KR102230533B1 - Surface-Enhanced Raman Spectroscopy based Protease Detection Sensor using Conductive Polymer Nanostructures and Metal Nanoparticle Complex - Google Patents

Abstract

The surface-enhanced Raman spectroscopy-based biosensor comprising a conductive polymer nanostructure according to the present invention and a metal nanoparticle complex to which a peptide consisting of a sequence cleavable by a Raman marker and a proteolytic enzyme is immobilized is used. It provides a highly sensitive platform that can quickly and accurately measure activity and inhibition of activity, and can be usefully used as a non-destructive cell-based, real-time measurement, and a body fluid-based field disease diagnosis kit in the future.

Description

Surface-Enhanced Raman Spectroscopy based Protease Detection Sensor using Conductive Polymer Nanostructures and Metal Nanoparticle Complex}

The present invention relates to a sensor for detecting a proteolytic enzyme based on surface-enhanced Raman spectroscopy using a conductive polymer nanostructure and a metal nanoparticle complex.

Proteins are one of the biological substances that make up our body, and are transcribed and translated from DNA in cells to perform various functions. In particular, specific overexpressed proteins are used as biomarkers in diagnosing and measuring diseases. Therefore, the development of protein biosensors has received great attention for its wide application in disease diagnosis and medicine. Among them, an enzyme-linked immunosorbent assay (ELISA) detection method based on an immune response was mainly used. The characteristic of this detection method is that it is advantageous to selectively measure a specific protein based on an antigen-antibody reaction, and there is no limitation on a protein-based target substance. However, it has a limitation in that it takes a lot of time and cost to produce an antibody, and the stability of the antibody is poor, which may cause a malfunction of the biosensor.

Numerous biomaterials have been used to overcome the problems of such immune response-based protein biosensors. Among disease-related proteins, some of the proteins having the property of enzymes have the property of degrading proteins, and in particular, they have the property of recognizing and decomposing a specific peptide sequence. Using the properties of these proteins, development of a peptide-cleavage based biosensor to which a specific peptide sequence is attached is in progress. This is attracting attention as an excellent biosensor detection method that can overcome disadvantages such as high cost, long time, and stability, which are the limitations of antibody-based biosensors. In addition, since the step of immobilizing additional signal substances required in the antibody-based biosensor can be omitted (label-free), it is expected that the user can easily and quickly measure protein.

When all materials are irradiated with light, light scattering occurs, and among them, measuring the scattering of light with different wavelengths is called Raman spectroscopy. Using this, a specific substance can be selectively measured, but there is a disadvantage in that it is difficult to measure due to a small Raman intensity. To compensate for this, surface-enhanced Raman spectroscopy is being applied to biosensors. This technology amplifies the Raman signal, and it is possible to amplify the signal by ^{approximately 10 to 11 times.} In order to induce this, nanostructures such as gold and silver, or carbon-based materials such as graphene and carbon nanotubes are mainly used. If a Raman marker having a specific Raman wavelength is used, sensitive measurement of disease-related biomaterials can be made possible. In order to sensitively measure biomaterials, it can be said that it is essential to fabricate a nanostructure capable of maximizing amplification of Raman signals.

The present inventors have tried to develop a biosensor that can effectively detect the presence, activity, and concentration of proteolytic enzymes. As a result, a conductive polymer nanostructure and a metal nanoparticle-peptide complex including a Raman marker on the surface were prepared, and the nanostructure and the metal nanoparticle-peptide complex amplify the signal of the Raman marker due to the electromagnetic effect of the surface, When the peptide was degraded by a proteolytic enzyme, it was confirmed that the distance between the nanostructure and the Raman marker was increased, and thus the signal of the Raman marker was decreased. Through this, it was found that proteolytic enzymes can be quickly and stably detected using conductive polymer nanostructures and metal nanoparticle-peptide complexes that induce amplification of Raman marker signals based on surface-enhanced Raman spectroscopy. Was completed.

Accordingly, an object of the present invention is to provide a sensor for detecting proteolytic enzymes.

Another object of the present invention is to provide a method of detecting a proteolytic enzyme using the sensor for detecting a proteolytic enzyme of the present invention.

Another object of the present invention is to provide a composition for screening a substance that inhibits the activity of a proteolytic enzyme comprising the sensor for detecting a proteolytic enzyme of the present invention.

According to one aspect of the invention, the present invention (a) a nanostructure made of a conductive polymer coated with metal nanoparticles; And (b) a metal nanoparticle complex comprising a metal nanoparticle, a Raman marker, and a peptide consisting of a sequence cleavable by a proteolytic enzyme.

The present inventors have tried to develop a biosensor that can effectively detect the presence, activity, and concentration of proteolytic enzymes. As a result, a conductive polymer nanostructure and a metal nanoparticle-peptide complex including a Raman marker on the surface were prepared, and the nanostructure and the metal nanoparticle-peptide complex amplify the signal of the Raman marker due to the electromagnetic effect of the surface, When the peptide was degraded by a proteolytic enzyme, it was confirmed that the distance between the nanostructure and the Raman marker was increased, and thus the signal of the Raman marker was decreased. Through this, it was found that proteolytic enzymes can be quickly and stably detected using a conductive polymer nanostructure and a metal nanoparticle-peptide complex that induces amplification of the Raman marker signal based on surface-enhanced Raman spectroscopy.

Raman spectroscopy is a phenomenon in which inelastic scattering occurs when the energy of incident light is applied to a molecular sieve, resulting in a change in energy. However, the Raman spectroscopy method has a disadvantage in that the signal strength is very weak and the reproducibility is low, which deteriorates the sensitivity and accuracy of analysis and diagnosis.

Surface-Enhanced Raman Spectroscopy refers to a phenomenon in which the Raman signal of the molecule is greatly increased when a target molecule is present around a metal nanostructure. By enhancing the signal compared to general Raman scattering through the surface treatment of nanoparticles, it is possible to detect a small amount of ultra-high sensitivity to a level that can technically measure single molecules. The intensity of the signal is amplified by utilizing the effect of Localized Surface Plasmon Resonance (LSPR) that the incident energy is amplified and released from metal nanostructures with various nanogaps, allowing for analysis and quantification of specific biomaterials or chemical molecules It is possible. In addition, since it has a molecular-specific Raman signal, multiple detection is possible, the Raman signal does not decrease with time, and is not sensitive to environmental influences such as temperature and humidity.

In the present invention, a substrate is prepared to amplify the Raman signal by synthesizing a conductive polymer into a nanostructure and coating the surface with metal nanoparticles, and a peptide, streptavidin, and a Raman marker that can be cleaved by a specific protease on the surface of the metal nanoparticles To prepare a metal nanoparticle composite immobilized. Cysteine and biotin were attached to both ends of the peptide, and the nanostructure and the metal nanoparticle complex were designed to be connected to each other by the peptide. When the nanostructure and the metal nanoparticle complex are connected by a peptide, the Raman signal on the surface of the metal nanoparticle is amplified by the characteristics of surface-enhanced Raman spectroscopy, and when the peptide is decomposed by reacting with a proteolytic enzyme, the nanostructure and the metal nanoparticle Since the Raman signal on the surface of the complex is distant, the Raman signal is reduced, so it can be used as a sensor for detecting proteases through this characteristic.

In the present invention, the metal nanoparticles included in the sensor for proteolytic enzyme detection may be selected from the group consisting of gold (Au), silver (Ag), copper (Cu), and platinum (Pt).

According to one embodiment of the present invention, in the nanostructure made of the conductive polymer coated with the metal nanoparticles, the metal nanoparticles are from the group consisting of gold (Au), silver (Ag), copper (Cu), and platinum (Pt). It may be selected, for example, may be silver (Ag).

According to an embodiment of the present invention, in the nanostructure made of the conductive polymer, the conductive polymer is polypyrrole (PPy), polyaniline (PANI), polythiophene (PT), polyacetylene, and PEDOT. It may be selected from the group consisting of (poly(3,4-ethylene dioxythiophene)), and may be, for example, polypyrrole (PPy).

The nanostructure made of the conductive polymer may be manufactured in a shape of a horn having nano-sized pores, but is not limited thereto.

According to an embodiment of the present invention, the metal nanoparticles in the metal nanoparticle composite may be selected from the group consisting of gold (Au), silver (Ag), copper (Cu), and platinum (Pt), for example For example, it could be gold (Au).

The size of the metal nanoparticles in the metal nanoparticle composite is 1 to 100 nm, 5 to 100 nm, 10 to 100 nm, 15 to 100 nm, 20 to 100 nm, 25 to 100 nm, 1 to 90 nm, 5 to 90 nm, 10 to 90 nm, 15 to 90 nm, 20 to 90 nm, 25 to 90 nm, 1 to 80 nm, 5 to 80 nm, 10 to 80 nm, 15 to 80 nm, 20 to 80 nm, 25 to 80 nm, 1 to 70 nm, 5 to 70 nm, 10 to 70 nm, 15 to 70 nm, 20 to 70 nm, 25 to 70 nm, 1 to 60 nm, 5 to 60 nm, 10 to 60 nm, 15 to 60 nm, 20 to 60 nm, 25 to 60 nm, 1 to 50 nm, 5 to 50 nm, 10 to 50 nm, 15 to 50 nm, 20 to 50 nm, 25 to 50 nm, 1 to 40 nm, 5 to 40 nm, 10 to 40 nm, 15 to 40 nm, 20 to 40 nm, 25 to 40 nm, 1 to 30 nm, 5 to 30 nm, 10 to 30 nm, 15 to 30 nm, 20 to 30 nm or 25 to 30 nm, for example, 30 nm.

According to one embodiment of the present invention, the metal nanoparticle composite may have a surface coated with streptavidin.

The peptide may be linked to the metal nanoparticle by introducing biotin at one end.

The peptide may be connected to a nanostructure made of the conductive polymer by introducing a thiol group (-SH) at one end, for example, a cysteine having a thiol group is introduced at one end of the peptide. It may be connected to the nanostructure made of the conductive polymer.

According to an embodiment of the present invention, the Raman marker is rhodamine B, rhodamine 6G, cyanine-3, cyanine-5, Cyanine-5.5, Cyanine 7, 4-aminothiophenol, 4-methylbenzenethiol, 2-naphthalenethiol, and rhodamine -5-(and-6)-isothiocyanate (rhodamine-5-(and-6)-isothiocyanate), tetramethylrhodamine-5-isothiocyanate, FAM and TAMRA It may be selected from the group consisting of, for example, may be rhodamine B (rhodamine B).

According to an embodiment of the present invention, in the sensor for detecting a proteolytic enzyme, the peptide may be a dipeptide, a tripeptide, an oligopeptide or a polypeptide depending on the number of amino acid residues, and, for example, 2 to 500 amino acids. It may be a composed peptide.

The peptide is a compound in which one or more amino acids are bound by a peptide bond, and is preferably a peptide substrate that is specifically degraded by a target proteolytic enzyme.

The proteolytic enzyme is a proteolytic enzyme that cleaves between two specific amino acids in a peptide sequence, such as caspase, trypsin, cathepsin, urokinase, matrix metalloproteinases (MMP), and thrombin. (thrombin), HIV-1 protease, HSV-1 protease, TEV protease, PSA (Prostate Specific Antigen) and secretase ( secretase) may be selected from the group consisting of, for example, caspase.

According to an embodiment of the present invention, the caspase may be caspase-3 or caspase-1, for example, caspase-3 3) can be.

According to an embodiment of the present invention, in the sensor for detecting proteolytic enzymes, the peptide may be a peptide consisting of a sequence cleavable by caspase-3, for example, consisting of a sequence represented by the first sequence of the sequence list. It may be a peptide.

According to another aspect of the present invention, the present invention comprises the steps of adding a target proteolytic enzyme to the sensor for detecting the proteolytic enzyme; And measuring a Raman signal from the sensor.

According to an embodiment of the present invention, by measuring the intensity of the unique Raman peak displayed by the sensor for detecting the protease, it is possible to easily analyze the presence, concentration, activity, and inhibition of activity of the protease. I can.

According to another aspect of the present invention, the present invention relates to a composition for screening a substance that inhibits the activity of proteolytic enzymes, including the sensor for detecting proteolytic enzymes.

Proteolytic enzymes play a role in regulating various cellular functions in a wide range, and since this is accomplished through the decomposition of physiologically active substances, the function and role of proteolytic enzymes in the life phenomena of all living organisms are very important. Deficiency, lack or overexpression of proteolytic enzymes can cause cancer, arthritis, neurodegenerative diseases, cardiovascular and autoimmune inflammatory diseases, and the like.

The sensor for detecting proteolytic enzymes of the present invention can easily determine the presence, activity, and inhibition of activity of proteolytic enzymes present in specific tissues or cells in a living body, so cell images, specific tissue images, drug delivery systems, etc. It can be used for a variety of purposes. The composition can be applied both in vivo and in vitro , and thus can be used for various purposes such as high-throughput screening method and early disease diagnosis required for new drug development.

The surface-enhanced Raman spectroscopy-based biosensor comprising a conductive polymer nanostructure according to the present invention and a metal nanoparticle complex to which a peptide consisting of a sequence cleavable by a Raman marker and a proteolytic enzyme is immobilized is used. It provides a highly sensitive platform that can quickly and accurately measure activity and inhibition of activity, and can be usefully used as a non-destructive cell-based, real-time measurement, and a body fluid-based field disease diagnosis kit in the future.

1 is a schematic diagram showing the prepared gold nanocomposite.
2 is a schematic diagram of fabrication of a silver/polypyrrole/silver nanostructure and a gold nanocomposite inducing a surface-enhancing Raman effect.
3A is a Raman spectrum for comparing Raman signals of polypyrrole and silver/polypyrrole/silver nanostructures.
3B is a bar graph showing specific peaks for comparing Raman signals of polypyrrole and silver/polypyrrole/silver nanostructures.
4 is a spectrum graph comparing Raman signals of silver/polypyrrole/silver nanostructures using silver/polypyrrole/silver nanostructures and gold nanocomposites.
5 is a Raman spectrum obtained after reacting caspase-3 at different concentrations on the prepared gold nanocomposite-silver/polypyrrole/silver nanostructure.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1: Preparation of gold nanoparticle-Raman marker-streptavidin-peptide complex

Gold nanoparticles with a size of 30 nm were used to amplify the Raman intensity of the Raman marker. Rhodamine B (10 mg/ml), one of Raman markers, was coated on the surface of gold nanoparticles (1 ml) by reacting for 10 hours or more at room temperature. This serves to amplify the inherent Raman signal of Rhodamine B.

A peptide composed of a specific sequence was used for the binding of gold nanoparticles and silver/polypyrrole/silver nanostructures. This specific sequence is a peptide containing DEVD that is degraded by selectively reacting to a target protein, and has a property of being selectively degraded by caspase-3. For the binding of gold nanoparticles and silver nanostructures, biotin and cysteine were designed to attach to both sides of the peptide, respectively. In addition, streptavidin (1 mg/ml) capable of binding with biotin was attached to the surface of the gold nanoparticles by reacting for 10 hours or more at room temperature. The biotin-attached peptide was reacted with the gold nanoparticles at room temperature for 1 hour to bind to each other, and then reacted with the silver/polypyrrole/silver nanostructure for 1 hour at room temperature to allow the gold nanoparticles to adhere well to the surface of the silver nanostructure. . After the reaction with the gold nanoparticles, all biomaterials are washed three times through a centrifuge and a washing solution (0.1% BSA, 0.05% Tween 20 in PBS, pH 7.4).

1 is a schematic diagram of a synthesized gold nanoparticle-peptide complex. The gold nanocomposite is composed of gold nanoparticles, Raman marker (Rhodamine B), streptavidin, and biotin-peptide, and the specific sequence of the peptide is as shown in the schematic diagram.

The surface of the gold nanoparticles is coated with Rhodamine B and streptavidin, and a peptide with biotin is selectively bound to streptavidin. The fabricated gold nanocomposite is bound to the surface of the silver/polypyrrole/silver nanostructure through the SH-Au reaction, and the Raman signal of Rhodamine B is expected to be amplified due to the double electromagnetic effect of the gold-silver nanostructure.

Example 2: Confirmation of the Raman amplification effect of the prepared silver/conductive polymer/silver nanostructure and gold nanocomposite

A silver/polypyrrole/silver nanostructure was fabricated to improve the Raman signal of the gold nanocomposite prepared above. Using the cleaned gold substrate as a working electrode, polypyrrole was electrochemically deposited. The deposited polypyrrole nanostructure was manufactured in a shape of a horn with a nano-sized hole in the middle, and through this, the effect of increasing the reaction surface area can be expected and it is expected that the signal of the biosensor can be improved. It is intended to induce a surface-enhanced Raman spectroscopy effect by reducing silver ions on the surface of the fabricated polypyrrole nanostructure. The Raman marker between the gold nanocomposite and the silver nanostructure is expected to improve the Raman signal due to the electromagnetic effect of the two metals.

2 is a schematic diagram of a silver/polypyrrole/silver nanostructure and a gold nanocomposite for protein measurement. First, silver/polypyrrole/silver nanostructures are fabricated by reducing silver ions to polypyrrole (conductive polymer) nanostructures made by an electro-deposition method. The gold nanocomposite prepared above is immobilized on the silver nanostructure to amplify the Raman signal. Subsequently, it was confirmed that the Raman signal was decreased by reacting a protein having hydrolytic properties to decompose femtide.

A gold nanocomposite with a peptide immobilized on a nanostructure made through reduction of silver ions is reacted at room temperature for 1 hour. The cysteine at the end of the peptide has a thiol group that can bind to the surface of the silver nanostructure, inducing the connection between the gold nanocomposite and the silver nanostructure. The fabricated nano-platform for protein measurement exhibits a strong Raman signal, and it is expected to be able to sensitively measure even a small concentration of protein using this. After reacting the caspase-3 protein, which can selectively decompose a specific peptide sequence designed in this sensor, at room temperature for 1 hour, washing the substrate and measuring the Raman signal, the Raman intensity decreases depending on the concentration of caspase-3. You can check the degree.

3 is a Raman measurement graph for confirming the Raman signal amplification of the silver nanostructure. The Raman signal obtained by attaching the peptide-Rhodamine B on the silver-reduced nanostructure and the non-silver-reduced flippyrrole nanostructure was compared. As can be seen from the figure, it was confirmed that the Raman intensity of the silver-reduced nanostructure was significantly higher than that of the polypyrrole nanostructure. In addition, when 1000 ng/ml of caspase-3 was reacted on each substrate, a change in Raman signal was not detected in the polypyrrole nanostructure, whereas the Raman signal was significantly reduced in the silver/polypyrrole/silver nanostructure. Through this, it was confirmed that silver/polypyrrole/silver nanostructures are essential for Raman-based biosensor fabrication.

4 is a graph showing Raman signals before and after immobilization of a gold nanocomposite on a silver/polypyrrole/silver nanostructure to confirm the surface-enhanced Raman effect of the gold nanocomposite. A total of 3 repeated experiments were conducted, and it was confirmed that the Raman signal using the gold nanocomposite exhibited an amplification effect of about 4 times or more compared to that of the other, as can be seen from the Raman spectrum. As mentioned before, this can be seen as a phenomenon in which rhodamine B receives an electromagnetic effect between gold nanoparticles and silver nanostructures at the same time and amplifies the Raman signal. Through this result, the surface enhancement Raman effect of the nanoplatform using the fabricated silver/polypyrrole/silver nanostructure and gold nanocomposite at the same time was confirmed.

Example 3: Invented silver/conductive polymer/silver nanostructure and gold nanocomposite-based protein measurement

The amplification effect of the Raman signal was confirmed using the prepared silver/polypyrrole/silver nanostructure and gold nanocomposite. Using this, we tried to measure caspase-3, one of the proteolytic enzymes, in a tactical way. In the peptide sequence connecting the silver nanostructure and the gold nanoparticle, there is a DEVD that is selectively degraded by caspase-3. When Caspase-3 decomposes the peptide, the gold nanocomposite with a Raman marker attached thereto falls off the surface of the silver/polypyrrole/silver nanostructure, and the Raman signal can be expected to decrease rapidly. This role is expected to enable sensitive measurement of caspase-3.

5 is a Raman spectrum graph showing changes in Raman signals for each concentration of caspase-3. The concentration of cCaspase-3 was measured from 10 pg/ml to 10 μg/ml, and the reaction conditions were 1 hour at room temperature. As can be seen from the spectrum, it can be seen that when the concentration of caspase-3 is high, the Raman signal drops sharply, and as the concentration is low, the drop size decreases. It was confirmed through the experimental results that caspase- can be successfully measured even at a small concentration of 10 pg/ml, and that the measurement range is relatively wide as ^{10 7.} Through this, it was confirmed that the fabricated nanotechnology-based platform has excellent sensitivity and measurement range.

<110> SOGANG UNIVERSITY RESEARCH FOUNDATION <120> Surface-Enhanced Raman Spectroscopy based Protease Detection Sensor using Conductive Polymer Nanostructures and Metal Nanoparticle Complex <130> PN190005 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Digestion peptide by Caspase-3 <400> 1 Ser Gly Asp Glu Val Asp Ser Gly 1 5

Claims (11)

(a) a nanostructure made of a conductive polymer coated with metal nanoparticles; And
(b) Metal nanoparticle complex comprising a peptide consisting of a metal nanoparticle, a Raman marker, and a sequence cleavable by a proteolytic enzyme
Including,
The Raman marker of (b) and the peptide consisting of a sequence cleavable by a proteolytic enzyme are immobilized on the surface of the metal nanoparticles of (b),
The metal nanoparticle complex of (b) is connected to the nanostructure of (a) by a thiol group (-SH) introduced at one end of the peptide, a sensor for proteolytic enzyme detection.
The proteolytic enzyme of claim 1, wherein the metal nanoparticles of (a) or (b) are selected from the group consisting of gold (Au), silver (Ag), copper (Cu), and platinum (Pt). Sensor for detection.
The sensor according to claim 1, wherein the metal nanoparticles of (a) are silver (Ag).
The sensor according to claim 1, wherein the metal nanoparticles of (b) are gold (Au).
delete The method of claim 1, wherein the metal nanoparticle of (b) is coated with streptavidin, and the peptide consisting of a sequence cleavable by the proteolytic enzyme is introduced with biotin at the other end thereof. The metal nanoparticles and the peptide are connected to each other, a sensor for proteolytic enzyme detection.
The method of claim 1, wherein the conductive polymer is polypyrrole (PPy), polyaniline (PANI), polythiophene (PT), polyacetylene, and poly(3,4-ethylene dioxythiophene) (PEDOT). ) That is selected from the group consisting of, a sensor for proteolytic enzyme detection.
The method of claim 1, wherein the Raman marker is rhodamine B, rhodamine 6G, cyanine-3, cyanine-5, and cyanine-5.5. (Cyanine-5.5), cyanine 7, 4-aminothiophenol, 4-methylbenzenethiol, 2-naphthalenethiol, rhodamine-5- From the group consisting of (and-6)-isothiocyanate (rhodamine-5-(and-6)-isothiocyanate), tetramethylrhodamine-5-isothiocyanate, FAM and TAMRA Which is selected, a sensor for proteolytic enzyme detection.
The method of claim 1, wherein the proteolytic enzyme is caspase, trypsin, cathepsin, urokinase, matrix metalloproteinases (MMP), thrombin, and HIV-1 protein degradation. Selected from the group consisting of enzymes (HIV-1 protease), HSV-1 proteases (HSV-1 protease), TEV proteases, PSA (Prostate Specific Antigen) and secretases Sensor for detecting phosphorus and proteolytic enzymes.
The step of adding a target proteolytic enzyme to the sensor for detecting a proteolytic enzyme according to any one of claims 1 to 4 and 6 to 9; And
Measuring a Raman signal from the sensor
Method for detecting a proteolytic enzyme comprising a.
A composition for screening a substance that inhibits the activity of a proteolytic enzyme, comprising the sensor for detecting a proteolytic enzyme according to any one of claims 1 to 4 and 6 to 9.

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