Single-particle micro-nano particle measurement device and use thereof

Abstract

The present application relates to a device for measuring micro-nano particles, and the related use thereof. The device comprises: a first chamber having a first electrode; a second chamber adjacent to the first chamber and having a second electrode; a substrate that separates the first chamber from the second chamber; a membrane covering the side of the substrate close to the first chamber, wherein the membrane has a micropore; the substrate has an electro-osmotic cell with an inner diameter larger than the pore diameter of the micropore and smaller than the inner diameter of the second chamber; the first chamber is in communication with the second chamber by means of the micropore and the electro-osmotic cell; inner walls of the electro-osmotic cell are provided with electro-osmotic cell modification layers, which have a specific Zeta potential at an interface when coming into contact with an electrolyte.

Description

A single-particle micro-nano particle detection device and its application Technical Field

[0001] The present application relates to the technical field of micro-nano particle detection, and specifically to a micro-nano particle detection device, a detection method, a nanopore chip, a method for preparing a nanopore chip, and related applications thereof. Background Art

[0002] Micro- and nanoparticles (particle sizes ranging from 1 nm to 10 μm, hereinafter referred to as micro- and nanoparticles) exhibit many unique physical behaviors due to size-dependent effects such as small size effects, surface effects, and quantum size effects. As particle size decreases, the surface area-to-volume ratio increases, allowing micro- and nanoparticles to exhibit unexpected properties and find widespread application in medicine, chemicals, materials, and other fields. Therefore, single-particle detection techniques that measure properties such as size, size distribution, particle concentration (also referred to as concentration or particle concentration value), and zeta potential are crucial in the application of micro- and nanoparticles.

[0003] Traditional single-particle detection technologies include electron microscopy, nanotracer technology (NTA), nanofluidic cytometry (NanoFCM), and resistive pulse sensing (RPS). Electron microscopy can visually measure the particle size of nanoparticles, but requires dry sample preparation before measurement and cannot display the sample's state in liquid. The basic principle of nanotracer technology (NTA) is to track the Brownian motion of nanoparticles in liquid through imaging, and infer the particle size based on the speed of Brownian motion. Nanotracer technology can be affected by the sample's hydrophilicity, light scattering intensity, and image acquisition accuracy, resulting in inaccurate particle size measurements. The basic principle of nanofluidic cytometry (NanoFCM) is to pass sample particles individually through a laser in a sheath flow pattern and infer the particle size by detecting the laser scattering signal. Nanofluidic cytometry is also affected by differences in the optical properties of the particles, resulting in inaccurate tests.

[0004] Nano-Coulter technology is a type of resistive pulse sensing (RPS). Its principle is to allow particles suspended in the electrolyte to pass through the small holes with the electrolyte, replacing the same volume of electrolyte. In the constant current design circuit, it causes the resistance between the two electrodes inside and outside the small hole tube to change instantaneously, generating a potential pulse. The size and number of pulse signals are proportional to the size and number of particles.

[0005] Patent CN211122429U describes a device for detecting nanoparticles using nano-Coulter technology, but the patent does not mention the source of the driving force for the nanoparticles to pass through the nanopores. At the same time, the patent relies on single-particle electrophoresis in an electrophoresis tank to test the potential, and needs to be tested under low sample concentrations (to prevent two particles from being in the electrophoresis tank at the same time), so the scope of application is small.

[0006] Patent CN105705934B describes a method for measuring the charge of micro- and nanoparticles using the nano-Coulter technique. The basic principle is to calculate the charge by determining the value of the electric velocity component, which represents the total velocity of the test particle, considering that the total velocity of the test particle includes a non-zero convective velocity component and an electric velocity component. Because the measured total velocity includes a non-zero convective velocity component, calculating the charge of the test particle is relatively complex.

[0007] Summary of the Invention

[0008] The present invention conducts an in-depth study on the driving force problem of the existing RPS detection technology and improves the device for detecting micro-nanoparticles so that it can drive the particles to be tested through the micropores to complete the detection of micro-nanoparticle information without the need for other external driving forces, simply by utilizing the potential difference between the two electrodes.

[0009] In the first aspect, the micro-nano particle detection device provided by the present application includes: a first chamber having a first electrode; a second chamber adjacent to the first chamber and having a second electrode; a substrate separating the first chamber and the second chamber; a membrane covering one side of the first chamber of the substrate (the membrane may also be referred to as a microporous membrane). Wherein: the membrane has micropores (also referred to as nanopores), the substrate has an electroosmotic groove, the inner diameter of the electroosmotic groove is larger than the pore diameter of the micropores and smaller than the inner diameter of the second chamber, the first chamber and the second chamber are connected through the micropores and the electroosmotic groove, and the inner wall of the electroosmotic groove has an electroosmotic groove modification layer, and the interface of the electroosmotic groove modification layer has a specific Zeta potential when in contact with the electrolyte. In a specific embodiment of the present application, the Zeta potential is a negative potential.

[0010] In a second aspect, the present application provides a method for measuring micro-nanoparticles, comprising: placing an electrolyte in the device disclosed in the first aspect of the present application; placing the micro-nanoparticles to be measured in the first chamber or the second chamber; applying voltage / current between the first electrode and the second electrode; testing the changes in the current / voltage signal flowing through the electrodes; and determining at least one of the particle size, concentration, and potential information of the micro-nanoparticles to be measured based on the changes in the current / voltage signal.

[0011] In a third aspect, the present application provides a nanopore chip suitable for the device in the first aspect and a preparation method thereof. The nanopore chip includes: a substrate (including an electroosmotic cell), a membrane (including micropores) and an electroosmotic cell modification layer. The substrate has a first surface and a second surface relative to each other, and has a cavity that passes from the second surface to the first surface, namely the electroosmotic cell; the membrane (also referred to as a microporous membrane) is formed on the first surface, and the membrane has at least one micropore (also referred to as a nanopore), the pore size of the micropore is smaller than the pore size of the electroosmotic cell, and the projections of all micropores on the first surface are covered by the projections of the electroosmotic cell on the first surface; the electroosmotic cell modification layer is formed on the side wall of the electroosmotic cell, and the interface of the electroosmotic cell modification layer has a specific Zeta potential when in contact with the electrolyte.

[0012] It should be understood that the above-mentioned nanopore chip can be configured to be placed vertically at the bottom of the measuring container and immersed in the electrolyte, which can divide the measuring container into a first chamber and a second chamber (corresponding to the first surface and the second surface of the nanopore chip); one of the first chamber and the second chamber is used to place the particles to be measured; the first electrode and the second electrode are respectively arranged on both sides of the first surface of the nanopore chip, and the first electrode and the second electrode are configured to input a preset voltage difference to form an electric field in the electrolyte.

[0013] In a specific embodiment of the present invention, the electroosmotic tank modification layer of the above-mentioned nanopore chip is configured so that the zeta potential of the interface immersed in the electrolyte is negative (therefore, the electroosmotic tank modification layer can also be called a negative potential membrane layer), so that the electrolyte in the electroosmotic tank has a positive potential. The electrolyte moves under the action of the electric field and drives the particles to be tested to flow from one chamber through the micropores to another chamber.

[0014] In a specific embodiment of the present invention, the preparation method of the above-mentioned nanopore chip includes: providing a substrate; forming a membrane on the first surface of the substrate, the membrane having at least one micropore; forming a cavity, namely an electroosmotic tank, which passes through the first surface on the second surface of the substrate, and forming an electroosmotic tank modification layer on the side wall of the electroosmotic tank; the electroosmotic tank modification layer is configured to have a negative zeta potential at the interface when immersed in an electrolyte; wherein the pore size of the micropore is smaller than the pore size of the electroosmotic tank, and the projections of all the micropores on the first surface are covered by the projections of the electroosmotic tank on the first surface.

[0015] In a fourth aspect, based on the device of the first aspect and the detection method of the second aspect, the present application provides a method for analyzing low-density lipoprotein cholesterol (LDL-C), the method comprising:

[0016] 1) Specific capture and separation of LDL-C in samples;

[0017] 2) Recording and measuring the current or voltage pulse signals generated on the electrodes when the LDL-C obtained in step 1) passes through the micropores of the device of the first aspect, and analyzing the particle size distribution of each LDL-C particle in the sample and the particle concentration within each particle size range based on the pulse signals. The micropores in the device have a pore diameter of no greater than 200 nm and a pore depth of no greater than 150 nm.

[0018] In one embodiment of the present application, magnetic beads conjugated with antibodies or aptamers that specifically bind to the surface membrane protein of the LDL-C to be detected are used to specifically capture LDL-C in the sample. After the magnetic beads are collected, they are eluted with a dissociation solution to separate the captured LDL-C from the magnetic beads.

[0019] Preferably, the magnetic beads are coated with antibodies against the ApoB100 protein.

[0020] Preferably, the dissociation solution contains trypsin, salicylic acid, glycine or citric acid.

[0021] In a fifth aspect, based on the apparatus of the first aspect and the detection method of the second aspect, the present application provides a method for tracing the particle concentration value of small-sized nanoparticles. The small-sized nanoparticles are nanoparticles with a particle size greater than or equal to 20 nm and less than or equal to 400 nm. The method includes designing several intermediate samples with unknown particle concentration values ​​and particle sizes between a particle sample with a known particle concentration value and a small-diameter nanoparticle sample to be tested, sorting the particle sample, a first intermediate sample, a second intermediate sample...the Nth intermediate sample, and the small-diameter nanoparticle sample to be tested in descending order of particle size, where N is a positive integer greater than or equal to 1; at the same time, the intermediate samples are designed so that the particle sizes of two adjacent samples after sorting meet the condition that they can be tested within the detection range of the same device provided in the first aspect; testing the two adjacent samples in descending order of particle size in the same device, and calculating the particle concentration value of the sample with an unknown particle concentration value in the adjacent samples based on the detection parameter values ​​of the adjacent samples combined with the particle concentration value of the sample with a known particle concentration value in the adjacent samples; and ultimately obtaining the particle concentration value of the small-diameter nanoparticle sample to be tested by analogy; wherein the particle concentration value calculation formula of the sample with an unknown particle concentration value in the adjacent samples is:

[0022] Among them, C 待测 is the particle concentration value of the sample with unknown particle concentration value in the adjacent sample, C 已知 is the particle concentration value of the sample with known particle concentration value among the adjacent samples, P 待测 P is the voltage / current pulse frequency of the unknown particle concentration value sample in the detection parameter value. 已知 The voltage / current pulse frequency of the sample with known particle concentration value in the detection parameter value;

[0023] The above conditions include:

[0024] 1) Among two adjacent samples, the particle size of the sample with the larger particle size is less than or equal to 60% of the pore size of the device;

[0025] 2) Among two adjacent samples, the baseline amplitude of the pulse signal of the sample with larger particle size is smaller than the pulse signal height of the sample with smaller particle size;

[0026] 3) The depth of the micropores of the above device is less than or equal to the pore diameter of the micropores and greater than or equal to one tenth of the pore diameter.

[0027] In the sixth aspect, the present application provides a method for calculating the Zeta potential of particles, by obtaining detection electric pulse signals of several particle samples with known particle size and Zeta potential and recording the pulse peak value and pulse width of the electric pulse signal, the detection electric pulse signal is obtained by the micropore measurement of the particles in the sample under the action of the electric field force through the device provided in the first aspect of the present invention; a particle Zeta potential calculation model is constructed based on the particle size of the particle and the pulse peak value and pulse width of its electric pulse signal.

[0028] In one embodiment of the present invention, constructing a particle Zeta potential calculation model based on the particle size and the pulse peak value and pulse width of the electric pulse signal of the particle includes: constructing a training sample set based on the particle size and the pulse peak value and pulse width of the electric pulse signal of the particle in combination with the Zeta potential of the particle; performing several types of model training based on the training sample set, and evaluating the several types of models after training, and confirming the optimal particle Zeta potential calculation model according to the model evaluation index.

[0029] In a specific embodiment of the present application, the method for calculating the zeta potential of the particles includes: 1) obtaining the detection electric pulse signals of a number of known particle samples with different particle sizes and the same zeta potential through the device disclosed in the first aspect of the present application and recording the pulse peak value and pulse width of the electric pulse signal, and obtaining a first relationship based on the particle size and the pulse peak value of the electric pulse signal, the first relationship being: A = a × D 3, where A is the pulse peak value of the particle, D is the particle size, and a is the particle size coefficient; based on the pulse peak value and pulse width of the particle's electric pulse signal, a second relationship is obtained, which is: W=b×A+Wn, where A is the pulse peak value of the particle, W is the particle's pulse width, b is the pulse coefficient, and Wn is the pulse width correction coefficient. 2) Using the device disclosed in the first aspect of the present application (the same as the device in step 1 above), the detection electric pulse signals of several known particle samples with the same particle size and different Zeta potential are obtained and the pulse width of the electric pulse signal is recorded. Based on the Zeta potential of the particle and the pulse width of its electric pulse signal, combined with the first and second relationship, a fourth relationship between the Zeta potential of the particle and the pulse peak value and pulse width of its electric pulse signal is obtained, which is: Z=d×(Wb×A) e , where Z is the absolute value of the particle Zeta potential, W is the particle pulse width, A is the particle pulse peak, b is the pulse coefficient, d is the potential coefficient, and e is the potential index.

[0030] Furthermore, by obtaining the particle Zeta potential calculation model based on the above-mentioned device, the Zeta potential of the particles to be measured can be calculated. In one embodiment of the present application, the micro-nano particle detection device provided in the first aspect of the present application is used to obtain the detection electric pulse signal of the particles to be measured when they pass through the detection area under the action of the electric field force; the pulse peak value and pulse width of the obtained electric pulse signal are input into the particle Zeta potential calculation model calculated by the calculation method above, and the Zeta potential of the particles to be measured can be obtained. In some specific embodiments, the particle Zeta potential calculation model is the fourth relationship mentioned above.

[0031] At the same time, based on the above-mentioned particle Zeta potential calculation model, the present application also provides a device for calculating the Zeta potential of the particle to be measured, which includes: a signal acquisition module and a signal processing module. The signal acquisition module is used to obtain the detection electric pulse signal when the particle to be measured passes through the detection area of ​​the micro-nano particle detection device provided in the first aspect under the action of the electric field force, including the pulse peak value and pulse width of the electric pulse signal. The signal processing module is used to input the obtained pulse peak value and pulse width into the particle Zeta potential calculation model obtained by the above-mentioned calculation method to obtain the Zeta potential of the particle to be measured.

[0032] In a seventh aspect, the present application provides a device for measuring micro-nanoparticles, comprising:

[0033] A sample chamber having a common electrode; at least two measurement chambers adjacent to the sample chamber, including a first measurement chamber having a first measurement electrode and a second measurement chamber having a second measurement electrode; substrates separating the sample chamber and the at least two measurement chambers, such as a first substrate and a second substrate; and membranes covering one side of the substrates, wherein the membrane on one side of the first substrate is a first membrane and the membrane on one side of the second substrate is a second membrane. The membranes have at least a first micropore and a second micropore, specifically, the first membrane has the first membrane on the first membrane and the second membrane has the second membrane on the second membrane. The substrates have a first electroosmotic cell and a second electroosmotic cell, specifically, the first substrate has the first electroosmotic cell and the second substrate has the second electroosmotic cell. The inner diameter of the first electroosmotic cell is larger than the aperture of the first micropore but smaller than the inner diameter of the first measurement chamber, the inner diameter of the second electroosmotic cell is larger than the aperture of the second micropore but smaller than the inner diameter of the second measurement chamber, the sample chamber is connected to the first measurement chamber through the first micropore and the first electroosmotic cell, and at the same time, the sample chamber is connected to the second measurement chamber through the second micropore and the second electroosmotic cell, the inner wall of the first electroosmotic cell has a first electroosmotic cell modification layer, and the interface of the first electroosmotic cell modification layer has a specific first Zeta potential when in contact with the electrolyte, and the inner wall of the second electroosmotic cell has a second electroosmotic cell modification layer, and the interface of the second electroosmotic cell modification layer has a specific second Zeta potential when in contact with the electrolyte.

[0034] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the application to be protected by the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG1 shows a schematic structural diagram of a micro-nano particle measuring device disclosed in the present application;

[0036] FIG2 is a schematic structural diagram of a nanopore chip in one embodiment of the present application;

[0037] FIG3 is a schematic structural diagram of a device assembled from nanopore chips in one embodiment of the present application;

[0038] FIG4 is a schematic diagram of a process of a manufacturing method provided in one embodiment of the present application;

[0039] FIG5 is a process diagram of a manufacturing method provided by an embodiment of the present application (I);

[0040] FIG6 is a schematic diagram of a manufacturing method according to an embodiment of the present application (II);

[0041] FIG7 is a schematic diagram of a manufacturing method according to an embodiment of the present application (III);

[0042] FIG8 is a process diagram (IV) of a manufacturing method provided by an embodiment of the present application;

[0043] FIG9 is a process diagram (V) of a manufacturing method provided by an embodiment of the present application;

[0044] FIG10 is a schematic diagram of a manufacturing method according to an embodiment of the present application (VI);

[0045] FIG11 is a schematic diagram of a pulse signal of a particle to be detected provided in one embodiment of the present application (I);

[0046] FIG12 is a schematic diagram of a pulse signal of a standard particle provided in one embodiment of the present application (I);

[0047] FIG13 is a schematic diagram of the micro-nano particle measuring device used in Example 3;

[0048] FIG14A shows that the width of the pulse signal of the silica microspheres to be tested in Example 3 is 0.2 mS;

[0049] FIG14B shows the 150 nm standard microsphere sample (average particle size 150 nm, concentration 3×10 9 The width of the pulse signal (particles / mL, average potential -30mV) is 0.4mS;

[0050] FIG15A shows the pulse signal of the 0.5 mm inner diameter electroosmotic cell detected in Example 4;

[0051] FIG15B shows the pulse signal of the 1 mm inner diameter electroosmotic cell detected in Example 4;

[0052] FIG15C shows the pulse signal of the 2 mm inner diameter electroosmotic cell detected in Example 4;

[0053] FIG15D shows the pulse signal of the 3 mm inner diameter electroosmotic cell detected in Example 4;

[0054] FIG15E shows the pulse signal of the 4 mm inner diameter electroosmotic cell detected in Example 4;

[0055] FIG16A shows the pulse signal of the 0.025 mm depth electroosmotic cell detected in Example 5;

[0056] FIG16B shows the pulse signal of the 0.1 mm depth electroosmotic cell detected in Example 5;

[0057] FIG16C shows the pulse signal of the 0.4 mm deep electroosmotic cell detected in Example 5;

[0058] FIG17 is a schematic diagram of the 200 nm nanopore measurement device in Example 6;

[0059] FIG18 is a bar graph showing LDL-C particle size and concentration in Example 6;

[0060] Figure 19 is a schematic diagram of the 150 nm nanopore measurement device in Example 7;

[0061] FIG20 is a bar graph showing LDL-C particle size and concentration in Example 7;

[0062] FIG21 shows the pulse width and peak value of raw data collected from via particles in a specific embodiment of the present application;

[0063] FIG22 is a linear fitting result diagram of W and A in Example 10 of the present invention;

[0064] FIG. 23 shows A and D in Example 10 of the present invention. 3 Linear fitting result graph of ;

[0065] FIG. 24 is a diagram showing the linear fitting results of the absolute value of the potential Z and Wn in Example 10 of the present invention.

[0066] Figure numerals: 1-substrate; 2-membrane (microporous membrane); 3-electroosmotic cell modification layer (negative potential membrane layer); 4-transition layer; 5-protective layer; 101-electroosmotic cell; 201-micropore; 301-passivation layer; 100-nanopore chip; 200-measuring container; 310-first electrode; 320-second electrode. DETAILED DESCRIPTION

[0067] The specific embodiments of the present application will be described in detail below. Although the present application will disclose various aspects and embodiments below, it is obvious that various equivalent changes and modifications can be made to it by those skilled in the art without violating the spirit and scope of the subject matter of the present application. The various aspects and embodiments disclosed in the present application are only for illustration and are not intended to limit the present application. The actual scope of protection of the present application is subject to the claims. Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present application belongs. All references, patents, and patent applications cited in this application are incorporated herein by reference in their entirety.

[0068] 1. Structure of the micro-nano particle detection device

[0069] The present application provides a device for detecting nanoparticles using Nanocoulter technology. Nanocoulter technology is based on the Nanocoulter principle. During the test, the particles to be tested are dispersed in an electrolyte. As they pass through a nanopore chip with a specific pore size, the particles occupy the same volume of electrolyte at the moment they pass through the micropore (also known as a nanopore). The resistance between the two electrodes inside and outside the pore (a circuit designed with a constant current) changes instantaneously, generating a potential pulse. The intensity and number of pulse signals are proportional to the size and number of particles passing through the nanopore. By directly converting the pulse signal, the particle size and particle count information of the particles in the detected sample can be obtained.

[0070] Based on the above principles, the present application provides a new micro-nano particle detection device, which can achieve detection by driving micro-nano particles through micropores by using only the potential difference between two electrodes without the need for other external driving forces through an electroosmotic tank modification layer provided on the electroosmotic tank. As shown in Figure 1, Figure 1 is a structural cross-sectional view of a device for measuring micro-nano particles according to an exemplary embodiment. The detection device includes a first chamber having a first electrode and a second chamber adjacent to the first chamber and having a second electrode. The first chamber and the second chamber are separated by a substrate. The substrate is covered with a membrane on one side of the first chamber. The membrane has micropores. The substrate has an electroosmotic tank, the inner diameter of which is larger than the pore diameter of the micropores and smaller than the inner diameter of the second chamber. The first chamber and the second chamber are connected through the micropores and the electroosmotic tank. The inner wall of the electroosmotic tank has an electroosmotic tank modification layer.

[0071] It should be noted that the substrate (including the electroosmotic cell), membrane (including micropores) and electroosmotic cell modification layer in the above device can together constitute a nanopore chip, the structure of which is shown in FIG2 .

[0072] 1. Substrate, membrane and electroosmotic tank modification layer (nanopore chip) of micro-nano particle detection device

[0073] The following describes in detail the structure and preparation methods of each part of the nanopore chip. It should be understood that as part of the device, the description of the nanopore chip and the related preparation methods below are also applicable to the micro-nano particle detection device in this application.

[0074] In some embodiments, the structure of the nanopore chip is shown in (A) of FIG2 , and the nanopore chip 100 may include: a substrate 1, a membrane 2, and an electroosmotic tank modification layer 3. The substrate 1 has a first surface and a second surface relative to each other, and the substrate 1 has a cavity, namely, an electroosmotic tank 101, which passes through from the second surface to the first surface. The membrane 2 is formed on the first surface, and the membrane 2 has at least one micropore 201. The pore size of the micropore 201 is smaller than the pore size of the electroosmotic tank 101. The micropore 201 is connected to the electroosmotic tank 101, and the projections of all micropores 201 on the first surface are covered by the projections of the electroosmotic tank 101 on the first surface. The electroosmotic tank modification layer 3 is formed on the side wall of the electroosmotic tank 101, and the interface of the electroosmotic tank modification layer 3 has a specific Zeta potential when in contact with the electrolyte. In a specific embodiment of the present application, the electroosmotic tank modification layer 3 is configured so that the zeta potential of the interface when immersed in the electrolyte is negative.

[0075] 1.1 Substrate and electroosmotic cell

[0076] It should be understood that the substrate of the nanopore chip has a structure that can isolate the first chamber and the second chamber and is made of a material on which a film covering the substrate can be grown. In some embodiments, the material forming the substrate is silicon (silicon substrate). In some embodiments, the material forming the substrate is boron nitride. In some embodiments, the thickness of the substrate is 25μm to 1mm, for example, 25μm, 50μm, 75μm, 100μm, 150μm, 200μm, 250μm, 300μm, 350μm, 400μm, 450μm, 500μm, 550μm, 600μm, 650μm, 700μm, 750μm, 800μm, 850μm, 900μm, 950μm, 1000μm or a value or range between any two of the above values. The thickness of the substrate described here refers to the distance in the direction in which the micro-nanoparticles move through the electroosmotic tank during the measurement process.

[0077] It should be understood that, depending on the function of the electroosmotic cell, the inner diameter and depth of the cell should be set to produce an electroosmotic effect on the electrolyte in the cell. In some embodiments, the shape of the electroosmotic cell is selected from the group consisting of a cylinder, a cuboid, a conical platform, and a trapezoidal platform.

[0078] In certain embodiments, the inner diameter of the electroosmotic cell is less than or equal to 3 mm, preferably 25 μm to 3 mm, for example, 25 μm, 50 μm, 75 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 1000 μm, 1500 μm, 2000 μm, 2500 μm, 3000 μm, or a value or range between any two of the above values. The inner diameter of the electroosmotic cell described herein refers to the distance of the electroosmotic cell in a direction perpendicular to the direction in which the micro-nanoparticles move through the electroosmotic cell during the measurement process. When the shape of the electroosmotic cell is not parallel to the direction in which the micro-nano particles move through the device during the measurement process, for example, when the shape of the electroosmotic cell is a conical table or a trapezoidal table, the inner diameter of the electroosmotic cell refers to the maximum distance of the electroosmotic cell in a direction perpendicular to the direction in which the micro-nano particles move through the electroosmotic cell during the measurement process.

[0079] 1.2 Membrane and micropores

[0080] In some embodiments, the film covering the substrate is an insulating layer formed of polysilicon, silicon nitride, silicon oxynitride, or silicon oxide.

[0081] It should be understood that, depending on the function of the micropore, the pore diameter and pore depth must be set based on the ability to detect the micro-nanoparticles to be measured using the Nano-Coulter Principle. In certain embodiments, the pore diameter of the micropore is 20 nm to 5 μm. In certain embodiments, the pore diameter of the nanopore is 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, or a value or range between any two of the above values. The pore diameter of the micropore described herein refers to the distance perpendicular to the direction in which the micro-nanoparticles move through the micropore during measurement. In certain embodiments, the micropore is circular in shape. When the micropore is not circular in shape, the pore diameter of the micropore refers to the maximum distance perpendicular to the direction in which the micro-nanoparticles move through the micropore during measurement. In some embodiments, the pore depth of the micropore is 20nm to 5μm. In some embodiments, the pore depth of the micropore is 20nm, 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 200nm, 300nm, 400nm, 500nm, 600nm, 700nm, 800nm, 900nm, 1μm, 2μm, 3μm, 4μm, 5μm or a numerical value or range between any two of the above values. In some embodiments, the pore depth of the micropore is determined by the thickness of the film covering the substrate. The pore depth of the micropore or the thickness of the film described here refers to the distance in the direction of the movement of the micro-nano particles through the micropore during the measurement process.

[0082] 1.3 Electroosmotic cell modification layer

[0083] 1.3.1 In certain embodiments, the electroosmotic cell modification layer of the device has an interface zeta potential more negative than -20 mV when in contact with the electrolyte. In certain specific embodiments, the material of the electroosmotic cell modification layer of the device is selected from the group consisting of amorphous carbon, carbon chain polymers, silicon oxide, silicon nitride, polycrystalline silicon, single crystal silicon, and adsorption layers formed by adsorption of anions thereon.

[0084] In certain embodiments, the zeta potential of the interface of the electroosmotic cell modification layer of the device when in contact with the electrolyte is -65±6 mV.

[0085] 1.3.2 In certain embodiments, the electroosmotic cell modification layer of the device has an interface zeta potential of no greater than 10 mV when in contact with the electrolyte. In certain specific embodiments, the material of the electroosmotic cell modification layer is selected from the group consisting of titanium oxide, aluminum oxide, and magnesium oxide. In certain embodiments, the electroosmotic cell modification layer of the device has an interface zeta potential of 0±3 mV when in contact with the electrolyte.

[0086] 1.3.3 In certain embodiments, the electroosmotic cell modification layer may also cover the interface between the membrane and the electroosmotic cell and the inner wall of the micropores.

[0087] 1.4 Transition layer and protective layer

[0088] In some embodiments, as shown in (B) and (D) in Figure 2, the nanopore chip 100 may further include a transition layer 4, which is formed between the membrane 2 and the substrate 1. The transition layer 4 has a hollow structure or through hole connecting the micropores 201 and the electroosmotic groove 101. The transition layer 4 ensures that the micropores 201 are connected to the electroosmotic groove 101. The transition layer 4 can be etched under the same mask as the electroosmotic groove 101. The transition layer 4 located on the second surface can serve as a hard mask when etching the electroosmotic groove 101.

[0089] In some embodiments, as shown in Figures 2(B) to (D), the nanopore chip 100 may further include a protective layer 5. The protective layer 5 is formed on the second surface and has a hollow structure or through-holes that connect to the electroosmotic cell 101. A transition layer 4 may also be formed between the protective layer 5 and the substrate 1. The protective layer 5 on the second surface may also serve as a hard mask when etching the electroosmotic cell 101.

[0090] That is, when etching to form the micropores 201 or the electroosmotic grooves 101 , the transition layer 4 or the protective layer 5 can serve as a hard mask, having a hollow structure or through holes corresponding to the upper and lower ports of the electroosmotic grooves 101 .

[0091] It should be understood that the transition layer and protective layer in the above-mentioned nanopore chip are applicable to electroosmotic cell modification layers of various materials. At the same time, the setting of the transition layer and protective layer is also applicable to the micro-nano particle detection device of the present application.

[0092] 1.5 Depth of electroosmotic cell and depth of micropores

[0093] It is understood that in some embodiments, the depth of the electroosmotic cell is determined by the thickness of the substrate. In some embodiments, the depth of the electroosmotic cell is 5 times or more the pore depth of the micropore. The examples of the present application show that when the depth of the electroosmotic cell is less than 5 times the pore depth, for example 4 times or 3 times, the measured pulse frequency is very low. In some embodiments, the depth of the electroosmotic cell is 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more the pore depth of the micropore.

[0094] 1.6 Preparation method

[0095] The following preparation method is described using the preparation of a nanopore chip as an example. The following preparation method is also applicable to micro-nano particle detection devices.

[0096] The preparation method of a nanopore chip includes: providing a substrate; forming a membrane on a first surface of the substrate, the membrane having at least one micropore; forming a cavity, namely an electroosmotic cell, which passes through to the first surface on the second surface of the substrate, and forming an electroosmotic cell modification layer on the side wall of the electroosmotic cell; the electroosmotic cell modification layer is configured so that the zeta potential of the interface when immersed in an electrolyte is negative; wherein the pore size of the micropore is smaller than the pore size of the electroosmotic cell, and the projections of all the micropores on the first surface are covered by the projections of the electroosmotic cell on the first surface.

[0097] The method for forming the electroosmotic cell modification layer may be adsorption, surface chemical modification, self-assembly, and the like.

[0098] In certain embodiments, the electroosmotic tank modification layer is a silicon oxide, silicon nitride, polysilicon or monocrystalline silicon layer. After the electroosmotic tank modification layer of the above-mentioned material is formed on the inner wall of the electroosmotic tank of the substrate during preparation, it is immersed in an aqueous solution containing anions to adsorb anions. The above-mentioned aqueous solution with anions can be an alkaline salt solution, a phosphate solution or a HEPES solution, for example, a NaCl aqueous solution, a NaOH aqueous solution, a PBS aqueous solution, a sodium phosphate aqueous solution, or a solution of sodium perchlorate. For example, a silicon nitride layer is plated on the inner wall of the electroosmotic tank, and then the silicon nitride layer is immersed in a sodium perchlorate solution, and after adsorbing anions, it can be used as the electroosmotic tank modification layer.

[0099] In some embodiments, the electroosmotic tank modification layer is titanium oxide, aluminum oxide, or magnesium oxide. For example, a silicon dioxide layer is plated on the inner wall of the electroosmotic tank as the electroosmotic tank modification layer.

[0100] It should be noted that, as shown in FIG2 , in some embodiments, the electroosmotic groove modification layer can be an amorphous carbon layer or a carbon chain polymer layer. The carbon layer or carbon chain polymer layer can be formed by deposition after ionization of hydrocarbons or fluorocarbons in plasma. Hydrocarbons or fluorocarbons can form carbon ions after ionization, and carbon ions can form amorphous carbon layer carbon chain polymers. For example, plasma fluorinated carbon (C4F8) can be used to passivate the side walls of the electroosmotic groove 101. C4F8 decomposes into ionic CF+x groups, CF-x groups and active F- groups under plasma conditions, wherein the CF+x groups and CF-x groups react with the silicon surface to form a (CF2)n polymer passivation film. The (CF2)n long chain is a fluorinated carbon polymer, which is a type of carbon chain polymer. For another example, it is formed by thermal decomposition or plasma deposition of hydrocarbon gas (such as methane, ethylene, etc.), and the carbon atoms or carbon ions ionized from the hydrocarbon gas in the plasma are then deposited on the surface of the substrate 1 to form an amorphous carbon layer.

[0101] Furthermore, in certain embodiments, when the electroosmotic channel modification layer is an amorphous carbon layer or a carbon chain polymer layer, the electroosmotic channel modification layer is formed after the electroosmotic channel. In its preparation method, the substrate is first patterned using wet etching to form an electroosmotic channel on the second surface of the substrate that extends through to the first surface. The sidewalls of the electroosmotic channel are then passivated using a hydrocarbon or fluorocarbon compound in plasma to form an amorphous carbon layer or a carbon chain polymer layer as the electroosmotic channel modification layer.

[0102] Furthermore, in certain embodiments, when the electroosmotic groove modification layer is an amorphous carbon layer or a carbon chain polymer layer, the electroosmotic groove modification layer is formed during the formation of the electroosmotic groove. The preparation method includes: an etching step, wherein the substrate is patterned using dry etching to form a groove body of a first depth on the second surface of the substrate; the first depth is less than the thickness of the substrate; a passivation step, wherein the sidewalls of the groove body are passivated using a hydrocarbon or fluorocarbon compound in a plasma to form an amorphous carbon layer or a carbon chain polymer layer as the electroosmotic groove modification layer; and the etching step and the passivation step are repeated until the depth of the groove body is equal to the thickness of the substrate, thereby forming an electroosmotic groove penetrating to the first surface and forming the electroosmotic groove modification layer on the sidewalls of the electroosmotic groove.

[0103] In certain specific embodiments, the preferred fluorocarbon is octafluorocyclobutane.

[0104] In certain embodiments, the nanopore chip is immersed in an alkaline salt solution, a phosphate solution, or a HEPES solution.

[0105] 2. Principles of Micro-Nano Particle Detection Device

[0106] The basic structure of the micro-nanoparticle detection device in this application is shown in Figure 1. In certain embodiments, the device further includes an electrolyte placed in the first chamber and the second chamber. Suitable electrolytes for this application include alkaline salt solutions, phosphate solutions, or HEPES solutions, such as aqueous NaCl solutions, aqueous NaOH solutions, PBS solutions, aqueous sodium phosphate solutions, and sodium perchlorate solutions.

[0107] In some embodiments, the device can be assembled using a nanopore chip. For example, as shown in FIG3 , an exemplary micro-nano particle detection device of the present application may include a measurement container 200, a first electrode 310, a second electrode 320, and the nanopore chip 100 described above. The nanopore chip 100 is configured to be placed vertically at the bottom of the measurement container 200 and immersed in an electrolyte, and the measurement container 200 is divided into a first chamber and a second chamber; a particle to be detected is placed in one of the first and second chambers. The measurement container 200 contains an electrolyte, which is a PBS solution.

[0108] At this time, in the measuring state, the first chamber, the electroosmotic tank and the second chamber are filled with electrolyte, the electrolyte in the first chamber or the second chamber contains micro-nanoparticles to be measured, and the micro-nanoparticles can pass through the electroosmotic tank and the micropores.

[0109] 2.1 In certain embodiments, the material of the electroosmotic tank modification layer is selected from the following group: amorphous carbon, carbon chain polymer, silicon oxide, silicon nitride, polycrystalline silicon, single crystal silicon, and adsorption layers formed by adsorption of anions thereof. In these embodiments, the electroosmotic tank modification layer has an interface zeta potential more negative than -20mV when in contact with the electrolyte. In certain embodiments, the zeta potential value of the interface of the electroosmotic tank modification layer when in contact with the electrolyte is -65±6mV. When the device is not in use, the electroosmotic tank modification layer is stored in a phosphate aqueous solution to keep the zeta potential value of the interface stable. For example, it can be stored in a PBS aqueous solution, a sodium phosphate aqueous solution, or other aqueous solutions containing phosphate anions. When the device is in use, the first chamber and the second chamber separated by the substrate, as well as the electroosmotic tank and the micropores are filled with electrolyte. At this time, the electroosmotic tank modification layer is negatively charged in the electrolyte, and due to the principle of electrical neutrality, the electrolyte in the electroosmotic tank is positively charged. When a voltage is applied to the first and second electrodes, an electric field is formed in the electrolyte. Under the influence of the electric field, the positively charged electrolyte in the electroosmotic cell moves toward the first or second electrode, thereby driving the liquid in the second or first chamber to replenish the electroosmotic cell, and in turn driving the micro-nanoparticles in the chamber through the micropores. Therefore, even without any other driving force between the first and second chambers, this mechanism can enable micro-nanoparticles in the chamber to pass through the micropores.

[0110] At this time, the amplitude of the pulse signal generated when the micro-nanoparticles pass through the micropores is proportional to the volume of the particles and proportional to the cube of the particle size. At the same time, the frequency of the pulse signal detected by the device is proportional to the concentration of the micro-nanoparticles. Utilizing this mechanism, the size and volume of the micro-nanoparticles to be tested can be confirmed in subsequent detection methods by testing standard microspheres (also known as microparticle standard substances) with determined particle size and concentration in the same device as the micro-nanoparticles to be tested.

[0111] 2.2 In some embodiments, the material of the electroosmotic tank modification layer is selected from the following group: titanium oxide, aluminum oxide, and magnesium oxide. In the above embodiment, the interface of the electroosmotic tank modification layer in contact with the electrolyte has a zeta potential with an absolute value of no more than 10mV. In some embodiments, the zeta potential value of the interface of the electroosmotic tank modification layer in contact with the electrolyte is 0±3mV. When the device is not in use, the electroosmotic tank modification layer is stored in a Tris solution to keep the zeta potential value of the interface stable. It can also be stored in a HEPES solution, a PBS aqueous solution or other pH buffer solution. When the device is in use, the first chamber and the second chamber separated by the substrate, as well as the electroosmotic tank and the micropores are filled with electrolyte. At this time, the electroosmotic tank modification layer is basically uncharged in the electrolyte, the electrolyte in the electroosmotic tank is basically electrically neutral, and the liquid in the electroosmotic tank basically does not flow. When a voltage is applied to the first and second electrodes (for example, the first electrode is grounded and the second electrode is connected to a negative voltage), an electric field is formed in the electrolyte. Under the action of the electric field, negatively charged particles in the electrolyte in all chambers move toward the first electrode, and positively charged particles in the electrolyte in all chambers move toward the second electrode. Therefore, when there are negatively charged particles in the second chamber, they will pass through the electroosmotic tank and then through the micropores under the action of the electric field; when there are positively charged particles in the first chamber, they will pass through the micropores under the action of the electric field. In the absence of any other driving force between the first and second chambers, this mechanism enables micro-nanoparticles to pass through the micropores.

[0112] Similarly, the amplitude of the electrical pulse signal generated when a micro-nano particle passes through the micropore is proportional to the particle volume and to the cube of the particle size. Furthermore, the frequency of the electrical pulse signal detected by the device is proportional to the concentration of the micro-nano particles. Similar to 2.1, this mechanism can be used to confirm the size and concentration of the micro-nano particles being tested in the same device by testing standard microspheres with determined particle size and concentration.

[0113] Furthermore, under this mechanism, the speed at which particles pass through the micropores is unaffected by liquid flow or other driving forces; they move solely under the influence of the electric field. The speed at which particles pass through the micropores is directly proportional to the particle's potential, while the time it takes for a particle to pass through the micropores is inversely proportional to the particle's potential. This mechanism can subsequently be used to determine the zeta potential of micro- and nanoparticles.

[0114] 2.3 In certain embodiments, the micro-nanoparticle detection device further includes a liquid driver that can drive the flow of the electrolyte. In some cases, the liquid driver can be combined with an electroosmotic tank modification layer to achieve no or weak flow of liquid within the micropores of the device. For example, if the electroosmotic tank modification layer has a zeta potential greater than 10 mV at its interface when in contact with the electrolyte, a pressure drive can be applied between the first chamber and the second chamber to offset the electroosmotic flow, allowing the micro-nanoparticles to pass through the micropores solely relying on the traction of the electric field force or under the dominance of the electric field force.

[0115] 2. Detection Methods and Applications of Micro-Nano Particles

[0116] In another aspect, the present application provides a method for measuring micro-nanoparticles using nano-Coulter and electroosmotic principles, comprising:

[0117] 1) placing the micro-nanoparticles to be tested in the device disclosed in this application;

[0118] 2) applying a voltage / current between the first electrode and the second electrode;

[0119] 3) Testing the change in current / voltage signal flowing through the electrodes; and

[0120] 4) Determining at least one of the particle size, concentration, or Zeta potential information of the micro-nano particles to be measured based on the change in the current / voltage signal.

[0121] In certain embodiments, the particle size of the micro-nanoparticles measured by the above method is between 1 nm and 2 μm, for example, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, 1600 nm, 1700 nm, 1800 nm, 1900 nm, 2000 nm or a value or range between any two of the above values.

[0122] In certain embodiments, the above-mentioned detection method further includes: using a standard microsphere with a determined particle size and concentration to obtain the peak value and frequency of the electric pulse signal generated by the standard microsphere in the above-mentioned device; testing the peak value and frequency of the electric pulse signal generated by the micro-nanoparticles to be tested in the device; and obtaining the particle size and concentration of the micro-nanoparticles to be tested by the ratio of the peak value and frequency of the electric pulse signals of the standard microsphere and the micro-nanoparticles to be tested.

[0123] The above detection method can be used to detect some micro-nanoparticles. For example, in one embodiment of the present application, the above device and detection method can be used to analyze LDL-C, thereby obtaining the particle size distribution of each LDL-C particle in the sample and the particle concentration within each particle size range. The provided method specifically includes the following steps:

[0124] 1) A sample containing the LDL-C to be measured is mixed with magnetic beads in a solution to allow for specific binding. The magnetic beads are bound to antibodies or aptamers that specifically bind to the surface membrane proteins of the LDL-C particles to be measured. These antibodies or aptamers can selectively bind to LDL-C particles among all lipoprotein particles. It should be understood that the function of these antibodies or aptamers is to capture LDL-C particles. To capture as many LDL-C particles as possible or to stabilize the capture efficiency at a high ratio, the binding of one or more antibodies or aptamers to the magnetic beads can be adjusted according to experimental conditions.

[0125] 2) The magnetic bead sample mixture bound to the sample LDL-C is placed in a magnetic field. The magnetic beads (including magnetic beads bound to LDL-C particles) are magnetically adsorbed to a local area in the magnetic field. A cleaning solution is then added to replace the magnetic bead sample mixture in the magnetic field area, thereby removing impurity particles in the sample that are similar in size to the LDL-C particle size.

[0126] The cleaning solution is a liquid that does not contain particles that interfere with particle detection, such as Tris, phosphate buffer, or other buffers. It does not contain surfactants or contains mild surfactants such as Tween20, Triton X, or F68. Its concentration is lower than the critical micelle concentration (CMC), and it does not produce micellar particles that interfere with the detection of target particles. This replacement process can be repeated multiple times, so that non-test particles in the sample are cleared as the magnetic bead sample mixture is replaced, resulting in a clear liquid in the magnetic field area containing only magnetic beads (including magnetic beads bound to LDL-C particles).

[0127] 3) Add a dissociation solution to the replaced supernatant. Dissociation solutions include, but are not limited to, proteases such as trypsin, papain, and proteinase K; suitable acidic or alkaline solutions such as NaOH, glycine, or citric acid; and denaturants such as DTT, guanidine hydrochloride, and β-mercaptoethanol.

[0128] Specifically, the dissociation solution can be trypsin, salicylic acid, glycine, citric acid, or other solutions capable of detaching LDL-C particles from the magnetic beads, dispersing the LDL-C particles into the mixed solution. Add one of these dissociation solutions to the magnetic beads bound to the LDL-C particles and mix thoroughly. Dissociate at 37°C for 30 minutes, then remove the solution after dissociation is complete.

[0129] 4) The mixed solution is subjected to magnetic force to cause the magnetic beads to settle or aggregate in a localized area, and the supernatant is collected from the area outside the aggregated area. The removal of the supernatant effectively separates the magnetic beads from the LDL-C particles, and the LDL-C particles separated from the magnetic beads are resuspended.

[0130] Furthermore, this step may specifically include:

[0131] (1) The magnetic beads are separated from the supernatant containing the dissociated LDL-C particles by magnetic adsorption.

[0132] (2) Use a small amount of new dissociation solution to resuspend the magnetic beads and mix thoroughly to wash off the remaining LDL-C particles adsorbed on the surface of the magnetic beads. Mix this part of the dissociation solution with the supernatant in the previous step (1). Repeat twice until the LDL-C particles become suspended particles again.

[0133] 5) Recording and measuring the current or voltage pulse signals generated on the electrodes when the LDL-C particles in the supernatant obtained in step 1 pass through the micropores. Specifically, the micro-nanoparticle detection device comprises a first chamber and a second chamber on either side of the micropore, each containing an electrode. The supernatant is added to the first chamber or the second chamber, and a voltage or current is applied to the two electrodes. The LDL-C particles in the sample pass through the micropores from the first chamber into the electroosmotic cell, or from the second chamber through the electroosmotic cell and the micropore into the first chamber, generating current or voltage pulses on the electrodes. By measuring the voltage or current pulse signals, the particle size distribution of the LDL-C particles in the sample and the particle concentration within each particle size range are obtained. The particle size resolution of the nanopores suitable for this application preferably reaches the 2 nm level. In the specific application of this analysis, the pore diameter of the micropore is no greater than 200 nm, and the pore depth of the micropore is no greater than 150 nm. It should be noted that, based on the function of the micropore, the pore diameter and pore depth must be set to accommodate the lipoprotein. However, taking into account the purpose of detection accuracy, in certain embodiments, the pore size of the micropore in step 4) is 200 nm or less, for example, 100-200 nm. In certain embodiments, the pore size of the micropore is 200 nm, 170 nm, 150 nm, 130 nm, 110 nm, 100 nm or a value or range between any two of the above values. In certain embodiments, the pore depth of the micropore is 150 nm or less. In certain embodiments, the pore depth of the micropore is 150 nm, 120 nm, 90 nm, 70 nm, 50 nm, 30 nm or a value or range between any two of the above values. In certain embodiments, the pore size of the micropore is 150 nm and the pore depth is 100 nm.

[0134] Table 1 below shows experimental results on the effects of micropores of varying pore diameters and depths on the repeatability of the Nano-Coulter technique used in this application. The test data was obtained using a diluted sample of 30 nm standard microspheres as the test sample, with the repeatability (CV) of 10 test concentrations used as the basis for determination.

[0135] Table 1 Comparison of the repeatability of the micropores with different pore depths and pore diameters for the same standard microsphere diluted sample

[0136] In the subsequent examples of this application, the lipoprotein concentrations in this example were detected by the nano-Coulter technique (the pore diameter of the nanopore in Example 6 was 200 nm and the pore depth was 150 nm; the pore diameter of the nanopore in Example 7 was 150 nm and the pore depth was 100 nm).

[0137] At the same time, in the specific embodiment of the present application, the particle concentration value of small-diameter nanoparticles can also be further traced based on the above-mentioned device and detection principle. With regard to small-diameter nanoparticles, since nanoparticles of 400nm and above can be detected by a direct method, the above-mentioned tracing method is mainly used for detecting small-diameter nanoparticles. The small-diameter nanoparticles refer to micro-nanoparticles with a particle size in the range of 400nm-20nm. These micro-nanoparticles are extremely difficult to observe under an optical microscope due to the wavelength of visible light. For example, the particle size range of small-diameter nanoparticles in the present application includes but is not limited to 370nm, 370nm, 350nm, 330nm, 310nm, 300nm, 270nm, 270nm, 250nm, 230nm, 210nm, 200nm, 170nm, 170nm, 150nm, 130nm, 110nm, 100nm, 90nm, 80nm, 70nm, 60nm, 50nm, 40nm, 30nm, and 20nm. In this traceability method, the particle sample with a known particle concentration value is a particle standard material. The particle standard material refers to a particle standard (also called a standard microsphere) with a known particle concentration value that can be used for calibration and verification, providing a reliable reference and calibration basis for scientific research and industrial applications in related fields. The height of the pulse signal refers to the peak value of the pulse signal. Regarding the baseline amplitude of the pulse signal, it should be noted that since the generated pulse signal has a certain amount of background noise interference, the amplitude of this noise is defined as the baseline amplitude of the pulse signal in this application. Those skilled in the art should understand that when the height of the effective signal is equivalent to the baseline amplitude, the effective signal will be submerged in the baseline. Therefore, in this application, the voltage / current pulse signal height of the sample with a smaller particle size needs to be higher than the baseline amplitude of the voltage / current pulse signal of the sample with a larger particle size. It should be understood that the liquid flows through the micropore at a steady flow rate, so the higher the concentration of the particles, the higher the number of pulses generated per unit time on the electrical signal. The pulse frequency per unit time is proportional to the particle concentration. Taking the electrical signal of a 5μm latex particle standard substance as an example, the amplitude of the baseline noise is about 100 when it is detected in a microporous particle detection device with a micropore aperture of 10μm. The smaller the particle size of the nanoparticles, the lower the amplitude of the generated electrical signal. If the height of the pulse signal generated by the sample being tested is less than 100 (for example, after testing, the height of this pulse signal corresponds to a particle size of approximately 800nm ​​in the micro-nano particle detection device), the pulse signal is already equivalent to the noise, and it is impossible to clearly distinguish between the real particle signal and the noise. The particle size at this time (800nm) is the lower limit of particle size detection that can be measured together with the 5μm latex particle standard substance. After research, it was also found that 60% of the aperture of the measuring device is roughly the upper limit of the particle detection particle size range.At the same time, it should be understood that since the detection range of the micropores of a specific micro-nano particle detection device is limited, different particles with large particle size differences are difficult to be detected in the same micro-nano particle detection device. Therefore, through the progressive method of the tracing method of this application, especially by using microparticle standard substances in combination with micro-nano particle detection devices with different detection ranges to calibrate the intermediate samples layer by layer, and finally confirm the particle concentration value of the sample to be tested, this tracing method has extremely high accuracy and can accurately trace the source. In the above tracing method, the device used includes a micropore and an electroosmotic tank connected to the micropore, and the inner wall of the electroosmotic tank includes an electroosmotic tank modification layer. When the electroosmotic tank modification layer contacts the electrolyte, the zeta potential of the interface is negative. Preferably, the electroosmotic tank modification layer is a silicon dioxide layer or a silicon nitride layer. In a specific embodiment, two adjacent samples are detected by the same device respectively.

[0138] In certain embodiments, for example, the detection method corresponding to the corresponding mechanisms in 2.2 and 2.3 includes: using a standard microsphere with a determined particle size, concentration and potential to obtain the peak value, frequency and pulse width of the electric pulse signal generated by the standard microsphere in the above-mentioned device; testing the peak value, frequency and pulse width of the electric pulse signal generated by the micro-nanoparticles to be tested in the device; and obtaining the particle size, concentration and Zeta potential of the micro-nanoparticles to be tested by the ratio of the peak value, frequency and pulse width of the electric pulse signals of the standard microsphere and the micro-nanoparticles to be tested.

[0139] It should be noted that, as mentioned in 2.2 and 2.3, the direct calculation of the Zeta potential of micro-nano particles can also be achieved. Specifically, the micro-nano particles obtained by the micro-nano particle detection device provided by the present application rely solely on the electric pulse signal generated by the micropores of the particle detection device under the action of the electric field force or under the dominance of the electric field force to calculate the Zeta potential of the micro-nano particles. The electric pulse signal can be a voltage signal or a current signal, and the specific form of this signal does not constitute a limitation to the present application. It should be understood that under the above mechanism, the speed at which the particles pass through the micropores will not be affected by the flow of liquid or other driving forces, and will only move under the dominance of the electric field force. At this time, the speed at which the particles pass through the micropores is directly proportional to the Zeta potential of the particles, and the passage time of the particles through the micropores is inversely proportional to the Zeta potential of the particles. The above-mentioned passage speed and passage time are closely related to the pulse width in the electric pulse signal of the particle. The present application also found through experiments that for particles of different potentials at the same particle size, the inverse ratio between the above-mentioned transit time and the particle Zeta potential is valid. However, for sample particles of different particle sizes and different potentials, the above-mentioned relationship is not completely valid. In fact, the size of the particle size will affect the pulse height (i.e., pulse peak value) of the collected electric pulse signal. At the same time, for particles of different particle sizes, the viscous resistance of the liquid in the hole to different particle sizes during the transit process is also different. Therefore, when calculating the particle Zeta potential by the transit time, it is necessary to correct the calculation result in combination with the particle size information. The particle size information is closely related to the pulse peak value of the electric pulse signal of the particle. Based on the above research results, especially the correlation obtained from each study, when a particle detection device is used to detect particles, the calculation model of the particle Zeta potential of the particle detection device can be constructed by the pulse width and pulse peak value of the electric pulse signal of particles with known particle size and Zeta potential. Here, it should be understood that the selection of different electrolytes in the particle detection device will affect the viscous resistance when passing through the hole, but when the same electrolyte is used for the experiment, this effect on the particles is consistent. Therefore, there will be certain differences in the Zeta potential correlation obtained when different electrolytes are selected for measurement, and those skilled in the art can make adjustments based on the selected electrolyte. Specifically, in this application, the construction of a particle Zeta potential calculation model based on the particle size and the pulse peak and pulse width of the electric pulse signal of the particle can be achieved in the following way. The particle size of particles with known particle size and Zeta potential and the pulse peak and pulse width of the electric pulse signal are used as modeling factors, and a training sample set is constructed in combination with the Zeta potential of the particle. In the same particle detection device, several types of model training are performed on the above-mentioned training sample set, and several types of models after training are evaluated, and the optimal particle Zeta potential calculation model is confirmed based on the model evaluation index.

[0140] It should be understood that in the actual Zeta potential measurement, by adding samples of different particle sizes with the same potential, when particles of different particle sizes pass through the hole, as shown in Figure 21 (the raw data pulse width and peak value collected from the particles passing through the hole), the correlation between the pulse peak value A of the particle and the particle size information is first analyzed, and then the pulse peak value A and the pulse width W are analyzed to obtain the corrected relationship between W and A. Based on the corrected relationship, when detecting the sample to be tested, the pulse width deviation caused by the inconsistent particle size can be corrected by the corrected relationship, and then the potential information of the single particle can be obtained by the single particle potential calculation equation. As mentioned above, in a specific embodiment of the present application, the calculation method provided by the present application specifically includes:

[0141] 1) Obtain detection electric pulse signals of several known particle samples with different particle sizes and the same zeta potential and record the pulse peak value and pulse width of the electric pulse signals.

[0142] (1) Based on the particle size of the known particles obtained above and the pulse peak value of the electric pulse signal, a first relational expression about the relationship between the particle size and the pulse peak value is obtained. The first relational expression is: A = a × D 3 , where A is the pulse peak value of the particle, D is the particle size of the particle, and a is the particle size coefficient. The first relationship shows that there is a linear relationship between the pulse peak value of the particle and the cube of its particle size.

[0143] (2) Based on the pulse peak value and pulse width of the electrical pulse signal of the above-mentioned particles, a second relationship about the relationship between pulse width and pulse peak value is obtained. The second relationship is: W = b × A + Wn, wherein A is the pulse peak value of the particle, W is the pulse width of the particle, b is the pulse coefficient, and Wn is the pulse width correction coefficient. The second relationship shows that there is a linear relationship between the pulse peak value of the particle and the cube of its particle size. It should be understood that since the particle size of each particle in the electrical pulse signal of the above-mentioned different particles is different, but the Zeta potential is the same, therefore, in the above-mentioned second relationship obtained from the particle samples with different particle sizes and the same Zeta potential, Wn is only related to the Zeta potential of the particle.

[0144] 2) Obtain the detection electrical pulse signals of several known particle samples of the same particle size but different zeta potentials and record the pulse width of the electrical pulse signals. It should be understood that from the analysis of the second relationship above, Wn is only related to the zeta potential of the particles. Therefore, when selecting known particle samples of the same particle size but different zeta potentials, the relationship between Wn and different zeta potentials can be obtained. This relationship can be expressed as: Z = d × (Wn) eSpecifically, based on the Zeta potential of the particle and the pulse width of its electric pulse signal in combination with the first and second relationship formulas, a fourth relationship formula between the Zeta potential of the particle and the pulse peak value and pulse width of its electric pulse signal is obtained. The fourth relationship formula is:

[0145] Z=d×(Wb×A) e , where Z is the absolute value of the particle's zeta potential, W is the particle's pulse width, A is the particle's pulse peak, b is the pulse coefficient, d is the potential coefficient, and e is the potential exponent. Therefore, in this application, the pulse peak value A is introduced to eliminate the effect of particle size on zeta potential.

[0146] It should be understood that for a specific micro-nano particle detection device, based on the fourth relationship obtained above, the device can be used to detect unknown particles and obtain the detection electric pulse signal of the particle under the action of the electric field force when the particle passes through the detection area. The pulse peak value and pulse width of the electric pulse signal in the detection result are substituted into the fourth relationship to obtain the zeta potential of the particle under test. In this case, the zeta potential of the particle can be directly measured and calculated, no longer relying on other standard particles, which is more direct and reliable.

[0147] In a specific embodiment of the present application, a preferred electroosmotic tank modification layer of a micro-nano particle detection device suitable for obtaining and applying the above-mentioned Zeta potential calculation model is a silicon dioxide layer or a silicon nitride layer. When the electroosmotic tank modification layer of the device is in contact with the electrolyte, the interface has a Zeta potential with an absolute value of no more than 10mV. Or when the interface of the electroosmotic tank modification layer in contact with the electrolyte has a Zeta potential with an absolute value greater than 10mV, a pressure drive can be applied between the first chamber and the second chamber of the device, and the pressure is used to offset the electroosmotic flow, so that the particles rely on the traction of the electric field force to pass through the micropores.

[0148] At the same time, in addition to the specific implementation methods described in detail above, those skilled in the art may, after reading this application, adopt other specific calculation or construction methods according to the principles and design ideas recorded in this application to realize the particle Zeta potential calculation model based on the pulse peak value and pulse width of the electric pulse signal.

[0149] 3. Expansion of the device

[0150] In another aspect, as an extension, the present application provides a device for measuring micro-nanoparticles using multiple micropores and measurement chambers. The device comprises a sample chamber having a common electrode and at least two measurement chambers adjacent to the sample chamber, including a first measurement chamber having a first measurement electrode and a second measurement chamber having a second measurement electrode. The sample chamber and the at least two measurement chambers are separated by a substrate, one side of which is covered by a membrane. The membrane has at least a first micropore and a second micropore. The substrate comprises a first electroosmotic cell and a second electroosmotic cell. The inner diameter of the first electroosmotic cell is larger than the pore diameter of the first micropore but smaller than the inner diameter of the first measurement chamber, and the inner diameter of the second electroosmotic cell is larger than the pore diameter of the second micropore but smaller than the inner diameter of the second measurement chamber. The sample chamber is connected to the first measurement chamber via the first micropore and the first electroosmotic cell. The sample chamber is connected to the second measurement chamber via the second micropore and the second electroosmotic cell. The inner wall of the first electroosmotic cell comprises a first electroosmotic cell modification layer, which has a specific first zeta potential at its interface when in contact with an electrolyte. The inner wall of the second electroosmotic tank is provided with a second electroosmotic tank modification layer, and the interface of the second electroosmotic tank modification layer has a specific second zeta potential when in contact with the electrolyte.

[0151] It is understood that the sample chamber and measurement chamber described herein may have the features of the first chamber and the second chamber described above. The substrate, membrane, first micropore, second micropore, first electroosmotic cell, second electroosmotic cell, first electroosmotic cell modification layer, and second electroosmotic cell modification layer described herein may have the features of the substrate, membrane, micropore, electroosmotic cell, and electroosmotic cell modification layer described above.

[0152] It is understood that the pore diameters of the first micropore and the second micropore described herein may be different. The inner diameters of the first electroosmotic cell and the second electroosmotic cell may be the same or different.

[0153] It will be appreciated that the membrane described herein can be applied to either the sample chamber side or the measurement chamber side of the substrate. When the membrane is applied to the sample chamber side of the substrate, the sample chamber and the measurement chamber are connected sequentially via the micropores and the electroosmotic cell. When the membrane is applied to the measurement chamber side of the substrate, the sample chamber and the measurement chamber are connected sequentially via the electroosmotic cell and the micropores.

[0154] It is understood that the first electroosmotic cell modification layer and the second electroosmotic cell modification layer described herein may be the same or different.

[0155] The present application will be further described in detail below through specific implementation methods in conjunction with the accompanying drawings.

[0156] Example 1:

[0157] As shown in FIG3 , an embodiment of the present application provides a method for manufacturing a nanopore chip 100 , which can be used to manufacture the nanopore chip 100 shown in FIG2 . The manufacturing method may include:

[0158] Step 1: Provide a substrate 1. In this application, substrate 1 serves as a support layer for membrane 2 and also serves as the sidewall of electroosmotic cell 101 for depositing electroosmotic cell modification layer 3. The material of substrate 1 is not limited in this application. While this application uses silicon wafers as an example, it does not exclude the applicability of other substrates.

[0159] For example, a double-sided polished silicon wafer is provided as the substrate 1 , and the thickness thereof may be 0.5 mm.

[0160] In some embodiments, after step 1, the following steps may also be included:

[0161] Step 100 , as shown in (A) and (C) of FIG. 5 , a transition layer 4 is formed on both sides of the substrate 1 , and the transition layer 4 is an insulating film layer.

[0162] The transition layer 4 can be silicon dioxide or other insulating materials. When the substrate 1 is a silicon substrate, a silicon dioxide layer with a thickness of 1 micron can be formed on the first surface and the second surface of the substrate 1 by a thermal oxidation process as the transition layer 4. The transition layer 4 is formed at least between the film 2 and the substrate 1.

[0163] Of course, the transition layer 4 can also be formed by physical vapor deposition or chemical vapor deposition. In this case, the transition layer 4 can be formed on the first surface, or on the first surface and the second surface.

[0164] Step 2: forming a film 2 on the first surface of the substrate 1 , wherein the film 2 has at least one micropore 201 .

[0165] Step 2 above may include:

[0166] Step 200 , as shown in (B) and (C) of FIG. 5 , deposit an insulating film layer as a film 2 on the first surface of the substrate 1 .

[0167] When the transition layer 4 is formed on the first surface of the substrate 1 , an insulating film layer is formed on the transition layer 4 on the first surface as the film 2 .

[0168] For example, as shown in (C) of FIG5 , a low stress chemical vapor deposition method (LPCVD) is used to deposit a silicon nitride layer of about 500 nm on each side of the transition layer 4 , one layer as the membrane 2 and the other as the protective layer 5 .

[0169] Step 210, as shown in FIG6, the membrane 2 is patterned to form at least one micropore 201, wherein the pore size of the micropore 201 is larger than the particle size of the particles to be measured. Generally, the particle size of the particles to be measured is at the nanometer level, and the corresponding pore size of the micropore 201 can be at the micrometer level.

[0170] Step 3, as shown in Figures 2, 8 and 9, an electroosmotic groove 101 is formed on the second surface of the substrate 1 and extends through the first surface, and an electroosmotic groove modification layer 3 is formed on the side wall of the electroosmotic groove 101; the electroosmotic groove modification layer 3 is configured so that the zeta potential of the interface when immersed in the electrolyte is negative.

[0171] The pore size of micropores 201 is smaller than that of electroosmotic cell 101, and the projections of all micropores 201 on the first surface are covered by the projections of electroosmotic cell 101 on the first surface. Electroosmotic cell modification layer 3 can be formed after electroosmotic cell 101 is formed; or, electroosmotic cell modification layer 3 can be formed during the formation of electroosmotic cell 101.

[0172] The embodiment of the present application focuses on forming an electroosmotic cell modification layer 3 on the sidewalls of the electroosmotic cell 101, and the zeta potential of the interface of the electroosmotic cell modification layer 3 when immersed in the electrolyte is negative. The specific method for forming the electroosmotic cell modification layer 3 is not limited, and the specific material of the electroosmotic cell modification layer 3 is not limited.

[0173] In some embodiments, the electroosmosis slot modification layer 3 may be an amorphous carbon layer or a carbon chain polymer layer; the electroosmosis slot modification layer 3 is formed after the electroosmosis slot 101 .

[0174] In step 3, forming the electroosmotic groove 101 on the second surface of the substrate 1 and penetrating to the first surface may include:

[0175] Step 301, as shown in Figure 7, wet etching is used to pattern substrate 1, etching the protective layer 5 and transition layer 4 (if any) on the second surface, and then etching substrate 1. Electroosmotic grooves 101 are formed on the second surface of substrate 1, extending through to the first surface. Transition layer 4 prevents etching of membrane 2, ensuring its integrity.

[0176] For example, the silicon substrate may be etched with KOH, and then the silicon oxide layer (transition layer 4) and the silicon nitride layer (film 2) on the first surface may be etched with BOE etching solution.

[0177] In the above step 3, forming the electroosmotic tank modification layer 3 on the sidewall of the electroosmotic tank 101 may include:

[0178] Step 302 : Passivate the sidewalls of the electroosmotic cell 101 using hydrocarbons or fluorocarbons in plasma to form an amorphous carbon layer or a carbon chain polymer layer as the electroosmotic cell modification layer 3 .

[0179] For example, a silicon substrate is placed in a C 4 F 8 reaction chamber containing plasma, and an amorphous carbon layer or a carbon chain polymer layer is formed on the sidewall of the electroosmosis groove 101 as the electroosmosis groove modification layer 3 .

[0180] Step 4: Slice the substrate 1 to obtain a plurality of nanopore chips 100 .

[0181] Step 5: Immerse the nanopore chip 100 in an alkaline salt solution, a phosphate solution, or a HEPES solution.

[0182] For example, a silicon wafer is sliced ​​using laser cutting to obtain a plurality of chips of a size of 10 mm×10 mm.

[0183] In some embodiments, the electroosmosis cell modification layer 3 may be an amorphous carbon layer or a carbon chain polymer layer; the electroosmosis cell modification layer 3 is formed during the formation of the electroosmosis cell 101 .

[0184] In step 3, forming an electroosmotic groove 101 on the second surface of the substrate 1 and penetrating to the first surface, and forming an electroosmotic groove modification layer 3 on the sidewall of the electroosmotic groove 101 may include:

[0185] In the etching step, as shown in (A) of FIG10 , the substrate 1 is patterned by dry etching to form a groove of a first depth on the second surface of the substrate 1 ; the first depth is less than the thickness of the substrate 1 .

[0186] The passivation step, as shown in (B) in Figure 10, uses plasma hydrocarbons or fluorocarbons to passivate the side walls of the groove body to form an amorphous carbon layer or a carbon chain polymer layer as the electroosmotic groove modification layer 3. At this time, the electroosmotic groove modification layer 3 has not been fully formed. The electroosmotic groove modification layer 3 in this state is defined as a passivation layer 301.

[0187] As shown in (C) to (E) in Figure 10 and Figure 9, the etching step and the passivation step are repeated until the depth of the groove body is equal to the thickness of the substrate 1, forming an electroosmotic groove 101 that penetrates to the first surface, and an electroosmotic groove modification layer 3 is formed on the side wall of the electroosmotic groove 101.

[0188] For example, a silicon substrate is dry-etched using SF6 plasma. The exposed sidewalls are then passivated using C4F8 plasma. As the groove deepens, the length of the passivation layer 301 on the sidewalls increases until it is completely deposited on the sidewalls, forming the electroosmotic groove finishing layer 3.

[0189] There is no limit to the number of times the above etching steps are repeated, or the first etching depth in each etching step can be different, and the depth of the groove body is continuously deepened by etching to form the final electroosmotic groove 101 and to form an electroosmotic groove modification layer 3 on the side wall of the electroosmotic groove 101.

[0190] Example 2:

[0191] Corresponding to the preparation method in Example 1, the nanopore chip 100 can be prepared using the following steps.

[0192] 1. A 0.5 mm thick 4-inch double-sided polished silicon wafer (substrate 1) was deposited on both sides by low-pressure chemical vapor deposition (LPCVD) to deposit a 30 nm thick silicon nitride layer (film 2 and protective layer 5).

[0193] 2. Spin-coat photoresist on the wafer and expose it through a photolithography process. Take a unit area of ​​5 mm × 5 mm and develop a circular pattern with a diameter of 20 nm to be etched. Use reactive ion etching (RIE) technology to etch a 20 nm circular hole (micropore 201) in the silicon nitride of the exposed area.

[0194] 3. Remove the photoresist on the top layer of the wafer and apply photoresist on the back of the wafer. Use exposure and development processes again to create a 2mm×2mm back cavity groove on the photoresist at the position corresponding to the circular hole on the back of the silicon substrate. Etch the silicon nitride through reactive ion etching (RIE) technology and remove the photoresist on the back of the wafer.

[0195] Silicon is released by dry etching to form an electroosmotic groove 101, forming a self-supporting SiNx suspended membrane. The dry etching method is to etch the silicon exposed by the groove in a plasma using the etching gas SF6 to react with the silicon, and then react and passivate it in the plasma C4F8 (octafluorocyclobutane). After the reaction, the C4F8 can form a carbon layer or carbon chain polymer (electroosmotic groove modification layer 3), which is deposited on the silicon surface to prevent the reaction between fluoride ions and silicon. Physical sputtering bombardment is performed in the depth direction, so that the carbon layer or carbon chain polymer in the depth direction is removed while the carbon layer or carbon chain polymer on the side can be retained, and the silicon in the depth direction is exposed and can be etched again. Through this periodic "etching-passivation-etching" cycle, dry deep silicon etching is achieved.

[0196] 4. Laser cut the silicon wafer into single chips of 5mm×5mm size.

[0197] 5. Soak the single chip in a 3 mol / L NaOH aqueous solution for 2 hours, then rinse with clean water and transfer to a PBS aqueous solution for storage.

[0198] The above-mentioned single chip was tested using electron microscope EDX and the elemental analysis results showed that there was a carbon layer on the silicon base surface.

[0199] The zeta potential of the carbon layer at the interface in PBS electrolyte was measured using an atomic force microscope and was -65 mV.

[0200] When the single chip is not used for testing, the chip / carbon layer (electroosmotic tank modification layer 3) is stored in a PBS aqueous solution to keep the zeta potential value of the interface stable.

[0201] As shown in Figure 3, the monolithic chip is placed vertically in a measurement container 200, which is then divided horizontally into two chambers: a first chamber and a second chamber. The first chamber contains a first electrode 310 and a second electrode 320, respectively. The first chamber, micropores, electroosmotic cell, and second chamber are all filled with PBS electrolyte. The PBS electrolyte in the first chamber contains the polyethylene microsphere particles to be measured.

[0202] In the electrolyte, the electroosmotic tank modification layer 3 is negatively charged. Due to the principle of electrical neutrality, the electrolyte in the electroosmotic tank 101 is positively charged. When a negative 200mV voltage is applied to the first electrode 310 and the second electrode 320 (such as the first electrode 310 is grounded and the second electrode 320 is connected to a negative voltage), an electric field is formed in the electrolyte. The positively charged electrolyte in the electroosmotic tank as a whole moves toward the second electrode 320 under the action of the electric field, thereby driving the liquid in the first chamber to pass through the nanopores and replenish the electroosmotic tank, and then driving the particles to be tested (such as polyethylene microspheres) in the first chamber to pass through the micropores 201. Therefore, under the condition that there is no other driving force between the first chamber and the second chamber, this mechanism can also be used to enable the nanoparticles in the first chamber to pass through the nanopores.

[0203] As shown in FIG11 , the current flowing through the electrodes is tested, and the pulse signal to the polyethylene microspheres is tested.

[0204] As shown in FIG12 , similarly, a pulse signal as shown was obtained in the measuring device using a 15 nm standard microsphere sample / standard particle (average particle size 15 nm, concentration 2E9 particle / mL).

[0205] According to the following formula, the amplitude of the pulse signal is proportional to the particle volume and proportional to the cube of the particle diameter.

[0206] Where ΔV is the pulse voltage amplitude, ρe is the resistivity of the electrolyte solution, I is the current size of the nanopore, π is the circumference, d is the particle size of the nanoparticle, and D is the pore size of the nanopore.

[0207] The average pulse peak value of the 15nm standard microsphere sample is 19000, and the average pulse peak value of the polyethylene microsphere to be tested is 5600. It can be calculated that the polyethylene microsphere to be tested is 10nm.

[0208] The flow rate passing through the nanopore per unit time is a constant, so the more particles there are in a unit volume, the more pulse signals are generated. The frequency of the pulse signals tested in the device is directly proportional to the particle concentration in the sample.

[0209] The pulse signal frequency measured for the 15nm standard microsphere sample is 90 / minute, and the pulse signal frequency measured for the polyethylene microsphere sample to be tested is 60 / minute. It can be calculated that the concentration of the polyethylene microsphere sample to be tested is 1.333E9particle / mL.

[0210] Example 3:

[0211] A 1µm polysilicon film was grown on a 5mm x 5mm, 0.5mm thick boron nitride substrate. A 1mm inner diameter groove was cut into the boron nitride substrate, extending 0.5mm deep into the polysilicon film to serve as an electroosmotic channel. The inner wall of the electroosmotic channel was coated with a titanium oxide layer, which served as a finishing layer. A micropore with an inner diameter of 600nm was located in the center of the polysilicon film, directly opposite the electroosmotic channel.

[0212] As shown in Figure 13, the boron nitride sheet was placed vertically in a plastic container, which was then divided horizontally into two chambers: a first chamber and a second chamber. The first and second chambers contained a first electrode, respectively. The first chamber, the nanopore, the electroosmotic cell, and the second chamber were all filled with PBS solution. The PBS electrolyte in the second chamber contained the silica microspheres to be tested.

[0213] The zeta potential of the interface of the electroosmotic cell modified layer in PBS solution was measured using an atomic force microscope and was -0.3 mV.

[0214] When the device is not in use, the electroosmotic cell modification layer is stored in a Tris solution to keep the zeta potential value of the interface stable.

[0215] As shown in Figure 13, the electroosmotic cell modification layer is basically uncharged in the PBS solution, the electrolyte in the electroosmotic cell is basically electrically neutral, and the liquid in the electroosmotic cell basically does not flow. When a voltage is applied to the first electrode and the second electrode (as shown in the figure, the first electrode is grounded and the second electrode is connected to a negative voltage), an electric field is formed in the electrolyte. Under the action of the electric field, the negatively charged particles in the electrolyte in all chambers move toward the first electrode, and the positively charged particles in the electrolyte in all chambers move toward the second electrode. Therefore, when there are negatively charged particles in the second chamber, they will pass through the electroosmotic cell under the action of the electric field and then through the nanopore; when there are positively charged particles in the first chamber, they will pass through the nanopore under the action of the electric field. In the absence of any other driving force between the first and second chambers, this mechanism enables the microsphere particles to pass through the nanopore.

[0216] At the same time, under the above mechanism, the speed at which particles pass through the nanopore will not be affected by liquid flow or other driving forces, and they only move under the action of electric field forces. The speed at which particles pass through the nanopore is directly proportional to the potential of the particles, and the time it takes for particles to pass through the nanopore is inversely proportional to the potential of the particles.

[0217] As shown in FIG14A , the width of the pulse signal of the silica microsphere to be tested is 0.2 mS.

[0218] When a standard microsphere sample with the same particle size of 150 nm as the silica to be tested (average particle size 150 nm, concentration 3×10 9 When the 400 μg / mL (500 μg / mL, average potential -30 mV) test was conducted, the width of the pulse signal was 0.4 mS ( FIG. 14B ).

[0219] It can be approximately calculated that the potential of the silica microspheres to be tested is -60mV.

[0220] Similarly, similar to Example 2, the particle size and concentration of the silica microspheres to be tested can be calculated based on the peak value and frequency of the pulse signal.

[0221] Example 4: Effect of the Inner Diameter of the Electroosmotic Cell on the Device

[0222] A 1µm polysilicon film was grown on a 5mm x 5mm, 0.5mm thick boron nitride substrate. A 1mm inner diameter groove was cut into the boron nitride substrate, extending 0.5mm deep into the polysilicon film to serve as an electroosmotic channel. The inner wall of the electroosmotic channel was coated with a titanium oxide layer, which served as a finishing layer. A micropore with an inner diameter of 600nm was located in the center of the polysilicon film, directly opposite the electroosmotic channel.

[0223] When the chip was used to test 200 nm polyethylene microspheres, the frequency data shown in Figures 15A-15E and Table 2 were obtained. It can be seen that when the inner diameter of the electroosmotic cell is larger than 3 mm, the measured pulse frequency is very low.

[0224] Table 2. Pulse frequencies measured for different electroosmotic cell inner diameters

[0225] Example 5: Effect of electroosmotic tank depth on the device

[0226] A 5µm thick silicon nitride membrane was grown on a 5mm x 5mm single-crystal silicon substrate with thicknesses of 25µm, 100µm, and 400µm. A 25µm inner diameter groove was cut into the silicon substrate, extending to depths of 25µm, 100µm, and 400µm, extending into the silicon nitride membrane to form an electroosmosis channel. A silicon dioxide layer was applied to the inner wall of the channel as a finishing layer. A nanopore with an inner diameter of 5µm was located in the center of the silicon nitride membrane, directly opposite the channel.

[0227] When the chip was used to test 800 nm polyethylene microspheres, the frequency data shown in Figures 16A-16C and Table 3 were obtained. It can be seen that when the depth of the electroosmotic cell is less than 5 times the pore depth, the measured pulse frequency is very low.

[0228] Table 3. Pulse frequencies measured at different electroosmotic cell depths

[0229] Example 6: Analysis of sdLDL Particle Concentration in Blood Samples

[0230] The blood to be tested was mixed with a solution of magnetic beads coated with specific binding antibodies at a volume ratio of 1:1. The surface of the magnetic beads used was coated with antibodies against protein ApoB100. In this example, the magnetic beads had a particle size of 1.6 μm and a concentration of 5.4×10 10 / ml. The mixed solution was incubated at 37°C and 1000 rpm for 30 minutes. After the reaction, the magnetic beads were separated from the supernatant by magnetic adsorption. The supernatant was discarded and the beads were washed three times with a washing solution. A dissociation solution containing trypsin was then added. After thorough mixing, the solution was incubated at 37°C and 1000 rpm for 15 minutes. The supernatant containing the specific low-density lipoprotein was collected after magnetic adsorption of the magnetic beads.

[0231] In this example, a micro-nano particle measurement device with a micropore diameter of 200 nm, as shown in Figure 17, was used. Specifically, a 150 nm polysilicon film was modified on a boron nitride substrate to serve as the membrane. A groove with an inner diameter of 3 mm was cut in the boron nitride substrate and a depth of 1 mm into the polysilicon film to serve as the electroosmotic cell. The inner wall of the electroosmotic cell was coated with a silicon nitride layer, which was then immersed in a sodium perchlorate solution to adsorb anions and serve as the electroosmotic cell modification layer. A micropore with an inner diameter of 200 nm was located in the center of the polysilicon film directly opposite the electroosmotic cell.

[0232] The boron nitride sheet was placed vertically in a plastic container, which was then divided horizontally into two chambers: a first chamber and a second chamber. The first and second chambers contained a first electrode, respectively. The first chamber, microwell, and electroosmotic cell were all filled with a NaCl solution, while the second chamber contained the nanoparticles to be tested in a PBS electrolyte.

[0233] The zeta potential of the electroosmotic cell modified layer in NaCl aqueous solution was measured to be -25 mV using an atomic force microscope.

[0234] When voltage is applied between the two electrodes (as shown in Figure 17, the first electrode is grounded and the second electrode is connected to a positive voltage), an electric field is formed in the electrolyte, and particles can pass through the nanopore through this mechanism without any other driving force.

[0235] The particle size distribution of the obtained LDL-C particles and the particle concentration within each particle size range were then detected using the above-mentioned Nano-Coulter technique, and a histogram of LDL-C particle size and concentration was obtained as shown in FIG18 .

[0236] In this example, parameters such as a particle size resolution of 2 nm and a particle size detection range of 20-34 nm were selected for detection. As shown in FIG18 , the number of particles tested within the range of 20-26 nm (the particle size distribution range of sdLDL particles) were 1.7E6, 2.4E6, and 4.3E6, respectively. The total number of particles in this range was 1.7E6+2.4E6+4.3E6=8.4E6, that is, the concentration of sdLDL particles that can be effectively detected in this blood sample within a specific time period is 8.4E6.

[0237] Example 7: Detecting the particle size distribution ratio of low-density lipoprotein particles in a blood sample

[0238] The blood to be tested was mixed with a solution of magnetic beads coated with specific binding antibodies at a volume ratio of 1:1. The surface of the magnetic beads used was coated with antibodies to protein ApoB100. The magnetic beads had a particle size of 2.4 μm and a concentration of 3.6×10 10 / ml. The mixed solution was incubated at 37°C at 1000 rpm for 15 minutes. After the reaction, the magnetic beads were separated from the supernatant by magnetic adsorption. The supernatant was discarded and the beads were washed three times with a washing solution. A dissociation solution containing glycine was then added. After thorough mixing, the solution was incubated at 37°C at 1000 rpm for 30 minutes. The supernatant containing the specific low-density lipoprotein was collected by magnetic adsorption of the magnetic beads.

[0239] In this example, a micro-nano particle measurement device with a micropore diameter of 150 nm, as shown in Figure 19, was used. Specifically, a 100 nm thick silicon nitride membrane was grown on a single-crystal silicon substrate. A groove with an inner diameter of 2 mm and a depth of 0.5 mm was cut into the silicon nitride membrane to serve as an electroosmotic cell. The inner wall of the electroosmotic cell was coated with a layer of silicon dioxide as a finishing layer. A micropore with an inner diameter of 150 nm was located in the center of the silicon nitride membrane, directly opposite the electroosmotic cell.

[0240] The silicon wafer was placed vertically in a plastic container, which was then divided horizontally into two chambers: a first chamber and a second chamber. The first and second chambers contained a first electrode, respectively. The first chamber, microwell, electroosmotic cell, and second chamber were all filled with PBS electrolyte. The PBS electrolyte in the first chamber contained sample particles to be tested.

[0241] The zeta potential of the interface of the electroosmotic cell modified layer in PBS electrolyte was measured using an atomic force microscope and was -65 mV.

[0242] When voltage is applied between the two electrodes (the first electrode is grounded and the second electrode is connected to a negative voltage), an electric field is formed in the electrolyte, and particles can pass through the nanopore through this mechanism without any other driving force.

[0243] The particle size distribution of LDL-C particles and the particle concentration within each particle size range were detected using the above RPS technology to obtain a bar graph of LDL-C particle size and concentration as shown in Figure 20, and the proportion of sdLDL particles in the total LDL-C particles was calculated.

[0244] In this example, parameters such as a particle size resolution of 6 nm and a particle size detection range of 20-38 nm were selected for detection. As shown in FIG20 , the number of particles tested within the range of 20-26 nm (the particle size distribution range of sdLDL particles) was 1.2E7, and the number of particles within the particle size range of 26-38 nm was 3.1E7. That is, the proportion of sdLDL particles detected in this blood sample within a specific time period in the total LDL-C particles was 1.2E7 / 3.1E7=0.387 (38.7%).

[0245] Example 8:

[0246] Take 50uL of the blood sample to be tested and the series of concentration calibrators, and mix them with 100uL of magnetic beads (same as Example 7) coated with specific binding antibodies. The mixed solution is incubated at room temperature for 10 minutes. After the reaction is completed, the magnetic beads are separated from the supernatant by magnetic adsorption, and the supernatant is discarded and the magnetic beads are washed twice with a cleaning solution. After washing, an eluent whose main component is citric acid is added, and after thorough mixing, the mixture is incubated at room temperature for 5 minutes. The supernatant of the eluate containing specific low-density lipoprotein is collected after magnetic adsorption of the magnetic beads. Finally, the sample is tested by the method of Example 7. The sdLDL particle concentration that can be detected in this reaction system is 4.2E8 / mL, and the molar concentration of sdLDL in the sample is determined to be 0.37mmol / L based on the established calibration curve. The establishment of the above-mentioned calibration curve is confirmed by the following method. The series of concentration calibrators provided by this detection method are a series of calibrators of known molar concentrations obtained by performing a concentration gradient dilution of the LDL-C standard substance calibrated with a certified peroxidase method kit. A corresponding relationship is established between the particle concentrations obtained from the series of concentration calibrants under this detection method, that is, a calibration curve of the LDL-C particle concentration and the molar concentration in the sample is obtained.

[0247] The blood samples were also tested using a certified peroxidase assay kit (Beijing Jiuqiang Biotechnology Co., Ltd., Cat. No. GSSD) at a concentration of 0.31 mmol / L, yielding consistent results. Unlike existing kits, the present method not only measures the molar concentration of sdLDL but also the concentration and size distribution of sdLDL particles. This method offers broader value in lipid research than existing single-concentration analysis.

[0248] Example 9. Method for Accurately Detecting the Concentration of Gold Nanoparticles with an Average Particle Size of 50 nm ± 6 nm

[0249] 1. Select a large-particle polystyrene microsphere sample. First, measure and statistically analyze its particle size using a scanning electron microscope. Counting the total number of particles in different fields of view is over 100, yielding an average particle size of approximately 2 μm. This is used to select an appropriate particle size range for the micro-nanoparticle detection device. Next, measure its particle concentration using a microscope counting method, calculating a particle concentration of 2.31E5 particles / mL. Separately, select polystyrene microsphere samples with average particle sizes of approximately 800 nm and 200 nm as intermediate concentration standards.

[0250] 2. The schematic diagram of the micro-nanoparticle device used in this example is the same as Figure 19 . A 400nm thick silicon nitride membrane is grown on a 5mm×5mm, 0.5mm thick single-crystal silicon substrate. A 2mm inner diameter groove is cut in the silicon substrate and 0.5mm deep into the silicon nitride membrane to serve as an electroosmotic cell. The inner wall of the electroosmotic cell is coated with a layer of silicon dioxide as a finishing layer. A micropore with an inner diameter of 4μm is located in the center of the silicon nitride membrane, directly opposite the electroosmotic cell. The silicon wafer is placed vertically in a plastic container, which is then divided horizontally into two chambers: a first chamber (left side) and a second chamber (right side). The first and second chambers contain a first electrode, respectively. The first chamber, micropore, electroosmotic cell, and second chamber are all filled with PBS electrolyte. The PBS electrolyte in the first chamber contains the microsphere particles to be tested.

[0251] The zeta potential of the interface of the electroosmotic cell modified layer in PBS electrolyte was measured to be -45mV using atomic force microscopy.

[0252] By analyzing the signal-to-noise ratio of the electrical signal and the parameters of the pore size, it was determined that this nanopore particle detection device can detect particles with a particle size measurement range of 500nm-2μm.

[0253] 3. Particle concentration was measured using the first-stage 4μm nanopore particle detection device described above. The pulse signal frequency measured in the device is directly proportional to the particle concentration in the sample. The average pulse signal frequency for 2μm microspheres with a particle concentration of 2.31E5 particles / mL was 82 pulses / minute. Simultaneously, an intermediate microsphere standard sample with a particle size of approximately 800nm ​​was tested, and its average pulse signal frequency was 367 pulses / minute. Using the same formula (1) as above, the particle concentration at this intermediate concentration was calculated to be 1.03E6 particles / mL, based on the proportional relationship between pulse signal frequency and particle concentration in the sample.

[0254] 4. The above process was also used to prepare a nanopore chip with a pore size of 1600 nm and a membrane thickness of 200 nm, obtaining a second-stage particle detection device (1600 nm nanopore particle detection device) with a particle size measurement range covering 200 nm-800 nm. Under this device, the microsphere standard sample with a particle size of about 800 nm was detected again, and its average pulse signal frequency was 47 / min. At the same time, the prepared intermediate microsphere standard sample with a particle size of about 200 nm was detected, and its average pulse signal frequency was 3290 / min. Therefore, the concentration of the intermediate microsphere standard sample with a particle size of about 200 nm was calculated to be 7.21E7 / mL based on the proportional relationship between the pulse signal frequency and the particle concentration in the sample according to the above formula 1.

[0255] 5. The above process was also used to prepare a nanopore chip with a pore size of 400 nm and a membrane thickness of 400 nm, resulting in a third-level particle detection device (400 nm nanopore particle detection device) with a particle size measurement range covering 40 nm-200 nm. Under this device, a microsphere standard sample with a particle size of about 200 nm was again detected, and its average pulse signal frequency was 92 pulses / minute. At the same time, a nanogold particle sample with an average particle size of 50 nm ± 6 nm was tested, and its average pulse signal frequency was 1312 pulses / minute. The same as the above formula 1, the concentration of these nanogold particles was calculated to be 1.03E9 particles / mL through the proportional relationship between the pulse signal frequency and the particle concentration in the sample, thereby achieving accurate detection of the concentration value of small-particle micro-nanoparticle samples of about 50 nm.

[0256] Example 10

[0257] Referring to FIG13 , under the action of the electric field, according to the principle of electrophoresis, the particles with positive Zeta potential on the left side move toward the negative electrode under the action of the electric field force, and according to the Coulter principle, a change in the electrical signal is generated when passing through the via.

[0258] In this example, the micro-nano particle detection device employed includes: a 1000nm thick polysilicon film (as a membrane) grown on a 5mm x 5mm, 0.5mm thick boron nitride substrate. A groove with an inner diameter of 1mm and a depth of 0.5mm extending into the polysilicon film is then cut into the boron nitride substrate, serving as an electroosmotic groove. The inner wall of the groove is coated with a titanium oxide layer, which serves as a finishing layer. A micropore with an inner diameter of 600nm is located in the center of the polysilicon film, directly opposite the electroosmotic groove.

[0259] The boron nitride sheet was placed vertically in a plastic container, which was then divided horizontally into two chambers: a first chamber and a second chamber. The first and second chambers contained a first electrode, respectively. The first chamber, microwell, and electroosmotic cell were all filled with PBS solution, while the second chamber contained the silica microspheres to be tested.

[0260] The zeta potential of the electroosmotic cell modified layer in PBS solution was measured using atomic force microscopy to be -0.3 mV. When the device is not in use, the electroosmotic cell modified layer can be stored in Tris solution to maintain a stable zeta potential.

[0261] In this embodiment, the raw data of the pulse width and pulse peak value of the test of the standard samples with the same Zeta potential (relatively close) of different particle sizes of 150nm, 200nm, and 300nm are shown in Table 4:

[0262] Table 4 Pulse peak value and pulse width of the original electric pulse signal of particles with known particle size in the same detection device

[0263] Analyzing the data in the table above, we can find that the pulse width of the particles is correlated with the particle size, and the pulse peak value of the measurement signal is proportional to the cube of the particle size. Specifically, the linear fitting results of W and A are shown in Figure 22, and A and D 3 The linear fitting results are shown in Figure 23.

[0264] In this example, the correlation function between the original data pulse width and pulse peak in Table 4 is analyzed as follows: W = 0.0006 × A + Wn, that is, the influence of the pulse peak on the pulse width is 0.0006 × A. A = 0.0006 × D 3 , that is, the pulse height is proportional to the cube of the particle size. The pulse peak value will eliminate the influence of the particle size and the pulse width correction value Wn. The pulse width calibration equation is: Wn = W-0.0006×A, or Wn = W-0.00000036×D 3 At this time, Wn is only related to the Zeta potential of the particles.

[0265] Then, samples with the same particle size D = 100 nm and different zeta potentials were measured. The original pulse width data W was used to calculate the pulse width correction value Wn according to the above pulse width calibration equation, where the pulse peak value is 600 (substitute A = 600 into Wn = W-0.0006×A or substitute D = 100 into Wn = W-0.00000036×D 3 ). The relationship obtained through comprehensive analysis, the actual measured data is shown in Table 5:

[0266] Table 5 Detection data and correction data of particles with the same particle size but different Zeta potential in the same particle detection device

[0267] The absolute value Z of the known potential of the above particle sample and the value Wn after pulse width correction in Table 5 are shown in Table 6:

[0268] Table 6 Corrected data and Zeta potential of particles of the same particle size in the same particle detection device

[0269] Combined with Table 6, the above analysis shows that the functional relationship between the absolute value of potential Z and Wn is: Z = 45.517Wn (-3.418) , and the relevant fitting relationship is shown in Figure 24.

[0270] In summary, when the particle detection device provided in this example is used for particle detection, the equation for calculating the absolute value of the particle Zeta potential, i.e., the particle Zeta potential calculation model of the particle detection device in this example, is: Z = 45.517 × (W - 0.0006 × A) (-3.418) , Z is the absolute value of the particle Zeta potential, W is the pulse width of the particle, A is the pulse peak of the particle, 0.0006 is the pulse coefficient, 45.517 is the potential coefficient, and -3.418 is the potential index.

[0271] Comparative Example:

[0272] The same particle detection device as in this example is used to detect the unknown particle sample 1. Specifically, the device is used to detect the sample 1. The electrode on the side where the unknown particle sample 1 is added is grounded, and a positive voltage is applied to the electrode on the side where the blank electrolyte is added. A pulse signal is detected, indicating that the particles in the unknown particle sample 1 are negatively charged.

[0273] Through testing, it can be found that the pulse peak value A in the unknown particle sample 1 is 1050 and the pulse width is 1.782. The absolute value calculation equation of the particle Zeta potential obtained above in this example is: Z = 45.517 × (W - 0.0006 × A) (-3.418) The particle Zeta potential is calculated to be -28 mV, and the particle size is also calculated to be 121 nm.

[0274] Electron microscopy of the particles in the unknown particle sample 1 was performed, and the surface potential of the particles was verified to be -30 mV using an ELS device, indicating that the measured value is very accurate. Therefore, the particle zeta potential calculation model obtained in this application can accurately and effectively calculate the zeta potential of the particles to be tested.

[0275] The above examples are used to illustrate the present invention, which are only used to help understand the present invention and are not intended to limit the present invention. Those skilled in the art can make several simple deductions, modifications or substitutions based on the concept of the present invention.

Claims

1. A device for measuring micro-nano particles, comprising: a first chamber having a first electrode; a second chamber adjacent to the first chamber and having a second electrode; a substrate separating the first chamber and the second chamber; a membrane covering the first chamber side of the substrate; Wherein: the membrane has micropores; the substrate has an electroosmotic groove, the inner diameter of the electroosmotic groove is larger than the pore diameter of the micropores and smaller than the inner diameter of the second chamber, the first chamber and the second chamber are connected through the micropores and the electroosmotic groove, and the inner wall of the electroosmotic groove has an electroosmotic groove modification layer, and the interface of the electroosmotic groove modification layer has a specific Zeta potential when in contact with the electrolyte.

2. The device according to claim 1, wherein the pore size of the micropore is 20 nm to 5 μm; Preferably, the inner diameter of the electroosmotic cell is less than or equal to 3 mm; Preferably, the depth of the electroosmotic cell is 5 times or more than the depth of the micropores.

3. The device according to claim 1, characterized in that wherein the interface of the electroosmotic slot modification layer has a Zeta potential more negative than -20 mV when in contact with the electrolyte; Preferably, the material of the electroosmotic slot modification layer is selected from the following group: amorphous carbon, carbon chain polymer, silicon oxide, silicon nitride, polycrystalline silicon, single crystal silicon and adsorption layers formed by adsorption of anions thereon; Preferably, the Zeta potential value of the interface of the electroosmotic tank modification layer when in contact with the electrolyte is -65±6mV; Preferably, the electroosmotic cell modification layer also covers the interface between the membrane and the electroosmotic cell and the inner wall of the micropore.

4. The device according to claim 1, wherein the interface of the electroosmotic slot modification layer and the electrolyte has a Zeta potential of an absolute value not greater than 10 mV when in contact with the electrolyte; Preferably, the material of the electroosmosis slot modification layer is selected from the following group: titanium oxide, aluminum oxide, and magnesium oxide. Preferably, the Zeta potential value of the interface of the electroosmotic cell modification layer when in contact with the electrolyte is 0±3 mV. Preferably, the electroosmotic cell modification layer also covers the interface between the membrane and the electroosmotic cell and the inner wall of the micropore.

5. The device according to claim 1 further comprises a liquid driver which can drive the flow of the electrolyte.

6. A method for measuring micro-nano particles, comprising: Placing an electrolyte in the device according to any one of claims 1 to 5; Placing the micro-nano particles to be measured in the first chamber or the second chamber; applying a voltage / current between the first electrode and the second electrode; Test the change of current / voltage signal flowing through the electrode; and At least one of the particle size, concentration or potential information of the micro / nano particles to be measured is determined according to the change of the current / voltage signal.

7. The method according to claim 6, comprising: Using a standard microsphere with a determined particle size and concentration, obtaining the peak value and frequency of the electric pulse signal generated by the microsphere in the device; Testing the peak value and frequency of the electric pulse signal generated by the micro-nano particles to be tested in the device; and The particle size and concentration of the micro-nano particles to be measured are obtained by the ratio of the peak values ​​and frequencies of the electric pulse signals of the standard microspheres and the micro-nano particles to be measured.

8. The method according to claim 6, comprising: Using a standard microsphere with a determined particle size, concentration and potential, obtaining the peak value, frequency and pulse width of the electric pulse signal generated by the microsphere in the device as claimed in claim 4; Testing the peak value, frequency and pulse width of the electric pulse signal generated by the micro-nanoparticles to be tested in the device; and The particle size, concentration and potential of the micro-nano particles to be measured are obtained by the ratio of the peak value, frequency and pulse width of the electric pulse signal of the standard microsphere and the micro-nano particles to be measured.

9. A nanopore chip, characterized in that: The invention comprises a substrate, a membrane and an electroosmotic groove modification layer; the substrate has a first surface and a second surface opposite to each other, and has an electroosmotic groove penetrating from the second surface to the first surface; A membrane is formed on the first surface, the membrane has at least one micropore, the pore size of the micropore is smaller than the pore size of the electroosmotic cell, and the projections of all the micropores on the first surface are covered by the projections of the electroosmotic cell on the first surface; the electroosmotic cell modification layer is formed on the side wall of the electroosmotic cell, and the interface of the electroosmotic cell modification layer has a specific Zeta potential when in contact with the electrolyte; Preferably, the electroosmotic cell modification layer is configured so that the zeta potential of the interface when immersed in the electrolyte is negative.

10. The nanopore chip according to claim 9, characterized in that: Also includes: A transition layer (4) is formed between the membrane (2) and the substrate (1), the transition layer (4) having a hollow structure or a through hole connecting the micropore (201) and the electroosmotic groove (101); and / or, A protective layer (5) is formed on the second surface, and the protective layer (5) has a hollow structure or a through hole connected to the electro-osmotic groove (101).

10. A method for preparing a nanopore chip, characterized in that: Providing a substrate (1); forming a film (2) on a first surface of the substrate (1), wherein the film (2) has at least one micropore (201); An electroosmotic groove (101) is formed on the second surface of the substrate (1) and penetrates to the first surface, and an electroosmotic groove modification layer (3) is formed on the side wall of the electroosmotic groove (101); the electroosmotic groove modification layer (3) is configured so that the Zeta potential of the interface immersed in the electrolyte is negative; The aperture of the micropores (201) is smaller than the aperture of the electroosmotic cell (101), and the projections of all the micropores (201) on the first surface are covered by the projections of the electroosmotic cell (101) on the first surface.

11. The manufacturing method according to claim 10, characterized in that: The electroosmosis slot modification layer (3) is an amorphous carbon layer or a carbon chain polymer layer; the electroosmosis slot modification layer (3) is formed after the electroosmosis slot (101); Wherein, forming an electroosmotic groove (101) on the second surface of the substrate (1) and penetrating to the first surface comprises: The substrate (1) is patterned by wet etching to form an electroosmotic groove (101) on the second surface of the substrate (1) that penetrates to the first surface; A negative electroosmotic tank modification layer (3) is formed on the side wall of the electroosmotic tank (101), comprising: The side wall of the electroosmotic groove (101) is passivated by using hydrocarbons or fluorocarbons in plasma to form an amorphous carbon layer or a carbon chain polymer layer as the electroosmotic groove modification layer (3).

12. The manufacturing method according to claim 10, characterized in that: The electroosmotic slot modification layer (3) is an amorphous carbon layer or a carbon chain polymer layer; the negative electroosmotic slot modification layer (3) is formed during the formation process of the electroosmotic slot (101); An electroosmotic groove (101) penetrating to the first surface is formed on the second surface of the substrate (1), and an electroosmotic groove modification layer (3) is formed on the side wall of the electroosmotic groove (101), comprising: The etching step w performs patterning on the substrate (1) by dry etching, so as to form a groove with a first depth on the second surface of the substrate (1); the first depth is less than the thickness of the substrate (1); A passivation step, using hydrocarbons or fluorocarbons in plasma to passivate the side walls of the groove body to form an amorphous carbon layer or a carbon chain polymer layer as the electroosmotic groove modification layer (3); The etching step and the passivation step are repeated until the depth of the groove body is equal to the thickness of the substrate (1), thereby forming an electroosmotic groove (101) penetrating to the first surface, and the electroosmotic groove modification layer (3) is formed on the side wall of the electroosmotic groove (101).

13. The manufacturing method according to claim 11 or 12, characterized in that: The fluorocarbon is octafluorocyclobutane.

14. The manufacturing method according to any one of claims 9 to 11, characterized in that: Also includes: The nanopore chip is immersed in an alkaline salt solution, a phosphate solution or a HEPES solution.

15. A method for analyzing low-density lipoprotein cholesterol, characterized in that: include: 1) Specific capture and separation of low-density lipoprotein cholesterol in samples; 2) recording and measuring the time when the low-density lipoprotein cholesterol particles obtained in step 1) pass through the micropores of the device according to any one of claims 1 to 5 A pulse signal of current or voltage is generated on the electrode, and the particle size distribution of each particle of low-density lipoprotein cholesterol in the sample and the particle concentration within each particle size range are analyzed according to the pulse signal; Wherein, the pore diameter of the micropore is not greater than 200 nm, and the pore depth of the micropore is not greater than 150 nm.

16. The analysis method according to claim 15, characterized in that In step 1), magnetic beads having antibodies or aptamers that specifically bind to surface membrane proteins of the low-density lipoprotein cholesterol to be detected are used to specifically capture the low-density lipoprotein cholesterol in the sample; Preferably, the antibody is an antibody against protein ApoB100; Preferably, after capture in step 1), the magnetic beads are magnetically adsorbed in a local area of ​​the magnetic field, and then a cleaning solution is added to replace the magnetic bead-sample mixture in the magnetic field area; Preferably, after replacing with the cleaning solution, a dissociation solution is added to the magnetic bead sample mixture to elute the magnetic beads, and the captured low-density lipoprotein cholesterol is separated from the magnetic beads; Preferably, the dissociation solution contains pancreatic enzyme, salicylic acid, glycine or citric acid; Preferably, the electroosmotic slot modification layer of the device is a silicon dioxide layer or a silicon nitride layer.

17. A method for tracing the particle concentration value of small-size nanoparticles, wherein the small-size micro-nanoparticles are nanoparticles with a particle size greater than or equal to 20 nm and less than or equal to 400 nm, characterized in that: The method comprises designing several intermediate samples with unknown particle concentration values ​​and particle sizes between the particle samples with known particle concentration values ​​and the small-diameter nanoparticle samples to be tested, and sorting the particle samples, the first intermediate sample, the second intermediate sample ... the Nth intermediate sample and the small-diameter micro-nanoparticle samples to be tested from large to small according to the particle sizes, where N is a positive integer greater than or equal to 1; at the same time, the design of the intermediate samples meets the condition that the particle sizes of the two adjacent samples after sorting meet the condition of being tested within the detection range of the same device as described in any one of claims 1 to 5; in descending order of particle sizes, the two adjacent samples are tested in the same device, and the particle concentration value of the sample with unknown particle concentration value in the adjacent samples is calculated according to the detection parameter values ​​of the adjacent samples combined with the particle concentration value of the sample with known particle concentration value in the adjacent samples; and by analogy, the particle concentration value of the small-diameter nanoparticle sample to be tested is finally obtained; wherein the particle concentration value calculation formula of the sample with unknown particle concentration value in the adjacent samples is: Among them, C 待测 is the particle concentration value of the sample with unknown particle concentration value in the adjacent samples, C 已知 is the particle concentration value of the sample with known particle concentration value among the adjacent samples, P 待测 P is the voltage / current pulse frequency of the unknown particle concentration value sample in the detection parameter value. 已知 The voltage / current pulse frequency of the sample with known particle concentration value in the detection parameter value; The conditions include: 1) Among two adjacent samples, the particle size of the sample with the larger particle size is less than or equal to 60% of the pore size of the device; 2) Among two adjacent samples, the baseline amplitude of the pulse signal of the sample with larger particle size is smaller than the pulse signal height of the sample with smaller particle size; 3) The depth of the micropores of the device is less than or equal to the pore diameter of the micropores and greater than or equal to one tenth of the pore diameter.

18. The tracing method according to claim 17, characterized in that: The particle sample with known particle concentration value is a particle standard substance; Preferably, the device comprises a micropore and an electroosmotic tank connected to the micropore, the inner wall of the electroosmotic tank comprises an electroosmotic tank modification layer, and the zeta potential of the interface when the electroosmotic tank modification layer contacts the electrolyte is a negative potential; Preferably, the electroosmotic slot modification layer is a silicon dioxide layer or a silicon nitride layer; Preferably, the two adjacent samples are detected by the same device respectively.

19. A method for calculating the Zeta potential of particles, characterized in that: Obtain detection electric pulse signals of several particle samples with known particle size and Zeta potential and record the pulse peak value and pulse width of the electric pulse signal The detection electric pulse signal is obtained by measuring the particles in the sample under the action of the electric field force through the detection area of ​​the device as claimed in any one of claims 1 to 5; A particle Zeta potential calculation model is constructed based on the particle size of the particles and the pulse peak value and pulse width of the electric pulse signal.

20. The calculation method according to claim 19, characterized in that: The method of constructing a particle Zeta potential calculation model based on the particle size and the pulse peak value and pulse width of the electric pulse signal of the particle includes: constructing a training sample set based on the particle size and the pulse peak value and pulse width of the electric pulse signal of the particle and the Zeta potential of the particle as modeling factors; performing several types of model training based on the training sample set, and evaluating the several types of models after training, and confirming the best particle Zeta potential calculation model according to the model evaluation index.

21. The calculation method according to claim 19, characterized in that: The calculation method includes: 1) Obtaining detection electric pulse signals of several known particle samples with different particle sizes and the same Zeta potential by the micro-nano particle detection device according to any one of claims 1 to 5 and recording the pulse peak value and pulse width of the electric pulse signal, and obtaining a first relationship based on the particle size of the particle and the pulse peak value of the electric pulse signal, wherein the first relationship is: A=a×D 3 , where A is the pulse peak value of the particle, D is the particle size of the particle, and a is the particle size coefficient; A second relationship is obtained based on the pulse peak value and pulse width of the electric pulse signal of the particle, and the second relationship is: W=b×A+Wn, wherein A is the pulse peak value of the particle, W is the pulse width of the particle, b is the pulse coefficient, and Wn is the pulse width correction coefficient; 2) Obtaining the detection electric pulse signals of several known particle samples with the same particle size and different Zeta potentials through the device in step 1) and recording the pulse width of the electric pulse signals, and obtaining a fourth relationship between the Zeta potential of the particles and the pulse peak value and pulse width of their electric pulse signals based on the Zeta potential of the particles and the pulse width of their electric pulse signals in combination with the first and second relationship, wherein the fourth relationship is: Z=d×(Wb×A) e , where Z is the absolute value of the particle Zeta potential, W is the pulse width of the particle, A is the pulse peak of the particle, b is the pulse coefficient, d is the potential coefficient, and e is the potential index.

22. The calculation method according to claim 21, characterized in that: The electroosmotic slot modification layer of the micro-nano particle detection device is a silicon dioxide layer or a silicon nitride layer; Preferably, the interface of the electroosmotic tank modification layer in contact with the electrolyte has a Zeta potential of an absolute value not greater than 10 mV; Preferably, when the interface of the electroosmotic cell modification layer in contact with the electrolyte has a Zeta potential with an absolute value greater than 10 mV, a pressure drive is applied between the first chamber and the second chamber, and the pressure is used to offset the electroosmotic flow, so that the particles rely on the traction of the electric field force to pass through the micropores.

23. A method for calculating the Zeta potential of a particle to be measured, characterized in that: Acquire a detection electric pulse signal when the particle to be detected passes through a detection area under the action of an electric field force by using a micro-nano particle detection device as described in any one of claims 1 to 5; Inputting the obtained pulse peak value and pulse width of the electric pulse signal into the particle Zeta potential calculation model calculated by the calculation method according to any one of claims 19 to 22 to obtain the Zeta potential of the particle to be measured; Preferably, the pulse peak value and pulse width of the obtained electric pulse signal are input into a fourth relationship to obtain the Zeta potential of the particle to be measured. The fourth relationship is Z=d×(Wb×A) e , where Z is the absolute value of the Zeta potential of the particle to be measured, W is the pulse width of the particle to be measured, A is the pulse peak value of the particle to be measured, b is the pulse coefficient, d is the potential coefficient, and e is the potential index.

24. A device for calculating the Zeta potential of particles to be measured, characterized in that: The computing device comprises: A signal acquisition module, used to acquire a detection electric pulse signal when the particle to be detected passes through the detection area of ​​the micro-nano particle detection device according to any one of claims 1 to 5 under the action of an electric field force, including a pulse peak value and a pulse width of the electric pulse signal; A signal processing module is used to input the obtained pulse peak value and pulse width into a particle Zeta potential calculation model calculated by the calculation method described in any one of claims 19-22 to obtain the Zeta potential of the particle to be measured.

25. A device for measuring micro-nano particles, comprising: a sample chamber having a common electrode; at least two measurement chambers adjacent to the sample chamber, including a first measurement chamber having a first measurement electrode, and a second measurement chamber having a second measurement electrode; a substrate separating the sample chamber and at least two measurement chambers; a film covering one side of the substrate; in: The membrane has at least a first micropore and a second micropore, The substrate has a first electroosmotic cell and a second electroosmotic cell, wherein the inner diameter of the first electroosmotic cell is larger than the aperture of the first micropore but smaller than the inner diameter of the first measurement chamber, and the inner diameter of the second electroosmotic cell is larger than the aperture of the second micropore but smaller than the inner diameter of the second measurement chamber. The sample chamber and the first measurement chamber are connected through the first micropore and the first electroosmotic cell. The sample chamber and the second measurement chamber are connected through the second micropore and the second electroosmotic cell. The inner wall of the first electroosmotic tank has a first electroosmotic tank modification layer, and the interface of the first electroosmotic tank modification layer has a specific first Zeta potential when in contact with the electrolyte, and The inner wall of the second electroosmotic cell has a second electroosmotic cell modification layer, and the interface of the second electroosmotic cell modification layer has a specific second Zeta potential when in contact with the electrolyte.

26. The device according to claim 25, characterized in that wherein the first micropore and the second micropore have different pore sizes; Preferably, the membrane covers the sample chamber side of the substrate; Preferably, the film covers the measurement chamber side of the substrate.

Images