e.g., 1 μL) of the mixed suspension using a micropipette and gently drop it onto the center of the pretreated silicon substrate. Subsequently, the sample is transferred to a controlled environment with low temperature (e.g., 4°C) and high humidity (relative humidity ≥70%) for static evaporation; the low temperature and high humidity aim to slow down the overall evaporation rate and reduce capillary flow intensity; this process does not require heating or vacuuming.

[0010] Step S3, Microscopic Imaging and Particle Counting: After evaporation until the solvent has completely evaporated, a solid sample uniformly distributed on the silicon wafer surface is obtained; the sample is placed under a scanning electron microscope, and the area with well-dispersed particles is located and imaged at an appropriate magnification (e.g., 3000-5000 times); the field of view is randomly selected, and the nanoparticles in the image are counted manually or with software assistance (e.g., ImageJ).

[0011] Step S4, Concentration Calculation: Based on the volume of the added suspension sample, the ratio of the area of ​​the counting field to the area of ​​the entire deposition zone, and the number of particles counted within the field of view, the molar concentration of the original nanoparticle suspension can be calculated using Avogadro's constant.

[0012] The core physical process of achieving uniform deposition in this invention lies in the preferential and rapid adsorption of surface-active free polyvinyl alcohol (PVA) molecules onto the entire gas-liquid interface, forming a continuous, viscoelastic, and mechanically strong polymer film. Simultaneously, PVA-modified particles, due to their surface properties, tend to migrate towards this interface and are captured or embedded in the film, forming a "particle-polymer" composite interface structure. This structure severely restricts the lateral migration ability of the particles. During evaporation, the gas-liquid interface area inevitably shrinks, and geometric constraints drive the interfacial material to compress and aggregate towards the contact line. However, by actively suppressing the evaporation rate under experimental conditions (low temperature and high humidity), the bulk capillary flow driven by evaporation compensation can be significantly weakened, thereby eliminating the main kinetic driving force leading to the coffee ring effect. Under these conditions, the composite interfacial film, with its inherent mechanical strength, can effectively resist the compressive stress caused by interfacial shrinkage and residual non-uniform flow, preventing irreversible enrichment of material towards the edges. Finally, after the solvent has completely evaporated, this structurally complete composite film is laid flat as a whole on the substrate, forming a deposition layer with relatively uniform thickness and composition distribution. The successful implementation of this process depends on polyvinyl alcohol having a sufficiently high molecular weight and concentration to form a strong interfacial film, and the particle concentration needing to be low enough to avoid disrupting the film's continuity. Simultaneously, strong adsorption or entanglement between the particles and the film is required to ensure the particles are effectively embedded within the film. Furthermore, hydrophobic substrates such as silicon facilitate overall droplet contraction rather than pinning evaporation, which also helps improve the uniformity of deposition.

[0013] The beneficial effects of this invention are as follows: This invention provides a method for measuring the concentration of nanoparticles in liquids for particle counter calibration. This method fundamentally alters the droplet drying kinetics by introducing polyvinyl alcohol with a specific degree of hydrolysis and precisely controlling the evaporation environment, resulting in a more uniformly distributed nanoparticle sample. The obtained sample can be directly used with high-end characterization equipment such as scanning electron microscopes, exhibiting good particle dispersion and no overlap, greatly facilitating morphology observation, size statistics, and particle counting. Furthermore, based on particle counting on a uniform sample, absolute concentration measurement of nanoparticle suspensions without standards can be achieved through simple geometric ratio conversion. This method requires no complex equipment, has no special requirements on the material, shape, or surface charge properties of the nanoparticles, is low-cost, and easily promoted in ordinary laboratories. The sample prepared by this method can be used as a standard or reference sample to calibrate other concentration measurement instruments based on counting principles (such as nanoparticle trackers, electrophoretic counting devices, and light scattering particle counters), improving their measurement reliability. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 This is a scanning electron microscope field of view of a randomly selected region in the method for uniform sample preparation and concentration measurement of polystyrene nanoparticles provided in Embodiment 2 of the present invention. Figure 2 This is a scanning electron microscope field of view of another randomly selected region in the method for uniform sample preparation and concentration measurement of polystyrene nanoparticles provided in Embodiment 2 of the present invention. Figure 3 This is an overview diagram of the entire droplet in a method for uniform sample preparation and concentration measurement of polystyrene nanoparticles provided in Embodiment 2 of the present invention. Figure 4 This is an overview diagram of the entire droplet in a method for uniform sample preparation and concentration measurement of polystyrene nanoparticles provided in Comparative Example 1 of the present invention. Figure 5 This is a partial droplet diagram from a method for uniform sample preparation and concentration measurement of polystyrene nanoparticles provided in Comparative Example 1 of this invention. Figure 6 This is an overview diagram of droplets with a final PVA concentration of 0.4 wt% in the mixed solution in the uniform sample preparation and concentration measurement method for polystyrene nanoparticles provided in Comparative Example 2 of the present invention. Figure 7 This is an overview diagram of droplets with a final PVA concentration of 0.01 wt% in the mixed solution in the uniform sample preparation and concentration measurement method for polystyrene nanoparticles provided in Comparative Example 3 of the present invention. Figure 8 This is a partial field-of-view image of a droplet in a method for uniform sample preparation and concentration measurement of polystyrene nanoparticles provided in Comparative Example 3 of the present invention. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0017] Example 1 This embodiment provides a method for measuring the concentration of nanoparticles in liquids for particle counter calibration. This method is mainly applicable to the measurement of nanoparticles from 50 nm to 2 μm. The nanoparticles are mixed with a polyvinyl alcohol solution and slowly evaporated on a silicon wafer in a low-temperature, high-humidity environment to form a uniform monolayer deposition. Scanning electron microscopy (SEM) is then used for imaging and counting. The molar concentration of the original suspension is calculated based on the added volume, dilution factor, and the ratio of the counting field of view.

[0018] The method includes the following steps: Step S1, Sample Pretreatment and Mixing: The flat silicon substrate is thoroughly cleaned. The nanoparticle suspension to be tested is mixed with a polyvinyl alcohol solution with a specific degree of hydrolysis (98%-99%) and ultrapure water in a certain proportion to form a homogeneous mixed suspension; Furthermore, the cleaning process for the silicon substrate is as follows: ultrasonic cleaning with isopropanol (IPA) for 5 minutes, ultrasonic cleaning with anhydrous ethanol for 5 minutes, and finally ultrasonic cleaning in ultrapure water for 5 minutes. Furthermore, after preparing a 1% PVA solution (with a hydrolysis degree of 98%-99%), it is mixed with ultrapure water and the nanoparticle suspension. Since the nanoparticle suspension needs to be diluted, the dilution factor depends on the concentration of the nanoparticle suspension. The dilution factor should be such that the particle solids content in the mixed suspension sample is within a certain range. %arrive %between.

[0019] Step S2, Controlled Droplet Evaporation Deposition: Accurately pipette a certain volume (e.g., 1 μL) of the mixed suspension and gently drop it onto the center of the pretreated silicon substrate. Then, transfer the sample to a controlled environment with low temperature (e.g., 4°C) and high humidity (relative humidity ≥70%) for static evaporation for 1-12 hours until the solvent is completely evaporated. The low temperature and high humidity aim to slow down the overall evaporation rate and reduce capillary flow intensity; this process does not require heating or vacuuming.

[0020] Step S3, Microscopic Imaging and Particle Counting: After evaporation until the solvent is completely evaporated, a solid sample with highly monodisperse nanoparticles uniformly distributed on the silicon wafer surface is obtained. The sample is placed under a scanning electron microscope, and areas with good particle dispersion are located and imaged at an appropriate magnification (e.g., 3000-5000x). Fields of view are randomly selected, and the nanoparticles in the image are counted manually or with software assistance.

[0021] Step S4, Concentration Calculation: Based on the volume of the added suspension sample, the ratio of the area of ​​the counting field to the area of ​​the entire deposition zone, and the number of particles counted within the field of view, the molar concentration of the original nanoparticle suspension can be calculated using Avogadro's constant.

[0022] Example 2 This embodiment provides a method for uniform sample preparation and concentration measurement of polystyrene nanoparticles (PS NPs), which is based on the method described in Embodiment 1; First, let me introduce the reagents and pretreatment procedures used in this embodiment: 200nm monodisperse polystyrene nanoparticle aqueous suspension (nominal concentration 1×10⁻⁶) 9 particles / mL, solid content is 4.4 × 10⁻⁶ -4 %, purchased from Thermo Fisher Scientific (China) Co., Ltd.; Polyvinyl alcohol (PVA, degree of hydrolysis 98-99%) was purchased from Aladdin and a 1 wt% polyvinyl alcohol aqueous solution was prepared.

[0023] Silicon wafer pretreatment: The silicon wafer is ultrasonically cleaned in isopropanol, ethanol and ultrapure water for 5 minutes each, and then dried with nitrogen.

[0024] The method includes: Step 1: Sample preparation; Mix a 200nm monodisperse polystyrene nanoparticle aqueous suspension, a 1% PVA solution, and ultrapure water at a volume ratio of 1:2:7. Specifically, mix 1 ml of the 200nm monodisperse polystyrene nanoparticle aqueous suspension, 2 ml of the 1% PVA solution, and 7 ml of ultrapure water, and gently vortex to mix. Use a pipette to take V... 滴加 =2.5μL of the mixture was dropped into the center of the pretreated silicon wafer; the silicon wafer was placed in a petri dish and placed in a 4℃ constant temperature oven (with a built-in humidity maintenance device to ensure high humidity, relative humidity of about 70%), and allowed to stand and evaporate until it was easy to completely evaporate to obtain the sample.

[0025] Step 2: SEM observation and counting; The dried sample was sputter-coated with gold using an MC1000 ion sputtering system and observed under a scanning electron microscope; the results were as follows: Figure 3 The results show that the entire droplet coverage area exhibits a uniform gray contrast, with no obvious annular accumulation; random selection Figure 1 and 2 The number of particles in each high-magnification field of view (3000x) shown was counted using ImageJ software, and the average value was taken.

[0026] Step 3: Concentration calculation; Measured area of ​​a single field of view: , Average number of particles in five fields of view: , Measurements using low-magnification SEM images show that the entire particle deposition zone is approximately elliptical, with an area of: , Calculate the total number of particles: indivual, Calculate the original particle number concentration: particles / mL The main sources of error in this method include: (1) The system error is lowered due to adsorption loss of particles during sample preparation, with an estimated loss rate of about 2%; (2) The relative standard deviation of a single count is approximately ±2% due to the particle identification threshold setting and edge particle judgment during SEM counting; (3) The volume error of the liquid transfer (calibration error of 2.5 μL liquid transfer operation is about ±1.5%) and the measurement error of the deposition area (about ±2%) introduced by the elliptical approximation jointly affect the concentration calculation; (4) The uneven distribution of particles in the sedimentation area is the main source of random error. The particle count in the five fields of view in this experiment was 34±3 (standard deviation), and its dispersion caused the average particle count to fluctuate by about ±8.8%. Taking all the above factors into account, the total relative uncertainty is calculated using the error propagation formula, the expression of which is: , Substitute the estimated value: , Therefore, the measured concentration was 0.95 × 10⁻⁶. 8 The practical range of particles / mL is approximately (0.86-1.04)×10⁻⁶. 8 particles / mL, compared to the nominal dilution concentration of 1.0 × 10⁻⁶ 8 The particles / mL were consistent within the error range, demonstrating the accuracy of the method provided by this invention.

[0027] The nanoparticle suspensions of known concentrations prepared using this method can be used as standard samples for particle counter calibration. Specifically, the standard sample with the concentration value measured by this method is imported into the particle counter to be calibrated (such as a nanoparticle tracking analyzer, an inductively coupled plasma particle counter, or a light scattering particle counter) for measurement. The instrument readings are recorded, and by comparing the instrument readings with the known concentration value of the standard sample, a correction factor can be calculated or a calibration curve can be plotted. In subsequent measurements of unknown samples, this correction factor is used to correct the instrument readings, effectively eliminating systematic errors and improving the accuracy and reliability of the measurement results. Compared to indirect calibration methods relying on other instruments, this method obtains concentration values ​​through direct microscopic counting and geometric proportional conversion, providing clear metrological traceability and making it particularly suitable for internal laboratory quality control and periodic calibration of particle counters.

[0028] Comparative Example 1 In this comparative example, except for the absence of polyvinyl alcohol and the change of the drying environment to room temperature (25°C, ~50% humidity), the remaining steps were exactly the same as in Example 1; after drying, SEM observation showed... Figure 4 and 5 As shown, the particles are highly concentrated at the edge of the droplet, forming a distinct coffee ring, while there are very few particles in the central area, making it impossible to count them effectively and calculate the concentration.

[0029] Comparative Example 2 In this comparative example, except that the final PVA concentration in the mixture is 0.4 wt%, the other steps are exactly the same as in Example 1.

[0030] The results are as follows Figure 6 As shown, the deposition area macroscopically presents a non-uniform film with significant macroscopic wrinkles and local ruptures, forming irregular polymer debris. The particles are encapsulated in the aforementioned non-uniform polymer matrix, and their distribution is completely dominated by the morphology of the film wrinkles and ruptures, resulting in an overall non-uniform distribution.

[0031] High concentrations of PVA lead to problems such as excessive thickness, wrinkles, and unevenness in the composite deposition film, making it impossible to perform effective counting and concentration calculation.

[0032] Comparative Example 3 In this comparative example, except that the final PVA concentration in the mixture is 0.01 wt%, the other steps are exactly the same as in Comparative Example 2.

[0033] The results are as follows Figure 7 As shown, the deposition pattern exhibits a distinct "coffee ring" characteristic, where particles form ring-shaped accumulations at the droplet edges; the results of high-magnification SEM are as follows. Figure 8As shown, further observation revealed that the particles were not uniformly distributed within the ring, but rather adhered only to discontinuous island-like or network-like PVA polymer residues on the substrate. In the substrate areas without PVA residues, almost no particles were observed. This phenomenon indicates that this concentration of PVA could neither form a continuous interfacial film to suppress the coffee ring effect caused by capillary flow, nor serve as a uniform template to guide particle deposition. Ultimately, the particle distribution depended on the distribution of discontinuous polymers, resulting in poor uniformity and making effective counting impossible.

[0034] Some steps in the embodiments of the present invention can be implemented using software, and the corresponding software program can be stored in a readable storage medium, such as an optical disc or a hard disk.

[0035] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for measuring the concentration of nanoparticles in liquid for calibrating a particle counter, characterized in that, The measurement method includes: Step S1: Prepare a mixed suspension sample and perform pretreatment; Step S2: Perform evaporation deposition on the sample prepared in step S1; Step S3: Observe the results after evaporation in step S2 under a microscope and count them; Step S4: Calculate the concentration of nanoparticles in the mixed suspension sample in step S1 based on the counting results in step S3.

2. The measurement method according to claim 1, characterized in that, The mixed suspension sample in step S1 is obtained by mixing polyvinyl alcohol solution, nanoparticle suspension and ultrapure water; The polyvinyl alcohol solution has a degree of hydrolysis of 98%-99% and a mass concentration of 1%. The nanoparticle suspension needs to be diluted, with the dilution factor depending on the concentration of the nanoparticle suspension. The dilution factor needs to ensure that the particle solids content in the mixed suspension sample is within a certain range. %arrive %between.

3. The measurement method according to claim 2, characterized in that, In step S2, the deposition process involves using a micropipette to aspirate 1 μL of a mixed suspension and drop it onto a cleaned silicon substrate for evaporation deposition. The evaporation is carried out statically at 4°C and relative humidity ≥70% for 1-12 hours until the solvent is completely evaporated.

4. The measurement method according to claim 3, characterized in that, In step S3, the sample after evaporation in step S2 is sputtered with gold, and the area with uniform particle dispersion is imaged at a magnification of 3000-5000 times. Several fields of view are randomly selected for counting.

5. The measurement method according to claim 4, characterized in that, In step S4, the number of nanoparticles in the entire deposition area is calculated by the volume of the mixed suspension sample added, the number of nanoparticles in the field of view, and the ratio of the area of ​​the counting field of view to the area of ​​the entire deposition area.

6. The measurement method according to claim 5, characterized in that, In step S4: The total number of particles within a single droplet of the mixed suspension sample is: ， Where, N 总 N is the total number of particles within a single droplet. 平均 It is the average number of particles counted across multiple high-magnification fields, S 视野 It is the field of view area of ​​a high-magnification field of view, S 总 It is the droplet area; The particle number concentration is: ， Among them, C 颗粒 It is the particle number concentration, V 滴加 It is the volume of the droplet added to the silicon wafer.

7. The measurement method according to claim 6, characterized in that, The pretreatment is to clean the silicon substrate. The cleaning process of the silicon substrate includes: first, ultrasonic cleaning with a 70% volume concentration isopropanol solution for 5 minutes, then ultrasonic cleaning with anhydrous ethanol for 5 minutes, and finally ultrasonic cleaning with ultrapure water for 5 minutes.

8. The measurement method according to claim 7, characterized in that, During the evaporation process: the surface-active free polyvinyl alcohol molecules of the mixed suspension adsorb onto the entire gas-liquid interface to form a polymer film, and the polyvinyl alcohol-modified nanoparticles of the mixed suspension are embedded into the polymer film to form a particle-polymer composite interface structure; the particle-polymer composite interface structure spreads on the substrate to form a deposition layer after the solvent has completely evaporated.

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