Micro-fluidic chip for cell electrical impedance detection and detection method

Abstract

The invention relates to a micro-fluidic chip for cell impedance detection and a detection method, and relates to the technical field of cell detection. The micro-fluidic chip comprises a transport layer, an excitation electrode layer, a flow channel layer and a detection electrode layer which are sequentially bonded together from top to bottom, the excitation electrode layer and the detection electrode layer form a differential electrode detection area with the upper portion and the lower portion facing each other, and cell sample flow to be detected and sheath fluid are injected into the flow channel layer through the transport layer. The positions of detected cells in the flow channel are adjusted through extrusion of sheath fluid, so that the cells stably close to the bottom detection electrode flow through the detection area, and the problem that the distribution positions of cell particles in the flow channel of the detection area are disordered is solved; the electric field of a detection area is more uniform by combining upper and lower opposite electrode configuration, the electrical impedance detection precision is improved by utilizing the particularity that higher signal amplitude can be obtained by approaching the detection electrode, the problems of non-uniform cell particle electrical impedance signals and low detection precision are solved, and the accurate and high-throughput detection of the cell electrical impedance signals is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell detection, in particular to a microfluidic chip and a detection method for cell impedance detection. BACKGROUND

[0002] At present, microfluidic impedance technology as a new technology can be used for cell counting and cell type identification, which can effectively make up for the shortcomings of traditional technology methods, and has the advantages of not being interfered by cell surface antigen heterogeneity, no labeling, convenience, high throughput, no inactivation, low cost and the like. The principle is mainly to detect cells by using the physical and electrical characteristics of different types of cells, such as cell size, cell dielectric properties, etc.; usually the cell membrane and the nuclear membrane can be regarded as having capacitance, and the cytoplasm and the internal genetic material of the nucleus exhibit conductivity. The alternating current signal of different frequencies can penetrate the cell membrane and the nuclear membrane to different degrees, and the dielectric properties inside the cell can be detected and studied, so that the cell type identification and counting can be realized by analyzing the measured impedance signals. Using this technology, circulating tumor cells and normal cells (white blood cells, red blood cells, etc.) or various tumor cells can be identified and classified.

[0003] In the prior art measurement, the microfluidic impedance detection signal is usually affected by the spatial position of the cells or particles flowing through the detection area, for example, small cells (or small particles) close to the detection electrode have the same amplitude of impedance signal as large particles (or large particles) far away from the detection electrode, thus interfering with the detection; and by changing the size of the narrow channel to be close to the size of the particles, the spatial position influence can be avoided, but often causes blockage and cannot realize high throughput detection. Therefore, it is difficult to balance the detection accuracy and detection throughput of cell label-free impedance detection. SUMMARY

[0004] The purpose of the present application is to provide a microfluidic chip and a detection method for cell impedance detection to solve the above problems.

[0005] The first aspect of the present application provides a microfluidic chip for cell impedance detection, which adopts the following technical scheme:

[0006] A microfluidic chip for cell impedance detection, comprising a transport layer, an excitation electrode layer, a flow channel layer and a detection electrode layer bonded together from top to bottom, the transport layer is used to input the cell sample to be tested and sheath liquid into the flow channel layer, the excitation electrode layer and the detection electrode layer are located on both sides of the flow channel layer and cooperate to form an upper and lower facing differential electrode detection area, the excitation electrode layer is used to apply an alternating current signal, and the detection electrode layer is used to collect the impedance signal of the cell sample to be tested in the flow channel layer.

[0007] By adopting the technical scheme, the excitation electrode layer and the detection electrode layer form a differential electrode detection area facing each other, the cell sample flow is injected into the flow channel layer through the transport layer, the sheath liquid is injected into the flow channel layer through the transport layer, the sheath liquid extrudes the cell sample flow to flow through the detection area near the center area of the bottom of the channel, the excitation electrode layer applies an alternating excitation signal, the detection electrode is connected to a lock-in amplifier to collect the cell impedance signal, the position of the measured cell in the flow channel is adjusted through the extrusion of the sheath liquid, so that the cell stably flows through the detection area near the bottom detection electrode, the problem of random distribution of the cell particles in the flow channel of the detection area is solved; and the electrodes arranged on the upper and lower surfaces are combined to make the electric field of the detection area more uniform, and the particularity of obtaining a higher signal amplitude near the detection electrode is ingeniously utilized to improve the impedance detection precision, the problems of non-uniform cell impedance signal and low detection precision are solved, and the accurate and high-throughput detection of the cell impedance signal is realized.

[0008] Preferably, the transport layer comprises a transfer layer and a flow transfer layer arranged in sequence from top to bottom, the transfer layer has, in sequence from left to right on the upper surface, a first sheath liquid inlet hole penetrating the upper and lower surfaces of the transfer layer, a cell sample liquid inlet hole, a waste liquid outlet hole and a second sheath liquid inlet hole, the lower surface of the transfer layer has a first sheath liquid flow channel and a second sheath liquid flow channel, the first sheath liquid flow channel is in communication with the first sheath liquid inlet hole and has two first sheath liquid outlet points, the second sheath liquid flow channel is in communication with the second sheath liquid inlet hole and has one second sheath liquid outlet point, and the length of the first sheath liquid flow channel is less than the length of the second sheath liquid flow channel.

[0009] By adopting the technical scheme, after the cell sample flow enters through the cell sample liquid inlet hole, the first sheath liquid enters through the first sheath liquid inlet hole and extrudes the cell sample flow horizontally to make it located in the center area of the channel, and the second sheath liquid enters through the second sheath liquid inlet hole and extrudes the cell sample flow vertically downward to make it close to the bottom area of the channel, so that the position of the measured cell in the flow channel is adjusted through three-dimensional focusing, and the cell stably flows through the detection area near the bottom detection electrode, thereby solving the problem of random distribution of the cell particles in the flow channel of the detection area.

[0010] Preferably, the transfer layer has a connecting layer on the upper surface, the connecting layer has screw holes in one-to-one communication with the cell sample liquid inlet hole, the first sheath liquid inlet hole, the second sheath liquid inlet hole and the waste liquid outlet hole, and the screw holes are used to connect external pipelines.

[0011] By adopting the technical scheme, the screw holes are designed to facilitate the connection of external pipelines for the injection and discharge of corresponding liquids, thereby improving the convenience of detection.

[0012] Preferably, the flow transfer layer has, from left to right, a cell flow injection through-hole penetrating through the upper and lower surfaces of the flow transfer layer, two first sheath liquid injection through-holes, a second sheath liquid injection through-hole, and a waste liquid discharge through-hole in sequence, the cell flow injection through-hole is in communication with the cell sample liquid inlet through-hole, the two first sheath liquid injection through-holes are respectively in communication with two first sheath liquid outlet points, the second sheath liquid injection through-hole is in communication with a second sheath liquid outlet point, and the waste liquid discharge through-hole is in communication with a waste liquid discharge hole.

[0013] Preferably, the excitation electrode layer has a pair of excitation electrodes connected together on the lower surface, and five flow through-holes penetrating through the excitation electrode layer on the upper surface, the five flow through-holes are respectively in communication with the cell flow injection through-hole, the two first sheath liquid injection through-holes, the second sheath liquid injection through-hole, and the waste liquid discharge through-hole.

[0014] Preferably, the flow channel layer has a cross-shaped flow channel penetrating through the flow channel layer on the upper surface, the cross-shaped flow channel is composed of a long flow channel extending from left to right and a short flow channel perpendicular to the long flow channel, the long flow channel has a cell flow injection end and a waste liquid discharge end at both ends respectively, and the short flow channel has two first sheath liquid injection ends at both ends, the cell flow injection end, the waste liquid discharge end, and the two first sheath liquid injection ends are respectively in communication with the cell flow injection through-hole, the waste liquid discharge through-hole, and the two first sheath liquid injection through-holes through corresponding flow through-holes, and the second sheath liquid injection through-hole is in communication with the middle part of the long flow channel through a corresponding flow through-hole.

[0015] Preferably, the detection electrode layer has a pair of independent and separate detection electrodes on the upper surface, the detection electrodes are connected with a lock-in amplifier and used to collect cell electrical impedance signals in the long flow channel.

[0016] Preferably, the transport layer is made of any one of PMMA material, PC, PS, resin or metal (aluminum, iron, copper, etc.), preferably PMMA material, with a thickness of 1-5 mm, preferably 3 mm; the flow transfer layer is made of UV double-sided adhesive or pressure-sensitive double-sided adhesive, preferably UV double-sided adhesive, with a thickness of 50-200 μm, preferably 100 μm; the excitation electrode layer is made of glass, the excitation electrodes on the excitation electrode layer are made of any one of ITO, gold, silver, platinum conductive materials, preferably ITO conductive material, the thickness of the excitation electrode layer is 0.8-2 mm, preferably 1.2 mm, the width of the excitation electrode is 60-150 μm, preferably 80 μm, and the spacing between two excitation electrodes is 60-150 μm, preferably 80 μm; the excitation electrode layer and the transport layer are bonded together through the flow transfer layer; the flow channel layer is made of pressure-sensitive double-sided adhesive or UV double-sided adhesive, with a thickness of 40-100 μm, preferably 50 μm, the width of the long flow channel is 60-200 μm, preferably 90 μm, and the width of the short flow channel is 90 μm; the detection electrode layer is made of glass, the detection electrodes on the detection electrode layer are made of any one of ITO, gold, silver, platinum conductive materials, preferably ITO conductive material, the thickness of the detection electrode layer is 0.8-2 mm, preferably 1.2 mm, the width of the detection electrode is 60-150 μm, preferably 80 μm, and the spacing between two detection electrodes is 60-150 μm, preferably 80 μm; the detection electrode layer and the excitation electrode layer are bonded together through the flow channel layer.

[0017] By adopting the technical scheme, the materials and sizes of the components are preferably selected, the cost is reduced, and the convenience of the microfluidic chip preparation is increased.

[0018] The second aspect of the application provides a detection method for cell impedance detection, which adopts the microfluidic chip described above and comprises the following steps:

[0019] S1, a cell sample solution resuspended by a PBS solution is prepared, and the sample solution is injected into the through hole by a pneumatic pump or a digital injection pump to form a cell sample flow;

[0020] S2, the PBS first sheath liquid is injected through the first sheath liquid inlet hole to horizontally extrude the cell sample flow to the center area of the channel, and then the PBS second sheath liquid is injected through the second sheath liquid inlet hole to vertically downwardly extrude the cell sample flow to the area close to the bottom of the channel;

[0021] S3, the three-dimensional focusing of the cell sample flow is realized by twice extrusion of the sheath liquid, so that the cells flow through the upper and lower facing differential electrode detection zones composed of the excitation electrode layer and the detection electrode layer and are located near the central area of the bottom of the channel;

[0022] S4, the excitation electrodes of the excitation electrode layer apply alternating excitation signals of different frequencies, and the differential mode is adopted to collect the cell electrical impedance signals, so that higher amplitude signals can be obtained by the detection electrodes near the bottom;

[0023] S5, the cell electrical impedance signals are collected by connecting the detection electrodes through the lock-in amplifier, and signal processing and machine learning are performed to realize cell type identification and counting;

[0024] S6, the fluid in the long flow channel is finally discharged through the waste liquid discharge hole.

[0025] Preferably, in the above detection method technical scheme, the flow rate of the cell sample flow is 30-150 μL / min, preferably 60 μL / min, and the flow rate ratio between the cell sample flow, the first sheath liquid and the second sheath liquid is cell sample flow: first sheath liquid: second sheath liquid = 1: (n-1): n or 1: (n-1): (n+1), wherein 1

[0026] In summary, the present application has at least one of the following beneficial technical effects:

[0027] 1. After the cell sample flow to be tested enters the through hole through the cell sample liquid to be tested, the first sheath liquid enters through the first sheath liquid inlet hole and extrudes the cell sample flow to be tested horizontally, so that it is located in the central area of the channel, and the second sheath liquid enters through the second sheath liquid inlet hole and extrudes the cell sample flow to be tested vertically downward, so that it is close to the bottom area of the channel. By adjusting the position of the measured cell in the flow channel, the cell stably flows through the detection area near the bottom detection electrode, thereby solving the problem of random distribution of cell particles in the detection area flow channel.

[0028] 2. By combining the upper and lower facing electrode configuration, the electric field of the detection zone is more uniform, and the speciality of obtaining higher signal amplitude near the detection electrode is utilized to improve the electrical impedance detection precision, thereby solving the problems of non-uniform cell particle electrical impedance signals and low detection precision, and realizing accurate and high-throughput detection of cell electrical impedance signals.

[0029] 3. The microfluidic chip provided by the present application is simple to prepare and has low cost, and the detection method provided by the present application is easy to operate and easy to popularize. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 is the overall structure schematic diagram of the microfluidic chip of the present application;

[0031] Figure 2 is the exploded view of the top view of the microfluidic chip of the present application;

[0032] Figure 3 is the exploded view of the bottom view of the microfluidic chip of the present application;

[0033] Figure 4 is the microsphere resistance impedance signal graph collected in the detection of application example 1 of the present application;

[0034] Figure 5 is the multi-frequency resistance impedance signal graph collected in the detection of application example 2 of the present application;

[0035] Figure 6 is the cell opacity scatter plot in application example 2 of the present application, wherein, Figure 6 (a) is the cell resistance impedance signal real part / imaginary part opacity scatter plot under the condition of applying 2V, 2MHz excitation signal, Figure 6 (b) is the cell resistance impedance signal real part / imaginary part opacity scatter plot under the condition of applying 2V, 4MHz excitation signal, Figure 6 (c) is the cell resistance impedance signal real part / imaginary part opacity scatter plot under the condition of applying 2V, 10MHz excitation signal.

[0036] Reference signs: 1, transport layer; 11, transport layer; 111, first sheath liquid inlet hole; 112, to-be-detected cell sample liquid inlet through hole; 113, waste liquid discharge hole; 114, second sheath liquid inlet hole; 12, flow transfer layer; 121, first blocking groove; 122, cell flow injection through hole; 123, first sheath liquid injection through hole; 124, second sheath liquid injection through hole; 125, waste liquid discharge through hole; 126, second blocking groove; 13, connecting layer; 131, threaded hole; 14, first sheath liquid flow channel; 141, first sheath liquid outflow point; 15, second sheath liquid flow channel; 151, second sheath liquid outflow point; 2, excitation electrode layer; 21, excitation electrode; 22, flow through hole; 3, flow channel layer; 31, cross-shaped flow channel; 32, long flow channel; 321, cell flow injection end; 322, waste liquid discharge end; 33, short flow channel; 331, first sheath liquid injection end; 4, detection electrode layer; 41, detection electrode. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical scheme and advantages of the present application more clear and understandable, the present application is further described in detail below in combination with examples and drawings. It should be understood that the specific examples described herein are only used to explain the present application and are not used to limit the present application. Those skilled in the art can modify or replace equivalently on the basis of understanding the technical scheme of the present application without departing from the spirit and scope of the technical scheme of the present application, which should be covered within the protection scope of the present application.

[0038] The reagents, instruments and equipment used in the following examples are conventional products that can be obtained by commercial purchase, all reagents and chemicals are used as received without further treatment, and other specific conditions not mentioned are carried out according to conventional conditions or manufacturer's recommended conditions.

[0039] The chemical abbreviations in this application are as follows:

[0040] Polymethyl methacrylate (PMMA), indium tin oxide (ITO), polycarbonate (PC), polystyrene (PS), phosphate buffer (PBS).

[0041] The embodiments of the present application disclose a microfluidic chip and a detection method for cell electrical impedance detection.

[0042] I. Embodiments

[0043] Embodiment 1

[0044] With reference to Figure 1 , Figure 2 and Figure 3 , a microfluidic chip for cell electrical impedance detection comprises a transport layer 1, an excitation electrode layer 2, a flow channel layer 3 and a detection electrode layer 4 bonded together from top to bottom, the transport layer 1 is used to input the cell sample to be tested and the sheath liquid into the flow channel layer 3, the excitation electrode layer 2 and the detection electrode layer 4 are located on both sides of the flow channel layer 3 and cooperate to form an upper and lower facing differential electrode detection area, the excitation electrode layer 2 is used to apply an alternating current signal, and the detection electrode layer 4 is used to collect the electrical impedance signal of the cell sample to be tested in the flow channel layer 3.

[0045] With reference to Figure 1 , Figure 2 and Figure 3 , the transport layer 1 comprises a transfer layer 11 and a flow transfer layer 12 arranged in sequence from top to bottom, the transfer layer 11 has a first sheath liquid inlet hole 111, a cell sample liquid inlet hole 112, a waste liquid outlet hole 113 and a second sheath liquid inlet hole 114 arranged in sequence from left to right on the upper surface of the transfer layer 11, the transfer layer 11 has a connecting layer 13, the connecting layer 13 has a threaded hole 131 in one-to-one communication with the cell sample liquid inlet hole 112, the first sheath liquid inlet hole 111, the second sheath liquid inlet hole 114 and the waste liquid outlet hole 113, and the threaded hole 131 is used to connect the external pipeline to facilitate the injection or discharge of fluid.

[0046] With reference to Figure 1 , Figure 2 and Figure 3The first sheath liquid flow channel 14 and the second sheath liquid flow channel 15 are arranged on the lower surface of the transport layer 11, the first sheath liquid flow channel 14 is communicated with the first sheath liquid inlet hole 111 and has two first sheath liquid flow-out points 141, and the second sheath liquid flow channel 15 is communicated with the second sheath liquid inlet hole 114 and has one second sheath liquid flow-out point 151, and the length of the first sheath liquid flow channel 14 is less than the length of the second sheath liquid flow channel 15.

[0047] The connecting layer 13 is made of PMMA material and has a thickness of 5 mm, and in other possible embodiments, the material for making the connecting layer 13 can also be replaced by any one of PC, PS, resin or metal (aluminum, iron, copper, etc.), and the thickness can be any one of integers from 3 to 10 mm.

[0048] The transport layer 11 is made of PMMA material and has a thickness of 3 mm, and in other possible embodiments, the material for making the transport layer 11 can also be replaced by any one of PC, PS, resin or metal (aluminum, iron, copper, etc.), and the thickness can be any one of integers from 1 to 5 mm; the transport layer 11 and the connecting layer 13 can be processed and made as a whole, or can be separately made, and when separately made, the lower surface of the connecting layer 13 is bonded to the upper surface of the transport layer 11 by UV light curing glue, and in this embodiment, the transport layer 11 and the connecting layer 13 are separately made.

[0049] Referring to Figure 1 , Figure 2 and Figure 3 , the flow transfer layer 12 has, from left to right, a first plugging groove 121 penetrating through the upper and lower surfaces of the flow transfer layer 12, a cell flow injection through hole 122, two first sheath liquid injection through holes 123, a second sheath liquid injection through hole 124, a waste liquid discharge through hole 125 and a second plugging groove 126, the first plugging hole is used for plugging the bottom end of the first sheath liquid inlet hole 111, the cell flow injection through hole 122 is communicated with the cell sample liquid inlet through hole 112, the two first sheath liquid injection through holes 123 are respectively communicated with the two first sheath liquid flow-out points 141, the second sheath liquid injection through hole 124 is communicated with the second sheath liquid flow-out point 151, the waste liquid discharge through hole 125 is communicated with the waste liquid discharge hole 113, and the second plugging groove 126 is used for plugging the bottom end of the second sheath liquid inlet hole 114.

[0050] The flow transfer layer 12 is made of UV double-sided adhesive, and has a thickness of 100 μm, and in other possible embodiments, the material for making the flow transfer layer 12 can also be replaced by pressure-sensitive double-sided adhesive, and the thickness can be any one of integers from 50 to 200 μm.

[0051] Referring to Figure 1 , Figure 2 and Figure 3The lower surface of the excitation electrode layer 2 has a pair of excitation electrodes 21 connected together, and the excitation electrode layer 2 further has five flow-through holes 22 penetrating the excitation electrode layer 2, which respectively communicate with the cell flow injection hole 122, the two first sheath liquid injection holes 123, the second sheath liquid injection hole 124 and the waste liquid discharge hole 125.

[0052] The excitation electrode layer 2 is made of glass, and the excitation electrode layer 2 and the transport layer 11 are bonded together by the flow transfer layer 12. The thickness of the excitation electrode layer 2 is 1.2 mm. The excitation electrodes 21 on the excitation electrode layer 2 are made of ITO conductive material, with a width of 80 μm, and the spacing between the two excitation electrodes 21 is 80 μm. In other feasible embodiments, the material for preparing the excitation electrodes 21 can also be replaced by any one of gold, silver and platinum. The thickness of the excitation electrode layer 2 is any one of 0.8-2 mm. The width of the excitation electrodes 21 is any one of 60-150 μm. The spacing between the two excitation electrodes 21 is any one of 60-150 μm.

[0053] Referring to Figure 1 , Figure 2 and Figure 3 , the flow channel layer 3 has a cross-shaped flow channel 31 penetrating the flow channel layer 3, which is composed of a long flow channel 32 extending from left to right and a short flow channel 33 perpendicular to the long flow channel 32. The two ends of the long flow channel 32 are respectively a cell flow injection end 321 and a waste liquid discharge end 322. The two ends of the short flow channel 33 are both a first sheath liquid injection end 331. The cell flow injection end 321, the waste liquid discharge end 322 and the two first sheath liquid injection ends 331 respectively communicate with the cell flow injection hole 122, the waste liquid discharge hole 125 and the two first sheath liquid injection holes 123 through the corresponding flow-through holes 22. The second sheath liquid injection hole 124 communicates with the middle part of the long flow channel 32 through the corresponding flow-through hole 22.

[0054] The flow channel layer 3 is made of pressure-sensitive double-sided adhesive, with a thickness of 50 μm. The width of the long flow channel 32 is 90 μm, and the width of the short flow channel 33 is 90 μm. In other feasible embodiments, the material for preparing the flow channel layer 3 can also be replaced by UV double-sided adhesive, with a thickness of any one of 40-100 μm. The width of the long flow channel 32 is any one of 60-200 μm.

[0055] Referring to Figure 1 , Figure 2 and Figure 3 , the detection electrode layer 4 has a pair of independent and separate detection electrodes 41. The detection electrodes 41 are connected with a lock-in amplifier and used for collecting the cell electrical impedance signal in the long flow channel 32.

[0056] The detection electrode layer 4 is made of glass, and the detection electrode layer 4 and the excitation electrode layer 2 are bonded together by the flow channel layer 3, and the detection electrode layer 4 and the excitation electrode layer 2 are located on both sides of the flow channel layer.

[0057] The thickness of the detection electrode layer 4 is 1.2 mm, the detection electrode 41 on the excitation electrode layer 4 is made of ITO conductive material, the width is 80 μm, and the spacing between the two detection electrodes 41 is 80 μm. In other feasible embodiments, the material for preparing the detection electrode 41 can also be replaced by any one of gold, silver, and platinum conductive materials. The thickness of the detection electrode layer 4 is any one of 0.8-2 mm, the width of the detection electrode 41 is any one of 60-150 μm, and the spacing between the two detection electrodes 41 is any one of 60-150 μm.

[0058] Embodiment 2

[0059] Referring to Figure 1 , Figure 2 and Figure 3 , a detection method for cell resistance impedance detection, using the microfluidic chip prepared in Embodiment 1, comprising the following steps:

[0060] S1, preparing a cell sample solution resuspended by a PBS solution, and injecting the sample solution into the cell sample liquid to be tested into the through hole 112 to form a cell sample flow by a pneumatic pump or a digital injection pump.

[0061] Specifically, the cell sample solution is injected through the threaded hole 131 in communication with the through hole 112 of the cell sample liquid to be tested, and the cell sample solution sequentially passes through the through hole 112 of the cell sample liquid to be tested, the cell flow injection hole 122, and the corresponding flow hole 22, and then enters the long flow channel 32 from the cell flow injection end 321 to form a cell sample flow.

[0062] S2, injecting the PBS first sheath liquid through the first sheath liquid inlet hole 111 to horizontally extrude the cell sample flow to the center area of the channel, and then injecting the PBS second sheath liquid through the second sheath liquid inlet hole 114 to vertically downwardly extrude the cell sample flow to the area close to the bottom of the channel.

[0063] Specifically, the PBS first sheath liquid is injected through the threaded hole 131 corresponding to the first sheath liquid inlet hole 111, enters the first sheath liquid flow channel 14 from the first sheath liquid inlet hole 111, and flows out from the two first sheath liquid outlet points 141 of the first sheath liquid flow channel 14, then enters the short flow channel 33 from the first sheath liquid injection end 331 after passing through the first sheath liquid injection through hole 123 and the corresponding flow through hole 22. At this time, the cell sample flow has passed the connection point of the short flow channel 33 and the long flow channel 32, and the PBS first sheath liquid flows and horizontally extrudes the cell sample flow to the center area of the channel.

[0064] The PBS second sheath liquid is injected through the threaded hole 131 corresponding to the second sheath liquid inlet hole 114, enters the second sheath liquid flow channel 15 from the second sheath liquid inlet hole 114, and flows out from the second sheath liquid outlet point 151 of the second sheath liquid flow channel 15, then enters the long flow channel 32 from the second sheath liquid injection end after passing through the second sheath liquid injection through hole 124 and the corresponding flow through hole 22. At this time, the cell sample flow is located below the second sheath liquid, and the PBS second sheath liquid flows and vertically extrudes the cell sample flow to the area close to the bottom of the channel.

[0065] S3, the two extrusions of the sheath liquid realize three-dimensional focusing of the cell sample flow, so that the cells are located in the center area close to the bottom of the channel and flow through the upper and lower facing differential electrode detection zones composed of the excitation electrode layer 2 and the detection electrode layer 4;

[0066] S4, the excitation electrode 21 of the excitation electrode layer 2 applies an alternating excitation signal of different frequencies, and the differential mode is adopted to collect the cell electrical impedance signal. The detection electrode 41 close to the bottom can obtain a signal with higher amplitude.

[0067] S5, the detection electrode 41 is connected through a lock-in amplifier to collect the cell electrical impedance signal, and signal processing and machine learning are performed to realize cell type identification and counting.

[0068] Specifically, an excitation signal is applied to the excitation electrode 21 located at the top, the differential mode is adopted to collect the electrical impedance signal, and the existing equipment is used for signal processing.

[0069] S6, the fluid in the long flow channel 32 is finally discharged through the waste liquid discharge hole 113.

[0070] Specifically, the fluid in the long flow channel 32 passes through the waste liquid discharge end 322, the corresponding flow through hole 22, the waste liquid discharge through hole 125, and the waste liquid discharge hole 113, and is discharged from the corresponding threaded hole 131.

[0071] The cell sample flow can be various particle size microspheres, various circulating tumor cells or tumor cells (such as A549, MCF-7, HCC827, etc.), various blood cells (WBC, RBC, etc.); in the embodiment, the flow rate of the cell sample flow is 30-150 μL / min, preferably 60 μL / min, and the flow rate ratio among the cell sample flow, the first sheath liquid and the second sheath liquid is cell sample flow: first sheath liquid: second sheath liquid = 1: (n-1): n or 1: (n-1): (n+1), wherein 1 < n < 10, preferably cell sample flow: first sheath liquid: second sheath liquid = 1:2:3.

[0072] II. Application Examples

[0073] Application Example 1

[0074] The microfluidic chip of Example 1 and the detection method of Example 2 are used for application experiments.

[0075] A 10 μm polystyrene microsphere sample solution resuspended by a PBS solution is prepared, with a concentration of 10 6 A digital injection pump is used to inject the microsphere sample solution into the chip through the threaded hole 131 in communication with the through hole 112 of the cell sample liquid to be detected, and a digital injection pump is also used to inject the first sheath liquid and the second sheath liquid into the chip; the flow rate of the cell sample flow is 50 μL / min, and the flow rate ratio of the cell sample flow: first sheath liquid: second sheath liquid is set to 1:2:4.

[0076] The top excitation electrode 21 applies an excitation signal of 2 V and 500 KHz, and the cell electrical impedance signal is collected in differential mode, Figure 4 The collected microsphere electrical impedance signal is further processed and calculated to obtain an electrical resistance signal CV value of 0.0469, and a microsphere particle size CV value of 0.0413.

[0077] The results show that the microfluidic chip and the detection method provided by the application can realize the electrical impedance data collection of high-concentration samples; the data collection is stable under the condition of high-concentration samples, has only a variation coefficient of 0.0469, and has a small difference from the microsphere particle size CV value, so that the information of the measured object can be accurately restored; therefore, the microfluidic chip and the detection method provided by the application can realize accurate and high-throughput detection of cell electrical impedance signals, and effectively solve the problems of uneven electrical impedance signals and low detection precision caused by the random distribution of cell particles in the detection area flow channel.

[0078] Application Example 2

[0079] The microfluidic chip of Example 1 and the detection method of Example 2 are used for application experiments.

[0080] The cell sample solution is prepared by resuspension in a PBS solution, and the cells can be selected from liver cancer cells (Mahlavu), lung cancer A549 cells, white blood cells, red blood cells and other types of cells for electric impedance signal data collection; further, lung cancer A549 cells, liver cancer cells (Mahlavu), white blood cells and inactivated liver cancer cells (Mahlavu) are selected for electric impedance data collection.

[0081] A digital injection pump is used to inject the cell sample solution into the chip through the threaded hole 131 in communication with the through hole 112 with the cell sample solution to be tested, and a digital injection pump is also used to inject the first sheath liquid and the second sheath liquid into the chip; preferably, the flow rate of the cell sample flow is 70 μL / min, and the flow rate ratio of the cell sample flow, the first sheath liquid and the second sheath liquid is set to 1:2:3.

[0082] The top excitation electrode 21 applies 2V, 500KHz, 2MHz, 4MHz and 10MHz excitation signals, and the electric impedance signals of the cells are collected by using a differential mode, Figure 5 The multi-frequency electric impedance signals collected are shown in the following table.

[0083] Further signal processing and calculation are performed, and the electric impedance signals, signal real part, signal imaginary part and phase are selected as characteristic parameters, Figure 6 The cell scatter plot is shown in the following table.

[0084] The neural network structure is set to 12*13*4, and the performance of the cell type classification model is verified.

[0085] 1000 electric impedance signal data of different cells are selected, 3000 data in a random sequence are selected as a training data set, and the remaining 1000 signals are selected as a test set.

[0086] After training and learning, the correct recognition rates of the model are 0.9963 (A549), 1.0000 (Mahlavu), 0.9847 (white blood cells) and 0.9958 (inactivated Mahlavu), all of which are greater than 98%, showing a very high accuracy, and the cell types can be accurately classified.

[0087] According to the results, the microfluidic chip and the detection method provided by the application can realize stable and effective collection of multi-frequency electric impedance data signals of cells, and further realize high-accuracy cell classification by machine learning using the data.

[0088] In summary, the application adjusts the position of the measured cells in the three-dimensional focusing flow channel, so that the cells stably flow through the detection area close to the bottom detection electrode, solves the problem of disorderly distribution of cell particles in the detection area flow channel; and the upper and lower surface electrodes are combined to make the electric field of the detection area more uniform, and the particularity of obtaining higher signal amplitude close to the detection electrode is ingeniously utilized to improve the electrical impedance detection precision, solve the problems of uneven cell particle electrical impedance signal and low detection precision, and realize accurate and high-throughput detection of cell electrical impedance signal.

Claims

1. A microfluidic chip for cell electrical impedance detection, characterized in that: The microfluidic chip comprises, from top to bottom, a transport layer, an excitation electrode layer, a flow channel layer and a detection electrode layer, the transport layer is used for inputting a cell sample and a sheath liquid into the flow channel layer, the excitation electrode layer and the detection electrode layer are located on both sides of the flow channel layer and cooperate to form an upper and lower facing differential electrode detection area, the excitation electrode layer is used for applying an alternating current signal, and the detection electrode layer is used for collecting an electrical impedance signal of the cell sample in the flow channel layer.

2. The microfluidic chip of claim 1, wherein: The transport layer comprises, from top to bottom, a transfer layer and a flow transfer layer, the transfer layer has, from left to right, a first sheath liquid inlet hole penetrating through the upper and lower surfaces of the transfer layer, a cell sample liquid inlet hole, a waste liquid outlet hole and a second sheath liquid inlet hole, the lower surface of the transfer layer has a first sheath liquid flow channel and a second sheath liquid flow channel, the first sheath liquid flow channel is communicated with the first sheath liquid inlet hole and has two first sheath liquid outlet points, the second sheath liquid flow channel is communicated with the second sheath liquid inlet hole and has one second sheath liquid outlet point, and the length of the first sheath liquid flow channel is less than the length of the second sheath liquid flow channel.

3. The microfluidic chip of claim 2, wherein: The transfer layer has a connecting layer thereon, the connecting layer has a threaded hole communicated with the cell sample liquid inlet hole, the first sheath liquid inlet hole, the second sheath liquid inlet hole and the waste liquid outlet hole one by one, and the threaded hole is used for connecting an external pipeline.

4. The microfluidic chip of claim 2, wherein: The flow transfer layer has, from left to right, a cell flow injection hole penetrating through the upper and lower surfaces of the flow transfer layer, two first sheath liquid injection holes, one second sheath liquid injection hole and a waste liquid outlet hole, the cell flow injection hole is communicated with the cell sample liquid inlet hole, the two first sheath liquid injection holes are respectively communicated with the two first sheath liquid outlet points, the second sheath liquid injection hole is communicated with the second sheath liquid outlet point, and the waste liquid outlet hole is communicated with the waste liquid outlet hole.

5. The microfluidic chip of claim 4, wherein: The lower surface of the excitation electrode layer has a pair of excitation electrodes connected together, and the upper surface of the excitation electrode layer further has five flow holes penetrating through the excitation electrode layer, and the five flow holes are respectively communicated with the cell flow injection hole, the two first sheath liquid injection holes, the second sheath liquid injection hole and the waste liquid outlet hole.

6. The microfluidic chip of claim 5, wherein: The flow channel layer has a cross-shaped flow channel penetrating through the flow channel layer, the cross-shaped flow channel is composed of a long flow channel extending from left to right and a short flow channel perpendicular to the long flow channel, two ends of the long flow channel are respectively a cell flow injection end and a waste liquid outlet end, and two ends of the short flow channel are both first sheath liquid injection ends, the cell flow injection end, the waste liquid outlet end and the two first sheath liquid injection ends are respectively communicated with the cell flow injection hole, the waste liquid outlet hole and the two first sheath liquid injection holes through corresponding flow holes, and the second sheath liquid injection hole is communicated with the middle part of the long flow channel through a corresponding flow hole.

7. The microfluidic chip of claim 6, wherein: The detection electrode layer has a pair of independent detection electrodes, the detection electrodes are connected with a lock-in amplifier and used for collecting a cell electrical impedance signal in the long flow channel.

8. The microfluidic chip according to claim 7, wherein: the transfer layer is made of any one of PMMA material, PC, PS, resin or metal (aluminum, iron, copper, etc.), preferably PMMA material, and has a thickness of 1-5 mm, preferably 3 mm; The flow transfer layer is made of UV double-sided adhesive or pressure-sensitive double-sided adhesive, preferably UV double-sided adhesive, with a thickness of 50-200 μm, preferably 100 μm; The excitation electrode layer is made of glass, and the excitation electrodes on the excitation electrode layer are made of any one of ITO, gold, silver, and platinum, preferably ITO conductive material, the thickness of the excitation electrode layer is 0.8-2 mm, preferably 1.2 mm, the width of the excitation electrode is 60-150 μm, preferably 80 μm, and the spacing between two excitation electrodes is 60-150 μm, preferably 80 μm; the excitation electrode layer and the transport layer are bonded together through the flow transfer layer; The flow channel layer is made of pressure-sensitive double-sided adhesive or UV double-sided adhesive, with a thickness of 40-100 μm, preferably 50 μm, the channel width of the long flow channel is 60-200 μm, preferably 90 μm, and the width of the short flow channel is 90 μm; The detection electrode layer is made of glass, and the detection electrodes on the detection electrode layer are made of any one of ITO, gold, silver, and platinum, preferably ITO conductive material, the thickness of the detection electrode layer is 0.8-2 mm, preferably 1.2 mm, the width of the detection electrode is 60-150 μm, preferably 80 μm, and the spacing between two detection electrodes is 60-150 μm, preferably 80 μm; the detection electrode layer and the excitation electrode layer are bonded together through the flow channel layer.

9. A method of detection for cell electrical impedance detection, characterized by: The microfluidic chip of any one of claims 1-8 comprises the following steps: S1, prepare a cell sample solution resuspended by a PBS solution, and inject the sample solution into the through hole to form a cell sample flow by a gas pressure pump or a digital injection pump; S2, inject the first sheath liquid into the first sheath liquid inlet hole to horizontally extrude the cell sample flow to the center area of the channel, and then inject the second sheath liquid into the second sheath liquid inlet hole to vertically downwardly extrude the cell sample flow to the area close to the bottom of the channel; S3, the two extrusions of the sheath liquid realize three-dimensional focusing of the cell sample flow, so that the cells flow through the upper and lower facing differential electrode detection zones composed of the excitation electrode layer and the detection electrode layer at a position close to the center area of the bottom of the channel; S4, the excitation electrodes of the excitation electrode layer apply alternating excitation signals of different frequencies, and the cell resistance impedance signals are collected by differential mode, and the detection electrodes close to the bottom of the cell can obtain higher amplitude signals; S5, connect the detection electrodes by a lock-in amplifier and collect the cell resistance impedance signals, and perform signal processing and machine learning to realize cell type identification and counting; S6, the fluid in the long flow channel is finally discharged through the waste liquid discharge hole.

10. The detection method of claim 9, wherein: The flow rate of the cell sample stream is 30-150 μL / min, preferably 60 μL / min, and the flow rate ratio between the cell sample stream, the first sheath liquid and the second sheath liquid is cell sample stream: first sheath liquid: second sheath liquid = 1:(n-1):n or 1:(n-1):(n+1), wherein 1 < n < 10, preferably cell sample stream: first sheath liquid: second sheath liquid = 1:2:

3. The flow rate of the cell sample stream is 30-150 μL / min, preferably 60 μL / min, and the flow rate ratio between the cell sample stream, the first sheath liquid and the second sheath liquid is cell sample stream: first sheath liquid: second sheath liquid = 1:(n-1):n or 1:(n-1):(n+1), wherein 1 < n < 10, preferably cell sample stream: first sheath liquid: second sheath liquid = 1:2:

3. The flow rate of

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