Single-particle micro-nano particle detection device

Abstract

The invention relates to a device for measuring micro-nano particles. The device comprises a first chamber with a first electrode and a second chamber adjacent to the first chamber and provided with 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 is provided with micropores, the substrate is provided with an electroosmosis groove, and the electroosmosis groove is communicated with the first chamber and the second chamber. The first chamber is provided with a micropore, the inner diameter of the electroosmosis tank is larger than the aperture of the micropore and smaller than the inner diameter of the second chamber, the first chamber is communicated with the second chamber through the micropore and the electroosmosis tank, the inner wall of the electroosmosis tank is provided with an electroosmosis tank modification layer, and the interface of the electroosmosis tank modification layer has a specific zeta potential when the electroosmosis tank modification layer is in contact with an electrolyte.

Description

Technical Field

[0001] The present application relates to the technical field of micro-nano particle detection, and in particular to a device and method for detecting micro-nano particles. Background Art

[0002] Micro-nano particles (particle size is 1nm~10um, hereinafter referred to as micro-nano particles) show many specific physical behaviors due to their size-dependent effects, such as small size effect, surface effect, quantum size effect, etc. As the particle size decreases, the ratio of its surface area to volume becomes larger and larger, making micro-nano particles show unexpected characteristics and can be widely used in medicine, chemical industry, materials and other fields. Therefore, single particle detection technology that measures the size, particle size distribution, particle concentration, zeta potential and other properties of micro-nano particles is very important in the application of micro-nano particles.

[0003] Traditional single particle detection technologies include: electron microscopy, nano-tracer technology (NTA), nano-flow cytometry (NanoFCM), and resistive pulse sensing (RPS). Electron microscopy can intuitively measure the particle size of nanoparticles, but dry sample preparation is required before measurement, and the state of the sample in liquid cannot be displayed. The basic principle of nano-tracer technology (NTA) is to track the Brownian motion rules of nanoparticles in liquid by imaging, and to infer the particle size of nanoparticles by the speed of Brownian motion. Nano-tracer technology is affected by the hydrophilicity of the sample, the light scattering intensity of the sample, and the accuracy of image acquisition, resulting in inaccurate particle size measurement. The basic principle of nano-flow cytometry (NanoFCM) is to pass the sample particles through the laser individually in a sheath flow manner, and infer the particle size of the particles by detecting the scattered signal of the laser. Nano-flow detection is also affected by the difference in the optical properties of the particles, resulting in inaccurate testing.

[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, the resistance between the two electrodes inside and outside the small hole tube changes 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 concentration conditions (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-nano particles using the nano-Coulter technique. The basic principle is to calculate the charge by determining the value of the electric velocity component indicating the total velocity of the test particle, taking into account that the total velocity of the test particle includes a non-zero convective velocity component and an electric velocity component. Since the measured total velocity includes a non-zero convective velocity component, the method for calculating the charge of the test particle is relatively cumbersome. Summary of the invention

[0007] In one aspect, the present application provides a single-particle nanoparticle detection device, which can drive nanoparticles through nanopores (when the zeta of the electroosmotic tank modification layer is high) by using only the potential difference between two electrodes without other external driving forces. Under certain conditions (when the zeta of the electroosmotic tank modification layer is very weak), it can also be used for zeta potential detection of nanoparticles in high-concentration samples.

[0008] In one embodiment, the nanoparticle detection device provided by the present application includes:

[0009] a first chamber having a first electrode;

[0010] a second chamber adjacent to the first chamber and having a second electrode;

[0011] a substrate separating the first chamber and the second chamber;

[0012] a membrane covering the first chamber side of the substrate;

[0013] in:

[0014] The membrane has micropores.

[0015] The substrate has an electroosmotic groove, the inner diameter of which is larger than the aperture of the micropores but smaller than the inner diameter of the second chamber.

[0016] The first chamber and the second chamber are connected through the micropore and the electroosmotic tank, and

[0017] The inner wall of the electroosmotic cell is provided with an electroosmotic cell modification layer, and the interface of the electroosmotic cell modification layer has a specific zeta potential when in contact with an electrolyte.

[0018] In certain embodiments, the pore size of the micropores is 20 nm to 5 um.

[0019] In certain embodiments, the inner diameter of the electroosmotic cell is less than 3 mm.

[0020] In certain 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.

[0021] In certain embodiments, the depth of the electroosmotic cell is 5 times or more than the depth of the micropores.

[0022] In some embodiments, the device further comprises an electrolyte placed in the first chamber and the second chamber. In some embodiments, the electrolyte is a PBS solution.

[0023] In some embodiments, the electroosmotic slot modification layer has an interface with a zeta potential more negative than -20mV when in contact with the electrolyte. In some embodiments, the material of the electroosmotic slot modification layer is selected from the group consisting of silicon oxide, silicon nitride, polycrystalline silicon, single crystal silicon, and an adsorption layer formed by the adsorption of anions. In some embodiments, the electroosmotic slot modification layer has an interface zeta potential of -65±6mV when in contact with the electrolyte.

[0024] In some embodiments, the interface of the electroosmotic slot modification layer in contact with the electrolyte has a zeta potential of no greater than 10 mV in absolute value. In some embodiments, the material of the electroosmotic slot modification layer is selected from the group consisting of titanium oxide, aluminum oxide, and magnesium oxide. In some embodiments, the interface of the electroosmotic slot modification layer in contact with the electrolyte has a zeta potential of 0±3 mV.

[0025] 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 micropore.

[0026] In certain embodiments, the device further comprises a liquid driver, which can drive the flow of the electrolyte.

[0027] In another aspect, the present application provides a method for measuring micro-nano particles, comprising:

[0028] Placing an electrolyte in the device disclosed in the present application;

[0029] Placing the micro-nano particles to be measured in the first chamber or the second chamber;

[0030] applying a voltage / current between the first electrode and the second electrode;

[0031] Testing the change in current / voltage signal flowing through the electrodes; and

[0032] 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.

[0033] In certain embodiments, the 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 device; testing the peak value and frequency of the electric pulse signal generated by the micro-nanoparticle to be tested in the device; and obtaining the particle size and concentration of the micro-nanoparticle to be tested by the ratio of the peak value and frequency of the electric pulse signal of the standard microsphere and the micro-nanoparticle to be tested.

[0034] In certain embodiments, the method further 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 device; testing the peak value, frequency and pulse width of the electric pulse signal generated by the micro-nanoparticle to be tested in the device; and obtaining the particle size, concentration and potential of the micro-nanoparticle to be tested by the ratio of the peak value, frequency and pulse width of the electric pulse signal of the standard microsphere and the micro-nanoparticle to be tested.

[0035] In another aspect, the present application provides a device for measuring micro-nano particles, comprising:

[0036] a sample chamber having a common electrode;

[0037] 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;

[0038] a substrate separating the sample chamber and at least two measurement chambers;

[0039] a film covering one side of the substrate;

[0040] in:

[0041] The membrane has at least a first micropore and a second micropore,

[0042] 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.

[0043] The sample chamber is connected to the first measurement chamber through the first micropore and the first electroosmotic cell.

[0044] The sample chamber is connected to the second measurement chamber through the second micropore and the second electroosmotic cell.

[0045] 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

[0046] 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.

[0047] In certain embodiments, the first micropores and the second micropores have different pore sizes.

[0048] In some embodiments, the membrane covers the sample chamber side of the substrate. In some embodiments, the membrane covers the measurement chamber side of the substrate.

[0049] In some embodiments, the materials of the first electroosmotic slot modification layer and the second electroosmotic slot modification layer are the same. In some embodiments, the materials of the first electroosmotic slot modification layer and the second electroosmotic slot modification layer are different.

[0050] 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 invention, which is intended to be protected by the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 A schematic diagram of the micro-nano particle measurement device disclosed in the present application is shown.

[0052] Figure 2 Schematic diagram of the micro-nano particle measuring device used in Example 1.

[0053] Figure 3A The pulse signal of the polyethylene microspheres tested in Example 1 is shown.

[0054] Figure 3B The 15 nm standard microsphere sample (average particle size 15 nm, concentration 2×10 9 particles / mL).

[0055] Figure 4 Schematic diagram of the micro-nano particle measuring device used in Example 2.

[0056] Figure 5A The pulse signal of the magnetic beads tested in Example 2 is shown.

[0057] Figure 5B The pulse signal of the 800nm ​​standard microsphere sample tested in Example 2 is shown.

[0058] Figure 6 Schematic diagram of the micro-nano particle measuring device used in Example 3.

[0059] Fig. 7A It shows that the width time of the pulse signal of the silica microsphere to be tested in Example 3 is 0.2 mS.

[0060] Figure 7B The 150 nm standard microsphere sample (average particle size 150 nm, concentration 3×10 9 The width time of the pulse signal (particles / mL, average potential -30mV) is 0.4mS.

[0061] Fig. 8A The pulse signal of the 0.5 mm inner diameter electroosmotic cell detected in Example 4 is shown.

[0062] Figure 8B The pulse signal of the 1 mm inner diameter electroosmotic cell detected in Example 4 is shown.

[0063] Figure 8C The pulse signal of the 2 mm inner diameter electroosmotic cell detected in Example 4 is shown.

[0064] Fig.8D The pulse signal of the 3 mm inner diameter electroosmotic cell detected in Example 4 is shown.

[0065] Fig. 8E The pulse signal of the 4 mm inner diameter electroosmotic cell detected in Example 4 is shown.

[0066] Fig. 9A The pulse signal of the 0.025 mm depth electroosmotic cell detected in Example 5 is shown.

[0067] Fig. 9B The pulse signal of the 0.1 mm depth electroosmotic cell detected in Example 4 is shown.

[0068] Fig. 9C The pulse signal of the 0.4 mm deep electroosmotic cell detected in Example 4 is shown. DETAILED DESCRIPTION

[0069] The specific embodiments of the present application will be described in detail below. Although the present application will disclose multiple 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 multiple 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 meanings as those generally understood by ordinary technicians in the field to which the present application belongs. All references, patents, and patent applications cited in this application are incorporated herein by reference in their entirety.

[0070] In one aspect, 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 will occupy the same volume of electrolyte at the moment they pass through the nanopore. The resistance between the two electrodes inside and outside the pore (circuit designed with constant current) will change 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 number information of the particles in the detected sample can be obtained.

[0071] Figure 1 is a cross-sectional view of a device for measuring micro-nanoparticles according to an exemplary embodiment, which can drive nanoparticles through a nanopore by using only the potential difference between two electrodes without any other external driving force. Under certain conditions, the device can also be used for zeta potential detection of nanoparticles in high-concentration samples.

[0072] like Figure 1 As shown, in an exemplary embodiment, the device for detecting nanoparticles 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 film on one side of the first chamber. The film has micropores. The substrate has an electroosmotic groove, and 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. The inner wall of the electroosmotic groove has an electroosmotic groove modification layer.

[0073] In the measurement state, the first chamber, the electroosmotic tank and the second chamber are filled with electrolyte, and 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. In some embodiments, the electrolyte is a PBS solution.

[0074] It should be understood that the substrate described in this embodiment has a structure that isolates the first chamber and the second chamber and is made of a material that can grow a film covering the substrate thereon. In some embodiments, the material forming the substrate is silicon. In some embodiments, the material forming the substrate is boron nitride. In some embodiments, the thickness of the substrate is 25um to 1mm, such as 25um, 50um, 75um, 100um, 150um, 200um, 250um, 300um, 350um, 400um, 450um, 500um, 550um, 600um, 650um, 700um, 750um, 800um, 850um, 900um, 950um, 1000um 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-nano particles move through the electroosmotic tank during the measurement process.

[0075] In some embodiments, the film covering the substrate is formed of silicon nitride. In some embodiments, the film covering the substrate is formed of polysilicon.

[0076] It should be understood that, according to the function of the micropore, the setting of its pore size and pore depth is based on the basic premise that the micro-nano particles to be measured can be detected by the nano-Coulter principle. In some embodiments, the pore size of the micropore is 20nm to 5μm. In some embodiments, the pore size of the nanopore is 20nm, 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 200nm, 300nm, 400nm, 500nm, 600nm, 700nm, 800nm, 900nm, 1μm, 2μm, 3μm, 4μm, 5um or a value or range between any two of the above values. The pore size of the micropore described here refers to the distance in the direction perpendicular to the direction in which the micro-nano particles move through the micropore during the measurement process. In some embodiments, the shape of the micropore is circular. When the shape of the micropore is not circular, the pore size of the micropore refers to the maximum distance of the micropore in the direction perpendicular to the direction in which the micro-nano particles move through the micropore during the measurement process.

[0077] In some embodiments, the pore depth of the micropore is 20nm~5μm. In some embodiments, the pore depth of the nanopore is 20nm, 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 200nm, 300nm, 400nm, 500nm, 600nm, 700nm, 800nm, 900nm, 1μm, 2μm, 3μm, 4μm, 5um or the numerical value or range between any two values ​​above. 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 micro-nano particles moving through the micropore during the measurement process.

[0078] It should be understood that, according to the function of the electroosmotic cell, the setting of its inner diameter and depth should be based on the basic premise of being able 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.

[0079] In some embodiments, the inner diameter of the electroosmotic cell is 25um to 3mm, such as 25um, 50um, 75um, 100um, 150um, 200um, 250um, 300um, 350um, 400um, 450um, 500um, 550um, 600um, 650um, 700um, 750um, 800um, 850um, 900um, 1000um, 1500um, 2000um, 2500um, 3000um or a value or range between any two of the above values. The inner diameter of the electroosmotic cell described here refers to the distance in the direction perpendicular to the direction in which the micro-nano particles 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.

[0080] It is understood that in some embodiments, the depth of the electroosmotic groove is determined by the thickness of the substrate. In some embodiments, the depth of the electroosmotic groove is 5 times or more than the hole depth of the micropore. The embodiment of the present application shows that when the depth of the electroosmotic groove is lower than 5 times of the hole depth, the pulse frequency tested is very low. In some embodiments, the depth of the electroosmotic groove is 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more than the hole depth of the micropore.

[0081] In some embodiments, the interface of the electroosmotic slot modification layer has a zeta potential more negative than -20mV when in contact with the electrolyte. In some embodiments, the zeta potential value of the interface of the electroosmotic slot modification layer when in contact with the electrolyte is -65±6mV. The electroosmotic slot modification layer is negatively charged in the electrolyte, and due to the principle of electrical neutrality, the electrolyte in the electroosmotic slot will be positively charged. When a voltage is applied to the first electrode and the second electrode, an electric field will be formed in the electrolyte, and the positively charged electrolyte in the electroosmotic slot will move toward the first or second electrode as a whole under the action of the electric field, thereby driving the liquid in the second or first chamber to be replenished in the electroosmotic slot, and then driving the micro-nano particles in the chamber to pass through the micropores. Thus, under the condition that there is no other driving force between the first chamber and the second chamber, the micro-nano particles in the chamber can also be enabled to pass through the micropores by this mechanism.

[0082] In some embodiments, the material of the electroosmotic slot modification layer is selected from the group consisting of silicon oxide, silicon nitride, polycrystalline silicon, single crystal silicon, and adsorption layers formed by adsorption of anions thereon. In some embodiments, the electroosmotic slot modification layer may also cover the interface between the microporous membrane and the electroosmotic slot and the inner wall of the micropores.

[0083] In some embodiments, the electroosmotic slot modification layer has an interface with an absolute value of no more than 10 mV when in contact with the electrolyte. In some embodiments, the electroosmotic slot modification layer has an interface with an absolute value of no more than 10 mV when in contact with the electrolyte. In some embodiments, the electroosmotic slot modification layer has an interface with a zeta potential of 0±3 mV when in contact with the electrolyte. In some embodiments, the material of the electroosmotic slot modification layer is selected from the group consisting of titanium oxide, aluminum oxide, and magnesium oxide.

[0084] In this embodiment, 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 does not flow. When a voltage is applied to the first electrode and the second electrode (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, and the negatively charged particles in the electrolyte in all chambers move in the direction of the first electrode under the action of the electric field, and the positively charged particles in the electrolyte in all chambers move in the direction of the second electrode under the action of the electric field. Thus, when there are negatively charged particles in the second chamber, they will pass through the electroosmotic tank under the action of the electric field, and then pass through the micropores; when there are positively charged particles in the first chamber, they will pass through the micropores under the action of the electric field. Under the condition that there is no other driving force between the first chamber and the second chamber, the micro-nano particles are realized through the micropores by this mechanism.

[0085] At the same time, under the above mechanism, the speed at which particles pass through the micropores 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 micropores is directly proportional to the potential of the particles, and the time it takes for particles to pass through the micropores is inversely proportional to the potential of the particles.

[0086] In certain embodiments, the device further comprises a liquid driver, which can drive the flow of the electrolyte.

[0087] In another aspect, the present application provides a device for measuring micro-nano particles using multiple micropores and measurement chambers, which includes a sample chamber with a common electrode and at least two measurement chambers adjacent to the sample chamber, including a first measurement chamber with a first measurement electrode, and a second measurement chamber with a second measurement electrode. The sample chamber and the at least two measurement chambers are separated by a substrate, and a membrane covering the substrate is provided on one side of the substrate. The membrane has at least a first micropore and a second micropore. The substrate has a first electroosmotic groove and a second electroosmotic groove, the inner diameter of the first electroosmotic groove is larger than the pore diameter of the first micropore and smaller than the inner diameter of the first measurement chamber, and the inner diameter of the second electroosmotic groove is larger than the pore diameter of the second micropore and 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 groove. The sample chamber is connected to the second measurement chamber through the second micropore and the second electroosmotic groove. The inner wall of the first electroosmotic groove has a first electroosmotic groove modification layer, and the interface of the first electroosmotic groove modification layer has a specific first zeta potential when in contact with an electrolyte. 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.

[0088] It is understood that the sample chamber and the measurement chamber described herein may have the features of the first chamber and the second chamber described above. The substrate, membrane, the first micropore, the second micropore, the first electroosmotic cell, the second electroosmotic cell, the first electroosmotic cell modification layer and the 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.

[0089] It is understood that the pore sizes 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.

[0090] It is understood that the membrane described herein can be covered on the sample chamber side of the substrate or the measurement chamber side of the substrate. When the membrane covers the sample chamber side of the substrate, the sample chamber and the measurement chamber are connected through the micropores and the electroosmotic groove in turn. When the membrane covers the measurement chamber side of the substrate, the sample chamber and the measurement chamber are connected through the electroosmotic groove and the micropores in turn.

[0091] It is to be understood that the first electroosmotic cell modification layer and the second electroosmotic cell modification layer described herein may be the same or different.

[0092] In another aspect, the present application provides a method for measuring micro-nano particles using nano-Coulter and electroosmotic principles, comprising:

[0093] 1) Placing the micro-nanoparticles to be tested in the device disclosed in the present application;

[0094] 2) applying a voltage / current between the first electrode and the second electrode;

[0095] 3) Testing the change of current / voltage signal flowing through the electrodes; and

[0096] 4) Determine the particle size, concentration or potential information of the micro-nano particles to be measured according to the change of the current / voltage signal.

[0097] In some embodiments, the particle size of the micro-nanoparticles measured by the method is between 1 nm and 2 um, 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.

[0098] In certain embodiments, the 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 device; testing the peak value and frequency of the electric pulse signal generated by the micro-nanoparticle to be tested in the device; and obtaining the particle size and concentration of the micro-nanoparticle to be tested by the ratio of the peak value and frequency of the electric pulse signal of the standard microsphere and the micro-nanoparticle to be tested.

[0099] In certain embodiments, the method further 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 device; testing the peak value, frequency and pulse width of the electric pulse signal generated by the micro-nanoparticle to be tested in the device; and obtaining the particle size, concentration and potential of the micro-nanoparticle to be tested by the ratio of the peak value, frequency and pulse width of the electric pulse signal of the standard microsphere and the micro-nanoparticle to be tested.

[0100] The present application is further described below by way of examples, but the present application is not limited to the scope of the examples. The experimental methods without specific conditions in the following examples are selected according to conventional methods and conditions, or according to the product instructions.

[0101] Example 1: Using the device disclosed in this application to detect the particle size and concentration of polyethylene microsphere particles

[0102] A 30nm silicon nitride film is grown on a single crystal silicon substrate with a size of 5mm×5mm and a thickness of 0.5mm. A groove with an inner diameter of 2mm is opened on the silicon substrate, and the groove depth is 0.5mm to the silicon nitride film as an electro-osmosis groove. A layer of silicon dioxide is plated on the inner wall of the electro-osmosis groove as a modification layer of the electro-osmosis groove. In the center of the silicon nitride membrane directly opposite the electro-osmosis groove, there is a nanopore with an inner diameter of 20nm.

[0103] like Figure 2 As shown, the silicon wafer is placed vertically in a plastic container, and the container is divided horizontally into two chambers, namely the first chamber and the second chamber, and the first chamber and the second chamber respectively contain the first electrode and the second electrode. The first chamber, the nanopore, the electroosmotic tank, and the second chamber are all filled with PBS electrolyte, and the PBS electrolyte in the first chamber contains polyethylene microsphere particles to be tested.

[0104] The zeta potential of the interface of the electroosmotic cell modified layer in PBS electrolyte was measured to be -65mV using an atomic force microscope.

[0105] When the device is not in use, the electroosmotic cell modification layer is kept in a phosphate aqueous solution to keep the zeta potential value of the interface stable.

[0106] like Figure 2 As shown, the modified layer of the electroosmotic tank is negatively charged in the electrolyte. Due to the principle of electrical neutrality, the electrolyte in the electroosmotic tank will be positively charged. 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 will be formed in the electrolyte. The positively charged electrolyte in the electroosmotic tank will move toward the second electrode as a whole under the action of the electric field, thereby driving the liquid in the first chamber to pass through the nanopores to replenish the electroosmotic tank, and then driving the polyethylene microspheres in the first chamber to pass through the nanopores. 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.

[0107] like Figure 3A As shown, the pulse signal of polyethylene microspheres was tested.

[0108] like Figure 3B As shown, the 15nm standard microsphere sample (average particle size 15nm, concentration 2×10 9 particles / mL) in the measuring device.

[0109] 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.

[0110]

[0111] 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.

[0112] The frequency of the pulse signal measured in the device is directly proportional to the particle concentration in the sample.

[0113] The pulse signal frequency measured by the 15nm standard microsphere sample is 90 / min, and the pulse signal frequency measured by the polyethylene microsphere sample to be tested is 60 / min. It can be calculated that the concentration of the polyethylene microsphere sample to be tested is 1.333×10 9 particles / mL.

[0114] Example 2: Using the device disclosed in this application to detect the particle size and concentration of magnetic bead microsphere particles

[0115] A 1um polysilicon film is grown on a boron nitride substrate with a size of 10mm×10mm and a thickness of 1mm. A groove with an inner diameter of 3mm is opened on the boron nitride substrate, and the groove depth is 1mm to the polysilicon film as an electro-osmosis groove. A layer of silicon nitride is plated on the inner wall of the electro-osmosis groove, and the silicon nitride layer is immersed in a sodium perchlorate solution to adsorb anions as an electro-osmosis groove modification layer. In the center of the polysilicon film facing the electro-osmosis groove, there is a nanopore with an inner diameter of 5um.

[0116] like Figure 4 As shown, the boron nitride sheet is placed vertically in a plastic container, and the container is divided horizontally into two chambers, namely the first chamber and the second chamber, and the first chamber and the second chamber respectively contain the first electrode and the second electrode. The first chamber, the nanopore, the electroosmotic tank, and the second chamber are all filled with NaCl aqueous solution, and the magnetic bead microsphere particles to be tested are contained in the PBS electrolyte in the second chamber.

[0117] The zeta potential of the electroosmotic cell modified layer in NaCl aqueous solution was measured to be -25 mV using an atomic force microscope.

[0118] When the device is not in use, the electroosmotic cell modification layer is stored in a sodium perchlorate aqueous solution to keep the zeta potential value of the interface stable.

[0119] like Figure 4As shown, in the NaCl aqueous solution, the modified layer of the electroosmotic tank is negatively charged. Due to the principle of electrical neutrality, the electrolyte in the electroosmotic tank will be positively charged. 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 positive voltage), an electric field will be formed in the electrolyte. The positively charged electrolyte in the electroosmotic tank will move toward the first electrode as a whole under the action of the electric field, thereby driving the liquid in the second chamber into the electroosmotic tank, and then driving the magnetic beads in the second chamber into the electroosmotic tank, and finally through the nanopore. 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 magnetic beads in the second chamber to pass through the nanopore.

[0120] like Figure 5A As shown, the pulse signal generated by the magnetic beads in the device was tested.

[0121] Figure 5B The pulse signal of the tested 800nm ​​standard microsphere sample is shown.

[0122] Similarly to Example 1, the particle size and concentration of the magnetic microspheres can be calculated.

[0123] Example 3: Detecting the zeta potential of silica microsphere particles using the device disclosed in this application

[0124] A 1um polysilicon film is grown on a boron nitride substrate with a size of 5mm×5mm and a thickness of 0.5mm. A groove with an inner diameter of 1mm is opened on the boron nitride substrate, and the groove depth is 0.5mm to the polysilicon film as an electro-osmosis groove. A titanium oxide layer is plated on the inner wall of the electro-osmosis groove, and the titanium oxide serves as the electro-osmosis groove modification layer. In the center of the polysilicon film facing the electro-osmosis groove, there is a nanopore with an inner diameter of 600nm.

[0125] like Figure 6 As shown, the boron nitride sheet is placed vertically in a plastic container, and the container is divided horizontally into two chambers, namely the first chamber and the second chamber, and the first chamber and the second chamber respectively contain the first electrode and the second electrode. The first chamber, the nanopore, the electroosmotic tank, and the second chamber are all filled with PBS solution, and the PBS electrolyte in the second chamber contains the silica microsphere particles to be tested.

[0126] The zeta potential of the electroosmotic cell modified layer in PBS solution was measured using an atomic force microscope and found to be -0.3 mV.

[0127] 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.

[0128] like Figure 6As shown, in the PBS solution, the electroosmotic tank modification layer is basically uncharged, 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 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 will be formed in the electrolyte, and the negatively charged particles in the electrolyte in all chambers will move toward the first electrode under the action of the electric field, and the positively charged particles in the electrolyte in all chambers will move toward the second electrode under the action of the electric field. Thus, when there are negatively charged particles in the second chamber, they will pass through the electroosmotic tank under the action of the electric field, and then pass 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. Under the condition that there is no other driving force between the first chamber and the second chamber, the microsphere particles can pass through the nanopore through this mechanism.

[0129] 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 force. 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.

[0130] like Fig. 7A As shown, the width time of the pulse signal of the silica microsphere to be tested is 0.2 mS.

[0131] When using a 150nm standard microsphere sample (average particle size 150nm, concentration 3 10 9 particles / mL, average potential -30mV) test, the width of the pulse signal was 0.4mS ( Figure 7B ).

[0132] It can be approximately calculated that the potential of the silica microspheres to be tested is -60mV.

[0133] Similarly, similar to Example 1, the particle size and concentration of the silica microspheres to be tested can be calculated through the peak value and frequency of the pulse signal.

[0134] Example 4: Effect of the inner diameter of the electroosmotic cell on the device

[0135] A 1um polysilicon film is grown on a boron nitride substrate with a size of 5mm×5mm and a thickness of 0.5mm. A groove with an inner diameter of 1mm is opened on the boron nitride substrate, and the groove depth is 0.5mm to the polysilicon film as an electro-osmosis groove. A titanium oxide layer is plated on the inner wall of the electro-osmosis groove, and the titanium oxide serves as the electro-osmosis groove modification layer. In the center of the polysilicon film facing the electro-osmosis groove, there is a nanopore with an inner diameter of 600nm.

[0136] When the above chip was used to test 200nm polyethylene microspheres, the following results were obtained: Figures 8A-8EAnd the frequency data shown in Table 1. It can be seen that when the inner diameter of the electroosmotic cell is greater than 3mm, the pulse frequency tested is very low.

[0137] Table 1. Pulse frequencies tested for different electroosmotic cell inner diameters

[0138] Inner diameter of electroosmosis cell 0.5mm 1mm 2mm 3mm 4mm Pulse signal frequency 35 pieces / minute 16 pieces / minute 6 pieces / minute 3 pieces / minute 1 piece / minute

[0139] Example 5: Effect of electroosmotic cell depth on the device

[0140] A 5um silicon nitride film is grown on a single crystal silicon substrate with a size of 5mm×5mm and a thickness of 25um, 100um, and 400um. A groove with an inner diameter of 25um is opened on the silicon substrate, and the groove depth is 25um, 100um, and 400um to the silicon nitride film as an electro-osmosis groove. A layer of silicon dioxide is plated on the inner wall of the electro-osmosis groove as a modification layer of the electro-osmosis groove. In the center of the silicon nitride membrane facing the electro-osmosis groove, there is a nanopore with an inner diameter of 5um.

[0141] When the above chip was used to test 800nm ​​polyethylene microspheres, the following results were obtained: Figures 9A-9C And the frequency data shown in Table 2. It can be seen that when the depth of the electroosmotic cell is less than 5 times the pore depth, the pulse frequency tested is very low.

[0142] Table 2. Pulse frequencies measured at different electroosmotic cell depths

[0143] Electroosmotic cell depth 25um 100um 400um Pulse signal frequency 3 pieces / minute 18 pieces / minute 96 pieces / minute

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; in: The membrane has micropores. The substrate has an electroosmotic groove, the inner diameter of which is larger than the aperture of the micropores but smaller than the inner diameter of the second chamber. The first chamber and the second chamber are connected through the micropore and the electroosmotic tank, and The inner wall of the electroosmotic cell is provided with an electroosmotic cell modification layer, and the interface of the electroosmotic cell modification layer has a specific zeta potential when in contact with an electrolyte. 2 . The device according to claim 1 , wherein the pore size of the micropore is 20 nm to 5 um.

3. The device according to claim 1, wherein the inner diameter of the electroosmotic cell is less than 3 mm. 4 . The device according to claim 1 , wherein the interface of the electroosmotic cell modification layer has a zeta potential more negative than −20 mV when in contact with the electrolyte. 5 . The device according to claim 4 , wherein the material of the electroosmotic slot modification layer is selected from the group consisting of silicon oxide, silicon nitride, polycrystalline silicon, single crystal silicon, and adsorption layers formed by adsorption of anions thereon. 6 . The device according to claim 4 , wherein the zeta potential value of the interface of the electroosmotic slot modification layer when in contact with the electrolyte is -65±6 mV.

7. The device according to claim 1, wherein the depth of the electroosmotic cell is 5 times or more than the pore depth of the micropore.

8. 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.

9. The device according to claim 8, wherein the material of the electroosmotic cell modification layer is selected from the group consisting of titanium oxide, aluminum oxide, and magnesium oxide. 10 . The device according to claim 8 , wherein the zeta potential value of the interface of the electroosmotic slot modification layer when in contact with the electrolyte is 0±3 mV.

11. The device according to claim 8, wherein the electroosmotic cell modification layer also covers the interface between the membrane and the electroosmotic cell and the inner wall of the micropore.

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

13. A method for measuring micro-nano particles, comprising: Placing an electrolyte in the device of claim 1; 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 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. 14 . The method according to claim 13 , wherein the interface of the electroosmotic cell modification layer has a zeta potential more negative than −20 mV when in contact with the electrolyte. 15 . The method according to claim 14 , wherein the material of the electroosmotic slot modification layer is selected from the group consisting of silicon oxide, silicon nitride, polycrystalline silicon, single crystal silicon, and adsorption layers formed by adsorption of anions thereon. 16 . The method according to claim 14 , wherein the zeta potential value of the interface of the electroosmotic cell modification layer when in contact with the electrolyte is −65±6 mV.

17. The method according to claim 14, 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.

18. The method according to claim 13, wherein the interface of the electroosmotic cell modification layer and the electrolyte has a zeta potential of an absolute value not greater than 10 mV when in contact with the electrolyte.

19. The method according to claim 18, wherein the material of the electroosmotic cell modification layer is selected from the group consisting of titanium oxide, aluminum oxide, and magnesium oxide. 20 . The method according to claim 18 , wherein the zeta potential value of the interface of the electroosmotic cell modification layer when in contact with the electrolyte is 0±3 mV.

21. The method of claim 18, comprising: Using a standard microsphere with a determined particle size, concentration and potential, the peak value, frequency and pulse width of the electric pulse signal generated by the microsphere in the device are obtained; 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.

22. 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. The device according to claim 22 , wherein the first micropore and the second micropore have different pore sizes.

24. The device of claim 22, wherein the membrane covers the sample chamber side of the substrate.

25. The device of claim 22, wherein the membrane covers the measurement chamber side of the substrate.

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