KR101633473B1 - Protease detection sensor - Google Patents

Abstract

The present invention relates to a protease for cleaving between two specific amino acids and a protease for detecting protease using a protease for cleaving the terminal amino acid. The sensor for detecting protease according to the present invention can be produced by using a target proteinase that cleaves between two specific amino acids in a peptide sequence and a protease that cleaves N-terminal or C-terminal amino acid, It exhibits an effect of detecting a target protein degrading enzyme with high sensitivity through an electrophoretic or electrochemical method.

Description

Protease detection sensor [0002]

The present invention relates to a protease for cleaving between two specific amino acids, and a sensor for detecting a target protease using a protease that cleaves a terminal amino acid.

A protease is an enzyme that selectively cleaves a specific sequence of a protein through hydrolysis. A particular amino acid, a specific sequence of multiple consecutive amino acids, or a peptide bond between two specific amino acids may be the cleavage sequence of a protein that specifically responds to proteolytic enzymes. Because these proteolytic enzymes are involved in metabolism in the body, their concentrations vary depending on the health condition of the organ that secretes proteolytic enzymes. In addition, in microorganisms and animals having toxins, proteolytic enzymes are used as means for destroying the protein tissue of living organisms. Therefore, it is very important to measure the concentration of proteolytic enzyme at high sensitivity for early detection of disease and accurate detection of toxic factors.

Proteolytic enzyme detection sensors developed so far use color detection, fluorescence detection, or electrochemical detection methods. Color or fluorescence detection methods are methods in which a protease digests a specific sequence of a peptide having a label capable of emitting color or fluorescence to obtain an absorption or fluorescence signal (Lou, X., Zhang, L., Qin, 2008, 80, 8509-8513 .; Zhao, Q., de Zoysa, RSS, Wang, D., Jayawardhana, DA, Guan, XJ Am. Chem. Soc., 2009, 131, 6324-6325.). Particularly, the fluorescence detection method utilizes fluorescence when fluorescence appears by a fluorescence resonance energy transfer (FRET) technique when a specific sequence of a peptide is cleaved, or fluorescence appears when a label attached to the peptide is cleaved. The electrochemical detection method is advantageous in that it can be easily detected with a small measuring instrument and a very low detection limit can be obtained. The electrochemical detection method of proteolytic enzyme is a method of analyzing the change of electrochemical signal generated when the protease is specifically bound to the electrode surface (Kerman, K., Mahmoud. KA, Kraatz, H.-B. Chem Sawole, M., Kraatz, H.-B. Analyst 2012, 137, 1120-1124. ) And the method of analyzing the change of the electrochemical signal caused by the peptide cleavage reaction of proteolytic enzyme. Here, the latter method can be subdivided as follows. (1) a method of removing a blocking membrane such as a gelatin film on the electrode surface using a proteolytic enzyme reaction (Ionescu, RE, Cosnier, S., Marks, RS Anal. Chem., 2006, 78, 6327-6331; D., Boyac1, IH Electroanalysis 2010, 22, 265-267 .; Zheng, X., Cook, JP, Watkinson, M., Yang, S., Douglas, I., Rawlinson, A., Krause, S. Faraday Actu 2012, 76, 43-47.), (2) proteolytic degradation, and (3) proteolytic degradation. (Swisher, LZ, Syed, LU, Prior, AM, Madiyar, FR, Carlson, KR, Nguyen, Ta, Hua, DH, Li, JJ Phys.Chem.C 2013, 117, 4268-4277 .; Shin, D.-S., Liu, Y., Gao, Y., Kwa, T., Matharu, Z. Ji, J., Gan, J., Kong, J., Yang, P., Liu, B., Ji, C. Electrochem. Commun 2012, 16, 53-56.), (3) a method of producing an electrochemically active substance such as 4-nitroaniline or 4-amino-2-chlorophenol through the reaction of a protease (Inoue, KY Ino, K., Shiku, H., Matsue, T. Electrochem. Commun., 2010, 12, 1066-1069 .; Thuerlemann, C., Haeberli, A., Alberio, Chem. 2009, 55, 505-512.), (4) a method of converting a polyionic peptide into a fragment of an amino acid using a protease (Gemene, KL, Meyerhoff, ME Anal. Biochem. 2011, 416, 67-73 Fordyce, K., Shvarev, A. Anal. Chem., 2008, 80, 827-833).

The fluorescence detection method and the electrochemical detection method (1), (2), and (4) using the FRET can be used for detecting a protease that cleaves between two specific amino acids. However, the detection method using fluorescence or color signals generated when the label attached to the peptide is cleaved and the electrochemical detection method (3) can detect the proteolytic enzyme which cleaves between two specific amino acids Can not be used to. In the detection of a proteolytic enzyme that cleaves between two specific amino acids, only the label can not be separated after cleavage by the proteolytic enzyme. Therefore, the present invention can be applied only to detection of a proteolytic enzyme that recognizes and cleaves a specific amino acid.

Therefore, a method of detecting a protease capable of cleaving between two specific amino acids with high sensitivity, based on a method of detecting using a fluorescent or color signal generated when a label attached to the peptide is cleaved or an electrochemical detection method (3) Is required to be developed.

KR 110-2008-0085497

When searching for a sensor for detecting a protease, the present inventors have found that when a protease that cleaves between two specific amino acids and a protease that cleaves a terminal amino acid are used, It was confirmed that protease can be detected with high sensitivity and the present invention has been completed.

Accordingly, the present invention provides a sensor for detecting a target protease using a protease that cleaves between two specific amino acids and a protease that cleaves a terminal amino acid, and a method for producing the same.

To achieve the above object,

The present invention

A sensor for detecting a protease having the following structural formula 1,

[Structural formula 1]

(1) (N) A-B-C (C); or

(2) (N) C-B-A (C);

Wherein A-B is a peptide consisting of a sequence cleavable by a target protease,

B is an amino acid cleaved from C by a second protease,

And C is a signal generating substance.

In addition,

A sensor for detecting the protease; And

A second protease, and a second protease.

In addition,

Adding a target proteinase and a second protease to the protease-detecting sensor; And

And measuring the color, fluorescence, or electrochemical signal from the sensor.

In addition,

(1) a step of reacting and binding a signal generating substance C to a peptide composed of sequence A-B cleavable by a target protease; And

(2) adding the ABC complex of the step (1) to the electrode.

Hereinafter, the present invention will be described in detail.

The present invention

A sensor for detecting a protease having the following structural formula 1,

[Structural formula 1]

(1) (N) A-B-C (C); or

(2) (N) C-B-A (C);

Wherein A-B is a peptide consisting of a sequence cleavable by a target protease,

B is an amino acid cleaved from C by a second protease,

And C is a signal generating substance.

In addition, the protease-detecting sensor has the following structural formula 2,

[Structural formula 2]

(1) (N) D-A-B-C (C); or

(2) (N) C-B-A-D (C);

And D is a protease-detecting sensor, wherein the protease inhibits the cleavage of the second protease.

The A-B may be a dipeptide, a tripeptide, an oligopeptide or a polypeptide according to the number of amino acid residues, and preferably a peptide consisting of 2 to 500 amino acids.

The peptide is preferably a peptide substrate in which one or more amino acids are bound by peptide bonds and is specifically degraded by the target proteinase. For example, the peptide substrate may be a target peptide such as MMP (matrix metalloproteinase), thrombin, caspase activated at apoptosis, protease, and the like. Depending on the specific protease, the peptide substrate used varies.

 The target proteinase is a proteolytic enzyme that cleaves between two specific amino acids in a peptide sequence, and is useful as a proteolytic enzyme for treating diseases caused by toxins such as botulinum neurotoxin (BoNT), matrix metalloproteinases (MMP), thrombin, FXIIIa (factor Xiiia) it is preferably one or more selected from the group consisting of caspase, urokinase plasminogen activator (uPA), HIV protease, DPP-IV (dipeptidyl peptidase) and proteasome, (BoNT).

The second protease may be an aminopeptidase or a carboxylpeptidase. Aminopeptidase and carboxypeptidase can be subdivided into leucyl-aminopeptidase, alanyl-aminopeptidase, carboxypeptidase A and B according to the amino acid showing activity. The aminopeptidase cleaves the N-terminal amino acid and the carboxypeptidase cleaves the C-terminal amino acid to remove all of the amino acids bound to the signal generating material, thereby generating a signal from the signal generating material.

The signal generating material preferably generates color, fluorescence, or electrochemical signals, but is not limited thereto.

The signal-generating material may include a color-changing agent, a fluorescent material, or an electrochemically active material capable of binding to the peptide, but is not limited thereto, and any material capable of generating a signal can be used.

Preferably, the signal generating material is selected from the group consisting of hydroquinone, aminophenol, diaminobenzene, dihydroxynaphthalene, aminonaphthol, diaminonaphthalene, benzoquinone, quinoneimine, naphthoquinone, naphthoquinone imine and Derivatives thereof, and may be at least one cyclic compound selected from the group consisting of naphthylamine, aminoisophthalin, coumarin, cyanine, fluorescein, tetramethylolamine, Alexa, Derivatives thereof, and may be para-nitro aniline or beta-naphthyl amine which exhibits a color change.

The electrochemical signal can be a direct redox reaction or a redox circulation reaction of the signal generating material. The redox circulation reaction can be performed by an electrochemical-enzymatic reaction, an electrochemical-chemical reaction, an electrochemical-chemical- An electrochemical-electrochemical reaction, and a combination thereof.

The D is not particularly limited so long as it is a substance that interferes with the cleavage action of the second protease. In one example, D may be carboxyethylamine or carboxypropylamine.

The detection limit for the target protease of the detection sensor is preferably 1 ng / mL or less. According to one embodiment of the present invention, the detection limit for the E-type botulinum neurotoxin light chain (BoNT / ELC) of the detection sensor is 200 pg / mL, 700 pg / mL in phosphate chloride buffer solution, tap water and commercial drinking water / mL and 700 pg / mL, respectively.

In addition,

A sensor for detecting the protease; And a second proteolytic enzyme.

In addition,

Adding a target proteinase and a second protease to the protease-detecting sensor; And measuring the color, fluorescence, or electrochemical signal from the sensor.

In addition,

(1) a step of reacting and binding a signal generating substance C to a peptide composed of sequence A-B cleavable by a target protease; And

(2) adding the A-B-C complex of the step (1) to the electrode.

The electrode may be a tin oxide electrode, a tin oxide electrode coated with graphene, a tin oxide electrode coated with carbon nanotube, a boron-doped diamond electrode, a diamond-like carbon electrode, , A gold electrode, a silver electrode, an Ag / AgCl electrode, a platinum electrode, and a combination thereof, but is not limited thereto.

The sensor for detecting a protease according to the present invention can be produced by using a proteolytic enzyme which cleaves between two specific amino acids and a proteolytic enzyme which cleaves the terminal amino acid, And exhibits an effect of detecting the enzyme with high sensitivity.

FIG. 1 is a conceptual diagram of a proteolytic enzyme-detecting sensor using an aminopeptidase cleaving a protease and an N-terminal amino acid between two specific amino acids (AB) in a peptide sequence bound to a signal generating substance C to be.
FIG. 2 is a conceptual diagram of a protease for detecting a protease using a carboxylpeptidase that cleaves a specific protease (AB) between two amino acids in the peptide sequence bound to the signal generating substance C and a C-terminal amino acid to be.
FIG. 3 is a graph showing the relationship between the amount of proteolytic enzymes that cleave between two specific amino acids (AB) in the peptide sequence bound to the signal generating substance C, and a substance D that interferes with the cleavage action of the second protease at the N-terminal of the peptide sequence And aminopeptidase which cleaves N-terminal amino acid.
FIG. 4 is a graph showing the relationship between the amount of proteolytic enzymes that cleave between two specific amino acids (AB) in the peptide sequence bound to the signal generating substance C, and a substance D that interferes with the cleavage action of the second protease at the C- And a carboxylpeptidase cleaving a C-terminal amino acid.
FIG. 5 is a graph showing the activity of the E-type botulinum neurotoxin light chain (BoNT / E LC) and 4-amino-1-naphthol, which cleaves between arginine and isoleucine (RI) (Aminopeptidase) cleaving an isoleucine linked to 4-amino-1-naphthol (4-amino-1-naphthol). Here, the product 4-amino-1-naphthol is electrochemically-chemically-chemically redox circulated to amplify the electric signal.
FIG. 6 is a graph showing the results of (a) phosphate buffer saline (pH 7.4), (b) tap water and (c) BoNT / E LC detection in commercial drinking water of the proteolytic enzyme detection sensor according to Example 1 Time versus charge (chronocoulometry).
7 is a graph showing corrected charges according to the BoNT / E LC concentration at a time of 100 seconds in time versus charge for BoNT / E LC detection in FIGS. 6A to 6C. FIG.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

1 and 3 are conceptual diagrams of a protease for detecting a protease using aminopeptidase for cleaving a protease and a N-terminal amino acid between two specific amino acids (A-B) in a peptide sequence. As shown in FIGS. 1 and 3, when the protease to be detected is cleaved between two amino acids A-B of a specific sequence, the amino-peptidase sequentially removes B, which is an N-terminal amino acid, to obtain a product C.

FIGS. 2 and 4 are conceptual diagrams of a proteolytic enzyme that cleaves between two specific amino acids (AB) in a peptide sequence, and a sensor that detects a protease using a carboxylpeptidase that cleaves a C-terminal amino acid. As shown in FIG. 2 and FIG. 4, when the protease to be detected is cleaved between two amino acids of a specific sequence, the product C can be obtained by sequentially removing the C-terminal amino acid B from the carboxypeptidase.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example 1. Preparation of a sensor for detecting protease

Figure 5 depicts the E-type botulinum neurotoxin light chain (BoNT / E LC) and 4-amino-1-naphthol cleavage between arginine and isoleucine (RI) (Aminopeptidase) cleaving an isoleucine, which is linked to a 1-naphthol. Here, the product, 4-amino-1-naphthol, was subjected to an electrochemical-chemical-chemical redox cycle to amplify the electric signal.

As shown in FIG. 5, when BoNT / E LC was reacted with arginine and isosin in the reaction product of an oligopeptide (IDTQNRQIDR-I) and 4-amino-1-naphthol When cleavage occurs between isoleucines, the aminopeptidase sequentially removes isoleucine to obtain the product, 4-amino-1-naphthol. Subsequently, the product, 4-amino-1-naphthol, Ru (NH ₃ ) ₆ ³⁺ and tris (2-carboxyethyl) phosphine, a reducing agent, dithiothreitol ), The current signal is amplified through an electrochemical-chemical-chemical redox cycle.

In this embodiment, in order to detect BoNT / ELC, a reaction product (IDTQNRQIDR-I-AN) in which a peptide and 4-amino-1-naphthol are bonded to an oligopeptide in a liquid sample in which BoNT / ELC is dissolved, aminopeptidase, Ru (NH ₃ ) ₆ ³⁺ and dithiothreitol, which is a reducing agent, were added at concentrations of 0.1 mM, 10 μg / mL, 1.0 mM and 2.0 mM, respectively, followed by reaction at 37 ° C. for 2 hours. In addition, indium-tin oxide (ITO) without pre-treatment such as a single film formation was used as an electrode.

Experimental Example 1. Detection of proteolytic enzyme

(A) phosphate buffer saline at pH 7.4, (b) tap water, and (c) commercial concentration of BoNT / E LC mixed with commercial drinking water in the sensor for detecting protease according to Example 1 The chronocoulometry for the time is shown in FIG. 6, and the corrected charge for the BoNT / E LC concentration at 100 seconds in FIG. 6 (a) - (c) is shown in FIG.

As shown in Figures 6 and 7, the detection limits of BoNT / E LC were 200 pg / mL, 700 pg / mL and 700 pg / mL, respectively, in phosphate buffered saline buffer, tap water and commercial drinking water, , The range of detectable concentrations was wider than that of tap water and commercial drinking water.

Claims (14)

A sensor for detecting a protease having the following structural formula 1,
[Structural formula 1]
(1) (N) ABC (C); or
(2) (N) CBA (C);
AB is a peptide consisting of a sequence cleavable by a target protease,
B is an amino acid cleaved from C by a second protease,
Wherein C is a signal generating substance which indicates a difference in color, fluorescence, or electrochemical signal between the state in which B is bonded and the state in which B is cleaved. The method according to claim 1,
The protein degradation inhibitor according to (1) or (2) above, wherein the protease inhibitor D, which interferes with the cleavage of the second protease, is bound to the (N) Sensor for enzyme detection. The method according to claim 1,
Wherein the AB is a peptide consisting of 2 to 500 amino acids. The method according to claim 1,
The target proteinase may be selected from the group consisting of botulinum neurotoxin (BoNT), matrix metalloproteinases (MMP), thrombin, FXIIIa (factor Xiiia), caspase, urokinase plasminogen activator, wherein the protease is at least one selected from the group consisting of uPA, HIV protease, dipeptidyl peptidase (DPP-IV), and proteasome. The method according to claim 1,
Wherein the second protease is an aminopeptidase or a carboxylpeptidase. 2. The sensor according to claim 1, wherein the second protease is an aminopeptidase or a carboxylpeptidase. The method according to claim 1,
Characterized in that the signal generating material generates a color, fluorescent or electrochemical signal. The method according to claim 1,
Wherein the signal generating material is selected from the group consisting of hydroquinone, aminophenol, diaminobenzene, dihydroxynaphthalene, aminonaphthol, diaminonaphthalene, benzoquinone, quinoneimine, naphthoquinone, naphthoquinone imine, Wherein the electrochemically active substance is an electrochemically active substance. The method according to claim 1,
Characterized in that the signal generating material is at least one phosphor selected from the group consisting of naphthylamine, aminoisophthalin, coumarin, cyanine, fluorescein, tetramethylolamine, alexa, Detecting enzyme for degrading enzyme. The method according to claim 6,
Wherein the electrochemical signal utilizes a direct redox reaction or a redox circulation reaction of the signal generating material. The method according to claim 1,
Wherein the detection limit of the target protease is 1 ng / mL or less. 11. A protease-detecting sensor according to any one of claims 1 to 10; And
A composition for detecting protease, comprising a second protease. 10. A method for detecting a protease comprising the steps of: adding a target proteinase and a second protease to a sensor for detecting a protease according to any one of claims 1 to 10; And
And measuring the color, fluorescence or electrochemical signal from the sensor. (1) A signal generating substance C, which shows a difference in color, fluorescence or electrochemical signal, between a state in which B is bound and a state in which B is cleaved is reacted with a peptide consisting of AB, which is a cleavable sequence by a target protease, Combining; And
(2) adding the ABC complex of the step (1) to the electrode. 14. The method of claim 13,
The electrode may be a tin oxide electrode, a tin oxide electrode coated with graphene, a tin oxide electrode coated with carbon nanotube, a boron-doped diamond electrode, a diamond-like carbon electrode, , Gold electrode, silver electrode, Ag / AgCl electrode, platinum electrode, and combinations thereof.

Images