WO2018186682A1 - 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.

The present invention to solve the above problems, the substrate portion; 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 substrate portion is made of glass, silicon and quartz It may include one or more selected from the group consisting of.

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

The present invention, a) preparing a substrate portion; b) reacting the prepared substrate portion with an amine-containing alkoxysilane-based compound; And c) introducing a norbornadiene compound to a substrate portion reacted with the amine-containing alkoxysilane compound to form a modified portion.

The amine-containing alkoxysilane compound is selected from the group consisting of (3-aminopropyl) trimethoxysilane and (3-aminopropyl) triethoxysilane ((3-Aminopropyl) triethoxysilane) It may be one or more.

The norbornadiene-based compound may be a compound represented by Formula 2 below.

[Formula 2]

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 is a reference diagram for explaining an experimental example 1 of the present invention.

7 is a reference diagram for explaining an experimental example 2 of the present invention.

<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 be made of a material known in the art. Specifically, the substrate portion 10 may be made of one or more selected from the group consisting of glass, silicon, and quartz.

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 modification 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 compound represented by the following Chemical Formula 1 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 coupled to the substrate portion 10 by a linker including an amine group on one side and a hydrophilic group on the other side. That is, the reforming unit 20 is composed of a linker and a norbonadiene-based derivative, the hydrophilic group of the linker is bonded to the surface of the substrate portion 10, the norbornadiene-based derivative is bonded to the amine group side of the linker is modified The portion 20 may be fixed to the surface of the substrate portion 10.

Specifically, the linker may have a structure including a repeating unit represented by Formula 3 below.

[Formula 3]

In Formula 3 n is an integer of 100 to 1,000,000.

For reference, in Formulas 2 and 3, * means a site where Formula 2 and Formula 3 are bonded to each other.

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 board | substrate part 10 is prepared. Preparation of the substrate portion 10 may be made by a conventionally known method.

b) reaction with amine-containing alkoxysilane-based compounds

The prepared substrate portion 10 is reacted with an amine-containing alkoxysilane compound. Specifically, the substrate portion 10 is immersed in a solution in which an amine-containing alkoxysilane compound and an organic solvent (for example, benzene, toluene, xylene, etc.) are mixed and reacted for a predetermined time to the surface of the substrate portion 10. Linkers capable of immobilizing derivatives of light-sensitive compounds are combined.

Although the amine-containing alkoxysilane-based compound is not particularly limited, (3-aminopropyl) trimethoxysilane and (3-aminopropyl) triethoxysilane ((3-Aminopropyl) triethoxysilane) It is preferably at least one compound selected from the group consisting of.

The reaction between the substrate portion 10 and the amine-containing alkoxysilane-based compound may be performed in the presence of an inert gas (eg, nitrogen or argon), and the reaction time may be 3 to 9 hours, although not particularly limited.

c) formation of the reforming portion 20

A norbornadiene-based compound is introduced into the substrate 10 reacted with the amine-containing alkoxysilane-based compound to form a reformed portion 20. Specifically, the substrate portion 10 reacted with the amine-containing alkoxysilane-based compound is subjected to a norbornenadiene-based compound, which is a light-sensitive compound, and an organic solvent (for example, dimethylformamide, dimethylacetamide, tetrahydrofuran, N- Methyl-2-pyrrolidone and the like) are immersed in the mixed solution and reacted for a predetermined time to fix the derivative of the norbornadiene compound on the linker bonded to the surface of the substrate portion 10.

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 (2).

[Formula 2]

The reaction time of the substrate unit 10 and the norbornadiene compound is not particularly limited, but may be 10 to 15 hours.

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 70 to 90 ° C. for 15 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).

By repeating the above step E), the present invention can produce a biosensor capable of probing and detecting various bio target materials.

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) and 3- (aminopropyl) trimethoxysilane (97%) 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).

Example 1

The glass substrate was immersed in a solution of toluene (20 ml) and 3- (aminopropyl) trimethoxysilane (10 µl), and shaken for 6 hours in a nitrogen atmosphere to give amine-functionalized glass. A substrate was obtained. Next, the glass substrate functionalized with amine was washed with toluene, and unreacted 3- (aminopropyl) trimethoxysilane was removed with a mixture of toluene and methanol in a volume ratio of 1: 1.

2.5-norbornadiene-2,3-dicarboxylic anhydride obtained in Synthesis Example 2 was dissolved in dimethylformamide. Next, the amine-functionalized glass substrate is immersed in a solution in which 2.5-norbornadiene-2,3-dicarboxylic acid anhydride is dissolved, and shaken for 12 hours to give 2.5-norbornadiene- to the amine-functionalized glass substrate. 2,3-dicarboxylic anhydride was introduced.

[Production Example 1]

Part of the surface of the glass substrate of Example 1 into which 2.5-norbornadiene-2,3-dicarboxylic anhydride was introduced was covered with a hand-made mask. Next, the surface of the glass substrate was irradiated with ultraviolet rays (= 300 nm) to inactivate the surface of the glass substrate not covered with a mask (quadricyclane form) so that a thiolation reaction did not occur (steps A and B)). .

Next, 10 μl of solution containing fluorescence labeled thiol-terminal bioprobe (peptide (HS-CDMSPPWHK-K-FITC, Lusen Sci.,)) And 1 ml of dimethyl After immersing the glass substrate on which only part of the surface was activated in the mixed solution of formamide and reacting for 12 hours, the mixture was washed with dimethylformamide and deionized water (C).

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

Thereafter, steps A) to D) were repeated to prepare another thiol-terminal bioprobe (thiol-terminal bioprobe) (Aptamer (HS-TATCAGTTCTTTGACCTTTGTCA-FAM-3 ', Bioneer)).

Experimental Example 1 Verification of Activation and Deactivation of Substrate

Part of the surface of the glass substrate of Example 1 into which 2.5-norbornadiene-2,3-dicarboxylic anhydride was introduced was covered with a hand-made mask. Next, the surface of the glass substrate was irradiated with ultraviolet light (= 300 nm) to inactivate the surface of the glass substrate not covered with a mask (quadricyclane form) so that a thiolation reaction did not occur.

Next, a glass substrate having an activation region and an inactivation region was immersed in a solution in which 4-bromobenzenethiol (5 mg) was dissolved in dimethylformamide (1 mL) and reacted at room temperature for 12 hours. The glass substrate was washed with dimethylformamide and deionized water.

The cleaned glass substrate was analyzed by x-ray photoelectron spectroscopy, and the results are shown in FIG. 6.

Referring to FIG. 6, it can be seen that in the region covered with the mask and maintained in the activated state, about 6 times more bromine is present than the inactivated region. This point is supported by the introduction of the light-sensitive compound 2,5-norbornadiene-2,3-dicarboxylic acid anhydride into the glass substrate to form an activation region and an inactivation region.

Experimental Example 2

In order to confirm that different types of thiol-terminal bioprobes were combined, the biosensor prepared in Preparation Example 1 was analyzed by a fluorescence microscope (EVOS® FL Cell Imaging System, ThermoFisher SCIENTIFIC). Is shown in FIG. 7.

Referring to Figure 7, it can be seen that different types of thiol-terminal bioprobe (thiol-terminal bioprobe) is combined.

Claims (11)

1. A substrate portion; 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 substrate portion is made of glass, silicon and quartz Biosensor substrate comprising one or more selected from the group consisting of.
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 portion;

   b) reacting the prepared substrate portion with an amine-containing alkoxysilane-based compound; And

   c) introducing a norbornadiene compound to a substrate portion reacted with the amine-containing alkoxysilane-based compound to form a reformed portion.
6. The method according to claim 5,

   The amine-containing alkoxysilane compound is selected from the group consisting of (3-aminopropyl) trimethoxysilane and (3-aminopropyl) triethoxysilane ((3-Aminopropyl) triethoxysilane) The manufacturing method of the biosensor substrate which is 1 or more types.
7. The method according to claim 5,

   Method of producing a biosensor substrate, wherein the norbornadiene compound is a compound represented by the following formula (2).

   [Formula 2]

   
8. 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.
9. The method according to claim 8,

   It includes a plurality of bio probes,

   The plurality of bio probes will probe different target materials from each other.
10. 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).
11. The method according to claim 10,

    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.

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