KR20080054955A - Polydiacetylene sensor chip comprising protein and manufacturing process thereof - Google Patents

Abstract

A polydiacetylene sensor chip comprising protein is provided to detect rapidly protein-protein or protein-cell reaction without labeling of the subject to be analyzed, and reduce the amount of polydiacetylene and subject to be analyzed. A polydiacetylene sensor chip comprises a substrate, a vesicle bound to the substrate and having polymerized diacetylene represented by the formula(1): A-(L1)d-(CH2)e-C=C-C=C-(CH2)f-(L2)g-B, and a probe protein bound to the vesicle, wherein a sum d and g is 0, 1 or 2; a sum of e and f is an integer from 2 to 50; e and f are each independently an integer of 1 or more; A and B are each independently methyl group, amine group, carboxyl group, hydroxyl group, maleimide group, biotin group, hydroxysuccinimide group, benzoic acid group or ester group; and L1 and L2 are each independently alkyl group having carbon number of 2 or more, at least one ethylene oxide group, amine group, amide group, ester group or carbonyl group.

Description

Polydiacetylene Sensor Chip Comprising Protein and Manufacturing Method Thereof {Polydiacetylene Sensor Chip Comprising Protein and Manufacturing Process}

Figure 1 is a schematic diagram of an embodiment for the production of the polydiacetylene sensor chip and analyte detection of the present invention.

Figure 2 is a photograph of one embodiment of the detection result of the protein (second antibody) of the present invention.

Figure 3 is a photograph of the detection results when there is no probe protein.

4 is a photograph of the detection result of the second antibody that does not bind to the probe protein.

Figure 5 is a picture taken an embodiment of the E. coli detection results of the present invention.

Figure 6 is a photograph of another embodiment of the E. coli detection results of the present invention.

Figure 7 is a picture taken an embodiment of the anthrax detection result of the present invention.

The present invention relates to a polydiacetylene sensor chip containing a protein and a method for manufacturing the same, wherein a polydiacetylene vesicle to which a probe protein corresponding to an analyte is bound is bound to a substrate. The present invention relates to a sensor chip and a method of manufacturing the same, which show a quick response time without a separate labeling treatment.

Nanobiosensors are biosensors that are improved by advanced nanotechnology, which converts reactions by binding to biocognitive materials into signals, and refers to sensors that can quickly test specific materials. This is the same principle as the enzyme-substrate complex of the biological concept, in which one ligand is only reactive with one substance having a specific component for the ligand and measures the degree of reactivity. Miniaturized biosensor using nanotechnology minimizes human injury and enables painless human diagnosis and has the advantage of directly analyzing single cells. In addition, biosensors with improved operating characteristics such as high stability, fast response time, high sensitivity, and high selectivity using nanotechnology enable continuous measurement of human diagnosis and single-molecule analysis.

On the other hand, conjugated polydiacetylene vesicles have been studied as a chemical or biological sensor because of their unique color development properties. Diacetylene monomers such as 10,12-pentacosadiynoic acid (PCDA) are polymerized by 1,4-addition reaction under ultraviolet irradiation, and their form is en- yne) A chain of polymerization in which bonds appear alternately, forming vesicles similar to liposomes.

The polydiacetylene vesicles have a color expression ability that changes from blue to red due to external disturbances such as heat, pH, physical or chemical stimuli, or solvents. Furthermore, red polydiacetylene vesicles have the property of expressing fluorescence, and have been developed as various sensors using such properties. For example, polydiacetylene sensors have successfully detected influenza viruses, cholera toxicants, E. coli, oligonucleotides, lipopolysaccharides, antibodies and antigens.

However, until now, the application of polydiacetylene as a biosensor has been mostly made in an aqueous solution, so that a large amount of vesicles, antibodies, and analytes were required. Due to these limitations, the possibility of biosensors, that is, biochips, in the form of fine patterns immobilized on solid materials is being explored.

The present invention has been made to solve the above problems, and an object thereof is to provide a sensor chip in which a polydiacetylene vesicle to which a probe protein corresponding to an analyte is bound is bonded to a substrate.

It is another object of the present invention to provide a method for manufacturing the polydiacetylene sensor chip.

Another object of the present invention is to provide a polydiacetylene sensor chip manufactured by the above manufacturing method.

In order to achieve the object as described above, the polydiacetylene sensor chip of the present invention,

Board;

Vesicles bonded to the substrate and polymerized with diacetylene having a structure of Formula 1; And

Probe protein bound to the vesicles

Characterized in that it comprises:

A- (L ₁ ) _d- (CH ₂ ) _e -C≡CC≡C- (CH ₂ ) _f- (L ₂ ) _g -B (1)

In the above formula

d + g is 0, 1 or 2,

e + f is an integer from 2 to 50,

e and f are integers greater than 1,

A and B are the same or different from each other and are a methyl group, an amine group, a carboxyl group, a hydroxyl group, a maleimide group, a biotin group, a hydroxysuccinimide group, a benzoic acid group or an ester group,

L ₁ and L ₂ are the same as or different from each other and are an alkyl group having 2 or more carbon atoms, at least one ethylene oxide group, an amine group, an amide group, an ester group or a carbonyl group.

In addition, it is preferable that some of said diacetylene is A or B is a biotin group or a hydroxysuccinimide group.

Further, between the substrate and the vesicle, between the vesicle and the probe protein, or between the substrate and the vesicle and between the vesicle and the probe protein, an avidin-biotin bond, streptavidin-biotin bond, or hydride It is preferable that it is an amine substituted bond by oxysuccinimide.

In addition, the diacetylene is 10,12-pentacosadiynoic acid (PCDA) or PCDA-aniline (PCDA-aniline), and 10,12-pentacosadiinoic acid-2,2 '. -(Ethylenedioxy) -bis- (ethylamide) -succinic anhydride-normal-hydroxysuccinimide [10,12-pentacosadiynoic acid-2,2 '-(ethylenedioxy) -bis- (ethylamide)- succinic anhydride-normal-hydroxysuccinimide. PCDA-EDEA-SA-NHS].

In addition, the molar ratio of PCDA (or PCDA-aniline) to PCDA-EDEA-SA-NHS is preferably 100: 5 to 25.

In addition, the diacetylene preferably further comprises 10,12-pentacosadiynoic acid-biotin (10,12-pentacosadiynoic acid-Biotin. PCDA-biotin).

In addition, the molar ratio of PCDA (or PCDA-aniline): PCDA-EDEA-SA-NHS: PCDA-biotin is preferably 100: 3 to 15: 2 to 10.

In addition, the diacetylene is preferably a mixture of PCDA-aminobutyric acid (PCDA-ABA), and PCDA-biotin.

In addition, the molar ratio of PCDA-ABA: PCDA-Biotin is preferably 100: 5 to 25.

In addition, it is preferable that a lipid is added to the diacetylene.

In addition, the lipid is preferably 1,2-dimyristoyl-lac-glycero-3-phosphocholine (1,2-dimyristoyl- rac -glycero-3-phosphocholine.DMPC).

In addition, the lipid is preferably added in a molar ratio of diacetylene: lipid = 100: 0.001 to 70.

In addition, the probe protein is preferably an antibody.

In addition, the substrate is preferably a substrate made of glass, metal or plastic material whose surface is modified with amine, aldehyde, carboxylic acid, ester, maleimide or carbohydrate.

On the other hand, the manufacturing method of the polydiacetylene sensor chip of the present invention,

(A) dissolving the diacetylene having the structure of Formula 1 in a hydrophobic solvent and mixing it with water or a hydrophilic solvent, or directly mixing with water or a hydrophilic solvent;

(B) sonicating and dispersing the mixed diacetylene mixture;

(C) spotting the dispersed diacetylene dispersion onto a substrate; And

(D) immobilizing the instilled diacetylene dispersion

Comprising, after the step (B),

(E) reacting a probe protein with the diacetylene; And

(F) polymerizing the diacetylene by exposing ultraviolet light to the dispersed diacetylene dispersion or to the substrate dipped with the diacetylene dispersion.

Characterized in that it further comprises.

In addition, the hydrophobic solvent of step (A) is chloroform, dimethylformamide (dimethylformamide. DMF), dimethylsulfoxide (dimethylsulfoxide. DMSO), dichloromethane, hexane, cyclohexane, tetrahydrofuran (THF), acetone, Preference is given to those selected from the group consisting of alcohols and mixtures thereof.

Further, after dissolving in the hydrophobic solvent in the step (A), before mixing it with water or a hydrophilic solvent,

Removing the hydrophobic solvent by injecting nitrogen or inert gas

It is preferable to further include.

In addition, the hydrophilic solvent preferably comprises a buffer.

In addition, after mixing in water or a hydrophilic solvent of step (A) and heating for 10 to 30 minutes at 70 to 95 ℃

It is preferable to further include.

On the other hand, the polydiacetylene sensor chip of the present invention is characterized in that it is manufactured by the above method.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. In addition, in the following description there are shown a number of specific matters, such as specific components, which are provided to help a more general understanding of the present invention that the invention may be practiced without these specific matters in the art It will be self-evident to those of ordinary knowledge. In describing the present invention, when it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the subject matter of the present invention, the detailed description thereof will be omitted.

The present invention is to provide a polydiacetylene sensor chip in which a probe protein is bound to a polydiacetylene vesicle which causes color transition from blue to red by an external stimulus and exhibits red fluorescence, and the vesicle is bound to a substrate. The purpose.

Here, polydiacetylene is prepared by aligning and polymerizing a diacetylene monomer having the structure of Chemical Formula 1.

The present invention not only expresses embolism and fluorescence, but also a part of the diacetylene is biotin group or hydride at the end thereof so that the polydiacetylene vesicles that structurally mediate the substrate and the probe protein can bind to the substrate and the protein, respectively. It is preferable to have a oxysuccinimide group.

In particular, between the substrate and the vesicles, between the vesicles and the probe protein, or between the substrate and the vesicles, and between the vesicles and the probe proteins, are combined with avidin-biotin bonds, streptavidin-biotin bonds, or hydroxysuccinimides. It is preferable to be bound by amine substitution. Through this, it is possible to increase the analysis efficiency by giving a certain direction to the probe protein.

Examples of diacetylene that binds the substrate and the probe protein by avidin-biotin bond, streptavidin-biotin bond, or amine substitution by hydroxysuccinimide include PCDA-EDEA-SA-NHS (Formula 2), And PCDA-biotin (Formula 3).

These binding die-acetylenes and other diacetylenes, such as PCDA (Formula 4), PCDA-aniline (Formula 5), PCDA-ABA (Formula 6), or PCDA-mBzA (Formula 7) by mixing the die for the defoaming of the present invention Prepare the acetylene mixture.

In the mixture, the biotin group of PCDA-biotin binds to the substrate or probe protein avidin (or streptavidin), thereby binding the vesicles to the substrate or probe protein, and the hydroxysuccinimide group of PCDA-EDEA-SA-NHS The vesicles bind to the substrate or probe protein while being substituted with an amine group of the substrate or probe protein.

The diacetylene mixture of the present invention may contain, for example, PCDA when the other diacetylene: bonding diacetylene molar ratio is 100: 5 to 25, and simultaneously contains diacetylene having a hydroxysuccinimide group and diacetylene having a biotin group. When the molar ratio of PCDA-EDEA-SA-NHS: PCDA-Biotin is 100: 3 to 15: 2 to 10, the binding and analytical efficiencies were found to be high. .

Furthermore, lipids such as 1,2-dimyristoyl-lac-glycero-3-phosphocholine (1,2-dimyristoyl- rac -glycero-3-phosphocholine.DMPC. When the (lipid) was added, it was confirmed that the time required for analysis was shortened and the embolism was more clearly expressed. This is because as the lipid is added to the vesicles, the alignment of the diacetylene is disturbed and the hydrogen bonding force is weakened, which causes the vesicles to be more sensitive to external stimuli. The amount of the lipid added is preferably added in a molar ratio of diacetylene: lipid = 100: 0.001 to 70.

The probe protein is not particularly limited as long as it can detect a protein or microorganism, but is preferably an enzyme or an antibody, and in particular, an antibody.

The substrate is preferably a glass, metal or plastic substrate whose surface is modified with amines, aldehydes, carboxylic acids, esters, maleimides or carbohydrates, and particularly preferably a glass substrate with surface modifications with amines.

On the other hand, the method for producing a polydiacetylene sensor chip of the present invention, first from the step of dissolving the diacetylene having a structure of Formula 1 in a hydrophobic solvent and mixed with water or a hydrophilic solvent, or directly mixed with water or a hydrophilic solvent Begins. At this time, after dissolving in the hydrophobic solvent, it is preferable to further include the step of removing the hydrophobic solvent by injecting nitrogen or an inert gas (inert gas) before mixing it with water or hydrophilic solvent.

The hydrophobic solvent is not limited as long as it can dissolve the diacetylene, and is generally selected from the group consisting of chloroform, DMF, DMSO, dichloromethane, hexane, cyclohexane, THF, acetone, alcohol and mixtures thereof, Chloroform is particularly preferable.

The hydrophilic solvent is 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (2- [4- (2-hydroxyethyl) -1-piperazinyl] ethansulfonic acid. ), Phosphate buffered saline (PBS), 2- (N-morpholino) ethanesulfonic acid [2- (N-Morpholino) ethanesulfonic acid. MES].

In addition, by mixing in water or a hydrophilic solvent and then heated for 10 to 30 minutes at 70 to 95 ℃ aligning the diacetylene monomers can be increased the polymerization degree during the polymerization by ultraviolet irradiation.

The mixed diacetylene mixture is then sonicated to disperse. The sonicated diacetylene dispersion is filtered and stabilized at low temperature. In this case, the dispersion may be directly irradiated with ultraviolet rays to photopolymerize, but in consideration of the polymerization efficiency, it is more preferable to perform polymerization after spotting on the substrate.

Fixing the die acetylene vesicles to the substrate is as follows.

First, when the substrate and the vesicles are connected by an avidin or streptavidin-biotin bond, a compound having a biotin group is reacted with a amine-modified substrate to expose the biotin group from the substrate, and then the exposed biotin group is avidin or Reacting with streptavidin causes the final exposure of avidin or streptavidin from the substrate. The previously prepared diacetylene dispersion is added to the substrate, and the vesicles are immobilized on the substrate by combining the biotin group of the diacetylene and avidin or streptavidin exposed from the substrate. It is particularly preferable that the compound having a biotin group is Biotin-normal-hydroxysuccinimide (Biotin-NHS).

In the case where the substrate and the vesicles are directly connected by the amine substitution bond of the hydroxysuccinimide, the immobilization is directly performed by replacing the hydroxysuccinimide group of the vesicle with the amine of the substrate.

On the other hand, the probe protein that reacts directly with the analyte can bind to any of the dispersed diacetylene dispersion, the diacetylene vesicle immobilized on the substrate, or the polydiacetylene vesicle formed by photopolymerization by UV irradiation, It is preferable in terms of binding efficiency to mix and bind the probe protein in the diacetylene dispersion state before immobilization.

Between the vesicle and the probe protein, the amine group of the probe protein is linked by substituting the hydroxysuccinimide group of the vesicle.

Or by reacting the probe protein with a biotin-containing compound such as biotin-NHS so that the biotin group is located at the end, and binding the separate avidin or streptavidin to the biotin group of the vesicle, resulting in It is also possible to bind the biotin of the probe. This binding method has the advantage that the reaction rate of avidin (or streptavidin) and biotin is fast, and that the orientation of the probe can be kept constant on the vesicles, thereby improving reactivity with the analyte and analyzing. This results in higher efficiency.

Figure 1 is a schematic diagram of an embodiment for the production of the polydiacetylene sensor chip and analyte detection of the present invention.

The vesicles at the top are vesicles before polymerization, in which the biotin group and the hydroxysuccinimide group are exposed, and do not develop color and fluorescence.

When this is combined with a primary antibody as a probe protein, the substrate exposed to avidin is irradiated and irradiated with ultraviolet light, and the biotin group of the vesicles binds to the avidin of the substrate, and the amine succinimide group of the vesicles is replaced by the amine group of the primary antibody. Combined, and the resulting vesicles became blue (interrupted).

Passing the secondary antibody as an analyte therein, the secondary antibody reacts with the primary antibody bound to the vesicles, as a result the vesicles are embolized in red, the fluorescence is expressed to enable analysis.

EMBODIMENT OF THE INVENTION Hereinafter, the Example of this invention is described.

Example

Example  1-1: PCDA - EDEA - SA - NHS  Produce

PCDA-EDEA-SA-NHS, one of the diacetylenes containing a hydroxysuccinimide group, was synthesized as follows.

PCDA-EDEA (J.-M. Kim, E.-K. Ji, S.-M. Woo, H.-W. Lee, terminally substituted with amine in 30 ml of dimethylformamide. , and DJ Ahn, “Immobilized polydiacetylene vesicles on solid substrates for use as chemosensors,” Advanced Materials , 15, 1118-1121 (2003)) A solution containing 1 g (1.98 mmol) was prepared first, which was also dissolved in 0.792 g (7.92 mmol) of succinic anhydride in 30 ml DMF. Dropwise into the solution and stir at room temperature for one day. The residue obtained by concentration in vacuo was then dissolved in ethyl acetate and extracted with water. The organic layer thus obtained was collected and concentrated to give a white powder (PCDA-EDEA-SA) (1.07 g, 89%).

The powder obtained (0.40 g, 0.66 mmol PCDA-EDEA-SA) was then dissolved in 30 ml chloroform, to which 0.23 g (1.98 mmol) normal-hydroxysuccinimide and 0.51 mg (2.64 mmol) ( 1-ethyldimethylaminopropyl) -carbodiimide ((1-ethyldimethylaminopropyl) -carbodiimide.EDC) was put together and stirred at 0 ° C. for 1 day. Also concentrated in vacuo, dissolved in ethyl acetate and the solution was extracted with water. The organic layer at that time was collected and concentrated to finally obtain PCDA-EDEA-SA-NHS powder (0.24 g, 51%).

Example  1-2: PCDA -Aniline manufacturing

One of the other diacetylenes, PCDA-aniline, was synthesized as follows.

0.5 g (1.33 mmol) of PCDA was added to 5 ml of methylene chloride, and 0.33 ml (3.99 mmol) of oxalyl chloride was added to the solution under nitrogen gas at room temperature for 30 minutes. Was dropped.

It was concentrated in vacuo and then dissolved in 5 ml of methylene chloride again. It was added dropwise to a solution of 0.3 ml (3.2 mmol) of aniline and 0.44 ml (3.2 mmol) of triethanolamine (TEA) in 20 ml of methylene chloride at room temperature and stirred overnight.

It was then concentrated in vacuo again and the resulting residue was dissolved again in methylene chloride and the solution was extracted with water. The resulting organic layer was collected and concentrated, and the residue was purified by chromatography using silica, eluted again with methylene chloride, and recrystallized to give 0.5 g (38.2%) of PCDA-aniline powder. .

Example  2-1: Parcel ( 小 胞 . vesicle Manufacturing (1)

PCDA (GFS chemicals), PCDA-EDEA-SA-NHS (Example 1-1), PCDA-Biotin (YK Jung, HG Park, and JM Kim, "Polydiacetylene (PDA) -based colorimetric detection of biotinstreptavidin interactions", Biosens .. Bioelectron, 21, 1536-1544 ( 2006)) of 8.5: 1 were mixed in a molar ratio of 0.5, it was dissolved in chloroform. Nitrogen was injected into the dissolved mixture to remove the chloroform. A buffer solution (HEPES, 5 mM, pH = 8.0) was added thereto so that the total concentration of PCDA and the like was 1.0 mM, and heated at 80 ° C. for 15 minutes. The mixture was sonicated for 15 minutes (Fisher Sonic Dismembrator Model 550 W, 25% of power), the monomers were aligned and filtered through a 0.8 μm PTFE filter and cooled at 4 ° C. for 12 hours.

Example  2-2: Parcel Manufacturing (2)

The same procedure as in Example 2-1 was performed, except that PCDA-aniline (Example 1-2) was used instead of PCDA.

Example  2-3: Parcel Manufacturing (3)

PCDA-s-Biotin (YK Jung, HG Park, and JM Kim, "Polydiacetylene (PDA) -based colorimetric detection of biotinstreptavidin interactions", Biosens. Bioelectron . , 21, 1536-1544 (2006)) 0.548325 mg and PCDA-ABA (J.-M. Kim, J.-S. Lee, H. Choi, D. Sohn, and DJ Ahn, "Rational design and in-situ FTIR analyzes of colorimetrically reversible polydiacetylene supramolecules", Macromolecules , 38, 9366-9376 (2005)) 4.48227 mg were mixed in 1 ml of DMSO (Junsei), and then dissolved by heating at 80 ° C. Separately, 3.051 mg of DMPC (Sigma) was added to an Erlenmeyer flask and dissolved in 1 ml of chloroform. When the DMPC was completely dissolved, nitrogen was blown to form a thin film at the bottom of the Erlenmeyer flask. Distilled water was filled to a concentration of 1 mM, followed by stirring in water at 80 ° C. for 15 minutes to disperse the monomer well. At this time, the prepared PCDA-s-Biotin solution and PCDA-ABA solution were added little by little (PCDA-ABA: DMPC: PCDA-s-Biotin = 6.5: 3: 0.5). At the end of the water bath, the solution in which the monomers were dispersed was sonicated for 15 minutes at a intensity of 10 W (Fisher Sonic Dismembrator Model 550 W, 25% of power). After the sonication was filtered with a nylon filter of 0.8 ㎛ and refrigerated at 4 ℃ for more than 8 hours.

Example  2-4: Parcel Manufacturing (4)

The same procedure as in Example 2-1 was performed, except that PCDA-biotin was not used (PCDA: PCDA-EDEA-SA-NHS = 9: 1).

Example  3: Glass slide ( Avidin ) Ready

1 mg Biotin-NHS (Pierce) was dissolved in 1 ml DMSO (Junsei) and then mixed with 3 ml PBS buffer (Pierce). The amine coated glass slide (NC-AMCS, Nuricell co.) Was immersed at room temperature for 4 hours. Separately, 0.1 mg of avidin (Sigma) was dissolved in 1 ml of a PBS solution to prepare an avidin solution, and the glass slide was immersed in an avidin solution at room temperature for 2 hours to prepare a glass slide for use in the present invention.

Example  4-1: Probe  Protein Binding and Immobilization (1)

To 200 µl of the vesicle solution of Example 2-1, 6 µl of a 1.0 mg / ml first antibody (E. coli surface protein detection rabbit-producing antibody. Fitzgerald Industries International, Inc.) solution was added and incubated at room temperature for 4 hours. . The vesicle solution to which the first antibody was bound was dipped into the avidin coated glass slide of Example 3 (Nano-plotter v 1.2, GeSim, German). After immobilization at 37 ° C. for 2 hours, the mixture was washed with distilled water for 1 minute, and then irradiated with 254 nm UV of 1 mW / cm 2 for 3 minutes to polymerize the antifoam monomer.

Example  4-2: Probe  Protein Binding and Immobilization (2)

To 200 μl of the vesicle solution of Example 2-2, 6 μl of a 1.0 mg / ml first antibody (anti-E. Coli antibody. Fitzgerald Industries International, Inc.) solution was added and incubated at room temperature for 4 hours. The vesicle solution to which the first antibody was bound was dipped into the avidin coated glass slide of Example 3 (Nano-plotter v 1.2, GeSim, German). After immobilization at 37 ° C. for 2 hours, the mixture was washed with distilled water for 1 minute, and then irradiated with 254 nm UV of 1 mW / cm 2 for 8 minutes to polymerize the antifoam monomer.

Example  4-3: Probe  Protein Binding and Immobilization (3)

The antifoam solution of Example 2-3 was added to the avidin coated glass slide of Example 3 (Nano-plotter v 1.2, GeSim, German), immobilized at 37 ° C. for 2 hours, and then washed with distilled water for 1 minute. 10 μl of a solution of the first antibody (E. coli antibody. Fitzgerald Industries International, Inc.) was sprinkled on the glass slide, covered with a cover glass, incubated at room temperature for 2 hours, and then washed again with distilled water. It was irradiated with 254 nm UV of 1 mW / cm 2 for 8 minutes to polymerize the antifoam monomer.

Example  4-4: Probe  Protein Binding and Immobilization (4)

The antifoam solution of Examples 2-4 was added dropwise to an amine coated glass slide (Nano-plotter v 1.2, GeSim, German), immobilized at 37 ° C. for 2 hours, and then washed with distilled water for 1 minute. It was irradiated with 254 nm UV of 1 mW / cm 2 for 3 minutes to polymerize the antifoam monomer. Here, 6 μl of a 2.0 mg / ml first antibody (anti- Bacillus antracis spore antibody.Abcam) solution was sprayed onto the glass slide, covered with a cover glass, incubated at room temperature for 1 hour, and then distilled water again. Washed with.

Example  5-1: Second antibody detection

7 μl of 150 μg / ml fluorescent isothiocyanate (FITC) -conjugated anti-rabbit second antibody (Sigma Aldrich Co.) was dispersed on the glass slide of Example 4-1. Covered with cover glass, incubated at room temperature for 4 hours, washed three times with distilled water and observed embolism and fluorescence (Olympus BX51).

Example  5-2: Coliform Detection (1)

E. coli (10 ⁸ CFU / ml) and Bacillus 7 μl of subtilis (10 ⁸ CFU / ml) was dispersed on the glass slide of Example 4-2, covered with cover glass, incubated at room temperature for 12 hours, washed three times with distilled water, and embolized and fluorescence Was observed (Olympus BX51).

Example  5-3: Coliform Detection (2)

E. coli , Bacillus subtilis , Salmonella 7 μl each of typhimurium , and Listeria monocytogenes (10 ⁸ CFU / ml each) was dispersed on the glass slide of Example 4-3, covered with cover glass, incubated at room temperature for 2 hours, and then washed three times with distilled water. Embolization and fluorescence were observed (Olympus BX51).

Example  5-4: Anthrax Detection

Bacillus 7 μl of anthracis (Athrax, ATCC 14578. 10 ⁸ CFU / ml spore state) and Bacillus subtilis (10 ⁸ CFU / ml spore state) were dispersed on the glass slide of Example 4-4, and then covered with cover glass. After incubation for 5 hours at room temperature, washed three times with distilled water and observed the embolism and fluorescence (Olympus BX51).

Comparative example  1: first antibody In case of lack

Examples 1-1, 2-1, 3, 4-1 and 5-1, but the reaction process of the vesicle solution and the first antibody solution in Example 4-1 was omitted.

Comparative example  2: Antibiotic mice  When using a second antibody

Example 1-1, 2-1, 3, 4-1 and 5-1, but in Example 5-1 anti-rabbit second antibody was used in place of the anti-rabbit second antibody.

Test Example  1: 1st antibody-in presence of 2nd antibody

Figure 2 is a photograph of the result of the process of Example 1-1, 2-1, 3, 4-1 and 5-1. Figure 2 (A) is a fluorescence picture before reacting the second antibody, it can be seen that no fluorescence appears. The second antibody of Example 5-1 is labeled with fluorescence and can be monitored with a green filter at 515 nm. Therefore, when the second antibody is reacted and then observed with a green filter, green fluorescence can be observed as shown in FIG. 2 (B). In addition, red fluorescence is also expressed as a result of disturbance of the vesicles of the present invention due to the binding of the second antibody. As a result of monitoring with a red filter at 580 nm, red fluorescence is observed as shown in FIG. 2 (C). Could. In addition, in order to confirm the immobilization of the vesicles of the present invention, the glass slide was heated for 5 minutes and observed with a red filter.

Test Example  2: first antibody In case of lack

As a result of monitoring the red filter at 580 nm after performing the procedure of Comparative Example 1, even after the second antibody treatment, it can be seen that no fluorescence was expressed as in Figure 3 (A). Heating for 5 minutes and monitoring with a red filter shows that the vesicle itself is immobilized as shown in FIG. 3 (B).

Test Example  3: Antibiotic mice  When using a second antibody

After performing the procedure of Comparative Example 2 was monitored with a green filter at 515 nm [Fig. 4 (A)] and a red filter at 580 nm [Fig. 4 (B)], no fluorescence was observed. Heating for 5 minutes and monitoring with a red filter shows that the vesicles themselves are immobilized as shown in FIG. 4 (C).

Test Example  4: 1st antibody-Escherichia coli present (1)

Figure 5 is a photograph of the result of the process of Example 1-2, 2-2, 3, 4-2 and 5-2. The photo on the upper left showed embolism in red as a result of disturbance of the vesicles of the present invention due to the binding of E. coli , but Bacillus In the photo on the upper right of the subtilis reaction, no binding to the bacterial cells occurs, indicating that the vesicles were not disturbed and the embolism did not occur. In order to confirm the fixation of the vesicles of the present invention, the glass slide was heated and observed for 5 minutes, and as shown in the bottom of FIG. 5, the color transition to red as a result of the vesicle disturbance was observed in both.

Test Example  5: primary antibody in the presence of E. coli (2)

Figure 6 is a photograph of the result of the process of Example 2-3, 3, 4-3 and 5-3. The photo on the upper left showed embolism in red as a result of disturbance of the vesicles of the present invention due to the binding of E. coli , but Bacillus subtilis , Salmonella typhimurium , or Listeria The rest of the photographs reacted with monocytogenes showed no binding to the bacterial cells, leading to no disturbance of the vesicles and consequently no embolism.

Test Example  6: 1st antibody-in presence of anthrax

Figure 7 is a photograph of the result of the process of Example 2-4, 4-4 and 5-4. The left picture is the Bacillus As a result of disturbance of the vesicles of the present invention due to the binding of anthracis , embolization occurred in red, but Bacillus In the photo on the right of the subtilis reaction, no binding to the bacterial cells was observed, indicating that the vesicles were not disturbed and the embolism did not occur.

According to the results of the test examples, the embolization and fluorescence expression of the vesicles can be caused only by specific binding of the first antibody and the second antibody or the first antibody and the microorganism, and thus the polydiacetylene sensor chip of the microarray type of the present invention May be utilized as a biosensor to detect protein-protein or protein-microbial level responses. In particular, anthrax is easy to obtain and resistant to disinfectants and environmental changes, so there is a high risk of being converted into biochemical weapons. Therefore, a rapid, simple and accurate analysis method has been strongly demanded. It seems to be able to meet.

In the present invention, the experiment was conducted with the first antibody and the second antibody or the first antibody and the bacteria, but if the protein is properly selected, cholera toxicants, phospholipases, HIV protease, MAP kinase (MAP) kinases, MEK kinases, Phosphoinositide 3-kinases, Factor X, PDGF, FGF, ICAM, VCAM, E-selectin, Thrombin ( thrombin), Bradykinin, proteins such as PGF2, Escherichia coli, Anthrax, Bacillus, Salmonella, Listeria, Mycobacterium tuberculosis, Brucellella, Legionella, Vibrio, Microorganism cells, Flu virus, Hepatitis virus, AIDS virus Viruses such as Hantan virus and rotavirus can be detected in the same manner.

Although the above has been illustrated and described with respect to the preferred embodiments of the present invention, the present invention is not limited to the specific embodiments described above, those skilled in the art without departing from the gist of the present invention various modifications Of course, implementation is possible. Therefore, the scope of the present invention should not be construed as being limited to the above embodiments, but should be defined by the claims below and equivalents thereof.

According to the present invention, a protein as a probe for detecting a protein or a cell can be bound to a polydiacetylene vesicle, and the sensor chip can be manufactured by dropping and immobilizing the vesicle in the form of a microarray on a glass slide. Only by specific binding of proteins and proteins (e.g., first and second antibodies) or proteins and cells (e.g., antibodies and cells), vesicle embolization and fluorescence can be induced, resulting in the microarray form of the present invention. The diacetylene sensor chip is capable of detecting protein-protein or protein-cell level reactions without a separate label for the analyte and with a much smaller amount of polydiacetylene and analyte than conventional liquid phase reactions. Furthermore, it also has the advantage of being portable while achieving miniaturization while showing high sensitivity. In particular, it is easy to obtain and strong resistance to disinfectants and environmental changes, it is confirmed that it is possible to accurately analyze anthrax, which is highly applicable to biochemical weapons in a short time, and thus it is considered to be very useful in diagnostic fields.

Claims (19)

Board; Vesicles bonded to the substrate and polymerized with diacetylene having a structure of Formula 1; And Probe protein bound to the vesicles Polydiacetylene sensor chip comprising: [Formula 1] A- (L ₁ ) _d- (CH ₂ ) _e -C≡CC≡C- (CH ₂ ) _f- (L ₂ ) _g -B (1) In the above formula d + g is 0, 1 or 2, e + f is an integer from 2 to 50, e and f are integers greater than 1, A and B are the same or different from each other and are a methyl group, an amine group, a carboxyl group, a hydroxyl group, a maleimide group, a biotin group, a hydroxysuccinimide group, a benzoic acid group or an ester group, L ₁ and L ₂ are the same as or different from each other and are an alkyl group having 2 or more carbon atoms, at least one ethylene oxide group, an amine group, an amide group, an ester group or a carbonyl group. The method of claim 1, Part of the diacetylene is a polydiacetylene sensor chip, characterized in that A or B is a biotin group or hydroxysuccinimide group. The method of claim 1, Between the substrate and the vesicle, between the vesicle and the probe protein, or between the substrate and the vesicle and between the vesicle and the probe protein, an avidin-biotin bond, streptavidin-biotin bond, or hydroxysuccine Polydiacetylene sensor chip, characterized in that the amine substituted bond by the imide. The method of claim 1, The diacetylene is 10,12-pentacosadiynoic acid (PCDA) or PCDA-aniline (PCDA-aniline), and 10,12-pentacosadiinoic acid-2,2 '-( Ethylenedioxy) -bis- (ethylamide) -succinic anhydride-normal-hydroxysuccinimide [10,12-pentacosadiynoic acid-2,2 '-(ethylenedioxy) -bis- (ethylamide) -succinic anhydride -normal-hydroxysuccinimide. PCDA-EDEA-SA-NHS]. The method of claim 4, wherein The polydiacetylene sensor chip, characterized in that the molar ratio of PCDA (or PCDA-aniline): PCDA-EDEA-SA-NHS is 100: 5 to 25. The method of claim 4, wherein The diacetylene is a poly acetylene sensor chip further comprises 10,12-pentacosadiinoic acid-biotin (10,12-pentacosadiynoic acid-Biotin. PCDA-biotin). The method of claim 6, Wherein the molar ratio of the PCDA: PCDA-EDEA-SA-NHS: PCDA-biotin is 100: 3 to 15: 2 to 10, characterized in that the poly acetylene sensor chip. The method of claim 1, The diacetylene is a polydiacetylene sensor chip, characterized in that a mixture of PCDA-aminobutyric acid (PCDA-ABA), and PCDA-biotin. The method of claim 8, The molar ratio of PCDA-ABA: PCDA-biotin is 100: 5 to 25, characterized in that the poly acetylene sensor chip. The method of claim 1, Polydiacetylene sensor chip, characterized in that the lipid (lipid) is added to the diacetylene. The method of claim 10, Wherein said lipid is 1,2-dimyristoyl-lac-glycero-3-phosphocholine (1,2-dimyristoyl- rac -glycero-3-phosphocholine. DMPC). The method of claim 10, The lipid is poly acetylene sensor chip, characterized in that the addition of a mole ratio of diacetylene: lipid = 100: 0.001 to 70. The method of claim 1, The probe protein is a polydiacetylene sensor chip, characterized in that the antibody. The method of claim 1, The substrate is a polydiacetylene sensor chip, characterized in that the substrate is a glass, metal or plastic material surface is modified with amine, aldehyde, carboxylic acid, ester, maleimide or carbohydrate. (A) dissolving diacetylene having a structure of Formula 1 in a hydrophobic solvent and mixing it with water or a hydrophilic solvent, or directly mixing with water or a hydrophilic solvent: [Formula 1] A- (L ₁ ) _d- (CH ₂ ) _e -C≡CC≡C- (CH ₂ ) _f- (L ₂ ) _g -B (1) In the above formula d + g is 0, 1 or 2, e + f is an integer from 2 to 50, e and f are integers greater than 1, A and B are the same or different from each other and are a methyl group, an amine group, a carboxyl group, a hydroxy group, a maleimide group, a biotin group, a hydroxysuccinimide group, a benzoic acid group or an activated ester group, L ₁ and L ₂ are the same as or different from each other and are an alkyl group having 2 or more carbon atoms, at least one ethylene oxide group, an amine group, an amide group, an ester group or a carbonyl group; (B) sonicating and dispersing the mixed diacetylene mixture; (C) spotting the dispersed diacetylene dispersion onto a substrate; And (D) immobilizing the instilled diacetylene dispersion Comprising, after the step (B), (E) reacting a probe protein with the diacetylene; And (F) polymerizing the diacetylene by exposing ultraviolet light to the dispersed diacetylene dispersion or to the substrate dipped with the diacetylene dispersion. Method for producing a polydiacetylene sensor chip further comprising. The method of claim 15, After dissolving in the hydrophobic solvent in step (A), before mixing it in water or hydrophilic solvent, Removing the hydrophobic solvent by injecting nitrogen or inert gas Method for producing a poly acetylene sensor chip, characterized in that it further comprises. The method of claim 15, The hydrophilic solvent is a method for producing a polydiacetylene sensor chip, characterized in that it comprises a buffer. The method of claim 15, Mixing with water or hydrophilic solvent of step (A) and then heating at 70-95 ° C. for 10-30 minutes Method for producing a poly acetylene sensor chip, characterized in that it further comprises. A polydiacetylene sensor chip manufactured by the method of any one of claims 15 to 18.

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