KR20080085618A - Array-based transglutaminase activity assay chip and transglutaminase activity assay method - Google Patents

Abstract

A method for analyzing transglutaminase activity is provided to reduce the analysis time by simply measuring the transglutaminase activity by using an array-based assay chip, and decrease the amount of sample used, so that the method is used for identifying mechanisms occurred in cells. A method for analyzing transglutaminase activity comprises the steps of: introducing alkoxysilane with a function group in the end to the surface of a substrate; attaching a tape with pores at regular distances to the substrate surface to prepare an array chip with a plurality of spots; introducing a substrate protein of transglutaminase in ends of the spots; adding dropwise of a sample containing transglutaminase for activity analysis, and analysis solution containing (5-(biotinamido)pentylamine) DTT(dithiothreitol) and CaCl2 on the substrate protein to induce transamidation of (5-(biotinamido)pentylamine) with the substrate protein; and introducing a target material for analysis to biotin located in the end by the transamidation, and analyzing the target material. Further, the alkoxysilane is selected from aminoalkyltrialkoxysilane, mercaptoalkyltrialkoxysilane or epoxyalkyltrialkoxysilane.

Description

Array-based transglutaminase activity assay chip and transglutaminase activity assay method}

1a and 1b are graphs showing the results of measuring the activity of the transglutaminase according to Example 1 of the present invention and the fluorescence intensity of the measured results.

2A and 2B are graphs showing the results of measuring the activity of transglutaminase and the fluorescence intensity of the measured results according to Example 2 of the present invention.

3A and 3B are graphs showing the results of measuring the activity of transglutaminase and the fluorescence intensity of the measured results according to Example 3 of the present invention.

4A and 4B are graphs showing the results of measuring the activity of transglutaminase in cell extracts isolated from cells according to Example 4 of the present invention.

5a and 5b are graphs showing the results of measuring the activity of the transglutaminase in the blood serum unfavorable according to Example 5 of the present invention.

The present invention relates to a technique for analyzing the activity of transglutaminase known to play a key role in various diseases, such as degenerative brain disease, degenerative arthritis, autoimmune diseases such as dementia, The present invention relates to a chip for measuring the activity of transglutaminase and a method for measuring the activity of transglutaminase, which enables not only environmentally friendly analysis but also high sensitivity analysis even with a small sample.

Transglutaminase (TGase) catalyzes the covalent interaction of lysine (or polyamine) with glutamine and is involved in a variety of diseases, particularly neurological diseases such as degenerative brain diseases such as dementia, degenerative arthritis and autoimmune diseases. It is known. TGase is an enzyme that acts as a protective agent for blood clotting and wound healing under normal circumstances.

However, TGase is known to play a major role in the pathological mechanism of many diseases when expression is unregulated. In particular, TGase activity is increased in diseases with inflammation, such as degenerative arthritis, diabetes mellitus, autoimmune myositis, arteriosclerosis, stroke, liver cirrhosis, malignant breast cancer, meningitis and inflammatory gastric ulcer. In addition, TGase activity has been reported to be elevated in dementia.

Therefore, it is very important to analyze the activity of TGase. Conventionally, TGase activity measurement method generally used is a method of using a radioisotope ³ H and Western blot (Western blot) method.

First, the method using the radioisotope ³ H is measured by activating TGase and measuring [ ³ H] when an amine exchange reaction occurs between a casein-like substrate protein (substrate) and [ ³ H] putrescine. By analyzing the TGase activity. However, this method of measuring TGase activity has a number of problems because it uses radioisotopes, which may cause environmental pollution and risks in the course of the experiment.

Western blot method amide transamidation of the substrate protein and 5- (biotinamido) pentylamine, which in turn is HRP-streptavidin (Horradish Peroxidase-streptavidin). And TGase activity through the detection of streptavidin. However, this method takes a long time, requires a large amount of sample, and difficult to analyze several samples simultaneously because it has to go through SDS-PAGE and immunooblot. In addition, due to its low sensitivity, TGase activity analysis is difficult at low concentrations.

Accordingly, the present invention is to produce an array chip to measure the activity of transglutaminase (TGase), and to analyze the activity of TGase using it, thereby analyzing a number of samples at the same time environmentally friendly In particular, a method of manufacturing a chip for measuring activity of transglutaminase, which can measure high sensitivity with only a small amount of sample and can drastically shorten the time required for the final analysis, and the transglutami produced by the method It is an object of the present invention to provide a chip for measuring the activity of Naase.

In addition, the present invention has another object to provide a method for measuring the activity of transglutaminase.

In order to achieve the above object, the present invention includes the steps of introducing an alkoxysilane having a functional group at the terminal to the surface of the substrate; Manufacturing an array chip having a plurality of spots formed by attaching a perforated tape at predetermined intervals on a surface of the substrate; It provides a method for producing a chip for transglutaminase activity measurement, comprising the step of introducing a substrate protein of trans glutaminase at the end of the spot.

In addition, the present invention is produced by the above production method, a plurality of spots are formed by attaching a perforated tape at predetermined intervals on the surface of the substrate introduced alkoxysilane having a functional group at the end, Provided is a chip for transglutaminase activity measurement, characterized in that by introducing a substrate protein of transglutaminase at the end.

In addition, the present invention comprises the steps of introducing an alkoxysilane having a functional group at the terminal to the surface of the substrate; Manufacturing an array chip having a plurality of spots formed by attaching a perforated tape at predetermined intervals on a surface of the substrate; Introducing a substrate protein of trans glutaminase at an end of said spot; A transglutaminase-containing sample for measuring activity and a measurement solution containing 5- (biotinamido) pentylamine and CaCl ₂ were added dropwise onto the substrate protein. Amine transamidation of 5- (biotinamido) pentylamine; Measuring the activity of transglutaminase by introducing an analytical labeling material into biotin positioned at the end by the amine exchange reaction and then measuring the activity of transglutaminase through its analysis; Provide a method.

Hereinafter, a chip for measuring activity and a method for measuring activity of a transglutaminase (TGase) according to the present invention will be described in more detail.

The present invention provides a chip-based measuring method that is completely different from the method of using ³ H, which is a conventionally used radioisotope, and a Western blot method to measure TGase activity.

To this end, the present invention provides a method for manufacturing a chip for measuring TGase activity consisting of a predetermined step.

To this end, a step of first introducing an alkoxysilane having a functional group at the end of the substrate surface.

Here, the alkoxysilane having a functional group at the terminal may be selected from aminoalkyltrialkoxysilane, mercaptoalkyltrialkoxysilane, or epoxyalkyltrialkoxysilane.

The aminoalkyltrialkoxysilanes are 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-2-2-aminoethylaminoethylaminopropyltrimethoxysilane (3- [2- (2-aminoethylamino) ethylamino] propyl-trimethoxysilane) may be used, and as mercaptoalkyltrialkoxysilane, 3-mercaptopropyltrimethoxysilane may be used. As the epoxy alkyltrialkoxysilane, 3-glycidoxypropyltrimethoxysilane may be used.

The alkoxysilane having a functional group at this end can be introduced to the surface of the substrate by applying various known methods. In the present invention, the alkoxysilane is dissolved in a common alcohol solvent such as ethanol, and then the substrate is deposited in this solution. An alkoxysilane having a functional group at the terminal was introduced to the surface of the substrate. At this time, in consideration of the convenience of operation, the alkoxysilane having a functional group in the ethanol was dissolved so as to contain 1 to 2% by weight, but the concentration is not necessarily limited.

Submersion time of the substrate is sufficient to carry out at least 2 hours, and after about 2 hours, the substrate is washed with ethanol, distilled water and the like and dried to introduce an alkoxysilane having a functional group at the end of the substrate.

In this case, a metal substrate or a slide glass substrate widely used in the art may be used, and the substrate may be used after being sufficiently cleaned before use. The cleaning method may be applied to a method generally used in the art, and in the present invention, a slide glass washed by dipping at least 10 minutes in a cleaning liquid mixed with 10 to 40 parts by weight of hydrogen peroxide and 10 to 40 parts by weight of ammonia with respect to 100 parts by weight of distilled water. Was used.

When an alkoxysilane having a functional group is introduced into the surface of the substrate, a perforated tape is attached to the surface at predetermined intervals to produce an array chip having a plurality of spots formed thereon.

The number of spots can be arbitrarily selected, and a tape with less reactive Teflon tape can be used.

Thereafter, the step of introducing the substrate protein of trans glutaminase at the end of the spot.

Substrate proteins of transglutaminase include casein, aldolase A, 3-glyaldehyde phosphate dehydrogenase, phosphorylase kinases Phosphorylase kinase, Crystallins, Glutathione S-transferase, Cytoskeletal proteins, Actin, Myosin, Troponin ), Beta-tubulin (β-Tubullin), Tau, Rho A, Rac1, Histone H2B, alphaoxoglutarate dehydrogenase (α-Oxoglutarate dehydrogenase), Cytocromes, Erythrocyte band III, CD38, CD4, Acetylcholine esterase, Collagen, Fibronectin, Fibrinogen, Vitronectin (Vitronectin), Osteopontin, Nidogen ogen), laminin (Laminin), El TV P 1 (LTBP-10, austenite O nektin (Osteonectin), osteocalcin (Osteocalcin), Sup Stan's P (Substance P), Phospholipase A ₂ (Phopholipase A _2), Mead Midkine, Wheat gliadin, references therein, whey proteins, soy protein, Pea legumin, Candia albicans surface protein Candida albicans surface proteins, HIV envelope glycoproteins gp120 and gp41, HIV aspartyl proteinase or Hepatitis C virus core protein) can be used as a substrate of a known transglutaminase. If necessary, a substance that can be used as a substrate for transglutaminase may be used in addition to the protein, and there is no need to limit the protein.

Various methods can be used to introduce the substrate protein. In the present invention, the substrate protein is introduced by dropping the substrate protein-containing solution prepared by dissolving the substrate protein in a spot. In this case, the reaction is performed by reacting a functional group of an alkoxysilane having a functional group at the terminal with a substrate protein. The reaction may be performed by dropping the substrate protein-containing solution for at least 2 hours. After two hours, the reaction is completed, washed with PBST or distilled water and dried, the substrate protein is introduced.

As a solution for dissolving the substrate protein, a buffer solution may be used typically, and various solutions may be applied as necessary. In the present invention, the phosphate buffer solution or Tris-HCl buffer solution prepared in the range of pH 4-9 was used, but this can be variously modified as necessary, and the concentration of the substrate protein need not be limited, but in the present invention, The dissolved material was used so as to contain 0.1 to 1% by weight.

If necessary, the medium may be reacted with an alkoxysilane in a spot formed by attaching a tape before introducing the substrate protein, and the substrate protein may be introduced onto the medium in order to facilitate the introduction of the substrate protein.

Here, the medium may be a carboxylate modified latex bead (carboxylate modified latex bead) or a compound having an aldehyde group that has a functional group at both ends and reacts with the substrate protein at at least one end.

First, using the latex bead modified with a carboxyl group as a medium will be described. The carboxyl group-modified latex beads may be purchased and used in general, and the size may be applied to those commonly used in the art, that is, in the field of protein analysis chips. For reference, commercially available modified latex beads include amine modified latex beads, carboxyl modified latex beads, sulfate modified latex beads, and the like. In the present invention, carboxyl modified latex beads having a size of 500 to 1000 nm (Molecular Probes, Eugene, OR, USA).

For smooth reaction, carboxyl-modified latex beads are reacted in the presence of N-hydroxysuccinimide and carbodiimide to be activated, and then the reaction solution is added dropwise onto the spot to react the latex beads at the amine end of the alkoxysilane. It is good to be introduced.

Here, carbodiimide plays a role of activating a carboxyl group as a catalyst so that N-hydroxysuccinimide can be easily substituted at the carboxyl group (-OH) of latex beads. The carbodiimide may be N-ethyl-N '-(3-dimethylaminopropyl) carbodiimide hydrochloride (N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC) generally used in the art. N, N'-dicyclohexyl carbodiimide (DCC) or diisopropylcarbodiimide may be used.

The reaction is carried out by adding a latex bead modified with a carboxyl group to a mixed solution of N-hydroxysuccinimide and carbodiimide, and then dropping the solution into a spot and reacting for about 20 minutes. At this time, the addition amount of the carboxyl group-modified latex beads added to the mixed solution of N-hydroxysuccinimide and carbodiimide is not necessarily limited, but in the present invention, the mixture of N-hydroxysuccinimide and carbodiimide 20 to 50 parts by weight of latex beads modified with a carboxyl group were added to 100 parts by weight of the solution. In addition, carbodiimide may be used by mixing N-hydroxysuccinimide with various equivalent ratios, and in the present invention, a mixture of 2 to 4: 1 equivalent ratios is used, but various changes are necessary. It is possible.

After the reaction, the latex beads introduced substrate is washed with PBST (Phosphate buffered saline with 0.1 wt% Tween 20; a solution prepared by adding 0.1 wt% of Tween 20 in phosphate buffered saline) or distilled water and dried. Latex beads are introduced into the amino group terminal of the alkoxysilane.

When the latex beads are introduced as described above, the substrate protein-containing solution is added dropwise onto the latex beads to introduce the substrate protein. At this time, the introduction of the substrate protein on the latex is made by the reaction of the amine group included in the substrate protein with the carboxyl group of the latex beads. Therefore, in order to introduce the matrix protein into the latex beads more easily in the same manner as the reaction between the amine group of the alkoxysilane group and the carboxyl group of the latex beads, a mixed solution of N-hydroxysuccinimide and carbodiimide is added dropwise onto the latex beads. After activating the carboxyl group of the beads, it is preferable to drop the dissolved substrate protein therein.

As a medium for introducing the substrate protein, a compound having an aldehyde group reacting with the substrate protein at at least one terminal while having functional groups at both ends may be used.

As the aldehyde compound of this kind, one selected from glutaraldehyde and benzene-1-4-dicarboxyaldehyde can be used.

As a method of introducing such an aldehyde compound, various methods may be applied. In the simplest method, a method prepared by diluting the aldehyde compound may be added dropwise to a spot to react. The reaction may be carried out for at least 30 minutes after dropping the solution. When the reaction is completed after about 30 minutes, the reaction may be easily carried out by washing with PBST or distilled water and then drying the aldehyde compound. As a solution for diluting the aldehyde compound, a buffer solution may be typically used, and various solutions may be applied as necessary. In addition, there is no need to limit the concentration of the aldehyde compound in the solution, but in the present invention, dissolved in an amount of 0.1 to 1% by weight was used.

As described above, when the introduction of the aldehyde compound is completed, the substrate protein-containing solution is added dropwise onto the aldehyde compound to introduce the substrate protein.

In this case, in order to increase the accuracy of the measured value, it is preferable to block the empty part of the spot to which the substrate protein is not bound after introducing the substrate protein. Blocking is generally carried out when analytical chips are manufactured, and a solution containing a common blocking substance such as bovine serum albumin (BSA), normal serum or skim milk may be added dropwise. After one hour after the addition of the reaction, the reaction is completed, washed with PBST or distilled water, and dried, and the blocking substance is introduced into the empty portion where the substrate protein is not introduced. This blocking material contributes to the increase of the accuracy of the measurement by inhibiting the non-specific reaction in the steps described later. As an example, in the present invention, a blocking solution obtained by dissolving 1 to 10% by weight of BSA in PBST (phosphate buffered saline with Tween 20) was added dropwise, and blocking was performed for 1 hour. Modifications are possible.

Array type chip in which the substrate protein is introduced as described above can be usefully used as a chip for measuring activity of transglutaminase through the following steps, and the user can simply transglutaminaa through the following steps. The activity of the agent can be measured.

Particularly, in the case of the chip in which the substrate protein is introduced, since it has an array type capable of measuring a plurality of samples at the same time, the activity analysis time of the transglutaminase is drastically shortened, and the amount of samples required for analysis is extremely low, Because it is high, not only can be utilized in analytical methods that can identify mechanisms occurring in the cells, but also has the advantage that it can be used for various disease tests using body fluids such as blood.

In order to measure the activity, a transglutaminase-containing sample and 5- (biotinamido) pentylamine (5- (biotinamido) pentylamine), DTT and CaCl ₂ , were added dropwise onto the substrate protein. The amine exchange process of the substrate protein and 5- (biotinamido) pentylamine is carried out.

Here, the activity-transfecting glutaminase-containing sample is a generic term for those that are isolated from various cells or those for which activity is to be measured, including blood samples. 5- (Biotinamido) pentylamine is activated by transglutaminase, causing an amine exchange reaction with the substrate protein, causing biotin to be placed at the end of the substrate protein. CaCl ₂ and DTT are added to aid in the activity of transglutaminase. Depending on the purpose of measurement, a cofactor such as Triton X-100 may be added to the measurement solution. As described above, the buffer solution may be a protein chip generally used in the art.

At this time, biotin, which is located at the end of the substrate protein by the amine exchange reaction between the substrate protein and 5- (biotinamido) pentylamine, is strongly associated with streptavidin or avidin, an analytical marker. As a binding substance, its analysis allows the activity of transglutaminase to be measured. That is, the degree of amine exchange reaction is determined according to the activity of the transglutaminase, and thus the amount of biotin located at the terminal of the substrate protein is determined. Eventually, the binding of streptavidin or avidin to biotin, which are widely used as markers, can be analyzed to determine the activity of transglutaminase.

The content and composition of the sample used to measure the activity of transglutaminase can be arbitrarily changed as necessary, but in the present invention, 5- (biotinamido) pentyl per 1 part by weight of the sample containing transglutaminase for activity measurement amine 5 to 7 parts by weight of CaCl ₂ 5 to 7 parts by weight, and DTT 28~45 parts by weight of buffer solution was used 400 to 600 parts by weight of a mixture. However, this content is found to be variously modified depending on the analysis conditions or analysis conditions.

When the mixed solution of this ratio is dropped on the substrate protein and left for 1 hour, an amine exchange reaction of the substrate protein with 5- (biotinamido) pentylamine occurs. Afterwards, after washing with PBST or distilled water and drying, Biotin is located at.

As described above, when positioned at the end by the amine exchange reaction, the analytical label is introduced into the biotin, and then the activity of the transglutaminase is measured through its analysis.

As described above, streptavidin or avidin may be used as a labeling material widely used in biotin. In particular, using a streptavidin or avidin (avidin) in combination with a fluorescent marker selected from Cy3, Cy5, Alexa, BODIPY, Rhodamine or Q-dot as the analytical marker, transglutamina It is good to be able to measure the activity of the agent. In addition to the analytical label, streptavidin (avitre) or avidin (avidin) labeled with an enzyme selected from alkaline phosphatase, horseradish peroxidase, or luciperase. ) And react with a chromogenic substrate to determine the activity of transglutaminase. In addition, various known analytical markers may be used as necessary.

By measuring the activity of trans glutaminase according to the method described above, it is possible to significantly reduce the analysis time compared to the method using ³ H, which is a commonly used radioisotope, and Western blot. It is possible to measure with high sensitivity while using a small amount of samples, and to simultaneously analyze multiple samples.

Hereinafter, the present invention will be described in more detail with reference to the following examples, which are only presented to aid the understanding of the present invention, but the present invention is not limited thereto.

<Example 1>

The slide glass was immersed in a solution prepared by dissolving 1.5% by weight of 3-aminopropyltrimethoxysilane in an ethanol solution for 2 hours, washed with ethanol and distilled water, and then washed in an oven at 110 ° C. for 1 hour. It was dried to introduce 3-aminopropyltrimethoxysilane into the slide glass surface. At this time, the slide glass was used by dipping for 10 minutes in a cleaning solution (H ₂ O ₂ : NH ₄ OH: dH ₂ O = 1: 1: 5) at 70 ℃.

A teflon tape having a spot of 1.5 mm in diameter was formed by using a punch on the slide glass surface into which the 3-aminopropyltrimethoxysilane was introduced to prepare an array chip having 200 (8 × 25) spots. Then, casein, one of the substrate proteins, casein was added dropwise to the phosphate buffer solution (pH 7.4) in a concentration of 1 mg / ml, and reacted in an incubator at 37 ° C. for 2 hours. After washing with PBS containing 0.1% by weight Tween 20 for 10 minutes, and then washed again with distilled water for 5 minutes, and air dried to introduce casein to the 3-aminopropyltrimethoxysilane-attached array chip.

A blocking solution in which 3% by weight of BSA was added to PBST (0.1% by weight Tween 20) was added dropwise onto the case where the casein was introduced and left in an incubator at 37 ° C for 1 hour. After washing with PBS containing 0.1 wt% Tween 20 for 10 minutes, and then again washed with distilled water for 5 minutes, and dried with air to prepare a chip for measuring transglutaminase activity of the array type.

Casein of the transglutaminase activity measurement chip prepared above was treated with 294 µg / ml CaCl ₂ and 328 µg / ml 5- (biotinamido) pentylamine (BAPA) in 24,220 µg / ml Tris-HCl buffer solution on casein. ), And a sample prepared by adding 1,542 µg / ml DTT and transglutaminase in an amount shown in FIG. 1 was added dropwise. After standing in an incubator at 37 ° C. for 1 hour, washed with PBS containing 0.1 wt% Tween 20 for 10 minutes, and then washed with distilled water for 5 minutes and dried while blowing air. After dropping the labeled streptavidin fluorescent substance Alexa546 bound to the biotin, and then measured the intensity by scanning with a fluorescence scanner (Scan Array Lite) and the results are shown in Figure 1a, the measurement shown in Figure 1a The fluorescence intensity of the result is graphically shown in FIG. 1B.

As shown in Figure 1a and Figure 1b it can be seen that the degree of activation of transglutaminase changes depending on the concentration of transglutaminase. The higher the intensity of fluorescence, the higher the activity of the enzyme. The lower the intensity, the lower the activity of the enzyme.

<Example 2>

The slide glass was immersed in a solution prepared by dissolving 1.5% by weight of 3-aminopropyltrimethoxysilane in an ethanol solution for 2 hours, washed with ethanol and distilled water, and then washed in an oven at 110 ° C. for 1 hour. It was dried to introduce 3-aminopropyltrimethoxysilane into the slide glass surface. At this time, the slide glass was used by dipping for 10 minutes in a cleaning solution (H ₂ O ₂ : NH ₄ OH: dH ₂ O = 1: 1: 5) at 70 ℃.

A teflon tape having a spot of 1.5 mm in diameter was formed by using a punch on the slide glass surface into which the 3-aminopropyltrimethoxysilane was introduced to prepare an array chip having 200 (8 × 25) spots.

The carboxyl group-modified latex beads of 500 ± 100 nm size washed with PBS were added to 50mM EDC and 200mM NHS mixed solution, and then added to the spot of the array chip and reacted for 20 minutes. After washing with PBS containing 0.1 wt% Tween 20 for 1 hour and then again washed with distilled water for 5 minutes, and dried with air to introduce latex beads to the amine group end of 3-aminopropyltrimethoxysilane.

200mM EDC and 50mM NHS mixed solution was added dropwise to the latex beads and left for 10 minutes, washed with distilled water and dried while blowing air. Then, casein was added dropwise to the phosphate buffer solution (pH 7.4) at a concentration of 1 mg / ml, and reacted in an incubator at 37 ° C. for 2 hours. After washing with PBS containing 0.1 wt% Tween 20 for 10 minutes and then again washed with distilled water for 5 minutes, and dried by air to introduce casein, one of the substrate proteins in the latex beads.

A blocking solution in which 3% by weight of BSA was added to PBST (0.1% by weight Tween 20) was added dropwise onto the case where the casein was introduced and left in an incubator at 37 ° C for 1 hour. After washing with PBS containing 0.1 wt% Tween 20 for 10 minutes, and then again washed with distilled water for 5 minutes, and dried with air to prepare a chip for measuring transglutaminase activity of the array type.

On the casein of the prepared chip for transglutaminase activity measurement, 50 µg / ml transglutaminase, 328 µg / ml 5- (biotinamido) pentyl in 24,220 µg / ml Tris-HCl buffer solution An amine (BAPA), 1,542 µg / ml DTT and the amount of CaCl ₂ added in the amount shown in FIG. 1 were added and mixed with the sample. After standing in an incubator at 37 ° C. for 1 hour, washed with PBS containing 0.1 wt% Tween 20 for 10 minutes, and then washed with distilled water for 5 minutes and dried while blowing air. Then, the fluorescent substance Alexa546 was dropped onto the labeled streptavidin to bind with biotin, and then the intensity was measured by scanning with a fluorescence scanner (Scan Array Lite), and the results are shown in FIG. 2A. The fluorescence intensity of the result is graphically shown in FIG. 2b.

As shown in Figure 2a and 2b it can be seen that the degree of activation of transglutaminase changes depending on the concentration of calcium ions. The higher the intensity of fluorescence, the higher the activity of the enzyme. The lower the intensity, the lower the activity of the enzyme.

<Example 3>

The slide glass was immersed in a solution prepared by dissolving 1.5% by weight of 3-aminopropyltrimethoxysilane in an ethanol solution for 2 hours, washed with ethanol and distilled water, and then washed in an oven at 110 ° C. for 1 hour. It was dried to introduce 3-aminopropyltrimethoxysilane into the slide glass surface. At this time, the slide glass was used by dipping for 10 minutes in a cleaning solution (H ₂ O ₂ : NH ₄ OH: dH ₂ O = 1: 1: 5) at 70 ℃.

 The array chip into which 3-aminotrimethoxysilane was introduced was dissolved in a phosphorylated buffer solution containing 1% by weight of glutamine aldehyde, and then immersed in a solution prepared for 30 minutes, washed with distilled water, and dried by air. Aldehydes were introduced into the array chip into which trimethoxysilane was introduced.

A teflon tape having a spot of 1.5 mm in diameter was formed on the surface of the slide glass into which the aldehyde was introduced by using a punch to produce an array chip having 200 (8 × 25) spots. Then, casein was added dropwise to the phosphate buffer solution (pH 7.4) at a concentration of 1 mg / ml, and reacted in an incubator at 37 ° C. for 2 hours. After washing for 10 minutes with PBS containing 0.1 wt% Tween 20 and then again with distilled water for 5 minutes, and air dried to introduce casein, one of the substrate proteins, into the array chip into which 3-aminopropyltrimethoxysilane was introduced. It was.

A blocking solution containing 3% by weight of BSA in PBST (0.1% by weight Tween 20) was added dropwise to the casein-introduced 3-aminopropyltrimethoxysilane array chip and left in an incubator at 37 ° C for 1 hour. . After washing with PBS containing 0.1 wt% Tween 20 for 10 minutes, and then again washed with distilled water for 5 minutes, and dried with air to prepare a chip for measuring transglutaminase activity of the array type.

Casein of the transglutaminase activity measurement chip prepared above was treated with 294 µg / ml CaCl ₂ and 328 µg / ml 5- (biotinamido) pentylamine (BAPA) in 24,220 µg / ml Tris-HCl buffer solution on casein. ), 1,542 µg / ml DTT and the amount of transglutaminase shown in FIG. 1 were added and mixed with the sample. After standing in an incubator at 37 ° C. for 1 hour, washed with PBS containing 0.1 wt% Tween 20 for 10 minutes, and then washed with distilled water for 5 minutes and dried while blowing air. Then, the fluorescent substance Alexa546 was dropped onto the labeled streptavidin to bind to biotin, and then the intensity was measured by scanning with a fluorescence scanner (Scan Array Lite), and the results are shown in FIG. 3A. The fluorescence intensity of the result is graphically shown in FIG. 3b.

As shown in Figure 3a and 3b it can be seen that the degree of activation of transglutaminase changes depending on the concentration of transglutaminase. The higher the intensity of fluorescence, the higher the activity of the enzyme. The lower the intensity, the lower the activity of the enzyme.

<Example 4>

As shown in FIG. 4, 250 µg / ml protein extract extracted from 10 kinds of commercially available cells, 1,542 µg / ml DTT and 294 µg / ml CaCl, 328 µg / ml 5- (biotinamido) pentylamine ₂ was added to 24,220 µg / ml Tris-HCl buffer solution, and the mixed TGase activity measurement solution was added dropwise onto the transglutaminase activity measurement chip prepared in Example 2. After standing in an incubator at 37 ° C. for 1 hour, washed with PBS containing 0.1 wt% Tween 20 for 10 minutes, and then washed with distilled water for 5 minutes and dried while blowing air. Then, the fluorescent substance Alexa546 was dropped onto the labeled streptavidin to bind to biotin, and then scanned with a fluorescence scanner (Scan Array Lite) to analyze TGase activity in blood cell extracts, and the results are shown in FIG. 4A. The fluorescence intensity of the measurement result shown in the graph is shown in Figure 4b.

As shown in Figure 4a and 4b by using the TGase activity measurement chip according to the present invention to measure the TGase activity in various cell extracts, TGase activity in a number of cells using the TGase activity measurement chip developed in the present invention It can be seen that it can measure at very high speed.

<Example 5>

As shown in FIG. 5, 10 kinds of patient blood extracts 10 μg / ml, 328 μg / ml 5- (biotinamido) pentylamine 1,542 μg / ml DTT, and 294 μg / ml CaCl ₂ were 24,220 μg / ml of Tris. The TGase activity measurement solution added and mixed with -HCl buffer solution was added dropwise onto the transglutaminase activity measurement chip prepared in Example 2. After standing in an incubator at 37 ° C. for 1 hour, washed with PBS containing 0.1 wt% Tween 20 for 10 minutes, and then washed with distilled water for 5 minutes and dried while blowing air. Then, the fluorescent substance Alexa546 was dropped onto the labeled streptavidin to bind to biotin, and then scanned with a fluorescence scanner (Scan Array Lite) to analyze TGase activity in blood cell extracts, and the results are shown in FIG. 5A. The fluorescence intensity of the measurement result shown in the graph is shown in Figure 5b.

As shown in Figure 5a and 5b by using the TGase activity measurement chip according to the present invention to measure the TGase activity in the blood extracts of several patients, the TGase activity measurement chip developed in the present invention in a plurality of patient blood extracts It can be seen that the TGase activity can be measured at very high speed.

As described above, the present invention, unlike the conventional method of measuring the activity of transglutaminase by using a radioisotope or Western blot, uses a simple transglue using an assay chip in the form of an array. It is possible to measure the activity of taminase, which can significantly reduce the analysis time, and to measure with high sensitivity even with a small sample, so that it can be used in analytical methods that can identify mechanisms occurring in cells. Not only can be used to study the regulation and function of the cells through the measurement, there is an effect that can be used for various disease tests using body fluids such as blood.

Claims (24)

Introducing an alkoxysilane having a functional group at the end to the surface of the substrate; Manufacturing an array chip having a plurality of spots formed by attaching a perforated tape at predetermined intervals on a surface of the substrate; Introducing a substrate protein of trans glutaminase at the end of the spot; A transglutaminase-containing sample for measuring activity and a measurement solution containing 5- (biotinamido) pentylamine DTT and CaCl ₂ were dropped on the substrate protein. Amine transamidation of 5- (biotinamido) pentylamine with; Measuring the activity of transglutaminase by introducing an analytical labeling material into biotin positioned at the end by the amine exchange reaction and then measuring the activity of transglutaminase through its analysis; Way. The method according to claim 1, The alkoxysilane having a functional group at the terminal introduced into the substrate surface is selected from aminoalkyltrialkoxysilane, mercaptoalkyltrialkoxysilane, or epoxyalkyltrialkoxysilane. Method for measuring activity of taminase. The method according to claim 2, The aminoalkyltrialkoxysilane may be 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, or 3-2-2-aminoethylaminoethylaminopropyltrimethoxy. The silane (3- [2- (2-aminoethylamino) ethylamino] propyl-trimethoxysilane) is used, the mercaptoalkyltrialkoxysilane uses 3-mercaptopropyltrimethoxysilane, and epoxy Alkyltrialkoxysilane is a method for measuring activity of transglutaminase, characterized in that 3-glycidoxypropyltrimethoxysilane is used. The method according to claim 1, Wherein the step of introducing a substrate protein: a method of measuring the activity of transglutaminase, characterized in that the alkoxysilane of the spot formed by attaching a tape and the medium, and introducing a substrate protein on the medium. The method according to claim 4, The method of measuring the activity of the transglutaminase, characterized in that the medium is a carboxylate modified latex bead (carboxylate modified latex bead). The method according to claim 5, The reaction of the alkoxysilane and the carboxyl group-modified latex beads is characterized in that the carboxyl-modified latex beads are reacted in the presence of N-hydroxysuccinimide and carbodiimide to activate and then the reaction solution is added dropwise to the alkoxysilane. Method for measuring activity of transglutaminase. The method according to claim 6, Introducing the substrate protein onto the latex beads: adding a mixed solution of N-hydroxysuccinimide and carbodiimide onto the latex beads to activate the carboxyl group of the latex beads and then dropping the substrate protein containing solution thereon. Method for measuring the activity of transglutaminase characterized in that. The method according to claim 4, The method of measuring the activity of transglutaminase, wherein the medium is a compound having an aldehyde group reacting with a substrate protein at at least one terminal while having a functional group in the sockdan. The method according to claim 8, Method for measuring the activity of transglutaminase, characterized in that the compound is selected from glutaraldehyde, benzene-1,4-dicarboxaldehyde. The method according to any one of claims 1 to 9, Wherein the step of introducing the substrate protein: Transglutaminase activity measuring method comprising the step of introducing the substrate protein and then blocking the blank portion of the spot that the substrate protein is not bonded with a blocking (blocking) solution . The method according to claim 1, Introducing a label for analysis into biotin; Method for measuring the activity of transglutaminase, characterized in that using a streptavidin or avidin conjugated with a fluorescent substance or enzyme. The method according to claim 11, The analytical labeling material uses streptavidin or avidin in combination with a fluorescent label selected from Cy3, Cy5, Alexa, BODIPY, Rhodamine or Q-dot, and transglutaminaa through fluorescence analysis. Method for measuring the activity of the transglutaminase, characterized in that for measuring the activity of the agent. The method according to claim 11, The marker for analysis is streptavidin or avidin labeled with an enzyme selected from alkaline phosphatase, horseradish peroxidase or luciperase. Using, and measuring the activity of the transglutaminase by reacting it with a chromogenic substrate (Chromogenic substrate). Introducing an alkoxysilane having a functional group at the end to the surface of the substrate; Manufacturing an array chip having a plurality of spots formed by attaching a perforated tape at predetermined intervals on a surface of the substrate; A method for manufacturing a chip for transglutaminase activity measurement, comprising the step of introducing a substrate protein of transglutaminase at the end of the spot. The method according to claim 14, The alkoxysilane having a functional group at the terminal introduced into the substrate surface is selected from aminoalkyltrialkoxysilane, mercaptoalkyltrialkoxysilane, or epoxyalkyltrialkoxysilane. Method for producing a chip for measuring the activity of Naze. The method according to claim 15, The aminoalkyltrialkoxysilane is 3-aminopropyltrimethoxysilane or 3-2-2-aminoethylaminoethylaminopropyltrimethoxysilane (3- [2- (2-aminoethylamino) ethylamino] propyl-trimethoxysilane) is used, the mercaptoalkyltrialkoxysilane is 3-mercaptopropyltrimethoxysilane, and the epoxyalkyltrialkoxysilane is 3-glycidoxypropyltrimethoxy Method for producing a chip for measuring the activity of transglutaminase, characterized in that using silane (3-Glycidoxypropyltrimethoxysilane). The method according to claim 14, The substrate protein introduction step: preparing a chip for measuring activity of a transglutaminase, characterized in that the alkoxysilane of the spot formed by attaching a tape and the medium react, and introduce a substrate protein on the medium Way. The method according to claim 17, The medium is a method for producing a chip for measuring activity of transglutaminase, characterized in that the carboxylate modified latex beads (carboxylate modified latex bead). The method according to claim 18, The reaction of the alkoxysilane and the carboxyl group-modified latex beads is performed by reacting the carboxyl-modified latex beads in the presence of N-hydroxysuccinimide and carbodiimide to activate the reaction solution, and then dropping the reaction solution into the alkoxysilane. The manufacturing method of the chip | tip for measuring the activity of the transglutaminase. The method according to claim 19, Introducing the substrate protein onto the latex beads: adding a mixed solution of N-hydroxysuccinimide and carbodiimide onto the latex beads to activate the carboxyl group of the latex beads and then dropping the substrate protein containing solution thereon. Method for producing a chip for measuring the activity of transglutaminase characterized in that. The method according to claim 17, The medium is a method for producing a chip for measuring activity of transglutaminase, characterized in that the compound having an aldehyde group reacting with the substrate protein at least one terminal having a functional group in the sockdan. The method of claim 20, Method for producing a chip for measuring the activity of the transglutaminase, characterized in that the compound is selected from glutaraldehyde or benzene-1,4-dicarboxaldehyde. The method according to any one of claims 14 to 22, The substrate protein introduction step: After the introduction of the substrate protein for measuring the activity of the transglutaminase comprising the step of blocking the blank portion of the spot that the substrate protein is not bonded with a blocking (blocking) solution Chip manufacturing method. A plurality of spots are formed by attaching a perforated tape at predetermined intervals to the surface of the substrate on which the alkoxysilane having a functional group is introduced, and a substrate protein of transglutaminase is introduced at the end of the spot. Transglutaminase activity measurement chip, characterized in that.

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