CN113804735A - High-performance single-cell impedance detection microelectrode and preparation method thereof - Google Patents

Abstract

The invention relates to a high-performance single-cell impedance detection microelectrode and a preparation method thereof.A stimulation electrode and a detection electrode are respectively arranged at two sides of a microfluidic channel, and the stimulation/detection electrodes can be one pair or a plurality of pairs; and a ground electrode is provided in a region other than the excitation electrode and the detection electrode. Compared with the traditional single-cell electrical impedance detection with coplanar electrodes, the symmetrical electrodes on the two sides can make the electric field distribution in the detection area more uniform, and improve the detection precision; and set up telluric electricity field concentration degree that telluric electricity field can improve the detection area, avoid the mutual interference of electric field between the many pairs of electrodes simultaneously, promote the signal strength that cell or granule detected. The method has very important practical value and innovative significance for accurately obtaining the single-cell or particle stable and high signal-to-noise ratio electrical impedance value, and can effectively solve the problems of unstable electrical impedance value and large noise interference of the single cell at present.

Description

High-performance single-cell impedance detection microelectrode and preparation method thereof

Technical Field

The invention relates to a cell electrical impedance detection technology in the technical field of micro-electro-mechanical engineering and biomedical engineering, in particular to a high-performance single-cell electrical impedance detection microelectrode design and preparation method based on the Coulter counting principle.

Background

The development and combination of micro-electro-mechanical systems (MEMS) technology and biological cell engineering provide important guarantee for the development of instruments for high-precision cell detection, counting and the like. The electrical impedance detection is to use the micro structure of the MEMS system to enable single cells or particles to sequentially pass through a detection area, the cells or particles can cause the electrical impedance change of the detection area, an impedance change curve can be obtained from a detection electrode, the cell types passing through the detection area are judged according to the amplitude and the span time of the impedance change, and the cell types are classified and counted. Electrical impedance detection and analysis are widely applied in the fields of drug screening, blood cell counting, food detection, environmental monitoring and the like. The cell-oriented electrical impedance detection counting has the advantages of rapidness, no mark, simple structure, small damage to cells and the like, and gradually becomes an effective analysis tool in biomedical research.

At present, the detection and counting research on single cells is not complete, and the single cell electrical impedance detection is often accompanied by the defects of high noise and instability. Therefore, the improvement of the arrangement scheme of the electrodes in the electrical impedance detection has important significance for improving the signal-to-noise ratio of the impedance value of the single cell.

D.s.de Bruijn et al, Biosensors & Bioelectronics 173 (2020): 112808 written, "Coocola suspension chromatography analysis by single-cell electrophoresis cell cytometry: towards single-cell PIC: POC measurements' adopt a single-side coplanar electrode to realize the identification of calcified algae cells. However, in order to reduce the influence of noise on the signal, the method needs to measure the electrical impedance characteristic of the target cell in advance, so that the method cannot identify the unknown cell; and the electric field generated by the single-side coplanar electrode is not uniform, and the distance between the cell and the electrode can have important influence on the electrical impedance signal. The patent CN109852542A discloses a micro-fluidic chip for single-cell electrical impedance detection and a processing method thereof, wherein the micro-fluidic chip adopts two pairs of symmetrical electrodes to carry out differential detection on the electrical impedance value of cells; the uniformity of the electric field of the symmetrical electrodes is better than that of the coplanar electrodes, but the electric fields generated by the two pairs of electrodes interfere with each other to influence the detection effect. J.Gonz a lez-Murillo et al at 2018Spanish Conference on Electron Devices, 2018: 1-4 written, "electric Impedance Spectroscopy microfluidics Cytometer for cell Impedance tests" proposes a method for improving the electric field concentration by adding a grounding electrode, but the micro-fluidic chip adopts coplanar electrodes and has poor electric field uniformity.

Therefore, the microelectrode design and preparation method with more uniform and concentrated electric field is developed, and has very important practical value and innovation significance for accurately detecting the electrical characteristics of cells.

Disclosure of Invention

Technical problem to be solved

The invention provides a high-performance single cell impedance detection microelectrode and a preparation method thereof, aiming at solving the problems of low signal-to-noise ratio, low precision and the like in the current single cell impedance detection.

Technical scheme

A high-performance single-cell impedance detection microelectrode is characterized by comprising an upper chip and a lower chip, wherein an excitation electrode is arranged on the upper chip, a detection electrode is arranged on the lower chip, grounding electrodes are arranged around the excitation electrode and the detection electrode, and the excitation electrode and the detection electrode are respectively positioned on the upper side and the lower side of a microfluidic channel; the excitation electrode and the detection electrode form a contact at the edge of the chip substrate, then gradually extend towards the center of the chip, and finally form rectangular electrode plates on the upper side and the lower side of a detection area; the lengths of the exciting electrode and the detecting electrode are 1.2-3 times of the maximum dimension of the cell or particle to be detected; the widths of the excitation electrode and the detection electrode need to completely cover the microfluidic channel and are 0.2-1 times larger than the width of the microfluidic channel.

The further technical scheme of the invention is as follows: the distance between the grounding electrode and the exciting electrode and between the grounding electrode and the detecting electrode is kept between 10 and 500 micrometers.

The further technical scheme of the invention is as follows: the excitation/detection electrodes may be in one or more pairs.

The further technical scheme of the invention is as follows: the upper chip and the lower chip are made of glass or polymethyl methacrylate (PMMA) or Polycarbonate (PS).

The further technical scheme of the invention is as follows: the excitation electrode and the detection electrode are made of metal.

The further technical scheme of the invention is as follows: the metal is Indium Tin Oxide (ITO), gold or platinum.

A preparation method of a high-performance single-cell impedance detection microelectrode is characterized by comprising the following steps:

step 1: preparing a chip substrate: firstly, carrying out metal coating patterning treatment on the surfaces of an upper layer chip substrate and a lower layer chip substrate, and forming an excitation electrode, a detection electrode and a grounding electrode by using a metal coating; punching a hole on the upper chip to form an inlet and an outlet of cells or particles;

step 2: preparing a micro-fluid channel: spin-coating photoresist with a certain thickness on the bottom surface of the lower chip substrate, and then forming a microfluidic channel structure through photoetching and developing;

and step 3: chip bonding: bonding the prepared upper chip substrate with the lower chip substrate with the channel structure to form the microfluidic chip with the upper, middle and lower three-layer structures.

Advantageous effects

According to the high-performance single-cell impedance detection microelectrode and the preparation method thereof, the electrode arrangement scheme can effectively reduce the mutual interference between adjacent detection areas and between the adjacent detection areas and an electric field of a peripheral detection circuit, and improve the strength of a detection signal; meanwhile, the uniformity of an electric field in a detection area is improved, and the detection precision is improved.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention provides that an excitation electrode and a detection electrode are designed on the upper side and the lower side of a micro-channel, a stable uniform electric field is formed in a detection area by utilizing the symmetry of an excitation electrode plate and a detection electrode plate, and the dependence of an impedance signal on the height of a cell (the distance between the cell and the detection electrode) when the cell passes through the detection area is greatly reduced; meanwhile, on the basis of the original excitation electrode and detection electrode, grounding electrodes are arranged outside the areas of the excitation electrode and the detection electrode at equal intervals, and by utilizing the zero potential characteristic of the grounding electrodes, the electric field distribution at the edge of the electrode can be greatly improved, so that the concentration ratio of an electric field is further improved, the mutual interference of the electric fields of adjacent detection areas can be avoided, and the accuracy and the signal-to-noise ratio of cell or particle impedance detection signals are fundamentally improved.

2. The invention designs a gradual change electrode structure (excitation/detection electrode) from big to small. The electrode structure contact is positioned at the edge of the upper chip substrate and the lower chip substrate, the contact is a square bonding pad area, the contact is used as the input of an excitation signal and the output of a detection signal, the size of the contact is slightly larger than that of a common signal input/output contact on the market, and the applicability is good. The excitation/detection electrode gradually shrinks inwards (in the middle of the chip) after passing through an arc region with equal width, and finally 70 multiplied by 50um is formed²The detection area of (1). The electrode transition section adopts smooth processing such as rounding off and chamfering, on one hand, the processing difficulty is reduced, on the other hand, the right-angle and obtuse-angle static concentration is avoided, and the service life of the chip electrode is prolonged. And arranging grounding electrodes at equal intervals outside the excitation/detection electrodes, wherein the grounding electrodes extend outwards to the bottom edge of the chip substrate. So far, the area of the grounding electrode area is larger than that of the exciting/detecting electrode, and the function of preventing the interference of an external electric field and an electric field of an adjacent detecting area can be achieved.

3. The invention designs the electrode structure on the surface of the upper and lower chips as central symmetry (the upper exciting electrode and the lower detecting electrode which form a detecting area are respectively arranged on two sides of the micro-fluid channel) to reduce the influence of the upper electrode signal on the lower electrode signal.

4. The electrode preparation method of the invention combines the traditional technologies of photoetching, alignment, metal sputtering and the like, and designs the electrode structure with the upper and lower centrosymmetry. The electrode preparation method can greatly improve the completeness and yield of electrode preparation by utilizing several mature micro-nano manufacturing processes.

Drawings

The drawings are only for purposes of illustrating particular embodiments and are not to be construed as limiting the invention, wherein like reference numerals are used to designate like parts throughout.

FIG. 1 is a cross-sectional view of an electrical impedance detection region of a preferred embodiment of the present invention.

Fig. 2 is a schematic diagram of a structure of a microfluidic chip based on electrical impedance cell detection and a schematic diagram of each chip level according to a preferred embodiment of the invention.

Fig. 3 is a structural diagram of an upper chip substrate of a microfluidic core based on electrical impedance cell detection according to a preferred embodiment of the present invention and a partially enlarged view, and a gray dotted line in an internal detection area of the partially enlarged view indicates a projection of a microfluidic channel in the upper chip substrate.

Fig. 4 is a structural diagram and a partial enlarged view of a microfluidic channel of the microfluidic chip based on electrical impedance cell detection according to the preferred embodiment of the present invention.

Fig. 5 is a structural diagram and a partially enlarged view of a lower chip substrate of a microfluidic chip based on electrical impedance cell detection according to a preferred embodiment of the present invention, and a gray dotted line in an internal detection area of the partially enlarged view indicates a projection of a microfluidic channel in the lower chip substrate.

Fig. 6 is a simulation diagram of electric field distribution with or without a ground electrode according to a preferred embodiment of the present invention and a partially enlarged view thereof.

FIG. 7 is a graph showing the impedance of cells or particles in a detection region I, II in accordance with a preferred embodiment of the present invention.

Fig. 8 is a schematic structural diagram and a partial enlarged view of a microfluidic chip including three detection regions according to a preferred embodiment of the present invention.

The scores in the figure are indicated as: the chip comprises a lower chip substrate 1, a microfluidic channel 2 positioned in a middle chip, an upper chip substrate 3, a grounding electrode 4, an excitation electrode 5, cells or particles to be detected 6, a detection electrode 7, a middle chip 8, an inlet 9, an outlet 10, a filtering cylinder 11, a microfluidic channel width 12 and an electrode extra microfluidic channel width 13.

Among them, a detection region composed of the excitation electrode 5 and the detection electrode 7 near the entrance is particularly referred to as a detection region I, and a detection region composed of the excitation electrode 5 and the detection electrode 7 near the exit is referred to as a detection region II. The detection area I and the detection area II jointly form a detection area of the microfluidic chip.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The invention provides an arrangement scheme of electrodes for high-performance electrical impedance detection, which comprises the steps of respectively arranging an excitation electrode and a detection electrode on the upper side and the lower side of a micro-channel, and arranging grounding electrodes around the excitation electrode and the detection electrode.

The arrangement scheme that the excitation electrode and the detection electrode are respectively arranged on the upper side and the lower side of the microchannel can enable the detection area in the microchannel to be always under a uniform electric field, the excitation electrode and the detection electrode firstly form contacts (respectively used for applying an excitation electric signal and reading a detection electric signal) on the edge of the chip substrate, then gradually extend towards the center of the chip, and finally form tiny rectangular electrode plates (an excitation electrode plate and a detection electrode plate) on the upper side and the lower side of the detection area. The length (along the flowing direction of cells or particles) of the excitation electrode slice and the detection electrode slice is 1.2-3 times of the maximum dimension of the cells or particles to be detected; the width (perpendicular to the cell or particle flow direction) of the excitation electrode plate and the detection electrode plate needs to completely cover the microfluidic channel, and the width of the extra electrode plate is 0.2-1 times of the width of the microfluidic channel, namely the width 13 of the extra electrode plate is 0.2-1 times of the width 12 of the microfluidic channel.

And the grounding electrodes are arranged around the excitation electrodes and the detection electrodes, and the distance between the grounding electrodes and the excitation electrodes and the distance between the grounding electrodes and the detection electrodes are kept at 10-500 micrometers, so that the electric field concentration is enhanced, and the crosstalk between adjacent electric fields is reduced.

The width and height of the microfluid channel in the detection area through which the cells or particles sequentially and singly pass are 1.2-3 times of the maximum dimension of the cells or particles to be detected. The height of the microfluidic channel outside the detection region is consistent with that of the microfluidic channel in the detection region, the width of the microfluidic channel in the detection region is wider than that of the microfluidic channel in the detection region, and the length of the microfluidic channel in the detection region is determined according to the excitation/detection electrode, the grounding electrode and the interval between the excitation/detection electrode and the grounding electrode.

The invention also provides a preparation method of the high-performance single-cell impedance detection microelectrode, which comprises the following steps:

step one, preparing a chip substrate: firstly, the surface of the upper and lower chip substrates is processed by a patterned metal coating, and an excitation electrode, a detection electrode and a grounding electrode are formed by the metal coating. And the upper chip is perforated to form the inlet and outlet of cells or particles.

Step two, preparing a micro-fluid channel: the bottom surface of the lower chip substrate is coated with photoresist with a certain thickness (height of the microfluidic channel) in a spin mode, and then the microfluidic channel structure is formed through operations such as photoetching, developing and the like.

Step three, chip bonding: bonding the prepared upper chip with the lower chip with the microfluidic channel structure to form the microfluidic chip with the upper, middle and lower three-layer structures.

In the three-layer chip preparation scheme, the electrode arrangement scheme in the upper and lower chip substrates is designed in a central symmetry mode, namely an upper excitation electrode and a lower detection electrode which form a detection area are respectively positioned on two sides of a microfluidic channel so as to reduce the influence of an upper electrode signal on a lower electrode signal.

Example 1:

in a specific implementation, the electrodes (excitation, detection, ground) near the electrical impedance detection region are arranged as described with reference to fig. 1. The whole chip structure is divided into three layers, and referring to fig. 2, the structure of each layer of the specific detection chip is as follows:

the upper chip substrate 3 is made of glass, and patterned metal sputtering is performed on the lower surface of the glass to finally form two excitation electrodes 5 and a grounding electrode 4, wherein the excitation electrodes 5 extend inwards from the edge of the chip gradually. Firstly, a 2X 2mm chip is formed at the edge of the chip²Then gradually extends inwards, finally protrudes to form a tiny rectangular electrode plate,the electrode plate covers the upper side of the micro-channel of the detection area of the middle chip and forms a stable uniform electric field together with the electrode plate of the lower detection electrode. The ground electrode 4 is laid over the rest of the upper chip (the region outside the excitation electrode), and is spaced from the excitation electrode 5 by a certain distance, and the distance between the ground electrode 4 and the excitation electrode 5 is maintained at about 100 μm. And laser drilling is performed on the upper chip as an inlet 9 and an outlet 10 for cells or particles to be detected, the specific structure of which is shown in fig. 3.

The chip 8 in the middle layer mainly aims at constructing a microfluidic channel 2, the whole microfluidic channel is of a rectangular channel structure with the length of 10.5mm and the width of 0.5mm, a micron-sized microfluidic channel with the width and the thickness only being 1.2-3 times of the maximum direction size of cells or particles is designed in the middle of the rectangular channel, and the cells or the particles sequentially and singly pass through the microfluidic channel by utilizing the tiny geometric dimension of a detection area. The circular structure with the diameter of 1mm is designed on both sides of the channel so as to match with the inlet hole and the outlet hole of the upper chip to realize the inflow and outflow of cells or particles. At the same time, the detection region is closely adjacent to the excitation/detection electrode above and below, so that a stable uniform electric field can be formed in the region. In particular, the present invention designs a filter cylinder 11 in the cell or particle inflow channel, and the purpose of the filter cylinder 11 is to filter large particle residues in the solution, prevent the blockage of the tiny detection area, and ensure the smooth passage of the cells or particles to be detected.

The size of the common cells is maintained between 1 and 20 microns, which has high requirements on the thickness of the middle chip 8 and the width of the channel. Therefore, the middle chip 8 adopts the photoetching process, i.e. photoresist is directly spin-coated on the structure of the lower chip substrate 1, and after alignment, exposure and development of a mask plate, the middle chip 8 with the thickness of 30um is formed, and the width of the microfluidic channel in the detection area is 30 um. The specific structure is shown in fig. 4.

The lower chip substrate 1 is made of glass, and patterned metal sputtering is performed on the upper surface of the glass. The structure of the lower chip detection electrode and the grounding electrode and the structure of the upper chip electrode are in a central symmetry structure, and the structure sizes are consistent. This prevents, to some extent, the upper excitation electrode signal from interfering with the lower detection electrode signal. The specific structure is shown in FIG. 5.

The middle layer chip 8 and the lower layer chip substrate 1 are bonded in the photoetching process, for the bonding of the middle layer chip 8 and the upper layer chip substrate 3, firstly, a plasma cleaning machine is used for surface modification, then, the bonding of the middle layer chip 8 and the upper layer chip substrate 3 is completed, and the whole chip structure is shown in figure 2.

Referring to fig. 2, two excitation electrodes 5 in the upper chip substrate 3 of the whole chip and corresponding detection electrodes 7 in the lower chip substrate 1 form dual detection areas, and particularly, a detection area near an inlet is defined as a detection area I, and a detection area near an inlet and an outlet is defined as a detection area II.

Referring to fig. 2, in the cell or particle detection in this embodiment, first, electrical signals are applied to two excitation electrodes 5 in the upper chip substrate 3, respectively, and the induced signals of the detection electrodes 7 in the lower chip substrate are connected to an electrical impedance analyzer, so that the upper layer and the lower layer form a uniform detection electric field, and the ground electrodes 4 of the upper (lower) chip substrate are grounded, thereby increasing the electric field concentration in the detection area. A suspension of cells or particles is passed in from an inlet 9 and out through an outlet 10, and the impedance signal of the cells is differentially amplified using two pairs of detection zones.

Referring to fig. 6, based on the electric field simulation of the present embodiment, it can be found that the electric field concentration can be greatly enhanced by providing the ground electrode, and the electric field distribution in the chip can be improved. Meanwhile, referring to fig. 7, it can be seen that, in the case where the ground electrode is provided, the amount of impedance change caused by the passage of the cells or the microparticles through the detection region is greater, which will greatly improve the signal strength of the electrical impedance detection.

Example 2:

in another embodiment, the material used for the chips in each layer may be replaced to achieve the same effect of enhancing the uniformity and concentration of the electric field. The design material of the upper and lower chips can be changed from glass to polymethyl methacrylate (PMMA), Polycarbonate (PS), etc., and the electrode material in the upper and lower chips can be Indium Tin Oxide (ITO), gold, platinum, etc.

Example 3:

in another embodiment, the number of the detection electrodes and the grounding electrodes can be increased or decreased, and in the electrical impedance detection of the cells, voltages with different frequencies are applied to the excitation electrode, so that more cell information can be obtained, and the identification and the classification counting of the cells at the later stage are facilitated. Therefore, in this embodiment, a three-detection-area design of three pairs of electrodes is designed, referring to fig. 8. The chip with the three detection areas is still formed by overlapping three layers of chip substrates, and three excitation electrodes extending from the edge to the inside and grounding electrodes surrounding the excitation electrodes are manufactured on the lower surface of the upper layer chip by metal sputtering; the middle chip designs a micro channel according to the size of the cell to be detected, so that the cell or the particle can pass through the detection area in sequence; the upper surface of the lower chip is provided with a detection electrode and a grounding electrode which are in a centrosymmetric structure with the upper chip. Thus, the superposition of three layers of chip substrates can form three pairs of excitation/detection electrodes above and below the detection area. Therefore, different detection signals can be applied to the three excitation electrodes to obtain more cell information, so that the purpose of improving the cell or particle electrical impedance detection performance is achieved.

Compared with the traditional single-cell electrical impedance detection with coplanar electrodes, the two symmetrical electrodes can make the electric field distribution in the detection area more uniform, and improve the detection precision; and set up telluric electricity field concentration degree that telluric electricity field can improve the detection area, avoid the mutual interference of electric field between the many pairs of electrodes simultaneously, promote the signal strength that cell or granule detected. The method has very important practical value and innovative significance for accurately obtaining the single-cell or particle stable and high signal-to-noise ratio electrical impedance value, and can effectively solve the problems of unstable electrical impedance value and large noise interference of the single cell at present.

While the invention has been described with reference to specific embodiments, the invention is not limited thereto, and various equivalent modifications or substitutions can be easily made by those skilled in the art within the technical scope of the present disclosure.

Claims (7)

1. A high-performance single-cell impedance detection microelectrode is characterized by comprising an upper chip and a lower chip, wherein an excitation electrode is arranged on the upper chip, a detection electrode is arranged on the lower chip, grounding electrodes are arranged around the excitation electrode and the detection electrode, and the excitation electrode and the detection electrode are respectively positioned on the upper side and the lower side of a microfluidic channel; the excitation electrode and the detection electrode form a contact at the edge of the chip substrate, then gradually extend towards the center of the chip, and finally form rectangular electrode plates on the upper side and the lower side of a detection area; the lengths of the exciting electrode and the detecting electrode are 1.2-3 times of the maximum dimension of the cell or particle to be detected; the widths of the excitation electrode and the detection electrode need to completely cover the microfluidic channel and are 0.2-1 times larger than the width of the microfluidic channel.

2. The high-performance single-cell impedance detection microelectrode of claim 1, wherein the distance between the grounding electrode and the excitation electrode and between the grounding electrode and the detection electrode is 10-500 μm.

3. The high-performance single-cell impedance detection microelectrode according to claim 1, wherein the excitation/detection electrode is one or more pairs.

4. The high-performance single-cell electrical impedance detection microelectrode according to claim 1, wherein the upper chip and the lower chip are made of glass, polymethyl methacrylate (PMMA), or Polycarbonate (PS).

5. The high-performance single-cell electrical impedance detection microelectrode of claim 1, wherein the material of the excitation electrode and the detection electrode is metal.

6. The high-performance single-cell electrical impedance detection microelectrode according to claim 5, wherein the metal is Indium Tin Oxide (ITO), gold or platinum.

7. A preparation method of the high-performance single-cell impedance detection microelectrode of claim 1 is characterized by comprising the following steps:

step 1: preparing a chip substrate: firstly, carrying out metal coating patterning treatment on the surfaces of an upper layer chip substrate and a lower layer chip substrate, and forming an excitation electrode, a detection electrode and a grounding electrode by using a metal coating; punching a hole on the upper chip to form an inlet and an outlet of cells or particles;

step 2: preparing a micro-fluid channel: spin-coating photoresist with a certain thickness on the bottom surface of the lower chip substrate, and then forming a microfluidic channel structure through photoetching and developing;

and step 3: chip bonding: bonding the prepared upper chip substrate with the lower chip substrate with the channel structure to form the microfluidic chip with the upper, middle and lower three-layer structures.

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