KR102211590B1 - Preparation method of nanowire array for diagnosis middle east respiratory syndrome virus - Google Patents

Abstract

The present invention relates to a method for manufacturing a nanowire array for diagnosis of MERS virus. More particularly, the present invention relates to a method for manufacturing a nanowire array for diagnosis of MERS virus, comprising the steps of: growing a nanowire on a substrate; introducing a functional group on the grown nanowire; and fixing a capture antibody that recognizes the MERS virus as an antigen. The nanowire array of the present invention has improved detection sensitivity, enabling more accurate diagnosis of MERS.

Description

Manufacturing method of nanowire array for diagnosis of MERS virus antigen {Preparation method of nanowire array for diagnosis middle east respiratory syndrome virus}

The present invention relates to a method of manufacturing a nanowire array for diagnosis of MERS virus antigens.

Fluorescence immunoassay is an analysis method that detects antigen-antibody reactions by using fluorescence. Fluorescence is a photonics, molecule including medicine, pharmacy, and genetics, including tracking the location of drugs and proteins in vivo and detecting biomolecules. It can be applied to various fields such as biology, materials science, and chemistry. The analysis method using fluorescence provides high detection sensitivity, but the limitation of quantum efficiency due to the indirect method of labeling, quenching due to aggregation between molecules, overlapping phenomenon due to fluorescence of substances other than fluorescent molecules themselves, and There are problems such as stability of the fluorescence signal. Fluorescence intensity of fluorescent molecules is a major factor that determines the performance of fluorescence-based technology, and nanomaterials utilization technology is being studied as a study to improve the fluorescence signal.

Nanomaterials are recently used in various technical fields as various types such as nanoparticles, nanowires, and nanotubes, using their unique physical, chemical, optical and electrical properties, and nanostructures are used in the composition, shape, and arrangement of nanoparticles. Various characteristics can be adjusted accordingly. Among them, nanowire, a one-dimensional nanomaterial, has excellent crystallinity from less than 10 nm to several hundred nm in diameter, and has high chemical reactivity, quantum confinement effect, and self-assembly due to its large specific surface area. It has features such as self-assembly) and stress relaxation. Nanowires can be synthesized based on various materials including Si, ZnO, GaN, and SnO ₂ , among which zinc oxide (ZnO) nanowires have excellent photoelectric properties, biocompatibility, low toxicity, and easy surface modification. It is attracting attention.

When nanowires are used in biodevices, a large surface area for immobilizing biomolecules can be provided to improve detection signals according to the immune response of biomolecules, and high sensitivity can be realized. Accordingly, technologies for improving fluorescence signals through precise immobilization of biomolecules on nanostructures have been developed. For example, Korean Patent Registration No. 10-1837827 includes a substrate; One or more nanostructures fixed to the substrate and grown vertically from the substrate; And an aptamer complex comprising at least one double-stranded DNA fixed to the surface of the nanostructure, capable of selectively binding to biochemical molecules, and intercalating a fluorescent dye that generates fluorescence. A molecular detection device is disclosed, and the binding efficiency of biochemical molecules can be increased by controlling the properties of nanostructures. In addition, Korean Patent Registration No. 10-1163535 maximizes the three-dimensional volume-to-surface area ratio by exposing nanowires on a nanoframe to connect biological nanoparticles, and directionally arranging antibodies on the nano-surface by physical bonding.

Meanwhile, Middle East Respiratory Syndrome (MERS) is an acute respiratory infection caused by MERS-CoV, a species of beta coronavirus, and is a new virus discovered in Saudi Arabia in 2012. The mortality rate reaches 20-55%, and it is accompanied by complications such as pneumonia, respiratory failure, septic shock, and acute renal failure as well as a high mortality rate. In addition to the Middle East, Korea is the region where MERS was the most prevalent.Of the MERS outbreaks by country, the number of officially infected people in Korea was the 2nd in the world, ranking first among non-dominant countries.In 2015, there were 186 confirmed cases and deaths in Korea. 38 (20.4%).

The most common gene detection-based MERS in vitro diagnostic device developed and marketed to date is designed to simultaneously or separate detection of the specific genes of MERS, upE, ORF1a or ORF1b. Rapid and accurate in vitro diagnostic technology is required to prevent the early spread of MERS. However, in the case of gene detection-based technology, while the accuracy is high, it takes a long time for confirmation, so improvement is needed in terms of speed.

Meanwhile, in the case of antigen-antibody-based MERS in vitro diagnostic technology, a product capable of on-site diagnosis based on immunochromatography using a sandwich ELISA technique and a product of an indirect ELISA technique using the N protein antigen of recombinant MERS-CoV were developed. , MERS diagnostic technology through detection of MERS-CoV antibodies based on immunofluorescence and protein microarrays was studied. Antigen-antibody-based diagnostic technology has the advantage of having a very short diagnostic time and more excellent specificity than gene detection-based, but it has a problem with low sensitivity and needs to be improved.

MERS outbreaks are repeatedly prevalent, and accordingly, it is necessary to develop a rapid and accurate diagnostic technology to prevent the spread of MERS.

Korean Patent Registration No. 10-1068972 Korean Patent Registration No. 10-1163535

A. Dorfman et al., “Nanoscale ZnO-enhanced fluorescence detection of protein interactions”, Advanced materials, 2006, 18(20), 2685-2690 L. E. Green et al., “Solution-grown zinc oxide nanowires”, Inorg. Chem., 2006, 45(19) 7535-7543

The present invention was conceived to solve the above problem, and by growing nanowires on a substrate to fix the antibody, antibody fixation and signal strength are improved, thereby producing a nanowire array for diagnosis of MERS virus antigens capable of highly sensitive diagnosis of MERS virus. It aims to provide a method.

The present invention to achieve the above object,

(a) preparing a nanowire seed solution;

(b) preparing a nanowire precursor solution;

(c) growing nanowires on the substrate;

(d) introducing a functional group to the nanowire;

It provides a method of manufacturing a nanowire array for diagnosis of MERS virus antigens comprising: (e) fixing the capture antibody to the functional group.

The nanowire is zinc oxide, and the diameter of the nanowire is 50 to 100 nm.

The substrate is a glass or silicon wafer.

The capture antibody recognizes the spike protein of the MERS virus as an antigen.

The step (c) is performed by a hydrothermal synthesis method, i) forming a seed layer by applying the zinc seed solution to a substrate; And ii) immersing the substrate in the zinc precursor solution with the seed layer facing downward and reacting at 95°C. The length of the nanowire can be adjusted by adjusting the reaction time in step (c). It is preferably synthesized for 3 to 5 hours, and more preferably synthesized for 5 hours to obtain a nanowire of 2 μm length having a diameter distribution of 50 to 100 nm.

The zinc seed solution contains zinc acetate dihydrate, and the zinc precursor solution contains zinc nitrate hexahydrate, hexamethylene tetramine, and polyethylenimine. do.

The step (d) includes i) immersing the substrate on which the nanowires are grown in a 3-aminopropyltriethoxysilane solution and reacting at room temperature for 2 hours; And ii) immersing the substrate in a glutaraldehyde solution and reacting at 4° C. for 2 hours.

In step (e), in step (e), the capturing antibody is cultured on the substrate to which the functional group is attached overnight at 4° C., 1 hour at 37° C., or 2 to 3 hours at room temperature and fixed.

In another aspect, the present invention provides a nanowire array for diagnosis of MERS virus antigens, characterized in that a capture antibody recognizing MERS virus antigen is immobilized on a nanowire grown on a substrate and prepared by the above manufacturing method. to provide.

In addition, the present invention in another aspect, the nanowire array; And a detection antibody to which a MERS virus antigen is recognized and a fluorescent marker is bound. Or it provides a MERS virus antigen diagnostic kit comprising; a primary antibody recognizing a MERS virus antigen and a secondary antibody in which a fluorescent marker recognizing the primary antibody is combined.

According to the present invention, as the antibody is immobilized by growing nanowires on a substrate, the antibody's immobilization power is improved, thereby increasing sensitivity, selectivity, and stability, thereby enabling rapid and accurate MERS virus antigen detection.

1 is a schematic diagram of the MERS virus antigen diagnostic kit according to the present invention.
2 shows a photograph of a nanowire array according to an embodiment of the present invention.
3 shows the measurement result of the SEM image of the nanowire according to an embodiment of the present invention.
4 shows the measurement result of an SEM image of a nanowire according to an embodiment of the present invention.
5 shows (a) fluorescence images and (b) fluorescence signal measurement results of a nanowire array according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail. In describing the present invention, when it is determined that a detailed description of a related known configuration or function may obscure the subject matter of the present invention, a detailed description thereof will be omitted.

The present invention provides a method of manufacturing a nanowire array for diagnosis of MERS virus antigens.

The manufacturing method of the nanowire array for diagnosis of MERS virus antigen according to the present invention comprises the steps of: (a) preparing a nanowire seed solution; (b) preparing a nanowire precursor solution; (c) growing nanowires on the substrate; (d) introducing a functional group to the nanowire; (e) immobilizing the capture antibody to the functional group; includes.

The nanowire is preferably zinc oxide, and zinc oxide has a very stable polar surface, so that it is easy to form various nanostructures. When the nanowires are grown on the substrate, a large surface area for immobilization of the antibody is provided, so it is effective as a substrate for an antigen-antibody immune response. On the other hand, when 0-dimensional nanoparticles are used instead of nanowires, there may be difficulties in realizing an accurate enhancement effect or reproducibility of a signal due to the wide size distribution of the nanoparticles and non-uniform distribution on the substrate. In addition, in the case of a two-dimensional plate, the degree of freedom of the antibody is lowered, which may lower antibody activity.

It is preferable that the diameter of the nanowire is 50 to 100 nm.

In the step (c), the nanowires can be grown using a hydrothermal synthesis method, and the step (c) includes: i) forming a seed layer by applying the zinc seed solution to a substrate; And ii) immersing the substrate in the zinc precursor solution with the seed layer facing downward and reacting at 95°C. At this time, the length of the nanowire can be adjusted by adjusting the reaction time between the zinc oxide seed layer and the zinc precursor solution.

The seed solution for forming the zinc oxide seed layer may include zinc acetate dihydrate.

The zinc precursor solution may include zinc nitrate hexahydrate, hexamethylene tetramine, and polyethylenimine. The hexamethylenetetramine and polyethyleneimine are capable of growing in a specific plane direction (one-dimensional direction) on a substrate when the zinc precursor is converted to zinc oxide, and affect the morphological characteristics of the nanowire. It serves to promote or inhibit the growth of That is, the zinc precursor is converted to oxide and plays a role of suppressing the lateral growth so that it grows into a one-dimensional structure of nanowires. At this time, the hexamethylenetetramine functions as a reducing agent, and polyethyleneimine induces the formation of an array of one-dimensional structures.

The capture antibody may be immobilized on the nanowire through a linker serving as an intermediate bridge. Random immobilization that does not take into account the orientation of the antibody, even if the amount of the immobilized antibody is large, can not be used for antigen binding or if the antigen binding site is not exposed to the outside, the performance of target detection is reduced. . The linker is for site-specific immobilization of the capture antibody to the nanowire by introducing a functional group into the nanowire, and the linker is preferably 3-aminopropyltriethoxysilane and glutaraldehyde.

The step (d) includes i) immersing the substrate on which the nanowires are grown in a 3-aminopropyltriethoxysilane solution and reacting at room temperature for 2 hours; And ii) immersing the substrate in a glutaraldehyde solution and reacting at 4° C. for 2 hours. By modifying the nanowire surface with 3-aminopropylethoxysilane, reacting with glutaraldehyde, and binding the capture antibody, the antibody can be easily immobilized, and the antibody's immobilization power can be improved. The capture antibody includes, but is not limited to, an antibody that recognizes MERS CoV Spike or nucleoprotein as an antigen.

The step (e) is preferably fixed by incubating the capture antibody on the substrate to which the functional group is attached overnight at 4°C, 1 hour at 37°C, or 2 to 3 hours at room temperature.

In the present invention, the substrate is preferably a glass or silicon wafer, but is not limited thereto.

In another aspect, the present invention is a nanowire array for diagnosis of MERS virus antigens, characterized in that a capture antibody that recognizes MERS virus antigen is immobilized on a nanowire prepared by the above manufacturing method and grown on a substrate. Provides.

In addition, in another aspect, the present invention is the nanowire array; And a detection antibody to which a MERS virus antigen is recognized and a fluorescent marker is bound. Or it provides a MERS virus antigen diagnostic kit comprising; a primary antibody that recognizes the MERS virus antigen and a secondary antibody in which a fluorescent marker that recognizes the primary antibody is combined.

1 is a schematic diagram of the MERS virus antigen diagnostic kit of the present invention. Referring to FIG. 1, the MERS virus antigen diagnostic kit of the present invention uses a substrate on which a nanowire has been grown and a MERS virus fixed on the nanowire as an antigen. It may be a configuration comprising a capture antibody that recognizes, a primary antibody that recognizes the antigen, and a secondary antibody to which a fluorescent marker that recognizes the primary antibody is bound,

In addition, it may include a substrate on which the nanowires are grown, a capture antibody that recognizes the MERS virus immobilized on the nanowires as an antigen, and a detection antibody to which a fluorescent marker that recognizes the antigen is bound.

On the surface of the Middle East Respiratory Syndrome Virus (MERS-CoV), there is a spike protein that plays an important role in penetrating and infecting the human body, and the Middle East Respiratory Syndrome virus is prevented through an antigen-antibody reaction that binds to the spike protein. Can be detected.

The capture antibody refers to an antibody immobilized on a nanowire, and has a characteristic of recognizing and binding to the spike protein of the Middle East Respiratory Syndrome virus as an antigen.

In addition, the primary antibody is an antibody that recognizes a spike protein or a nucleoprotein of the Middle East Respiratory Syndrome virus as an antigen, and binds to the antigen bound to the capture antibody.

In addition, the secondary antibody is an antibody specific to the primary antibody, and a fluorescent marker is bound. By labeling the secondary antibody, it does not affect the immune response of the primary antibody, and the sensitivity is improved by amplifying the signal, enabling more accurate detection.

In addition, the detection antibody is an antibody that recognizes the spike protein of the Middle East Respiratory Syndrome virus as an antigen, and a fluorescent marker is bound. The detection antibody binds to the target antigen bound to the capture antibody, and the fluorescent signal labeled on the detection antibody can be detected directly.

Specifically, looking at the MERS virus antigen detector, a target antigen is reacted to a capture antibody immobilized on a nanowire grown on a substrate, and a primary antibody that specifically interacts with the target antigen and a fluorescently labeled 2 By reacting the secondary antibody, the fluorescence signal is detected, and the presence or absence of the viral antigen is finally detected.

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

Example One. Nanowire Array of Manufacturing example

1-1. Preparation of nanowire seed solution

A 0.01 M Zinc acetate dihydrate in methanol (Zn(CH ₃ COO) _{2 ·} 2H ₂ O) solution and a 0.03 M sodium hydroxide in methanol solution were heated to 60°C, respectively, and a drop of the Zinc acetate dihydrate solution was added to the sodium hydroxide solution. Slowly dropping each and stirred at 200 rpm. Thereafter, the mixture was heated while stirring until the volume of the mixture was half.

1-2. Preparation of nanowire precursor solution

Zinc nitrate hexahydrate (Zn(NO ₃ ) ₂ ·6H ₂ O), Hexamethylene tetramine (C ₆ H ₁₂ N ₄ ) and polyethylenimine (H(NHCH ₂ CH ₂ ) _n NH ₂ ) were added to deionized water and stirred at 200 rpm. Then, a precursor solution of 25 mM Zinc nitrate hexahydrate, 25 mM Hexamethylene tetramine, and 5 mM polyethylenimine was prepared.

1-3. Nanowire synthesis

The seed solution was dropped onto the substrate, applied thinly, washed with ethanol before drying, and dried with nitrogen using an air gun, and this process was repeated three times. Thereafter, the substrate was heated at 350° C. for 5 minutes using a hot plate to form a seed layer. A polyimide tape having a desired pattern was attached to the completely cooled substrate to expose only the portion to be synthesized. The substrate was placed in a container with the surface of the substrate facing down, and the precursor solution was filled so that the substrate was completely immersed, and then put in a convection oven to synthesize nanowires at 95° C. for 5 hours. After 5 hours, the substrate on which the nanowires were synthesized was taken out, the tape was removed, washed with deionized water, and dried with nitrogen. At this time, the nanowire synthesis time may vary depending on the desired length of the nanowire.

1-4. Formation of functional groups for antibody immobilization

The substrate on which the nanowire was synthesized was immersed in an ethanol-based 4% 3-aminopropyltriethoxysilane solution and then reacted at room temperature for 2 hours. Then, the substrate was immersed in a PBS-based 2% glutaraldehyde solution and reacted at 4° C. for 2 hours. After sufficiently washing with deionized water, nitrogen was blown and dried with an air gun. See L. Wang et. al., “surface plasmon resonance biosensor based on water-soluble ZnO-Au nanocomposites,” Analytica Chimica Acta, 2009, 653, 109-115.

1-5. Antibody fixation

The capture antibody (1 mg/mL diluted 1:1000) was incubated at 4℃ overnight, 37℃ for 1 hour, or at room temperature for 2~3 hours to attach the capture antibody to the substrate on which the functional group was formed, and then 0.05% tween Washed with ®20 in Tris-buffered saline (TBS-T) washing solution. After reacting MERS CoV S antigens of different concentrations (0.01, 0.05, 0.1, 0.5, 1 μm), a primary antibody (1 mg/mL diluted 1:500) was reacted to the substrate at room temperature for 2 hours. After attaching, it was washed using the washing solution. A secondary antibody to which fluorescent particles are attached (1 mg/mL diluted 1:200) was reacted to the substrate for 2 hours and attached thereto, followed by washing with the washing solution. At this time, the diluted solution was Invitrogen Coating Buffer B, 0.1% skim milk in TBS-T.

Example 2. Nanowire diameter And length characteristics evaluation

As shown in the SEM images of FIGS. 3 and 4, the diameter of the nanowire synthesized by the hydrothermal synthesis method was 50 to 100 nm. The length of the nanowire was 2 μm when synthesized for 5 hours, and the length of the nanowire can be adjusted according to the synthesis time.

Example 3. MERS CoV spike protein antigen detection

After attaching the capture antibody on the nanowire substrate and reacting with MERS CoV Spike antigens of different concentrations (0.01, 0.05, 0.1, 0.5, 1 μm), the primary antibody and the secondary antibody with fluorescent markers were reacted under a microscope. The fluorescence signal was observed with the image. As shown in FIG. 5, as a result of measuring the fluorescence signal intensity at each concentration, when the antigen of 0 to 1 μg/mL was reacted, it was possible to confirm a bright signal from the concentration of 50 ng/mL, thereby reducing the detection limit. It can be seen that the sensitivity is excellent.

As can be seen from the results of the above examples, the nanowire array for diagnosing MERS virus antigens of the present invention increases the fixation power of the antibody by growing the nanowires on the substrate, and accordingly, the signal sensitivity is enhanced, resulting in a low detection limit of 50 ng/mL. Was shown, and it is possible to diagnose MERS virus with high sensitivity. In addition, rapid diagnosis was also possible by diagnosing using an antibody to which a fluorescent marker was bound, thus securing excellence in diagnosis of MERS virus.

In the above, the present invention has been exemplarily described, and various modifications may be made without departing from the essential characteristics of the present invention to those of ordinary skill in the art. Accordingly, the embodiments disclosed in the present specification are not intended to limit the present invention, but to explain the present invention, and the spirit and scope of the present invention are not limited by these embodiments. The scope of protection of the present invention should be interpreted by the claims below, and all technologies within the scope equivalent thereto should be construed as being included in the scope of the present invention.

Claims (14)

(a) preparing a nanowire seed solution;
(b) preparing a nanowire precursor solution;
(c) growing nanowires on the substrate;
(d) introducing a functional group to the nanowire;
(e) fixing the capture antibody to the functional group; Including,
The capture antibody recognizes the spike protein of the MERS virus as an antigen,
The step (d) includes i) immersing the substrate on which the nanowires are grown in a 4% 3-aminopropyltriethoxysilane solution and reacting at room temperature for 2 hours; And ii) immersing the substrate in a 2% glutaraldehyde solution and reacting at 4° C. for 2 hours; a method of manufacturing a nanowire array for diagnosis of MERS virus antigens comprising:
The method of claim 1, wherein the nanowire is zinc oxide.
The method of claim 1, wherein the diameter of the nanowire in step (c) is 50 to 100 nm.
The method of claim 1, wherein the substrate is a glass or silicon wafer.
delete delete The method of claim 1, wherein the zinc seed solution contains zinc acetate dihydrate.
The nanowire of claim 1, wherein the zinc precursor solution comprises zinc nitrate hexahydrate, hexamethylene tetramine, and polyethylenimine. Array manufacturing method
The method of claim 1, wherein the step (c) is performed by a hydrothermal synthesis method.
The method of claim 1, wherein step (c) comprises: i) forming a seed layer by applying the zinc seed solution to a substrate; And ii) immersing the substrate in the zinc precursor solution with the seed layer facing down, and reacting at 90 to 95°C; a method of manufacturing a nanowire array for diagnosis of MERS virus antigens comprising:
The method of claim 1, wherein the length of the nanowire is adjusted by adjusting the reaction time of step (c) for 3 to 5 hours.
The method of claim 1, wherein in the step (e), the capturing antibody is cultured on the substrate to which the functional group is attached overnight at 4°C, 1 hour at 37°C, or 2 to 3 hours at room temperature and fixed. Method of manufacturing nanowire array for diagnosing virus antigen
A nanowire array for diagnosis of MERS virus antigen, characterized in that a capture antibody that recognizes MERS virus antigen is immobilized on a nanowire grown on a substrate and prepared by the manufacturing method according to claim 1
The nanowire array of claim 13; And a detection antibody to which a MERS virus antigen is recognized and a fluorescent marker is bound. Or a primary antibody that recognizes the MERS virus antigen and a secondary antibody in which a fluorescent marker that recognizes the primary antibody is combined; a MERS virus antigen diagnostic kit comprising

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