KR101643453B1 - Proteins Immobilization Method Using Polymer Based Funtional Group - Google Patents

Abstract

The present invention relates to a process for preparing a dispersion, comprising: preparing a dispersion solution by dissolving a polymer having a functional group capable of covalently bonding with an amine group in an organic solvent; Forming a polymer coating layer having a functional group by coating the solution on a substrate surface; Attaching the protein to the coating layer; To a protein immobilization method using a polymer having a functional group. The present invention also provides a method and kit for detecting a target protein using a polymer having a functional group. When the protein immobilization method using the functional group-containing polymer according to the present invention is used, the protein can be immobilized by a simple method of dotting on the surface, and time is saved.

Description

[0001] The present invention relates to a protein immobilization method using a functional group-based polymer,

The present invention relates to a protein immobilization method using a functional group-based polymer.

The most widely used technique to combine inorganic and organic materials is the silane coupling agent. In this method, a hydroxyl group is formed on a surface of a substrate to be immobilized by a plasma treatment or the like, and a silane coupling agent is reacted on the hydroxyl group. Then, a crosslinking agent is bonded to each silane such as glutaraldehyde, and the functional group possessed by the crosslinking agent and the amine group of the protein bind to each other to immobilize the protein on the surface (Non-Patent Document 1). However, this method requires several steps of pretreatment and takes a long time because it requires several steps to fix the protein. In addition, when the surface of the material can not form a hydroxyl group, or when a hydroxyl group is not formed even through the pretreatment, the protein can not be immobilized, and it is disadvantageous that the probability of occurrence of errors can be increased due to a lot of cleaning steps .

On the other hand, many items have been analyzed for the supply of safe water at the collection point, but the analysis of new trace pollutants has still been detected by methods such as chromatography. However, this method requires more than half a day from sample collection to analysis, and it is difficult to take immediate action if new trace pollutants are generated in the source.

To this end, sensor technology is needed to detect new trace pollutants on the spot.

(Non-Patent Document 1) Simone Karrasch et al., Covalent Binding of Biological Samples to Solid Supports for Scanning Probe Microscopy in Buffer Solution, Biophysical Journal Volume 65 December 1993 2437-2446.

An object of the present invention is to provide a method for instantly detecting new micropollutants in the field, and a method for immobilizing proteins using functional group-based polymers capable of binding with amine groups.

Another object of the present invention is to provide a protein detection method using a functional group-based polymer.

It is another object of the present invention to provide a kit comprising a functional group-based polymer.

Although many items have been analyzed for supply of safe water at the collection point, the analysis of new trace pollutants (Estrogen, PCBs, Geosmin) is still being detected by methods such as chromatography. However, the above-mentioned methods such as chromatography require more than half a day from sample collection to analysis, and it is difficult to take immediate action if new trace pollutants are generated from the collection source. In order to achieve this, a sensor technology capable of instantly detecting new trace contaminants in the field is needed. Among them, sensor technology using an antigen / antibody reaction is actively under investigation so as to accurately measure contaminants to trace amounts. In the case of a sensor using an antigen / antibody reaction, how much antibody is attached to the chip surface is the most important part in developing the sensor. How much of the antibody is attached correctly can greatly affect the ultimate measurement concentration and sensitivity of the sensor.

Accordingly, the present invention provides a method of immobilizing proteins including antibodies in a sensor and various industrial fields in a shorter time than in the case of immobilizing proteins on the surface.

Hereinafter, the present invention will be described in detail.

The present invention relates to a process for preparing a dispersion, comprising: preparing a dispersion solution by dissolving a polymer having a functional group capable of covalently bonding with an amine group in an organic solvent; Forming a polymer coating layer having a functional group by coating the solution on a substrate surface; And attaching the protein to the coating layer. The present invention also provides a method for immobilizing a protein using a polymer having a functional group.

As used herein, the term " functional group " refers to an organic compound having a functional atomic group capable of covalent bonding with an amine group of a protein for protein immobilization. In one embodiment of the present invention, the functional group is a group consisting of a formyl, a thiol, an isocyanate, a sulforyl chloride and a chlorocarbonyl, , And may include both functional groups capable of bonding with amine groups (Fig. 1).

The type of the substrate used in the present invention is preferably glass, alumina, ceramic, carbon, gold, silver, copper, aluminum, compound semiconductor and silicon. Preferably, glass can be used. Since a hydroxyl group is not required, a solid substrate for protein immobilization commonly used in the art can be used without limitation.

A method to be proposed in the present invention is to coat a polystyrene-based polymer having a functional group capable of bonding with an amine on a surface of a substrate. This makes it possible to immobilize the protein by forming a functional group capable of reacting with the amine group of the protein on the surface while sufficiently utilizing it on the surface without hydroxyl groups. This is simpler because it is possible to reduce the number of steps required for immobilization than the conventional method and does not require much time and allows the fixation of the protein in one treatment.

In addition, the conventional method takes more than 4 hours and takes 12 hours. However, since the method of the present invention takes 30 minutes or less, the time required until the same protein immobilization efficiency can be reduced by 3 hours or more have.

In one embodiment of the present invention, the isocyanate functionality is attributed to the results of one of several functional groups capable of reacting with the amine group. However, if a plurality of functional groups capable of reacting with an amine group and a polymer having the functional group are used, a function similar to that of the present invention can be performed. Such a functional group may be one or more selected from the group consisting of formyl, thiol, isocyanate, sulforyl chloride and chlorocarbonyl, and mixtures thereof. And may include both functional groups capable of bonding with amine groups (Fig. 1).

The isocyanate may be selected from the group consisting of 1,5-naphthalene diisocyanate, benzyl isocyanate, 2,4-toluene diisocyanate (TDI), 2,6-toluene diisocyanate, 2,4'-diphenylmethane diisocyanate (MDI) '- diphenylmethane diisocyanate, 4,4'-diphenylmethane diisocyanate, and mixtures thereof; MDI having two or more isocyanate groups in the molecule and oligomers thereof; Or a diphenylmethane diisocyanate comprising at least one member selected from urethane, allophanate, urea, buret, carbodiimide, uretonimine, isocyanurate groups and mixtures thereof, and isocyanate Based polystyrene-based polymer having a " group "

In one embodiment of the present invention, the solvent may be selected from the group consisting of chloroform, tetrachlorethylene, carbon tetrachloride, dichloromethane, dichloroethane, toluene, and mixtures thereof, but is not limited thereto.

The polymer having a functional group capable of forming a covalent bond with the amine group of the present invention may be contained in an amount of 1 to 20% by weight, preferably 2 to 10% by weight, and most preferably 5% by weight based on the total weight of the total solution have. When the amount of the polymer is less than 1% by weight, the surface of the substrate is unevenly formed, resulting in poor protein immobilization efficiency. When the amount of the polymer is more than 20% by weight, excess polymer is used excessively.

In one embodiment of the present invention, the protein may be an antibody or an antigen, but is not limited thereto, and the protein may be attached with a fluorescent substance.

In one embodiment of the present invention, the fluorescent material is a fluorescein-based TRITC (Tetramethylrhodamine-5- (and 6) -isothiocyanate), FITC (Fluorescein-5-isothiocyanate), DAPI (4,6- 2-phenylindole), pyrene, propidium iodide, and RITC (Rhodamine isothiocyanate).

The present invention also provides a method for preparing a polymer electrolyte membrane, comprising: preparing a dispersion solution by dissolving a polymer having a functional group capable of covalently bonding with an amine group in an organic solvent; Forming a polymer coating layer having a functional group by coating the solution on a substrate surface; Binding the target protein to the coating layer; Attaching an antibody having a fluorescent substance to the protein; And analyzing the fluorescence signal; The present invention provides a method for detecting a target protein using a polymer having a functional group.

As used herein, the term "target protein" is a protein to be detected whether or not it is present, specifically, a protein capable of specifically binding to and detecting a substrate having a polymer coating layer having the functional group, The kind is not limited as long as it is a protein commonly known in the art. Specifically, the target protein may be an antigen, preferably an environmental pollutant. The environmental pollutants may be one or more selected from environmental estrogens, PCBs, Geosmin, DDT, Progesterone, bisphenol A, and mixtures thereof, And may include all the new trace amounts of environmental pollutants.

Those skilled in the art will appreciate that in order to detect a target protein, an indirect-competitive assay format, a direct-competitive assay format, or a sandwich assay format It can be easily understood that the detection can be performed.

For example, the target protein detection method of the present invention comprises the steps of: preparing a dispersion solution by dissolving a polymer having a functional group capable of covalently bonding with an amine group in an organic solvent; Forming a polymer coating layer having a functional group by coating the solution on a substrate surface; Attaching a bioreceptor to the coating layer; Binding the target antigen to the bioreceptor; Attaching an antibody having a fluorescent substance to the antigen; Analyzing the fluorescence signal; , ≪ / RTI > Wherein the bioreceptor may be a protein, an antigen, or an antibody.

The present invention also provides a semiconductor device, A coating layer formed on the substrate, the coating layer comprising a polymer having a functional group capable of covalently bonding with an amine group; And a protein attached to the coating layer.

The functional group may be one or more selected from the group consisting of an isocyanate group, a sulfonyl chloride group, a chlorocarbonyl group, a thiol group, a formyl group, and a mixture thereof, and may include both functional groups capable of binding with an amine.

When a kit prepared using the protein immobilization method of the present invention is used, it can be widely applied not only to fixation of an antibody but also to a combination of an inorganic substance and an organic substance in various fields.

When a protein immobilization method using a functional group-based polymer capable of binding to an amine group according to the present invention is used, a polymer layer having a functional group capable of binding to a protein is formed on a substrate surface. Since the functional group coating layer thus formed easily reacts with the amine group of the protein to form a urea bond and is immobilized, the protein can be immobilized by a simple method of dotting on the surface, and time is saved.

Figure 1 shows various functional groups capable of reacting with amine groups.
2 is a principle of a protein immobilization method using a polymer having a functional group of the present invention.
3 is a schematic diagram of a protein immobilization method using a polymer having a functional group of the present invention.
FIG. 4 is an FT-IR graph showing whether or not a polymer having a functional group according to an embodiment of the present invention is attached and protein immobilized.
5 is a graph comparing the protein immobilization efficiency using the conventional isocyanate silane and APTES and the protein immobilization efficiency using the isocyanate and sulfonyl group-containing polymer according to one embodiment of the present invention.
FIG. 6 shows the surface morphology of a substrate according to the concentration of a polymer having a functional group according to an embodiment of the present invention. The polymer having functional groups was added in an amount of 1 wt%, 5 wt%, 10 wt%, and 20 wt% of the whole solution, and the shape and thickness of the substrate surface were analyzed by AFM (Atomic Force Microscope).
FIG. 7 is a graph showing the optimal time (left) and temperature (right) necessary for fixing a protein according to an embodiment of the present invention (left: fluorescence intensity was measured at 15 minute intervals until the first 1 hour after protein immobilization, After 1 hour, the fluorescence intensity was measured once every 30 minutes, and the protein immobilization efficiency was observed by setting the temperature at a low temperature (4 ° C), a middle temperature (25 ° C), and a high temperature (50 ° C).
8 is a graph comparing the environmental pollutant-antibody immobilization efficiency according to an embodiment of the present invention (BLANK: no protein attached: BSA: control protein attached: PCB-antibody and estrogen-antibody: Environmental pollutants - when the antibody is attached).
9 is a graph comparing the protein immobilization efficiency according to the substrate material. (Slide glass: a polymer solution having a functional group according to an embodiment of the present invention is coated on a slide glass. Paper: a functional group according to one embodiment of the present invention APTES-Slide glass: APTES is processed in a slide glass; APTES-Paper: APTES is processed in a paper).
10 is a graph showing an experiment for water resistance of an isocyanate group according to an embodiment of the present invention. Blocking: Observation of protein immobilization efficiency by blocking water in an isocyanate group Experiment without moisture blocking: n = 5: Experiment 5 times).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by one of ordinary skill in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Example  1. Isocyanate groups The polymer  Protein immobilization

Example  1-1. Isocyanate The polymer  Surface treatment and protein immobilization using

In this study, a polystyrene-based polymer (Isocyanate, polymer-bound (473658-5G), purchased from Sigma Aldrich) containing isocyanate was used as a functional group capable of bonding with the amine group (NH2) of the protein. The isocyanate polymer was dissolved in dichloromethane to 5% by weight based on the total weight of the solution. The solution was coated on the surface of a substrate to be immobilized with a protein by a dipping method. As a substrate, a microscope slides glass was used. The slide glass was first cleaned. This is a process to remove oil film and contaminants that might occur in the production of slide glass and circulation storage. The slide glass was immersed in ethanol, NaOH 1M, HCl 1M solution for 1 hour and rinsed with DI water Dried and stored. The dip coating process was dipped for 5 minutes and a vacuum desiccator was used to evaporate the solvent so as to avoid contact with outside air. Protein dotting was performed using BSA-FITC. A sufficient reaction time of 1 hour or more was given for protein immobilization. As a result, a polystyrene layer having functional groups capable of binding to proteins was formed on the substrate surface. Since the functional group coating layer thus formed easily reacts with the amine group of the protein to form a urea bond, the protein can be immobilized by the most basic method of dotting on the surface. Subsequently, the substrate was washed three times using PBST, and the fluorescence intensity was measured using a microarray scanner (FIGS. 2 and 3).

Example  1-2. Isocyanate Polymer  Confirmation of attachment and protein immobilization

The chemical bond was confirmed by FT-IR to determine whether the isocyanate polymer was well coated on the surface and whether the protein was stably fixed through chemical bonding with the isocyanate formed by the coating. When the isocyanate polymer was coated on the surface, the peak was observed at wavenumber 2270 and 2800 ~ 3000. 2270 is isocyanate, and 2800-3000 is derived from dichloromethane which is a solvent. Through this, it was confirmed that the isocyanate polymer was fixed on the surface well. Thereafter, the protein was immobilized on the surface and the surface was measured. In the case of proteins, amine and isocyanate bonds were identified using amino acids having amine groups. As a result, it was confirmed that a peak was formed at 1720 due to the C = O bond formed by the urea bond, and a peak was formed by the H-N bond at 3100 to chemically bond the isocyanate and the amine (FIG. 4).

Example  2. Sulfonyl  Having The polymer  Protein immobilization

Among the functional groups capable of binding to the amine group (NH2) of proteins, sulfonyl chloride-based polymer (sulfonyl chloride, polymer bound (516228-5G) was dissolved in dichloromethane at 5% by weight in a polystyrene-based polymer The solution was coated on the surface of the substrate to be immobilized with a protein by a dipping method to form a polystyrene layer having a functional group capable of binding to the protein on the surface of the substrate. The protein can be immobilized only by the most basic method of dotting on the surface because it reacts easily with a urea bond to form a urea bond.

Example  3. Comparison of protein immobilization efficiency

Example  3-1. Functional group  Have The polymer  Comparison of protein immobilization efficiency

The efficiency of the protein immobilization method of Examples 1-1 and 2 and the efficiency of the protein immobilization method using the conventional silane coupling agent were compared. To this end, proteins (BSA, purchased from Sigma Aldrich, A9771-50 mg) conjugated with a fluorescent dye (FITC) were immobilized on a substrate pretreated with each method at the same concentration. Thereafter, the fluorescence intensity of the surface was measured to compare the immobilization efficiency of the protein. As a control, pretreatment was carried out on a substrate using isocyanate silane (purchased from Gelest, SII6455.0-100G) and APTES (3-Aminopropyltriethoxysilane, purchased from Tokyo chemical industry co., Inc., 213-048-4) Protein immobilization was performed using a silane coupling method (see Non-Patent Document 1). As a protein, fluorescence intensity was measured after dotting using BSA-FITC. As a result, when the protein immobilization method using the isocyanate and the sulfonyl polymer of the present invention was used as compared with the conventional method, there was no significant difference in the protein immobilization efficiency compared with the conventional method using the silane coupling agent. In particular, when isocyanate was used, the protein immobilization efficiency using the isocyanate silane and APTES as control groups was superior (FIG. 5).

Example  3-2. Functional group  contain Of polymer  Efficiency by concentration

In order to screen the efficiency of the functional group-containing polymer by the coating concentration, the concentration was adjusted to contain 1% by weight, 5% by weight, 10% by weight and 20% by weight of the polymer. After the surface was treated by the method of Example 1-1, the morphology of the surface was measured by AFM (Atomic Force Microscope). As a result of the measurement, it was confirmed that the surface was unevenly formed at 1 wt% or more and 10 wt% or more, and the surface of the coating layer was formed flat at 5 wt%. Thus, it was confirmed that 5 wt% was the optimum coating concentration (Fig. 6).

Example  3-3. Comparison of time and temperature conditions for protein fixation

The optimal time and temperature for protein fixation were tested. The surface of the substrate was treated with the method of Example 1-1 and the protein immobilization efficiency was compared with time and temperature. Fluorescence intensity was measured every 15 minutes after the first hour and every 30 minutes after 1 hour. As a result, the maximum fluorescence intensity was observed at 4 hours, and after that, the fluorescence intensity was saturated and the optimum time required for protein fixation was 4 hours. In the case of the prior art, the protein immobilization time is 12 hours longer. In the case of using the method of the present invention, the time required for exhibiting protein immobilization efficiency can be reduced.

On the other hand, the protein immobilization efficiencies were compared with different temperature conditions. As a result, fluorescence intensity was saturated in 4 hours at low temperature (4 ℃), middle temperature (25 ℃) and high temperature (50 ℃), and no significant difference was observed at each temperature and temperature influence on protein immobilization was considered to be small (Fig. 7).

Example  3-4. New trace contaminants - Antibody immobilization efficiency comparison

Protein immobilization efficiencies were compared using BSA and a new micronutrient - antibody as a protein. To this end, a protein (BSA) conjugated with a fluorescent dye (FITC) was immobilized on a substrate surface-treated by the method of Example 1-1 at the same concentration as a control. Also, Estrogen-antibody (purchased from Abcam, # ab54122) and PCB-antibody (purchased from Fitzgerald, # 10R-P115A), to which FITC was conjugated, were immobilized on the substrate pretreated with the method of Example 1-1 Respectively. Thereafter, the fluorescence intensity of the surface was measured to compare the immobilization efficiency of the protein. As a result, the immobilization efficiency of the novel trace contaminant-antibody also showed a high efficiency similar to that using BSA (FIG. 8).

Example  3-5. Comparison of protein immobilization efficiency on hydroxyl-free surface

The disadvantage of the conventional method of immobilizing proteins using silane is that a hydroxyl group must be present on the surface of the protein. In contrast, the present invention can immobilize proteins without the presence of hydroxyl groups on the surface, and the protein immobilization efficiency is compared using a paper without a hydroxyl group as a substrate for comparison.

The surface of the substrate was treated using the method of Example 1-1 using paper instead of the slide glass as a substrate to confirm whether or not the protein was immobilized. As a control group, a protein immobilization method using APTES (3-Aminopropyltriethoxysilane, purchased from Tokyo chemical industry co., Ltd., 213-048-4) was used. As a result, when the method of the present invention, which does not require a hydroxyl group, was used, protein immobilization efficiency was also good on paper. On the other hand, in the case of APTES in the prior art, protein immobilization was not observed on paper without hydroxyl groups (Fig. 9).

Example  4. Isocyanate Polymer  Evaluation of moisture resistance of substrate

Since isocyanate reacts readily with water vapor in the atmosphere, it is easily deteriorated, and moisture resistance evaluation is performed. 4.8 g (0.055 mol) of methyl ethyl ketone was added to 10 g of isocyanate under a nitrogen free atmosphere. The isocyanate group was blocked by mixing isocyanate with methyl ethyl ketone at 80 占 폚. The blocking isocyanate was protein-immobilized using the method of Example 1-1, and the five protein immobilization experiments were repeated to calculate the average.

As a result, there was no significant difference in the fluorescence intensity between the blocked surface and the non-blocked surface (Fig. 10). This is because the substrate pretreated with the isocyanate polymer of the present invention did not undergo deformation due to moisture in the atmosphere more than expected. This suggests that the protein immobilization method using the isocyanate polymer of the present invention is resistant to moisture to some extent.

Claims (11)

i) dissolving a polystyrene-based polymer having a functional group capable of covalently bonding with an amine group in an organic solvent to prepare a dispersion solution;
ii) forming a polystyrene-based polymer coating layer having a functional group capable of covalently bonding with the amine group by coating the solution on the substrate surface; And
iii) attaching the protein to the coating layer;
A protein immobilization method comprising:
Wherein in the step ii), the substrate is not coated with a hydroxyl group, and the dispersion solution is directly dipped on the substrate. The method according to claim 1,
Wherein the functional group is selected from the group consisting of an isocyanate group, a sulfonyl chloride group, a chlorocarbonyl group, a thiol group, a formyl group, and a mixture thereof. The method according to claim 1,
Wherein the solvent is selected from the group consisting of chloroform, tetrachlorethylene, carbon tetrachloride, dichloromethane, dichloroethane, toluene, and mixtures thereof. The method according to claim 1,
Wherein the protein is an antigen or an antibody. The method according to claim 1,
Wherein the protein has a fluorescent substance attached thereto. i) dissolving a polystyrene-based polymer having a functional group capable of covalently bonding with an amine group in an organic solvent to prepare a dispersion solution;
ii) forming a polystyrene-based polymer coating layer having a functional group capable of covalently bonding with the amine group by coating the solution on the substrate surface;
iii) binding the target protein to the coating layer;
iv) attaching an antibody having a fluorescent substance to the protein; And
v) analyzing the fluorescence signal;
A target protein detection method comprising the steps of:
Wherein in the step ii), the substrate is not coated with a hydroxyl group and is directly dipped in the dispersion solution on the substrate. The method according to claim 6,
Wherein the protein is an antigen. The method according to claim 6,
Wherein the protein is an environmental pollutant. 9. The method of claim 8,
Wherein the environmental pollutants are selected from environmental estrogens, PCBs, Geosmin, DDT, Progesterone, Bisphenol A, and mixtures thereof. Board;
A coating layer formed on the substrate, the coating layer comprising a polystyrene-based polymer having a functional group capable of covalently bonding with an amine group; And
A protein attached to the coating layer;
, ≪ / RTI >
Wherein the substrate is free of hydroxyl groups on the surface of the substrate. 11. The method of claim 10,
Wherein the functional group is selected from the group consisting of an isocyanate group, a sulfonyl chloride group, a chlorocarbonyl group, a thiol group, a formyl group, and a mixture thereof.

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