CN111220443A - Weak contact sample concentration and purification method and application - Google Patents
Weak contact sample concentration and purification method and application Download PDFInfo
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- CN111220443A CN111220443A CN202010192149.XA CN202010192149A CN111220443A CN 111220443 A CN111220443 A CN 111220443A CN 202010192149 A CN202010192149 A CN 202010192149A CN 111220443 A CN111220443 A CN 111220443A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4022—Concentrating samples by thermal techniques; Phase changes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4022—Concentrating samples by thermal techniques; Phase changes
- G01N2001/4033—Concentrating samples by thermal techniques; Phase changes sample concentrated on a cold spot, e.g. condensation or distillation
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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Abstract
According to the weak contact sample concentration and purification method and the application thereof, the weak contact sample concentration and purification method comprises the following steps: step 1, collecting a sample stock solution with internal free or suspended components, and then dripping the sample stock solution on the surface of a supercooling bottom plate to obtain liquid drops on the surface of the bottom plate; step 2, performing solidification nucleation on the liquid drops on the supercooling bottom plate, wherein the solidification nucleation occurs on a solid-liquid contact surface, and target components in the liquid drops are gathered in the top area of the liquid drops due to solidification segregation; step 3, controlling the temperature of the supercooling bottom plate, and suspending the moving of the solidification interface; step 4, the concentrate at the top of the droplet is extracted. Compared with the prior art, the concentration and purification of the weak contact sample are synchronously carried out in the method for concentrating and purifying the weak contact sample, the extraction of a chemical solvent is not needed, the contact surface between the sample and a container is small, the sample is not easy to be polluted, a strong physical field and relative motion are not needed, the activity of a target component is not damaged in the whole process at low temperature, and the method is particularly suitable for the sample with small dose and biochemical activity.
Description
Technical Field
The invention belongs to the technical field of concentration and purification, and particularly relates to a concentration and purification method of a weak contact sample and application thereof.
Background
At present, in biochemical detection, because the concentration of target components in a sample is low and a reaction signal is weak, the phenomenon that the detection period is prolonged and the detection effectiveness is even influenced exists widely. Taking the clinically prevailing immunity and nucleic acid detection as examples, the detection samples are blood and saliva extracts respectively. In the early stage of the disease, the concentrations of antigen, antibody and virus DNA are very low, and the events causing the disease delay can often occur due to the large diagnosis difficulty coefficient.
At present, concentration and purification technologies for the samples are not provided, so that corresponding pretreatment is mostly not carried out, and the extracted samples are directly detected.
But the conventional centrifugation and electrophoresis techniques have limited applicability. The centrifugal machine utilizes the density difference of components to separate, the separation effect on the components with similar density or with the particle size of nanometer scale is not obvious, and strong relative motion causes damage to active components. Electrophoresis can only drive the separation of charged components, and local electric fields can affect the distribution of functional groups on the surfaces of the components and can also damage active components.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a novel method for concentrating and purifying a liquid sample, which is applied to the pretreatment of a biochemical detection sample, so that the detection process is accelerated and the detection accuracy is improved.
The invention provides a novel sample pretreatment technology from the aspects of micro-nano hydrodynamics and multi-interface composite mechanics, and aims to realize the purposes of accelerating detection and improving accuracy by carrying out nondestructive concentration on a detected sample, screening out interference components and amplifying reaction signals through weak contact dropwise solidification and accurate segregation. The technology can be widely applied to the concentration, purification and separation of biochemical and medical clinical detection samples, and is simultaneously suitable for various fields of food engineering, chemical engineering, environmental detection and the like.
The invention provides a method for concentrating and purifying a weak contact sample, which is characterized by comprising the following steps: step 1, collecting a sample stock solution with internal free or suspended components, and then dripping the sample stock solution on the surface of a supercooling bottom plate to obtain liquid drops on the surface of the bottom plate; step 2, performing solidification nucleation on the liquid drops on the supercooling bottom plate, wherein the solidification nucleation occurs on a solid-liquid contact surface, and target components in the liquid drops are gathered in the top area of the liquid drops due to solidification segregation; step 3, controlling the temperature of the supercooling bottom plate, and suspending the moving of the solidification interface; step 4, the concentrate at the top of the droplet is extracted.
The method for concentrating and purifying a weakly contacted sample provided by the invention can also have the following characteristics: the stock solution medium is water, an organic solvent or liquid metal, and the target components and the interference components in the stock solution comprise any one or a mixture of a plurality of micro-nano particles, acid-base salt ions, organic macromolecules, biological tissues, cells and viruses.
In addition, the method for concentrating and purifying a weakly contacted sample provided by the present invention may further have the following characteristics: in the step 1, a micro-injection pump is used for dripping stock solution to be treated to the surface of the supercooling bottom plate through a capillary, and the distance between an outlet of the capillary and the surface of the supercooling bottom plate is 5-20 mm.
In addition, the method for concentrating and purifying a weakly contacted sample provided by the present invention may further have the following characteristics: wherein the volume of the liquid drop is 15-20 mu L.
In addition, the method for concentrating and purifying a weakly contacted sample provided by the present invention may further have the following characteristics: wherein, the bottom plate is made of smooth heat conducting material, and the initial temperature is set to be 8-12 ℃ lower than the freezing point of the matrix medium in equilibrium state.
In addition, the method for concentrating and purifying a weakly contacted sample provided by the present invention may further have the following characteristics: wherein in the step 1, the steady-state contact angle of the liquid drop on the surface of the bottom plate is 110-130 degrees.
In addition, the method for concentrating and purifying a weakly contacted sample provided by the present invention may further have the following characteristics: in step 2, the target component is separated by adopting liquid drop solidification segregation.
In addition, the method for concentrating and purifying the weak contact sample further comprises a step 5 of heating the bottom plate and then extracting residual liquid, wherein a solidification phase in the liquid drop is melted after the temperature of the bottom plate is raised to room temperature, and the residual liquid in the liquid drop is recovered through a residual liquid capillary channel.
The invention provides an application of a method for concentrating and purifying a weak contact sample, which is characterized by comprising the following steps: the method for concentrating and purifying the weakly contacted sample is applied to concentration, purification and separation of biochemical and medical clinical detection samples, or applied to food engineering, chemical engineering and environmental detection.
Action and Effect of the invention
According to the weak contact sample concentration and purification method provided by the invention, through weak contact dropwise solidification and accurate segregation, the detection sample is subjected to nondestructive concentration, interference components are screened out, and a reaction signal is amplified, so that the purposes of accelerating detection and improving accuracy are achieved.
The scheme provided by the invention has the following advantages:
(1) the concentration rate is high, and 3 to 4 orders of magnitude of concentration increase can be realized by overlapping use;
(2) the method has strong pertinence, and interference components can be screened out while concentration is carried out through accurate control of interface rate;
(3) the sample is not easy to be polluted, chemical solvent extraction is not needed, and the contact surface of the sample and the container is small;
(4) the target component is not damaged, strong physical field and relative movement do not exist, the target component is not damaged by low-temperature protection, and the method is more suitable for components with biochemical activity such as viruses, enzymes, bacteria and the like;
(5) the dosage is adjustable, and the method can be suitable for the micro sample amount from nanoliter to picoliter.
Drawings
FIG. 1 is a schematic diagram of step 1 in the first embodiment of the present invention;
FIG. 2 is a schematic diagram of step 2 in the first embodiment of the present invention;
FIG. 3 is a schematic diagram of step 4 in the first embodiment of the present invention;
FIG. 4 is a schematic diagram of step 5 in the first embodiment of the present invention;
FIG. 5 is a schematic view of step 1 in the second embodiment of the present invention;
FIG. 6 is a schematic diagram of step 2 in the second embodiment of the present invention;
FIG. 7 is a schematic view of step 4 in the second embodiment of the present invention;
FIG. 8 is a diagram illustrating step 5 in the second embodiment of the present invention.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the following embodiments are combined with the drawings to specifically describe the concentration and purification method of the weak contact sample and the application thereof.
During the solidification of the matrix medium, other components such as ions, particles, macromolecules (e.g., proteins and DNA) and cells, etc., which are free or suspended within the matrix medium, are screened from the continuous solidification (ice) phase by interfering with the molecular lattice matching of the base medium (water). Although all other components except the base medium molecule are screened out in the thermodynamic equilibrium state, there is a corresponding critical rate of displacement for each particular component. If the solidification interface displacement velocity is less than this value, the component is screened out into the retained liquid phase, i.e., concentrated; if the solidification interface advancing speed is higher than this value, the component is coated with the advancing solidification phase, and the spatial distribution thereof is not significantly deviated. The critical rate of travel of a certain component depends on particle size, shape, surface groups, etc. The invention utilizes the characteristic segregation behavior to concentrate and purify the target component in the complex component liquid by adjusting the advancing speed of the solidification interface.
Example one
The method for concentrating and purifying a liquid sample of the present embodiment includes the steps of:
all the following steps are carried out in a closed bin to isolate outside water vapor and floating dust. The inside of the closed bin is dry air, nitrogen or inert gas, the ambient temperature in the bin is room temperature, and the pressure is kept balanced with the external atmosphere through a one-way regulating valve.
Step 1, treating a stock solution, wherein a stock solution medium is water, an organic solvent or liquid metal, and in the embodiment, the stock solution of the influenza virus nucleic acid detection sample is pretreated.
As shown in FIG. 1, a stock solution to be treated is dripped to the surface of a supercooling bottom plate 30 through a capillary 10 by using a micro-syringe pump to obtain a droplet 20 on the surface of the bottom plate, the droplet 20 contains a plurality of viruses 21 and sputum components 22, and a residual liquid capillary channel 31 communicated with the surface of the bottom plate 30 is arranged on the bottom plate 30.
The stock solution is the respiratory tract extract of the patient, and the main components are water (matrix medium), sputum 22 (interfering component) and virus 21 (target component).
In the embodiment, the stock solution passing through the capillary 10 is a respiratory tract extract droplet, the outer diameter of the selected capillary 10 is 500 μm, the volume of the droplet 20 is 15-20 μ L, the flow rate of the micro-injection pump is set to be 1-10 μ L/min, and the distance from the capillary 10 to the surface of the bottom plate 30, namely the height of the droplet 20, is 5-20 mm and needs to be larger than the diameter of the droplet 20.
The volume of the single liquid drop 20 can be adjusted according to the surface tension, viscosity and pipe diameter of capillary, the liquid drop 20 collides with the surface of the bottom plate 30 at low speed, the subsequent spreading and retraction processes are negligible, the final steady contact angle of the liquid drop 20 is 110-130 °, and in the embodiment, the steady contact angle after the liquid drop 20 is dropped is about 125 °. The steady state contact angle is defined as the angle between the outer edge of the droplet and the contact surface, and when the droplet strikes the supercooled surface at low velocity, the contact angle achieved is typically greater than 90 °. The advantage of using a large contact angle is that the corresponding actual contact area is small, making the solid/liquid contact surface displacement speed easier to control.
And 2, carrying out solidification nucleation, wherein the solidification nucleation occurs at a solid/liquid contact surface, and then the solidification interface gradually moves upwards.
The target component is concentrated in the concentrated phase 24 at the top of the droplet 20 due to coagulation segregation, as shown in fig. 2, the droplet 20 comprises the concentrated phase 24 at the top and the coagulated phase 23 at the lower part thereof, the virus 21 has a diameter of about 100nm and is completely screened out by the coagulated interface, and the sputum component 23 has no obvious segregation behavior.
Thus, the interfering components are uniformly distributed without solidification segregation, and the liquid droplet solidification segregation can be used to separate the components.
The bottom plate 30 is a smooth heat conducting material with an initial temperature reference value of 8-12 deg.c below the equilibrium freezing point of the matrix medium, in the example 10 deg.c below the equilibrium freezing point of the matrix medium. The initial temperature of the soleplate 30 is set to-10 ℃ for the water-based medium, and heterogeneous nucleation is ensured to occur at a solid-liquid contact point.
Experiments show that the moving speed of the solidification interface is lower than the critical moving speed of the target component and lower than the critical moving speed of the interference component so as to ensure that the target component is screened by the solidification interface and the spatial distribution of the interference component is unchanged. And for a single-component stock solution, the moving speed of the solidification interface is reduced as much as possible.
And 3, suspending the movement of the solidification interface through temperature control, and extracting a concentrated and purified product by using a micro-injection pump. Or adopting a time matching program to dynamically extract the concentrated and purified product in the interface moving process.
Among them, there are many general methods for controlling the specific temperature, such as using a thermoelectric chip, that is, directly adjusting the applied voltage and current; such as refrigerant channels, the flow rate is directly adjusted. The solidification time of a single liquid drop is several seconds to dozens of seconds, and the temperature of the bottom plate is adjusted during the solidification time, so that the solid/liquid interface is fixed at a certain position, and the concentrated product in the step 4 can be conveniently extracted.
As shown in FIG. 2, after 4/5 of the volume of the droplet 20 has coagulated, the concentration of the virus 21 in the concentrate is increased by 5 times, the concentration of the sputum 22 is reduced to 1/5 of the original concentration, and only a part of the sputum component 22 is contained in the coagulated phase 23.
Step 4, the concentrate at the top of the droplet is extracted.
In an embodiment, as shown in fig. 3, the concentrated phase 24 is extracted through the capillary channel 40 using a micro-syringe pump.
If the temperature control is adopted to suspend the moving of the solidification interface, the extraction flow rate of the concentrated and purified product can be flexibly adjusted.
Preferably, dynamic extraction is performed in the interface shifting process by adopting a time matching program, and a large extraction flow rate of 100-1000 mu L/min needs to be applied.
And 5, heating the surface of the bottom plate to melt the solidification phase, and pumping and extracting residual liquid by using a micro-injection pump.
The surface of the bottom plate 30 is heated to a temperature slightly above the equilibrium freezing point to recover the residual liquid. The melted solidification phase is recovered through the bottom residual night recovery passage 31, and the surface temperature of the base plate 30 can be controlled using the thermoelectric refrigerating sheet.
In the embodiment, as shown in FIG. 4, the temperature of the bottom plate 30 is raised to room temperature, the solidified phase 23 is melted, and the residual liquid 60 is recovered through the residual capillary channel 31, and the residual liquid 60 contains the same concentration of the interfering components 23 as the original liquid.
Under special conditions, if the target component has no obvious segregation behavior, the residual liquid is a purified product and is concentrated by combining other methods.
And (3) repeating the steps 1-5 on the extracted concentrate, continuously performing re-concentration and purification on the concentrate, further increasing the concentration of the virus 21 in the concentrate, and reducing the concentration of the sputum 22.
Example 2
The stock solution in this example is a mixed nanofluid, with the main components being water (matrix medium), polystyrene particles (interfering component), gold particles (target component).
The method for separating the polystyrene nanoparticles and the silver nanoparticles with the same particle size of 50nm from the stock solution comprises the following steps:
step 1, as shown in fig. 5, a stock solution to be processed is dropped on the surface of the supercooling bottom plate 30 through a capillary 100 by using a micro-syringe pump to obtain a droplet 200 on the surface of the bottom plate, the droplet 200 contains a plurality of polystyrene nanoparticles 202 and silver nanoparticles 201 with the same particle size of 50nm, and the bottom plate 30 is provided with a residual liquid capillary channel 31 communicated with the surface of the bottom plate 30.
The mixed nanofluid is injected through the stock solution capillary channel, the outer diameter of the selected capillary tube 100 is 500 mu m, the volume of the liquid drop 200 is about 7-10 mu l, and the steady-state contact angle of the dropped liquid drop 200 is about 125 degrees.
Step 2, the temperature of the soleplate 30 is set to-10 ℃.
The target component is aggregated in the concentrated phase 204 at the top of the droplet 200 due to solidification segregation, as shown in fig. 6, the droplet 200 comprises the concentrated phase 204 at the top and the solidified phase 203 at the lower part thereof, the silver nanoparticles 201 are completely screened out by the solidification interface, and the polystyrene nanoparticles 202 have no obvious segregation behavior. Thus, liquid droplet solidification segregation can be used to separate the components.
And 3, after 4/5 volume of the liquid drop 200 is solidified, the concentration of the silver nanoparticles 201 in the concentrate is increased by 5 times, the concentration of the polystyrene nanoparticles 202 is reduced to 1/5 of the original concentration, and the solidified phase 203 only contains part of the polystyrene nanoparticles 202.
Step 4, the concentrate at the top of the droplet is extracted.
In an embodiment, as shown in fig. 7, the concentrated phase 204 is extracted through the capillary channel 400 using a micro-syringe pump.
And 5, heating the surface of the bottom plate to melt the solidification phase, and pumping and extracting residual liquid by using a micro-injection pump.
In the embodiment, as shown in fig. 8, the temperature of the bottom plate 30 is raised to room temperature, the solidification phase is melted, and the residual liquid 600 containing the polystyrene nanoparticles 202 in the same concentration as the stock solution is recovered through the residual capillary channel 31.
The extract concentrate is repeated in steps 1-5 to further separate the silver nanoparticles 201 and the polystyrene nanoparticles 202.
Effects and effects of the embodiments
According to the method for concentrating and purifying the weak contact sample, the detection sample is subjected to nondestructive concentration, interference components are screened (separated) and reaction signals are amplified through weak contact dropwise solidification and precise segregation, so that the purposes of accelerating detection and improving accuracy are achieved.
Has the following advantages:
(1) the concentration rate is high, and 3 to 4 orders of magnitude of concentration increase can be realized by overlapping use;
(2) the method has strong pertinence, and interference components can be screened out while concentration is carried out through accurate control of interface rate;
(3) the sample is not easy to be polluted, chemical solvent extraction is not needed, and the contact surface of the sample and the container is small;
(4) the target component is not damaged, strong physical field and relative movement do not exist, the target component is not damaged by low-temperature protection, and the method is more suitable for components with biochemical activity such as viruses, enzymes, bacteria and the like;
(5) the dosage is adjustable, and the method can be suitable for the micro sample amount from nanoliter to picoliter.
The above embodiments are preferred examples of the present invention, and are not intended to limit the scope of the present invention.
Claims (9)
1. A method for concentrating and purifying a weakly contacted sample is characterized by comprising the following steps:
step 1, collecting a sample stock solution with internal free or suspended components, and then dripping the sample stock solution on the surface of a supercooling bottom plate to obtain liquid drops on the surface of the bottom plate;
step 2, the liquid drops are subjected to solidification nucleation on the supercooling bottom plate, the solidification nucleation occurs on a solid-liquid contact surface, and target components in the liquid drops are gathered in the top areas of the liquid drops due to solidification segregation;
step 3, controlling the temperature of the supercooling bottom plate, and suspending the moving of a solidification interface;
and 4, extracting the concentrate at the top of the liquid drop.
2. The method for concentrating and purifying a weakly contacted sample according to claim 1, wherein:
wherein the stock solution medium is water, an organic solvent or liquid metal,
the target component and the interference component in the stock solution comprise any one or a mixture of a plurality of micro-nano particles, acid-base salt ions, organic macromolecules, biological tissues, cells and viruses.
3. The method for concentrating and purifying a weakly contacted sample according to claim 1, wherein:
wherein, in the step 1, a micro-injection pump is used for dripping stock solution to be treated to the surface of the supercooling bottom plate through a capillary,
the distance from the outlet of the capillary tube to the surface of the bottom plate is 5-20 mm.
4. The method for concentrating and purifying a weakly contacted sample according to claim 3, wherein:
wherein the volume of the liquid drop is 15-20 mu L.
5. The method for concentrating and purifying a weakly contacted sample according to claim 1, wherein:
wherein, the bottom plate is made of smooth heat conducting material, and the initial temperature is set to be 8-12 ℃ lower than the freezing point of the matrix medium in an equilibrium state.
6. The method for concentrating and purifying a weakly contacted sample according to claim 1, wherein:
wherein in step 1, the steady-state contact angle of the liquid drop on the surface of the bottom plate is 110-130 °.
7. The method for concentrating and purifying a weakly contacted sample according to claim 1,
wherein, in step 2, the target component is separated by adopting liquid drop solidification segregation.
8. The method for concentrating and purifying a weakly contacted sample according to claim 1, further comprising:
step 5, heating the bottom plate, extracting residual liquid,
wherein the solidified phase in the liquid drop is melted after the temperature of the bottom plate is raised to the room temperature, and the residual liquid of the liquid drop is recovered through the residual liquid capillary channel.
9. The application of the method for concentrating and purifying the weakly-contacted sample is characterized in that:
the method for concentrating and purifying a weakly contacted sample according to any one of claims 1 to 8, which is used for concentrating, purifying and separating biochemical and medical clinical test samples,
or in food engineering, chemical engineering and environmental testing.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112108086A (en) * | 2020-09-24 | 2020-12-22 | 上海理工大学 | Directional solidification segregation device and method for colloidal particle system |
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