KR20150121379A - Development of hydrogel microarray biosensor utilizing metal enhanced fluorescence effect - Google Patents

Abstract

The present invention relates to a nanocomposite which includes a silver nanoparticle core/silica shell, and to a biosensor which includes an enzyme-immobilized hydrogel. It is possible to provide a biosensor capable of detecting substrate concentration more sensitively, stably, and easily by means of metal enhanced fluorescence (MEF).

Description

[Background Art] [0002] Development of a biosensor using a hydrogel microarray based on fluorescence signal amplification effect [

The present invention relates to a microarray-based biosensor for detecting glucose, and a nanocomposite comprising a silver nanoparticle core / silica shell and an enzyme are immobilized in a hydrogel, and a metal enhanced fluorescence (MEF) And more particularly, to a biosensor capable of detecting a concentration of a substrate with high sensitivity, stability, and ease.

Many studies now focus on the development of glucose sensors that can perform screening more quickly, accurately and economically. Conventional optical sensors use an enzyme, glucose oxidase, to analyze the components, but this has limited optical detection.

Meanwhile, one of the biggest research trends of biosensor research is biosensor, which is increasingly used as a bio-chip using microarrays, because of the fusion of technologies related to cutting-edge research such as biology, medicine, chemistry, electronics, and mechanical engineering. It is getting better. Considering this point, the development of a glucose sensor enables the analysis and apparatus to be miniaturized and obsessed quickly and simply by using only the sample of the minimum circle, which requires complex and long time, It has the advantage of real-time analysis in the field of analysis, which requires immediate information acquisition, such as environmental survey or industrial survey, because it is portable.

In the conventional optical sensor, a method of attaching a fluorescent marker to a biological material and then optically measuring the biological material is used, but this has limitations in optical measurement.

In addition, enzymes react sensitively from physical impacts and chemical reactions and are easily denatured. In order to measure these enzymes, substances such as fluorescent dyes, which can be easily confirmed, are required.

The present invention provides a biosensor comprising a nanocomposite including a silver nanoparticle core / silica shell and a hydrogel to which an enzyme is immobilized.

In addition, the present invention provides a method of manufacturing a biosensor including a step of curing a mixture of a nanocomposite including a silver nanoparticle core / silica shell, an enzyme, a polymer and a photoinitiator by UV irradiation.

In addition, the present invention provides a method for detecting glucose using the above-described biosensor.

The biosensor according to the present invention can fix the enzyme inside the hydrogel to maintain the activity of the enzyme for a long time and reuse of the sensor is possible.

In addition, the biosensor according to the present invention can measure a high sensitivity glucose according to the concentration of the enzyme, the nanocomposite and the thickness of the silica shell.

In addition, in the present invention, it is possible to manufacture a microarray including other components on a substrate, thereby enabling multi-sensing.

In addition, the biosensor according to the present invention can quickly and simply process the analysis requiring complex and long time by using only a small sample due to miniaturization and integration of the analyzer, and also can reduce power consumption and maintenance cost It has the advantage of being portable.

In addition, the biosensor according to the present invention can be used in various fields such as photonics, molecular biology, materials science, and chemistry as well as a biosensor because a high sensitivity signal can be detected.

1 is an SEM photograph of silver nanoparticles prepared according to an embodiment of the present invention.
2 is a TEM photograph of a nanocomposite according to TEOS concentration.
3 is a UV-VIS graph of a nanocomposite according to TEOS concentration.
4 is a schematic view showing a method of manufacturing a hydrogel array according to the present invention.
5 is a view showing a measuring method of glucose.
6 is a photograph (a) and a graph (b) showing the effect of amplifying the fluorescence signal of the nanocomposite according to the concentration of TEOS.
FIG. 7 is a photograph (a) and a graph (b) showing the amplification effect of the formation signal according to the concentration of the nanocomposite when the TEOS concentration is 0.3 mg / mL.
8 is a photograph (a) and a graph (b) showing the effect of amplifying the forming signal using the hydrogel microarray.

The present invention relates to a biosensor comprising a nanocomposite comprising a silver nanoparticle core / silica shell and a hydrogel immobilized with an enzyme.

Hereinafter, a biosensor according to the present invention will be described in detail.

As described above, the biosensor according to the present invention includes a nanocomposite including a silver nanoparticle core / silica shell and a hydrogel to which an enzyme is immobilized.

In the present invention, a nanocomposite including a silver nanoparticle core / silica shell (which may be referred to as a nanocomposite) may serve to amplify fluorescence emitted from the biosensor.

Silver nanoparticles have a plasmon resonance phenomenon. When the silver nanoparticles have a certain distance from the fluorescent marker, amplification of fluorescence intensity may occur. However, when the distance between the silver nanoparticles and the fluorescent label is too long, there is no fluorescence signal amplification phenomenon. If the distance between the fluorescent labels is too close, the silver nanoparticle acts as an electron acceptor and the fluorescent label may lose fluorescence. Therefore, it is important to maintain a constant distance between the silver nanoparticles and the fluorescent marker.

In the present invention, by coating the silver nanoparticles with the silica shell, the distance between the silver nanoparticles and the fluorescent marker can be controlled, and further, the fluorescence intensity of the fluorescent indicator can be controlled.

That is, since the nanocomposite of the present invention has a silver nanoparticle core / silica shell structure, the intensity of fluorescence generated in the biosensor can be easily controlled.

The particle diameter of the silver nanoparticles in the nanocomposite is not particularly limited as long as it can cause fluorescence intensity amplification phenomenon. For example, the silver nanoparticle may have a particle diameter of 1 to 1000 nm, 1 to 100 nm, 1 to 50 nm, or 10 to 20 nm .

The thickness of the silica shell is not particularly limited as long as the distance between the silver nanoparticle and the fluorescent marker can be adjusted so that the silver nanoparticle can exhibit the fluorescence intensity effect. For example, the silica shell may have a thickness of 1 to 1000 nm, 50 nm.

The kind of the enzyme in the present invention is not particularly limited as long as it can be used for the detection of the desired substrate. In one embodiment, the enzyme may be used to detect glucose, wherein the enzyme may be glucose oxidase, alcohol oxidase, or cholesterol oxidase.

The above-described nanocomposite and enzyme have a structure fixed to a hydrogel.

The enzyme can be easily denatured by reacting sensitively from physical impact and chemical reaction. Thus, by fixing the enzyme to a polymer hydrogel having a high water content and suitable for a living body, the activity of the enzyme can be maintained for a long time and the reaction with the substrate can be more stably performed.

The kind of such hydrogel is not particularly limited and may be derived from, for example, a polymer selected from the group consisting of polyethylene glycol (PEG), polyethylene, polyvinyl alcohol, polylactic coglycolic acid, collagen and hyaluronic acid, Specifically, it may be derived from polyethylene glycol (PEG).

The molecular weight (Mw) of the hydrogel may be 200 to 20,000, and the molecular weight of the hydrogel may be adjusted to control the mesh size of the hydrogel.

In the present invention, the enzyme is immobilized by the hydrogel to prevent denaturation of the enzyme, the activity of the enzyme can be maintained for a long time, and the biosensor can be reused.

The hydrogel in which the nanocomposite and the enzyme are immobilized in the biosensor may be used in a biosensor in the form of an array, specifically a microarray.

The hydrogel in which the nanocomposite and the enzyme are immobilized in the biosensor may be located in two or more channels. Specifically, the biosensor may include one or more sample injection channels to be measured and one or more standard channels, the width of the channels may be 50 to 200 μm, and the interval may be 50 to 200 μm.

The present invention also relates to a method of manufacturing a biosensor comprising curing a mixture of a nanocomposite comprising a silver nanoparticle core / silica shell, an enzyme, a polymer and a photoinitiator by UV irradiation.

In the present invention, the nanocomposite can be prepared by reacting silver nanoparticles and a silica precursor.

The silver nanoparticles can be prepared by methods known in the art and in one embodiment can be prepared by reacting silver nitrate (AgNO ₃ ) with a polymer, specifically polyvinylpyrrolidone (PVP) have. In addition, silver nanoparticles such as gold and platinum may be used in place of the silver nanoparticles.

The type of the silica precursor is not particularly limited as long as it can form a silica shell. For example, tetraethylorthosilicate (TEOS) or Si (OR) ₄ can be used. In the Si (OR) ₄ , R may be an alkyl group having 1 to 5 carbon atoms, and may specifically be methyl, ethyl or propyl. The thickness of the silica shell can be controlled according to the concentration of the silica precursor. In the present invention, the content ratio of the silver nanoparticles and the silica precursor (silver nanoparticles / Silica precursor) can be adjusted to 0.005 to 0.1 v / v%.

The preparation of the silica shell by the reaction of the silver nanoparticles and the silica precursor can be carried out through the stober method.

The kind of the polymer in the polymer solution is not particularly limited and may be selected from the group consisting of polyethylene glycol, polyethylene, polyvinyl alcohol, polylactic acid, glycolic acid, collagen and hyaluronic acid.

When the enzyme and the photoinitiator are mixed with the nanocomposite and polymer solution and UV irradiation is performed, a hydrogel in which the nanocomposite and the enzyme are immobilized is formed. In the present invention, a hydrogel can be formed by using a photolithography method. Hydrogels having a uniform shape and shape can be produced according to the design of a photomask.

In the present invention, the fluorescence intensity of a biosensor manufactured according to the concentration of the nanocomposite and the enzyme may be varied. In order to improve the fluorescence intensity of the biosensor, the concentration of the nanocomposite may be adjusted to 0.01 to 1.0 mg / mL or 0.1 to 0.5 mg / mL, and the concentration of the enzyme may be adjusted to 0.0001 to 0.1 mg / mL or 0.001 to 0.1 mg / mL. < / RTI >

The wavelength range of UV to be irradiated in the present invention may be 300 to 400 nm, 350 to 370 nm, or 365 nm.

Polymer exposed to ultraviolet rays forms a network structure due to radical polymerization reaction. Specifically, when PEG (polyethylene glycol) -DA having a vinyl group at both ends is exposed to ultraviolet rays, radicals generated from the photoinitiator break the carbon double bond of the vinyl group to form cross-linking and form a network structure. Enzyme and fluorescent material can be fixed within the network structure.

The present invention also relates to a method for detecting a substrate using the above-described biosensor. In one embodiment, the biosensor can be used to detect glucose.

The detection of the glucose can be performed by reacting the biosensor according to the present invention with glucose and a fluorescent marker.

In this case, as a fluorescent marker, a substance showing fluorescence in the detection reaction of glucose can be used. Examples of the fluorescent marker include Amplelred, Peroxyresorufin-1 (PR1), Peroxyfluor- 1, PF1), peroxyxanthone-1, 2'-7'-dichlorodihydrofluorescein diacetate (DCFDA), dihydrorhodamine 123 123, DHR) or dihydrocyanine (PX1) can be used.

In the present invention, the biosensor reacts with glucose to generate fluorescence. Specifically, the glucose oxidase in the biosensor reacts with glucose to form hydrogen peroxide. The hydrogen peroxide reacts with amplex red and peroxidase to undergo the oxidation process. At this time, the colorless amplex red reacts with resoloxol having fluorescence .

That is, fluorescence occurs due to a specific reaction between glucose and glucose oxidase, and as the concentration of glucose increases, the intensity of fluorescence becomes stronger through the reaction with AmplexRed.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples of the present invention and Comparative Examples which are not based on the present invention, but the scope of the present invention is not limited by the following embodiments.

Example 1. Synthesis of silver nanoparticles

In an Erlenmeyer flask, 75 mL of ethanol was added, and 0.8 g of PVP (polyvinylpyrrolidone) was dissolved, followed by reaction at 500 rpm for 5 minutes. Thereafter, 0.05 g of AgNO ₃ (silver nitrate) was added, and the reaction was allowed to proceed at 900 rpm for 30 minutes (after the reaction, the color changed to dark brown). After the reaction, the Erlenmeyer flask was maintained at 120 ° C for 2 hours to prepare silver nanoparticles.

In the present invention, FIG. 1 is a SEM photograph of the silver nanoparticles prepared in Example 1. As shown in FIG. 1, silver nanoparticles having a size of 10 to 20 nm are prepared by the above-described manufacturing method.

Example 2. Synthesis of nanocomposite comprising nanoparticle core / silica shell

The nanocomposites were synthesized using the silver nanoparticles synthesized in Example 1.

1 mL of ammonia was added to a mixed solution containing 45 mL of ethanol and 3 mL of silver nanoparticles at 0.3 mg / mL, and the mixture was reacted for 5 minutes. Then, 5 mL of 0.1, 0.2, 0.3, or 0.5 mg / mL TEOS (Tetraethyl orthosilicate) was added and reacted.

2 shows a TEM photograph (FIG. 2) and a UV-VIS graph (FIG. 3) of a nanocomposite prepared according to the concentration of TEOS which is a silica precursor, and Table 1 below shows a nanocomposite Of the shell thickness of the shell.

TEOS concentration Shell thickness 0.1 mg / mL 10 nm 0.2 mg / mL 15 nm 0.3 mg / mL 20 nm 0.5 mg / mL 35 nm

As shown in Table 1 and FIG. 2, it can be seen that as the concentration of TEOS increases, the thickness of the silica shell increases. In addition, it can be seen from FIG. 3 that as the concentration of TEOS increases, the absorbance of the produced nanocomposite increases.

Example 3. Hydrogel microarray fabrication

1 mL of the nanocomposite solution prepared in Example 2, 1 mL of PEG-DA (poly (ethylene glycol) diacrylate, MW: 575) and 1 mL of glucose oxidase (concentration: 1 mg / mL) 2-dimethoxy-2-phenyl-acetophenone was added to prepare a PEG precursor solution. The PEG precursor solution was poured into a substrate and exposed to 100 W ultraviolet rays using a photomask.

The PEG precursor solution exposed to ultraviolet rays forms a network structure due to the radical polymerization reaction. Specifically, when PEG-DA having a vinyl group at both ends is exposed to ultraviolet rays, a radical generated from the photoinitiator breaks the carbon double bond of the vinyl group to form cross-linking, thereby forming a network structure. Enzyme and fluorescent material can be fixed within the network structure.

In the present invention, a microarray having various shapes and sizes can be produced. This can be performed by controlling the mesh size by changing the molecular weight of PEG-DA.

4 is a schematic view showing a method of manufacturing a hydrogel array according to the present invention. As shown in FIG. 4, the microarray can be prepared by applying a solution containing a nanocomposite on a substrate, and then exposing it to ultraviolet rays using a photomask.

Silver nanoparticles of the nanocomposite may not exhibit the fluorescence signal amplification effect if the distance between the fluorescent materials is too large. If the distance between the fluorescent materials is close to that of the nanoparticles, the nanoparticles may act as electron acceptors and lose fluorescence of the fluorescent material.

Accordingly, in the present invention, the fluorescence intensity of the fluorescent material can be controlled by controlling the thickness of the silica shell by the distance between the silver nanoparticles and the fluorescent material.

Example 5 Confirmation of amplification effect of fluorescent signal according to silica shell thickness

A nanocomposite having a different silica shell thickness was prepared by varying TEOS concentration as in Example 2, and 1 mL of the nanocomposite (concentration: 0.3 mg / mL) was used to prepare a nanocomposite according to the method of Example 3 A hydrogel microarray was prepared.

Glucose was measured using the above-prepared hydrogel microarray. The measurement method of the above-mentioned glucos is as follows.

1 ml of glucose (concentration: 10 mM), 500 uL of peroxidase (concentration: 1 mg / ml) and 300 uL of AmplexRad (concentration: 0.1 mM) were mixed and reacted with the microarray for 1 minute.

In the present invention, Fig. 5 shows a method of measuring glucose. First, glucose is oxidized by oxygen oxidase and water and microarray of glucose oxidase to produce gluconic acid and hydrogen peroxide. The hydrogen peroxide produced here is oxidized by encountering amplex red and peroxidase. In this oxidation process, the colorless AmplexRed transforms into a fluorescent substance called resorufin. By measuring the fluorescence of this resolubilin, the activity of the enzyme can be measured.

FIG. 6 is a photograph (a) and a graph (b) showing the effect of amplifying the fluorescence signal when the thickness of the silica shell is changed by varying the TEOS concentration. In FIG. 6, 0 nm indicates that nanoparticles are substituted for silver nanoparticles, 10 nm indicates that TEOS concentration is 0.1 mg / mL, 15 nm indicates that TEOS concentration is 0.2 mg / mL, and 20 nm indicates TEOS When the concentration is 0.3 mg / mL, 35 nm is the TEOS concentration of 0.5 mg / mL, and Only enzyme is the non-nanocomposite.

As a result of the above experiment, when the TEOS concentration was set to 0.3 mg / mL, that is, when the silica shell thickness was 20 nm, the best fluorescence intensity was obtained.

Example 6. Confirmation of amplification effect of fluorescent signal according to concentration of nanocomposite

In Example 2, a nanocomposite was prepared with a TEOS concentration of 0.3 mg / mL, and 1 mL of the nanocomposite was used to prepare a hydrogel microarray according to the method of Example 3 described above. At this time, a hydrogel microarray was prepared by varying the concentration of the nanocomposite at 0, 0.05, 0.1, 0.2, 0.3 and 0.5 mg / mL.

Glucose was measured using the above-prepared hydrogel microarray, and the method of measuring the glucos was as follows.

1 ml of glucose (concentration: 10 mM), 500 uL of peroxidase (concentration: 1 mg / ml) and 300 uL of AmplexRad (concentration: 0.1 mM) were mixed with a microarray containing nanocomposite having different concentrations And then reacted for 5 minutes.

FIG. 7 is a photograph (a) and a graph (b) showing the amplification effect of the forming signal according to the concentration of the nanocomposite when the TEOS concentration is 0.3 mg / mL in the present invention.

As shown in FIG. 7, when the concentration of the nanocomposite is 0.3 mg / mL or more, excellent fluorescence intensity can be obtained.

Example 7 Preparation of Hydrogel Microarrays Containing Enzyme, Silver Nanoparticle, or Enzyme + Nanocomposite, respectively and Confirmation of Fluorescence Signal Amplification Effect

(2) a hydrogel microarray containing silver nanoparticles and glucose oxidase; (3) a concentration of TEOS of 0.3 mg / ml; and (3) mL and a microarray containing glucose oxidase were prepared.

Glucose was measured using the above-prepared hydrogel microarray, and the method of measuring the glucos was as follows.

(Concentration: 1 mg / ml) and ampel red (concentration: 1, 2, 4, 6, 8 or 10 mM) : 0.1 mM) were mixed and reacted for 1 minute.

FIG. 8 is a photograph (a) and a graph (b) showing amplification effect of a forming signal using the hydrogel microarray fabricated by the above. 8, the microarray of (1) is the only enzyme, the microarray of (2) is the SNPs or the only SNPs, and the microarray of (3) is the silica coated SNPs or the silica coated SNPs + enzyme.

As shown in FIG. 8, the microarray 2 containing only silver nanoparticles showed insufficient fluorescence. Fluorescence was observed in microarrays of (1) and (3), that is, microarrays containing only enzymes and enzymes and nanocomposites, which were 2.8 times higher fluorescence than microarrays of microarray (1) , And the microarray of (2) showed fluorescence reduction about 4 times as much as the microarray of (3).

Further, it can be confirmed that the higher the concentration of glucose is, the better the fluorescence intensity is exhibited.

Claims (12)

Wherein the biosensor comprises a nanocomposite comprising a silver nanoparticle core / silica shell and a hydrogel to which the enzyme is immobilized.
The biosensor according to claim 1, wherein the silver nanoparticles have a particle diameter of 1 to 1000 nm.
The biosensor according to claim 1, wherein the silica shell has a thickness of 1 to 1000 nm.
The biosensor according to claim 1, wherein the enzyme is glucose oxidase, alcohol oxidase or cholesterol oxidase.
The biosensor according to claim 1, wherein the hydrogel is derived from a polymer selected from the group consisting of polyethylene glycol, polyethylene, polyvinyl alcohol, polylactic coglycolic acid, collagen and hyaluronic acid.
The biosensor according to claim 1, wherein the biosensor is reusable.
The biosensor according to claim 1, wherein the hydrogel is fixed in an array form.
Comprising the step of curing a mixture of a nanocomposite comprising an nanoparticle core / silica shell, an enzyme, a polymer and a photoinitiator by UV irradiation.
The method of claim 8, wherein the nanocomposite is prepared by reacting silver nanoparticles and a silica precursor, wherein the silica precursor is tetraethylorthosilicate (TEOS) or Si (OR) ₄ , wherein R is an alkyl group of 1 to 5 carbon atoms A method of manufacturing a biosensor.
The method of manufacturing a biosensor according to claim 9, wherein the content ratio of the silver nanoparticles and the silica precursor is 0.005 to 0.1 v / v%.
The method of manufacturing a biosensor according to claim 8, wherein the concentration of the nanocomposite is 0.01 to 1.0 mg / mL.
A method for detecting glucose using the sensor of any one of claims 1 to 7.

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