KR20200047436A - Polydiacetylene Based Biosensor Chip and Method for Detecting Influenza Virus Using the Same - Google Patents

Abstract

The present invention relates to a polydiacetylene (PDA) based biosensor chip which comprises: a polymer substrate; a PDA-N-hydroxysuccinimide (NHS) linker bound to the polymer substrate; and a nucleoprotein antibody immobilized on the PDA-NHS linker. Moreover, the present invention relates to a manufacturing method of the PDA based biosensor chip comprises the steps of: forming the PDA-based biosensor chip bound to the polymer substrate by mixing the polymer substrate, a diacetylene monomer, a diacetylene-NHS monomer, and phospholipids to irradiate with ultraviolet rays; and immobilizing the nucleoprotein antibody on the PDA-NHS linker.

Description

Polydiacetylene-based biosensor chip and method for detecting influenza virus using the same

The present invention relates to a polydiacetylene-based biosensor chip capable of determining the presence or absence of an influenza virus by detecting the color change of the biosensor chip with the naked eye even with a very short detection time and a method for detecting the influenza virus using the same.

A biosensor can selectively detect a substance to be analyzed / detected by converting biological interactions and recognition reactions into electrical or optical signals by combining biological receptors that have a recognition function for a specific substance with electrical or optical transducers. The bioreceptor is a biomolecule that selectively recognizes a substance to be analyzed / detected and simultaneously generates a signal that a transducer can measure. Enzymes, proteins, DNA, cells, hormones, biomembrane, You can use a variety of tissues.

In particular, among biosensors, a chip in the form of a chip is called a biosensor chip (or biochip), and electrochemical, optical, magnetic, piezoelectric, and electronics are used to convert generated biosignals or recognition reactions into useful signals. Various physical and chemical methods are applied, and ultimately an electrical signal can be obtained.

Influenza is a disease commonly known as 'flu', which is an acute respiratory disease caused by the influenza virus. Influenza is a pandemic of small and large worldwide every year, and it is a highly contagious disease that infects 10 to 20% of the population within 2 to 3 weeks when the epidemic begins. In particular, when a new virus appears through mutation, the prevalence and mortality rate have increased significantly, and the influenza surveillance system is operating worldwide to prepare for the epidemic.

Therefore, rapid and sensitive detection is needed so that the influenza virus can be diagnosed as soon as the clinical trial is conducted in the field, ie around a patient suspected of infection. Therefore, in the present invention, a biosensor chip based on a colorimetric method for point-of-care testing (POCT), also called field inspection, was developed.

The present invention is intended to provide a biosensor chip capable of visually observing a color change of a chip and a method for detecting an influenza virus using the same, when detecting the influenza virus as described above, a rapid and sensitive detection is performed even in the field.

One aspect of the present invention, a polymer substrate; A polydiacetylene-NHS (N-hydroxysuccinimide) linker bound to the polymer substrate; It provides a polydiacetylene (PDA) -based biosensor chip comprising; and a nuclear protein (Nucleoprotein) antibody immobilized on the polydiacetylene-NHS linker.

In addition, an aspect of the present invention, mixing the polymer substrate, diacetylene monomer, diacetylene-NHS monomer, and phospholipids to ultraviolet irradiation to form a polydiacetylene-NHS linker bound to the polymer substrate; And immobilizing a nucleoprotein antibody on the polydiacetylene-NHS linker. A polydiacetylene (PDA) -based biosensor chip manufacturing method is provided.

In addition, another aspect of the present invention, processing the sample to the above-described polydiacetylene (PDA) -based biosensor chip; And determining that an influenza virus is present when the color of the biosensor chip is changed from blue to red.

The present invention immobilizes an antibody that specifically binds to the nuclear protein of the influenza virus to a polydiacetylene-NHS linker prepared by controlling the reaction ratio of diacetylene-NHS monomer, diacetylene monomer, and phospholipid to a specific level. By using the biosensor chip manufactured by the presbyopia, it is possible to detect / diagnose the influenza virus by simply observing the color change (blue → red) with the naked eye in the presence of various concentrations of the influenza virus.

1 is a schematic view showing a method for detecting an influenza virus according to an embodiment of the present invention.
Figure 2 is a view showing the results of FT-IR and ¹ H-NMR analysis for PCDA-NHS and PCDA prepared according to an embodiment of the present invention.
3 is a diagram showing the results of FT-IR analysis of PVDF films according to Experimental Example 1 of the present invention.
4 is a view showing a scanning electron microscope (SEM) photograph of PVDF films according to Experimental Example 2 of the present invention.
5 is a diagram showing the color change result according to the pH of the PDA-PVDF membrane according to Experimental Example 3 of the present invention.
6 is a view showing a color change according to the temperature of the PDA-PVDF membrane according to Experimental Example 4 of the present invention.
7 is a view showing the color change of the biosensor chip of Examples and Comparative Examples according to Experimental Example 5 of the present invention.
8 is a diagram showing color changes of biosensor chips of Examples and Comparative Examples according to Experimental Example 6 of the present invention.
9 is a view showing the binding force of the influenza A virus to the nuclear protein antibody used in the present invention.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention provides a polydiacetylene-based biosensor chip.

The polydiacetylene-based biosensor chip includes a polymer substrate; A polydiacetylene-NHS (N-hydroxysuccinimide) linker bound to the polymer substrate; And a nucleoprotein antibody immobilized on the polydiacetylene-NHS linker.

First, the polymer substrate to which the polydiacetylene-NHS linker is bonded is formed by photopolymerizing a diacetylene monomer, a diacetylene-NHS monomer, a phospholipid, and a polymer substrate. At this time, diacetylene monomers, diacetylene-NHS monomers, phospholipids and a polymer substrate may be mixed and then irradiated with ultraviolet light (UV) to photopolymerize. The polymer substrate to which the photopolymerized polydiacetylene-NHS is bound is blue.

The diacetylene monomer may contain a carboxyl group at the terminal in the molecule.

Specifically, the diacetylene monomer may include a compound represented by Formula 1 below.

[Formula 1]

In Chemical Formula 1,

n and m are the same or different from each other by the repeating number of units in parentheses, and each independently is a number of 0 to 20.

When n and / or m is 0, it means a direct bond.

The n and m may be the same or different from each other, and each independently may be 1 to 15, for example, 5 to 12. Further, n + m may be 10 to 30, or 10 to 20.

The diacetylene monomer may be 10,12-pentacosadiynoic acid (PCDA).

The diacetylene-NHS monomer may be formed by an esterification reaction of a diacetylene monomer and N-Hydroxysuccinimide (NHS). Specifically, a carboxyl group of a diacetylene monomer may be formed by reacting a hydroxy group of N-Hydroxysuccinimide (NHS).

The diacetylene monomer and N-hydroxysuccinimide (NHS) may react at a molar ratio of 1: 1 to 1: 2, and specifically, may be 1: 1 to 1: 1.2. If the reaction molar ratio of the diacetylene monomer and N-hydroxysuccinimide is less than 1: 1, a problem may occur in the detection efficiency of the influenza virus to be detected, and when it exceeds 1: 2, the remaining amount of N-hydroxysuccine Problems may arise where imide induces a non-specific reaction.

The contents of the diacetylene monomers described above may be applied to the diacetylene monomers.

The phospholipid may serve to enable the occurrence of discoloration well in the presence of the influenza virus because polydiacetylene is well docked and / or inserted into the polymer substrate. The phospholipid may contain a tetravalent amine group at the terminal. Specifically, a tetravalent amine group may be bound to the phosphoric acid group of the phospholipid.

In addition, the fatty acid portion of the phospholipid may have 5 to 20 carbon atoms, and specifically 10 to 15 carbon atoms.

The phospholipid may form a single layer or may form a double layer.

Specifically, the phospholipid may include 1,2-dimyristoyl-sn-glycero-3-phosphocholine (1,2-dimyristoyl-sn-glycero-e-phosphocholine).

As shown in Figure 9, the antibody may be an antibody specifically binding to the nuclear protein of influenza A virus among influenza viruses. Specifically, the antibody is immobilized on the polydiacetylene-NHS linker, and can specifically bind to the nuclear protein of the influenza A virus. Specifically, the influenza A virus is not limited as long as it is a commonly known influenza A virus, and may be, for example, influenza A virus such as H1N1, H3N2, H1N2, H3N8, H6N5, pH1N1, or H9N2.

In addition, the antibody is an antibody that specifically binds to the nuclear protein of the influenza A virus. Unlike conventionally known influenza virus antibodies, they are reactive with influenza A and B viruses and react with nuclear proteins. For example, it does not specifically react to influenza B virus.

The polymer may include polyvinylidene fluoride (PVDF). In particular, in the case of a cellulose substrate, aggregation occurs on the substrate during accumulation (staining) of polydiacetylene (PDA) particles, and red discoloration may occur even without binding of antibodies or viruses. Therefore, polyvinylidene fluoride (PVDF) base is preferred for impregnation of polydiacetylene (PDA) polymer base.

The polydiacetylene-based biosensor chip is a solid-state sensor, and has improved storage performance and is ideal as a diagnostic sensor for POCT compared to a liquid-phase sensor using a polydiacetylene liposome solution based on a conventional colorimetric method.

The polydiacetylene-NHS linker-binding polymer substrate formed as described above is blue. In addition, even when the antibody is immobilized on the polydiacetylene-NHS linker, the color of the membrane remains blue.

The polydiacetylene-based biosensor chip has no color change when only the antibody is immobilized as above, and the color change from blue to red can be observed only when the antigen, ie, the influenza virus, binds to the antibody. In the case of using an acetylene-based biosensor chip, the influenza virus can be diagnosed by observing color changes with the naked eye.

Specifically, when the antigen, i.e., influenza virus, is bound to the polydiacetylene-based biosensor chip, as the electronic state of the conjugated backbone of the polydiacetylene is changed by weakening the interaction between the chains, The HOMO-LUMO energy gap may widen, causing a color change from blue to red.

The polydiacetylene-based biosensor chip may be used as a biosensor chip of various types, for example, may be in the form of a paper chip.

In addition, one aspect of the present invention provides a method for manufacturing a polydiacetylene (PDA) based biosensor chip.

The manufacturing method of the polydiacetylene-based biosensor chip is a polydiacetylene-NHS bound to the polymer substrate by ultraviolet irradiation by mixing a polymer substrate, a diacetylene monomer, a diacetylene-NHS monomer, and a phospholipid. Forming a linker; And immobilizing a nucleoprotein antibody on the polydiacetylene-NHS linker.

The aforementioned contents of the diacetylene monomer, diacetylene-NHS monomer, phospholipid, and polymer may be applied in the same manner.

The polydiacetylene-NHS linker may be combined with a diacetylene monomer, diacetylene-NHS monomer, phospholipid, and polymer substrate, and then irradiated with ultraviolet (UV) light to photopolymerize to bond to the polymer substrate. Based on 1 mole of diacetylene-NHS monomer, it may be formed by reacting 4 to 6 moles of diacetylene monomers and 3 to 5 moles of phospholipids.

If the diacetylene-based monomer is less than 4 mol based on 1 mol of the diacetylene-NHS monomer, a problem may occur in which the binding strength of the antibody decreases, and if it is more than 6 mol, the photopolymerization reaction does not occur and color change does not occur. Problems may arise. In addition, if the phospholipid is less than 3 mol based on 1 mol of the diacetylene-NHS monomer, polydiacetylene may not be docked / inserted well into the polymer substrate, and if it is more than 5 mol, the biosensor chip There may be problems that do not develop blue.

The polydiacetylene-NHS linker is mixed with a diacetylene monomer, a diacetylene-NHS monomer, a phospholipid, and a polymer substrate, and then irradiated with ultraviolet rays (UV) having a wavelength of 200 to 300 nm, thereby polymerizing polydiacetylene. A leu-NHS linker can be linked. At this time, the polydiacetylene-NHS linker may be chemically and / or physically bound to the polymer substrate, and specifically, may be bound in an adsorbed form. When the wavelength of the ultraviolet rays does not satisfy the above range, a problem may occur in that the polydiacetylene-based biosensor chip does not develop blue.

The ultraviolet irradiation time may be 10 seconds to 5 minutes, specifically 10 seconds to 3 minutes, or 30 seconds to 2 minutes. When the time for irradiating the ultraviolet rays is less than 10 seconds, a problem that color development of the polydiacetylene-based biosensor chip does not occur in blue may occur, and when it is longer than 5 minutes, the biosensor chip is generated by the energy of ultraviolet rays. Problems that turn red can occur.

The step of mixing the diacetylene monomer, diacetylene-NHS monomer, phospholipid, and polymer substrate is to immerse the polymer substrate in a mixed solution of the diacetylene monomer, diacetylene-NHS monomer, and phospholipid. It may include steps. For example, after immersing the polymer substrate in the mixed solution, it may include drying at room temperature for 5 to 7 hours.

The mixed solution may further include a polar solvent, and the polar solvent may include, for example, chloroform.

The antibody can be immobilized on the polydiacetylene-NHS linker after first binding a polydiacetylene-NHS linker to the polymer substrate. Accordingly, when the polydiacetylene-NHS linker is bonded to the polymer substrate, the ultraviolet light irradiated is not affected, so that the antibody may not be damaged.

The antibody may be immobilized on the NHS portion of the polydiacetylene-NHS linker. Specifically, the step of immobilizing the nucleoprotein antibody to the polydiacetylene-NHS linker may include culturing the polymer substrate to which the polydiacetylene-NHS linker is bound to a solution containing the nucleoprotein antibody. have. The antibody and the polydiacetylene-NHS linker are immobilized by incubating the polymer substrate to which the polydiacetylene-NHS linker is bound in a solution containing the antibody at a temperature of 3 to 10 ° C for 18 to 36 hours. I can do it. When the culture temperature and time conditions as described above are satisfied, the efficiency of immobilizing the nucleoprotein antibody on the polydiacetylene-NHS linker increases.

In addition, after the step of immobilizing the nucleoprotein antibody to the polydiacetylene-NHS linker, the step of inactivating the NHS portion not bound to the nucleoprotein antibody may be further included.

After the antibody is immobilized on a polydiacetylene-NHS linker, it can be washed and / or dried to finally form a polydiacetylene-based biosensor chip.

Another aspect of the invention provides a method of detecting influenza virus.

The method for detecting the influenza virus may include: processing a sample on the aforementioned polydiacetylene (PDA) -based biosensor chip; And when the color of the biosensor chip changes from blue to red, determining that an influenza virus is present.

As described above, the polydiacetylene-based biosensor chip changes color from blue to red in an environment in which an influenza virus is present, and the color change can be observed with the naked eye.

Therefore, the method for detecting the influenza virus as described above has an advantage in that it can be diagnosed within a short period of time near a suspected influenza virus infection, and has the advantage of being easy to carry by using only a simple biosensor chip without sophisticated or complicated equipment. .

Meanwhile, the polydiacetylene-based biosensor chip may include processing a sample in an environment of pH 3 to 9. The polydiacetylene-based biosensor chip may have a color change under strong base conditions (pH 10 to 13). Basic conditions the hydroxyl anion (OH ^-) because the poly-di-acetylenic compounds rearrangement of the alkyl chain of polydioxanone acetylenic compounds in accordance with the resistance of the trapped protons of the carboxyl group with a carboxylate group of the negatively charged can take place in .

In addition, the polydiacetylene-based biosensor chip may include processing a sample at a temperature condition of 20 to 45 ° C. The polydiacetylene-based biosensor chip may have a color change under a temperature condition of more than 45 ° C. This is presumed to be due to the widening of the HOMO-LUMO energy gap as the electron state of the polydiacetylene backbone changes at temperatures above 45 ° C.

Therefore, in the detection method of the influenza virus, in the environment of pH 3 to 9 and 20 to 45 ° C, the color change from blue to red can be observed only when the antigen (ie, influenza virus) is bound to the antibody. In the same environment, the polydiacetylene-based biosensor chip can be used to diagnose an infection from a suspected influenza virus infection patient.

In addition, the sample may further include the step of standing the biosensor chip treated at a temperature of 35 ℃ to 45 ℃ for 1 minute to 10 minutes. If the sample further includes the step of standing the biosensor chip treated with the sample at a temperature of 35 ° C to 45 ° C for 1 minute to 10 minutes, color change can be observed even with a very short time (within about 10 minutes). It has the advantage of being able to detect the influenza A virus very quickly and with high reliability.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are only for understanding the present invention, and the scope of the present invention is not limited to these examples in any sense.

<Example>

1.PCDA-NHS synthesis

First, 10,12-pentacosadiynoic acid (PCDA) (2.7 mmol, 1 g), N-Hydroxysuccinimide (NHS) (2.9 mmol, 337.5 mg) And 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (3.2 mmol, 615 mg) in 10 mL of dichloromethane ( dichloromethane, DCM). Then, the reaction product was stirred at room temperature for 4 hours. The organic solvent in the reaction product was evaporated under vacuum, and the residue was extracted with ethyl acetate (EA) and distilled water (DI). The organic solvent layer was then filtered using magnesium sulfate anhydride, and the solvent was concentrated in vacuo. The resulting white powder (PCDA-NHS) was confirmed by thin-layer chromatography (hexane: ethyl acetate = 3: 1). FT-IR (Bruker optics Alpha-P) (Bruker, Billerica, MA, USA) and ¹ H-NMR (JEOL JNM-AL 400) (JEOL, Tokyo, Japan) analysis of the prepared PCDA-NHS, respectively It is shown in (a) and (b) of FIG. 2.

According to (a) of FIG. 2, PCDA showed a carboxyl group (-COOH) peak at 1698 cm ⁻¹ , but PCDA-NHS did not show this peak. In addition, a new peak at 1729 cm ^-1 corresponding to the ester group (-COO-) was observed in PCDA-NHS. Also, according to FIG. 2 (b), it was confirmed that PCDA-NHS in the ¹ H-NMR spectrum showed a peak due to NHS at 2.8 ppm, but not PCDA.

2. Formation of PDA-based biosensor chip

10,12-pentacosadiynoic acid (PCDA) (0.06 mol), previously prepared PCDA-NHS (0.012 mol) and 1,2-dimyristoyl-sn-glycero-3 -Phosphocholine (1,2-dimyristoyl-sn-glycero-e-phosphocholine, DMPC) (0.048 mmol) powder was mixed with 20 mL of chloroform to prepare a mixed solution.

Polyvinylidene fluoride (PVDF) membranes were cut into square shapes using scissors. The above PVDF membrane was immersed in the previously prepared mixed solution and dried at room temperature for 6 hours. A PDA-PVDF film was prepared by irradiating ultraviolet light (UV) onto a PVDF film on which the above mixed solution was immersed for 1 minute using a LAB 24 UV headlight of 254 nm wavelength (Dong seo, Seoul, Korea).

Next, after the PDA-PVDF membrane was incubated in a solution containing NP12 antibody at 4 ° C. for 24 hours to immobilize the antibody, the PDA-PVDF membrane immobilized with the above antibody was incubated with 18.3 nM ethanolamine for 1 hour to retain all The NHS site was inactivated. Then, washed with deionized water and dried to prepare a PDA-based biosensor chip.

<Comparative Example>

In the above example, a PDA-based biosensor chip was prepared in the same manner as in the above example, except that a solution containing a human IgG antibody was used instead of the NP12 antibody.

The components used in the above Examples and Comparative Examples are as follows.

(1) 10,12-Pentacosadiynoic acid (PCDA): Alfa Aesar (Haverhill, MA, USA)

(2) 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC): Avanti Polar Lipids (Alabaster, AL, USA)

(3) N-Hydroxysuccinimide (NHS): Sigma Aldrich (St. Louis, MO, USA)

(4) ethanolamine: Sigma Aldrich (St. Louis, MO, USA)

(5) IgG: Sigma Aldrich (St. Louis, MO, USA)

(6) 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC): Thermo Fisher Scientific (Waltham, MA, USA)

(7) PVDF membrane (0.2 μm): EMD Millipore (Burlington, MA, USA)

(8) Nucleoprotein antibody (NP12): Anti-Influenza A Virus Nucleoprotein antibody (ab128193), Bio Nano Health Guard Research Center (H-GUARD)

<Experimental Example 1>

FT-IR (chemical composition for the biosensor chip (red), bare PVDF film (black), PDA-PVDF film before ultraviolet irradiation (green), and PDA-PVDF film (blue) after ultraviolet irradiation prepared according to the above embodiment Bruker optics Alpha-P) (Bruker, Billerica, MA, USA) analysis results are shown in FIG. 3.

According to Fig. 3 (a), characteristic absorption bands were observed at 1400 cm ^-1 and 1180 cm ^-1 corresponding to -CF ₂ stretching vibrations of all PVDF membranes. On the other hand, according to FIG. 3 (b), after coating the PDA on the PVDF membrane, the weak absorption band of C = O stretching vibration related to the carboxyl group in PCDA was found to be 1735 cm ^-1 . In particular, C = N and C = O stretching vibration of the amide bond was observed only in the FT-IR spectrum of the biosensor chip (red) of the example, indicating that the antibody was well immobilized on the PDA-PVDF membrane.

<Experimental Example 2>

Biosensor chip prepared according to the above embodiment (Fig. 4 (d)), PDA-PVDF membrane after binding of H1N1 antigen to the biosensor chip prepared according to the above embodiment (Fig. 4 (e)), bare PVDF The surface pore structure of the PVDF membrane was injected into the membrane (Fig. 4 (a)), the PDA-PVDF membrane before UV irradiation (Fig. 4 (b)), and the PDA-PVDF membrane after UV irradiation (Fig. 4 (c)). The results observed through an electron microscope (SEM) are shown in FIG. 4.

4 (b) and (c) compared to (a), the pore structure and pore size of the PDA-PVDF membrane before and after UV irradiation did not change significantly compared to the pore structure and pore size of the bare PVDF membrane. On the other hand, since the biosensor chip prepared according to the example was blocked by a high molecular weight conjugated antibody, narrow pores were observed after antibody immobilization.

<Experimental Example 3>

For the PDA-PVDF membrane prepared in the above example, the color change at pH 3 to 13 was observed, and the results of the red color change (RCS (%)) according to pH are shown in FIG. 5.

According to FIG. 5, the PDA-PVDF membrane was maintained in blue under acidic and neutral conditions (pH 3 to 9), but gradually changed to red under strong base conditions (pH 10 to 13).

<Experimental Example 4>

For the PDA-PVDF membrane prepared in the above example, color changes at 25 ° C, 37 ° C, 40 ° C, 50 ° C, 60 ° C, and 70 ° C were observed, and red chromatic shift (RCS) %)) The results are shown in FIG. 6.

According to FIG. 6, it was confirmed that the color of the PDA-PVDF film gradually changed to red from a temperature of 50 ° C. or higher, and the PCS (%) value was also increased.

<Experimental Example 5>

Using the biosensor chip (indicated as PDA-Paper chip) prepared in the above example and the biosensor chip (indicated as Control Ab-paper) prepared in the comparative example, influenza A virus sample (at 25 ° C) The results of observing the color change after contacting the Bio Nano Health Guard Research Center (H-GUARD) are shown in FIG. 7. At this time, the concentration of the influenza A virus sample was tested with TCID ₅₀ of 0, 10 ³ , 5Χ10 ³ , 10 ⁴ , 5Χ10 ⁴ , 10 ⁵ .

According to FIG. 7, the biosensor chip prepared in the comparative example did not exhibit a color change regardless of the concentration of the influenza A virus sample, whereas the biosensor chip prepared in the example had no influenza A virus sample. Although it showed blue, it was confirmed that the red color change (RCS (%)) increased as the concentration of influenza A virus increased.

<Experimental Example 6>

In Experimental Example 5, after contacting an influenza A virus sample (Bio Nano Health Guard Research Center (H-GUARD)) at 25 ° C., the result of observing color change after standing at 40 ° C. for 10 minutes is shown in FIG. 8. Shown.

In the case of Experimental Example 5 (FIG. 7) in which the PDA-based biosensor chip of the present invention was treated with virus and dried at room temperature (RT), it could be observed that the color gradually changed to purple after about 3 hours, but the Experimental Example When the process of standing at 40 ° C. for 10 minutes as in 8 was observed, it turned blue in about 9 minutes.

On the other hand, even if the biosensor chip of the comparative example in which the IgG antibody is bound is left at 40 ° C after room temperature treatment or before / after H1N1 virus treatment, it is impossible to discriminate the color change with the naked eye and thus, H1N1 virus detection is impossible. I could confirm.

In addition, even if there is no difference in the image identified by the naked eye, the redness (a *) of the PDA-based biosensor chip (PDA-Paper chip) according to the present invention at a concentration of 5 X 10 ³ TCID ₅₀ is a comparative example (Control Ab-paper) Redness (a *) of about 2 times higher than the measurement was confirmed. Redness (a *) was measured and analyzed with a spectrophotometer (CM-2600d, KONICA MINOLTA, Tokyo, Japan) and Color Data Software (CM-S100w, KONICA MINOLTA, Tokyo, Japan).

As a result, the present invention, in order to improve the detection ability of the PDA-based biosensor chip, when stored at about 40 ℃ after H1N1 virus treatment, PDA-based biosensor chip (PDA-Paper chip) with very fast and high reliability ), The influenza A virus could be detected.

Claims (17)

Polymer substrates;
A polydiacetylene-NHS (N-hydroxysuccinimide) linker bound to the polymer substrate; And
Nucleoprotein antibody immobilized on the polydiacetylene-NHS linker;
Polydiacetylene (PDA) -based biosensor chip comprising a. The method according to claim 1,
The nuclear protein antibody is a polydiacetylene (PDA) based biosensor chip that is specific for the nuclear protein of the influenza A virus. The method according to claim 1,
The polydiacetylene-NHS linker bound to the polymer substrate is a polydiacetylene (PDA) based bio which is formed by mixing ultraviolet rays by mixing diacetylene monomers, diacetylene-NHS monomers, phospholipids, and polymer substrates. Sensor chip. The method according to claim 3,
The diacetylene monomer is a polydiacetylene (PDA) -based biosensor chip comprising a compound represented by Formula 1 below:
[Formula 1]

In Chemical Formula 1,
n and m are the same or different from each other by the repeating number of units in parentheses, and each independently is a number of 0 to 20. The method according to claim 3,
The diacetylene monomer is a polydiacetylene (PDA) -based biosensor chip that includes 10,12-pentacosadiynoic acid (PCDA). The method according to claim 3,
The diacetylene-NHS monomer is a diacetylene monomer and N-hydroxysuccinimide (N-Hydroxysuccinimide) (NHS) is formed by the esterification reaction of polydiacetylene (PDA) based biosensor chip. The method according to claim 3,
The phospholipid is a polydiacetylene (PDA) based biosensor chip that contains a tetravalent amine group at the terminal. The method according to claim 7,
The tetravalent amine group is a polydiacetylene (PDA) based biosensor chip that is bound to the phosphoric acid group of the phospholipid. The method according to claim 3,
The phospholipid is polydiacetylene (PDA), which includes 1,2-dimyristoyl-sn-glycero-3-phosphocholine (1,2-dimyristoyl-sn-glycero-e-phosphocholine, DMPC). Based biosensor chip. The method according to claim 1,
The antibody is a polydiacetylene (PDA) based biosensor chip that specifically binds to the nuclear protein of the influenza virus. The method according to claim 1,
The polymer is a polydiacetylene (PDA) -based biosensor chip that includes polyvinylidene fluoride (PVDF). Mixing a polymer substrate, a diacetylene monomer, a diacetylene-NHS monomer, and a phospholipid to irradiate with ultraviolet rays to form a polydiacetylene-NHS linker bound to the polymer substrate; And
Immobilizing a nucleoprotein antibody on the polydiacetylene-NHS linker;
Polydiacetylene (PDA) based biosensor chip manufacturing method comprising a. The method according to claim 12,
The polydiacetylene-NHS linker is a polydiacetylene (PDA) based biosensor formed by reacting 4 to 6 moles of diacetylene monomers and 3 to 5 moles of phospholipids based on 1 mole of diacetylene-NHS monomer. Method of manufacturing chips. The method according to claim 12,
The step of immobilizing the nucleoprotein antibody on the polydiacetylene-NHS linker comprises culturing the polymer substrate to which the polydiacetylene-NHS linker is bound in a solution containing the nucleoprotein antibody ( PDA) based biosensor chip manufacturing method. The method according to claim 12,
After the step of immobilizing a nucleoprotein antibody to the polydiacetylene-NHS linker, a polydiacetylene (PDA) -based biosensor comprising the step of inactivating the non-nuclear protein bound NHS portion. Method of manufacturing chips. Processing a sample on the polydiacetylene (PDA) based biosensor chip of any one of claims 1 to 11; And
Determining that an influenza virus is present when the color of the biosensor chip changes from blue to red;
Influenza virus detection method comprising a. The method according to claim 16,
The method for detecting an influenza virus further comprising the step of allowing the sample-treated biosensor chip to stand at a temperature of 35 ° C to 45 ° C for 1 minute to 10 minutes.

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