CN112229761A - Cell density measuring method - Google Patents
Cell density measuring method Download PDFInfo
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- CN112229761A CN112229761A CN202011089615.8A CN202011089615A CN112229761A CN 112229761 A CN112229761 A CN 112229761A CN 202011089615 A CN202011089615 A CN 202011089615A CN 112229761 A CN112229761 A CN 112229761A
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- 238000000034 method Methods 0.000 title claims abstract description 20
- 239000007788 liquid Substances 0.000 claims abstract description 8
- 239000002245 particle Substances 0.000 claims abstract description 7
- 238000005516 engineering process Methods 0.000 claims description 4
- 239000011859 microparticle Substances 0.000 claims description 4
- 241000270295 Serpentes Species 0.000 claims 1
- 239000000835 fiber Substances 0.000 claims 1
- WABPQHHGFIMREM-UHFFFAOYSA-N lead(0) Chemical compound [Pb] WABPQHHGFIMREM-UHFFFAOYSA-N 0.000 claims 1
- 230000010354 integration Effects 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 13
- 239000012530 fluid Substances 0.000 description 9
- 238000005259 measurement Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 4
- 239000004005 microsphere Substances 0.000 description 4
- 238000001739 density measurement Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000011324 bead Substances 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 238000007599 discharging Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000004163 cytometry Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000013307 optical fiber Substances 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000012798 spherical particle Substances 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N9/00—Investigating density or specific gravity of materials; Analysing materials by determining density or specific gravity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1434—Optical arrangements
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Abstract
The invention provides a cell density measuring method, which adopts an integrated flow counting device to count the number of cells in a certain time; the flow rate of the liquid is calculated using a flow measuring device. According to the cell density measuring method, the solution flow channel, the counting electrode and the flow measuring electrode are arranged on the micro-fluidic chip, so that the density of particles in the solution is calculated on the micro-fluidic chip. The method obtains the cell density of the solution through the real-time flow rate of the solution in the micro-flow channel and the number of the cells flowing through the micro-flow channel correspondingly. The cell density measuring method has the advantages of high efficiency, convenience, integration and the like.
Description
Technical Field
The invention relates to the field of cell quantitative analysis and cell engineering experimental devices, in particular to a novel cell density measuring method.
Background
In the past decade, the demand for calculating and detecting the density of solution microparticles in medical, chemical and biological fields has increased, and the demand has also increased. When the cell density in a solution is calculated, the traditional particle counting detection method mainly adopts labor-intensive manual counting which consumes much time; in the traditional microfluidic channel for cell counting by using an impedance method on a chip, the probability of cell blockage is high in practical use, and the cell counting work is seriously influenced.
In order to obtain a certain stage of fluid volume, a fixed flow rate is usually set by using a syringe pump in the conventional method, but the flow rate may be unstable due to external influence, and if the calculation is performed according to the set fixed flow rate, the calculated cell density is not accurate, and the method is inconvenient for system integration; in addition, the solution flow rate measurement in the conventional cell density measurement method is expensive and has low precision.
Disclosure of Invention
Aiming at the technical defects of the prior art, the invention aims to provide a cell density measuring method which is accurate in counting, accurate in flow velocity measurement and wide in applicability.
The first aspect of the present invention provides a cell density measuring method, which uses an integrated flow counting device to count the number of cells in a certain time; the flow rate of the liquid is calculated using a flow measuring device. The integrated flow counting device for counting the number of cells can adopt an electrical impedance technology and/or an integrated optical fiber technology.
In a specific implementation, an integrated flow counting device for counting the number of cells may use a counter electrode, and the counter electrode is connected to a flow sensor of a flow measurement device on a microfluidic chip. Wherein the flow sensor for calculating the liquid flow may be a temperature measuring electrode.
The invention provides a micro-fluidic chip for measuring cell density, which comprises an inlet, an inlet end micro-fluidic channel, a counting channel, an outlet end micro-fluidic channel, an outlet, a counting electrode and a temperature measuring electrode.
Wherein the microfluidic channel provides a solution treatment operation space, has a length of 24mm and a width of 10-5000 μm, and has a main function of providing an operation space for liquid to be treated. The inlet and the outlet are symmetrical based on the microfluidic channel, the width of the inlet/outlet is 10-5000 μm, the counting channel has the main function of being used as a channel for injecting and discharging cell solution, the counting channel is also of a rectangular structure, the length of the counting channel is 3-1000 μm, and the width of the counting channel is 10-100 μm. The main functions are to limit the passage of single cells and reduce the impedance peak width caused by the passage of cells, and to facilitate cell counting and circulation.
The counting electrodes are two and are perpendicular to the counting channel and are arranged at equal intervals, the width of each single electrode is 10-5000 mu m, and the length of each single electrode is 200-500 mu m. The spacing between the two electrodes was 200-595 μm. The two electrodes are externally connected with leads, the leads are connected to an external impedance instrument for observing the change of impedance in the counting channel, and the cells are counted by observing the number of impedance peak values.
The temperature measuring electrodes are arranged perpendicular to the outlet end microflow channel at equal intervals, the width of the electrodes is 10-20 mu m, and the length is 2000-10000 mu m. The resistance of the electrode temperature measuring part accounts for 60-90% of the resistance of the whole electrode, and the temperature measuring electrode has the main function of obtaining the flow rate of the fluid by detecting the temperature change of the temperature measuring part. The shape of the temperature measuring part of the temperature measuring electrode is designed to be snakelike as shown in figure 1, and the purpose is to better correlate the flow velocity with the temperature and the resistance value.
The third aspect of the invention provides an integrated flow counting and flow measuring device, which comprises the microfluidic chip, an impedance meter and an external measuring circuit. The impedance meter is connected with the counting electrode by a lead, and the external measuring circuit is connected with the temperature measuring electrode by a lead.
The solution enters the inlet end microflow channel through the inlet in any dripping or flowing mode, the particles in the solution pass through the counting channel in a single mode, and when the particles flow between the two counting electrodes, the impedance meter generates wave peaks for counting.
It is understood by those skilled in the art that the method for measuring cell density or the microfluidic chip or the integrated flow counting and flow measuring device for detecting solution microparticle density is also within the protection scope of the present invention.
The invention has the beneficial effects that:
(1) the cell density measuring method uses an integrated flow counting device to count the number of cells in a certain time; the flow measuring device is adopted to calculate the flow of the liquid, the counting is accurate, the use is convenient, the integration of subsequent systems is convenient, and a good development prospect is shown.
(2) The micro-fluidic chip system used by the invention has the functions of recording the number of cells in real time and measuring the flow rate of the fluid in real time, and can accurately obtain the cell density in a certain time period.
(3) The integrated flow type counting and flow measuring device used by the invention has no complex mechanical structure and wide use scene, and can be further used for calculating and detecting the density of solution micro particles in the fields of medicine, chemistry and biology.
Drawings
FIG. 1 is a schematic diagram of an integrated flow counting and flow measuring device of the present invention.
FIG. 2 shows the phenomena observed by a microscope and an impedance meter in the fluorescent microsphere experiment of example 2 of the present invention.
FIG. 3 shows the observation of a microscope and an impedance meter in the Hela cell count test in example 3 of the present invention.
Fig. 4 is a schematic diagram of a constant temperature difference circuit of a flow sensor in embodiment 4 of the present invention.
Fig. 5 is an output characteristic of a flow sensor in embodiment 4 of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. If no special description is provided, the experimental reagents and consumables used in the examples are all sold in the market;
example 1
The schematic diagram of the cell density measuring device is shown in fig. 1, wherein the total length of the microfluidic chip is 32mm, the total width of the chip is 200 μm, and the depth of the chip is 300 μm; comprises a microfluidic channel, a counting electrode and a temperature measuring electrode.
The microfluidic channel is a rectangular structure, the length of the microfluidic channel is 24mm, the width of the microfluidic channel is 200 microns, the depth of the microfluidic channel is 100 microns, and the microfluidic channel mainly has the function of providing an operation space for liquid to be processed. The inlet and outlet are symmetrical based on the microfluidic channel, the inlet/outlet width is 200 μm, and the inlet/outlet mainly functions as a channel for injecting and discharging cell solution. The counting channel is also rectangular in structure, the length of the counting channel is 1000 μm, and the width of the counting channel is 10 μm. The main functions are to limit the passage of single cells and reduce the impedance peak width caused by the passage of cells, and to facilitate cell counting and circulation.
Impedance cytometry is a method in which cells flowing through a microfluidic channel are reduced into tiny, uniformly insulated spherical particles, and an alternating current signal or a direct current signal with a proper frequency is applied to an external electrode beside the fluidic channel, so that when the cells flow through a specific area, the current of the area changes, and therefore the impedance of the area also changes. By this principle, the corresponding impedance information can be observed to count the cells.
The counting electrodes are two and are perpendicular to the microfluidic channel and are arranged at equal intervals, the width of each single electrode is 100 mu m, the length of each single electrode is 500 mu m, and the distance between the two electrodes is 595 mu m. The two electrodes are externally connected with leads, the leads are connected to an external impedance instrument for observing the change of impedance in the counting channel, and the cells are counted by observing the number of impedance peak values.
The temperature measuring electrodes are arranged perpendicular to the outlet end microflow channel at equal intervals, the width of the electrodes is 20 μm, and the length of the electrodes is 3580 μm. The resistance of the electrode temperature measuring part accounts for 85% of the whole electrode resistance, and the temperature measuring electrode has the main function of obtaining the flow rate of the fluid by detecting the temperature change of the temperature measuring part. After a fixed voltage is applied to the temperature measuring electrode, when fluid flows through the temperature measuring resistor of the temperature measuring electrode, the resistance value of the fluid changes. When the flow rate is slow, the temperature of the temperature measuring part is higher, and the resistance value is larger; when the flow rate is fast, the temperature of the temperature measuring part is lower, and the resistance value is smaller. In view of this purpose, the temperature measuring part of the temperature measuring electrode is designed into the pattern as shown in fig. 1, and the temperature measuring resistor is designed in a serpentine shape, so that the relationship among the flow velocity, the temperature and the resistance value is enhanced.
Example 2 fluorescent microsphere counting experiment
A particle count experiment was performed on 20 micron fluorescent microspheres using the chip of example 1. The chip was placed on an electron microscope with the left channel as the inlet and DI water and microsphere aqueous solution were separately introduced using a needle tube built on a micropump at a flow rate of 5. mu.L/min. The observation under microscope and impedance meter is shown in FIG. 2.
It can be seen from the experimental phenomenon that each time the fluorescent beads flow between the two counting electrodes, a peak appears on the impedance meter, and the number of the beads flowing through is recorded by the value of the peak.
Example 3 Hela cell count experiment
The chip is placed on an electron microscope, two channels on the left side are inlets, a needle tube built on a micropump is used for respectively introducing Hela cell culture solution and PBS solution, the flow rate is 5 mu L/min, and the influence of low-frequency drift is eliminated by adopting a differential measurement method. The phenomena observed under the microscope and the impedance meter are shown in FIG. 3.
It can be seen from the experimental phenomenon that each time a cell passes between the counter electrodes, a corresponding peak appears on the impedance meter, and the number of the flowing cells can be obtained through the number of the upward peaks.
Example 4
The flow sensor uses a constant temperature difference circuit (see fig. 4) to read the fluid flow, and the output characteristic is shown in fig. 5. Fluid volume calculation formula: v ═ the cross-sectional area of the microfluid x flow rate x time; through multiple tests, the counting error of the volume of the fluid is within 5% under the condition of sufficient mixing.
Example 5 cell Density determination
Cell density in solution was calculated using a commercial counting device and the integrated flow counting and measuring device described in the present invention.
The 2 cells of different densities were mixed well. Cells were uniformly distributed in the liquid, measured using a commercial instrument (Countess II FL) and the apparatus of the present invention, and the density was calculated, and the experimental results are shown in table 1. The experimental result shows that the cell density measuring device provided by the invention can meet the requirement of cell density measurement with common instruments, can be automatically integrated into the whole cell production line, and can automatically realize the measurement of cell density through counting and volume measurement; commercial optical methods counter, need manual operations such as application of sample and must the sample of fixed volume, reduced precision and convenience.
TABLE 1 results of cell Density measurements for different devices
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. The cell density measuring method is characterized in that an integrated flow counting device is adopted to count the number of cells in a certain time; calculating the flow of the liquid using a flow measuring device, wherein the integrated flow counting device uses electrical impedance technology and/or integrated fiber optic technology.
2. The method according to claim 1, wherein the counter electrode of the integrated flow counter is provided on the same member as the thermometric electrode of the flow measuring device.
3. The method of claim 2, wherein the same component is a microfluidic chip.
4. The microfluidic chip for measuring the cell density is characterized by comprising an inlet, an inlet end microfluidic channel, a counting channel, an outlet end microfluidic channel and an outlet in the order of solution passing; wherein the width of the inlet end micro-flow channel and the outlet end micro-flow channel is 10-5000 μm.
5. The microfluidic chip according to claim 4, wherein 2 counter electrodes are respectively connected to: the joint of the counting channel and the inlet end microfluidic channel and the joint of the counting channel and the outlet end microfluidic channel; the width of a single electrode is 10-5000 μm, the length is 200-595 μm, and the distance between two electrodes is 200-595 μm.
6. The microfluidic chip according to claim 4, wherein the temperature measuring electrode is perpendicular to the outlet-end microfluidic channel, the width of the electrode is 10-20 μm, and the length is 2000-10000 μm; the resistance of the temperature measuring part is that the snake shape accounts for 60-90% of the whole temperature measuring electrode.
7. An integrated flow counting and flow measuring device, comprising the microfluidic chip of claim 4 or 5, an impedance meter and an external measuring circuit.
8. An integrated flow counting and flow measuring device according to claim 7, wherein the impedance meter is connected to the counter electrode using a lead wire.
9. An integrated flow counting and flow measuring device according to claim 8, wherein the solution is introduced into the inlet end microfluidic channel via the inlet port in any drop or flow pattern, particles in the solution pass through the counting channel in individual form, and the impedance meter peaks as the particles flow between the two counting electrodes for counting.
10. Use of the method for measuring cell density according to any one of claims 1 to 3 or the microfluidic chip according to any one of claims 4 to 6 or the integrated flow counter and flow measuring device according to any one of claims 7 to 9 for detecting the density of solution microparticles.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202011089615.8A CN112229761A (en) | 2020-10-13 | 2020-10-13 | Cell density measuring method |
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| CN202011089615.8A CN112229761A (en) | 2020-10-13 | 2020-10-13 | Cell density measuring method |
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1745292A (en) * | 2002-12-09 | 2006-03-08 | 先进流体实验室股份有限公司 | Male fertility assay method and device |
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2020
- 2020-10-13 CN CN202011089615.8A patent/CN112229761A/en active Pending
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