WO2018186683A1 - Biosensor substrate, method for producing same, and biosensor comprising same - Google Patents

Abstract

The present invention relates to a biosensor substrate, a method for producing same, and a biosensor comprising same.

Description

Biosensor substrate, manufacturing method thereof and biosensor comprising same

The present invention relates to a biosensor substrate and a method of manufacturing the same used for manufacturing a multi-diagnosis biosensor.

To date, various bio / chemical sensors have been developed, among which biosensors (eg PCR, diagnostic kits, etc.) that detect hazards in the healthcare diagnostic industry show excellent results in accuracy and precision. It is widely used for industrial purposes.

The substrate (platform) of the biosensor is made of a material such as glass, silicon, or polymer, and the material of the substrate is determined according to the use of the biosensor. For example, biosensors such as real-time PCs (RT-PCR) and biochips are made of glass or silicon substrates.

The measurement of these biosensors is based on hydrodynamics, so the performance of the biosensors varies markedly with the surface properties of the substrate. For example, biochips have different flow rates depending on whether their surfaces are hydrophilic or hydrophobic, resulting in differences in reaction rates, which have a significant effect on the response time and sensitivity of the biosensor. Therefore, in order to improve the performance of the biosensor, it is important to consider what kind of surface treatment substrate is used.

However, there is a limitation in obtaining a multi-diagnosis biosensor as the current state of the technology development of selective surface functionalization of the substrate is insufficient in manufacturing a biosensor substrate. In addition, as a complex post-treatment process is required to attach a bioprobe (antigen, aptamer, protein, etc.) for detecting a target substance to a substrate, there is a problem in that manufacturing efficiency of the biosensor is deteriorated.

In order to solve the above problems, an object of the present invention is to provide a biosensor substrate that can efficiently provide multiple diagnostic biosensors.

Another object of the present invention is to provide a method for manufacturing the biosensor substrate.

Another object of the present invention is to provide a biosensor comprising the biosensor substrate.

Another object of the present invention is to provide a method of manufacturing the biosensor.

In order to solve the above problems, the present invention, the substrate portion containing a polymer; And a modification unit coupled to the surface of the substrate unit and including a light sensitive derivative, wherein the light sensitive derivative is a norbornadiene-based derivative.

The norbornadiene derivative may have a structure represented by Formula 1 below.

[Formula 1]

The polymer is polystyrene (PS), polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polyester (PE), polycarbonate (PC), polyimide (PI), polyurethane (PU), Polyvinylidene fluoride (PVDF), polyamide (PA), polyethersulfone (PES), polytetrafluoroethylene (PTFE) and polymethyl methacrylate (PMMA) Can be.

The modified part may be coupled to a plurality of surfaces of the substrate part.

The present invention, a) preparing a substrate portion containing a polymer; b) reacting the prepared substrate part with a catecholamine-based compound; c) introducing a amine-containing compound in which a norbornadiene derivative is bonded to a molecular terminal to a substrate portion reacted with the catecholamine-based compound to form a modified portion. .

The catecholamine-based compound may be dopamine.

The amine-containing compound may be a compound represented by the following formula (2).

[Formula 2]

In Formula 2 n is an integer of 100 to 1,000,000.

C), c-1) synthesizing the amine-containing compound by reacting the norbornadiene-based compound with the amine-containing linker compound; And c-2) reacting the synthesized amine-containing compound with the substrate portion.

In addition, the step c), c-1) reacting the substrate portion with an amine-containing linker compound; And c-2) reacting the substrate portion reacted with the amine-containing linker compound with the norbornadiene-based compound.

The norbornadiene-based compound may be a compound represented by Formula 3 below.

[Formula 3]

The amine-containing linker compound includes polyethyleneimine, tris (2-aminoethyl) amine and 2,2 '-(ethylenedioxy) bis (ethylamine) (2,2' -(ethylenedioxy) bis (ethylamine)) may be one or more selected from the group consisting of.

The present invention, the biosensor substrate; And a bio probe unit coupled to a reforming unit of the bio sensor substrate.

The bioprobe unit may include a plurality, and the plurality of bioprobes may probe different target materials from each other.

The present invention, A) preparing the biosensor substrate; B) masking a portion of the biosensor substrate and irradiating light to form an activated reformed portion and an inactivated modified portion; C) binding a bioprobe to the activated reforming unit; D) activating the deactivated reformate; And E) combining the bioprobe unit that probes a different target material with the bioprobe unit coupled in step C) to the reformer activated in step D).

The activation of the reformed portion deactivated in step D) may be performed by heat treatment or reaction with a transition metal.

Since the biosensor substrate according to the present invention includes a reforming unit selectively activated or deactivated by light, the biosensor substrate may efficiently provide multiple diagnostic biosensors when the biosensor is manufactured using the biosensor substrate.

1 and 2 are reference diagrams for explaining the biosensor substrate of the present invention.

3 is a reference diagram for explaining a method of manufacturing the biosensor substrate of the present invention.

4 is a reference diagram for explaining the biosensor of the present invention.

5 is a reference diagram for explaining a method of manufacturing a biosensor according to the present invention.

6 to 8 are reference diagrams for explaining Experimental Examples 1 to 3 of the present invention, respectively.

<Description of the code>

10: substrate portion

20, 20a, 20b: reforming part

100: biosensor substrate

200, 200a, 200b: bio probe part

Hereinafter, the present invention will be described.

In the present invention, in the modification of the surface of the biosensor substrate, which is the base substrate of the biosensor, unlike the conventional method (for example, plasma treatment) in which the surface is modified in whole or in a batch, the surface is selectively modified so that various bio Characterized in that it can be combined with the probe, it will be described in detail with reference to the drawings as follows.

1. Biosensor Substrate

Referring to FIG. 1, the biosensor substrate of the present invention includes a substrate portion 10 and a reforming portion 20.

The substrate unit 10 included in the biosensor substrate of the present invention serves as a base of the biosensor substrate, and may include a polymer.

Specifically, the substrate unit 10 may be made of polystyrene (PS), polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polyester (PE), polycarbonate (PC), polyimide (PI), poly 1 selected from the group consisting of urethane (PU), polyvinylidene fluoride (PVDF), polyamide (PA), polyethersulfone (PES), polytetrafluoroethylene (PTFE) and polymethyl methacrylate (PMMA) It may consist of more than one species of polymer.

The reforming unit 20 included in the biosensor substrate of the present invention is present in combination with the surface of the substrate unit 10. The reforming unit 20 is formed by undergoing a surface modification process of the substrate unit 10 in the manufacturing process of the biosensor substrate, and includes a light-sensitive derivative.

The light-sensitive derivative is a norbornadiene-based derivative, and may be a norbornadiene-based derivative including a norbornadiene group. The modifying unit 20 may be selectively activated or deactivated when light (for example, ultraviolet rays) is irradiated by the norbornadiene-based derivative.

The norbornadiene-based derivative is not particularly limited, but preferably has a structure represented by the following formula (1). This is because the norbornadiene-based derivative has a structure represented by the following Chemical Formula 1, which is excellent in reactivity with light and can easily induce activation and deactivation of the reforming unit 20 according to specific conditions.

[Formula 1]

The norbornadiene-based derivative may be bonded to the substrate portion 10 by a linker including a structure in which a polymer (eg, polydopamine) and an amine-containing linker compound of the catecholamine-based compound are bonded. That is, the reforming portion 20 is composed of a linker and a norbonadiene derivative, the polymer of the catecholamine-based compound included in the linker is bonded to the surface of the substrate portion 10, the amine-containing linker compound included in the linker Norbornadiene-based derivative (* in the structure of Formula 1 means a position bonded to the molecular terminal of the amine-containing linker compound) is bonded to the molecular terminal of the modified portion 20 is the surface of the substrate portion 10 Can be fixed.

Meanwhile, as illustrated in FIG. 2, the modifying unit 20 may be coupled to the surface of the substrate unit 10 in plurality.

2. Manufacturing Method of Biosensor Substrate

The present invention provides a method of manufacturing the above-described biosensor substrate, which will be described in detail with reference to FIG. 3 as follows.

a) Preparation of the board | substrate part 10

First, the substrate unit 10 including the polymer is prepared. Preparation of the substrate portion 10 may be made by a conventionally known method.

b) reaction with catecholamine compounds

The prepared substrate portion 10 is reacted with a catecholamine compound (first linker compound). Specifically, the substrate 10 is immersed in a salt mixed catecholamine-based compound (eg, dopamine hydrochloride) and a buffer solution (eg, Tri-HCl, etc.) for a predetermined time. By coupling a portion (L ₁ ) of the linker capable of fixing the derivative of the light-sensitive compound on the surface of the substrate portion 10.

The catecholamine-based compound is not particularly limited, but is preferably dopamine.

The reaction between the substrate unit 10 and the catecholamine-based compound may be performed at room temperature, and the reaction time is not particularly limited, but may be 2 to 4 hours.

c) formation of the reforming portion 20

The modified portion 20 is formed by introducing an amine-containing compound in which at least one norbornadiene-based derivative is bonded to a molecule terminal to the substrate portion 10 reacted with the catecholamine-based compound.

Formation of the modified portion 20 is a reaction of the amine-containing compound in which at least one norbornadiene-based derivative is bonded at the molecular end directly with the substrate portion 10, or the amine-containing linker compound (second linker compound) The bonadiene compound may be formed by sequentially reacting with the substrate portion 10.

Specifically, after reacting the norbornadiene compound and the amine-containing linker compound to synthesize the amine-containing compound, the modified amine-containing compound is reacted with the substrate portion 10 reacted with the catecholamine-based compound to modify the modified portion 20 Alternatively, the substrate 10 reacted with the catecholamine-based compound may be reacted with the amine-containing linker compound to form the remainder of the linker, and then the reacted portion 20 may be formed with the norbornadiene-based compound. .

The linker compound refers to a reactant used to form a linker that serves as a bridge to connect (bond) the norbornadiene compound to the reforming unit 20.

The norbornadiene-based compound may include a norbornadiene group.

The norbornadiene-based compound is not particularly limited, but is preferably a compound represented by the following formula (3).

[Formula 3]

The amine-containing linker compound is not particularly limited, polyethyleneimine (M _w : 100,000 or less), Tris (2-aminoethyl) amine (M _w : 800 or less) and 2,2 '-(ethylenedioxy) bis (ethylamine) (2,2'- (ethylenedioxy) bis (ethylamine)) (M _w : 25,000 or less) is preferably one or more selected from the group consisting of, and more preferably polyethyleneimine. Specifically, the amine-containing compound is not particularly limited, but is preferably a compound represented by the following formula (2).

[Formula 2]

In Formula 2, n is an integer of 100 to 1,000,000.

3. biosensor

The present invention provides a biosensor capable of probing (detecting) various bio target materials, which will be described in detail with reference to FIG. 4.

The biosensor of the present invention includes a biosensor substrate 100 and a bioprobe unit 200.

The biosensor substrate 100 included in the biosensor of the present invention serves as a base substrate of the biosensor. It is the same as that described in "Bio-Sensor Substrate" and will be omitted.

The bioprobe 200 included in the biosensor of the present invention is to be coupled to the modified portion 20 of the biosensor substrate 100 (specifically, to the norbornadiene derivative of the modified portion 20), Probe and detect bio targets (eg, target nucleic acids, blood glucose, glycated proteins, etc.). The bioprobe 200 may be a functional group (eg, -S-, etc.) coupled with the reforming unit 20 of the biosensor substrate 100 and a reactor (eg, an antigen) capable of binding to a biotarget material. , Aptamer, protein, etc.) is not particularly limited.

Meanwhile, when there are a plurality of reformers 20 on the biosensor substrate 100, a plurality of bioprobes 200 included in the biosensor may also be provided. In this case, the plurality of bio probes may probe different bio target materials from each other, and accordingly, the present invention may provide a multi-diagnosis bio sensor.

4. Manufacturing method of bio sensor

The present invention provides a method of manufacturing the above-described biosensor, which will be described in detail with reference to FIG. 5 as follows.

A) Preparation of the biosensor substrate 100

First, the biosensor substrate 100 described above is prepared.

B) Formation of Activated Reform 20a and Deactivated Reform 20b

A portion of the prepared biosensor substrate 100 is masked and irradiated with light (for example, ultraviolet rays) to form the activated reformed portion 20a and the inactivated modified portion 20b.

Specifically, after selecting the region to which the bio probe unit 200 is to be coupled in the biosensor substrate 100, the mask is placed on the selected region and irradiated with light, and the modified portion 20a of the selected region is maintained in an active state. In addition, the reformed portion 20b of the non-selected region may be placed in an inactive state by the reaction of the light-sensitive derivative with light to form the activated reformed portion 20a and the inactivated modified portion 20b.

C) bonding of the bioprobe unit 200a

The bio probe 200a is coupled to the activated reforming unit 20a. The bio probe 200a may be combined by a conventionally known method (for example, bio-thiolation).

D) Activation of the disabled reformer 20b

After combining the bioprobe 200a, the deactivated reforming unit 20b is activated. The method of activating the deactivated reforming unit 20b is not particularly limited, but may be performed by heat treatment or reaction with a transition metal.

The heat treatment condition of the reforming unit 20b is not particularly limited, but may be performed at 50 to 80 ° C. for 12 to 24 hours.

Although the reaction between the modified part 20b and the transition metal is not particularly limited, the biosensor substrate 100 is immersed in a solution containing silver (Ag), cobalt (Co), or tin (Sn) and reacted for 10 to 14 hours. It can be made to.

E) binding of the bioprobe unit 200b

The bio-probe 200b for probing different bio target materials from the bio-probe 200a coupled in the step C) is coupled to the reformed portion 20b activated through the step D).

Specifically, the reforming unit 20a to which the bio probe unit 200a is coupled, and a reforming unit to couple the new bio probe unit 200b to probe a different bio target material from the bio probe unit 200a to which the bio probe unit 200a is coupled ( 20b) masking the region and irradiating light (for example, ultraviolet rays) to deactivate the unmasked region, and then combining the new bioprobe 200b to process the bio-bonded in step C). The bio probe 200b may be combined with the probe 200a to probe different bio target materials.

Combination of the bioprobe 200a and the bioprobe 200b for probing a different bio target material may be generally performed by a known method (eg, bio-thiolation).

According to the present invention, a biosensor substrate and a biosensor using a light sensitive compound capable of reversible reaction which is inactivated when irradiated with light and activated by specific conditions (eg, heat treatment, reaction with a transition metal, etc.) Since the manufacturing of the biosensor to include can increase the manufacturing efficiency of the multi-diagnostic biosensor.

Specifically, in the present invention, in irradiating light to a biosensor substrate including a modified portion combined with a light-sensitive derivative, the biosensor substrate is selectively activated or deactivated by irradiating light for each desired region. As the biosensor is manufactured by combining various bioprobes in the region, multiple diagnostic biosensors may be efficiently manufactured.

In addition, the present invention can easily induce the activation and deactivation of the biosensor substrate (region-by-region) using a mask, it is possible to easily manufacture a biosensor having a micro-miniature pattern.

Hereinafter, the present invention will be described in detail with reference to the following Examples. However, the following Examples are merely to illustrate the present invention, the present invention is not limited by the following Examples.

[Raw materials and devices]

Dicyclopentadiene, 2-butynedioic acid, 1,4-dioxane, N, N'-dicyclohexylcarbodiimide (N, N ') -Dicyclohexylcarbodiimide) were each purchased from Aldrich and used without purification. A JEOL 3700 was used as an instrument.

Synthesis Example 1 Synthesis of Norbornadiene-2,3-dicarboxylic Acid

Dicyclopentadiene (7 mL, 52 mmol) was distilled off to obtain monomeric cyclopentadiene (monomeric cyclopentadiene). The resulting monomeric cyclopentadiene (2.0 g, 30 mmol) was mixed with 1,4-dionic acid (20 mL) and 2-butyndioic acid (3.0 g, 26.3 mmol) under an ice-water bath. To the solution. Next, the solution containing monomeric cyclopentadiene was stirred at room temperature overnight, and then hexane (5 mL) was added and collected to obtain a solid precipitate (4.18 g, yield: 88%).

Synthesis Example 2 Synthesis of 2,5-norbornadiene-2,3-dicarboxylic acid anhydride (2,5-Norbornadiene-2,3-dicarboxylic acid anhydride)

The mixture of norbornadiene-2,3-dicarboxylic acid (500 mg, 2.78 mmol, 1.0 eq) and acetone (28 mL) synthesized in Synthesis Example 1 was mixed with N, N'-dicyclohexylcarbodiimide (573). mg, 2.78 mmol, 1.0 eq) was added to the solution. Then, the solution to which the mixture was added was stirred at room temperature overnight, and then, the product was obtained by filtration and evaporating process. The obtained product was purified by short column chromatography to give the purified product (215 mg, yield: 43%).

¹ H-NMR (CDCl ₃ ): d 2.71 (s, 2H), 3.99 (s, 2H), 6.98 (s, 2H).

Synthesis Example 3 Synthesis of Amine-Containing Compound

2,5-norbornadiene-2,3-dicarboxylic anhydride (100 mg) and Polyethyleneimine (300 mg) synthesized in Synthesis Example 2 were dissolved in dimethylformamide (10 ml), mixed and mixed at room temperature for 1 hour. Norbonadiene-containing solution was prepared by agitation. Next, dimethylformamide was removed from the solution by rotary evaporation and dried in vacuo without further purification to obtain an amine-containing compound (yield: 100%).

Example 1

The solution was prepared by dissolving dopamine hydrochloride (2 mg / ml) in 10 mM Tri-HCl buffer solution (pH 8.5). After immersing the polystyrene substrate (polystyrene film) in the prepared solution and shaking for 3 hours at room temperature to obtain a substrate functionalized with polydopamine.

The substrate functionalized with polydopamine was then washed with deionized water and dried with nitrogen gas.

Subsequently, the substrate functionalized with polydopamine was immersed in a mixture of the amine-containing compound (100 mg) synthesized in Synthesis Example 3 and a 10 mM Tri-HCl buffer solution (pH 8.5, 20 ml), and then kept at room temperature overnight. Shaking. The amine containing compound was then introduced to the substrate functionalized with polydopamine by washing with deionized water and drying with nitrogen gas.

Example 2

An amine-containing compound was introduced into a polydopamine-functionalized substrate in the same manner as in Example 1 except that a substrate made of polypropylene was applied instead of a substrate made of polystyrene.

Example 3

An amine-containing compound was introduced into a polydopamine functionalized substrate in the same manner as in Example 1 except that a substrate made of polyvinyl chloride was applied instead of a substrate made of polystyrene.

Example 4

The solution was prepared by dissolving dopamine hydrochloride (2 mg / ml) in 10 mM Tri-HCl buffer solution (pH 8.5). After immersing the polystyrene substrate (polystyrene film) in the prepared solution and shaking for 3 hours at room temperature to obtain a substrate functionalized with polydopamine. The substrate functionalized with polydopamine was then washed with deionized water and dried with nitrogen gas.

Next, a polydopamine-functionalized substrate was immersed in a solution mixed with polyethyleneimine (1 g) and 10 mM Tri-HCl buffer solution (pH 8.5, 20 ml), and shaken overnight at room temperature. . Subsequently, the substrate was washed with deionized water and dried with nitrogen gas to introduce polyethyleneimine into the substrate functionalized with polydopamine.

Next, polyethyleneimine was introduced into a solution obtained by dissolving 2,5-norbornadiene-2,3-dicarboxylic anhydride (100 mg) synthesized in Synthesis Example 2 in dimethylformamide (10 ml). The substrate was immersed and shaken overnight at room temperature.

Subsequently, the substrate was washed with dimethylformamide and deionized water and dried with nitrogen gas to introduce an amine containing compound into the substrate functionalized with polydopamine.

[Production Example 1]

Part of the surface of the polystyrene substrate of Example 1 in which the amine containing compound was introduced was covered with a hand-made mask. Next, the surface of the substrate was irradiated with ultraviolet light (≤300 nm) to inactivate the surface of the substrate not covered with a mask (quadricyclane form) so that a thiolation reaction did not occur (steps A and B).

Next, only a few surfaces in 10 μl solution containing fluorescence labeled thiol-terminal bioprobe (peptide (HS-CDMSPPWHK-K-FITC, Lusen Sci.,)) The activated substrate was immersed and reacted for 12 hours, followed by washing with deionized water (C).

Subsequently, the substrate reacted with thiol-terminal bioprobe was immersed in a mixed solution of AgClO ₄ and methanol and reacted for 12 hours to activate the surface of the substrate which was inactivated (step D).

Thereafter, steps A) to D) were repeated to prepare a biosensor in which another thiol-terminal bioprobe (thiol-terminal bioprobe) (HS-TATCAGTTCTTTGACCTTTGTCA-FAM-3 ', Bioneer) was combined.

[Production Example 2]

A biosensor was manufactured in the same manner as in Preparation Example 1, except that the polypropylene substrate of Example 2 was applied instead of the polystyrene substrate of Example 1.

[Production Example 3]

A biosensor was manufactured in the same manner as in Preparation Example 1, except that the polyvinyl chloride substrate of Example 3 was applied instead of the polystyrene substrate of Example 1.

Experimental Example 1

Emission of the fluorescent label at the end of the thiol-terminal bioprobe to confirm whether the thiol-terminal bioprobe is bound to the polypropylene substrate prepared in Preparation Example 2 Was confirmed by fluorescence microscopy (EVOS FL cell imaging system, Thermo Fisher scientific) step by step, the results are shown in FIG.

Referring to Figure 6, it can be seen that different thiol-terminal bioprobe (thiol-terminal bioprobe) is well coupled to the polypropylene substrate.

Experimental Example 2

In order to check whether the thiol-terminal bioprobe (peptide (HS-CDMSPPWHK-K-FITC, Lusen Sci.,)) Is bound to the polypropylene substrate obtained by proceeding to step C) in Preparation Example 2 Fluorescence was measured using a plate reader, and the results are shown in Fig. 7. At this time, the polypropylene substrate of Example 2 before the thiol-terminal bioprobe was combined was compared.

Referring to FIG. 7, it can be seen that the FITC absorbance appears on a polypropylene substrate having a thiol-terminal bioprobe. This supports the incorporation of thiol-terminal bioprobes to polypropylene substrates incorporating polydopamine and amine containing compounds.

[Production Example 4]

A bioprobe was prepared by combining a bioprobe in which the S. aureus electric field dielectric having -SH is bonded to the polypropylene substrate (PP) of Example 2 in a conventional manner.

Production Example 5

A biosensor was prepared by combining a bioprobe in which the S. aureus electric field dielectric having -SH is bonded to the polyvinyl chloride substrate (PVC) of Example 3 in a conventional manner.

Experimental Example 3

Using the biosensors prepared in Preparation Example 3 and Preparation Example 4 was carried out the nucleic acid isothermal amplification experiment in a conventional manner, the results are shown in FIG.

Referring to Figure 8, it can be seen that each substrate is showing a positive result for the S. aureus bacteria.

Claims (15)

1. A substrate portion including a polymer; And

   It is coupled to the surface of the substrate portion, including a modified portion containing a light-sensitive derivative;

   The light-sensitive derivative is a biosensor substrate that is a norbornadiene (Norbornadiene) derivative.
2. The method according to claim 1,

   The norbonadiene-based derivative is a biosensor substrate having a structure represented by the following formula (1).

   [Formula 1]

   
3. The method according to claim 1,

   The polymer is polystyrene (PS), polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polyester (PE), polycarbonate (PC), polyimide (PI), polyurethane (PU), At least one selected from the group consisting of polyvinylidene fluoride (PVDF), polyamide (PA), polyethersulfone (PES), polytetrafluoroethylene (PTFE) and polymethyl methacrylate (PMMA) A biosensor substrate.
4. The method according to claim 1,

   The biosensor substrate is coupled to a plurality of the modified portion on the surface of the substrate portion.
5. a) preparing a substrate part including a polymer;

   b) reacting the prepared substrate part with a catecholamine-based compound;

   c) introducing a amine-containing compound in which a norbornadiene derivative is bonded to a molecular terminal to a substrate portion reacted with the catecholamine compound to form a modified portion.
6. The method according to claim 5,

   The catecholamine compound is a dopamine manufacturing method of the biosensor substrate.
7. The method according to claim 5,

   Method for producing a biosensor substrate, wherein the amine-containing compound is a compound represented by the following formula (2).

   [Formula 2]

   

   In Formula 2 n is an integer of 100 to 1,000,000.
8. The method according to claim 5,

   C),

   c-1) synthesizing the amine-containing compound by reacting the norbornadiene compound with the amine-containing linker compound; And

   c-2) reacting the synthesized amine-containing compound with the substrate portion.
9. The method according to claim 5,

   C),

   c-1) reacting the substrate portion with an amine-containing linker compound; And

   c-2) reacting the substrate portion reacted with the amine-containing linker compound with the norbornadiene-based compound.
10. The method according to claim 8 or 9,

    Method of producing a biosensor substrate, wherein the norbornadiene compound is a compound represented by the following formula (3).

    [Formula 3]

    
11. The method according to claim 8 or 9,

    The amine-containing linker compound includes polyethyleneimine, tris (2-aminoethyl) amine and 2,2 '-(ethylenedioxy) bis (ethylamine) (2,2' -(ethylenedioxy) bis (ethylamine)) is a method for producing a biosensor substrate that is at least one selected from the group consisting of.
12. A biosensor substrate according to any one of claims 1 to 4; And

    And a bioprobe unit coupled to the reforming unit of the biosensor substrate.
13. The method according to claim 12,

    It includes a plurality of bio probes,

    The plurality of bio probes will probe different target materials from each other.
14. A) preparing a biosensor substrate according to any one of claims 1 to 4;

    B) masking a portion of the biosensor substrate and irradiating light to form an activated reformed portion and an inactivated modified portion;

    C) binding a bioprobe to the activated reforming unit;

    D) activating the deactivated reformate; And

    E) combining the bio-probe to probe the target material different from the bio-probe unit coupled in the step C) to the reformer activated in the step D).
15. The method according to claim 14,

    Activation of the reformed portion deactivated in the step D) is a method of manufacturing a biosensor made by a heat treatment or a reaction with a transition metal.

Images