CN112730560A

CN112730560A - Micro-fluidic impedance cell instrument and preparation method thereof

Info

Publication number: CN112730560A
Application number: CN202011464532.2A
Authority: CN
Inventors: 倪中华; 项楠; 朱树
Original assignee: Southeast University
Current assignee: Southeast University
Priority date: 2020-12-10
Filing date: 2020-12-10
Publication date: 2021-04-30
Anticipated expiration: 2040-12-10
Also published as: CN112730560B

Abstract

The invention provides a microfluidic impedance cytometer and a preparation method thereof, wherein the microfluidic impedance cytometer comprises an upper electrode layer, a flow channel layer and a lower electrode layer, wherein a focusing flow channel is arranged on the flow channel layer, an excitation electrode is arranged on the upper electrode layer, and an induction electrode is arranged on the lower electrode layer; the upper electrode layer and the lower electrode layer are made of ITO conductive thin film materials with PET as a substrate, the flow channel layer is made of AB double-sided adhesive tape materials with PET as a substrate, and the upper surface and the lower surface of the flow channel layer are respectively adhered with the upper electrode layer and the lower electrode layer to seal the focusing flow channel. By cutting the ITO conductive film, an upper insulating groove is cut on the upper electrode layer and a lower insulating groove is cut on the lower electrode layer respectively, the upper insulating groove surrounds and forms an excitation electrode independent of the rest part of the upper electrode layer, the lower insulating groove surrounds and forms an induction electrode independent of the rest part of the lower electrode layer, and the excitation electrode and the induction electrode are in butt joint to form a three-dimensional differential electrode.

Description

Micro-fluidic impedance cell instrument and preparation method thereof

Technical Field

The invention relates to the technical field of microfluidic cell detection, in particular to a microfluidic impedance cell instrument and a preparation method thereof.

Background

Tumor cells (also called circulating tumor cells) in peripheral blood are often used for predicting survival of cancer patients, and can also be used for guiding cancer diagnosis and prognosis evaluation, so that a thought is provided for developing anti-cancer drugs. Therefore, the ability to rapidly and efficiently obtain tumor cells from peripheral blood would be of great significance for cancer diagnosis and treatment.

The existing circulating tumor cell sorting and detecting device usually adopts an immunomagnetic bead marking and fluorescent staining mode to capture and detect tumor cells. Wherein, the captured circulating tumor cells lose biological activity and can not be used for subsequent clinical diagnosis, drug resistance detection and the like. In addition, such devices based on immunomagnetic bead labeling and fluorescent staining for capturing and detecting circulating tumor cells are often very expensive to use due to the high price of magnetic beads and fluorescent stains.

With the development of the microfluidic technology, the manufacturing method of the microfluidic resistance pit detection instrument mostly adopts a soft lithography process, and the chip manufactured by the method has good tightness, but the method is applied to equipment such as an ultraviolet lithography machine, a spin coater and the like, so that the preparation cost is increased, and the application range of the method is limited. The materials such as PDMS, gold-plated glass and the like are needed, the price of the consumable is high, and in addition, only one upper surface of the consumable is transparent, which is not beneficial to observation, optical analysis and identification and the like.

Disclosure of Invention

The invention provides a microfluidic impedance cytometer and a preparation method thereof, which can realize the functions of cell counting, volume measurement, focus position monitoring and the like, have simple preparation process and easily-obtained materials and low cost, and solve the technical problems of complicated preparation process and high cost in the prior art.

The technical scheme adopted by the invention is as follows:

a micro-fluidic impedance cytometer comprises an upper electrode layer, a flow channel layer and a lower electrode layer which are sequentially stacked, wherein a focusing flow channel is arranged on the flow channel layer, an excitation electrode and an outlet hole are arranged on the upper electrode layer, and an induction electrode and an inlet hole are arranged on the lower electrode layer; the upper electrode layer and the lower electrode layer are made of ITO conductive thin film materials with PET as a substrate, the flow channel layer is made of AB double-sided adhesive tape materials with PET as a substrate, the upper surface and the lower surface of the flow channel layer are respectively adhered to the upper electrode layer and the lower electrode layer, the focusing flow channel is sealed, meanwhile, the inlet end of the focusing flow channel is communicated with the inlet hole, and the outlet end of the focusing flow channel is communicated with the outlet hole.

The ITO conductive film is cut, an upper insulating groove is cut in the upper electrode layer, a lower insulating groove is cut in the lower electrode layer, the upper insulating groove surrounds the excitation electrode independent of the rest of the upper electrode layer, the lower insulating groove surrounds the induction electrode independent of the rest of the lower electrode layer, and the excitation electrode and the induction electrode are in butt joint to form a three-dimensional differential electrode.

The excitation electrode and the induction electrode are overlapped at the butt joint part to form a non-uniform electric field which is vertical to the tail section of the focusing flow channel close to the outlet end.

The upper electrode layer, the flow channel layer and the lower electrode layer are provided with positioning holes corresponding in position.

The AB double-sided adhesive tape with the PET as the substrate comprises three layers, wherein the middle layer is the PET substrate layer, and the upper layer and the lower layer are acrylic adhesive layers; the ITO conductive film with the PET as the substrate comprises two layers, wherein the lower layer is the PET substrate layer, and the upper layer is the ITO conductive film.

The upper electrode layer and the lower electrode layer are stacked in a staggered mode, and the flow channel layer is adhered to the overlapping position between the upper electrode layer and the lower electrode layer.

The focusing flow channel is a micro snake-shaped flow channel.

A preparation method of the microfluidic impedance cytometer comprises the following steps:

s1: manufacturing an upper electrode layer and a lower electrode layer:

s11: selecting two ITO conductive films with PET as a substrate, cutting an excitation electrode and an induction electrode on the two ITO conductive films respectively, and simultaneously forming an upper insulation groove and a lower insulation groove respectively, wherein the bottoms of the upper insulation groove and the lower insulation groove are the PET substrate, so that the excitation electrode and the induction electrode form independent electrode areas;

s12: respectively cutting through holes on the two ITO conductive films with PET as the substrates processed in S11 to form an outlet hole, an upper positioning hole, a corresponding inlet hole and a corresponding lower positioning hole;

s2: manufacturing a flow channel layer:

selecting an AB double-sided adhesive tape taking PET as a substrate, cutting a through focusing flow channel, an inlet end and an outlet end of the focusing flow channel, and then cutting a middle positioning hole corresponding to the upper positioning hole and the lower positioning hole;

s3: assembling:

and firmly adhering the upper electrode layer and the lower electrode layer on the upper side and the lower side of the flow channel layer, sealing the flow channel focusing flow channel, and simultaneously butting the inlet end with the inlet hole, butting the outlet end with the outlet hole, and butting the excitation electrode with the induction electrode.

And a laser cutting process is adopted during cutting.

The invention has the following beneficial effects:

the microfluidic impedance cytometer of the invention has the advantages of easily obtained preparation materials, simple preparation process, reliable electrode forming, reasonable structure and good flow passage sealing performance. The preparation process consumes time which is reduced from 3 to 5 days to 5 minutes, and greatly accelerates the production efficiency. The adopted preparation materials and the laser cutting process method both promote the cost to be greatly reduced.

The electrode forming process is simple and reliable in quality.

The microfluidic impedance cytometer of the invention is light and thin in material, has good transparency, and is beneficial to observation, optical analysis and identification and the like.

The microfluidic impedance cytometer of the invention is used for detecting cells and particles, corresponding particle and cell information is obtained through electrical impedance signals, and the experimental structure is accurate and reliable. Therefore, the method is expected to be produced in large scale and can be made into disposable biological and medical equipment to meet the future demand for rapid in vitro detection.

Drawings

FIG. 1 is an exploded view of the microfluidic impedance cytometer of the present invention.

Fig. 2 is a schematic structural diagram of the microfluidic impedance cytometer of the present invention.

Fig. 3 is a schematic structural diagram of the upper electrode layer according to the present invention.

Fig. 4 is a schematic structural diagram of a flow channel layer according to the present invention.

FIG. 5 is a schematic structural diagram of a lower electrode layer according to the present invention.

Fig. 6 is a schematic process diagram of preparing the upper electrode layer of the microfluidic impedance cytometer of the present invention.

FIG. 7 is a schematic diagram of the process of preparing the flow channel layer of the microfluidic impedance cytometer of the present invention.

FIG. 8 is a schematic diagram of the assembly of the microfluidic impedance cytometer of the present invention.

FIG. 9 is a graph showing the experimental effect of particle focusing in the microfluidic impedance cytometer of the present invention.

FIG. 10 is a graph showing the experimental effect of impedance signal measurement in the microfluidic impedance cytometer of the present invention.

In the figure: 1. an upper electrode layer; 2. a flow channel layer; 3. a lower electrode layer; 11. an upper positioning hole; 12. an excitation electrode; 13. an upper insulating trench; 14. an outlet aperture; 21. a middle positioning hole; 22. an inlet end; 23. a focusing flow channel; 24. an outlet end; 31. a lower positioning hole; 32. an inlet aperture; 33. a lower insulating trench; 34. and a sensing electrode.

Detailed Description

The following describes embodiments of the present invention with reference to the drawings.

As shown in fig. 1 and fig. 2, the microfluidic impedance cytometer of the present embodiment includes, from top to bottom, an upper electrode layer 1, a flow channel layer 2, and a lower electrode layer 3.

As shown in fig. 3 to 5, the focusing flow channel 23 is provided in the flow channel layer 2; the upper electrode layer 1 and the lower electrode layer 2 are respectively provided with an excitation electrode 12 and an induction electrode 34 to form an electric field in the flow channel.

The cells or particles are focused in the focusing channel 23, and the cells or particles pass through the electric field area to cause the change of the electric field, so that the related information of the cells or particles can be measured.

Specifically, the upper electrode layer 1 and the lower electrode layer 3 are made of ITO conductive films of a PET substrate, and the flow channel layer 2 is made of AB double-sided adhesive tapes of the PET substrate.

As shown in fig. 3, the upper electrode layer 1 is provided with an upper positioning hole 11, an excitation electrode 12, an upper insulating groove 13, and an outlet hole 14.

As shown in fig. 4, the flow channel layer 2 is provided with a center positioning hole 21, an inlet end 22, a focusing flow channel 23, and a flow channel outlet end 24.

As shown in fig. 5, the lower electrode layer 3 is provided with an inlet hole 32, a lower positioning hole 31, a lower insulation groove 33, and an induction electrode 34.

In particular, the upper insulating trench 13 is not conductive, and the excitation electrode 12 is separated from other areas of the upper electrode layer 1 by the upper insulating trench 13, i.e. the excitation electrode 12 is not conductive to other areas of the upper electrode layer 1.

Specifically, the lower insulation groove 33 is not conductive, and the sensing electrode 34 is separated from other regions of the lower electrode layer 3 by the lower insulation groove 33, i.e., the sensing electrode 34 is not conductive with other regions of the lower electrode layer 3. The sensing electrode 34 is angularly coincident with the flow path portion of the excitation electrode 12 in the plane of fig. 4.

Specifically, the outlet end 24 of the focusing flow field 23 is connected to the outlet hole 14 of the upper electrode layer 1, and the inlet end 22 is connected to the inlet hole 32 of the lower electrode layer 3.

The focusing flow channel 23 is a cell focusing flow channel, and the end section of the direct flow channel shape is perpendicular to the butt joint part of the excitation electrode 12 and the induction electrode 34.

The upper positioning hole 11, the middle positioning hole 21, and the lower positioning hole 31 coincide on a plane.

The preparation method of the microfluidic impedance cytometer of the present embodiment, as shown in fig. 6 and 7, includes the following steps:

s1: two ITO conductive film materials with PET as a substrate are selected and respectively used for manufacturing an upper electrode layer 1 and a lower electrode layer 3.

The ITO conductive film material with PET as the substrate comprises two layers, namely an ITO conductive thin layer with a slightly dark color in figure 6 and a PET substrate layer with a slightly light color.

S11: setting laser power, and respectively cutting an excitation electrode 12 and an induction electrode 34 on the ITO conductive film without damaging the PET substrate layer;

as shown in the upper part of fig. 6, the laser spot-cut regions form non-conductive grooves, i.e., the excitation electrode insulation groove 13 and the sensing electrode insulation groove 33. The PET substrate layer is exposed at the bottom of the groove; this process separates the excitation and

sensing electrodes

12, 34 from their surrounding thin conductive regions of ITO, thereby forming separate electrode regions.

S12: and adjusting the laser power, and based on S11, as shown in the lower part of fig. 6, continuing to cut the upper positioning hole 11, the outlet hole 14, and the lower positioning hole 31, the inlet hole 32 corresponding to the upper positioning hole 11, respectively, in which both the ITO conductive layer and the PET substrate layer are laser cut off during the cutting process, so as to form a through hole.

In the processing processes of the steps S11 and S12, different laser processing powers are respectively saved, repeated positioning is not needed during processing, and form and position errors are avoided.

S2: and selecting an AB double-sided adhesive tape material taking PET as a substrate for manufacturing the flow channel layer 2.

The AB double-sided adhesive tape with PET as a substrate comprises three layers, wherein the upper layer and the lower layer are acrylic adhesive layers with slightly dark colors in a figure 7, and the middle layer is a PET substrate layer with lighter colors in the figure.

The laser power is set, and the outlines of the pilot hole 21, the inlet end 22, the focusing flow channel 23 and the outlet end 24 are cut on the AB double-sided adhesive tape material taking PET as a substrate. In the processing process, three layers of the AB double-sided tape taking PET as a substrate are all penetrated by laser, so that the interiors of the middle positioning hole 21, the inlet end 22, the focusing flow channel 23 and the outlet end 24 are in an island shape, the island after laser cutting is abandoned, and the corresponding shapes of the middle positioning hole 21, the inlet end 22, the focusing flow channel 23 and the outlet end 24 are formed. When assembling, as shown in fig. 1, the double-sided adhesive force of the AB double-sided tape using PET as a substrate firmly adheres to the upper electrode layer 1 and the lower electrode layer 2 on the upper and lower sides, thereby sealing the focusing flow channel 23. The flow of cutting the focusing flow path 23 is shown in fig. 7, in which the upper half of fig. 7 is a state diagram of cutting "island" and the lower half is a state diagram of discarding "island".

S3: assembling:

the upper electrode layer 1 and the lower electrode layer 3 are firmly adhered to the upper and lower sides of the flow channel layer 2, the flow channel focusing flow channel 23 is sealed, the inlet end 22 is butted with the inlet hole 32, the outlet end 24 is butted with the outlet hole 14, and the exciting electrode 12 and the sensing electrode 34 are butted.

And positioning is carried out by depending on respective positioning holes of each layer in the assembling process.

In particular, the laser control system may employ an ultraviolet or femtosecond laser control system.

The specific experimental application of the microfluidic impedance cytometer of this example, which is simple and convenient to manufacture, is as follows:

the focusing flow channel 23 in this embodiment is an asymmetric sinusoidal flow channel, and the cells or particles are focused in a row in the flow channel by the combined action of dean force and inertial lift force when flowing in the flow channel. The particles are first focused at the end of the focusing flow path 23, passing through the electrode area one by one. As shown in FIG. 9, the particles are focused on the upper middle position of the focusing flow channel 23, and it is found during the experiment that the particles and cells with the particle size of 10-20 μm can be focused at the flow rate of 700 μ L/min. As shown in fig. 10, when a focused cell or a focused cell flows through an electric field region formed by the excitation electrode 12 and the sensing electrode 34, a pair of antisymmetric peaks are generated due to a change in the electric field, and information such as the size, position, and internal structure of the cell or particle can be obtained by analyzing these signals.

As shown in fig. 8, it can be seen from the real object diagram of the microfluidic impedance cytometer of the present embodiment that the real object is relatively light and thin and has good transparency. The perpendicularity of the laser cut edge is also better.

In this embodiment, the excitation electrode 12 and the sensing electrode 34 together form a facing three-dimensional differential electrode. In the area perpendicular to the focusing flow channel 23, the overlapped portion of the exciting electrode 12 and the sensing electrode 34 forms a non-uniform electric field, and the electrical impedance signals generated by the cells or particles flowing through this area are differentiated to improve the signal quality.

The preparation method of the microfluidic impedance cytometer in the embodiment is extremely quick, the material cost is low, and the structure and the manufacturing process are simple. Compared with the traditional microfluidic impedance cytometry, the manufacturing time of the embodiment is shortened from 3-5 days to 5 minutes, and the production efficiency is greatly accelerated. The cost of the similar microfluidic impedance cytometer is also high, generally about 1000-2000 yuan, while the cost of the microfluidic impedance cytometer of the present embodiment is less than 10 yuan. Therefore, the microfluidic impedance cytometer of the embodiment is expected to be produced in large scale and batch, and can be made into disposable biological and medical equipment to meet the future demand for rapid in vitro detection, which is not possessed by other similar devices.

Claims

1. The microfluidic impedance cytometer comprises an upper electrode layer (1), a flow channel layer (2) and a lower electrode layer (3) which are sequentially stacked, wherein a focusing flow channel (23) is arranged on the flow channel layer (2), an excitation electrode (12) and an outlet hole (14) are arranged on the upper electrode layer (1), and an induction electrode (34) and an inlet hole (32) are arranged on the lower electrode layer (3);

the upper electrode layer (1) and the lower electrode layer (3) are made of ITO conductive thin film materials with PET as a substrate, the flow channel layer (2) is made of AB double-sided adhesive tape materials with PET as a substrate, the upper surface and the lower surface of the flow channel layer are respectively adhered to the upper electrode layer (1) and the lower electrode layer (3), the focusing flow channel (23) is sealed, meanwhile, an inlet end (22) of the focusing flow channel (23) is communicated with the inlet hole (32), and an outlet end (24) of the focusing flow channel is communicated with the outlet hole (14).

2. The microfluidic impedance cytometer of claim 1, wherein an upper insulating groove (13) is cut in the upper electrode layer (1) and a lower insulating groove (33) is cut in the lower electrode layer (3) by cutting the ITO conductive film, the upper insulating groove (13) surrounds the excitation electrode (12) independent of the rest of the upper electrode layer (1), the lower insulating groove (33) surrounds the sensing electrode (34) independent of the rest of the lower electrode layer (3), and the excitation electrode (12) and the sensing electrode (34) are connected in opposite to form a stereo differential electrode.

3. The microfluidic impedance cytometer of claim 2, wherein the excitation electrode (12) and the sensing electrode (34) overlap at a butt joint to form a non-uniform electric field perpendicular to the end of the focusing flow channel (23) near the outlet end (24).

4. The microfluidic impedance cytometer according to claim 1, wherein the upper electrode layer (1), the flow channel layer (2) and the lower electrode layer (3) are provided with positioning holes corresponding to the positions of the positioning holes.

5. The microfluidic impedance cytometer of any one of claims 1-4, wherein the PET-based AB double-sided tape comprises three layers, a PET-based layer in the middle, and an acrylic adhesive layer on the top and bottom layers; the ITO conductive film with the PET as the substrate comprises two layers, wherein the lower layer is the PET substrate layer, and the upper layer is the ITO conductive film.

6. The microfluidic impedance cytometer of claim 5, wherein the upper electrode layer (1) and the lower electrode layer (3) are stacked in a staggered manner, and the flow channel layer (2) is adhered to the upper electrode layer (1) and the lower electrode layer (3) at the overlapping position.

7. The microfluidic impedance cytometer of claim 5, wherein the focusing flow channel (23) is a micro serpentine flow channel.

8. A method of making the microfluidic impedance cytometer of claim 1, comprising the steps of:

s1: manufacturing an upper electrode layer (1) and a lower electrode layer (3):

s11: selecting two ITO conductive films with PET as a substrate, cutting an excitation electrode (12) and an induction electrode (34) on the two ITO conductive films respectively, and simultaneously forming an upper insulation groove (13) and a lower insulation groove (33) respectively, wherein the bottoms of the upper insulation groove (13) and the lower insulation groove (33) are the PET substrate, so that the excitation electrode (12) and the induction electrode (34) form independent electrode areas;

s12: respectively cutting through holes on the two ITO conductive films with the PET as the base processed in the step S11 to form an outlet hole (14), an upper positioning hole (11), an inlet hole (32) and a lower positioning hole (31);

s2: production of a flow channel layer (2):

selecting an AB double-sided adhesive tape taking PET as a substrate, cutting a through focusing flow channel (23) on the AB double-sided adhesive tape, and cutting an inlet end (22) and an outlet end (24) of the focusing flow channel (23) into middle positioning holes (21) corresponding to the upper positioning hole (11) and the lower positioning hole (31);

s3: assembling:

and the upper electrode layer (1) and the lower electrode layer (3) are firmly adhered to the upper side and the lower side of the flow channel layer (2), the flow channel focusing flow channel (23) is sealed, the inlet end (22) is butted with the inlet hole (32), the outlet end (24) is butted with the outlet hole (14), and the excitation electrode (12) and the induction electrode (34) are butted.

9. The method for preparing a microfluidic impedance cytometer of claim 8, wherein a laser cutting process is used for cutting.