KR101686120B1 - Nanostructured electrode chip, a preparation method thereof and Methods for cell growth using thereof - Google Patents

Abstract

The present invention relates to a nano-structured microelectrode chip, a method of manufacturing the same, and a method of growing a cell using the nanoparticle structure microelectrode chip. More particularly, the present invention relates to a nano- The present invention relates to a nanoparticle-type microelectrode chip capable of studying the response of a nerve cell to a topological structure of the nanoparticle-type microelectrode chip, a method for manufacturing the same, and a cell growth method using the same.

Description

TECHNICAL FIELD [0001] The present invention relates to a nanostructure-typed microelectrode chip, a method of manufacturing the same, and a nanostructured electrode chip using the same,

The present invention relates to a nano-structured microelectrode chip, a method of manufacturing the same, and a method of growing a cell using the nanoparticle structure microelectrode chip. More particularly, the present invention relates to a nano- The present invention relates to a nanoparticle-type microelectrode chip capable of studying the response of a nerve cell to a topological structure of the nanoparticle-type microelectrode chip, a method for manufacturing the same, and a cell growth method using the same.

Microelectrode Arrays (MEAs) are microelectrode arrays (MEAs) on which microelectrodes are arranged on a glass plate. Neuron cells are cultured on microelectrode chips for neuroelectrophysical studies. It is used to measure simultaneously or to apply electrical stimulation.

The nervous system in the body has a nano-level topographical structure formed by various kinds of cells and various kinds of extracellular matrix. It is known that such a geographical structure can be a signal involved in the formation of neurons constituting the nervous system, and the exact mechanism is still being reported.

However, the flat plate type microelectrode chip up to now has a disadvantage that it can not express an extracellular matrix having a topographically curved structure.

In order to solve the above-mentioned problems, the inventor of the present invention measured the electrical activity of nerve cells and formed a topographical structure of several hundred nanometers in a microelectrode chip capable of applying stimulation, A nanoparticle structure microelectrode chip capable of measuring the influence, a method for manufacturing the same, and a cell growth method using the same.

Korean Patent No. 10-1262256

SUMMARY OF THE INVENTION

A nanoparticle structure microelectrode chip, a manufacturing method thereof, and a cell growth method using the same.

In order to achieve the above object,

An adhesive layer formed on the substrate;

An electrode layer formed on the adhesive layer; And

And an insulating layer formed on the substrate and the electrode layer,

At least a part of the electrode layer is exposed to the outside,

Wherein the insulating layer has a concavo-convex structure.

According to the present invention,

An adhesive layer formed on the substrate;

An electrode layer formed on the adhesive layer;

An insulating layer formed on the substrate and the electrode layer; And

And a cell-affinity polymer layer coated on the insulating layer,

At least a part of the electrode layer is exposed to the outside,

Wherein the insulating layer has a concavo-convex structure.

According to the present invention,

Forming an adhesive layer on the substrate (step 1);

Forming an electrode layer on the adhesive layer (step 2);

Patterning the adhesive layer and the electrode layer (step 3);

Depositing an insulating layer on the substrate of step 3 (step 4); And

And patterning at least a part of the insulating layer deposited in step 4 to expose a part of the electrode layer to the outside (step 5).

According to the present invention,

Forming an adhesive layer on the substrate (step 1);

Forming an electrode layer on the adhesive layer (step 2);

Patterning the adhesive layer and the electrode layer (step 3);

Depositing an insulating layer on the substrate of step 3 (step 4);

Exposing a part of the electrode layer to the outside by patterning at least a part of the insulating layer deposited in the step 4 (step 5);

And coating the cell affinity polymer on the substrate of step 5 to cultivate the cell (step 6). The present invention also provides a method of growing a cell using the nanoparticle structure microelectrode chip.

The nano-structured microelectrode chip, the method of manufacturing the same, and the cell growth method using the same according to the present invention are characterized in that nano scale irregularities are formed on a substrate to form a geomorphic structure to grow cells in various geographical structures, The morphological and electrophysiological effects of the microelectrode chips on the structure can be measured, and thus various studies of neurons according to the geographical structure can be made possible.

FIG. 1 is a cross-sectional view illustrating a nano-structured microelectrode chip according to an embodiment of the present invention.
FIG. 2 is a cross-sectional view showing a step 1 of a method of manufacturing a nanoporous-type microelectrode chip according to an embodiment of the present invention.
FIG. 3 is a cross-sectional view illustrating a step 2 of a method of manufacturing a nanoporous-type microelectrode chip according to an embodiment of the present invention.
4 is a cross-sectional view showing a step 3 of a method of manufacturing a nanoporous-type microelectrode chip according to an embodiment of the present invention.
FIG. 5 is a cross-sectional view showing a step 4 of a method of manufacturing a nanoporous-type microelectrode chip according to an embodiment of the present invention.
FIG. 6 is a cross-sectional view showing a step 5 of a method of manufacturing a nanoporous-type microelectrode chip according to an embodiment of the present invention.
7 is a view showing a nanoritic structure microelectrode chip manufactured according to the first embodiment of the present invention.
8 is a view showing a nanoritic structure microelectrode chip manufactured according to the second embodiment of the present invention.
9 is a view showing a nanoritic structure microelectrode chip manufactured according to the third embodiment of the present invention.
10 is a view showing a nanoritic structure microelectrode chip manufactured according to the fourth embodiment of the present invention.
11 is a photograph of a circular protrusion having a diameter of 3 탆 of a nanogranular-shaped microelectrode chip manufactured according to Example 1 of the present invention with a scanning electron microscope.
12 is a photograph of a nerve cell cultured on top of a nanorail type microelectrode chip of Example 1 of the present invention by a scanning electron microscope.
13 is a photograph of a nerve cell cultured on the upper part of Comparative Example 1 by scanning electron microscope.
FIG. 14 is a photograph of a nerve cell cultured on top of a nanoparticle structure microelectrode chip of Example 1 of the present invention by a scanning electron microscope. FIG.
FIG. 15 (a) is a graph showing the number of cells per unit area of neuron cultured on the nanoritic structure microelectrode chip of Example 1 and Comparative Example 1 of the present invention.
FIG. 15 (b) is a graph showing the cell survival rate of nerve cells cultured on the nanoritic structure microelectrode chip of Example 1 and Comparative Example 1 of the present invention.

According to an aspect of the present invention,

An adhesive layer formed on the substrate;

An electrode layer formed on the adhesive layer;

An insulating layer formed on the substrate and the electrode layer; And

And a cell-affinity polymer layer coated on the insulating layer,

At least a part of the electrode layer is exposed to the outside,

Wherein the insulating layer has a concavo-convex structure.

For example, FIG. 1 shows a cross-sectional view of a nanoporous-type microelectrode chip. Hereinafter, a nanoporous-type microelectrode chip according to the present invention will be described in detail.

Referring to FIG. 1, a nanoritic microelectrode chip according to the present invention may include a substrate 110, an adhesive layer 120, an electrode layer 130, an insulating layer 140, and a cell affinity polymer layer 150. have.

The substrate 110 may be any type of plate capable of culturing cells. Specifically, the substrate 110 may be selected from the group consisting of glass, plastic, metal, silicon, Su-8, polydimethylsiloxane (PDMS) However, the type of the substrate 110 is not limited thereto.

An adhesive layer 120 is formed on the substrate 110 so as to be spaced apart from each other. The adhesive layer 120 may be spaced apart from the substrate 110 and may be formed to increase the adhesive force between the electrode layer 130 and the substrate 110 to be formed thereafter. The adhesive layer 120 may be formed of at least one selected from the group consisting of Pt, Pd, Au, Ag, Ti, Cr, Al, Cu, , Molybdenum (Mo), ruthenium (Ru), and indium (In). The adhesive layer 120 may be formed on the substrate 110 by a physical or chemical vapor deposition method such as a pulsed laser deposition method, a sputtering method, and a sol-gel method. The adhesive layer 120 is preferably formed within a range of 10 nm to 500 nm. If the thickness of the adhesive layer 120 is less than 10 nm, the bonding strength between the electrode layer 130 and the substrate 110 may be reduced and the stability may be deteriorated. If the thickness of the adhesive layer 120 exceeds 50 nm, .

The electrode layer 130 is formed on the adhesive layer 120 and is formed of platinum Pt, Pd, Au, Ag, Ti, Cr, Al ), Copper (Cu), tin (Sn), molybdenum (Mo), ruthenium (Ru), and indium (In). The electrode layer 130 may be formed on the adhesive layer 120 by a physical or chemical vapor deposition method such as a pulse laser deposition method, a sputtering method, and a sol-gel method. The electrode layer 130 is preferably formed within a range of 100 nm to 300 nm. When the thickness of the electrode layer 130 is less than 100 nm, the electrode layer 130 does not have a sufficient thickness and the irregular structure of the insulating layer 140 can not be formed. When the thickness of the electrode layer 130 is 300 nm The thickness of the electrode layer 130 is excessively large, so that the concavo-convex structure of the insulating layer 140 can not be formed.

Insulating layer 140 is a layer for forming the formed concavo-convex (凹凸) structure on a substrate 110, a silicon nitride (SiNx), silicon oxide (SiO _2), aluminum oxide (Al ₂ O _3), BZN oxide (Bismuth -Zinc-Niobium Oxide), titanium oxide, hafnium oxide, zirconium oxide, tantalum oxide, and lanthanum oxide. The insulating layer 140 may be formed by chemical vapor deposition (CVD), and the insulating layer 140 may be formed in a shape in which protrusions are arranged in a dot, a line, or a combination thereof . When the insulating layer 140 has a concavo-convex structure, the mechanism of cells that grow differently depending on the terrain can be known. The insulating layer 140 may be formed to a thickness of 200 to 1000 nm, but is not limited thereto.

The cell affinity polymer layer 150 may be, but is not limited to, polydopamine, polylysine, laminin, or fibronectin. The cell-affinity polymer layer 150 may be coated on the insulating layer 140 having a concavo-convex structure to cultivate any one of microorganisms, animal and plant cells and neurons, have. The nerve cells can be cultured on the insulating layer 140 having a concavo-convex structure to effectively study cell growth and signal transmission characteristics according to various concavo-convex structures.

In order to manufacture the nanoporous-type microelectrode chip,

Forming an adhesive layer on the substrate (step 1);

Forming an electrode layer on the adhesive layer (step 2);

Patterning the adhesive layer and the electrode layer (step 3);

Depositing an insulating layer on the substrate and the electrode layer of step 3 (step 4); And

Exposing a part of the electrode layer to the outside by patterning at least a part of the insulating layer deposited in the step 4 (step 5);

The present invention also provides a method of manufacturing a nanoporous-type microelectrode chip.

For example, FIG. 2 shows a schematic view of a method of manufacturing a nanoripoid-shaped microelectrode chip, and a method of manufacturing the nanoritic microelectrode chip according to the present invention will be described in detail.

In the method of manufacturing a nanoritual type microelectrode chip according to the present invention, step 1 is a step of forming an adhesive layer on a substrate.

The substrate may be selected from the group consisting of glass, plastic, metal, silicon, Su-8, polydimethylsiloxane (PDMS) or nitrogen silicon, The type of the substrate is not limited to this.

The adhesive layer formed on the substrate may be formed by a physical or chemical vapor deposition method such as a pulse laser deposition method, a sputtering method and a sol-gel method. The adhesive layer may be formed of platinum (Pt), palladium (Pd) ), Silver (Ag), titanium (Ti), chromium (Cr), aluminum (Al), copper (Cu), tin (Sn), molybdenum (Mo), ruthenium (Ru) And may include at least one kind selected. The adhesive layer is preferably formed within 10 nm to 500 nm. If the thickness of the adhesive layer is less than 10 nm, the bonding strength between the electrode layer and the substrate 110 may be reduced to cause a problem in stability. If the thickness of the adhesive layer exceeds 50 nm, the quality of the electrode may deteriorate.

In the method of manufacturing a nanoritual type microelectrode chip according to the present invention, step 2 is a step of forming an electrode layer on the adhesive layer.

The electrode layer formed on the adhesive layer may be at least one selected from the group consisting of Pt, Pd, Au, Ag, Ti, Cr, Al, Cu, Sn), molybdenum (Mo), ruthenium (Ru), and indium (In). The electrode layer 130 may be formed on the adhesive layer by a physical or chemical vapor deposition method such as a pulsed laser deposition method, a sputtering method, and a sol-gel method. The electrode layer may be formed within a range of 100 nm to 300 nm. When the thickness of the electrode layer is less than 100 nm, the electrode layer does not have a sufficient thickness, so that the irregular structure of the insulating layer can not be formed. When the thickness of the electrode layer is more than 300 nm, Can not be formed.

In the method of manufacturing a nanoritual type microelectrode chip according to the present invention, step 3 is a step of patterning the adhesive layer and the electrode layer.

The patterning may be performed by methods such as nanoimprint lithography, interference lithography, self-assembled copolymer pattern transfer, electron beam lithography, focused ion beam milling, photolithography, reactive ion etching, wet-etching, And may preferably be patterned by a photolithographic method, but is not limited thereto.

According to the method of step 3, the adhesive layer and the electrode layer can be patterned into a shape in which dots and lines, or a combination thereof, are arranged, and the topography Can be formed.

In the method of manufacturing a nanoritge type microelectrode chip according to the present invention, the step 4 is a step of depositing on the substrate and the electrode layer of the step 3.

In the step 3, the edge portions of the adhesive layer and the electrode layer patterned are sharpened. A chemical vapor deposition (CVD) method insulating layer is deposited to form such sharp edges as gentle edge portions. The chemical vapor deposition (CVD) method is preferably performed by a plasma enhanced chemical vapor deposition (PECVD) method. An insulating layer is deposited by a plasma enhanced chemical vapor deposition (PECVD) method, and the boundary between the protruded portion and the protruded portion of the insulating layer can be continuously formed by the plasma enhanced vapor deposition (PECVD) method.

In the method of manufacturing a nanoritge type microelectrode chip according to the present invention, step 5 is a step of exposing a part of the electrode layer to the outside by patterning at least a part of the insulating layer deposited in step 4.

The electrode layer may be formed by depositing an insulating layer on the top of the electrode layer to form a concave-convex structure, as well as to measure the electrical activity transferred to the cells formed on the insulating layer of the concave-convex structure, An electrode connected to the cell is required. Accordingly, in step 5, at least a part of the insulating layer is patterned to remove a part of the electrode layer, and the electrode layer under the removed insulating layer is exposed to the outside to form an electrode.

Further, according to the present invention,

Forming an adhesive layer on the substrate (step 1);

Forming an electrode layer on the adhesive layer (step 2);

Patterning the adhesive layer and the electrode layer (step 3);

Depositing an insulating layer on the substrate and the electrode layer of step 3 (step 4);

Exposing a part of the electrode layer to the outside by patterning at least a part of the insulating layer deposited in the step 4 (step 5); And

And coating the cell affinity polymer on the substrate of step 5 to cultivate the cell (step 6). The present invention also provides a method of growing a cell using the nanoparticle structure microelectrode chip.

In the case of the above steps 1 to 5, since they can be formed through the same process as described above, a detailed description will be omitted.

In the method of manufacturing a nanoritge type microelectrode chip according to the present invention, step 6 is a step of coating a cell affinity polymer on the substrate of step 5 to cultivate the cells.

The cell affinity polymer may be coated on the insulating layer of step 5 and the cell affinity polymer may be polydopamine, polylysine, laminin, or fibronectin, but is not limited thereto Do not.

The cells may be selected from the group consisting of microorganisms, animal and plant cells, and nerve cells, preferably neurons.

The cell affinity polymer is coated on the insulating layer of the concavo-convex structure, and cells can be cultured by the cell affinity polymer. The cells may be formed on a substrate having a topographically curved structure by an insulating layer having a concave-convex structure, whereby the electrophysiological effects of the topographic structure on the cells can be measured.

Hereinafter, the present invention will be described more specifically with reference to Examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

≪ Example 1 >

Step 1: A titanium (Ti) adhesive layer was formed on the glass substrate by evaporation deposition to a thickness of 20 nm.

Step 2: A gold (Ag) electrode layer was formed on the titanium (Ti) adhesive layer by evaporation deposition to a thickness of 200 nm.

Step 3: The adhesive layer and the electrode layer are patterned by a photolithography method in a circular shape arranged apart from each other. 16 rounds of a diameter of 30 탆 and 600 rounds of a diameter of 3 탆 were arranged as shown in Fig.

Step 4: A silicon nitride (Si ₃ N ₄ ) insulating layer was deposited on the substrate and the electrode layer by plasma enhanced chemical vapor deposition (PECVD), and the thickness was 500 nm.

Step 5: Silicon nitride (Si ₃ N ₄ ) insulating layer formed on the upper part of 16 circles having a diameter of 30 탆 formed in step 3 was removed by photolithography to expose 16 electrode layers having a diameter of 30 탆 to the outside to form electrodes Respectively.

≪ Example 2 >

In step 3, the adhesive layer and the electrode layer were coated with a circular dot having a diameter of 30 占 퐉, a thickness of 3 占 퐉 in the longitudinal direction, a length of 800 占 퐉, a line spaced 3 占 퐉 apart from each other, ), Except that the film was formed by the same method as in Example 1.

≪ Example 3 >

In step 3, the adhesive layer and the electrode layer are laminated in a circular shape having a diameter of 30 mu m, a thickness of 3 mu m in the longitudinal direction, a length of 800 mu m and a thickness of 3 mu m in the transverse direction, And patterned in a line having a length of 800 탆 and spaced apart by 3 탆 from each other.

<Example 4>

In step 3, the adhesive layer and the electrode layer are formed on four substrates by forming four layers of adhesive layer and electrode layer patterned in a dot, a line, and a combination thereof as shown in FIG. 7 and forming four kinds of topography on one substrate Was prepared in the same manner as in Example 1.

&Lt; Comparative Example 1 &

An adhesive layer, an electrode layer, and an insulating layer were not formed on the upper surface of the glass substrate, and the state of the substrate was used.

<Experimental Example 1>

In order to compare Example 1 of the present invention and Comparative Example 1, the surface of the nanoragery type microelectrode chip manufactured by the method of Example 1 was observed through a scanning electron microscope, and it is shown in FIG. The neurons cultured on the nanogranular-shaped microelectrode chip prepared by the method of Example 1 were observed through a scanning electron microscope and are shown in Figs. 12 and 14. Fig. In addition, the neurons cultured on the substrate of Comparative Example 1 were observed through a scanning electron microscope, which is shown in Fig.

FIGS. 15A and 15B are graphs showing the results of observing the characteristics of neurons cultured on the substrate prepared in Example 1 and Comparative Example 1. FIG. Fig. 15 (a) shows the number of cells per unit area grown on the substrate of Example 1 and Comparative Example 1, Fig. 15 (b) shows the percentage of surviving cells cultured on the substrate of Example 1 and Comparative Example 1 Fig.

According to the analysis results shown in FIGS. 12, 14, 15A and 15B, the nerve cells cultured on the substrate are contacted with the nanoparticle structure by the neuronal cell bodies and neuron- . In addition, the adsorption of the neuron is the adsorption of the first embodiment of the case about 400Cells / mm ² cells as compared to Comparative Example 1 about 1000Cells / mm ² of a somewhat low, but, the viability of nerve cells in the case of the first embodiment from about from about 0.45% Was about 0.1% higher than that of Comparative Example 1 by about 0.35%.

As described above, the nanoparticle-type microelectrode chip according to the present invention, the method for producing the nanoparticle-type microelectrode chip, and the cell growth method using the nanoparticle structure can morphologically observe the effect of cell growth on a specific geographical structure and can be measured electrophysiologically Can be used as biologically significant research cell chips.

110: substrate
120: adhesive layer
130: electrode layer
140: insulating layer
150: Cell-affinity polymer layer

Claims (16)

An adhesive layer formed on the substrate;
An electrode layer formed on the adhesive layer; And
And an insulating layer formed on the substrate and the electrode layer,
The adhesive layer and the electrode layer form a pattern,
At least a part of the electrode layer is exposed to the outside,
Wherein the insulating layer has a concavo-convex structure,
The concavo-convex structure corresponds to the pattern,
Wherein the protrusions and protrusions have protrusions formed on the patterns of the adhesive layer and the electrode layer, and non-protrusions are formed on portions where the adhesive layer and the electrode layer are not present.
An adhesive layer formed on the substrate;
An electrode layer formed on the adhesive layer;
An insulating layer formed on the substrate and the electrode layer; And
And a cell-affinity polymer layer coated on the insulating layer,
The adhesive layer and the electrode layer form a pattern,
At least a part of the electrode layer is exposed to the outside,
Wherein the insulating layer has a concavo-convex structure,
The concavo-convex structure corresponds to the pattern,
Wherein the protrusions and protrusions have protrusions formed on the patterns of the adhesive layer and the electrode layer, and non-protrusions are formed on portions where the adhesive layer and the electrode layer are not present.
3. The method according to claim 1 or 2,
The concavo-
And an insulating layer is deposited on the adhesive layer and the electrode layer by a chemical vapor deposition (CVD) method.
3. The method according to claim 1 or 2,
The concavo-
Wherein the electrode is formed in a shape in which dots and lines or a combination thereof are arranged.
3. The method according to claim 1 or 2,
Wherein:
Wherein the nanostructured microelectrode includes at least one selected from the group consisting of glass, plastic, metal, and silicon.
3. The method according to claim 1 or 2,
The adhesive layer
A metal such as platinum Pt, Pd, Au, Ag, Ti, Cr, Al, Cu, Sn, Mo, And at least one selected from the group consisting of ruthenium (Ru) and indium (In).
3. The method according to claim 1 or 2,
The adhesive layer
And a thickness of 10 to 50 nm.
Claim 8 has been abandoned due to the setting registration fee. 3. The method according to claim 1 or 2,
Wherein,
A metal such as platinum Pt, Pd, Au, Ag, Ti, Cr, Al, Cu, Sn, Mo, And at least one selected from the group consisting of ruthenium (Ru) and indium (In).
3. The method according to claim 1 or 2,
Wherein,
Wherein the nanostructure-structured microelectrode chip is formed to a thickness of 100 to 300 nm.
Claim 10 has been abandoned due to the setting registration fee. 3. The method according to claim 1 or 2,
Wherein the insulating layer
Silicon nitride (SiNx), silicon oxide (SiO _2), aluminum oxide (Al ₂ O _3), BZN oxide (Bismuth-Zinc-Niobium Oxide) , titanium, hafnium oxide, zirconium oxide, the group consisting of tantalum oxide and the oxide is thallium Wherein the nanoparticle-structured microelectrode chip comprises at least one selected from the group consisting of nanoparticles and nanoparticles.
3. The method according to claim 1 or 2,
Wherein the insulating layer
Wherein the nanostructured microelectrode chip is formed to a thickness of 200 to 1000 nm.
3. The method of claim 2,
Wherein the cell affinity polymer layer comprises
A nanoparticle structure microelectrode chip comprising at least one selected from the group consisting of polydopamine, polylysine, laminin, and fibronectin.
Forming an adhesive layer on the substrate (step 1);
Forming an electrode layer on the adhesive layer (step 2);
Patterning the adhesive layer and the electrode layer (step 3);
Depositing an insulating layer on the substrate of the step 3 by a plasma vapor deposition method to form an insulating layer having a concavo-convex structure on the surface, wherein the adhesive layer and the electrode layer form protrusions on the patterned portion, (Step 4); And
And patterning at least a part of the insulating layer deposited in step 4 to expose a part of the electrode layer to the outside (step 5).
Forming an adhesive layer on the substrate (step 1);
Forming an electrode layer on the adhesive layer (step 2);
Patterning the adhesive layer and the electrode layer (step 3);
Depositing an insulating layer on the substrate of the step 3 by a plasma vapor deposition method to form an insulating layer having a concavo-convex structure on the surface, wherein the adhesive layer and the electrode layer form protrusions on the patterned portion, (Step 4);
Exposing a part of the electrode layer to the outside by patterning at least a part of the insulating layer deposited in the step 4 (step 5);
And coating the cell-affinity polymer on the substrate of step 5 to cultivate the cell (step 6).
15. The method of claim 14,
Preferably,
Wherein the nanoparticle-type microelectrode chip comprises at least one selected from the group consisting of microorganisms, animal and plant cells, and nerve cells.
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