KR20210035160A - Microneedle, patch comprising microneedle and manufacturing method of the microneedle - Google Patents

Abstract

The present invention relates to a microneedle, a patch comprising the same, and a manufacturing method thereof. According to the present invention, the microneedle has a needle shape including a plurality of nanopores formed on a surface, and at least one of an antibody, an enzyme, and a DNA molecule, which specifically are combined with a target biomarker, is bound to the surface. The present invention provides the microneedle capable of rapidly detecting a sub-nanogram level target material.

Description

Microneedle, a patch including the same, and a method for manufacturing the same TECHNICAL FIELD {MICRONEEDLE, PATCH COMPRISING MICRONEEDLE AND MANUFACTURING METHOD OF THE MICRONEEDLE}

The present invention relates to a microneedle, and more particularly, to a microneedle capable of rapidly detecting a target material of a nanogram level or less, and a method of manufacturing the same.

Point-of-care testing (POCT) is a clinical pathology test conducted in a place close to the location where the patient is treated, and is used for immediate diagnosis and response in the field. POCT can be largely classified into invasive and non-invasive. The invasive method is mainly used to analyze blood by collecting blood with a syringe, and it can be accurately diagnosed. However, there is a drawback that it causes pain and discomfort to the patient during the blood collection process and requires a specialist. The non-invasive method diagnoses a disease with body secretions such as blood pressure, body temperature, electrocardiogram (ECG), saliva or sweat, and has the advantage of being able to diagnose quickly and without pain. However, the non-invasive method has the disadvantage that it is affected by environmental factors and is less accurate than the diagnostic method using blood.

Microneedles are needles having a length in micron units and are used to help transdermal delivery of drugs by inducing local or systemic drug absorption by forming microchannels through the skin. The microneedle is a minimally invasive system that makes it easy and patient-friendly to administer drugs. The microneedles penetrate the skin and pass through the stratum corneum to the epidermis, avoiding nerve fibers and blood vessels in the dermal layer. Interstitial fluid (ISF) may be collected by micro holes formed by the microneedles, and the microneedles inserted into the skin may directly contact the biomarkers in the ISF. Therefore, the microneedle can be used as a diagnostic means that invades minimally without pain.

Conventional microneedles used for diagnostic purposes include swellable microneedles that are inserted into the skin to absorb biomarkers along with body fluids, and solid microneedles on which a probe that selectively binds to a target biomarker is treated on the surface. However, in the case of swellable microneedles, the swelling behavior is based on biomarker absorption, so it has a disadvantage that it takes a relatively long time for sensing.In the case of solid microneedles, a fast sensing ability has been proven, but biomarkers at the level of nanograms in the body The detection limit is not sufficient for detection. Therefore, there is a need for research and development on a new material capable of rapidly detecting a very small amount of biomarkers in the body.

On the other hand, preeclampsia (PE) is a pregnancy-specific hypertension syndrome that affects about 2-7% of pregnant women around the world, and is a type of pregnancy addiction. Symptoms of preeclampsia appear only after 20 weeks of pregnancy. Symptoms include hypertension, placental hypoxia, proteinuria, endothelial dysfunction, terminal organ ischemia, and increased vascular permeability. These symptoms of preeclampsia are a major cause of maternal and fetal mortality.

Although there have been studies on preeclampsia before, the exact pathogenic mechanisms are not yet known, and it is widely accepted that placental dysfunction can lead to preeclampsia. In the case of normal pregnancy, proper cytotrophic invasion forms helical arteries, allowing the placenta to exchange gas at high flow and low resistance. However, preeclampsia begins in early pregnancy with incomplete cytotrophic membrane invasion, which prevents proper placenta formation. In preeclampsia placenta, a decrease in cytotrophic invasion at the decidual and endometrial levels and failure of the cytotrophic cells that replace the helical arteries appear. In preeclampsia, high intensity and low blood flow occur, which reduces blood flow to the uterine valve. Incomplete placenta also increases placental oxidative stress, which contributes to the development of systemic endothelial dysfunction in the later stages of the disease.

Since drug treatment for the prevention of preeclampsia is effective at 16 weeks or before pregnancy, there is a need for a fast, non-invasive or minimally invasive diagnostic method capable of early diagnosis.

An object of the present invention is to provide a microneedle capable of rapidly detecting a target material of a nanogram or less level.

Another object of the present invention is to provide a microneedle patch including the microneedle.

Another object of the present invention is to provide a method for manufacturing the microneedle.

For one object of the present invention, the microneedle for detecting a biomarker of a nanogram level or less is a needle shape including a plurality of nanopores formed on the surface, and antibodies and enzymes that specifically bind to the target biomarker on the surface And at least one of the DNA molecules is bound.

In one embodiment, the nanopores are vertically aligned cylinders or irregular sponge-shaped porous structures, and may be formed entirely on the needle surface.

In one embodiment, the concentration of the biomarker detectable by the microneedle may be 50 pg/ml or more.

In one embodiment, the biomarker may include at least one of metabolites, proteins, cell by-products such as exosomes, and genes such as DNA and RNA.

In this case, the biomarker may include estrogen. In this case, the microneedle may be a microneedle for diagnosing preeclampsia.

In one embodiment, the microneedle may be inserted into the body to detect the biomarker in the body.

A microneedle patch for another object of the present invention includes a patch layer; And at least one of the microneedles of the present invention described above disposed on one surface of the patch layer.

In one embodiment, the biomarker may be detected by attaching the microneedle patch to the skin so that the microneedles disposed on one surface of the patch layer penetrate the skin.

A method of manufacturing microneedles for another object of the present invention comprises the steps of anodizing metal microneedles; And binding an antibody that specifically binds the target biomarker to the anodized metal microneedle.

In one embodiment, the metal microneedle may be a microneedle formed of at least one of aluminum, titanium, zirconium, and niobium.

In one embodiment, the anodizing step may be performed for more than 1 hour and less than 16 hours.

In one embodiment, prior to the anodizing step, the metal microneedle may further include polishing.

In one embodiment, in the anodizing step, a plurality of nanopores are formed on the surface of the metal microneedles, and before the step of binding the antibody, expanding the nanopores of the anodized metal microneedles It may contain more.

In this case, the step of expanding the nanopores may be performed by immersing the microneedles in an acid solution.

In this case, the nanopores are vertically aligned cylinders or irregular sponge-shaped porous structures, and may be formed entirely on the needle surface.

A method for detecting a biomarker in a body for another object of the present invention includes the steps of inserting any one of the microneedles of the present invention described above into the body; Removing the microneedles inserted into the body; Reacting the microneedle removed from the body with a target biomarker and reacting with a fluorescently labeled secondary antibody; And measuring the fluorescence intensity of the microneedle reacted with the secondary antibody.

According to the present invention, it is possible to provide a microneedle having a nanoporous structure capable of rapidly capturing biomarkers present in a concentration of nanograms or less in the body with minimal invasiveness. The microneedle of the present invention includes a nanopore structure of a uniform size formed on a surface through a controlled anodic oxidation process, and can be functionalized by immobilizing a biomarker-specific antibody capable of detecting a target biomarker on the surface. The functionalized microneedles can be inserted into the body to detect biomarkers present in the body very quickly and precisely. For example, estrogen, known as a biomarker of pregnancy addiction, can be detected in 1 minute. That is, the microneedle of the present invention and a patch including the same can be configured as a highly sensitive and rapid biomarker detection platform, and through this, can be applied as a small diagnostic device for clinical diagnosis of a disease in the field.

1 is a view for explaining a mechanism for detecting a biomarker of a microneedle and a patch including the same according to an embodiment of the present invention.
2 is a view for explaining an aluminum alloy microneedle having a porous structure in the form of a sponge according to an embodiment of the present invention.
3 is a view for explaining high-purity aluminum microneedles including nanopores in the form of cylinders perpendicular to the surface by performing anodization with oxalic acid and phosphoric acid solutions.
4 is a diagram for explaining a surface change of a microneedle according to an anodization time.
5 is a view for explaining a nanopore structure of a microneedle according to an embodiment of the present invention.
6 is a view for explaining the insertion force of the microneedle according to an embodiment of the present invention.
7 is a view for explaining the antibody functionalized microneedle according to an embodiment of the present invention.
8 is a view for explaining the surface charge of the antibody functionalized microneedle according to an embodiment of the present invention.
9 is a view for explaining the E2 detection characteristics of the microneedle according to an embodiment of the present invention.
10 and 11 are diagrams for explaining E2 detection characteristics of the microneedle according to the present invention.
12 and 13 are views for explaining the characteristics of the microneedle according to the concentration of E2 according to the present invention.
14 to 17 are views for explaining the E2 detection characteristics according to the culture time of the microneedle according to the present invention.
18 and 19 are views for explaining the selective coupling characteristics of the microneedle according to the present invention.
20 is a view for explaining the E2 detection characteristics in the body of the microneedle of the present invention.
21 is a view for explaining the E2 detection characteristics in the body of the microneedle of the present invention.
22 is a view for explaining the E2 detection characteristics in the body of the microneedle of the present invention.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the present invention, various modifications may be made and various forms may be applied, and specific embodiments will be illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to a specific form disclosed, it should be understood to include all changes, equivalents, or substitutes included in the spirit and scope of the present invention. In describing each drawing, similar reference numerals have been used for similar elements.

The terms used in the present application are only used to describe specific embodiments and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In the present application, terms such as "comprise" or "have" are intended to designate the presence of features, steps, actions, components, parts, or combinations thereof described in the specification, but one or more other features or steps. It is to be understood that it does not preclude the possibility of addition or presence of, operations, components, parts, or combinations thereof.

Unless otherwise defined, all terms used herein including technical or scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms as defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and should not be interpreted as an ideal or excessively formal meaning unless explicitly defined in the present application. Does not.

The microneedle of the present invention has a needle shape including a plurality of nanopores on the surface, and an antibody, enzyme, or DNA molecule (ssDNA, single-stranded DNA) that specifically binds to a target biomarker is bound to the surface. It is done.

Microneedles are needles having a size in micrometers, and due to their fine size, they rarely cause pain when inserted into the body and are minimally invasive. The microneedle of the present invention has a shape of a needle having a base portion and a tip portion disposed above the base portion. The tip portion refers to a tip, that is, a portion including a pointed tip (tip), and the tip portion may have a sharpness of a degree capable of penetrating the stratum corneum of the skin when the microneedle is applied to the skin. The base portion may mean a portion that is integrally connected to the tip portion in a cylindrical or truncated shape to form the body of the microneedle. For example, the microneedle of the present invention may have a shape similar to a bullet. For example, the maximum diameter of the tip portion of the microneedle may be 3 to 18 μm, the diameter of the base portion may be 200 to 250 μm, and the height of the microneedle may be 700 to 750 μm. Although the length and diameter of the microneedle have been exemplarily mentioned above, the present invention is not limited thereto.

The microneedle of the present invention may be disposed on a flat layer (patch layer) including a flat surface to constitute a microneedle patch. The microneedle patch of the present invention includes a patch layer in which the microneedles of the present invention described above are disposed on one surface. At this time, single or multiple microneedles are arranged regularly or irregularly. The microneedle patch of the present invention may detect a biomarker of a nanogram level or less by attaching one surface of the patch layer on which the microneedles are disposed to the skin so that the microneedles disposed on one surface penetrate the skin. At this time, in the present invention, when applied to the skin, the patch layer is not inserted into the skin and is located on the skin surface. The microneedle of the present invention may be a microneedle formed of a metal, and for example, may be formed of at least one of aluminum (Al), titanium (Ti), zirconium (Zr), and niobium (Nb). For example, when the microneedle of the present invention is a metal microneedle, the nanopores may be formed by an anodizing process. A detailed description of this will be described later in more detail below.

The microneedle of the present invention includes a plurality of nanopores on the needle surface. In this case, as an example, the nanopores may have a cylindrical shape formed in a direction perpendicular to the needle surface. Alternatively, the microneedle of the present invention may have a porous structure in which a plurality of nanopores form a regular or irregular sponge shape. Since the microneedle of the present invention has a large number of nanopores of the above-described shape, the nanopores in the form of a sponge or cylinder can easily absorb liquid by capillary phenomenon, and through this, the needle surface can be easily functionalized or for a short period of time. The target biomarker can be absorbed and captured in the inside, and at the same time, it has an excellent surface area, thereby providing a sufficient binding site to detect even a small amount of target. That is, the microneedle of the present invention can quickly and accurately detect a very small amount of biomarker. At this time, the concentration of the biomarker detectable by the microneedle of the present invention may be less than or equal to nanograms. A detailed description of this will be described in more detail below.

That is, the microneedles of the present invention can accurately measure the presence and concentration of the biomarkers in a very small amount within a short period of time. Therefore, the microneedle of the present invention and a patch including the same can be used for clinical diagnosis of POCT as a portable handheld analysis system requiring fast and accurate diagnosis.

The microneedle of the present invention binds an antibody, enzyme, and/or DNA molecule (ssDNA) capable of specifically binding to a target biomarker on the surface on which a number of nanopores are formed, and thus metabolites, proteins, and exosomes present in the body Biomarkers such as genes such as DNA and RNA can be detected, such as cell by-products and/or DNA. For example, the biomarker may be estrogen, and the microneedle of the present invention may detect estrogen and use it to diagnose preeclampsia (PE).

Specifically, examples of biochemical markers that can be used to diagnose preeclampsia in the early stages include water-soluble fms-like tyrosine kinase 1 (sFlt-1), placental growth factor (PlGF)), adrenocorticotropic hormone ( adrenocorticotropic hormone (ACTH), human chorionic gonadotropin (hCG), estrogen, progesterone, and preeclampsia-related RNA. In particular, among them, estrogen is a major female hormone responsible for the development and regulation of the female reproductive system, and occurs naturally in women in the form of estrone (E1), estradiol (E2), estriol (E3), and esterol (E4). do. E2 is a predominant estrogen during fertility development in terms of estrogen activity.In a recently published study, the serum concentration of E2 in preeclampsia women was 5 times lower than that of normal pregnant women (PE = 2.9 ㅁ 1.9 ng/ml, normal = 10.5 ㅁ 1.8 ng/ml), which suggests that estrogen has the potential as a biomarker for the diagnosis of preeclampsia. However, since estrogen is present at the level of nanograms per ml in body fluid, it is difficult to detect the concentration thereof quickly with a general antibody-antigen reaction system. However, the microneedle of the present invention can quickly and easily capture a very small amount of biomarker through a cylindrical nanopore structure formed on the surface, and visualize it to confirm the presence and concentration of the biomarker. Therefore, the microneedle of the present invention can be functionalized as an antibody capable of selectively capturing estrogen (E2) as a biomarker, and applied to the body to diagnose preeclampsia, a type of pregnancy addiction.

FIG. 1 specifically shows an antibody-functionalized microneedle patch to diagnose preeclampsia by detecting estrogen (E2), which is a hormone biomarker of preeclampsia, and hereinafter, referring to FIG. 1, the biomarker of the microneedle of the present invention is detected. Let's explain the mechanism.

1 is a view for explaining a mechanism for detecting a biomarker of a microneedle and a patch including the same according to an embodiment of the present invention.

Referring to FIG. 1, the microneedle of the present invention has a number of nanopores formed on the surface and an antibody that specifically binds to a target biomarker bound to the surface, so that when inserting the microneedle into the body, the body fluid is capillary. As a result, it is rapidly absorbed by the microneedle so that the biomarkers present in the body fluid can bind to the antibody. After removing the microneedle inserted into the body (a microneedle patch attached to the skin in the case of a patch) from the skin, specifically binding the microneedle with a biomarker and reacting a fluorescently labeled secondary antibody, a fluorescently labeled secondary antibody Is combined with the biomarker to show fluorescence. Accordingly, the presence or absence of a biomarker can be confirmed by measuring the fluorescence intensity of the microneedle of the present invention reacted with the secondary antibody, and at the same time, the concentration of the biomarker can also be quickly and accurately detected according to the fluorescence intensity. For example, the microneedle of the present invention can analyze the presence or absence of estrogen in the body in 1 minute, and the concentration of estrogen below the nanogram level can be analyzed within 10 to 30 minutes.

Meanwhile, the microneedle of the present invention can be manufactured by anodizing a metal microneedle.

At this time, before the microneedles are anodized, the microneedles may be polished. Polishing of the microneedles may planarize the surface of the microneedles so that nanopores having a uniform shape and size are formed during anodization. However, when polishing is performed for a long time, the length of the microneedles may be shortened or the tip angle may be increased, which may reduce the penetration force into the skin. Therefore, the polishing can be performed so that the tip of the microneedle is sharp and the shape of the microneedle does not change significantly. For example, the polishing may be electrolytic polishing, and the electrolytic polishing of the microneedles may be performed in a mixed solution of ethanol and perchloric acid. For example, when the microneedles have a tip diameter of 15 μm and an overall length of about 750 μm, it may be desirable to perform polishing of the microneedles for 30 seconds.

The metal microneedles may be high-purity aluminum, titanium, zirconium, niobium, or an alloy thereof. For example, the metal microneedle of the present invention may be an aluminum microneedle formed of an aluminum alloy or high-purity aluminum, and the anodic oxidation of the aluminum microneedles may be performed in an oxalic acid or phosphoric acid solution. When the aluminum microneedles are anodized, a layer of anodized alumina is formed on the surface, and a number of nanopores in the shape of a sponge or cylinder perpendicular to the surface of the needle are formed. At this time, in the case of the metal alloy microneedle, nanopores in the form of a sponge may be formed, and in the case of the high purity metal microneedles, nanopores in the form of a cylinder may be formed.

As the anodization time increases, the size of the nanopores formed on the surface of the microneedles increases, but the microneedles may be damaged. Therefore, when the electrolyte is oxalic acid, the anodic oxidation of the microneedles may be preferably performed for more than 1 hour and less than 6 hours, more preferably for 3 hours. In addition, nanopores having a size of several hundreds of nanometers can be produced through anodization using a phosphoric acid solution. The anodic oxidation through the phosphoric acid solution is performed by controlling the concentration of the solution or the voltage to be treated, and the anodizing treatment is preferably performed for less than 16 hours. Thereafter, in order to have the pores in the form of a cylinder that is more uniformly vertically erected, a more uniform vertical cylinder can be obtained by secondary anodic oxidation after removing the oxide layer. Depending on the secondary oxidation time, a nanoporous layer having a desired length can be prepared.

In addition, after the anodic oxidation process, a process of expanding the formed nanopores may be performed. In this case, the nanopore expansion process may be performed by immersing the microneedles in a phosphoric acid solution.

Hereinafter, a method of manufacturing a microneedle of the present invention, a microneedle of the present invention, and characteristics thereof will be described in detail by way of a more specific embodiment.

Preparation of a patch containing microneedles containing nanopores

An aluminum alloy or high-purity aluminum (Al) microneedle patch in which a plurality of bullet-shaped microneedles were arranged was cleaned by ultrasonic treatment in acetone for 5 minutes. Then, the electrolytic polishing of the microneedle patch was performed in a two-electrode configuration using an electrolyte composed of a volume ratio of 4:1 ethanol (99.9%, Duksan Chem.) and perchloric acid (70%, Samchun Chem.). At this time, the microneedle patch was used as an anode and a carbon plate was used as a counter electrode. Electrolytic polishing was performed at 7° C. for 30 seconds at a constant voltage of 20 V using a DC power source. Then, anodization was performed using oxalic acid. The oxalic acid anodic oxidation was carried out for 3 hours at 40 V at 15° C. in a 0.3 M oxalic acid (99%, Samchun Chem.) solution in the same two-electrode configuration. Subsequently, by immersing in a 0.1 M phosphoric acid (85%, Samchun Chem.) solution at 30° C. for 25 minutes to expand the pores, a microneedle having a nanoporous structure on the surface and a microneedle containing the same according to Example 1 of the present invention A needle patch was produced.

In addition, in order to fabricate a porous microneedle having a pore size of several hundred nanometers and a thickness of an alumina layer, anodic oxidation was performed using a phosphoric acid solution on a high-purity aluminum microneedle patch that had undergone the same electropolishing process as above. The anodic oxidation was carried out twice in the same two-electrode configuration, and first, it was firstly performed in a 0.1 M phosphoric acid solution at 0° C. and 195 V for 16 hours. After the first anodization, the previously formed porous alumina layer having a non-uniform structure was removed by immersing it in a mixed solution of 1.8 wt% chromic oxide (chromium oxide VI, Junsei Chem.) and 6 wt% phosphoric acid for 6 hours. . Subsequently, the secondary anodic oxidation was carried out for 2 minutes under the same conditions as the first anodic oxidation. Subsequently, the pores were expanded by immersing in a 0.1 M phosphoric acid solution at 30° C. for 90 minutes, and according to Example 2 of the present invention, a microneedle having a nanoporous structure on its surface and a microneedle patch including the same were prepared.

Microneedle structure including nanopores

The surface and cross-sectional area of the microneedle (patch) including nanopores according to Examples 1 and 2 of the present invention was confirmed with an operating voltage of 10 kV using a scanning electron microscope (FE-SEM, Hitachi S-4700, Japan). I did. At this time, the microneedles were coated with platinum for 40 seconds using an ion sputter coating machine (Hitachi E-1010, Japan). The results are shown in FIGS. 2 and 3.

2 and 3 are views for explaining a microneedle according to an embodiment of the present invention.

First, FIG. 2 is an SEM image of an aluminum alloy microneedle subjected to anodization in an oxalic acid solution according to the present invention.

In FIG. 2, a is a surface SEM image of an aluminum alloy microneedle (patch) including nanopores according to Example 1 of the present invention, and b is a cross-sectional SEM image.

Referring to FIG. 2, it can be seen that a number of nanopore structures are formed on the surface of the aluminum alloy microneedle subjected to anodization treatment according to the present invention, and the size of the formed pores is about 35 nm, and the aluminum alloy microneedle is anodic oxidation. The thickness of the porous alumina layer formed on the surface is about 15 μm.

3 is a SEM image of high-purity aluminum microneedles subjected to anodization in an oxalic acid or phosphoric acid solution according to the present invention.

In FIG. 3, a and c are surface SEM images of high-purity aluminum microneedles including cylindrical nanopores perpendicular to the anodic oxidation in oxalic acid and phosphoric acid solutions, respectively, and b and d are cross-sectional SEM images. .

Referring to FIG. 3, it can be seen that a plurality of nanoporous structures are formed on the surface of high-purity aluminum microneedles that have been anodized in oxalic acid and phosphoric acid solutions according to the present invention. The pore size formed is about 35 nm when oxalic acid is used, and about 180 nm when phosphoric acid is used. The thickness of the porous alumina layer formed on the surface of the microneedle by anodic oxidation is about 15 μm when oxalic acid is used, and about 350 nm when phosphoric acid is used.

In addition, the surface change of the microneedles according to the anodic oxidation time was confirmed. The results are shown in FIG. 4.

4 is a diagram for explaining a surface change of a microneedle according to an anodization time.

Referring to FIG. 4, when anodizing was performed using oxalic acid for 1 hour, nanoporous structures were not well formed on the surface of the microneedle, and this was a short time even though the thickness of the porous alumina layer could be controlled by adjusting the anodic oxidation time. Indicates that no pore structure is formed. On the other hand, when oxalic acid anodizing, a crack may be formed on the surface of the microneedles made of aluminum alloy, and it can be seen that the microneedles are severely damaged when oxalic acid anodizing for 6 hours. On the other hand, when the oxalic acid anodization is performed for 3 hours, it can be seen that the microneedles are not significantly damaged, and a porous structure is formed in the microneedles. Accordingly, it can be seen that when anodizing the aluminum alloy microneedle patch with oxalic acid according to the present invention, the anodization time is preferably greater than 1 hour and less than 6 hours.

In addition, when oxalic acid anodization was performed for 3 hours, the thickness of the formed porous layer was about 15 μm and the pore size was about 25 nm. This indicates that the pore size determined with reference to FIG. 2 is smaller than 35 nm, that is, the pores on the microneedle surface are expanded through the pore expansion process after anodization according to Example 1 of the present invention.

That is, referring to FIG. 4 together with FIG. 2, the microneedle including the nanopore structure according to Example 1 of the present invention, which performed the pore expansion process after oxalic acid anodization, has a porous layer having a thickness of about 15 μm, and about It can be seen that it includes a large number of pores having a size of 35 nm, and the distance between pores and pores is about 75 nm. Through this pore expansion process, the microneedles of the present invention can absorb antibodies or biomarkers through the porous layer.

In addition, the length, tip angle, tip diameter, and base diameter of the microneedle including and without nanopore structure were confirmed with an optical microscope (Eclipse TS100, Nikon, Japan).

The surface area (A _nano ) of the microneedle (nMN) including the nanopore structure according to the present invention was estimated using the unit cell structure of an anodic aluminum oxide (AAO), and the surface area was calculated through the unit cell structure. Is as shown in the following formula and FIG. 5.

5 is a diagram for explaining a nanoporous structure of a microneedle according to an embodiment of the present invention, and shows a schematic diagram of a hexagonal AAO unit cell.

In FIG. 5, r, h, and d are the unit cell parameters of the nanostructure related to the surface area, and represent the pore radius, the thickness of the pore layer, and the pore-pore distance, respectively.

[expression]

In the above formula, A _cell denotes the surface area of a hexagonal unit cell having a cylindrical pore structure, N _cell denotes the number of unit cells on the microneedle surface, A _non denotes the surface area of a non-porous microneedle, and r, h, And d represents the pore radius, the thickness of the pore layer, and the pore-pore distance (see Fig. 4).

Referring to FIG. 5 and the above formula, the surface area ( A _non ) of the microneedle not including the nanopore structure is approximately 4.02 X 10 ⁵ µm ^{2, and the microneedle (nMN} ) Has a surface area ( A _nano ) of about 1.37 X 10 ⁸ µm ² , which is about 340 times the surface area of the corresponding non-porous microneedle ( A _non).

In vitro ( ex vivo ) Insertion test

In order to confirm the effect of the morphological change in the microneedle penetrating the skin, the insertion force before and after formation of the nanopore structure in the microneedle was measured. A displacement-force test station (\Universal Testing Machine, A&D 5000H, A&D Sales Corp.) was used to measure the force applied to the needle and the needle part. The needle was inserted into pig skin having a structure similar to human skin (ie, stratum corneum), and the applied speed was 10 mm/min. At this time, the pig skin was used after drying for 15 minutes at 37 °C so as to have similar humidity and elasticity to human skin. The results are shown in FIG. 6.

6 is a view for explaining the insertion force of the microneedle according to an embodiment of the present invention.

Referring to FIG. 6, the measured insertion force is represented by about 0.3-0.4 N/needle both before (Non-porous MN in FIG. 6) and after (Nanoporous MN in FIG. 6) forming a nanoporous structure. It is suitable for penetrating the stratum corneum of the. That is, it can be seen that even if the nanoporous structure is formed on the surface of the microneedle through anodic oxidation according to the present invention, the microneedles of the present invention are sufficient to be inserted into the skin.

Preparation of Patch Containing Antibody Functionalized Microneedle

A capture antibody against E2 (hereinafter, anti-E2 antibody) was electrostatically attached to the surface of the microneedle using surface modification. Specifically, in order to immobilize the anti-E2 antibody on the microneedle surface, the microneedle patch was washed with acetone for 10 minutes. Then, the microneedle _{was treated with O 2} plasma to generate a negative charge on the surface of the microneedle. Subsequently, the negatively charged microneedle was added to a positively charged poly(allylamine hydrochloride) (poly(allylamine hydrochloride), PAH, Mw: 15000 g mol-1, Sigma-Aldrich) aqueous solution (0.6 wt/vol%) for 10 minutes. It was coated by immersion. Thereafter, the microneedles were immersed in deionized water for 10 minutes _{, dried with N 2,} ^{and then negatively charged anti-E2 antibody (1 st} Ab, 17-βestra) in carbonate buffer (0.2 M sodium carbonate/bicarbonate buffer pH 9.4). Diol antibody (5E500), Santa Cruz Biotechnology Inc., USA) was cast to each microneedle (2 μl per microneedle) and incubated for 1 hour. Subsequently, the microneedle patch was washed 6 times with phosphate buffered physiological saline (PBST) containing 0.05 wt% tween 20 to remove antibodies that could not bind to the needle surface. To reduce non-specific binding, each microneedle was treated with 2 μl of 5% bovine serum albumin (BSA in PBST blocking buffer) and incubated for 1 hour. Then, the microneedle was washed 6 times with PBST to prepare an antibody functionalized microneedle patch according to the present invention.

The microneedle surface in each coating process step was photographed with FE-SEM (Hitachi S-4700, Japan), and the results are shown in FIG. 7.

7 is a view for explaining the antibody functionalized microneedle according to an embodiment of the present invention.

Referring to FIG. 7, it can be seen that the pores on the microneedle surface gradually become smaller after coating ^{PAH, 1 st} Ab, and BAS/1 ^st Ab, respectively, according to the present invention. This means that each material is bonded to the surface of the microneedle.

Surface charge measurement

In order to confirm the surface charge of the oxygen plasma treated, PAH coated, and anti-E2 antibody (1 ^st Ab) coated microneedles according to the present invention, a nanoporous aluminum sheet having the same chemical composition was used instead of the microneedles. , Zeta potential was measured with a zeta potential measurement system (ELS-Z 2000, Otsuka Electronics, Japan) and Malvern zetasizer (Zetasizer Nano ZS, Malvern instruments, UK) under various pH conditions. The results are shown in FIG. 8.

8 is a view for explaining the surface charge of the antibody functionalized microneedle according to an embodiment of the present invention.

Referring to FIG. 8, first, it can be seen that the surface charge is negatively charged to about -1.2 mV during the _{O 2 plasma treatment.} In addition, it can be seen that when the PAH having a positive charge is coated on the _{O 2 plasma-treated surface, it is positively charged to about 1.2 mV.}

On the other hand, in ^{the case of 1 st} Ab, a specific charge did not appear under the condition of pH 7.4 (in the PBS solution), but it was confirmed that a negative charge was exhibited on the surface in the carbonate buffer of pH 9.4 (-8.1 mV). The surface charge of the PAH-coated surface measured at pH 9.4 is about 5.1 mV because the PAH-coated surface is treated with ^{1 st} Ab in a carbonate buffer with a pH of 9.4. ^{That is, this indicates that 1 st} Ab is immobilized on the surface of the microneedle including the PAH-coated nanopore structure by electrostatic attraction.

Optimization of microneedles for E2 capture

(1) 1 for E2 ^st Ab concentration

To confirm the appropriate concentration of 1 ^st Ab, a microneedle patch containing a PAH-coated nanopore structure was ^{treated with 2 μl of 1 st} Ab (10, 20 and 50 μg/ml) at various concentrations per needle, respectively, and 1 Incubated for hours. The microneedle patch was then washed 6 times with PBST and treated with 2 μl of BSA per needle. Subsequently, the microneedles were thoroughly washed with PBST, treated with 2 μl of E2 solution per needle (dissolved at 10 ng/ml in 0.1% DMSO in PBS) and incubated for 30 minutes. The microneedles were washed with PBST to remove all residues, and then each microneedle was washed with 20 μg/ml of Texas Red-labeled goat-derived anti-mouse IgG antibody (Texas Red-conjugated goat anti-mouse) in PBS. IgG antibody, TR-2 ^nd Ab, Invitrogen Corp., USA) (2 μl per needle) and incubated for 1 hour. Then, the microneedle patch was washed 6 times with PBST to remove unbound antibody. Subsequently, the prepared microneedle patch was confirmed with a fluorescence microscope (Eclipse TS100, Nikon, Japan), and the fluorescence intensity was measured using image analysis software (ImageJ, NIH, Bethesda, MD, USA).

9 is a view for explaining the E2 detection characteristics of the ^{microneedle according to an embodiment of the present invention. TR-2 nd} Ab on the surface of the microneedle including the nanoporous structure of the present invention according to the concentration of ^{1 st Ab} Indicate the fluorescence intensity.

Referring to FIG. 9, it can be seen that the microneedle of the ^{present invention exhibits fluorescence intensity at 1 st Ab at all concentrations.} This means that 1 ^st Ab is bound to the microneedle of the present invention and is captured when E2 is added, and the captured E2 binds with TR-2 ^nd Ab, resulting in fluorescence. That is, it can be seen that the microneedles according to the present invention can detect E2 at the level of 10 ng/ml, which is the concentration of E2 in normal pregnant women. In addition, fluorescence in the microneedle of the present invention shows that biomolecules, 1 ^st Ab, BSA, E2, TR-2 ^nd Ab, are stably bound to the microneedle surface even after washing 6 times.

In addition, the ^{microneedles exhibited excellent fluorescence intensity at all concentrations of 1 st Ab, but the microneedles treated with 1 st} Ab at concentrations of 20 and 50 μg/ml were higher than those treated with ^{1 st} Ab at a concentration of 10 μg/ml. It can be seen that the fluorescence intensity appears. This increases the concentration of the 1 ^st Ab increases the 1 ^st Ab amount adhering to the surface, and increasing this amount of E2 coupled to the 1 ^st Ab by addition and, by the combined E2 combined with TR-2 ^nd Ab, as a result Means high fluorescence intensity.

On the other hand, 20 in the 1 ^st Ab in μg / ml, and to determine a represents a fluorescence intensity similar to the 1 ^st Ab of 50 μg / ml, which limited the amount of since the surface area of the micro-needle is fixed, immobilized available 1 ^st Ab Means. That is, in the size of the microneedle having a nanoporous structure prepared according to Example 1, ^{it can be confirmed that the amount of 1 st} Ab that can be immobilized is 20 μg/ml. Accordingly, in the following test, 1 ^st Ab is 20 μg/ml It was used at a concentration of.

(2) E2 incubation time

In order to confirm the E2 capture ability of the microneedles containing the nanoporous structure according to the present invention according to the E2 culture time, the ^{microneedles containing the nanoporous structure treated with BSA/1 st} Ab (20 μg/ml of 1 ^st Ab And BSA) each was treated with 1 and 10 ng/ml of E2 solution (2 μl per needle) and incubated for 1, 10 and 30 minutes, respectively. Subsequently, the microneedle containing the nanopore structure was washed ^{, treated with TR-2 nd} Ab, and washed 6 times with PBST. The results are shown in Figs. 10 and 11.

10 and 11 are diagrams for explaining the E2 detection characteristics of the microneedle according to the present invention, FIG. 10 is a graph showing fluorescence intensity according to incubation time, and FIG. 11 is a fluorescence image according to incubation time (scale bar = 200 μm).

Referring to Figures 10 and 11, although the fluorescence intensity increases as the incubation time increases, it can be seen that the fluorescence intensity increases in proportion to the amount of E2 even in a short incubation time of 1 minute, which is visible through fluorescence images. It can also be confirmed by (see Fig. 11).

That is, it can be seen that the microneedles of the present invention can quickly capture and detect a low concentration of E2 within a short time.

E2 in the body ( in vitro ) Capture test

(1) E2 concentration

To confirm the detection limit of the microneedles, 2 μl of E2 solution per needle (0.5 -1000 ng/ml of E2 in 0.1% DMSO in PBS) was added to the microneedle patch containing the nanoporous structure treated with ^{BSA/1 st Ab.} Lysis) was treated and incubated for 1 minute. Then, it was washed ^{, treated with TR-2 nd} Ab, and washed 6 times with PBST.

The fluorescence intensity and fluorescence images of the prepared microneedles were confirmed, and the results are shown in FIGS. 12 and 13.

12 and 13 are diagrams for explaining the characteristics of the microneedle according to the concentration of E2 according to the present invention, FIG. 12 is a graph showing the fluorescence intensity according to the concentration of E2, and FIG. 13 is a fluorescence image according to the concentration of E2 ( Scale bar = 200 μm).

Referring to FIGS. 12 and 13, it can be seen that the fluorescence intensity increases as the concentration of E2 increases in the microneedle (Nanoporous MN in FIGS. 12 and 13) including the nanoporous structure according to the present invention. In particular, it can be seen that the microneedle of the present invention exhibits a significant difference even in the E2 concentration of 1, 10 ng/ml, which is the standard of preeclampsia.

On the other hand, in the non-porous microneedles (Non-porous MN in FIGS. 12 and 13), it can be seen that there is little difference in fluorescence intensity according to the E2 concentration even when the E2 is treated at a concentration of 1000 ng/ml.

In addition, the microneedle treated with ^{1 st} Ab, BSA, E2, TR-2 ^nd Ab at various E2 concentrations (0.05-10 ng/ml) as above, except that the incubation time is 10 minutes and 30 minutes. Was prepared. The fluorescence intensity and fluorescence images of the prepared microneedles are shown in FIGS. 14 to 17.

14 to 17 are diagrams for explaining E2 detection characteristics according to the culture time of the microneedle according to the present invention, and FIGS. 14 and 15 respectively show fluorescence intensity and fluorescence images when the culture time is 10 minutes, and FIG. 16 and 17 show fluorescence intensity and fluorescence images when the incubation time is 30 minutes, respectively. In each figure, the scale bar is 200 μm.

Referring to FIGS. 14 to 17, it can be seen that fluorescence appears even at a very low concentration of 50 pg/ml after 10 minutes incubation. In addition, it can be confirmed that fluorescence appears at a very low concentration of 50 pg/ml even after incubation for 30 minutes, which can be confirmed to show a higher fluorescence intensity than fluorescence at 10 minutes incubation. This means that the microneedle of the present invention can detect the E2 concentration according to the culture time, and it can be seen that even a very low concentration of E2 can be detected by extending the culture time.

Therefore, the microneedles including the nanopore structure according to the present invention can detect biomarkers at the level of nano- and micro-grams in the body within 1 minute or biomarkers at the level of nanograms or less within 10 to 30 minutes, and Accordingly, it may be applied to a POCT device requiring quick diagnosis.

(2) Checking the trapping in the microneedle

To verify the selective combination of trapping and 2 ^nd Ab of your fluorophore cracks of the ^{micro-needle, BSA / 1 st Ab TR-} 2 nd of 2 μl needle per the microneedle patch containing the treatment nanoporous structure Ab and Texas Red-labeled dextran (TR-dex, Mw. 40 kDa, Invitrogen Corp., USA) (solutions of 1, 10, and 1000 ng/ml, respectively) were treated. Then, the microneedle patch was incubated for 1 hour and washed with PBST. The results are shown in Figs. 18 and 19.

18 and 19 are views for explaining the selective binding characteristics of the microneedles of the present invention, and FIG. 18 shows a fluorescence image of the microneedles treated with ^{TR-2 nd Ab, and FIG. 19 is} A fluorescence image of the microneedle is shown. In each figure, the scale bar is 200 μm.

Referring to FIGS. 18 and 19, it can be seen that fluorescence does not appear in both the microneedles treated with ^{TR-2 nd Ab and TR-dex.} This means that TR-2 ^nd Ab and TR-dex were not trapped in the cracks in the microneedle. Furthermore, it can be seen that in the absence of E2, TR-2 ^nd Ab is not bound to the microneedle, and TR-dex is not bound to ^{1 st Ab as well.}

In other words, when a target substance (biomarker) is present, the microneedle of the present invention can exhibit fluorescence by performing a selective immune reaction, which means that the reliability of the microneedle of the present invention is excellent.

(3) Normal and PE-induced rat serum test

To confirm the capturing ability of E2 present in the mouse serum of the microneedles, 2 μl of each of the microneedles and non-porous microneedles containing the nanoporous structure treated with ^{BSA/1 st Ab were added to 2 μl of normal and PE-induced rat serum.} It was treated with. Then, it was incubated for 1 minute and washed with PBST. Then, the microneedle was washed and ^{treated with TR-2 nd} Ab, and then washed 6 times with PBST.

In addition, in order to confirm the concentration of E2 in the serum, a competitive enzyme-linked immunosorbent assay (ELISA, 582251, Cayman Chemical Company) was performed. After serum was added to a 96-well plate, the plate was incubated with Ellman's reagent at room temperature for 1 hour.

Then, each optical density value was photometrically measured at 420 nm, and the measured fluorescence intensity (final concentration) was calculated using standard curve analysis. The results are shown in FIG. 20.

Figure 20 is a diagram for explaining the E2 detection characteristics in the body of the microneedle of the present invention, ELISA kit, microneedles according to the present invention (Nanoporous MN), E2 concentration detected by non-porous microneedles (Non-porous MN) Represents.

Referring to FIG. 20, E2 was not detected in the non-porous microneedle, and the ELISA kit and the microneedle according to the present invention showed that the serum E2 level in the preeclampsia-induced rat was lower than that in the normal rat. I can.

Specifically, comparing the E2 concentration measured by the ELISA kit and the microneedle according to the present invention, the E2 level measured by the ELISA kit was 0.89 nm/ml in normal mice and 0.43 ng/ml in preeclampsia-induced mice, Similar to the ELISA kit, the level of E2 measured according to the present invention was 2.68 ng/ml in normal mice and 0.34 ng/ml in preeclampsia-induced mice.

That is, by using the microneedle according to the present invention, it is possible to quickly determine whether or not there is a risk of preeclampsia depending on the E2 concentration of the pregnant rat, and based on this characteristic, the microneedle of the present invention is a POCT requiring rapid biomarker diagnosis. It can be confirmed that it is applicable to.

(4) E2-loaded hydrogel insertion test

In order to check whether it can be used for E2 detection in the body, microneedles containing nanoporous structures were applied to the E2 containing hydrogel.

First, microneedles were inserted into agarose and gelatin hydrogels, and then microneedles were removed from the hydrogel, washed, and then treated with ^{TR-2 nd Ab.} The results are shown in Fig. 21.

21 is a view for explaining the E2 detection characteristics in the body of the microneedle of the present invention. In FIG. 21, the scale bar is 200 μm.

Referring to Figure 21, in the case of agarose gel, into the gel and the gel is broken away Loose the needle by the micro-needle is TR-2 ^nd Ab is absorbed or trapped, it can be confirmed that exhibiting fluorescence. However, in the case of gelatin gel, it can be confirmed that the gel does not appear because it has more elasticity than the agarose gel, and thus does not show fluorescence even after treatment with ^{TR-2 nd Ab.}

Based on these results, the microneedles including the nanopore structure of the present invention and the non-porous microneedles not including the nanopore structure were applied to the E2 containing and non-containing gelatin hydrogels, respectively. Hydrogels were prepared from 20 wt% gelatin similar to human skin, and each hydrogel contained 0, 1 to 10 ng/ml of E2. Each of the microneedle patch and non-porous microneedle patch including the BSA/1 ^st Ab treated nanoporous structure was inserted into each hydrogel for 1 minute, and then the patch was removed from the hydrogel and washed 6 times with PBST. The microneedle patch and non-porous microneedle patch including the washed nanoporous structure were again ^{treated with TR-2 nd} Ab, and then washed 6 times with PBST. The results are shown in Fig. 22.

22 is a view for explaining the E2 detection characteristics in the body of the microneedle of the present invention.

22A is a picture of the setup of the hydrogel insertion test, and b is a diagram showing the concentration of E2 trapped in the hydrogel using a microneedle including a nanoporous structure and a non-porous microneedle. In FIG. 22, the scale bar is 200 μm.

Referring to FIG. 22, in the case of a non-porous hydrogel, E2 is hardly detected in the E2-containing gel, whereas in the case of the microneedle of the present invention, the amount of E2 detected increases as the concentration of E2 dissolved in the hydrogel increases. can confirm. That is, based on this, it can be confirmed that the microneedle of the present invention can be used to detect E2 in the body.

Therefore, as examined above, according to the present invention, it is possible to provide a microneedle including a plurality of nanopores on the surface, and the microneedles according to the present invention have an improved surface area, so that an antibody against a target biomarker is formed on the surface. When attached, low concentration biomarkers can be quickly captured and their concentrations can be accurately detected. In particular, when the microneedles according to the present invention are functionalized with an E2 capture antibody, the microneedles of the present invention can detect E2 quickly in 1 minute, and when applied for 30 minutes, a very low concentration of E2 can be detected. In addition, the microneedles of the present invention can detect E2 quickly and precisely in the body through hydrogel and rat serum tests, and accordingly, the microneedles of the present invention are applied to the POCT platform requiring rapid diagnosis with minimal invasiveness to the human body. It will be possible.

Although the above has been described with reference to preferred embodiments of the present invention, those skilled in the art will be able to variously modify and change the present invention without departing from the spirit and scope of the present invention described in the following claims. You will understand that you can.

Claims (10)

It is a needle shape including a number of nanopores formed on the surface,
The nanopore is characterized in that it is configured to cause a capillary phenomenon,
At least any one of antibodies, enzymes, and DNA molecules that specifically bind to a target biomarker is bound to the surface of the nanopores,
Microneedle for detecting biomarkers at the level of nanograms or less.
The method of claim 1,
The nanopores are characterized in that formed by anodic oxidation,
Micro needle.
The method of claim 1,
The nanopores are in the form of a cylinder or a sponge, and are formed entirely on the surface of the needle,
Microneedle.
The method of claim 1,
The concentration of the biomarker detectable by the microneedle is 50 pg/ml or more,
Microneedle.
The method of claim 1,
The biomarker is characterized in that it contains at least one of a metabolite, a protein, a cell by-product, and a gene,
Microneedle.
The method of claim 5,
The biomarker is characterized in that it contains estrogen,
Microneedle.
The method of claim 6,
The microneedle is characterized in that it is a microneedle for diagnosing preeclampsia,
Microneedle.
The method of claim 1,
Characterized in that by inserting the microneedle into the body to detect the biomarker in the body,
Microneedle.
Patch layer; And one or more microneedles of any one of claims 1 to 8 disposed on one surface of the patch layer,
Microneedle patch for detecting biomarkers at the level of nanograms or less.
The method of claim 9,
A biomarker is detected by attaching the microneedle patch to the skin so that the microneedles disposed on one surface of the patch layer penetrate the skin,
Microneedle patch.

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