KR102347808B1 - Complexes for detecting viruses based conjugated polymer - Google Patents

Abstract

The present invention relates to a complex for detecting viruses based on a conjugated polymer. The complex for detecting viruses has characteristics that an absorption wavelength changes when combined with a virus, the presence or absence of the virus can be checked through the change of the absorption wavelength, and the virus can be quantified by quantifying the change in the absorption wavelength.

Description

Conjugated polymer-based virus detection complex {COMPLEXES FOR DETECTING VIRUSES BASED CONJUGATED POLYMER}

The present invention relates to a conjugated polymer-based virus detection complex, and specifically, a virus detection complex using a change in the optical signal of a conjugated polymer supported in the complex when the virus detection complex and the virus are combined.

Diseases caused by viral infections, including COVID-19, are on the rise. Among diseases caused by viruses, influenza (influenza), commonly known as the 'flu', is an acute respiratory disease caused by influenza virus. Influenza causes large and small epidemics worldwide, and is very contagious enough to infect 10-20% of the general population within 2-3 weeks after the outbreak begins.

When infected with influenza virus, a healthy person shows symptoms such as chills, fever, headache, and cough for several days and then recovers. Every year, 5-10% of the adult population and 20-30% of the pediatric population are infected with influenza virus, reaching the level of 1 billion people, of which 150,000 to 500,000 die.

Accordingly, various technologies capable of detecting viruses have been developed. For example, Korean Patent Registration No. 10-2050213 discloses a bimolecular fluorescent probe system for in situ detection of influenza virus having neuraminidase, and the bimolecular fluorescent probe system is sial by neuraminidase in the presence of influenza virus. By measuring the change in fluorescence generated by separating galactose from the ric acid and galactose-coupled structure and binding to the bimolecular fluorescent protein probe structure, the presence of influenza virus can be detected quickly and with high sensitivity in the field. However, since the above method can only confirm the presence of influenza virus, a gene amplification test such as polymerase chain reaction must be additionally performed to quantify the virus.

In addition, although a method for diagnosing a visible virus infection using gold nanoparticles and chromatography enables rapid detection, it is difficult to quantitatively detect a target virus and there is a need to solve the non-specific reaction of gold nanoparticles.

In this situation, the present inventors have completed a method for detecting and quantifying a virus using the fact that when the nanocarrier and the virus-derived peptide bind, an optical change occurs due to the non-covalent interaction of the conjugated polymer supported inside the nanocarrier.

An object of the present invention is to provide a nanocarrier comprising a first block copolymer polymer and a second block copolymer polymer including a carboxyl group, a conjugated polymer supported inside the nanocarrier; And to provide a complex for virus detection comprising a virus-specific peptide linked by a peptide bond with the carboxyl group of the second block copolymer polymer and its use.

In order to achieve the above object, an aspect of the present invention is

(a) a nanocarrier comprising a first block copolymer polymer and a second block copolymer polymer containing a carboxyl group;

(b) a conjugated polymer supported inside the nanocarrier; and

(c) provides a virus detection complex comprising a virus-specific peptide linked to the carboxyl group of the second block copolymer polymer by a peptide bond.

As used herein, the term "block copolymer" refers to a copolymer made repeatedly in such a way that one type of monomer is polymerized to form a block, and then another monomer is polymerized to form a block, and such a block copolymer is Materials that can be made are called block copolymer polymers.

According to one embodiment of the present invention, the first block copolymer polymer is methoxypolyethylene glycol-block-polyamino acid (methoxy-poly(ethylene glycol)-block-poly(amino acid)) or methoxypolyethylene glycol-block -lactic acid (methoxy-poly(ethylene glycol)-block-lactic acid, mPEG-b-PLA). In addition, the methoxypolyethylene glycol-block-polyamino acid is methoxypolyethylene glycol-block-polyphenylalanine (mPEG-b-pPhe), methoxypolyethylene glycol-block-polyleucine (mPEG-b-pLeu), methoxypolyethylene It is selected from the group consisting of glycol-block-polytryptophan (mPEG-b-pTrp) and methoxypolyethylene glycol-block-polylysine (mPEG-b-pLys), preferably methoxypolyethylene glycol-block-polyphenylalanine ( mPEG-b-pPhe).

According to one embodiment of the present invention, the second block copolymer polymer includes a carboxyl group so that the virus-specific peptide can be linked to the nanocarrier by a peptide bond. The second block copolymer includes carboxymethyl polyethylene glycol-block-polyamino acid (carboxymethyl-poly(ethylene glycol)-block-poly(amino acid)) or carboxymethyl polyethylene glycol-block-lactic acid (carboxymethyl-poly(ethylene glycol) )-block-lactic acid, CM-PEG-b-PLA). The carboxymethyl polyethylene glycol-block-polyamino acid is carboxymethyl polyethylene glycol-block-polyphenylalanine (CM-PEG-b-pPhe), carboxymethyl polyethylene glycol-block-polyleucine (CM-PEG-b-pLeu), carboxy It is selected from the group consisting of methyl polyethylene glycol-block-polytryptophan (CM-PEG-b-pTrp) and carboxymethyl polyethylene glycol-block-polylysine (CM-PEG-b-pLys), preferably carboxymethyl polyethylene glycol -block-polyphenylalanine (CM-PEG-b-pPhe).

The first and second block copolymer polymers exhibit self-assembly characteristics due to repulsion and attractive force between polymer chains, and the first and second block copolymer polymers are mixed with each other to form a structure such as micelles. . At this time, in both the first and second block copolymers, amino acids are located inside the nanocarrier, the first block copolymer is methoxypolyethylene glycol and the second block copolymer is carboxymethylpolyethyleneglycol to the outside of the nanocarrier. .

In the present invention, mPEG-b-pPhe and CM-PEG-b-pPhe were dispersed in chloroform, the solution was added to demineralized water to react, and then chloroform was evaporated to obtain a nanocarrier composed of a block copolymer polymer. Thereafter, a conjugated polymer was supported on the inside of the nanocarrier.

In the present specification, the term "conjugated polymer" is a polymer having a π-conjugated structure formed due to the repetition of single bonds and double bonds, and shows a lower band gap energy than conventional polymers, resulting from this Electrical conductivity is the main characteristic. In addition, it shows a unique optical signal change in the visible light region due to oxidation-reduction reactions, changes in the ubiquity of electrons in polymers, and non-covalent interactions between polymers.

According to one embodiment of the present invention, the conjugated polymer is a π-conjugated structural monomer polymerized, and the π-conjugated structural monomer is an aniline derivative, a thiophene derivative, a pyrrole derivative, 3, 4-ethylenedioxythiophene derivatives, azepine derivatives, indole derivatives, carbazole derivatives, fluorine derivatives, phenylene derivatives, pi It may be at least one selected from the group consisting of pyrene derivatives and azulene derivatives.

Preferably, as the conjugated polymer, polyaniline in which aniline is polymerized and polypyrrole in which pyrrole is polymerized may be used, and aniline and pyrrole may be supported on the nanocarrier in a molar ratio of 20:1 to 5:1. More preferably, aniline and pyrrole may be supported on the nanocarrier in a molar ratio of 15:1 to 7:1, most preferably 9:1. When the content of pyrrole in the above ratio is decreased, the stability of the conjugated polymer is lowered when the conjugated polymer is polymerized inside the nanocarrier.

According to one embodiment of the present invention, when the virus detection complex binds to the virus, the distance between the virus detection complexes becomes close, and a non-covalent interaction occurs between the conjugated polymers supported inside the complex, resulting in an optical signal (absorption wavelength change). ) occurs. This change in absorption wavelength is measured in the visible light region, and for example, when polyaniline and polypyrrole are used as conjugated polymers, the change in absorption wavelength of 430 nm and 630 nm is large.

In the present invention, the virus detection peptide is influenza A virus, influenza B virus, dengue virus, human respiratory syncytial virus (RSV), noro Viruses (norovirus), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and SARS-CoV-2 (SARS-CoV-2, COVID-) 19) may be derived from a virus selected from the group consisting of.

Influenza A virus is a virus causing influenza and its subtype is determined by hemagglutanin (HA) antigen and neuramidase (NA) antigen, which are surface antigens. HA antigens are known up to H1-H18, and NA antigens are N1-N9 is known until The subtypes that cause human-to-human infection include H1N1, H2N2, and H3N2, and the subtypes that cause avian influenza include H5N1, H7N9, H5N8, and H5N6.

The peptide for virus detection is preferably a sequence specific to each virus for the sake of accuracy of detection, and for the specific peptide sequence, for example, a sequence known in a public database such as NCBI may be used.

According to one embodiment of the present invention, the peptide for virus detection derived from influenza A virus may use the sequence represented by SEQ ID NO: 1.

The present inventors have studied a method for simultaneously performing detection and quantification of virus. As a result, when the complex and the target virus to be detected are brought into contact, the target virus binds to the virus-specific peptide on the surface of the complex and the complex aggregates, It was confirmed that the presence or absence of a virus can be detected because the change in the absorption wavelength occurs due to the non-covalent interaction of the conjugated polymer. In addition, the virus can be quantified by quantifying the change in the absorption wavelength for each concentration of the virus.

Accordingly, another aspect of the present invention provides a composition for confirming virus infection comprising the complex for virus detection as an active ingredient.

In addition, another aspect of the present invention is

(a) contacting a sample to be checked for the presence or absence of a virus with a composition for confirming virus infection; and

(b) measuring the absorbance in the resultant of step (a); provides a method of providing information for diagnosing a virus infection, including.

According to one embodiment of the present invention, step (a) may be carried out for a certain time so that the virus detection complex can bind to the virus, for example, 5 to 40 minutes, more specifically, 10 minutes to This can be done for 30 minutes.

In addition, the step of measuring the absorbance of step (b) may be performed at an absorption wavelength in the range of 400 nm to 800 nm.

When the complex for detecting virus according to an embodiment of the present invention is used, the presence or absence of a virus can be confirmed through the change in the absorption wavelength shown by the binding of the complex to the target virus, and the change in the absorption wavelength can be quantified to quantify the virus. can

1 is a schematic diagram of a virus detection principle using a polymer nanocarrier-peptide complex (CPNP-PEP) supported with a conjugated polymer.
2 is a schematic diagram of a method for synthesizing a polymer nanocarrier-peptide complex (CPNP-PEP) supported with a conjugated polymer according to an example of the present invention.
3 is a result of comparing the absorbance change according to concentration dilution in a CPNP sample (A) in which a polymer nanocarrier (CPNP) supported with a conjugated polymer exists as a single particle (A) and a CPNP-Linker sample (B) in which CPNP is combined as a linker. .
4 is a result of confirming the size distribution of nanoparticles in a CPNP sample and a CPNP-Linker sample with a dynamic light scattering (DLS).
5 is a graph showing the absorption reaction values according to the content of carboxymethyl polyethylene glycol-block-polyphenylalanine in the CPNP sample and the CPNP-Linker sample.
6 is a graph showing the change in the absorption wavelength after reacting the conjugated polymer-supported polymer nanocarrier (A) and the conjugated polymer-supported polymer nanocarrier-peptide complex (B) with influenza A virus subtype CA04. .
7 is a graph showing the absorbance reaction values after reacting the conjugated polymer-supported polymer nanocarrier (CPNP) and the conjugated polymer-supported polymer nanocarrier-peptide complex (CPNP-PEP) with influenza A virus subtype CA04. to be.
8 is a graph showing the absorbance value after reacting the conjugated polymer-supported polymer nanocarrier-peptide complex with influenza A virus subtypes CA04 (A) and H3N2 (B), respectively.
9 is a graph showing the absorbance value after reacting the conjugated polymer-supported polymer nanocarrier-peptide complex (CPNP-PEP) with influenza A virus subtypes CA04 (A) and H3N2 (B), respectively.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are for illustrative purposes of one or more embodiments, and the scope of the present invention is not limited to these examples.

Preparation Example 1: CPNP-PEP synthesis

1-1. Synthesis of polymer nanocarriers supported with conjugated polymers

mPEG-b-pPhe (poly(ethylene glycol)-block-poly(phenylalanine)), a block copolymer polymer of methoxy-poly(ethylene glycol) and phenylalanine, is known in the art. It was synthesized according to the method. CM-PEG-b-pPhe, a block copolymer polymer of carboxymethyl-poly(ethylene glycol) and phenylalanine, was synthesized in the same manner.

5 mg of the synthesized mixture of mPEG-b-pPhe and CM-PEG-b-pPhe is dispersed in 1 ml of chloroform, and the chloroform in which the block copolymer polymer is dispersed in a vial containing 10 ml of deionized water (DIW) 1 ml of the solution was added. In an open state, the vial was stirred at 600 rpm for 24 hours to evaporate chloroform (DIW-chloroform 2-phase system → DIW 1-phase system).

After chloroform was evaporated, 20.25 μl (0.225 mmol) of aniline and 1.75 μl (0.025 mmol) of pyrrole were added at a molar ratio of 9:1 and stirred for 2 hours. In this process, the solution becomes transparent yellow as a whole. While stirring, 0.5 ml of a 1M ammonium persulfate (APS) solution was added. Upon addition of APS, the color of the solution immediately changes from a clear yellow to an opaque brown. The resultant was stored in a refrigerator for 48 hours and then washed three times by centrifugation (8000 RPM, 10 minutes).

Hereinafter, the polymer nanocarrier on which the conjugated polymer is supported will be referred to as "CPNP (Conjugated polymer encapsulated nanoparticle)".

1.2 Combination of conjugated polymer-supported polymer nanocarriers and peptides

EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) and Sulfo-NHS (N-hydroxysulfosuccinimide) solutions were prepared at 5 mg/1.3 ml and 10 mg/1.3 ml concentrations, respectively. 4 ml of CPNP was stirred at 400 rpm, and 400 μl of the EDC solution (5 mg/1.3 ml) and Sulfo-NHS (10 mg/1.3 ml) solution were added at an interval of 1 hour for a total of 2 times. after. Influenza A virus subtype CA04 (H1N1) specific peptide (2 mg/1 ml in 20% DMSO) was added 800 μl, stirred overnight, and washed 3 times by centrifugation (8000 RPM, 10 minutes). The CA04(H1N1) specific peptide (Fmoc-ARLSPTMVHPNGAQP-NH ₂ ) was purchased from Peptron Co., Ltd. (Daejeon, Korea).

Hereinafter, a polymer nanocarrier having a virus detection peptide bound to the surface and having a conjugated polymer supported therein will be described as "CPNP-PEP". Figure 2 schematically shows the synthesis method of CPNP-PEP.

Experimental Example 1: Confirmation of the difference between a single CPNP sample and a CPNP-Linker sample

In order to confirm the change in absorbance due to the distance between nanoparticles, the change in absorbance according to dilution was compared between the CPNP sample in which CPNP is present as a single particle and the sample in which CPNP is combined as a linker (CPNP-Linker). As the linker, hexamethylenediamine containing amine groups on both sides was used, and the CPNP was artificially aggregated by linking the carboxymethyl functional groups exposed on the surface of the CPNP containing CM-PEG-b-pPhe. CPNP and linker were linked to each other by the EDC/Sulfo-NHS method used to prepare CPNP-PEP.

As a result of analyzing the absorption wavelength by diluting each sample by 1/2 from 0.25 mg/ml to 0.00782 mg/ml concentration, it was found that the change in the absorption wavelength (430 nm and 630 nm) was large in the CPNP sample (Fig. 3) A). On the other hand, in the CPNP-Linker sample, the change in the distance between the particles due to dilution was limited, so the change in the absorption wavelength was small (FIG. 3B). Through this result, the relationship between the distance between nanoparticles and the change in absorption wavelength was confirmed.

In addition, in order to confirm the degree of aggregation of CPNP, the CPNP sample and the CPNP-Linker sample were analyzed by dynamic light scattering (DLS). As a result of the analysis, it was confirmed that a wider particle distribution appeared in the CPNP-Linker sample, and it was found that the particles were aggregated in the CPNP-Linker sample, but existed in the form of single particles in the CPNP sample (FIG. 4).

Spectral changes according to the concentrations of the CPNP sample and the CPNP-Linker sample were quantified according to Equation 1 and displayed as a graph. First, as a result of confirming the spectral change according to the percentage of CM-PEGb-pPhe contained in CPNP, it was found that the spectral difference was most clearly revealed when 80% of CM-PEG-b-pPhe was contained (Fig. 5C; A to D, respectively, the percentage of CM-PEGb-pPhe is 20%, 50%, 80% and 100%). In addition, compared to the CPNP-Linker sample, as a result of quantifying and plotting the large spectrum change in the CPNP sample that exists as a single particle, the particle concentration that shows the greatest difference in reactivity depending on the presence or absence of CPNP binding by the linker is It was confirmed that it was 0.3125 mg/ml (FIG. 5C). The above concentration is a suitable concentration for maximizing reactivity.

[Equation 1]

Optical Response (OR) = (I ₄₃₀ -I ^* ₄₃₀ ) + (I ₄₃₀ -I ^* ₄₃₀ ) ( I ^* =initial value)

Experimental Example 2: Confirmation of binding of CPNP-PEP and virus

Influenza A virus samples (subtype CA04(H1N1): HA titer 2^10) diluted with CPNP-PEP (0.625 mg/ml) or CPNP (0.625 mg/ml) at various concentrations were 1:1 at room temperature for 20 minutes. The reaction was carried out, and the absorbance was measured to quantify the optical response according to Equation 1.

As a result of the confirmation, when the CPNP and the influenza A virus sample were reacted, the change in the absorption wavelength did not occur, so it was found that the CPNP and the influenza A virus did not bind. However, when CPNP-PEP and influenza A virus sample were reacted, an absorbance change occurred in proportion to the virus titer, which means that CPNP-PEP and influenza A virus were combined ( FIGS. 6A and 6B ).

In addition, as a result of displaying the absorbance response value as a graph, it was found that the optical response increased in proportion to the virus titer when the CPNP-PEP and the influenza A virus sample were reacted ( FIG. 7 ). This result means that particle aggregation due to virus-peptide binding appears as an absorption signal.

From the results of this experimental example, it can be seen that the CPNP-PEP of the present invention can specifically detect influenza A virus.

Experimental Example 3: Confirmation of Virus Detection Using CPNP-PEP

CPNP-PEP (0.0625 mg/ml) was reacted with influenza A virus samples diluted to various concentrations 1:1 at room temperature for 20 minutes, and the absorbance was measured and quantified according to Equation 1. For influenza A virus samples, subtype CA04 (H1N1) and subtype H3N2, which have a lot of nucleotide sequence difference from each other, were used.

As a result, when CPNP-PEP and CPNP were reacted with influenza A virus subtype H3N2 sample, there was no change in absorption wavelength, but when CPNP-PEP and influenza A virus subtype CA04 (H1N1) sample were reacted, it was proportional to the virus titer. It was found that an absorption change occurred (A and B in FIGS. 8A and 8B).

In addition, as a result of displaying the absorbance value as a graph, it was found that when the CPNP-PEP and influenza A virus subtype CA04 (H1N1) sample were reacted, the optical response increased in proportion to the virus titer (FIG. 9).

From the results of this experimental example, it can be seen that the CPNP-PEP of the present invention can bind to a specific virus according to the specificity of the peptide, and selective and quantitative virus detection is possible by quantifying the change in the absorption wavelength caused by the binding.

<110> UNIVERSITY-INDUSTRY FOUNDATION, YONSEI UNIVERSITY KNU-Industry Cooperation Foundation Korea University Research and Business Foundation, Sejong Campus <120> COMPLEXES FOR DETECTING VIRUSES BASED CONJUGATED POLYMER <130> DP-2020-1234_P20U16C1853 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 15 <212> PRT <213> Influenza A virus <400> 1 Ala Arg Leu Ser Pro Thr Met Val His Pro Asn Gly Ala Gln Pro 1 5 10 15

Claims (11)

(a) a nanocarrier consisting of a first block copolymer polymer and a second block copolymer polymer including a carboxyl group;
(b) a conjugated polymer supported inside the nanocarrier; and
(c) a virus detection complex comprising a virus-specific peptide linked to the carboxyl group of the second block copolymer polymer by a peptide bond. According to claim 1, wherein the first block copolymer is methoxypolyethylene glycol-block-polyamino acid (methoxy-poly(ethylene glycol)-block-poly(amino acid)) or methoxypolyethylene glycol-block-lactic acid ( methoxy-poly(ethylene glycol)-block-lactic acid, mPEG-b-PLA), a complex for virus detection. According to claim 2, wherein the methoxypolyethylene glycol-block-polyamino acid is methoxypolyethylene glycol-block-polyphenylalanine (mPEG-b-pPhe), methoxypolyethylene glycol-block-polyleucine (mPEG-b-pLeu) , Methoxypolyethylene glycol-block-polytryptophan (mPEG-b-pTrp) and methoxypolyethylene glycol-block-polylysine (mPEG-b-pLys), which is selected from the group consisting of, the complex for virus detection. According to claim 1, wherein the second block copolymer is carboxymethyl polyethylene glycol-block-polyamino acid (carboxymethyl-poly(ethylene glycol)-block-poly(amino acid)) or carboxymethyl polyethylene glycol-block-lactic acid ( carboxymethyl-poly(ethylene glycol)-block-lactic acid, CM-PEG-b-PLA), a complex for virus detection. 5. The method of claim 4, wherein the carboxymethyl polyethylene glycol-block-polyamino acid is carboxymethyl polyethylene glycol-block-polyphenylalanine (CM-PEG-b-pPhe), carboxymethyl polyethylene glycol-block-polyleucine (CM-PEG- b-pLeu), carboxymethyl polyethylene glycol-block-polytryptophan (CM-PEG-b-pTrp) and carboxymethyl polyethylene glycol-block-polylysine (CM-PEG-b-pLys) , a complex for virus detection. According to claim 1, wherein the conjugated polymer is a π- conjugated structure monomer is polymerized, the virus detection complex. 7. The method of claim 6, wherein the π-conjugated structural monomer is an aniline derivative, a thiophene derivative, a pyrrole derivative, a 3,4-ethylenedioxythiophene derivative, an aze From the group consisting of azepine derivatives, indole derivatives, carbazole derivatives, fluorine derivatives, phenylene derivatives, pyrene derivatives and azulene derivatives Any one or more species selected, the complex for virus detection. According to claim 1, wherein the virus detection peptide is influenza A virus, influenza B virus, dengue virus, human respiratory syncytial virus, norovirus, MERS coronavirus, SARS coronavirus and SARS coronavirus-2 from the group consisting of A complex for virus detection, which is derived from the selected virus. The complex for virus detection according to claim 8, wherein the peptide for virus detection derived from influenza A virus comprises the sequence represented by SEQ ID NO: 1. A composition for confirming virus infection comprising the complex for virus detection of claim 1 as an active ingredient. (a) contacting a sample to be checked for the presence or absence of a virus with the composition for confirming virus infection of claim 10; and
(b) measuring the absorbance in the resultant of step (a); including
How to provide information for diagnosing a viral infection.

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