Biosensor and Preparation Method Thereof

Abstract

A biosensor with improved detection sensitivity of a target substance, a method for preparing a biosensor with improved detection sensitivity, and a method for detecting a target substance using the biosensor.

Claims

1. A biosensor comprising: a base material; a support layer formed on a first side of the base material; and a first antibody immobilized in the support layer, wherein the support layer formed on the first side of the base material is a self-assembled monolayer formed by a vapor deposition method.

2. The biosensor according to claim 1, wherein an absorbance of the biosensor is greater than or equal to 0.15 at a maximum absorption wavelength measured using a UV-Vis spectrophotometer after contacting 100 μL of a solution containing 20 ng/mL to 40 ng/mL of a target substance with the support layer in which the first antibody is immobilized, and then contacting a second antibody capable of binding to the target substance and bound by an enzyme reacting with TMB (3,3′,5,5′-tetramethylbenzidine).

3. The biosensor according to claim 2, wherein the biosensor is configured to detect the target substance at a concentration in a range of 0.6 ng/mL to 1.0 ng/mL.

4. The biosensor according to claim 1, wherein the base material is a polymer substrate, a metal substrate, a metal oxide substrate or a glass substrate.

5. The biosensor according to claim 1, wherein a forming material of the support layer formed on the first side of the base material is a compound represented by:
A(R.sub.1).sub.p(OR.sub.2).sub.q(R.sub.3NH.sub.2).sub.r wherein, A is a carbon atom, a silicon atom or a germanium atom, R.sub.1 is a first alkyl group having 1 to 5 carbon atoms, R.sub.2 is second alkyl group having 1 to 5 carbon atoms, R.sub.3 is a third alkylene group having 1 to 12 carbon atoms or a phenylene group, p is an integer between 0 to 2, r is a natural number between 1 to 3, q is a natural number between 1 to 3, and a sum of p, q and r equals 4.

6. The biosensor according to claim 5, wherein the compound represented by Formula 1 above is (4-aminobutyl)triethoxysilane, (4-aminobutyl)trimethoxysilane, aminophenyltriethoxysilane, aminophenyltrimethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxy silane or (3-aminopropyl)dimethylethoxysilane.

7. The biosensor according to claim 1, wherein the support layer formed on the first side of the base material has a density of 800 pieces/μm.sup.2 to 2,000 pieces/μm.sup.2.

8. A method for preparing a biosensor comprising: forming a support layer, which is a self-assembled monolayer, on a first side of a base material by a vapor deposition method; and subsequently immobilizing a first antibody in the support layer.

9. The method for preparing a biosensor according to claim 8, wherein forming the support layer is performed at a temperature of between 40° C. to 200° C.

10. A method for detecting a target substance comprising: contacting a test subject substance comprising a target substance, a substrate, and a second antibody in which an enzyme reacting with the substrate is immobilized, with a biosensor including a base material, a support layer formed on a first side of the base material, and a first antibody immobilized in the support layer, wherein the support layer formed on the first side of the base material is a self-assembled monolayer formed by a vapor deposition method; and measuring changes in the substrate in contact with the biosensor.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0066] FIG. 1 is a conceptual diagram illustrating exemplary vapor deposition equipment that may be used in a method for preparing a biosensor.

[0067] FIG. 2 is an exemplary conceptual diagram showing a series of processes for manufacturing the biosensor of Example.

[0068] FIG. 3 is a graph showing absorbance at the maximum absorption wavelength (wavelength of about 450 nm) measured with a UV-Vis spectrophotometer according to concentrations of a target substance using the biosensor prepared through Example.

[0069] FIG. 4 is a graph showing absorbance at the maximum absorption wavelength (wavelength of about 450 nm) measured with a UV-Vis spectrophotometer according to concentrations of a target substance using the biosensor prepared through Comparative Example.

DETAILED DESCRIPTION

[0070] Hereinafter, the present application will be described in detail with reference to examples, but the scope of the present application is not limited by the following examples.

Absorbance Measurement

[0071] The biosensors prepared through examples and comparative examples were washed four times with 1% PBS and 0.05% Tween 20, blocked with 1% BSA (bovine serum albumin) for 1 hour, and then washed four times.

[0072] Thereafter, about 100 μL of IgE (NIBSC, 11/234) (20 ng/mL) as a target substance was added thereto, reacted for 1 hour, and then washed four times, and about 100 μL of an anti-human IgE antibody (manufacturer: Goma Biotech, product name: IgE, Anti-Human, HRP) (1 μg/mL), in which HRP (horseradish peroxidase) was immobilized, as a second antibody was added thereto, reacted for 1 hour, and then washed four times. Then, about 100 μL of TMB (3,3′,5,5′-tetramethylbenzidine) (0.1 mg/mL) as a substrate was added thereto, reacted for 15 minutes, and then about 100 μL of 2M H.sub.2SO.sub.4 was added thereto.

[0073] Thereafter, the absorbance was measured using a UV-Vis spectrophotometer (Agilent, Cary 8454).

Example

Forming Material of Biosensor

[0074] Base material: A glass substrate (8 cm×2 cm) (Sewon Tech, Soda-lime glass) was used as the base material.

[0075] Forming material of support layer: (3-aminopropyl)trimethoxysilane (APTMS) (Sigma-Aldrich, 281778) was used.

[0076] Linking medium: p-phenylene diisocyanate (Sigma-Aldrich, 258555) was used.

[0077] First antibody: Anti-human IgE antibody (ThermoFisher Scientific, A18797) was used.

Manufacture of Biosensor

[0078] Formation of support layer on base material:

[0079] The glass substrate was safely placed in the reaction chamber of the vapor deposition equipment configured as shown in FIG. 1, the second valve was opened to form the reaction chamber in a vacuum atmosphere, and the temperature of the reaction chamber was maintained at about 100° C. using the second heat source. Furthermore, (3-aminopropyl)trimethoxysilane (APTMS), which was a forming material of the support layer, was charged in the reaction tank, and then APTMS was vaporized by heating the temperature of the reaction tank to 100° C. using the first heat source. The first valve was opened for about 60 seconds so that the vaporized APTMS flowed into the reaction chamber, and again, the first valve was closed and then held for 10 minutes to deposit APTMS on the glass substrate, thereby forming a support layer as a self-assembled monolayer.

[0080] The second valve was opened and the temperature of the reaction chamber was lowered to room temperature. Thereafter, the second valve was closed and the third valve was opened to convert the pressure of the reaction chamber to normal pressure, and then the base material, on which the support layer was formed, was collected from the reaction chamber.

[0081] Immobilization of First Antibody in Support Layer:

[0082] The glass substrate, on which the support layer was formed, was supported on a 0.2% p-phenylene diisocyanate solution (in DMF/pyridine 9:1) for 2 hours.

[0083] Thereafter, about 100 μL of the first antibody (1 μg/mL) was reacted for 1 hour to immobilize the antibody in the support layer.

[0084] FIG. 2 is an exemplary conceptual diagram showing a series of processes for manufacturing a biosensor of an example. The absorbance was measured using the biosensor of Example prepared by the same method as above.

Comparative Example

[0085] Forming Material of Biosensor

[0086] The base material, the forming material of the support layer and the first antibody, which were the same as those of Example 1, were used.

[0087] Manufacture of Biosensor

[0088] Formation of Support Layer on Base Material:

[0089] Using a liquid phase deposition method of a glass substrate, the support layer was formed on the glass substrate. Specifically, the glass substrate was immersed in ethanol containing 1% (3-aminopropyl)trimethoxysilane (APTMS), which was a forming material of a support layer, for about 2 hours to form a support layer on the base material. Thereafter, the base material, on which the support layer was formed, was collected by washing it with ethanol for about 10 seconds.

[0090] Immobilization of first antibody in support layer: The first antibody was immobilized in the support layer in the same manner as in Example.

[0091] The absorbance was measured using the biosensor of Comparative Example prepared in the same method as above.

Evaluation Results

[0092] FIG. 3 is a graph showing absorbance at the maximum absorption wavelength (wavelength of about 450 nm) measured with a UV-Vis spectrophotometer (path length: 1 cm) according to concentrations of a target substance using the biosensor prepared through Example, and FIG. 4 is a graph showing absorbance at the maximum absorption wavelength (wavelength of about 450 nm) measured with a UV-Vis spectrophotometer (path length: 1 cm) according to concentrations of a target substance using the biosensor prepared through Comparative Example.

[0093] As shown in FIGS. 3 and 4, as a result of analyzing the absorbance of the biosensor prepared in Example, the absorbance was found to be high as about 0.175 at 20 ng/mL of the target substance. However, as a result of analyzing the absorbance of the biosensor prepared in Comparative Example, the absorbance was found to be low as about 0.08 at 20 ng/mL of the target substance. Through this, it could be confirmed that the detection sensitivity of the biosensor prepared in Example was significantly higher than the detection sensitivity of the biosensor prepared in Comparative Example.