Cell flow impedance detection micro-fluidic chip based on side wall electrode and preparation method thereof

Abstract

The invention relates to a cell flow impedance detection micro-fluidic chip based on side wall electrodes and a preparation method thereof, and relates to the technical field of cell detection. Comprising the steps of PCB processing, middle layer preparation, channel groove box side wall electrode preparation, side wall electrode post-processing, chip packaging and the like. A PCB copper wire is adopted as a side wall electrode base material, a middle layer is prepared on the surface of the PCB copper wire, and a CNC machining process is adopted for milling to obtain a channel groove and a side wall electrode at the same time, so that the problem that the side wall electrode is difficult to prepare is solved, and meanwhile the cost is reduced; silver is plated on the surface of the copper side wall electrode, the oxidation corrosion resistance and the conductivity are enhanced, the micro-fluidic chip is used for detecting particle flow impedance data with position height difference, an impedance signal with a low variable coefficient can be acquired, and information of a to-be-detected object can be truly and accurately reflected, so that the cell impedance detection side wall electrode is conveniently prepared at low cost, and the cell impedance detection efficiency is improved. And high-quality, accurate and stable electrical impedance signals are acquired.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell detection, in particular to a cell flow impedance detection microfluidic chip based on a side-wall electrode and a preparation method thereof. BACKGROUND

[0002] 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. 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, and the principle is mainly to detect cells by using physical and electrical characteristics of different types of cells, such as cell size and cell dielectric properties. At present, commonly used microfluidic impedance detection electrodes can be divided into coplanar, upper and lower facing and side-wall electrodes. Generally, corresponding channel structure design is carried out according to different electrode configurations to realize cell impedance detection.

[0003] The existing microfluidic impedance detection electrode is usually a gold electrode, which needs to be prepared by using a photolithography process, and the cost of preparing one electrode is relatively high. In order to meet the subsequent requirements of chip preparation and packaging, the number of electrodes in one electrode plate is usually only 3-4 pieces, and the smaller the size of the electrode, the higher the cost. Therefore, the price of a single electrode plate is about one thousand yuan, which has a high cost in actual application.

[0004] In addition, the coplanar electrode and the upper and lower facing electrode are affected by the height of the cells flowing through the detection area when detecting the impedance signal of the cells. 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 from the detection electrode, so as to interfere with the detection. The side-wall electrode has no position dependence on the channel height, which can effectively avoid the shortcomings of the first two electrodes, and obtain high-quality, accurate and stable impedance signals. However, the preparation of the side-wall electrode is difficult. SUMMARY

[0005] The present application aims at the above problems, and provides a cell flow impedance detection microfluidic chip based on a side-wall electrode and a preparation method thereof.

[0006] The first aspect of the present application provides a preparation method of a cell flow impedance detection microfluidic chip based on a side-wall electrode, which adopts the following technical scheme: comprising the following steps:

[0007] S1, PCB circuit board processing: drawing an installation area on the circuit board, the installation area being in a cross shape, laying a copper wire at one end of the length direction of the installation area, the extension direction of the copper wire being perpendicular to the length direction of the installation area, and setting two groups of wiring holes on the circuit board, the two groups of wiring holes being located on both sides of the copper wire and connected with the copper wire, and the wiring holes being used for welding a radio frequency wire seat;

[0008] S2, preparing the intermediate layer: preparing the intermediate layer in the installation area along the length direction, the intermediate layer covering on the copper wire;

[0009] S3, preparing the channel groove and the side wall electrode: using the CNC milling machine equipment to process the channel groove on the intermediate layer, the extension direction of the channel groove being consistent with the extension direction of the intermediate layer, the channel groove being composed of the asymmetric serpentine structure and the straight channel, the straight channel being located at both ends of the asymmetric serpentine structure, the end of the straight channel away from the copper wire being the cell flow inlet, the end of the straight channel away from the cell flow inlet being the waste liquid outlet, the intermediate layer being milled by the milling cutter to obtain the channel groove with the asymmetric serpentine structure, the copper wire being milled by the milling cutter to form the side wall electrode on both sides of the straight channel;

[0010] S4, post-processing of the side wall electrode: removing the burrs on the edge of the copper wire side wall and plating silver on the surface of the side wall electrode;

[0011] S5, chip packaging: first preparing the packaging layer, the packaging layer having two through holes penetrating the upper and lower surfaces of the packaging layer at both ends, the two through holes being in communication with the cell flow inlet and the waste liquid outlet of the channel groove respectively, and the packaging layer being adhered to the intermediate layer in the installation area by the UV glue to obtain the cell flow type electrical impedance detection microfluidic chip based on the side wall electrode.

[0012] By using the above technical scheme, the single PCB circuit board has a low cost, and in combination with the intermediate layer and the CNC processing, the existing microfluidic electrical impedance detection electrode adopts the gold electrode, and the price of the single electrode plate is relatively high. In comparison, the preparation cost of the preparation method provided in the present application only accounts for one percent of the existing cost. In addition, the copper wire is selected as the side wall electrode, and the electrical conductivity is better than that of the gold electrode, so that a side wall electrode with higher electrical conductivity can be obtained. The channel groove with the asymmetric serpentine structure can realize the inertial focusing of cells or particles, and the cells are focused at two positions, forming two focused flow lines. The microfluidic chip with the structure can remove the position dependence in the height direction of the channel, and the cell electrical impedance signals collected are not affected by the height of the flow through the detection area. In addition, the preparation method is simple and convenient, so that the microfluidic chip prepared by using the method not only has improved detection accuracy, but also improves the convenience of preparation and reduces the cost.

[0013] Preferably, in the above preparation method technical scheme, in S1, two copper wires are provided, the two copper wires being arranged adjacent to each other, the line width of the copper wire being 50-150 μm, preferably 90 μm, the thickness of the copper wire being 30-100 μm, preferably 40 μm, and the spacing between the two copper wires being 50-250 μm, preferably 150 μm.

[0014] Preferably, in the above preparation method, in S2, the intermediate layer material is SU-8 photoresist, and the thickness of the intermediate layer is 30-150 μm, preferably 40 μm.

[0015] Preferably, in the above preparation method, in S3, the asymmetric serpentine structure comprises 5-20 asymmetric serpentine structure units, preferably 12, and the width of the straight channel is 60-200 μm, preferably 100 μm.

[0016] Preferably, each of the serpentine structure units is composed of a first circular arc unit and a second circular arc unit connected in a head-to-tail manner, the first circular arc unit is composed of a first upper circular arc and a second upper circular arc with non-coincident centers, the center distance is 130 μm, the radius of the first upper circular arc is 320 μm, and the radius of the second upper circular arc is 200 μm, the second circular arc unit is composed of a first lower circular arc and a second lower circular arc with coincident centers, the radius of the first lower circular arc is 160 μm, and the radius of the second lower circular arc is 270 μm, the first upper circular arc is connected with the first lower circular arc, and the second upper circular arc is connected with the second lower circular arc.

[0017] Preferably, in the above preparation method, S4 is specifically as follows:

[0018] S4, side wall electrode post-processing: first, remove the burrs on the edge of the copper wire side wall by PCB environmentally friendly etchant, the etching time is 1-8 min, preferably 5 min, then treat the copper side wall electrode by using a metal surface treatment agent, and then use silver chemical plating liquid to plate a layer of silver film on the surface of the copper side wall electrode, the silver plating time is 1-10 min, preferably 3 min.

[0019] Preferably, in the above preparation method, in S5, the thickness of the packaging layer is 4-15 mm, preferably 8 mm, and the lower surface of the packaging layer is bonded to the upper surface of the intermediate layer by UV double-sided adhesive, and the thickness of the UV double-sided adhesive is 50-200 μm, preferably 100 μm.

[0020] Preferably, the packaging layer comprises a cover plate and a connecting plate bonded together, the outer contour length*width size of the packaging layer is 37 mm*10 mm, the cover plate is connected to the intermediate layer, two through holes are provided on the cover plate, the through hole size is 1.2 mm, the connecting plate is located above the cover plate, the connecting plate has a threaded hole corresponding to the through hole in one-to-one correspondence, the threaded hole is used for connecting an external pipeline, and the threaded hole size is M2-M5, preferably M3 threaded hole.

[0021] Preferably, the cover plate and the connecting plate are made of any one of PMMA, PC, PS, resin or metal material (aluminum, iron, copper, etc.), preferably PMMA material, the thickness of the cover plate is 1-5mm, preferably 3mm, and the thickness of the connecting plate is 3-10mm, preferably 5mm.

[0022] By adopting the technical scheme, the specific steps and parameters of the preferred preparation method, a microfluidic chip with better performance is obtained.

[0023] The second aspect of the application provides a cell flow-type electrical impedance detection microfluidic chip based on a side wall electrode, which is prepared by the above preparation method.

[0024] In summary, the present application includes at least one of the following beneficial technical effects:

[0025] 1. The single-piece PCB circuit board has a low cost, and in combination with the intermediate layer and the CNC machining, the existing microfluidic electrical impedance detection electrode usually adopts a gold electrode, which is relatively expensive. In comparison, the preparation cost of the preparation method provided by the present application is only one percent of the existing cost, and the preparation method is simple and convenient, thereby greatly reducing the preparation cost of the cell flow-type electrical impedance detection microfluidic chip based on the side wall electrode.

[0026] 2. By selecting a copper wire with better conductivity than a gold electrode as a side wall electrode, a side wall electrode with higher conductivity can be obtained, and a channel groove with an asymmetric serpentine structure can realize inertial focusing of cells or particles, with the cells being focused at two upper and lower positions to form two focused flow lines. The microfluidic chip with this structure can remove the position dependence on the height of the detection area, and the cell electrical impedance signals collected are not affected by the flow through the detection area, thereby improving the accuracy of cell detection.

[0027] 3. The present application uses a PCB copper wire as a side wall electrode substrate, and a channel structure and a side wall electrode are simultaneously milled by preparing an intermediate layer on the surface thereof and using a CNC machining process, thereby solving the problem of difficult preparation of the side wall electrode, making the preparation process more simple, fast and low-cost. Silver is plated on the surface of the copper side wall electrode to enhance the anti-oxidation corrosion resistance and conductivity. The cell flow-type electrical impedance detection microfluidic chip based on the side wall electrode of the present application can detect particle flow electrical impedance data with position height differences, and can collect low-coefficient-of-variation electrical impedance signals to truly and accurately reflect the information of the measured substance. Therefore, the preparation method provided by the present application can realize low-cost and convenient preparation of cell electrical impedance detection side wall electrodes, and can collect high-quality, accurate and stable electrical impedance signals. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is a structural diagram of a circuit board in the present application;

[0029] Figure 2 is a structural diagram of the intermediate layer and the channel groove in the application;

[0030] Figure 3 is Figure 2 is an enlarged schematic view of part A in the application;

[0031] Figure 4 is a position schematic view of the channel groove and the copper wire in the application;

[0032] Figure 5 is an exploded view of the packaging layer in the application;

[0033] Figure 6 is the interface profile of the sidewall electrode and the sidewall electrode and the channel groove in the application;

[0034] Figure 7 is a physical picture of the microfluidic chip prepared in the application example;

[0035] Figure 8 is a microsphere electrical impedance signal diagram collected in the application example.

[0036] The drawings show that: 1, circuit board; 11, mounting area; 12, copper wire; 13, wiring hole; 2, intermediate layer; 3, channel groove; 31, asymmetric serpentine structure; 32, straight channel; 33, cell inlet; 34, waste liquid outlet; 4, serpentine structure unit; 41, first circular arc unit; 411, first upper circular arc; 412, second upper circular arc; 42, second circular arc unit; 421, first lower circular arc; 422, second lower circular arc; 5, packaging layer; 51, through hole; 52, cover plate; 53, connecting plate; 531, threaded hole. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application will be 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 the equivalent without departing from the spirit and scope 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 purchased on the market, all reagents and chemicals are used as received without further treatment, and other specific conditions not specified are carried out according to conventional conditions or manufacturer's recommended conditions.

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

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

[0041] The application discloses a cell flow type resistance impedance detection microfluidic chip based on a side wall electrode and a preparation method thereof.

[0042] The application discloses a cell flow type resistance impedance detection microfluidic chip based on a side wall electrode and a preparation method thereof.

[0043] S1, PCB circuit board 1 processing: a mounting area 11 is drawn on the circuit board 1, the mounting area 11 is in a cross shape, a copper wire 12 is laid at one end of the length direction of the mounting area 11, the extension direction of the copper wire 12 is perpendicular to the length direction of the mounting area 11, two groups of wiring holes 13 are arranged on the circuit board 1 and are located on the two sides of the copper wire 12 and connected with the copper wire 12, and the wiring holes 13 are used for welding a radio frequency wire seat.

[0044] Referring to Figure 1 , specifically, the PCB board is drawn and processed, the surface of the circuit board 1 is not covered with solder resist ink, two copper wires 12 are arranged, the two copper wires 12 are arranged adjacently and will be processed into side wall electrodes later, the two groups of wiring holes 13 are located on the two sides of the copper wire 12 respectively and each group includes two wiring holes 13 corresponding to the two copper wires 12 one by one, a wire for indicating height can also be laid in the mounting area 11, the line width of the copper wire 12 is 50-150 μm, preferably 90 μm, the thickness of the copper wire 12 is 30-100 μm, preferably 40 μm, and the spacing between the two copper wires 12 is 50-250 μm, preferably 150 μm.

[0045] S2, preparing the intermediate layer 2: the intermediate layer 2 is prepared in the mounting area 11 along the length direction, and the intermediate layer 2 covers the copper wire 12.

[0046] Specifically, the material of the intermediate layer 2 is SU-8 photoresist, and the thickness of the intermediate layer 2 is 30-150 μm, preferably 40 μm.

[0047] S3, preparing the channel groove 3 and the side wall electrode: the CNC milling machine equipment is used to process the channel groove 3 on the intermediate layer 2, the extension direction of the channel groove 3 is consistent with the extension direction of the intermediate layer 2, the channel groove 3 is composed of an asymmetric serpentine structure 31 and a straight channel 32, the straight channel 32 is located at the two ends of the asymmetric serpentine structure 31, one end of the straight channel 32 away from the copper wire 12 is a cell flow inlet 33, and one end of the straight channel 32 away from the cell flow inlet 33 is a waste liquid outlet 34, the intermediate layer 2 is milled by a milling cutter to obtain the channel groove 3 with the asymmetric serpentine structure 31, and the copper wire 12 is milled by the milling cutter to form the side wall electrode on the two side walls of the straight channel 32.

[0048] With reference to Figure 2 , Figure 3 and Figure 4 , specifically, the asymmetric serpentine structure 31 comprises 5-20 asymmetric serpentine structure units 4, preferably 12, each serpentine structure unit 4 is composed of a first circular arc unit 41 and a second circular arc unit 42 connected end to end, the first circular arc unit 41 is composed of a first upper circular arc 411 and a second upper circular arc 412 with non-coincident centers, the center distance is 130 μm, the radius of the first upper circular arc 411 is 320 μm, and the radius of the second upper circular arc 412 is 200 μm, the second circular arc unit 42 is composed of a first lower circular arc 421 and a second lower circular arc 422 with coincident centers, the radius of the first lower circular arc 421 is 160 μm, and the radius of the second lower circular arc 422 is 270 μm.

[0049] With reference to Figure 2 , Figure 3 and Figure 4 , the first upper circular arc 411 and the second upper circular arc 412 close to the cell flow inlet 33 are connected with the straight channel 32, the first lower circular arc 421 and the second lower circular arc 422 close to the waste liquid outlet 34 are connected with the straight channel 32, the first upper circular arc 411 in the middle part is connected with the first lower circular arc 421, and the second upper circular arc 412 is connected with the second lower circular arc 422; the width of the straight channel 32 is 60-200 μm, preferably 100 μm.

[0050] S4, side wall electrode post-processing: removing burrs on the side wall edge of the copper wire 12 and plating silver on the surface of the side wall electrode.

[0051] Specifically, first, remove the burrs on the side wall edge of the copper wire 12 by PCB environmentally friendly etchant, the etching time is 1-8 min, preferably 5 min, then use a metal surface treatment agent to treat the copper side wall electrode, and after treatment, use silver chemical plating liquid to plate a layer of silver film on the surface of the copper side wall electrode, the silver plating time is 1-10 min, preferably 3 min.

[0052] S5, chip packaging: first, prepare a packaging layer 5, the packaging layer 5 has through holes 51 penetrating the upper and lower surfaces of the packaging layer 5 at both ends, the two through holes 51 are respectively communicated with the cell flow inlet 33 and the waste liquid outlet 34 of the channel groove 3, and the packaging layer 5 is adhered to the middle layer 2 located in the mounting area 11 by UV glue, to obtain a cell flow type electrical impedance detection microfluidic chip based on a side wall electrode.

[0053] With reference to Figure 4 and Figure 5Specifically, the packaging layer 5 comprises a cover plate 52 and a connecting plate 53, the cover plate 52 is connected with the intermediate layer 2, two through holes 51 are arranged on the cover plate 52, the connecting plate 53 is arranged above the cover plate 52, the connecting plate 53 is provided with threaded holes 531 corresponding to the through holes 51, and the threaded holes 531 are used for connecting external pipelines.

[0054] The cover plate 52 and the connecting plate 53 are made of any one of PMMA, PC, PS, resin or metal material (aluminum, iron, copper and the like), preferably PMMA material, the thickness of the cover plate 52 is 1-5 mm, preferably 3 mm, the thickness of the connecting plate 53 is 3-10 mm, preferably 5 mm, the cover plate 52 and the connecting plate 53 can be bonded by UV light curing glue after being prepared separately, and can also be prepared together as a whole.

[0055] The lower surface of the prepared packaging layer 5 is bonded with the upper surface of the intermediate layer 2 by UV double-sided adhesive to obtain a cell flow type resistance impedance detection microfluidic chip based on a side wall electrode, the thickness of the UV double-sided adhesive is 50-200 μm, preferably 100 μm, and the prepared physical map is as shown in Figure 6 .

[0056] A cell flow type resistance impedance detection microfluidic chip based on a side wall electrode prepared by the above preparation method can be used in the field of cell detection.

[0057] I. Embodiment

[0058] Embodiment 1

[0059] A preparation method of a cell flow type resistance impedance detection microfluidic chip based on a side wall electrode, comprising the following steps:

[0060] S1, PCB circuit board 1 processing: drawing an installation area 11 on the circuit board 1, the installation area 11 is in the shape of a cross, laying two copper wires 12 at one end of the length direction of the installation area 11, the two copper wires 12 are arranged adjacent to each other and the extension direction is perpendicular to the length direction of the installation area 11, arranging two groups of wire holes 13 on the circuit board 1, the two groups of wire holes 13 are respectively located on the two sides of the copper wires 12 and connected with the copper wires 12, the wire holes 13 are used for welding radio frequency wire seats, the line width of the two copper wires 12 is 90 μm, the thickness is 40 μm, and the spacing is 100 μm.

[0061] S2, preparing the intermediate layer 2: preparing the intermediate layer 2 in the installation area 11 along the length direction, the material of the intermediate layer 2 is selected from SU-8 photoresist, the thickness is 45 μm, and the intermediate layer 2 covers the copper wires 12.

[0062] S3, preparation of channel groove 3 and side wall electrode: CNC milling machine equipment is used to process channel groove 3 on middle layer 2, the extension direction of channel groove 3 is consistent with the extension direction of middle layer 2, channel groove 3 is composed of asymmetric serpentine structure 31 and straight channel 32, straight channel 32 is located at both ends of asymmetric serpentine structure 31, and the end of straight channel 32 away from copper wire 12 is cell flow inlet 33, and the end of straight channel 32 away from cell flow inlet 33 is waste liquid outlet 34.

[0063] Asymmetric serpentine structure 31 includes 12 asymmetric serpentine structure units 4, each serpentine structure unit 4 is composed of first circular arc unit 41 and second circular arc unit 42 connected end to end, first circular arc unit 41 is composed of first upper circular arc 411 and second upper circular arc 412 with different centers, the center distance is 130 μm, the radius of first upper circular arc 411 is 320 μm, and the radius of second upper circular arc 412 is 200 μm, second circular arc unit 42 is composed of first lower circular arc 421 and second lower circular arc 422 with the same center, the radius of first lower circular arc 421 is 160 μm, and the radius of second lower circular arc 422 is 270 μm.

[0064] First upper circular arc 411 and second upper circular arc 412 close to cell flow inlet 33 are connected with straight channel 32, first lower circular arc 421 and second lower circular arc 422 close to waste liquid outlet 34 are connected with straight channel 32, first upper circular arc 411 in the middle part is connected with first lower circular arc 421, and second upper circular arc 412 is connected with second lower circular arc 422; the width of straight channel 32 is 100 μm.

[0065] After milling cutter milling of middle layer 2, channel groove 3 with asymmetric serpentine structure 31 is obtained, and after milling cutter milling of copper wire 12, side wall electrode is formed on both sides of straight channel 32, Figure 6 The side wall electrode interface profile and the side wall electrode and channel groove are shown in the top view.

[0066] S4, side wall electrode post-processing: removing copper wire 12 side wall edge burr and silver plating on side wall electrode surface.

[0067] First, remove the copper wire 12 side wall edge burr by PCB environmentally friendly etchant, etching time is 3 min, then use metal surface treatment agent to treat copper side wall electrode, after treatment, use silver chemical plating liquid to plate a layer of silver film on the surface of copper side wall electrode to increase the anti-oxidation corrosion resistance and conductivity, silver plating time is 3 min.

[0068] S5, chip packaging: first, prepare the packaging layer 5, the packaging layer 5 has a through hole 51 penetrating the upper and lower surfaces of the packaging layer 5 at both ends, and the two through holes 51 are respectively communicated with the cell inlet 33 and the waste liquid outlet 34 of the channel groove 3, the packaging layer 5 includes a cover plate 52 and a connecting plate 53 bonded together, the cover plate 52 is connected with the intermediate layer 2, the two through holes 51 are arranged on the cover plate 52, the connecting plate 53 is located above the cover plate 52, the connecting plate 53 has a threaded hole 531 corresponding to the through hole 51 in communication, the threaded hole 531 is used for connecting the external pipeline, the cover plate 52 and the connecting plate 53 are integrally formed, the cover plate 52 and the connecting plate 53 are prepared by using PMMA material, the thickness of the cover plate 52 is 3mm, and the thickness of the connecting plate 53 is 5mm.

[0069] The packaging layer 5 is adhered to the intermediate layer 2 located in the mounting area 11 by 100 mu m UV glue to obtain a cell flow type resistance impedance detection microfluidic chip based on a side wall electrode, Figure 7 The physical map of the prepared microfluidic chip.

[0070] Example 2

[0071] A preparation method of a cell flow type resistance impedance detection microfluidic chip based on a side wall electrode, which is different from example 1, the line width of the two copper wires 12 is 90 mu m, the thickness is 45 mu m, and the spacing is 150 mu m; the thickness of the intermediate layer 2 is 50 mu m.

[0072] The side wall electrode preparation method has low cost, and the cost of single PCB circuit board is only 10 yuan. Combined with the preparation of the intermediate layer 2 and CNC processing, the total cost is less than 20 yuan. Compared with the existing microfluidic resistance impedance detection electrode which usually uses gold electrode and the price of single electrode plate is about 2000 yuan, the preparation cost of the present application is only about one percent. In addition, the silver conductivity is 6.3*10 7 S / m, the copper conductivity is 5.96*10 7 S / m, and the gold conductivity is 4.1*10 7 S / m, and the side wall electrode has higher conductivity and better detection performance.

[0073] The present application adopts PCB process and CNC machining process to prepare the side wall electrode, and the preparation process is more simple, fast and low cost compared with photolithography. Therefore, the cell flow type resistance impedance detection microfluidic chip and method based on the side wall electrode of the present application realize low-cost and convenient preparation of cell resistance impedance detection side wall electrode, and solve the problem of difficult preparation of side wall electrode.

[0074] II. Application Example

[0075] Application Example 1

[0076] The microfluidic chip prepared by the preparation method of example 1 is used for application experiments.

[0077] A 10 mu m polystyrene microsphere sample solution with a concentration of 10 6 The microsphere sample solution is injected into the chip through the threaded hole 531 corresponding to the cell flow inlet 33 by using a digital injection pump, and the flow rate of the sample flow is 500 mu L / min.

[0078] Two wire connection holes 13 located on one side of the channel groove 3 are selected as electrode connection ports of the excitation electrode, the two copper side wall electrodes located on this side are connected together by wires and a 2V, 500KHz excitation signal is applied, and the other side corresponding thereto is a detection electrode, which is connected with a lock-in amplifier for collecting signals at the rear end, and the cell electrical impedance signal is collected in a differential mode, Figure 8 The microsphere electrical impedance signal collected.

[0079] Further signal processing is performed to remove the baseline and calculate the electrical resistance signal CV value as 0.0794, and the microsphere particle size CV value is 0.0760.

[0080] As shown in the figure, the data collection is stable under the condition of high concentration sample, has only a variation coefficient of 0.0794, and the difference with the microsphere particle size CV value is small, so that the information of the measured object can be accurately restored.

[0081] In summary, the PCB copper wire is used as the side wall electrode base material, a middle layer is prepared on the surface thereof, and the channel structure and the side wall electrode are milled by using the CNC processing technology, so that the problem of difficult preparation of the side wall electrode is solved, the preparation process is more simple, fast and low in cost, the copper side wall electrode is plated with silver to enhance the anti-oxidation corrosion resistance and conductivity, the particle flow electrical impedance data with position height difference is detected by using the cell flow electrical impedance detection microfluidic chip based on the side wall electrode, the low variation coefficient electrical impedance signal can be collected and obtained, and the measured object information can be truly and accurately reflected, so that the cell electrical impedance detection side wall electrode can be prepared at low cost and conveniently, and the high-quality, accurate and stable electrical impedance signal can be collected and obtained.

Claims

1. A method for fabricating a microfluidic chip for cell flow cytometry impedance detection based on sidewall electrodes, characterized in that: Includes the following steps: S1. PCB Circuit Board Processing: Draw the mounting area on the circuit board. The mounting area is cross-shaped. Lay copper wires at one end of the length direction of the mounting area. The extension direction of the copper wires is perpendicular to the length direction of the mounting area. Set two sets of wiring holes on the circuit board. The two sets of wiring holes are located on both sides of the copper wires and connected to the copper wires. The wiring holes are used to solder RF wire sockets. S2. Preparation of intermediate layer: An intermediate layer is prepared in the mounting area along the length direction, and the intermediate layer covers the copper conductor; S3. Preparation of channel grooves and sidewall electrodes: Channel grooves are machined on the intermediate layer using a CNC milling machine. The extension direction of the channel grooves is consistent with the extension direction of the intermediate layer. The channel grooves consist of an asymmetric serpentine structure and straight channels. The straight channels are located at both ends of the asymmetric serpentine structure. The end of the straight channel away from the copper wire is the cell inlet, and the end of the straight channel away from the cell inlet is the waste liquid outlet. After the intermediate layer is milled by a milling cutter, a channel groove with an asymmetric serpentine structure is obtained. After the copper wire is milled by a milling cutter, sidewall electrodes are formed on both sides of the straight channel. S4. Post-treatment of sidewall electrodes: Remove burrs from the edge of the copper wire sidewall and plate silver on the surface of the sidewall electrodes; S5. Chip Packaging: First, prepare a packaging layer. Both ends of the packaging layer have through holes that penetrate the upper and lower surfaces of the packaging layer. The two through holes are connected to the cell inlet and waste liquid outlet of the channel groove, respectively. The packaging layer is then bonded to the intermediate layer located in the mounting area using UV adhesive to obtain a cell flow impedance detection microfluidic chip based on sidewall electrodes.

2. The method of claim 1, wherein: In S1, there are two copper wires arranged adjacent to each other. The line width of the copper wire is 50-150μm, preferably 90μm, and the thickness of the copper wire is 30-100μm, preferably 40μm. The distance between the two copper wires is 50-250μm, preferably 150μm.

3. The method of claim 1, wherein: In S2, the intermediate layer material is SU-8 photoresist, and the thickness of the intermediate layer is 30-150 μm, preferably 40 μm.

4. The method of claim 1, wherein: In S3, the asymmetric serpentine structure includes 5 to 20 asymmetric serpentine structure units, preferably 12, and the width of the straight channel is 60-200 μm, preferably 100 μm.

5. The method of claim 4, wherein: Each of the serpentine structural units is composed of a first arc unit and a second arc unit connected end to end. The first arc unit consists of a first upper arc and a second upper arc whose centers do not coincide, with a center-to-center distance of 130 μm. The radius of the first upper arc is 320 μm, and the radius of the second upper arc is 200 μm. The second arc unit consists of a first lower arc and a second lower arc whose centers coincide. The radius of the first lower arc is 160 μm, and the radius of the second lower arc is 270 μm. The first upper arc is connected to the first lower arc, and the second upper arc is connected to the second lower arc.

6. The preparation method according to claim 1, characterized in that: S4, side wall electrode post-processing: first, the PCB environmental etchant is used to remove the burrs on the side wall of the copper conductor, the etching time is 1-8 min, preferably 5 min, then the metal surface treatment agent is used to treat the copper side wall electrode, and a layer of silver film is plated on the surface of the copper side wall electrode by using silver chemical plating solution, the silver plating time is 1-10 min, preferably 3 min.

7. The method of claim 1, wherein: In S5, the thickness of the packaging layer is 4-15 mm, preferably 8 mm, and the lower surface of the packaging layer and the upper surface of the intermediate layer are bonded together by UV double-sided adhesive, the thickness of the UV double-sided adhesive is 50-200 μm, preferably 100 μm.

8. The method of claim 7, wherein: The packaging layer comprises a cover plate and a connecting plate bonded together, the outer contour length and width of the packaging layer is 37 mm*10 mm, the cover plate is connected with the intermediate layer, two through holes are arranged on the cover plate, the size of the through hole is 1.2 mm, the connecting plate is located above the cover plate, the connecting plate has a threaded hole corresponding to the through hole in one-to-one correspondence, the threaded hole is used to connect the external pipeline, the size of the threaded hole is M2-M5, preferably M3 threaded hole.

9. The method of claim 8, wherein: The cover plate and the connecting plate are made of any one of PMMA, PC, PS, resin or metal material (aluminum, iron, copper, etc.), preferably PMMA material, the thickness of the cover plate is 1-5 mm, preferably 3 mm, and the thickness of the connecting plate is 3-10 mm, preferably 5 mm.

10. A sidewall electrode-based cell flow impedance detection microfluidic chip, characterized in that: The preparation method is prepared by using the preparation method of any one of the above claims 1-9.

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