WO2008114935A1

WO2008114935A1 - Chip and method for determining transglutaminase activity

Info

Publication number: WO2008114935A1
Application number: PCT/KR2008/000844
Authority: WO
Inventors: Kwon-Soo Ha; Jae-Wan Jung
Original assignee: Knu-Industry Cooperation Foundation; Korea Clinical Medicin Center
Priority date: 2007-03-19
Filing date: 2008-02-13
Publication date: 2008-09-25
Also published as: WO2008114935A9

Abstract

The present invention relates to a technique for analysis of transglutaminase activity. Specifically, provided is a chip and method capable of simply and conveniently measuring transglutaminase activity, using an analysis chip in the form of an array. Therefore, the present invention provides various advantages such as significant reduction of an analysis period, high-sensitivity analysis even with a tiny amount of a sample, applicability in analysis methods for elucidation of intracellular mechanisms, applicability in cellular control and functional study via measurement of intracellular activity of a target enzyme, and applicability in examination of various diseases using body fluids such as blood.

Description

CHIP FOR MEASURING ACTIVITY OF TRANSGLUTAMINASE AND METHOD OF MEASURING ACTIVITY OF TRANSGLUTAMINASE

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to a method for analysis of activity of transglutaminase which is known to play a crucial role in the pathogenesis of various diseases such as degenerative brain diseases (e.g. dementia), degenerative arthritis, autoimmune diseases and the like. More specifically, the present invention relates to a chip and method for measuring activity of transglutaminase, which not only allows for rapid and environmentally-friendly analysis of a plurality of samples, but also is capable of achieving high-sensitivity analysis of a target enzyme even with a small amount of a sample.

Description of the Related Art Transglutaminase (TGase) is an enzyme which catalyzes the crosslinking of a glutamine residue to a lysine (or polyamine) residue. It is known that TGase is implicated in the development and/or incidence of various diseases, particularly neurological diseases such as neurodegenerative brain diseases (e.g. dementia), degenerative arthritis, autoimmune diseases, and the like. Further, TGase is a protective enzyme which is responsible for blood clotting or wound healing under normal conditions.

However, TGase is also reported to play an important role in the pathological mechanisms of various diseases in the absence of regulatory control in the level of expression thereof. The expression of TGase increases particularly upon the occurrence of various inflammatory diseases, including degenerative arthritis, diabetes, autoimmune myositis, arteriosclerosis, cerebral apoplexy, hepatocirrhosis, malignant breast cancer, cerebral meningitis, inflammatory gastric ulcer, etc. Recently, TGase activity is known to increase in dementia patients.

For these reasons, accurate analysis of TGase activity is very important for diagnosis or prognosis of diseases. Conventionally and widely used methods of measuring TGase activity may be broadly divided into a method using a radioisotope ³H and a Western blot analysis.

The former method using the radioisotope ³H is a method for analysis of TGase activity, which involves measuring a concentration of [³H] when activation of TGase results in transamidation between a substrate (such as casein) and [³H] putrescine. However, such a method poses the risk of environmental pollution due to use of radioisotopes, and safety concerns during experimental processes.

The Western blot analysis is a method for analysis of TGase activity, which involves transamidation of a substrate with 5-(biotinamido)pentylamine, reaction of the reaction product with horseradish peroxidase-streptavidin (HRP-streptavidin) and detection of streptavidin. However, this method still suffers from various disadvantages, such as a relatively long analysis period associated with a need for SDS-PAGE and immunoblotting, necessity of large amounts of samples, and difficulty in simultaneous analysis of multiple samples. Further, this method has a shortcoming that it is difficult to assay TGase activity at a low concentration of a target enzyme, due to poor sensitivity thereof.

SUMMARY OF THE INVENTION

Therefore, the present invention has been made in view of the above problems, and it is an object of the present invention to provide a method of fabricating a chip for measuring TGase activity, which involves fabricating an array chip to measure TGase activity and assaying the TGase activity using the chip. Accordingly, the present invention provides a method of fabricating a chip for measuring TGase activity, which is environmentally friendly, allows for simultaneous analysis of a plurality of samples and particularly high-sensitivity analysis even with a small amount of a sample, and is capable of remarkably reducing a period of time taken until final analysis. It is another object of the present invention to provide a chip for measuring TGase activity, which is fabricated by the aforementioned method.

It is a further object of the present invention to provide a method for measuring TGase activity.

In accordance with an aspect of the present invention, the above and other objects can be accomplished by the provision of a method of fabricating a chip for measuring transglutaminase (TGase) activity, comprising: introducing functional group-terminated alkoxysilane into a surface of a substrate; attaching a regularly punched tape to the substrate surface to fabricate an array chip having a plurality of spots formed thereon; and introducing a substrate for TGase into ends of spots. In accordance with another aspect of the present invention, there is provided a chip for measuring transglutaminase (TGase) activity, which is prepared by the aforesaid method comprising: attaching a regularly punched tape to a surface of a substrate with introduction of functional group-terminated alkoxysilane to form a plurality of spots; and introducing a substrate for TGase into ends of spots. In accordance with yet another aspect of the present invention, there is provided a method for measuring transglutaminase (TGase) activity, comprising: introducing functional group-terminated alkoxysilane into a surface of a substrate; attaching a regularly punched tape to the substrate surface to fabricate an array chip having a plurality of spots formed thereon; introducing a substrate for TGase into ends of spots; dropping a TGase-containing sample for measuring enzymatic activity and an assay solution containing 5- (biotinamido)pentylamine and CaCl₂ on the substrate to induce transamidation of the substrate with 5-(biotinamido)pentylamine; and introducing an assay label into biotin which was terminally-positioned via transamidation and assaying the labeled biotin to measure TGase activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates measurement results for TGase activity of individual samples (A) and fluorescence intensity versus TGase concentration (B), as measured according to Example 1 of the present invention; FIG. 2 illustrates measurement results for TGase activity of individual samples (A) and fluorescence intensity versus Ca²⁺ (B), as measured according to Example 2 of the present invention;

FIG. 3 illustrates measurement results for TGase activity of individual samples (A) and fluorescence intensity versus TGase concentration (B), as measured according to Example 3 of the present invention;

FIG. 4 illustrates measurement results for intracellular TGase activity of cell culture extracts of various cell lines, as measured according to Example 4 of the present invention; and

FIG. 5 illustrates measurement results for serum TGase activity of various blood samples, as measured according to Example 5 of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, a chip and method for measuring TGase activity in accordance with the present invention will be described in more detail. The present invention provides a chip-based method of measuring TGase activity, which is distinctly different from conventional methods using a radioisotope ³H and Western blot analysis.

To this end, the present invention provides a method of fabricating a chip for measuring TGase activity, comprising of given steps which will be illustrated hereinafter.

For this purpose, functional group-terminated alkoxysilane is first introduced into a surface of a substrate.

The functional group-terminated alkoxysilane may be one selected from aminoalkyltrialkoxysilane, mercaptoalkyltrialkoxysilane and epoxyalkyltrialkoxysilane. Examples of aminoalkyltrialkoxysilane may include 3- aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane and 3-[2-(2- aminoethylamino)ethylamino]propyl-trimethoxysilane. An example of mercaptoalkyltrialkoxysilane may be 3-mercaptopropyltrimethoxysilane, and an example of epoxyalkyltrialkoxysilane may be 3-glycidoxypropyltrimethoxysilane. The functional group-terminated alkoxysilane may be introduced into the substrate surface using a variety of conventional methods known in the art. In the present invention, alkoxysilane is dissolved in a conventional alcohol solvent such as ethanol, and a substrate is dipped in the resulting solution to allow introduction of functional group-terminated alkoxysilane into the surface of the substrate. For convenience of operation, 1 to 2% by weight of the functional group-terminated alkoxysilane is dissolved in ethanol, even though the alkoxysilane concentration is not limited thereto.

A dipping time of the substrate may be about 2 hours. After 2 hours, the substrate is washed with ethanol or distilled water and then dried to result in introduction of the functional group-terminated alkoxysilane into the substrate surface. The substrate may be a metal substrate or slide glass substrate which is conventionally and widely used in the art. The substrate is thoroughly cleaned prior to use. Cleaning of the substrate may be carried out by any conventional method known in the art. In the present invention, a slide glass as the substrate is dipped and cleaned for at least 10 min in a mixed cleaning solution of 10 to 40 parts by weight of hydrogen peroxide and 10 to

40 parts by weight of aqueous ammonia, based on 100 parts by weight of distilled water.

After introduction of the functional group-terminated alkoxysilane into the substrate surface, a regularly punched tape is attached to the substrate surface to fabricate an array chip having a plurality of spots formed thereon. There is no particular limit to the number of spots. The tape may be a Teflon tape with low reactivity.

Thereafter, a substrate for TGase is introduced into ends of the spots.

Examples of the substrate for TGase may include, but are not limited to, casein, aldolase A, glyceraldehyde-3 -phosphate dehydrogenase, phosphorylase kinase, crystallins, glutathione S-transferase, cytoskeletal protein, actin, myosin, troponin, β-tubulin, Tau, Rho

A, Racl, histone H2B, α-oxoglutarate dehydrogenase, cytochromes, erythrocyte band III,

CD38, acetylcholine esterase, collagen, fibronectin, fibrinogen, vitronectin, osteopontin, nidogen, laminin, LTBP-I, osteonectin, osteocalcin, Substance P, phospholipase A₂, midkine, wheat gliadin, whey proteins, soy protein, pea legumin, Candida albicans surface proteins, HIV envelope glycoproteins gpl20 and gp41, HIV aspartyl proteinase and

Hepatitis C virus core protein, which are well known in the art. However, a substrate for

TGase is not limited to proteins. If necessary, other materials may be used as the TGase substrate.

Introduction of the substrate may be carried out by various conventional methods known in the art. In the present invention, a substrate-containing solution, which is prepared by dissolving the substrate in a suitable solvent, is dropped to react with spots to thereby result in introduction of the substrate into spot ends. The reaction takes place by reaction of functional groups of the functional group-terminated alkoxysilane with the substrate. For this purpose, the substrate-containing solution is dropped and allowed to react with spots for at least 2 hours. After the reaction is complete, the substrate is washed with PBST or distilled water and then dried to result in introduction of the substrate.

Even though a solution for dissolution of the substrate may be typically a buffer, various solutions may also be applied if necessary. The present invention employs PBS or Tris-HCl buffer having a pH value of 4 to 9 which may vary if necessary. Even though there is no particular limit to the substrate concentration, the present invention uses 0.1 to 1% by weight of the substrate dissolved in the solvent.

If necessary, in order to ensure easier introduction of the substrate, a medium may be reacted with alkoxysilane of the spots formed by tape attachment prior to introduction of the substrate, followed by introduction of the substrate into the medium. The medium that can be used in the present invention may be carboxylate-modified latex beads, or a compound which has functional groups at both terminals and a substrate- reactive aldehyde group at at least one terminal.

The carboxylate-modified latex beads as the medium will be briefly illustrated hereinafter. The carboxylate-modified latex beads may be commercially available and have a size conventionally used in the art, e.g. protein analysis chip technology. As examples of commercially available modified latex beads used to impart the reactivity, mention may be made of amine-modified latex beads, carboxylate-modified latex beads, sulfate-modified latex beads, and the like. The present invention employs carboxylate-modified latex beads having a diameter of 500 to 1000 ran (Molecular Probes, Eugene, OR, USA). For smooth reaction, preferably carboxylate-modified latex beads are activated through the reaction of beads with N-hydroxysuccinimide in the presence of carbodiimide. Then, the resulting reaction solution is dropped to react with spots to result in introduction of latex beads into terminal amine groups of alkoxysilane. In this connection, carbodiimide serves as a catalyst. That is, carbodiimide activates carboxyl groups to facilitate easy substitution of N-hydroxysuccinimide at carboxyl terminals (-OH) of latex beads. Carbodiimide may be one conventionally used in the art. Examples of carbodiimide may include N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDC) hydrochloride, N,N'-dicyclohexyl carbodiimide (DCC) and diisopropylcarbodiimide. The reaction is made by adding the carboxylate-modified latex beads to a mixed solution of N-hydroxysuccinimide and carbodiimide, and then dropping the resulting solution on spots, followed by reaction for about 20 min. Even though there is no particular limit to the amount of carboxylate-modified latex beads added to the N- hydroxysuccinimide/carbodiimide mixture, the present invention uses 20 to 50 parts by weight of carboxylate-modified latex beads, based on 100 parts by weight of the N- hydroxysuccinimide/carbodiimide mixture. Additionally, carbodiimide may be used in admixture with N-hydroxysuccinimide in a range of various equivalent ratios. The present invention employs a mixture of carbodiimide and N-hydroxysuccinimide in an equivalent ratio of 2 to 4:1, but such a mixing ratio may be appropriately adjusted if necessary. After the reaction is complete, the latex bead-introduced substrate is washed with

PBST (Phosphate buffered saline with 0.1% by weight of Tween 20) or distilled water and then dried to result in introduction of latex beads into terminal amine groups of alkoxysilane.

After introduction of latex beads is complete, a substrate-containing solution as described above is dropped on the latex beads to result in introduction of a substrate. Introduction of the substrate into the latex is made via the reaction of amine groups of the substrate with carboxyl groups of latex beads. Therefore, similar to the reaction of amine groups of alkoxysilane with carboxyl groups of latex beads, in order to achieve easier introduction of the substrate into latex beads, a mixed solution of N-hydroxysuccinimide and carbodiimide is preferably dropped on latex beads to thereby activate carboxyl groups of latex beads, followed by addition of the substrate-containing solution.

The medium that can be used in the introduction of substrate may be a compound which has functional groups at both terminals and a substrate-reactive aldehyde group at at least one terminal.

Such an aldehyde compound may be selected from glutaraldehyde and benzene- 1 ,4-dicarboxaldehyde.

Introduction of the aldehyde compound may be carried out by various conventional methods known in the art. The simplest method is to drop a diluted solution of the aldehyde compound on spots, followed by reaction. For this purpose, the solution is dropped and allowed to react with spots for about 30 min. After the reaction is complete, the substrate is washed with PBST or distilled water and then dried to result in convenient introduction of the aldehyde compound. Even though a solution for dilution of the aldehyde compound may be typically a buffer, various solutions may also be applied if necessary. Further, there is no particular limit to a concentration of the aldehyde compound in the solution, but the present invention uses 0.1 to 1% by weight of the aldehyde compound dissolved in the solvent. After introduction of the aldehyde compound is complete, a substrate-containing solution as described above is dropped on the aldehyde compound to result in introduction of a substrate.

In order to improve measurement accuracy in accordance with the present invention, unoccupied parts of spots to which the substrate was not bound is preferably blocked after introduction of the substrate. Blocking is a process which is typically carried out upon fabrication of an analysis chip. For this purpose, a solution containing a conventional blocking agent, such as bovine serum albumin (BSA), normal serum or skim milk, may be dropped. When the reaction is complete after about 1 hour, the chip is washed with PBST or distilled water and then dried to result in introduction of the blocking agent into the unoccupied parts with no introduction of the substrate. These blocking agents will contribute to inhibition of non-specific reaction in a subsequent step, thereby improving the measurement accuracy. In one embodiment of the present invention, a blocking solution which was obtained by dissolving 1 to 10% by weight of BSA in PBST (phosphate buffered saline with Tween 20) is dropped, followed by allowing to stand at room temperature for 1 hour. However, various modifications may also be made if necessary.

The resulting array chip with introduction of the substrate can be usefully employed as a chip for measuring TGase activity, through a subsequent step. Users or consumers can simply and conveniently measure TGase activity according to the following steps.

Particularly in the case of substrate-introduced chip, it has an array structure that enables simultaneous measurement of a plurality of samples. Therefore, such a chip has various advantages such as a significantly reduced period of time taken for analysis of TGase activity, consumption of a very small amount of a sample, and high-sensitivity analysis. As a result, the chip and method of the present invention can be practically applied to analysis methods for elucidation of intracellular mechanisms, as well as examination of various diseases using body fluids such as blood.

Next, in order to measure an enzymatic activity, a TGase-containing sample for measuring enzymatic activity and an assay solution containing 5-(biotinamido)pentylamine, dithiothreitol (DTT) and CaCl₂ are dropped on the substrate to induce transamidation between the substrate and 5-(biotinamido)pentylamine. As used herein, the term "TGase-containing sample for measuring enzymatic activity" generally refers to a sample for TGase activity assay, including those isolated from a variety of cell line cultures and blood samples of subjects. 5-(biotinamido)pentylamine is activated by TGase, and then consequently undergoes the transamidation with the substrate, resulting in positioning of biotin into terminals of the substrate. CaCl₂ and DTT are added to facilitate activation of TGase. Depending upon desired assay purposes and applications, cofactors such as Triton X-IOO may be further added to the assay solution. The buffer may be one conventionally used in protein chip technology.

As well known in the art, biotin, which is positioned at terminals of the substrate via transamidation between the substrate and 5-(biotinamido)pentylamine, is a material that strongly binds to streptavidin or avidin which is an assay label. Therefore, it is possible to measure TGase activity by assay of streptavidin or avidin-bound biotin. That is, a degree of transamidation is determined depending upon the TGase activity, and therefore an amount of biotin which is positioned on terminals of the substrate will be determined. In conclusion, it is possible to measure TGase activity by conjugation of biotin with streptavidin or avidin which is widely used for labeling of biotin, and analysis of the streptavidin or avidin-bound biotin.

Even though amounts and components of samples used to measure TGase activity may arbitrarily vary if necessary, the present invention employs a mixture of 5 to 7 parts by weight of 5-(biotinamido)pentylamine, 5 to 7 parts by weight of CaCl₂, 28 to 45 parts by weight of DTT, and 400 to 600 parts by weight of a buffer, relative to 1 part by weight of the TGase-containing sample for measuring enzymatic activity. However, it should be understood that such a content ratio may vary depending upon a variety of analysis conditions or situations. When the mixed solution thus obtained is dropped on the substrate which is then allowed to stand at room temperature for 1 hour, transamidation takes place between the substrate and 5-(biotinamido)pentylamine. Thereafter, the reaction product is washed with PBST or distilled water and then dried to result in terminal positioning of biotin. When transamidation is complete with terminal positioning of biotin into the substrate, an assay label is incorporated into biotin, followed by fluorescence analysis to measure TGase activity.

As described above, streptavidin or avidin may be used as the label which is widely used for labeling of biotin. Particularly, it may be preferred to measure TGase activity by fluorescence analysis using streptavidin or avidin tagged with the assay label, e.g. a fluorescent dye selected from Cy3, Cy5, Alexa, BODIPY, Rhodamine and Q-dots.

Alternatively, TGase activity may be measured by labeling streptavidin or avidin with the assay label, e.g. an enzyme selected from alkaline phosphatase, horseradish peroxidase and luciferase, and then reacting the enzyme-labeled streptavidin or avidin with a chromogenic substrate. If necessary, a variety of assay labels known in the art may also be utilized.

When TGase activity is measured according to the above-mentioned method, it is possible to achieve various advantages such as significant reduction of an analysis period, high-sensitivity analysis even with a tiny amount of a sample, and simultaneous measurement of multiple samples, as compared to a conventional method using a radioisotope ³H and Western blot analysis method.

EXAMPLES

Now, the present invention will be described in more detail with reference to the following Examples. These examples are provided only for illustrating the present invention and should not be construed as limiting the scope and spirit of the present invention. Example 1

A slide glass was dipped in a solution of 1.5% by weight of 3- aminopropyltrimethoxysilane in ethanol for 2 hours, washed with ethanol and distilled water, and dried in an oven at 110^°C for 1 hour to result in introduction of 3- aminopropyltrimethoxysilane into the slide glass surface. The slide glass to be used was thoroughly cleaned in a cleaning solution (H₂O₂ !NH₄OH :dH₂O = 1:1:5) at 70 ^°C for 10 min.

A Teflon tape, which has spots with a diameter of 1.5 mm formed using a punch, was attached to the slide glass surface with introduction of 3-aminopropyltrimethoxysilane to thereby fabricate an array chip having 200 (8x25) spots. Then, a solution of a substrate

(casein) (1 mg/niL) in PBS (pH 7.4) was dropped onto spots, and the chip was reacted in an incubator at 37^°C for 2 hours. Thereafter, the chip was washed with PBS containing 0.1% by weight of Tween 20 for 10 min, then distilled water for 5 min, and dried in air to result in introduction of casein into the array chip to which 3-aminopropyltrimethoxysilane was attached.

A blocking solution with addition of 3% by weight of BSA to PBST (containing

0.1% by weight of Tween 20) was dropped onto the casein-introduced spots which were then placed in an incubator at 37^°C for 1 hour. Thereafter, the chip was washed with PBS containing 0.1% by weight of Tween 20 for 10 min, then distilled water for 5 min, and dried in air to fabricate an array chip for measuring TGase activity.

To casein of the fabricated array chip was dropped a sample with addition of 294

/Λg/mL Of CaCl₂, 328 βglmL of 5-(biotinamido)pentylamine (BAPA), 1,542 μg/mL ofDTT and TGase (at varying concentrations as indicated in FIG. IA) to 24,220 //g/mL of Tris-HCl buffer. The array chip was placed in an incubator at 37^°C for 1 hour, washed with PBS containing 0.1% by weight of Tween 20 for 10 min, then distilled water for 5 min, and air- blow dried. Then, fluorophore Alexa 546-labeld streptavidin was dropped and bound to biotin. Fluorescence intensity was measured using a fluorescence scanner (ScanArray Lite). The results obtained are shown in FIG. IA. The measurement results for fluorescence intensity of FIG. IA are graphed in FIG. IB. As shown in FIGS. IA and IB, it was confirmed that a degree of activation of

TGase varies with varying concentrations of TGase. That is, higher fluorescence intensity represents increases in the enzymatic activity, whereas lower fluorescence intensity represents decreases in the enzymatic activity.

Example 2

A slide glass was dipped in a solution of 1.5% by weight of 3- aminopropyltrimethoxysilane in ethanol for 2 hours, washed with ethanol and distilled water, and dried in an oven at 110^°C for 1 hour to introduce 3-aminopropyltrimethoxysilane into the slide glass surface. The slide glass to be used was thoroughly cleaned in a cleaning solution (H₂O₂INH₄OHIdH₂O = 1 : 1 :5) at 70 ^°C for 10 min.

A Teflon tape, which has spots with a diameter of 1.5 mm formed using a punch, was attached to the slide glass surface with introduction of 3-aminopropyltrimethoxysilane to thereby fabricate an array chip having 200 (8><25) spots.

PBS-washed carboxylate-modified latex beads having a diameter of 500±100 nm were placed in a mixed solution of 50 mM EDC and 200 mM NHS, and the resulting mixture was dropped to react with spots of the array chip for 20 min. Thereafter, the chip was washed with PBS containing 0.1% by weight of Tween 20 for 1 hour, then distilled water for 5 min, and dried in air to result in introduction of latex beads into terminal amine groups of 3-aminopropyltrimethoxysilane. A mixed solution of 200 niM EDC and 50 mM NHS was dropped on the latex beads, allowed to stand at room temperature for 10 min, washed with distilled water and dried with air blowing. Then, a solution of casein (1 mg/mL) in PBS (pH 7.4) was dropped onto spots, and the chip was reacted in an incubator at 37 ^°C for 2 hours. Thereafter, the chip was washed with PBS containing 0.1% by weight of Tween 20 for 10 min, then distilled water for 5 min, and dried in air to result in introduction of a substrate (casein) into the latex beads.

A blocking solution with addition of 3% by weight of BSA to PBST (containing 0.1% by weight of Tween 20) was dropped onto the casein-introduced spots which were then placed in an incubator at 37 ^°C for 1 hour. Thereafter, the chip was washed with PBS containing 0.1% by weight of Tween 20 for 10 min, then distilled water for 5 min, and dried in air to fabricate an array chip for measuring TGase activity.

To casein of the fabricated array chip was dropped a sample with addition of 50 μg/mL of TGase, 328 /zg/mL of 5-(biotinamido)pentylamine (BAPA), 1,542 μg/mL ofDTT and CaCl₂ (at varying concentrations as indicated in FIG. 2A) to 24,220 /zg/rnL of Tris-HCl buffer. The array chip was placed in an incubator at 37^°C for 1 hour, washed with PBS containing 0.1% by weight of Tween 20 for 10 min, then distilled water for 5 min, and air- blow dried. Then, fluorophore Alexa 546-labeld streptavidin was dropped and bound to biotin. Fluorescence intensity was measured using a fluorescence scanner (ScanArray Lite). The results obtained are shown in FIG. 2A. The measurement results for fluorescence intensity of FIG. 2 A are graphed in FIG. 2B.

As shown in FIGS. 2 A and 2B, it was confirmed that a degree of activation of TGase varies with varying concentrations of calcium ions. That is, higher fluorescence intensity represents increases in the enzymatic activity, whereas lower fluorescence intensity represents decreases in the enzymatic activity. Example 3

A slide glass was dipped in a solution of 1.5% by weight of 3- aminopropyltriniethoxysilane in ethanol for 2 hours, washed with ethanol and distilled water, and dried in an oven at 110^°C for 1 hour to introduce 3-aminopropyltrimethoxysilane into the slide glass surface. The slide glass to be used was thoroughly cleaned in a cleaning solution (H₂O₂INH₄OHIdH₂O = 1:1:5) at 70 ^°C for 10 min.

The 3-aminopropyltrimethoxysilane-introduced array chip was dipped in a solution of 1% by weight of glutaraldehyde in PBS for 30 min, washed with distilled water, and dried in air to result in introduction of aldehyde groups into the array chip into which 3- aminopropyltrimethoxysilane was introduced.

A Teflon tape, which has spots with a diameter of 1.5 mm formed using a punch, was attached to the slide glass surface with introduction of aldehyde to thereby fabricate an array chip having 200 (8><25) spots. Then, a solution of casein (1 mg/mL) in PBS (pH 7.4) was dropped onto spots, and the chip was reacted in an incubator at 37^°C for 2 hours.

Thereafter, the chip was washed with PBS containing 0.1% by weight of Tween 20 for 10 min, then distilled water for 5 min, and dried in air to result in introduction of a substrate (casein) into the array chip into which 3-aminopropyltrimethoxysilane was introduced.

A blocking solution with addition of 3% by weight of BSA to PBST (containing 0.1% by weight of Tween 20) was dropped onto the casein-introduced, 3- aminopropyltrimethoxysilane array chip which was then placed in an incubator at 37^°C for 1 hour. Thereafter, the chip was washed with PBS containing 0.1% by weight of Tween 20 for 10 min, then distilled water for 5 min, and dried in air to fabricate an array chip for measuring TGase activity. To casein of the fabricated array chip was dropped a sample with addition of 294 //g/mL Of CaCl₂, 328 βglmL of 5-(biotinamido)pentylamine (BAPA), 1,542 //g/mL of DTT and TGase (at varying concentrations as indicated in FIG. IA) to 24,220 //g/mL of Tris-HCl buffer. The array chip was placed in an incubator at 37^°C for 1 hour, washed with PBS containing 0.1% by weight of Tween 20 for 10 min, then distilled water for 5 min, and air- blow dried. Then, fluorophore Alexa 546-labeld streptavidin was dropped and bound to biotin. Fluorescence intensity was measured using a fluorescence scanner (ScanArray Lite). The results obtained are shown in FIG. 3A. The measurement results for fluorescence intensity of FIG. 3A are graphed in FIG. 3B. As shown in FIGS. 3 A and 3B, it was confirmed that a degree of activation of

Example 4

As shown in FIGS. 4A and 4B, a TGase activity-assay solution containing a mixture of each 250 //g/mL of protein extracts isolated from cell cultures of 10 commercially available cell lines, 328 //g/mL of 5-(biotinamido)pentylamine, 1,542 //g/mL of DTT and 294 //g/mL of CaCl₂ in 24,220 μg/mL of Tris-HCl buffer was dropped to the TGase activity-measuring chip fabricated in Example 2. The chip was placed in an incubator at 37^°C for 1 hour, washed with PBS containing 0.1% by weight of Tween 20 for 10 min, then distilled water for 5 min, and air-blow dried. Then, fluorophore Alexa 546-labeld streptavidin was dropped and bound to biotin. TGase activity in individual cell culture extracts was assayed using a fluorescence scanner (ScanArray Lite). The results obtained are shown in FIG. 4A. The measurement results for fluorescence intensity of FIG. 4A are graphed in FIG. 4B.

As shown in FIGS. 4 A and 4B, it was confirmed that when TGase activity in a variety of different cell culture extracts is assayed using the TGase activity-measuring chip in accordance with the present invention, it is possible to achieve rapid assay of TGase activity in a plurality of cell culture samples, using a chip for measuring an enzymatic activity developed by the present invention.

Example 5 As shown in FIGS. 5A and 5B, a TGase activity-assay solution containing a mixture of each 10 μg/mL of individual blood extracts collected from 10 patients, 328 μg/mL of 5-(biotinamido)pentylamine, 1,542 μg/mL of DTT and 294 μg/mL of CaCl₂ in 24,220 μg/mL of Tris-HCl buffer was dropped to the TGase activity-measuring chip fabricated in Example 2. The chip was placed in an incubator at 37^°C for 1 hour, washed with PBS containing 0.1% by weight of Tween 20 for 10 min, then distilled water for 5 min, and air-blow dried. Then, fluorophore Alexa 546-labeld streptavidin was dropped and bound to biotin. TGase activity in individual blood extracts was assayed using a fluorescence scanner (ScanArray Lite). The results obtained are shown in FIG. 5A. The measurement results for fluorescence intensity of FIG. 5 A are graphed in FIG. 5B. As shown in FIGS. 5 A and 5B, it was confirmed that when TGase activity in a variety of patient blood extracts is assayed using the TGase activity-measuring chip in accordance with the present invention, it is possible to achieve rapid assay of TGase activity in a plurality of patient blood extracts, using a chip for measuring an enzymatic activity developed by the present invention. As apparent from the above description, the present invention provides a method capable of simply and conveniently measuring TGase activity using an analysis chip in the form of an array, unlike conventional methods of measuring TGase activity via utilization of radioisotopes or Western blotting. Therefore, the present invention provides various advantages such as a significant reduction of an analysis period, high-sensitivity analysis even with a tiny amount of a sample, applicability in analysis methods for elucidation of intracellular mechanisms, applicability in cellular control and functional study via measurement of intracellular activity of a target enzyme, and applicability in examination of various diseases using body fluids such as blood.

Claims

WHAT IS CLAIMED IS:

1. A method for measuring transglutaminase (TGase) activity, comprising: introducing functional group-terminated alkoxysilane into a surface of a substrate; attaching a regularly punched tape to the substrate surface to fabricate an array chip having a plurality of spots formed thereon; introducing a substrate for TGase into ends of spots; dropping a TGase-containing sample for measuring enzymatic activity and an assay solution containing 5-(biotinamido)pentylamine, DTT and CaCl₂ on the substrate to induce transamidation of the substrate with 5-(biotinamido)pentylamine; and introducing an assay label into biotin which was terminally-positioned via transamidation and assaying the labeled biotin to measure TGase activity.

2. The method according to claim 1, wherein the functional group-terminated alkoxysilane is selected from aminoalkyltrialkoxysilane, mercaptoalkyltrialkoxysilane and epoxyalkyltrialkoxysilane.

3. The method according to claim 2, wherein the aminoalkyltrialkoxysilane is selected from 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane and 3-[2-(2- aminoethylamino)ethylamino]propyl-trimethoxysilane, the mercaptoalkyltrialkoxysilane is 3-mercaptopropyltrimethoxysilane, and the epoxyalkyltrialkoxysilane is 3- glycidoxypropyltrimethoxysilane.

4. The method according to claim 1 , wherein the introduction of the substrate includes reacting a medium with alkoxysilane of the spots formed by tape attachment, and introducing the substrate into the medium.

5. The method according to claim 4, wherein the medium is carboxylate-modified latex beads.

6. The method according to claim 5, wherein the reaction between alkoxysilane and carboxylate-modified latex beads includes reacting the carboxylate-modified latex beads with N-hydroxysuccinimide in the presence of carbodiimide to activate latex beads, and dropping the resulting reaction solution to alkoxysilane.

7. The method according to claim 6, wherein the introduction of the substrate into latex beads includes dropping a mixed solution of N-hydroxysuccinimide and carbodiimide on latex beads to activate carboxyl groups of latex beads, and dropping a substrate-containing solution onto the beads.

8. The method according to claim 4, wherein the medium is a compound which has functional groups at both terminals and a substrate-reactive aldehyde group at at least one terminal.

9. The method according to claim 8, wherein the compound is selected from glutaraldehyde and benzene- 1 ,4-dicarboxaldehyde.

10. The method according to any one of claims 1 to 9, wherein the introduction of the substrate includes blocking of unoccupied parts of spots to which the substrate was not bound after introduction of the substrate, using a blocking solution.

11. The method according to claim 1 , wherein the incorporation of an assay label into biotin is carried out using fluorescent dye or enzyme-conjugated streptavidin or avidin.

12. The method according to claim 11, wherein the assay label is streptavidin or avidin conjugated with a fluorescent dye selected from Cy3, Cy5, Alexa, BODIPY, Rhodamine and

Q-dots, and TGase activity is measured by fluorescence analysis.

13. The method according to claim 11, wherein the assay label is streptavidin or avidin labeled with an enzyme selected from alkaline phosphatase, horseradish peroxidase and luciferase, and TGase activity is measured by reacting the labeled streptavidin or avidin with a chromogenic substrate.

14. A method of fabricating a chip for measuring transglutaminase (TGase) activity, comprising: introducing functional group-terminated alkoxysilane into a surface of a substrate; attaching a regularly punched tape to the substrate surface to fabricate an array chip having a plurality of spots formed thereon; and introducing a substrate for TGase into ends of spots.

15. The method according to claim 14, wherein the functional group-terminated alkoxysilane is selected from aminoalkyltrialkoxysilane, mercaptoalkyltrialkoxysilane and epoxyalkyltrialkoxysilane.

16. The method according to claim 15, wherein the aminoalkyltrialkoxysilane is 3- aminopropyltrirnethoxysilane or 3-[2-(2-aminoethylamino)ethylamino]propyl- trimethoxysilane, the mercaptoalkyltrialkoxysilane is 3-mercaptopropyltrimethoxysilane, and the epoxyalkyltrialkoxysilane is 3-glycidoxypropyltrimethoxysilane.

17. The method according to claim 14, wherein the introduction of the substrate includes reacting a medium with alkoxysilane of the spots formed by tape attachment, and introducing the substrate into the medium.

18. The method according to claim 17, wherein the medium is carboxylate-modified latex beads.

19. The method according to claim 18, wherein the reaction between alkoxysilane and carboxylate-modified latex beads includes reacting the carboxylate-modified latex beads with N-hydroxysuccinimide in the presence of carbodiimide to activate latex beads, and dropping the resulting reaction solution to alkoxysilane.

20. The method according to claim 19, wherein the introduction of the substrate into latex beads includes dropping a mixed solution of N-hydroxysuccinimide and carbodiimide on latex beads to activate carboxyl groups of latex beads, and dropping a substrate-containing solution onto the beads.

21. The method according to claim 17, wherein the medium is a compound which has functional groups at both terminals and a substrate-reactive aldehyde group at at least one terminal.

22. The method according to claim 20, wherein the compound is selected from glutaraldehyde and benzene- 1,4-dicarboxaldehyde.

23. The method according to any one of claims 14 to 22, wherein the introduction of the substrate includes blocking of unoccupied parts of spots to which the substrate was not bound after introduction of the substrate, using a blocking solution.

24. A chip for measuring transglutaminase (TGase) activity, which is prepared by attaching a regularly punched tape to a surface of a substrate with introduction of functional group- terminated alkoxysilane to form a plurality of spots and introducing a substrate for TGase into ends of spots.