CN111330659B - Micro-fluidic chip based on electrical signals and blood cell analysis device and method - Google Patents
Micro-fluidic chip based on electrical signals and blood cell analysis device and method Download PDFInfo
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Abstract
A blood cell analysis device and method based on electrical signal, the micro-fluidic chip includes an insulation carrier and an insulation substrate, wherein, the insulation carrier includes a cell solution injection channel, a cell compression channel, a cell solution cracking pool, a cell nucleus compression channel and a cell nucleus recovery channel; the insulating substrate comprises metal electrodes, and the metal electrodes are respectively connected with outlets of each side compression passage of the cell compression passage and the cell nucleus compression passage. The invention can detect the cell nucleus at the same time of detecting the cell by extracting the cell nucleus through the serpentine channel, and compared with the prior method, the invention eliminates the influence that different types of cells cannot be distinguished because the cell volumes are the same; the invention can accurately acquire the inherent size information of the cells and the cell nucleuses through the four T-shaped compression channels, and compared with the prior method, the invention improves the accuracy of measurement, thereby improving the accuracy of blood cell analysis.
Description
Technical Field
The invention belongs to the field of biomedical detection, and particularly relates to a blood cell analysis device and method based on electrical signals.
Background
Blood cell analysis is a technique for analyzing white blood cells and the like by detection with some instruments. The change of various parameter indexes such as leucocyte is analyzed to prompt and early warn clinically, thereby providing basis for the diagnosis of clinical diseases and having important significance. The differential counting of blood cells is the core part and key link of blood cell analysis, however, the bottleneck of current research is the inherent property of lacking effective single cell characterization tools to collect a large amount of single blood cells. Single cell and nucleus size information, as an intrinsic property of single cells, has been shown to be useful for distinguishing blood cells and the like. Therefore, the detection of the cell and the cell nucleus having the same characteristics for blood cell analysis has great research significance for being rapidly and accurately applied to clinical diagnosis and treatment.
The conventional method of blood cell analysis mainly applies the coulter principle. The coulter principle means that when white blood cells suspended in electrolyte pass through a small hole along with the electrolyte, the white blood cells replace the electrolyte with the same volume, the resistance between electrodes on two sides of the small hole is instantaneously changed in a constant current designed circuit, and potential pulses are generated, wherein the size and the frequency of pulse signals are in direct proportion to the size and the number of the white blood cells. At low frequencies, differential white blood cell counts are determined from the conversion of the pulse signal generated by the cells through the pores into cell volumes. Furthermore, in order to differentiate the leukocyte population to a greater extent, hemolytic agents are used to differentially act on the leukocyte plasma membrane, the shrinkage of the lymphocyte plasma membrane results in its cell volume becoming smaller, the granulocytes are protected from the contractile action of hemolytic agents, and the monocyte volume shrinkage is instead smaller than the granulocytes volume but remains somewhat stable and larger than the lymphocyte volume. When the cells are analyzed, the pulse of each cell is distributed in the corresponding volume channel according to the volume size of the pulse, the data collected by each volume channel is counted to obtain a relative number, the distribution of the white blood cells is preliminarily determined to be a three-way group, a small volume area is mainly lymphocytes, a middle volume area is mainly monocytes, and a large volume area is mainly neutrophils. Although this method can be applied to the classification of leukocytes, the measured cell volume is not the physiological volume of the cells, and cannot characterize the intrinsic characteristics of leukocytes. And because the pulse signals generated by different types of cells with the same volume have the same amplitude and cannot be distinguished, the subsequent development simultaneously applies high-frequency signals to compensate the defect. The problem that low-frequency current cannot pass through cell membranes is solved by the high-frequency current, and white blood cells are distinguished according to different impedances of cell nuclei to the high-frequency current due to different sizes and densities. This method, while allowing further subdivision of leukocytes, also fails to characterize the intrinsic properties of the nucleus.
Therefore, it is very useful to develop a novel apparatus and method for analyzing blood cells, which can realize more accurate blood cell analysis by using the inherent characteristics of cells.
Disclosure of Invention
In view of the above, one of the main objectives of the present invention is to provide a micro-fluidic chip module based on electrical signals, a method for manufacturing the same, a blood cell analysis device and a method thereof, so as to at least partially solve at least one of the above technical problems.
In order to achieve the above object, as an aspect of the present invention, there is provided a microfluidic chip module including an insulating carrier and an insulating substrate, wherein the insulating carrier includes:
the cell solution injection channel is used for injecting cell solution to be detected;
a cell compressing channel for compressing cells flowing into the cell solution injecting channel;
the cell solution cracking pool comprises a cell lysate injection channel and a cell nucleus extraction channel, and the compressed cells enter the cell solution cracking pool to react and crack with the cell lysate injected through the cell lysate injection channel;
a cell nucleus compression channel for compressing the cell nucleus formed after the lysis; and
a cell nucleus recovery channel for recovering the compressed cell nucleus;
the insulating substrate comprises metal electrodes, and the metal electrodes are respectively connected with outlets of each side compression passage of the cell compression passage and the cell nucleus compression passage.
As another aspect of the present invention, there is also provided a method for preparing a microfluidic chip module, the method for preparing the microfluidic chip module described above, including:
forming a chromium mark on a substrate;
forming a seed layer on the glass sheet on which the chromium mark is formed;
preparing a microfluidic channel male die on the seed layer;
preparing an insulating bearing body containing the microfluidic channel on the microfluidic channel male die;
preparing a metal electrode layer on the other substrate, and stripping to obtain an on-chip electrode;
and punching holes at corresponding positions of the microfluidic channels of the insulating bearing body, and bonding the holes with an insulating substrate containing an upper electrode to obtain the microfluidic chip.
As another aspect of the present invention, there is also provided a microfluidic chip module, which contains at least one microfluidic chip as described above or a microfluidic chip obtained by the above preparation method.
As still another aspect of the present invention, there is also provided a blood cell analysis apparatus including:
a microfluidic chip module as described above;
the pressure control module is connected with the cell solution injection channel and the cell lysate injection channel and is used for driving cells to enter the cell compression channel or driving cell nuclei to enter the cell nucleus compression channel; and
and the impedance measuring module is respectively connected with each cell side compression channel and each cell nucleus side compression channel and is used for detecting the change of impedance when the cells pass through the cell side compression channels and the change of impedance when the cell nuclei pass through the cell nucleus side compression channels.
Based on the technical scheme, the micro-fluidic chip module based on the electrical signal, the preparation method thereof, the blood cell analysis device and the blood cell analysis method have at least one of the following advantages compared with the prior art:
1. the invention can detect the cell nucleus at the same time of detecting the cell by extracting the cell nucleus through the serpentine channel, and compared with the prior method, the invention eliminates the influence that different types of cells cannot be distinguished because the cell volumes are the same; the existing applied method for distinguishing the white blood cells, such as distinguishing by using low-frequency signals, can only distinguish the white blood cells according to small, medium and large volume areas, and the result has errors inevitably;
2. the invention can accurately acquire the inherent size information of the cells and the cell nucleuses through the four T-shaped compression channels, and compared with the prior method, the invention improves the accuracy of measurement, thereby improving the accuracy of blood cell analysis; the current method for analyzing leucocytes uses a method of combining low-frequency and high-frequency signals, and the measured cell and cell nucleus volumes are not the physiological volume of cells but relative quantities, so that the leucocytes cannot be accurately characterized and analyzed.
Drawings
FIG. 1 is a schematic structural view of a blood cell analyzer according to an embodiment of the present invention;
FIG. 2 is a schematic structural diagram of a microfluidic chip module according to an embodiment of the present invention;
FIG. 3 is a flow chart of the microfluidic chip module according to an embodiment of the present invention;
FIG. 4 is a graph of the cell and nucleus stretch length calculation model and the corresponding impedance magnitude in the embodiment of the present invention.
Description of reference numerals:
100-cell solution injection channel; 200-cell compression channel; 210-cell major compression channel; 211-first cell-side compression channel; 212-second cell side compression channel; 213-third cell side compression channel; 214-fourth cell-side compression channel; 300-cell solution lysis cell; 400-cell lysate injection channel; 500-cell nucleus extraction channel; 600-nuclear compaction channel; 610-nuclear major compression channel; 611-a first nucleus side compression channel; 612-second nucleus side compression channel; 613-third nucleus side compression channel; 614-fourth nuclear side compression channel; 700-cell nucleus recovery channel; 801-a first cell electrode; 802-a second cell electrode; 803-a third cell electrode; 804-a fourth cell electrode; 811-first nuclear electrode; 812-a second nuclear electrode; 813-a third nuclear electrode; 814-fourth nuclear electrode.
Detailed Description
In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.
The invention discloses a micro-fluidic chip based on electrical signals, which comprises an insulating carrier and an insulating substrate, wherein the insulating carrier comprises:
the cell solution injection channel is used for injecting cell solution to be detected;
a cell compressing channel for compressing cells flowing into the cell solution injecting channel;
the cell solution cracking pool comprises a cell lysate injection channel and a cell nucleus extraction channel, and the compressed cells enter the cell solution cracking pool to react and crack with the cell lysate injected through the cell lysate injection channel;
a cell nucleus compression channel for compressing the cell nucleus formed after the lysis; and
a cell nucleus recovery channel for recovering the compressed cell nucleus;
the insulating substrate comprises metal electrodes, and the metal electrodes are respectively connected with outlets of each side compression passage of the cell compression passage and the cell nucleus compression passage.
In some embodiments of the present invention, the cell compression passages include a cell main compression passage and a cell side compression passage, and the cell side compression passage is a first cell side compression passage, a second cell side compression passage, a third cell side compression passage and a fourth cell side compression passage in this order along the cell main compression passage;
the first cell side compression passage and the fourth cell side compression passage are arranged on the same side of the cell main compression passage, and the second cell side compression passage and the third cell side compression passage are arranged on the other side of the cell main compression passage at the same time.
In some embodiments of the invention, the cell main compression channel has a cross-sectional width of 5 to 20 microns;
in some embodiments of the invention, each of the cell-side compression channels has a cross-sectional width of 3 to 5 micrometers;
in some embodiments of the present invention, the cell main compression passage and each of the cell side compression passages have the same cross-sectional height.
In some embodiments of the present invention, the cell nucleus compression passages include a cell nucleus main compression passage and a cell nucleus side compression passage, and the cell nucleus side compression passage is a first cell nucleus side compression passage, a second cell nucleus side compression passage, a third cell nucleus side compression passage and a fourth cell nucleus side compression passage along the cell nucleus main compression passage in sequence;
the first cell nucleus side compression passage and the fourth cell nucleus side compression passage are arranged on the same side of the cell nucleus main compression passage, and the second cell nucleus side compression passage and the third cell nucleus side compression passage are arranged on the other side of the cell nucleus main compression passage at the same time.
In some embodiments of the invention, the nuclear main compression channel has a cross-sectional width of 4 to 12 microns;
in some embodiments of the invention, each of the nucleus-side compression passages has a cross-sectional width of 3 to 5 microns;
in some embodiments of the present invention, the cross-sectional heights of the nucleus main compression passage and each of the nucleus side compression passages are the same.
In some embodiments of the invention, the shape of the cell nucleus extraction channel comprises a serpentine shape.
The invention also discloses a preparation method of the microfluidic chip, which is used for preparing the microfluidic chip and comprises the following steps:
forming a chromium mark on a substrate;
forming a seed layer on the glass sheet on which the chromium mark is formed;
preparing a microfluidic channel male die on the seed layer;
preparing an insulating bearing body containing the microfluidic channel on the microfluidic channel male die;
preparing a metal electrode layer on the other substrate, and stripping to obtain an on-chip electrode;
and punching holes at corresponding positions of the microfluidic channels of the insulating bearing body, and bonding the holes with an insulating substrate containing an upper electrode to obtain the microfluidic chip.
The invention also discloses a micro-fluidic chip module which contains at least one micro-fluidic chip or the micro-fluidic chip prepared by the preparation method.
In some embodiments of the present invention, a plurality of the microfluidic chips are connected in series or in parallel.
The present invention also discloses a blood cell analysis apparatus, comprising:
a microfluidic chip module as described above;
the pressure control module is connected with the cell solution injection channel and the cell lysate injection channel and is used for driving cells to enter the cell compression channel or driving cell nuclei to enter the cell nucleus compression channel; and
and the impedance measuring module is respectively connected with each cell side compression channel and each cell nucleus side compression channel and is used for detecting the change of impedance when the cells pass through the cell side compression channels and the change of impedance when the cell nuclei pass through the cell nucleus side compression channels.
In one exemplary embodiment, the present invention is an apparatus and method for blood cell analysis using electrical signals. Mainly comprises a hardware system (a micro-fluidic chip module, a pressure control module and an impedance measurement module) which is necessary for realizing the method, and a realization method for obtaining blood cell analysis according to the hardware system. When the micro-fluidic chip works, the pressure control module is used for controlling the sample introduction speed of the cell solution and the cell lysate entering the micro-fluidic channel. The method comprises the steps that cells firstly pass through a four-T-shaped compression channel, impedance information of cells with or without cell passing is obtained through an impedance measurement module, then two phases of a cell solution and a cell lysate simultaneously pass through a snake-shaped channel, the two-phase laminar flow is changed into a turbid flow to generate diffusion, the extraction of cell nuclei is achieved, finally the extracted cell nuclei pass through the four-T-shaped compression channel, and the impedance information of the cells with or without cell passing is obtained through the impedance measurement module again. On the basis of the impedance information, the size information of the cells and the cell nucleuses can be obtained by combining the algorithm provided by the invention, and finally, the blood cell analysis is realized. Compared with the existing method, the invention utilizes the inherent characteristics of cells and cell nucleuses to more accurately perform blood cell analysis.
The technical solution of the present invention is further illustrated by the following specific embodiments in conjunction with the accompanying drawings. It should be noted that the following specific examples are given by way of illustration only and the scope of the present invention is not limited thereto.
The blood cell analysis apparatus based on electrical signals of this embodiment is shown in fig. 1, and mainly includes a microfluidic chip module, an impedance measurement module, and a pressure control module.
The microfluidic chip module is a core module in a hardware device and is formed by bonding an insulating carrier and an insulating substrateThe structure is schematically shown in figure 2. The insulating bearing body of the microfluidic chip module sequentially comprises a cell solution injection channel 100, a cell compression channel 200 (comprising a cell main compression channel 210, a first cell side compression channel 211, a second cell side compression channel 212, a third cell side compression channel 213 and a fourth cell side compression channel 214), a cell solution cracking cell 300 (comprising a cell lysate injection channel 400 and a snake-shaped cell nucleus extraction channel 500), a cell nucleus compression channel 600 (comprising a cell nucleus main compression channel 610, a first cell nucleus side compression channel 611, a second cell nucleus side compression channel 612, a third cell nucleus side compression channel 613 and a fourth cell side compression channel 614) and a cell nucleus recovery channel 700. Specifically, the cell solution injection channel 100 and the cell lysate injection channel 400 are structurally characterized in that the cross-section is much larger than the cell diameter to ensure the normal flow of cells, and the minimum dimension of the cross-section height of the channel is about 40 micrometers (the diameter of white blood cells is about 6-25 micrometers). The cell compression passage 200 is a four-T structure and comprises a cell main compression passage 210 and four cell side compression passages, wherein the cell main compression passage 210 is structurally characterized in that the cross section is smaller than the cell cross section so as to compress the cells flowing through, and the width and the height of the cross section are both about 5-20 micrometers; the cell side compression channel is characterized in that the cross sectional area is smaller than the cross sectional area of the side edge of the cell stretched in the main compression channel, the width of the cross sectional area of the channel is about 3-5 microns, and the height of the cross sectional area is the same as that of the cell main compression channel; the distance L between the first cell-side compression path 211 and the second cell-side compression path 21211A distance L from the third cell-side compression passage 213 and the fourth cell-side compression passage 21412Are different in length from one another, e.g. L12＝2L11While L is11And L12Distance L between13The minimum length is greater than the cell stretch length; the first cell side compression passage 211 and the fourth cell side compression passage 214 are on the same side of the cell main compression passage 210, and the second cell side compression passage 212 and the third cell side compression passage 213 are on the same side of the cell main compression passage 210.
The structural features of the cell lysate injection channel 400 and the serpentine nucleus extraction channel 500 are the same as those of the cell solution injection channel 100.
The structure of the cell nucleus compression passage 600 is similar to the structure of the cell compression passage 200, and is a four-T type structure, which comprises a cell nucleus main compression passage 610 and four cell nucleus side compression passages (including a first cell nucleus side compression passage 611, a second cell nucleus side compression passage 612, a third cell nucleus side compression passage 613 and a fourth cell nucleus side compression passage 614), wherein the structure characteristic of the cell nucleus main compression passage 610 is that the cross section is as small as possible than the cross section of the cell nucleus so as to compress the cell nucleus flowing through, and the size of the cross section is about 4-12 microns; the structure of the nucleus side compression passage is characterized in that the cross sectional area is smaller than the side cross sectional area of the nucleus stretched in the nucleus main compression passage 610, the width of the cross sectional area of the passage is about 3-5 microns, and the height of the cross sectional area is the same as that of the nucleus main compression passage 610; the distance L between the first nucleus-side compression passage 611 and the second nucleus-side compression passage 61221And a distance L between the third cell-nucleus side compression passage 613 and the fourth cell-nucleus side compression passage 61422Different, e.g. L22＝2L21While L is21And L22Distance L between23Is greater than the stretched length of the nucleus; while the first nucleus side compression passage 611 is on the same side of the nucleus main compression passage 610 as the fourth nucleus side compression passage 614, the second nucleus side compression passage 612 and the third nucleus side compression passage 613 are on the other side of the nucleus main compression passage 610. The structural characteristics of the nucleus recovery channel 700 are the same as those of the cell solution injection channel 100.
The glass insulating substrate of the micro-fluidic chip module mainly comprises metal electrodes, namely, the first cell side compression channel 211 and the second cell side compression channel 212 are in conductive connection with the impedance measurement module through the first cell electrode 801 and the second cell electrode 802 on the chip, the third cell side compression channel 213 and the fourth cell side compression channel 214 are in conductive connection with the impedance measurement module through the third cell electrode 803 and the fourth cell electrode 804 on the chip, the first cell nucleus electrode 811 and the second cell nucleus electrode 812 on the chip are in conductive connection with the first cell nucleus side compression channel 611 and the second cell nucleus side compression channel 612 and the impedance measurement module, the third cell nucleus electrode 813 and the fourth cell nucleus electrode 814 are in conductive connection with the third cell nucleus side compression channel 613 and the fourth cell nucleus side compression channel 614 and the impedance measurement module, wherein the overlapping area of the side channels and the on-chip electrodes cannot be too small, so as to prevent the capacitance of the electric double layer capacitor connected in series in the detection system from influencing the measurement effectiveness too much. And bonding the subsequent glass insulating substrate and the insulating bearing body to obtain the microfluidic chip.
The microfluidic chip module processing flow is shown in fig. 3. Specifically, as shown in a-b of fig. 3, firstly, an AZ 1500 photoresist is spin-coated on a glass sheet sputtered with chromium (Cr), a mask with a chromium mark is formed after pre-baking, exposure, development and hardening, then chromium outside the mark is removed by using a chromium etching solution, the photoresist of the remaining mask is removed, and finally, the chromium mark is formed on the glass sheet, as shown in fig. 3-c. And then spin-coating a layer of SU 8-2 on the chromium glass plate, and forming a seed layer after pre-baking, flood exposure and post-baking hardening, as shown in figure 3-d. Next, spin-coating a layer of SU 8-5 on the seed layer, and pre-baking, exposing and post-baking as shown in figure 3-e; then, a layer of SU 8-25 is coated in a spinning mode, and the required micro-fluidic channel male die is formed by pre-baking, exposure, post-baking, developing and film hardening, as shown in the attached drawing 3-g. And then pouring a PDMS and curing agent mixed solution with a certain thickness by using the prepared mould, as shown in the attached drawing 3-h, and demoulding after curing to obtain the PDMS containing the microfluidic channel. Then spin-coating AZ 1500 on the glass substrate, prebaking, exposing, developing to remove the photoresist at the electrode position, and sputtering chromium/gold (Cr/Au) on the metal electrode as shown in FIG. 3-j, and then performing a lift-off operation to obtain the on-chip electrode as shown in FIG. 3-k. And finally, punching a hole at the corresponding position of the obtained microfluidic channel, and bonding the punched hole with a glass substrate containing an on-chip electrode to obtain a complete microfluidic chip, as shown in the attached figure 3-1.
Impedance measurement modules are well known in the art and include lock-in amplifiers. According to the requirement of the embodiment, the existence of the impedance change can be accurately detected, the output frequency is 100000 sampling points/second, and the interface connected with the microfluidic chip module is a metal clamp or other metal clamps.
Pressure control modules are well known in the art and include micro-syringe pumps, syringes, and teflon tubing. The micro-injection pump is used for controlling the sample introduction speed of the solution in the experiment and is connected with the micro-fluidic chip module through a tetrafluoroethylene tube.
The specific implementation method of the embodiment includes an experimental operation for acquiring the original data and a method for processing the original data:
in the experimental operation of this embodiment, the microfluidic chip module, the impedance measurement module and the pressure control module are connected first. One end of the impedance measuring module is connected with an on-chip first electrode 801 corresponding to the first cell side compression channel 211 of the cell compression channel, an on-chip electrode 804 corresponding to the fourth cell side compression channel 214 of the cell compression channel, an on-chip first electrode 811 corresponding to the first cell nucleus side compression channel 611 of the cell nucleus compression channel, an on-chip electrode 814 corresponding to the fourth cell nucleus side compression channel 614 of the cell nucleus compression channel, and the other end is connected with an on-chip first electrode 802 corresponding to the second cell side compression channel 212 of the cell compression channel, an on-chip electrode 803 corresponding to the third cell side compression channel 213 of the cell compression channel, an on-chip first electrode 812 corresponding to the second cell nucleus side compression channel 612 of the cell nucleus compression channel, and an on-chip electrode 813 corresponding to the third cell nucleus side compression channel 613 of the cell nucleus compression channel; the micro-injection pump of the pressure control module is connected to the cell solution injection channel 100 and the cell lysate injection channel 400 of the micro-fluidic chip module through an injector (the influence of the discharged bubbles on the flow of the cells or cell nuclei is eliminated) containing the cell solution and the cell lysate. Then, a micro-injection pump controls a cell solution and a cell lysate to enter a micro-fluidic chip channel according to a certain sample introduction speed, cells enter a cell compression channel under the pushing of positive pressure, and an impedance measurement module is used for detecting impedance data when whether cells pass between electrodes or not; then the cell solution and the cell lysate pass through the serpentine channel simultaneously, so that the two-phase laminar flow is changed into turbid flow to generate diffusion, and the extraction of cell nucleus is realized; and finally, the extracted cell nucleus passes through a cell nucleus compression channel, and the impedance measurement module is used again to detect whether the cell nucleus passes between the electrodes or not, wherein the impedance data are used as original data of an experiment.
The method for processing the original data of the embodiment mainly calculates the cell and nucleus stretching length, namely the cell and nucleus size.
Considering the cell compression channel L11And L12When the cell passes through, the impedance amplitude (or phase) changes, and then the cell stretching length can be calculated. The calculation model of cell elongation is shown in FIG. 4, L in FIG. 4-a11Is the distance, L, between the first cell-side compression passage 211 and the second cell-side compression passage 21212Is the distance, L, between the third cell-side compression passage 213 and the fourth cell-side compression passage 21413Is L11And L12The spacing therebetween; when the cell passes to the position shown in the attached figure 4-I-b, the cell does not block the impedance line, and the impedance amplitude is unchanged; when the cell passes between the positions shown in FIGS. 4-I-b and 4-I-c, the cell blocks the electric field lines between the first cell-side compression passage 211 and the second cell-side compression passage 212, and the impedance rises in magnitude; when a cell passes between FIGS. 4-I-c and 4-I-d, since the second cell electrode 802 and the third cell electrode 803 are connected to have equal potential, and no electric field line exists between the second cell-side compression passage 212 and the third cell-side compression passage 213, the cell does not block the electric field lines and the impedance amplitude is unchanged; when the cell passes between FIGS. 4-I-d and 4-I-e, the electric field lines between the third cell side compression passage 213 and the fourth cell side compression passage 214 are blocked by the cell, and the impedance rises in magnitude.
Considering the nuclear compaction channel L21And L22When the nucleus passes through the space, the impedance amplitude (or phase) changes, and then the nucleus stretching length can be calculated. The calculation method is the same as the calculation method of the cell stretching length. The calculation model of the nuclear elongation is shown in FIG. 4, L in FIG. 4-f21Is the distance, L, between the first cell nucleus side compression passage 611 and the second cell nucleus side compression passage 61222A distance, L, between the third cell nucleus side compression passage 613 and the fourth cell nucleus side compression passage 61423Is L21And L22The spacing therebetween; when the nucleus has traveled to the position shown in FIG. 4-I-g, it is thinThe nucleus does not block the impedance line, and the impedance amplitude is unchanged; when the cell nucleus passes between the positions shown in FIGS. 4-I-g and 4-I-h, the electric field lines between the first cell nucleus side compression passage 611 and the second cell nucleus side compression passage 612 are blocked by the cell nucleus, and the impedance amplitude rises; when the cell nucleus passes between the cell nucleus electrodes as shown in FIGS. 4-I-h and 4-I-I, since the second cell nucleus electrode 812 and the third cell nucleus electrode 813 are connected to have equal potential, no electric field line exists between the second cell nucleus side compression passage 612 and the third cell nucleus side compression passage 613, so that the electric field line is not blocked by the cell nucleus, and the impedance amplitude is not changed; when the cell nucleus passes between the third and fourth cell nucleus side compression passages 613 and 614 as shown in FIGS. 4-I-I and 4-I-j, the electric field lines between the third and fourth cell nucleus side compression passages 613 and 614 are blocked by the cell nucleus, and the impedance amplitude rises.
According to the above analysis, when the cell passes through the positions shown in FIGS. 4-I-b-e and the cell nucleus passes through the positions shown in FIGS. 4-I-g-j, the impedance amplitude will show the curve shown in FIG. 4-II, wherein the positions of the cell and the cell nucleus in FIGS. 4-I-b-e correspond to those of the cell nucleus in FIG. 4-II, and the cell passing time t is formed11And t12And cell nucleus transit time t21And t22。
Considering that the cell main compression passage 210 is sufficiently small in length, it is considered that the cells maintain uniform motion during the passage between the first cell side compression passage 211 and the second cell side compression passage 212 and the passage between the third cell side compression passage 213 and the fourth cell side compression passage 214:
solving to obtain the cell stretching length LcComprises the following steps:
further equivalent calculating according to the volume equivalent to obtain the cell diameter DcIn which S is1Cross-sectional area of the main cell compression channel:
considering that the length of the nucleus main compression passage 610 is sufficiently small, it can be considered that the nucleus keeps moving at a constant speed during passing between the first nucleus side compression passage 611 and the second nucleus side compression passage 612 and passing between the third nucleus side compression passage 613 and the fourth nucleus side compression passage 614:
solving to obtain the cell nucleus stretching length LnComprises the following steps:
further performing equivalent calculation according to the volume equivalent to obtain the cell nucleus diameter DnIn which S is2Cross-sectional area of the main compression channel of the nucleus:
up to this point, the present embodiment has been described in detail with reference to the accompanying drawings. From the above description, those skilled in the art should clearly understand that the device and method for analyzing blood cells using electrical signals in the present invention.
In this embodiment, the substrate is made of glass, and it should be clear to those skilled in the art that, besides glass, the substrate may be a sheet material such as a silicon wafer, a Polymethyl methacrylate (PMMA, also called acryl, Acrylic, or plexiglass), or a Polydimethylsiloxane (PDMS) sheet.
In this embodiment, the material of the supporting body is PDMS. It will be clear to those skilled in the art that the carrier can be formed from materials other than PDMS, such as glass, SU-8, silicon, etc.
The structure of the microfluidic chip demonstrated in the invention is a basic unit of the method, and can conveniently carry out parallel and serial arrangement in the cell or cell nucleus passing direction, and even the combination of certain structures can bring different effects.
The cross section of the channel in the microfluidic chip is rectangular, and the channel can be replaced by a round or semicircular shape and the like, so that the realization of the basic function is not influenced.
The length and the number of the snake-shaped channels in the microfluidic chip can be changed without influencing the realization of basic functions.
The invention forms the channel by using the sealing mode of the cover plate and the substrate, and can be realized by etching in the materials such as glass and the like, and also can realize the required functions.
In this invention positive pressure is used to drive the cell solution and cell lysate through the channel, but other means such as applying negative pressure at the end of the cell nucleus recovery channel may be used.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. An electrical signal-based microfluidic chip comprising an insulating carrier and an insulating substrate, wherein the insulating carrier comprises:
the cell solution injection channel is used for injecting cell solution to be detected;
a cell compressing channel for compressing cells flowing into the cell solution injecting channel; wherein the cell compression passage comprises a cell main compression passage and a cell side compression passage; the cell compression channel comprises a cell main compression channel and a cell side compression channel, and the cell side compression channel is sequentially provided with a first cell side compression channel, a second cell side compression channel, a third cell side compression channel and a fourth cell side compression channel along the cell main compression channel; the first cell side compression passage and the fourth cell side compression passage are arranged on the same side of the cell main compression passage, and the second cell side compression passage and the third cell side compression passage are simultaneously arranged on the other side of the cell main compression passage;
the cell solution cracking pool comprises a cell lysate injection channel and a cell nucleus extraction channel, and the compressed cells enter the cell solution cracking pool to react and crack with the cell lysate injected through the cell lysate injection channel;
a cell nucleus compression channel for compressing the cell nucleus formed after the lysis; the cell nucleus compression passage comprises a cell nucleus main compression passage and a cell nucleus side compression passage, and the cell nucleus side compression passage sequentially comprises a first cell nucleus side compression passage, a second cell nucleus side compression passage, a third cell nucleus side compression passage and a fourth cell nucleus side compression passage along the cell nucleus main compression passage; the first cell nucleus side compression passage and the fourth cell nucleus side compression passage are arranged on the same side of the cell nucleus main compression passage, and the second cell nucleus side compression passage and the third cell nucleus side compression passage are arranged on the other side of the cell nucleus main compression passage at the same time; and
a cell nucleus recovery channel for recovering the compressed cell nucleus;
the insulating substrate comprises metal electrodes, and the metal electrodes are respectively connected with outlets of each side compression passage of the cell compression passage and the cell nucleus compression passage;
the cross section area of the cell main compression channel is smaller than that of the cell, and the cross section area of the cell nucleus main compression channel is smaller than that of the cell nucleus; and
the cross-sectional area of the cell-side compression passage is smaller than the cross-sectional area of the side edge of the cell stretched in the cell main compression passage, and the cross-sectional area of the nucleus-side compression passage is smaller than the cross-sectional area of the side edge of the nucleus stretched in the nucleus main compression passage.
2. The microfluidic chip according to claim 1,
the cross-sectional width of the cell main compression channel is 5 to 20 micrometers;
the cross section width of each cell side compression channel is 3-5 micrometers;
the cross-sectional heights of the cell main compression passage and each cell side compression passage are the same.
3. The microfluidic chip according to claim 1,
the cross-sectional width of the nucleus main compression channel is 4 to 12 microns;
the cross section width of each cell nucleus side compression channel is 3-5 microns;
the cross-sectional heights of the nucleus main compression passage and each nucleus side compression passage are the same.
4. The microfluidic chip according to claim 1,
the shape of the cell nucleus extraction channel comprises a serpentine shape.
5. A method for preparing a microfluidic chip according to any one of claims 1 to 4, comprising:
forming a chromium mark on a substrate;
forming a seed layer on the glass sheet on which the chromium mark is formed;
preparing a microfluidic channel male die on the seed layer;
preparing an insulating bearing body containing the microfluidic channel on the microfluidic channel male die;
preparing a metal electrode layer on the other substrate, and stripping to obtain an on-chip electrode;
and punching holes at corresponding positions of the microfluidic channels of the insulating bearing body, and bonding the holes with an insulating substrate containing an upper electrode to obtain the microfluidic chip.
6. A microfluidic chip module, which contains at least one microfluidic chip according to any one of claims 1 to 4 or the microfluidic chip obtained by the preparation method according to claim 5.
7. The microfluidic chip module of claim 6,
the microfluidic chips are connected in series or in parallel.
8. A blood cell analysis apparatus comprising:
the microfluidic chip module of claim 6 or 7;
the pressure control module is connected with the cell solution injection channel and the cell lysate injection channel and is used for driving cells to enter the cell compression channel or driving cell nuclei to enter the cell nucleus compression channel; and
and the impedance measuring module is respectively connected with each cell side compression channel and each cell nucleus side compression channel and is used for detecting the change of impedance when the cells pass through the cell side compression channels and the change of impedance when the cell nuclei pass through the cell nucleus side compression channels.
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