A method for measuring the mass of single particles / cells based on bubble micromotor

Abstract

The application discloses a method for measuring the mass of single particles / cells based on bubble micromotor. The application introduces transient inertia effect through micro-bubble collapse, and inverses the mass / density of cells based on the mechanical response of the cells under the inertia effect. High-speed microscopic imaging and handle control can be used to control the motion of the bubble micromotor in real time, so that the measurement of the micro particles to be measured can be rapidly completed, and the application has the advantages of strong operability, rapid sensitivity and high resolution. The application can provide a mass resolution with a precision of sub-nanogram (about 10 ‑10 g) order and a density resolution of 0.05 g / cm 3 .

Description

Technical Field

[0001] This invention belongs to the field of microfluidics applications in the measurement of the mass / density of tiny objects, specifically relating to a method for measuring the mass of a single particle / cell based on a bubble micromotor. Background Technology

[0002] The mass and density of single particles are crucial for quality control of synthesized micro / nanoparticle products. Changes in cell mass and density are also closely related to overall health and metabolic function. While we weigh objects daily using methods like balances, weighing very small biological samples such as single cells is extremely difficult. This is because these samples are typically on the scale of 1–100 μm and have a mass range of 10 pg–100 ng, far below the weighing limit of balances and existing in liquid environments incompatible with them. Nevertheless, it is essential to develop a method for measuring single-cell mass in liquid environments. For example, based on single-cell mass information, we can identify cell growth stages, monitor cell health, and perform drug sensitivity tests on cells.

[0003] Currently, microcantilever beam sensors operating in resonance mode are among the few methods for weighing minute masses. The weighing principle is based on the dependence of the total mass of the cantilever on its resonant frequency; the smaller the mass of the cantilever, the more sensitive it is to changes in added mass. However, this method is severely limited in fluid environments, especially liquid environments. This is because viscous damping plays a dominant role, significantly reducing measurement accuracy and limiting the lower limit of detectable masses. The resonance mode microcantilever beam sensor weighing method has an indirect mapping relationship with the measured cell mass; it obtains the mass increment of the cantilever, i.e., the buoyancy or "dry mass" of the cell, not the true mass or inertial mass. Secondly, manufacturing the cantilever beam sensor involves an exceptionally complex microfabrication process, and when a pressure gradient is applied at both ends of the microchannel to form a flow pair for cell loading, the cell will experience excessively high pressure and shear forces. Furthermore, the cantilever beam sensor needs to operate in a vacuum environment, which necessitates that it be a closed measurement system, making integration with other technologies difficult. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for measuring the mass of a single particle / cell based on a bubble micromotor. This invention introduces instantaneous inertial action through microbubble collapse, and inversely calculates the mass / density of the cell based on its mechanical response under inertial action. High-speed microscopic imaging and handheld manipulation allow for real-time control of the bubble micromotor's movement, enabling rapid measurement of the microparticles being measured. This method offers advantages such as high operability, rapid sensitivity, and high resolution. This invention can provide measurements down to sub-nanocels (~10). -10 g) level precision in mass resolution and 0.05 g / cm3 Density resolution.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A method for measuring the mass of a single particle / cell based on a bubble micromotor includes the following steps:

[0007] Step 1: Fabrication of a magnetic micromotor, including: first, preparing a dilute solution of SiO2 colloidal particles; second, using a spin coating technique, spin coating the dilute solution of SiO2 colloidal particles onto a single-polished superhydrophilic silicon wafer to obtain a single layer of densely distributed SiO2 colloidal particles; finally, using electron beam evaporation deposition technology or magnetron sputtering deposition technology, sequentially depositing a 20nm Ni layer and a 20nm Pt layer on the surface of the SiO2 colloidal particles to obtain a magnetic micromotor with an asymmetric structure.

[0008] Step 2: Perform bubble-driven operation of the magnetic micromotor, including: placing the magnetic micromotor prepared in Step 1 in a hydrogen peroxide solution of appropriate concentration, and generating a single bubble through a chemical reaction on the Pt side, thereby driving the micromotor to move.

[0009] Step 3: Observe and control the movement of the bubble-driven magnetic micromotor, including: placing the bubble-driven magnetic micromotor under a microscope equipped with a high-speed camera for motion observation, and using a handle to change the direction of the magnetic field to adjust the direction of the micromotor's movement in real time.

[0010] Step 4: Measure the mass / density of microparticles / cells based on bubble micromotors, including: observing the relative position of the bubble-driven magnetic micromotor and the microparticles / cells to be measured under a microscope platform; adjusting the movement direction of the magnetic micromotor in real time through a handle so that the bubbles generated by the micromotor can effectively push the microparticles / cells to be measured; and recording the displacement changes of the microparticles / cells to be measured under the push of the bubbles with a high-speed camera.

[0011] Step 5: Process the displacement velocity of the microparticles / cells to be tested, including: using a high-speed camera under a microscope platform to record the motion information of the microparticles / cells to be tested, and using image processing software and particle motion analysis software to analyze the motion of the microparticles / cells to be tested, thereby obtaining the velocity Vp of the cells / particles to be tested during the bubble collapse process as a function of time t.

[0012] Step 6: Invert the density / mass of the particles / cells to be tested by resolving the speed;

[0013] Step 7: Obtain the unknown parameter τ of the target particle by fitting the experimental data. p The initial velocity of the fluid uf(0) is used; the relaxation time τ of the target particle is obtained through two-parameter fitting.p The density ρ of microparticles / cells to be measured p By τ p =2R p 2 ρ p The inertial mass is calculated as / 9μ, which is the volume of the microparticle / cell being measured multiplied by the density ρ. p get.

[0014] Furthermore, in step 1, the particles are microparticles of different materials, including polystyrene, glass microspheres, and TiO2, with a particle diameter of 5-100 μm.

[0015] Furthermore, in step 2, the suitable concentration is 2%-15%.

[0016] Furthermore, in step 3, a uniform magnetic field in three-dimensional space is provided by setting up a three-dimensional Helmholtz coil on the microscopic platform, and magnetic field commands are integrated through the handle to control the magnetic micromotor driven by the bubble.

[0017] Furthermore, in step 4, the cells are cultured in a buffer environment, then diluted before testing, mixed with a low-concentration hydrogen peroxide solution, and then subjected to rapid experimental measurements.

[0018] Further, step 6 includes: establishing a kinetic equation for the phases of the movement of the microparticles / cells to be tested driven by bubble collapse, resulting in:

[0019]

[0020] Where m p Rp and Vp are the inertial mass, radius, and velocity of the particle / cell being tested, respectively, and μ is the viscosity of the surrounding solution. f (t) is the velocity of the fluid surrounding the target object, which varies with time t;

[0021] u f (t)=(c1e -t / τ1 +c2e -t / τ2 (2)

[0022] Wherein, parameters c1 and c2 are the initial velocities of the fluid during the bubble collapse stage (pullback and forward push phases), respectively, determined by the initial fluid velocity at the time of measurement, uf(0)=c1+c2, and the parameters are fitted according to the measurement; τ1 and τ2 are the characteristic times of the pullback phase and the forward push phase, respectively; where τ1≈0.5τ b ,τ b The characteristic time for bubble fusion, R bρ is the maximum radius of the bubble, γ is the surface tension coefficient of the bubble, ρ is the density of the solution; τ2 is 100-200 μs;

[0023] τ p =2R p 2 ρ p / 9μ (3)

[0024] Substituting formulas (2) and (3) into formula (1), we get:

[0025]

[0026] in, t is the measurement time, C is a constant, and Vp and τ p The velocity and relaxation time of the particles / cells to be tested are defined as follows, while other parameters are defined in the same way as above.

[0027] Furthermore, the particles are based on silica or hollow glass microspheres, with a chemically reacted metal layer and a magnetic layer covering half of the microspheres.

[0028] Furthermore, the diameter of the magnetic micromotor is 5μm to 100μm, the thickness of the Pt metal layer is 5-20nm, and the thickness of the Ni magnetic layer is 10-100nm. The reaction layer can also be Mg.

[0029] Furthermore, the magnetic field control of the bubble micromotor is based on the real-time control of the magnetic field direction by the handle, thereby realizing the real-time control of the bubble micromotor.

[0030] Furthermore, the bubble drive of the target microparticle is achieved by adjusting the movement direction of the bubble micromotor so that the bubble side is located between the micromotor and the target microparticle, thereby driving the target microparticle with the bubble.

[0031] Furthermore, the motion information of the microparticles is recorded based on the position information of the target particles driven by the bubble micromotor in the microscopic platform by a high-speed camera. The frame rate is 100,000 to 450,000 frames per second, according to the bubble collapse characteristic time and the particle relaxation characteristic time.

[0032] The beneficial effects of this invention are as follows:

[0033] 1. This invention proposes a new method for measuring the mass and density of single particles and cells, replacing the original microcantilever beam measurement method. It features simple design, strong operability, and high accuracy.

[0034] 2. This invention introduces considerable inertial forces at the microscale, which helps improve the differentiation of particles in microfluidic systems, thereby enabling precise manipulation of individual test particles. By changing the properties of the bubbles generated in the reaction, such as generating carbon dioxide bubbles in an acidic solution, in-situ measurement of single cells can be achieved in different systems;

[0035] 3. This invention utilizes a game controller to switch the magnetic field direction in real time, controlling the movement direction and mode of the magnetic micromotor, thereby enabling rapid target particle retrieval and improving measurement efficiency.

[0036] This invention differs from previous methods for measuring the mass / density of microparticles / single cells using microcantilever sensors. It introduces instantaneous inertial forces through microbubble collapse, and inversely calculates the mass / density of cells based on their mechanical response under inertial influence. High-speed microscopic imaging and handheld manipulation allow for real-time control of the bubble micromotor, enabling rapid measurement of the microparticles being measured. This invention offers advantages such as high operability, rapid sensitivity, and high resolution. It can provide measurements down to sub-nanocels (~10). -10 g) level precision in mass resolution and 0.05 g / cm 3 Density resolution. Attached Figure Description

[0037] The present invention will now be described in further detail with reference to the accompanying drawings and specific implementation methods.

[0038] Figure 1 This is a schematic diagram illustrating the principle of a method for measuring the mass of a single particle / cell based on a bubble micromotor according to the present invention.

[0039] Figure 2 This is a fabrication diagram of a magnetic micromotor.

[0040] Figure 3 This is a schematic diagram of bubble driving and control using a micro motor.

[0041] Figure 4 This is a verification diagram based on standard particles for this method.

[0042] Figure 5 This is a schematic diagram illustrating the actual density measurement of particles of different sizes and densities. Detailed Implementation

[0043] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Specific descriptions are provided below with reference to examples. The implementation conditions used in the embodiments can be adjusted according to experimental requirements; implementation conditions not specified are typically those used in conventional experiments.

[0044] like Figure 1As shown, the principle of the present invention, a method for measuring the mass of a single particle / cell based on a bubble micromotor, lies in manipulating the bubble micromotor to apply a transient high-speed force to the target particle, and then inverting the density and mass based on the velocity-inertial response of the target. This embodiment applies a platinum-hollow glass microsphere (Pt-HGM) bubble micromotor to measure the density and mass of inorganic particles and embryos. Specifically, it includes the following steps:

[0045] Step 1: Fabrication of Pt-HGM Magnetic Micromotors: First, a hollow glass microsphere (HGM) particle solution was prepared. Then, using a spin-coating technique, the dilute HGM particle solution was spin-coated onto a single-layer, superhydrophilic silicon wafer to obtain a single layer of densely distributed HGM particles. Finally, Ni and Pt layers were sequentially deposited on the surface of the HGM particles using electron beam evaporation deposition technology to obtain an asymmetric magnetic micromotor, such as... Figure 2 As shown. Preferably, the HGM particles have a diameter of about 10 micrometers, and the Ni layer and Pt layer are 20 nm and 10 nm, respectively.

[0046] Step 2: Bubble Drive of the Pt-HGM Magnetic Micromotor: The Pt-HGM magnetic micromotor prepared in Step 1 is placed in a hydrogen peroxide (H2O2) solution. A single bubble is generated on the Pt side through a chemical reaction, thereby driving the micromotor's movement. Preferably, the concentration of H2O2 is in the range of 2%-5%.

[0047] Step 3: Motion Observation and Control of the Bubble Micromotor: The bubble-driven Pt-HGM magnetic micromotor described above was placed under a microscope equipped with a high-speed camera (Phantom TMX7510) for motion observation. A self-made three-dimensional Helmholtz coil was installed on the microscope platform to provide a uniform magnetic field in three-dimensional space. The magnetic field commands were integrated into the handle, allowing control of the bubble-driven Pt-HGM magnetic micromotor. Preferably, the shooting frame rate was 450,000 fps, the observation objective was 20x, the NA was 0.7, the image resolution was 0.54 micrometers, and the magnetic field strength was approximately 20 mT. The motion control of the bubble micromotor is as follows: Figure 3 As shown. Figure 3 In the diagram, the spiral represents the trajectory of the micromotor without an applied magnetic field, the straight line represents the trajectory of the micromotor with an applied magnetic field, and the arrow indicates the direction of the applied uniform magnetic field.

[0048] Step 4: Measure the mass / density of microparticles using a bubble-driven Pt-HGM magnetic micromotor: Select homopolymer styrene (PS) particles with a known density of 1.05 g / cm³. 3The method was calibrated, and the diameters of the PS particles were observed under a microscope to be 19.8 μm, 25.4 μm, and 35.1 μm. The relative positions of the bubble-driven Pt-HGM magnetic micromotor and the test particles were observed under a microscope platform. The movement direction of the bubble-driven Pt-HGM magnetic micromotor was adjusted in real time via a handle, ensuring that the bubbles generated by the micromotor could effectively push the test particles, i.e., the bubbles were positioned between the PS particles and the Pt-HGM magnetic micromotor. The displacement changes of the test particles under the pushing of the bubbles were recorded using a high-speed camera (450,000 fps).

[0049] Step 5: The motion of the PS particles under test can be analyzed using particle motion analysis software (Video SpotTracker) to process the displacement velocity of the particles: the initial frame of the particles is set to (x k0 ,y k0 ), the position coordinates of the next frame (x k1 ,y k1 Similarly, the instantaneous velocity of the particles can be calculated by removing bits between two adjacent frames, using the time interval between the two frames. This allows us to obtain the velocity Vp of the tested cell / particle as a function of time t during the bubble collapse process. Figure 4 As shown, the scatter plot represents the measurements taken in the experiment.

[0050] Step 6: Invert the density / mass of the particle under test using the resolution rate:

[0051] According to the theoretical velocity curve of formula (4):

[0052]

[0053] in, t is the measurement time, C is an experimentally determined constant, and Vp and τ p The velocity and relaxation time of the target particle / cell are defined as follows, while other parameters are defined as described above; since the size density is known, the known relaxation time τ of the target microparticle / cell is used. p Substituting the initial velocity c1 when the bubble pulls back the microparticle / cell stage, the initial velocity c2 when the bubble pushes forward the microparticle / cell stage, and the characteristic times τ1 and τ2 of the two stages into the above formula, we can obtain the theoretical curve. It can be seen that the theoretical curve agrees well with the experimental data.

[0054] Step 7: Measurement of positional particle density: Only two unknown parameters are obtained by fitting the experimental data: the relaxation time τ of the target particle. p And the initial velocity of the fluid uf(0). τ can be obtained through two-parameter fitting. p Density ρ of microparticles / cells to be tested p By τ p =2Rp 2 ρ p / 9μ is calculated (Rp is the radius of the microparticle / cell being measured), and the inertial mass is calculated by multiplying the volume of the target particle by its density ρ. p The results are as follows. In this embodiment, HGM particles of different sizes were selected as the test particles, and the results are as follows. Figure 5 As shown, the theoretical prediction and the measured density are consistent.

[0055] Of course, the above description is not a limitation of the present invention, and the present invention is not limited to the examples given above. Any changes, modifications, additions or substitutions made by those skilled in the art within the scope of the present invention are also within the protection scope of the present invention.

Claims

1. A method for measuring the mass of a single particle / cell based on a bubble micromotor, characterized in that, Includes the following steps: Step 1: Fabrication of a magnetic micromotor, including: first, preparing a dilute solution of SiO2 colloidal particles; second, using a spin coating technique, spin coating the dilute solution of SiO2 colloidal particles onto a single-polished superhydrophilic silicon wafer to obtain a single layer of densely distributed SiO2 colloidal particles; finally, using electron beam evaporation deposition technology or magnetron sputtering deposition technology, sequentially depositing a 20nm Ni layer and a 20nm Pt layer on the surface of the SiO2 colloidal particles to obtain a magnetic micromotor with an asymmetric structure. Step 2: Perform bubble-driven operation of the magnetic micromotor, including: placing the magnetic micromotor prepared in Step 1 in a hydrogen peroxide solution of appropriate concentration, and generating a single bubble through a chemical reaction on the Pt side, thereby driving the micromotor to move. Step 3: Observe and control the movement of the bubble-driven magnetic micromotor, including: placing the bubble-driven magnetic micromotor under a microscope equipped with a high-speed camera for motion observation; Step 4: Measure the mass / density of microparticles / cells based on bubble micromotors, including: observing the relative position of the bubble-driven magnetic micromotor and the microparticles / cells to be measured under a microscope platform; controlling the movement direction of the magnetic bubble micromotor in real time through a handle, so that the bubbles generated by the micromotor can effectively push the microparticles / cells to be measured; and recording the displacement changes of the microparticles / cells to be measured under the push of the bubbles with a high-speed camera. Step 5: Process the displacement velocity of the microparticles / cells to be tested, including: recording the motion information of the microparticles / cells under a microscope platform using a high-speed camera; and processing the relaxation time τ based on the different relaxation times of the microparticles / cells. p =2R p 2 ρ p / 9μ,R p and ρ p The radius and density of the microparticles / cells to be tested are respectively, and μ is the viscosity of the environmental solution of the microparticles / cells to be tested. The motion of the microparticles / cells to be tested is analyzed using image processing software and particle motion analysis software, thereby obtaining the velocity Vp of the cell / particles to be tested as a function of time t during the bubble collapse process. Step 6: Determine the density / mass of the particles / cells under test by resolving velocity, including: establishing kinetic equations for the stages of particle / cell motion driven by bubble collapse, resulting in: （1） Where, m p Rp and Vp are the inertial mass, radius, and velocity of the particle / cell being tested, respectively, and μ is the viscosity of the surrounding solution. f (t) is the velocity of the fluid surrounding the target object, which varies with time t; u f (t) = (c1e -t / τ1 +c2e -t / τ2 ) (2) Wherein, parameters c1 and c2 are the initial velocities of the fluid during the bubble collapse phase (pullback and forward push phases), respectively, determined by the initial fluid velocity at the time of measurement, uf(0) = c1 + c2, and the parameters are fitted according to the measurement; τ1 and τ2 are the characteristic times of the pullback phase and the forward push phase, respectively; where τ1≈0.5τ b , τ b The characteristic time for bubble fusion, ,R b ρ is the maximum radius of the bubble, γ is the surface tension coefficient of the bubble, ρ is the density of the solution; τ2 is 100-200 μs; relaxation time τ of the target particle p The formula is: t p = 2R p 2 r p / 9m (3) Substituting formulas (2) and (3) into formula (1), we get: （4） in, t is the measurement time, C is a constant, and Vp and τ p The velocity and relaxation time of the particles / cells to be tested; Step 7: Obtain the unknown parameter τ of the target particle by fitting the experimental data. p The initial velocity of the fluid uf(0) is used; the relaxation time τ of the target particle is obtained by two-parameter fitting. p The density ρ of microparticles / cells to be measured p By τ p =2R p 2 ρ p The inertial mass is calculated by multiplying the volume of the microparticle / cell being measured by the density ρ. p get.

2. The method for measuring the mass of a single particle / cell based on a bubble micromotor according to claim 1, characterized in that, In step 1, the particles are microparticles of different materials, including polystyrene, glass microspheres, and TiO2, with a particle diameter of 5-100 μm.

3. The method for measuring the mass of a single particle / cell based on a bubble micromotor according to claim 1, characterized in that, In step 2, the appropriate concentration is 2%-15%.

4. The method for measuring the mass of a single particle / cell based on a bubble micromotor according to claim 1, characterized in that, In step 3, a uniform magnetic field in three-dimensional space is provided by setting up a three-dimensional Helmholtz coil on the microscopic platform. The magnetic field command is integrated through the handle, and the magnetic micro motor driven by the bubble is controlled through the handle.

5. The method for measuring the mass of a single particle / cell based on a bubble micromotor according to claim 1, characterized in that, In step 4, the cells are cultured in a buffer environment, then diluted before testing, mixed with a low-concentration hydrogen peroxide solution, and then subjected to rapid experimental measurements.

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