CN117664944A - Cell detection method and device based on micro-fluidic chip - Google Patents
Cell detection method and device based on micro-fluidic chip Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 40
- 210000004027 cell Anatomy 0.000 claims abstract description 297
- 238000000926 separation method Methods 0.000 claims abstract description 211
- 210000000601 blood cell Anatomy 0.000 claims abstract description 73
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- 238000000838 magnetophoresis Methods 0.000 claims description 9
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Abstract
The invention discloses a cell detection method and a cell detection device based on a microfluidic chip, and relates to the technical field of cell detection, wherein the cell detection method based on the microfluidic chip comprises the steps of carrying out first separation treatment on a sample to be detected based on a first separation module to obtain a first target cell solution; performing secondary separation on cells in the first target cell solution based on a secondary separation module to obtain a second target cell solution; and separating the cells in the second target cell solution for three times based on the three-time separation module to obtain a third target cell solution. According to the technical scheme, the blood is separated from the blood cells and the blood plasma through the primary separation module, so that the primary separation of the blood is finished, and the secondary separation module and the tertiary separation module are used for carrying out secondary separation and tertiary separation on the blood cells respectively, so that the blood cells are fully separated, the accuracy of rapid and accurate separation of the cells is ensured, and the physiological activity of the cells is not influenced.
Description
Technical Field
The invention relates to the technical field of cell detection, in particular to a cell detection method and device based on a microfluidic chip.
Background
With the continuous development of micro-nano processing technology, the cell screening technology based on the micro-fluidic chip is widely focused and approved, the channel structure of the micro-fluidic chip can be designed at will, and the sensitivity of cell screening is improved, but as the cell separation and quantity statistics methods in the prior art are imperfect, and most of cell separation is only carried out through one-time separation or two-time separation, the separation statistics accuracy is low easily caused, and the purity of the separated cells is low and the post-treatment is complex.
Disclosure of Invention
The invention mainly aims to provide a microfluidic chip and a detection method thereof, and aims to realize the separation of blood cells and plasma in blood through a primary separation module, so as to finish the primary separation of the blood, and then the secondary separation module and a tertiary separation module are used for respectively carrying out the secondary separation and the tertiary separation on the blood cells, so that the sufficient separation of the blood cells is realized, the accuracy of the rapid and accurate separation of the cells is ensured, and the physiological activity of the cells is not influenced.
In order to achieve the above object, the method for detecting cells based on a microfluidic chip according to the present invention comprises:
Placing collected blood into a microfluidic chip-based device through a sample collection port, and performing primary separation treatment on a sample to be detected based on a primary separation module to obtain a first target cell solution;
performing secondary separation on cells in the first target cell solution based on a secondary separation module to obtain a second target cell solution;
performing three times of separation on the cells in the second target cell solution based on the three times of separation module, and marking the separated cells based on a preset fluorescent marker to obtain a third target cell solution;
collecting fluorescent images of the third target cell solution based on the image collecting module;
based on a data processing module, carrying out quantitative analysis processing on the fluorescent image acquired by the image acquisition module to obtain the quantity and proportion of various blood cells in a blood sample, and outputting a statistical result;
wherein, based on the secondary separation module, the secondary separation is carried out to the cells in the first target cell solution, obtains the step of second target cell solution, includes:
applying magnetophoresis force to the first target cell solution based on a secondary separation module so as to enable different cell types in the first target cell solution to be separated for the second time in the microfluidic chip, thereby obtaining a second target cell solution;
The step of separating the cells in the second target cell solution for three times based on the three times separation module, and labeling the separated cells based on a preset fluorescent marker to obtain a third target cell solution comprises the following steps:
and applying dielectrophoresis force to the second target cell solution based on the third separation module so as to perform third separation on the obtained second target cell solution and perform fluorescent marking on the cells separated for the third time to obtain a marked third target cell solution.
In an alternative embodiment, the step of placing the collected blood in a microfluidic chip-based device through a sample collection port, and performing a first separation process on a sample to be tested based on a primary separation module to obtain a first target cell solution includes:
inputting collected blood into a microfluidic chip-based device through a sample collection port, and applying ultrasonic pulses to a sample to be detected by the primary separation module according to separation frequencies corresponding to cell types in the sample to be detected so as to separate cells in the blood in the microfluidic chip to obtain a first target cell solution;
the obtained first target cell solution flows into each channel in the microfluidic chip respectively.
In an alternative embodiment, the step of collecting the fluorescence image of the third target cell solution based on the image collecting module includes:
and imaging and acquiring the fluorescence image of the marked third target cell solution through an image acquisition module.
In an optional embodiment, the step of quantitatively analyzing the fluorescent image collected by the image collecting module based on the data processing module to obtain the number and the proportion of various blood cells in the blood sample and outputting the statistical result includes:
detecting the fluorescence image to obtain the fluorescence color, fluorescence intensity and fluorescence wavelength corresponding to the fluorescent marker on the cell;
the cell type is determined from the fluorescent color, the fluorescent intensity, and the fluorescent wavelength.
The invention also provides a cell detection device based on the micro-fluidic chip, which comprises;
the micro-channel piece is provided with a first channel, a second channel, a third channel and a fourth channel which are sequentially arranged and communicated along the length direction of the micro-channel piece;
the primary separation module is arranged in the first channel to perform primary separation treatment on a sample to be detected to obtain a first target cell solution, and the first target cell solution enters the second channel;
The secondary separation module is arranged in the second channel to separate different cell types in the first target cell solution so as to obtain a second target cell solution;
the third separation module is arranged in the second channel and on one side of the second separation module so as to separate different cell types in the obtained second target cell solution, and the cell solution after the third separation enters the third channel and is subjected to fluorescent marking in the third channel to obtain a marked third target cell solution;
the image acquisition module is arranged in the fourth channel and comprises a fluorescence microscope and a camera, and the marked third target cell solution presents different fluorescence colors under the imaging of the fluorescence microscope and captures fluorescence images of different cell types through the camera;
the data processing module comprises an image processor, wherein the image processor is electrically connected with the camera so as to enable fluorescent images obtained by the camera to be transmitted to the image processor, and the image processor is used for carrying out quantitative analysis on the collected fluorescent images.
In an alternative embodiment, the first channel includes a flow cavity, a first channel through which blood flows is provided in the flow cavity, the primary separation module is disposed on an outer peripheral side of the flow cavity, and the primary separation module is used for generating a surface acoustic wave and acting on the flow cavity, so that blood in the flow cavity separates plasma from blood cells, the first channel includes an inflow region, a narrow region and an outflow region, the narrow region is disposed on one side of the inflow region, the outflow region is disposed on one side of the narrow region away from the inflow region, the outflow region is provided with a first outlet and a second outlet, and the first outlet and the second outlet are disposed at intervals in a width direction of the flow cavity.
In an alternative embodiment, the second channel comprises an inlet area and a sorting area which are sequentially connected, and the inlet area is communicated with the second discharge outlet;
the sorting area comprises a first liquid outlet channel and a second liquid outlet channel which are arranged at an included angle;
the secondary separation module is a magnet generating piece, the magnet generating piece is arranged in the inlet area, and the magnet generating piece sorts blood cells passing through the inlet area;
The third separation module is respectively arranged on the first liquid outlet channel and the second liquid outlet channel so as to carry out secondary separation on the blood cells passing through the separation area.
In an alternative embodiment, the third channel includes two third liquid outlet channels disposed at an included angle and two fourth liquid outlet channels disposed at an included angle, both the third liquid outlet channels are all communicated with the first liquid outlet channel, each of the third liquid outlet channels is provided with a first liquid outlet, both the fourth liquid outlet channels are communicated with the second liquid outlet channel, and the fourth liquid outlet channel is provided with a second liquid outlet;
the three-time separation module comprises four microelectrode generating parts, wherein every two microelectrode generating parts are arranged on two sides of the first liquid outlet channel, and the other two microelectrode generating parts are arranged on two sides of the second liquid outlet channel, so that blood cells entering the first liquid outlet channel and the second liquid outlet channel are separated and discharged through two third liquid outlet channels and two fourth liquid outlet channels respectively;
the fourth channels are four, and each fourth channel is respectively communicated with each first liquid outlet and each second liquid outlet.
The invention provides a cell detection method and a cell detection device based on a microfluidic chip, and relates to the technical field of cell detection, wherein the cell detection method based on the microfluidic chip comprises the steps of placing collected blood into a device based on the microfluidic chip through a sample collection port, and carrying out first separation treatment on a sample to be detected based on a first separation module to obtain a first target cell solution; performing secondary separation on cells in the first target cell solution based on a secondary separation module to obtain a second target cell solution; thirdly, separating the cells in the second target cell solution based on the third separation module, and marking the separated cells based on a preset fluorescent marker to obtain a third target cell solution; meanwhile, collecting fluorescent images of the third target cell solution based on an image collecting module; based on a data processing module, carrying out quantitative analysis processing on the fluorescent image acquired by the image acquisition module to obtain the quantity and proportion of various blood cells in a blood sample, and outputting a statistical result; wherein, based on the secondary separation module, the secondary separation is carried out to the cells in the first target cell solution, obtains the step of second target cell solution, includes: applying magnetophoresis force to the first target cell solution based on a secondary separation module so as to enable different cell types in the first target cell solution to be separated for the second time in the microfluidic chip, thereby obtaining a second target cell solution; the step of separating the cells in the second target cell solution for three times based on the three times separation module, and labeling the separated cells based on a preset fluorescent marker to obtain a third target cell solution comprises the following steps: and applying dielectrophoresis force to the second target cell solution based on the third separation module so as to perform third separation on the obtained second target cell solution and perform fluorescent marking on the cells separated for the third time to obtain a marked second target cell solution. The blood is firstly separated from blood cells and plasma in the blood through the primary separation module, so that the primary separation of the blood is completed, the blood cells are fully separated through the secondary separation module and the tertiary separation module, and finally the blood cells after the tertiary separation are counted through the data processing module. The primary separation module, the secondary separation module and the tertiary separation module are used for realizing the full separation of blood cells in blood, ensuring the accuracy of rapid and accurate separation of the cells, not affecting the physiological activity of the cells and improving the statistical accuracy.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to the structures shown in these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic flow chart of a cell detection method based on a microfluidic chip of the present invention;
FIG. 2 is a schematic structural diagram of an embodiment of a microfluidic chip according to the present invention;
fig. 3 is a block diagram of a cell detection device based on a microfluidic chip according to a first embodiment of the present invention.
Reference numerals illustrate:
the achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It should be noted that all directional indicators (such as up, down, left, right, front, and rear … …) in the embodiments of the present invention are merely used to explain the relative positional relationship, movement, etc. between the components in a particular posture (as shown in the drawings), and if the particular posture is changed, the directional indicator is changed accordingly.
In the present invention, unless specifically stated and limited otherwise, the terms "connected," "affixed," and the like are to be construed broadly, and for example, "affixed" may be a fixed connection, a removable connection, or an integral body; can be mechanically or electrically connected; either directly or indirectly, through intermediaries, or both, may be in communication with each other or in interaction with each other, unless expressly defined otherwise. The specific meaning of the above terms in the present invention can be understood by those of ordinary skill in the art according to the specific circumstances.
Furthermore, descriptions such as those referred to as "first," "second," and the like, are provided for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying an order of magnitude of the indicated technical features in the present disclosure. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In addition, the technical solutions of the embodiments may be combined with each other, but it is necessary to base that the technical solutions can be realized by those skilled in the art, and when the technical solutions are contradictory or cannot be realized, the combination of the technical solutions should be considered to be absent and not within the scope of protection claimed in the present invention.
The invention provides a microfluidic chip and a detection method thereof, which aim at separating blood cells from blood plasma in blood through a primary separation module, so as to finish primary separation of the blood, and then respectively carrying out secondary separation and tertiary separation on the blood cells through a secondary separation module and a tertiary separation module, so as to realize full separation of the blood cells, ensure the accuracy of rapid and accurate separation of the cells, and not influence the physiological activity of the cells.
As can be seen, the microfluidic chip-based cell detection device may include: a processor, such as a central processing unit (Central Processing Unit, CPU), a communication bus, a user interface, a network interface, a memory. Wherein the communication bus is used to enable connection communication between these components. The user interface may comprise a Display screen (Display) and the optional user interface may also comprise a standard wired interface, a wireless interface, for which the wired interface may be a USB interface in the present invention. The network interface may optionally include a standard wired interface, a Wireless interface (e.g., a Wireless-Fidelity (Wi-Fi) interface). The memory may be a high-speed random access memory (Random Access Memory, RAM) or a stable memory (NVM), such as a disk memory. The memory may alternatively be a storage device separate from the aforementioned processor.
Referring to fig. 1 to 3, a specific structure of a microfluidic chip 10 according to the present invention will be described in the following, and in one embodiment of the present invention, a cell detection method based on a microfluidic chip includes:
step S10: the collected blood is placed in a microfluidic chip-based device through a sample collection port, and a sample to be detected is subjected to primary separation treatment based on a primary separation module, so that a first target cell solution is obtained.
It should be noted that, the execution body of this embodiment may be a cell detection device based on a microfluidic chip, where the cell detection device includes a sample shunt tube, a reaction chamber, a waste liquid chamber, a detection chamber, and a culture chamber, where the detection chamber may be further divided into a plurality of detection areas according to a cell detection requirement, where the detection device includes an electrical impedance identification area, an ultrasonic generating device, and a dye storage area, where the cell detection device is connected to a monitoring imaging device, where the monitoring imaging device may be a computer, a mobile phone, a tablet, etc., and this embodiment is not limited thereto. The cell detection method based on the microfluidic chip in the scheme is suitable for cell detection based on the microfluidic chip in various scenes, wherein the blood is whole blood, and the blood contains all components of blood cells and blood plasma. The blood cells comprise erythrocytes, leukocytes and platelets. In the case of cell counting, the cells such as white blood cells, red blood cells, and circulating tumor cells are mainly counted.
It will be appreciated that the first target cell solution is obtained by placing the collected blood through a sample collection port in a microfluidic chip-based device for blood cell capture separation. Wherein the first target cell solution is divided into a white blood cell solution and a small particle cell solution such as red blood cells, plasma, platelets and the like.
Specifically, the sample to be tested is subjected to the first separation treatment by applying the acoustic pulse to the sample to be tested through the preset acoustic pulse emission area, so that different types of cells can be separated into different liquid flows, and the different directions are shifted, so that the first target cell solution can be obtained.
It should be noted that, the acoustic wave device may refer to a device connected to the cell detection device and used for receiving an acoustic wave pulse signal and displaying a pulse frequency, where the device is connected to the monitoring imaging device, and the cell detection device includes an acoustic wave pulse emitting area, where the area may be disposed on two sides of the micro-channel and used for sending an acoustic wave pulse to the cell, so that the cell performs a corresponding offset to achieve an effect of cell separation, for example: in small particle cell solutions of red blood cells, plasma, platelets and the like after preliminary separation, due to different sound waves which can be born by different cells, different kinds of cells can be separated into different liquid flows and deflected in different directions by applying sound wave pulses to the cell solutions, so that the effect of cell separation is achieved.
Step S20: and carrying out secondary separation on the cells in the first target cell solution based on a secondary separation module to obtain a second target cell solution.
Specifically, the step of secondarily separating the cells in the first target cell solution based on the secondary separation module to obtain a second target cell solution includes:
step S201: and applying magnetophoretic force to the first target cell solution based on a secondary separation module so as to enable different cell types in the first target cell solution to be separated for the second time in the microfluidic chip, thereby obtaining a second target cell solution.
Meanwhile, a magnetophoretic force is applied to the once separated cell solution through a preset magnetophoretic force emission area, so that different types of cells can be separated into different liquid flows and offset in different directions, further, different cell types in the first target cell solution are separated for the second time in the microfluidic chip, and further, a second target cell solution can be obtained.
The higher the gradient of the magnetic flux density, the larger the magnetophoretic force to which the cell was subjected. Because of the presence of hemoglobin in erythrocytes, the magnetic susceptibility of erythrocytes is significantly higher than other blood cells (white blood cells, platelets, etc.). The magnetophoretic properties of red blood cells in the magnetophoretic separation zone are different from those of other blood cells. Erythrocytes subjected to positive magnetophoretic forces will be attracted to the side of the main channel close to the ferromagnetic body, because there the gradient of the magnetic flux density is higher, eventually flowing into the second outlet channel. Other cells (white blood cells and platelets) except red blood cells are subjected to the action of negative magnetophoresis, and the particle size of the white blood cells is larger, so that the negative magnetophoresis is stronger, and finally flows into the first liquid outlet channel; the particle size of the platelets is smaller, the effect of the negative magnetophoresis force is weaker, and the platelets and the red blood cells finally flow into the second liquid outlet channel together, so that separation is realized, namely, the separation of the red blood cells and the platelet mixed sample and the white blood cell mixed sample is realized.
Step S30: and carrying out three times of separation on the cells in the obtained second target cell solution based on the three times of separation module, and marking the separated cells based on a preset fluorescent marker to obtain a third target cell solution.
Specifically, the step of separating the cells in the obtained second target cell solution for three times based on the three times separation module, and labeling the separated cells based on a preset fluorescent marker to obtain a third target cell solution includes:
step S301: and applying dielectrophoresis force to the second target cell solution based on the third separation module so as to perform third separation on the obtained second target cell solution and perform fluorescent marking on the cells separated for the third time to obtain a marked third target cell solution.
Specifically, in order to further separate blood cells to obtain a more accurate separation result, the second target cell solution after the secondary separation is separated for the third time, that is, when the second target cell solution after the secondary separation passes through the third separation module, the second target cell solution is separated under the action of the third separation module, so that different types of cells can be separated into different liquid flows, and the different directions are deviated, so that the third target cell solution is obtained.
Meanwhile, the separated cells are marked by fluorescent markers to obtain a third target cell solution, so that the follow-up image acquisition of the third target cell solution is facilitated.
In the alternating electric field, the direction in which the cells are subjected to alternating-dielectrophoresis is related to the alternating frequency. By adjusting the frequency of the alternating current electric field in the first liquid outlet channel, white blood cells in the first liquid outlet channel are acted by positive alternating current-dielectrophoresis force and flow out from the first liquid outlet. And the circulating tumor cells flow out from the second liquid outlet under the action of negative communication-dielectrophoresis force, so that the separation of the white blood cells and the circulating tumor cells is realized. By adjusting the frequency of the alternating current electric field in the second liquid outlet channel, platelets in the second liquid outlet channel are acted by negative alternating current-dielectrophoresis force and flow out from the third liquid outlet. And the red blood cells flow out from the fourth liquid outlet under the action of the orthogonal flow-dielectrophoresis force, so that the separation of the platelets and the red blood cells is realized.
Step S40: and acquiring a fluorescence image of the third target cell solution based on the image acquisition module.
Step S50: and (3) quantitatively analyzing and processing the fluorescent image acquired by the image acquisition module based on the data processing module to obtain the quantity and proportion of various blood cells in the blood sample, and outputting a statistical result.
In the embodiment, collected blood is placed in a microfluidic chip-based device through a sample collection port, and a sample to be detected is subjected to primary separation treatment based on a primary separation module, so that a first target cell solution is obtained; performing secondary separation on cells in the first target cell solution based on a secondary separation module to obtain a second target cell solution; performing three times of separation on the cells in the second target cell solution based on the three times of separation module, and marking the separated cells based on a preset fluorescent marker to obtain a third target cell solution; the method comprises the steps of collecting fluorescent images of a third target cell solution based on an image collecting module, carrying out quantitative analysis processing on the fluorescent images collected by the image collecting module based on a data processing module to obtain the quantity and proportion of various blood cells in a blood sample, outputting a statistical result, separating the blood cells from plasma in the blood through a primary separating module, completing primary separation of the blood, and fully separating the blood cells through a secondary separating module and a tertiary separating module.
It will be appreciated that the predetermined fluorescent markers are predetermined fluorescent markers for labeling the cells after three separations, the fluorescent markers having different excitation wavelengths, so that the statistics of the cell number at a later stage can be facilitated by labeling the cells in the cell solution after three separations. And the third target cell solution comprises a solution obtained by labeling all the separated cell solutions with fluorescent substances, and the corresponding cell types are determined according to the different excitation wavelengths corresponding to the fluorescent markers because the fluorescent markers selected by different cells are different.
Referring to fig. 1 to 3, in an alternative embodiment, the step of placing collected blood in a microfluidic chip-based device through a sample collection port, and performing a first separation process on a sample to be tested based on a primary separation module to obtain a first target cell solution includes:
inputting collected blood into a microfluidic chip-based device through a sample collection port, and applying ultrasonic pulses to a sample to be detected by the primary separation module according to separation frequencies corresponding to cell types in the sample to be detected so as to separate cells in the blood in the microfluidic chip to obtain a first target cell solution;
The obtained first target cell solution flows into each channel in the microfluidic chip respectively.
It should be noted that, based on the fact that the primary separation module in the detection device has different acoustic wave modes, where the acoustic wave modes include surface acoustic waves and bulk acoustic waves, different acoustic wave forces and acoustic wave radiation are corresponding to the different acoustic wave modes, so in order to avoid rupture of cells caused by excessive acoustic wave forces, the acoustic wave emitting device may determine the acoustic wave frequency according to the cell type in the first target cell solution, so that an ultrasonic pulse is applied to the first target cell according to the acoustic wave frequency, and the cell offset angle is determined according to the received signal reflected after cell offset. It should be understood that the separation frequency refers to the lowest sonic frequency that cells in a cell solution can withstand, for example: and placing the white blood cell solution, the red blood cells, the plasma and the platelet cell solution obtained by performing primary cell separation on blood in each reaction cavity according to the type of the cells to be captured to wait for secondary separation operation, wherein in order to accurately obtain the number of the cells of the target type, the separation frequency can be set to be the lowest sonic frequency corresponding to the type of the cells to be captured so as to avoid damaging the cells by sonic waves, thereby being convenient for counting the cell markers in the later stage.
It will be appreciated that the angle of deflection of the cells at different acoustic frequencies is different, and thus the cell is ready for further manipulation by diverting the deflected cells into different chambers. For example: the red blood cells, the plasma and the platelet cell solution are applied with ultrasonic pulses, the red blood cells and other cells deflect in different directions, signals reflected after the deflection of the cells are received, and the cell deflection angle is determined according to the signals.
Referring to fig. 1 to 3, in an alternative embodiment, the step of acquiring the fluorescence image of the third target cell solution based on the image acquisition module includes:
and imaging and acquiring the fluorescence image of the marked third target cell solution through an image acquisition module.
Referring to fig. 1 to 3, in an alternative embodiment, the step of quantitatively analyzing the fluorescent image collected by the image collecting module based on the data processing module to obtain the number and the proportion of various blood cells in the blood sample and outputting the statistical result includes:
detecting the fluorescence image to obtain the fluorescence color, fluorescence intensity and fluorescence wavelength corresponding to the fluorescent marker on the cell;
the cell type is determined from the fluorescent color, the fluorescent intensity, and the fluorescent wavelength. And counting and classifying blood cells by an image processing algorithm, and analyzing and counting data obtained after image processing to obtain the quantity and proportion of various blood cells in the blood sample.
The invention also provides a cell detection device 10 based on a micro-fluidic chip, wherein the cell detection device 10 based on the micro-fluidic chip comprises;
a microchannel member 1, wherein the microchannel member 1 is provided with a first channel 11, a second channel 12, a third channel 13 and a fourth channel 14 which are sequentially arranged and communicated along the length direction thereof;
the primary separation module 2 is arranged in the first channel 11 to perform primary separation treatment on a sample to be detected to obtain a first target cell solution, and the first target cell solution enters the second channel 12;
a secondary separation module 3, wherein the secondary separation module 3 is arranged in the second channel 12 to separate different cell types in the first target cell solution to obtain a second target cell solution;
a third separation module 4, wherein the third separation module 4 is arranged in the second channel 12 and is arranged at one side of the second separation module 3, so as to separate different cell types in the obtained second target cell solution, and the cell solution after the third separation enters the third channel 13 and is subjected to fluorescent marking in the third channel 13, so as to obtain a marked third target cell solution;
The image acquisition module 5 is arranged in the fourth channel 14, the image acquisition module 5 comprises a fluorescence microscope and a camera, the marked third target cell solution presents different fluorescence colors under the imaging of the fluorescence microscope, and fluorescent images of different cell types are captured through the camera;
the data processing module 6, the data processing module 6 includes an image processor, the image processor is electrically connected to the camera, so that the fluorescence image obtained by the camera is transmitted to the image processor, and the image processor is used for performing quantization analysis on the collected fluorescence image.
Referring to fig. 2, in this embodiment, in order to facilitate separation of plasma and blood cells in blood, the microchannel member 1 is provided with a first channel 11, a second channel 12, a third channel 13 and a fourth channel 14 which are sequentially arranged and communicated along a length direction thereof, wherein the first channel 11, the second channel 12, the third channel 13 and the fourth channel 14 are sequentially arranged after the blood enters the microchannel member 1. Meanwhile, under the action of the first channel 11, plasma in blood and blood cells are effectively separated, so that pure blood cells are obtained, and further separation of the blood cells by the subsequent second channel 12 and third channel 13 is facilitated.
Specifically, in order to facilitate separation of blood, the first channel 11 is provided with the primary separation module 2, the second channel 12 is provided with the secondary separation module 3 and the tertiary separation module 4, so that blood enters the microchannel device 1, after first being subjected to separation treatment of the primary separation module 2, plasma and blood cells in the blood are separated, so that the blood entering the second channel 12 does not contain plasma, further separation of the secondary separation module 3 on residual blood cells is facilitated, a second target cell solution is obtained, and finally the tertiary separation module 4 is used for carrying out tertiary separation on the second target cell solution, so that each cell classification in the blood cells is obtained. The separated plasma may be collected in a sample tube or the like. The image acquisition module 5 is arranged in the fourth channel 14 to present different fluorescent colors to the marked third target cell solution under the imaging of the fluorescent microscope, and captures fluorescent images of different cell types through the camera. The data processing module 6 comprises an image processor electrically connected to the camera for transmitting fluorescence images obtained by the camera to the image processor for performing a quantitative analysis of the collected fluorescence images.
It should be noted that the image capturing module 5 further includes a light source for providing light to the camera, so that the camera can accurately capture fluorescent images of different cell types.
Referring to fig. 2, in an alternative embodiment, the first channel 11 includes a flow chamber 111, a first channel 11 through which blood flows is disposed in the flow chamber 111, the primary separation module 2 is disposed on an outer peripheral side of the flow chamber 111, the primary separation module 2 is configured to generate a surface acoustic wave and act on the flow chamber 111 to separate plasma from blood cells in the flow chamber 111, the first channel 11 includes an inflow region 112, a narrow region 113, and an outflow region 114, the narrow region 113 is disposed on one side of the inflow region 112, the outflow region 114 is disposed on a side of the narrow region 113 away from the inflow region 112, the outflow region 114 is provided with a first outlet 1141 and a second outlet 1142, the first outlet 1141 and the second outlet 1142 are disposed at intervals in a width direction of the flow chamber 111, and the flow chamber 111 is disposed within a propagation range of the surface acoustic wave so as to ensure that when blood cells in the blood flow through the first channel 11, the blood cells in the blood are subjected to the acoustic radiation of the primary separation module 2 to form a specific blood alignment and plasma alignment and separation rules.
In this embodiment, in order to facilitate the primary separation module 2 to sufficiently separate the blood plasma and the blood cells in the blood, the first channel 11 includes a flow chamber 111, a first channel 11 through which the blood flows is provided in the flow chamber 111, the primary separation module 2 is disposed on an outer peripheral side of the flow chamber 111, the primary separation module 2 is configured to generate a surface acoustic wave and acts on the flow chamber 111, and the primary separation module 2 is configured to set the first channel 11 in a propagation range of the primary separation module 2, so that the blood in the flow chamber 111 separates the blood plasma from the blood cells, and the primary separation module 2 generates a certain acoustic radiation force, so that the blood plasma and the blood cells are separated and the blood cells are arranged in a row under the action of the acoustic radiation force, thereby realizing the separation of the blood cells and the blood plasma in the blood. It should be noted that the primary separation module 2 is in the prior art, and the description is not repeated here.
Specifically, the first channel 11 includes an inflow region 112, a narrow region 113, and an outflow region 114, the narrow region 113 is disposed on one side of the inflow region 112, the outflow region 114 is disposed on one side of the narrow region 113 away from the inflow region 112, the outflow region 114 is provided with a first discharge port 1141 and a second discharge port 1142, the first discharge port 1141 and the second discharge port 1142 are disposed at intervals in the width direction of the flow chamber 111, and further the primary separation module 2 is matched with the narrow region 113 and the outflow region 114 to achieve thorough separation of plasma and blood cells. It should be noted that, the acoustic radiation force of the primary separation module 2 covers the inflow region 112, the narrow region 113 and the outflow region 114, so as to ensure that the blood cells in the blood are acted on by the acoustic radiation force to form a specific regular arrangement when flowing through these positions. The width of the inflow region 112 is larger than that of the narrow region 113, and the width of the narrow region 113 is smaller than that of the outflow region 114, so that when blood flows in from the inflow region 112, blood cells are more rapidly focused into a particle beam through the extrusion of the narrow region 113, and the effective separation of blood plasma and blood cells is facilitated. The junction between the inflow region 112 and the narrow region 113 is an inclined plane, the inclined plane is gradually inclined downwards from one side of the inflow region 112 towards the direction of the narrow region 113, and the inclined angle of the inclined plane is 30 degrees, so that when blood enters into the flow channel, the blood can enter into the propagation range of the sound surface wave, and is arranged into a strip parallel to the length direction of the first channel 11 under the action of the sound radiation force, the strip formed by blood cells deviates from one side of the primary separation module 2 under the driving of the inclined plane, and the blood cells can be smoothly separated from the blood plasma under the extrusion of the narrow region 113, and finally the separated blood plasma and blood cells respectively flow out to the first channel 11 after passing through the outflow region 114, so that the separation of the blood cells and the blood plasma in the blood is realized.
Specifically, the junction of the outflow region 114 and the narrow region 113 is an inclined plane, the inclined plane is gradually inclined upwards from one side of the narrow region 113 towards the direction of the outflow region 114, and the inclined angle of the inclined plane is 30 degrees, so that when blood passes through the separation of the narrow region 113, plasma and blood cells can be rapidly discharged from the first channel 11 and enter the second channel 12, and separation of the plasma and the blood cells can be rapidly realized under the effective cooperation of the narrow region 113 and the outflow region 114, so that the subsequent separation of the blood cells is facilitated.
Referring to fig. 2, in an alternative embodiment, the second channel 12 includes an inlet area 121 and a sorting area 122 connected in sequence, and the inlet area 121 is communicated with the second discharge port 1142;
the sorting area 122 includes two first liquid outlet passages 1221 and second liquid outlet passages 1222 disposed at an angle;
the secondary separation module 3 is a magnet generating member 31, the magnet generating member 31 is disposed in the inlet region 121, and the magnet generating member 31 sorts blood cells passing through the inlet region 121;
the third separation module 4 is disposed in the first liquid outlet passage 1221 and the second liquid outlet passage 1222, respectively, so as to perform the second separation of the blood cells passing through the separation area 122.
In this embodiment, in order to facilitate separation of various types of cells in blood cells, the sorting area 122 includes two first liquid outlet channels 1221 and second liquid outlet channels 1222 that are disposed at an included angle, the secondary separation module 3 is a magnet generating element 31, the magnet generating element 31 is disposed at the inlet area 121, and further, the blood cells are further rapidly separated under the influence of the magnetic force of the magnet generating element 31, wherein white blood cells and circulating tumor cells that are subjected to stronger negative magnetophoresis force are repelled by the magnet generating element 31 to flow into the first liquid outlet channels 1221, and red blood cells that are subjected to positive magnetophoresis force and platelets that are subjected to weaker negative magnetophoresis force flow into the second liquid outlet channels 1222, so that under the cooperation of the magnet generating element 31, various cells in the blood cells are respectively enabled to enter different liquid outlet channels, and then, under the effect of the tertiary separation module 4, the third cell separation is performed.
Referring to fig. 2, in an alternative embodiment, the third channel 13 includes two third liquid channels 131 disposed at an angle and two fourth liquid channels 132 disposed at an angle, the two third liquid channels 131 are all communicated with the first liquid channel 1221, each third liquid channel 131 is provided with a first liquid outlet, the two fourth liquid channels 132 are communicated with the second liquid channel 1222, and the fourth liquid channel 132 is provided with a second liquid outlet;
The third separation module 4 includes four microelectrode generating elements 41, each two microelectrode generating elements 41 are disposed on two sides of the first liquid outlet channel 1221, and the other two microelectrode generating elements 41 are disposed on two sides of the second liquid outlet channel 1222, so that the blood cells entering the first liquid outlet channel 1221 and the second liquid outlet channel 1222 are separated and discharged through the two third liquid outlet channels 131 and the two fourth liquid outlet channels 132 respectively;
four fourth channels 14 are provided, and each fourth channel 14 is respectively connected to each first liquid outlet and each second liquid outlet.
In this embodiment, the third channel 13 includes two third liquid channels 131 disposed at an included angle and two fourth liquid channels 132 disposed at an included angle, where the two third liquid channels 131 are all communicated with the first liquid channels 1221, each third liquid channel 131 is provided with a first liquid outlet, the two fourth liquid channels 132 are communicated with the second liquid channels 1222, the fourth liquid channels 132 are provided with a second liquid outlet, and further white blood cells, circulating tumor cells, platelets, red blood cells and the like separated through the inlet area 121 can be rapidly separated, and sequentially discharged through the two third liquid channels 131 and the two fourth liquid channels 132.
Specifically, in order to facilitate separation of various cells in the blood cells from each outlet in sequence, the third separation module 4 includes microelectrode generating elements 41, where four microelectrode generating elements 41 are provided, each two microelectrode generating elements 41 are provided on two sides of the first liquid outlet channel 1221, and the other two microelectrode generating elements 41 are provided on two sides of the second liquid outlet channel 1222, so that the cells flowing into the first liquid outlet channel 1221 and the second liquid outlet channel 1222 will be separated and flow into different liquid outlets under the influence of the microelectrode elements. White blood cells in the first liquid outlet passage 1221 flow out of the first liquid outlet of the third liquid outlet passage 131 under the action of positive alternating current-dielectrophoresis force, and circulating tumor cells flow out of the first liquid outlet of the other third liquid outlet passage 131 under the action of negative alternating current-dielectrophoresis force; platelets in the second fluid channel 1222 flow out of the second fluid outlet of one fourth fluid channel 132 under the action of negative ac-dielectrophoresis force, and red blood cells flow out of the second fluid outlet of the other fourth fluid channel 132 under the action of orthogonal flow-dielectrophoresis force. Thus realizing continuous separation of circulating tumor cells, red blood cells, platelets and white blood cells in the blood sample without marking the cell sample in advance and affecting the physiological activity of the cells. And, after the circulating tumor cells, the red blood cells, the platelets and the white blood cells separated for three times enter the third channel respectively, fluorescence labeling is carried out, and then a third target cell solution after labeling is obtained.
Referring to fig. 2, in an alternative embodiment, in order to facilitate the image acquisition of the third target cell solution after three separations by the image acquisition module 5, four fourth channels 14 are provided, each fourth channel 14 is respectively connected to the first liquid outlet and the second liquid outlet, and further, the labeled circulating tumor cells, erythrocytes, platelets and leukocytes enter each fourth channel 14 respectively, so that the third target cell solution labeled by the image acquisition module 5 can conveniently present different fluorescent colors under the imaging of the fluorescent microscope, and fluorescent images of different cell types are captured by the camera, and finally, the collected fluorescent images are used for quantization analysis by the image processor, so as to obtain the number and proportion of various blood cells in the blood sample, and output statistical results.
The foregoing description is only of the preferred embodiments of the present invention and is not intended to limit the scope of the invention, and all equivalent structural changes made by the description of the present invention and the accompanying drawings or direct/indirect application in other related technical fields are included in the scope of the invention.
Claims (8)
1. The cell detection method based on the micro-fluidic chip is characterized by comprising the following steps of:
placing collected blood into a microfluidic chip-based device through a sample collection port, and performing primary separation treatment on a sample to be detected based on a primary separation module to obtain a first target cell solution;
performing secondary separation on cells in the first target cell solution based on a secondary separation module to obtain a second target cell solution;
performing three times of separation on the cells in the second target cell solution based on the three times of separation module, and marking the separated cells based on a preset fluorescent marker to obtain a third target cell solution;
collecting fluorescent images of the third target cell solution based on the image collecting module;
based on a data processing module, carrying out quantitative analysis processing on the fluorescent image acquired by the image acquisition module to obtain the quantity and proportion of various blood cells in a blood sample, and outputting a statistical result;
wherein, based on the secondary separation module, the secondary separation is carried out to the cells in the first target cell solution, obtains the step of second target cell solution, includes:
Applying magnetophoresis force to the first target cell solution based on a secondary separation module so as to enable different cell types in the first target cell solution to be separated for the second time in the microfluidic chip, thereby obtaining a second target cell solution;
the step of separating the cells in the second target cell solution for three times based on the three times separation module, and labeling the separated cells based on a preset fluorescent marker to obtain a third target cell solution comprises the following steps:
and applying dielectrophoresis force to the second target cell solution based on the third separation module so as to perform third separation on the obtained second target cell solution and perform fluorescent marking on the cells separated for the third time to obtain a marked third target cell solution.
2. The method for detecting cells based on a microfluidic chip according to claim 1, wherein the step of placing the collected blood into the microfluidic chip-based device through the sample collection port and performing a first separation process on the sample to be detected based on the primary separation module to obtain the first target cell solution comprises the steps of:
inputting collected blood into a microfluidic chip-based device through a sample collection port, and applying ultrasonic pulses to a sample to be detected by the primary separation module according to separation frequencies corresponding to cell types in the sample to be detected so as to separate cells in the blood in the microfluidic chip to obtain a first target cell solution;
The obtained first target cell solution flows into each channel in the microfluidic chip respectively.
3. The microfluidic chip-based cell detection method according to claim 1, wherein the step of collecting the fluorescent image of the third target cell solution based on the image collection module comprises:
and imaging and acquiring the fluorescence image of the marked third target cell solution through an image acquisition module.
4. The method for detecting cells based on a microfluidic chip according to claim 1, wherein the step of quantitatively analyzing the fluorescent image collected by the image collection module based on the data processing module to obtain the number and the proportion of various blood cells in the blood sample and outputting the statistical result comprises the steps of:
detecting the fluorescence image to obtain the fluorescence color, fluorescence intensity and fluorescence wavelength corresponding to the fluorescent marker on the cell;
the cell type is determined from the fluorescent color, the fluorescent intensity, and the fluorescent wavelength.
5. A cell detection device based on a microfluidic chip, characterized in that the cell detection device based on the microfluidic chip comprises;
The micro-channel piece is provided with a first channel, a second channel, a third channel and a fourth channel which are sequentially arranged and communicated along the length direction of the micro-channel piece;
the primary separation module is arranged in the first channel to perform primary separation treatment on a sample to be detected to obtain a first target cell solution, and the first target cell solution enters the second channel;
the secondary separation module is arranged in the second channel to separate different cell types in the first target cell solution so as to obtain a second target cell solution;
the third separation module is arranged in the second channel and on one side of the second separation module so as to separate different cell types in the obtained second target cell solution, and the cell solution after the third separation enters the third channel and is subjected to fluorescent marking in the third channel to obtain a marked third target cell solution;
the image acquisition module is arranged in the fourth channel and comprises a fluorescence microscope and a camera, and the marked third target cell solution presents different fluorescence colors under the imaging of the fluorescence microscope and captures fluorescence images of different cell types through the camera;
The data processing module comprises an image processor, wherein the image processor is electrically connected with the camera so as to enable fluorescent images obtained by the camera to be transmitted to the image processor, and the image processor is used for carrying out quantitative analysis on the collected fluorescent images.
6. The microfluidic chip-based cell testing device according to claim 5, wherein the first channel comprises a flow chamber, a first channel through which blood flows is provided in the flow chamber, the primary separation module is provided on an outer peripheral side of the flow chamber, the primary separation module is used for generating surface acoustic waves and acting on the flow chamber to separate plasma from blood cells from the blood in the flow chamber, the first channel comprises an inflow region, a narrow region and an outflow region, the narrow region is provided on one side of the inflow region, the outflow region is provided on one side of the narrow region away from the inflow region, the outflow region is provided with a first outlet and a second outlet, and the first outlet and the second outlet are provided at intervals in a width direction of the flow chamber.
7. The microfluidic chip-based cell detection device according to claim 6, wherein the second channel comprises an inlet region and a sorting region connected in sequence, the inlet region being communicated with the second discharge port;
The sorting area comprises a first liquid outlet channel and a second liquid outlet channel which are arranged at an included angle;
the secondary separation module is a magnet generating piece, the magnet generating piece is arranged in the inlet area, and the magnet generating piece sorts blood cells passing through the inlet area;
the third separation module is respectively arranged on the first liquid outlet channel and the second liquid outlet channel so as to carry out secondary separation on the blood cells passing through the separation area.
8. The microfluidic chip-based cell detection device according to claim 7, wherein the third channel comprises two third liquid outlet channels arranged at an included angle and two fourth liquid outlet channels arranged at an included angle, the two third liquid outlet channels are all communicated with the first liquid outlet channel, each third liquid outlet channel is provided with a first liquid outlet, the two fourth liquid outlet channels are communicated with the second liquid outlet channel, and the fourth liquid outlet channel is provided with a second liquid outlet;
the three-time separation module comprises four microelectrode generating parts, wherein every two microelectrode generating parts are arranged on two sides of the first liquid outlet channel, and the other two microelectrode generating parts are arranged on two sides of the second liquid outlet channel, so that blood cells entering the first liquid outlet channel and the second liquid outlet channel are separated and discharged through two third liquid outlet channels and two fourth liquid outlet channels respectively;
The fourth channels are four, and each fourth channel is respectively communicated with each first liquid outlet and each second liquid outlet.
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