KR20230075112A - Kit for diagnosing virus infection comprising hydrogel sensor - Google Patents

Abstract

The present invention relates to a kit for diagnosing viral infection including a hydrogel sensor, wherein the hydrogel sensor includes polyethyleneglycol diacrylate (PEGDA) and polystyrene nanoparticles (PSNPs) to detect antigens. Since the detection sensitivity is increased by immobilizing the protein, it can be effectively used for virus detection and diagnosis of infection.

Description

Kit for diagnosing virus infection comprising hydrogel sensor}

The present invention relates to a kit for diagnosing viral infection including a hydrogel sensor, and more particularly, to a hydrogel containing polyethyleneglycol diacrylate (PEGDA) and polystyrene nanoparticles (PSNPs). It relates to a kit for diagnosing a virus infection comprising a sensor and an antigen protein immobilized on the hydrogel sensor.

The existing method for detecting COVID-19 is an analysis method using reverse transcription polymerase chain reaction (RT-PCR). These methods have limitations in that expensive analysis equipment must be used, pretreatment is essential, and that skilled experts must perform the analysis over a long period of time.

On the other hand, in the case of using the lateral flow immunoassay (Immunoassay lateral flow strip) method, which is developed on a strip (Point-of-care testing (POCT) device) using an antigen-antibody reaction, the cost required is low and no pretreatment is required. Since the detection method is simple, anyone can easily operate it, and the result is quickly obtained.

In order to apply this method, a method of application to lateral flow immunoassay by using an antibody targeting detection of SARS-COV-2 spike protein (S protein) was studied.

In this method, hydrophilic nitrocellulose is used as a material for the membrane where the reaction mainly proceeds. When proteins are immobilized on membranes by simple adsorption, proteins are mainly immobilized through electrostatic attraction. In the case of hydrophobic proteins or high molecular weight proteins, they may have low binding capacity to membranes, and for this reason, relatively low detection sensitivity.

Therefore, the present inventors used hydrogel at the test line position in the detection kit to increase the binding site for immobilizing the spike protein (S protein), thereby quickly and easily cross-linking. It was confirmed that crosslinking was achieved, biologically stable, physical manipulation was easy, and detection sensitivity was increased by increasing the surface area.

Accordingly, an object of the present invention is to provide a kit for diagnosing viral infection including a hydrogel sensor including polyethyleneglycol diacrylate (PEGDA) and polystyrene nanoparticles (PSNPs).

Another object of the present invention is to use a hydrogel sensor containing polyethylene glycol diacrylate and polystyrene nanoparticles to detect whether or not a reaction occurs when a sample separated from a subject is contacted, infection by a virus. It is a method of providing information for diagnosis.

Another object of the present invention relates to the use of a hydrogel sensor including polyethylene glycol diacrylate and polystyrene nanoparticles for diagnosing infection by a virus.

The present invention relates to a kit for diagnosing viral infection including a hydrogel sensor, and since the hydrogel sensor according to the present invention can immobilize both hydrophilic and hydrophobic proteins, it shows high detection sensitivity for various biomarkers.

The present inventors immobilized the virus spike protein (S protein) as a diagnostic antigen in a PEGDA (polyethyleneglycol diacrylate) hydrogel containing polystyrene nanoparticles (PSNPs) to obtain a kit for diagnosing viral infection. It was confirmed that the detection sensitivity increased when used.

Hereinafter, the present invention will be described in more detail.

One aspect of the present invention is a kit for diagnosing viral infection comprising a hydrogel sensor containing PEGDA and PSNPs.

In the present invention, the hydrogel sensor may contain 2 to 15% by weight of PEGDA, preferably 2 to 12% by weight, 2 to 10% by weight, 2 to 8% by weight, 5 to 15% by weight, 5 to 12% by weight, 5 to 10% by weight, 5 to 8% by weight, 8 to 15% by weight, or 8 to 12% by weight, for example, 8 to 10% by weight, but is not limited thereto. .

In the present invention, the hydrogel sensor may contain 0.01 to 15.00% by weight of PSNPs, preferably 1.00 to 15.00% by weight, 1.00 to 10.00% by weight, 1.00 to 5.00% by weight, 2.00 to 15.00% by weight, 2.00% by weight. to 10.00% by weight, 2.00 to 5.00% by weight, 5.00 to 15.00% by weight, or 10.00 to 15.00% by weight, for example, 12.00 to 15.00% by weight, but is not limited thereto,

The diameter of the PSNPs may be 100 to 10000 nm, but is not limited thereto.

In the present invention, the hydrogel sensor may include a photoinitiator and be cured by UV irradiation.

The hydrogel sensor may contain 0.1 to 5.0% by weight of a photoinitiator, for example, 0.5 to 2.0% by weight, but is not limited thereto.

The photoinitiator may be 2-hydroxy-2-methylpropiophenone, but is not limited thereto.

In the present invention, the hydrogel sensor may be in the form of an antigen protein immobilized. The antigenic protein may be a viral surface protein, and the viral surface protein includes a viral spike protein, an envelope protein, a nucleocapsid protein, hemagglutinin (HA), and an F protein (F protein). protein) may be one or more selected from the group consisting of, for example, it may be a virus spike protein, but is not limited thereto.

In the present invention, the hydrogel sensor may be provided in the form of curing after applying a hydrogel containing PEGDA and PSNPs to a strip.

In one embodiment of the present invention, the hydrogel sensor is applied to the test line of the strip, so that the detection kit operates according to the principle of fixing the target material in the sample to the test line as the sample spreads along the strip.

The strip may be a prefabricated strip having the same configuration as a commonly used one consisting of a sample pad, a reaction pad (a washing pad, a reaction membrane, mainly made of NC membrane), and a wick pad. In this case, a sample pad and an absorption pad are attached to both ends centered on the reaction pad, and when the sample is spread in the direction from the sample pad to the absorption pad, it can be used according to the process of confirming the detection result from the test line displayed in the center of the reaction pad. can

In addition, the strip may be an integral strip having a configuration implemented as a single part. In this case, the sample contact part at one end of the strip is put into or brought into contact with the place where the sample is located to be absorbed, and the absorbed sample is separated from the sample contact part. If it is expanded to the located inspection line, it can be used according to the process of confirming the detection result from the inspection line.

The detection kit may additionally include a detection antibody specific to a biomarker corresponding to the target. The detection antibody may be previously impregnated into a strip and applied in a dried state, or may be composed of a separate solution and developed on the strip simultaneously or sequentially with the sample, but is not limited thereto.

In order to facilitate confirmation of the detection result measured by the detection kit, the detection antibody may be a color enzyme, a fluorescent material, a magnetic material, a dye material, a quantum dot, a super paramagnetic particle, a superparamagnetic particle ( ultrasuper paramagnetic particles), and may be labeled with at least one label selected from the group consisting of radioactive isotopes, for example, may be labeled with a chromogenic enzyme, but is not limited thereto.

Another aspect of the present invention provides information for diagnosing infection by a virus, comprising a detection step of confirming whether a sample isolated from a subject reacts upon contact using a hydrogel sensor containing PEGDA and PSNPs way.

In the present invention, the hydrogel sensor may contain 2 to 15% by weight of PEGDA, preferably 2 to 12% by weight, 2 to 10% by weight, 2 to 8% by weight, 5 to 15% by weight, 5 to 12% by weight, 5 to 10% by weight, 5 to 8% by weight, 8 to 15% by weight, or 8 to 12% by weight, for example, 8 to 10% by weight, but is not limited thereto.

In the present invention, the hydrogel sensor may contain 0.01 to 15.00% by weight of PSNPs, preferably 1.00 to 15.00% by weight, 1.00 to 10.00% by weight, 1.00 to 5.00% by weight, 2.00 to 15.00% by weight, 2.00% by weight. to 10.00% by weight, 2.00 to 5.00% by weight, 5.00 to 15.00% by weight, or 10.00 to 15.00% by weight, for example, 12.00 to 15.00% by weight, but is not limited thereto,

In the present invention, the hydrogel sensor may include a photoinitiator and be cured by UV irradiation.

The hydrogel sensor may contain 0.1 to 5.0% by weight of a photoinitiator, for example, 0.5 to 2.0% by weight, but is not limited thereto.

The photoinitiator may be 2-hydroxy-2-methylpropiophenone, but is not limited thereto.

In the present invention, the hydrogel sensor may be in the form of an antigen protein immobilized. The antigen protein may be a viral surface protein, and the viral surface protein may be at least one selected from the group consisting of a virus spike protein, envelope protein, nucleocapsid protein, hemagglutinin, and F protein, For example, it may be a viral spike protein, but is not limited thereto.

In the present invention, the hydrogel sensor may be provided in a form in which a hydrogel containing PEGDA and PSNPs is applied to a strip and then cured, and the strip may be assembled or integrated.

In the present invention, the detecting step may be performed by determining whether a biomarker in a sample isolated from a subject binds to a hydrogel sensor or an antigen protein immobilized on the hydrogel sensor.

The detection kit may additionally include a detection antibody specific to a biomarker corresponding to the target. The detection antibody may be previously impregnated into a strip and applied in a dried state, or may be composed of a separate solution and developed on the strip simultaneously or sequentially with the sample, but is not limited thereto.

In order to facilitate confirmation of the detection result measured by the detection kit, the detection antibody is selected from the group consisting of a chromogenic enzyme, a fluorescent material, a magnetic material, a dye material, a quantum dot, a paramagnetic particle, a superparamagnetic particle, and a radioactive isotope. It may be labeled with one or more types of labeling substances, for example, it may be labeled with a chromogenic enzyme, but is not limited thereto.

The sample isolated from the subject may be at least one selected from the group consisting of serum, cells, and tissue, but is not limited thereto.

The biomarker in the sample may be an antibody specific to an antigen protein derived from a target virus, for example, a neutralizing antibody, but is not limited thereto.

The present invention relates to a kit for diagnosing viral infection including a hydrogel sensor, wherein the hydrogel sensor includes polyethyleneglycol diacrylate (PEGDA) and polystyrene nanoparticles (PSNPs) to detect antigens. Since the detection sensitivity is increased by immobilizing the protein, it can be effectively used for virus detection and diagnosis of infection.

1A is a photograph showing protein detection results of an alginate-nickel hydrogel prepared according to an embodiment of the present invention.
Figure 1b is a photograph showing the protein detection results using agarose and Ni-NTA resin hydrogel prepared according to an embodiment of the present invention.
1c is a photograph showing a protein detection result using a polyethyleneglycol diacrylate (PEGDA) hydrogel prepared according to an embodiment of the present invention.
Figure 2a is a photograph of the surface of a PEGDA hydrogel containing polystyrene nanoparticles (PSNPs) prepared according to an embodiment of the present invention at a magnification X 1,000 using a scanning electron microscope (SEM). It is a picture.
Figure 2b is a photograph of the surface of the PEGDA hydrogel containing PSNPs prepared according to an embodiment of the present invention using SEM at a magnification X 30,000.
Figure 3a is a graph comparing the virus spike protein detection intensity according to the presence or absence of PSNPs of the PEGDA hydrogel prepared according to an embodiment of the present invention.
Figure 3b is a graph confirming the minimum content of PSNPs for detecting the virus spike protein of the PEGDA hydrogel prepared according to an embodiment of the present invention.
Figure 3c is a graph comparing the detection intensity of the spike protein-specific antibody according to the PSNPs content of the PEGDA hydrogel prepared according to an embodiment of the present invention.
4 is a schematic diagram of a strip (top) and a strip (bottom) including a PEGDA hydrogel sensor including PSNPs prepared according to an embodiment of the present invention.
5 is a photograph confirming the detection sensitivity of a spike protein-specific antibody using a general strip (left) prepared according to an embodiment of the present invention and a strip (right) containing a PEGDA hydrogel sensor containing PSNPs.

Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight/weight)% for solids/solids, (weight/volume)% for solids/liquids, and liquid/liquid is (volume/volume) %.

Example 1: Search for a suitable hydrogel material

1-1. Alginate-nickel hydrogel

A 2% alginate solution was prepared and placed in a 96-well plate, and a nickel nitrate solution synthesized from nickel oxide and nitric acid was added to the plate to induce ionic crosslinking. A phosphate buffered saline (PBS) solution containing spike protein (S protein) at a concentration of 1.0 ug/ml was added to each well, and a blocking buffer was added to prevent non-specific adsorption at 20-minute intervals. It was carried out over time.

Then, a spike protein neutralizing antibody (S protein neutralizing Ab, Y biologics), which is a specific primary antibody for spike protein, and a secondary antibody specific for the primary antibody to which HRP is conjugated (HRP Anti-Nab conjugate , Y biologics) were treated for 1 hour, respectively, and washed 5 times with a PBST (phosphate buffer saline + tween-20) solution between each step. In order to observe the color change to confirm the result, a TMB (3,3',5,5'-tetramethylbenzidine) solution was reacted to cause a color change, and the reaction was terminated by HCl treatment after 10 minutes.

As can be seen in FIG. 1A, the alginate-nickel hydrogel exhibited degradation by the PBS solution, and was judged to be unsuitable for use because the background color was too dark.

1-2. Agarose and Ni-NTA resin hydrogel

Agarose and Ni-NTA beads were mixed in TAE buffer, heated, and cooled in a 96-well plate to form a gel. Thereafter, PBS solutions containing spike protein (S protein) at concentrations of 0.5 and 1.0 ug/ml were added to each well, respectively. Then, an antibody reaction against the spike protein was performed in the same manner as in Example 1-1.

As can be seen in FIG. 1b, the agarose and Ni-NTA resin hydrogels were not deteriorated by the PBS solution, but the background color was too dark as in the case of the alginate-nickel hydrogel of Example 1-1. It was judged to be unsuitable for use.

1-3. PEGDA Hydrating Gel

An 8% aqueous solution of PEGDA (polyethyleneglycol diacrylate) containing 1% of 2-hydroxy-2-methylpropiophenone, a photoinitiator, was added to a 96-well plate and maintained for 1 minute. A hydrogel was prepared by irradiating UV through a UV lamp with a wavelength of 365 nm to perform UV crosslinking. Thereafter, spike protein (S protein) was added to each well at various concentrations including 0.5 and 1.0 ug/mL. Then, an antibody reaction against the spike protein was performed in the same manner as in Example 1-1.

As can be seen in FIG. 1c, the PEGDA hydrogel was not deteriorated by the PBS solution, but the binding to fix the protein was weak due to the hydrophobic interaction. The background color appeared light, but there was a problem that the color intensity was also weak.

However, unlike other hydrogels, it was determined that PEGDA hydrogel has potential for use due to its inert property that does not show cross-reactivity with HRP-conjugated antibodies (Ab).

Example 2: Preparation and confirmation of optimized hydrogel

As an additive, polystyrene nanoparticles (PSNPs), which can be easily bound to hydrogel, were selected. Since proteins can be immobilized by physicochemical forces, and in particular by hydrophobic interactions, polystyrene, which is durable and chemically and immunologically inert, was used to increase the protein binding force of the hydrogel.

In this case, it was expected that the target protein lacking a hydrophilic site that is not effectively immobilized on the hydrogel could be immobilized by hydrophobic interaction by the PSNPs included in the hydrogel.

Specifically, a hydrogel was prepared by mixing 8% of PEGDA and 2% of PSNPs to a final concentration, and a UV lamp with a wavelength of 365 nm was applied to a solution in which 1% of 2-hydroxy-2-methylpropiophenone was added as a photoinitiator. It was cured through a scanning electron microscope (Scanning Electron Microscope; SEM) and observed at magnifications of X 1,000 and X 30,000.

As can be seen in FIGS. 2a and 2b, it was confirmed that the hydrogel fixed the polystyrene nanoparticles by dispersing and curing the polystyrene nanoparticles in the hydrogel.

Subsequently, the ELISA test was performed in the following manner by changing the manufacturing conditions of the hydrogel.

35 uL of each PEGDA gel containing or not containing PSNPs was added to each of the two well plates, cured, and washed 5 times with distilled water. Spike protein was added at each concentration, fixed by adsorption for 2 hours, and treated with a blocking buffer for 2 hours to prevent non-specific adsorption. Then, an antibody reaction against the spike protein was performed in the same manner as in Example 1-1.

First, in order to confirm the detection intensity of spike protein according to the presence or absence of PSNPs, using a hydrogel containing 15% by weight of PSNPs, when the primary antibody is 0, 200 or 400 ng / ml, 0.0, 0.5, and 1.0 ug The detection intensity of the spike protein corresponding to the concentration of /ml was confirmed.

As can be seen in Figure 3a, when the concentration of the primary antibody is the same, the detection intensity of the spike protein using the hydrogel containing PSNPs was higher than that of the hydrogel without PSNPs.

It was confirmed that the hydrogel containing PSNPs immobilized the spike protein better, as it showed stronger detection intensity under the same antibody and antigen conditions.

Second, in order to search for the minimum concentration of PSNPs included in the hydrogel for the detection of spike protein, the content of PSNPs was set to 0.0, 0.1, 0.3, 0.6, 1.0, 1.5 and 2.0% by weight, respectively, and spike protein 1 ug/ ml, and the degree of detection was confirmed under the conditions of 200 ng/ml of the primary antibody.

As can be seen in Figure 3b, the PSNPs included in the hydrogel were detectable from the concentration of 0.1% by weight, and the detection intensity increased in a concentration-dependent manner.

Since the detection intensity of spike protein increased in a concentration-dependent manner of PSNPs under the same antibody concentration conditions, it was confirmed that the hydrogel containing PSNPs immobilized spike protein better.

Third, in order to search for the optimal concentration of PSNPs included in the hydrogel, 2 ug/mL of spike protein and 0, 5, 10, 30, 50, The degree of detection was confirmed under the conditions of 100 and 200 ng/mL.

As can be seen in FIG. 3c, the detection limit concentration when the concentration of PSNPs contained in the hydrogel was 15% by weight was lower than that when the concentration of PSNPs contained in the hydrogel was 1% by weight, and even at least 5 ng/ml was visible to the naked eye. found to be detectable.

It was confirmed that the higher the concentration of PSNPs in the hydrogel under the same spike protein concentration condition, the lower the detection limit concentration of the detectable antibody.

Example 3: Preparation and confirmation of strips to which the optimized hydrogel was applied

A test line and a control line were marked on a nitrocellulose membrane (NC membrane).

Specifically, in preparing the test group strip, 2 uL of hydrogel was applied to the position of the inspection line on the NC membrane, cured with a UV lamp of 365 nm wavelength, and meticulously distilled water to remove unfixed particles and polymers. After washing, it was dried. After the strip was completely dried, spike protein was applied on the test line and dried again to fix the protein. Anti-NAb antibody (Y biologics) was applied to the position of the control line and then dried and fixed.

In preparing the control strip, it was performed in the same manner as in the method of manufacturing the test group strip, but the test line was marked in such a way that the protein was applied and dried without applying the hydrogel.

The assembly of the strip was performed by sequentially attaching a sample pad, a washing pad, and a wick pad so that a certain portion of each pad overlapped at each attachment site at the end of the NC membrane.

The sample pad was previously blocked with a BSA solution to prevent non-specific adsorption, and after the test line and the control line were completely dried, it was attached to one end of the NC membrane and used. The reaction pad was also subjected to a blocking treatment and attached so as to partially overlap the sample pad. Subsequently, a kit was prepared as shown in FIG. 4 by attaching an absorbent pad to the other end of the NC membrane so as to overlap a certain portion thereof.

40 μL of S protein neutralizing antibody solution was poured into the sample pad of each prepared strip to react on the reaction pad, and the secondary antibody and TMB solution were sequentially flowed, and detection was observed after 15 minutes.

As can be seen in FIG. 5, a clear band was observed at the test line position in the test group strip under the condition of the same antibody concentration of 500 ng/ml, but was faintly observed in the control strip.

Claims (12)

A kit for diagnosing viral infection including a hydrogel sensor containing polyethyleneglycol diacrylate (PEGDA) and polystyrene nanoparticles (PSNPs). The kit for diagnosing viral infection according to claim 1, wherein the hydrogel sensor contains 2 to 15% by weight of PEGDA. The kit for diagnosing viral infection according to claim 1, wherein the hydrogel sensor contains 0.01 to 15.00% by weight of PSNPs. The kit for diagnosing viral infection according to claim 1, wherein the hydrogel sensor includes a photoinitiator and is cured by UV irradiation. The kit for diagnosing viral infection according to claim 1, wherein the hydrogel sensor is in the form of an antigen protein immobilized. The kit for diagnosing viral infection according to claim 1, wherein the hydrogel sensor is provided in a form in which a hydrogel containing PEGDA and PSNPs is applied to a strip and then cured. The kit for diagnosing viral infection according to claim 6, wherein the strip is assembled or integral. Using a hydrogel sensor containing polyethyleneglycol diacrylate (PEGDA) and polystyrene nanoparticles (PSNPs), a detection step of checking whether or not a reaction occurs when a sample separated from the object is contacted Including, a method for providing information for diagnosing infection by a virus. The method of claim 8, wherein the hydrogel sensor includes a photoinitiator and is cured by UV irradiation. The method of claim 8, wherein the hydrogel sensor has an antigen protein in a fixed form. The method of claim 8, wherein the hydrogel sensor is provided in a form in which a hydrogel containing PEGDA and PSNPs is applied to a strip and then cured. [Claim 12] The method of claim 11, wherein the strip is assembled or integral.

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