KR20180021402A - Immobilization of Pathogens Using Hybrid Nanostructures Having Nanoweb Structure - Google Patents

Abstract

According to a specific embodiment of the present disclosure, various pathogens are immobilized using a hybrid nanostructure in which a nanoweb formed of a second polymer material is formed on the surface of a nanopillar array formed of a first polymer material. A method and a detection system for enabling specific or nonspecific detection in a subsequent procedure are disclosed.

Description

Immobilization of Pathogens Using Hybrid Nanostructures Having Nano-Web Structure {Immobilization of Pathogens Using Hybrid Nanostructures Having Nanoweb Structure}

The present disclosure relates to the fixation of pathogens using a complex nanostructure having a nanowire structure. More particularly, the present disclosure relates to a method of fixing various pathogens by using a complex nanostructure in which a nanoporous material of a second polymer material is formed on the surface of a nanopillar array of a first polymer material, And more particularly to a method and a detection system capable of detecting a nonspecifically or non-nonspecifically.

Nanostructures or nanoscale structures are currently being used in electronic materials (e.g., electrodes, light emitting diodes (LEDs) and solar cells), semiconductor manufacturing processes, data storage devices, wearable devices, Sensors, electrochemical sensors, and biosensors). Particularly, nanoscale structures such as nano-rod, nano-pillar or nano-wire have inherent optical and electrical properties and can be applied to various electronic devices and optical devices . These nanoscale fine patterns are important factors for imparting new functions in addition to improving time and space efficiency, and there is an increasing demand for nanostructures in conformity thereto.

Currently, nanostructure fabrication methods using photolithography techniques are widely used, but recently, techniques for fabricating nanostructured structures in a simpler and less costly manner have been actively studied. In this regard, nanopatterns, especially nano-pillar structures, fabricated in electronic devices (e.g., LEDs), semiconductor manufacturing processes, etc. are generally fabricated using an inorganic material (e.g., Si, GaN) (for example, Korean Patent Laid-Open No. 2008-30042) or a top-down method (for example, Korean Patent Laid-Open No. 2007-63731).

On the other hand, attempts have been made to apply structures having nanopatterned surfaces to new applications (for example, biomimetic fields). For example, pathogens are widely distributed in the surrounding environment. Specifically, , Animal organs, water contaminated by animal stools, and the like. The human body also has more than 150 types of bacteria in and out of the body on average, many of which are harmless to humans, but some are botulism, cholera, diarrhea, emesis, Pneumonia, typhoid, and the like. Especially, more than 200 kinds of diseases can be transmitted through food and drinking water alone. For example, E. coli strains do not cause major problems when consumed, but some are known as pathogens that produce toxins that cause diarrhea and the like.

Especially, the pathogenic Escherichia coli O157: H7 is widely known as enterohemorrhagic Escherichia coli as a causative organism of food poisoning, and it is a problem in the world. In addition, infections lead to hemolytic uremic syndrome, hemorrhagic colitis, diarrhea, kidney failure, seizures, and, in severe cases, death (Reisner, A. et al. , 2006. Journal of Bacteriology. 188, 3572-3581 ; Barrientos, RM et al. , 2009. Brain, Behavior, and Immunity, 23, 450-454; Schrag, SJ et al. , 2006. PEDIATRICS 118, 570-576). Salmonella choleraesuis (Salmonella choleraesuis, Salmonella bongori, Salmonella typhimurium) are also typical pathogens causing food poisoning. They are causative bacteria of typhoid fever and paratyphi and may be contaminated with various livestock and animals. In addition, if adequate cleaning, processing temperatures and storage conditions are not followed, Salmonella may multiply and cross contaminate with other foods through such contaminated food.

These pathogens can easily infect humans through contaminated soils and the water environment. Because the propagation rate of pathogens in a suitable environment is very fast, even a small number of pathogens can quickly grow to a level that can threaten human health if they enter the body once they are suitable for their growth environment. Especially, the skin of the human body, for example, a large number of pathogens is present on the hands, and contact between the human body is also one of the main pathogens propagating or infecting pathogens.

Techniques using nano-particles have been reported for the purpose of securing antibacterial properties for removing such pathogens. Specifically, in the case of silica nanoparticles, it is possible to provide an effective method for removing bacteria by transporting nitrogen oxide (NO) into cells. In addition, silver (Ag) and copper (Cu) nanoparticles also exhibit their ability to remove from contact with bacteria. In particular, silver (Ag) is known to utilize the oxidation and denaturing action of bacteria, and silver nanoparticles generate reactive acidic groups in air or water to affect the cell wall of bacteria in a manner similar to hydrogen peroxide To kill bacteria.

As another antibacterial mode, it is possible to exemplify inhibiting the growth of bacterial, yeast, virus or virus infected cells, cancer cells and / or protozoa, or preventing the fouling phenomenon thereof . In this regard, U.S. Patent Publication No. 2014-0290732 discloses a biomimetic technique that provides a repulsive surface by having a specific range of equilibrium contact angles. That is, a substrate comprising a network of antimicrobial and / or biofouling inhibiting polymers attached in a covalent manner to inhibit attachment of pathogens such as bacteria. In addition, U.S. Patent Publication No. 2013-0196365 also discloses a technique for preventing problems such as surface corrosion due to biofilms formed by bacteria, algae, protists, etc. on metal and non-metal surfaces. However, in the case of the above-mentioned prior art, techniques for preventing the fixation or adherence of pathogens are contrary to the approach adopted in the present invention, as described below, for the most part.

On the other hand, in the case of nanostructures that have been studied recently, their physical properties and behavior depend on the molecular constituents and precise spatial positioning, and are not predicted by controlled morphology in large scale devices It is known that it can provide characteristics. Particularly, since the main path of propagation of a pathogen is caused by physical contact between humans or rubbing or rubbing, a more fundamental technical solution for effective detection of a pathogen by using a nanostructure is required.

Particularly, in the case of a conventional method for detecting a pathogen of an antigen-antibody and gene amplification, it is difficult to rapidly detect and prevent diffusion of a contact / air-mediated high-risk pathogen and is also poor in stability. It is impossible to cope with direct and indirect infection pathways such as radio waves.

In this disclosure, unlike a conventionally known approach, it captures in a manner that promotes physical attachment (or immobilization) of pathogens onto the surface of the structure, and is not only easily applicable to ductile materials such as fibers, gloves, And the like, or to be able to detect specifically by appropriate subsequent reforming.

In particular, the present disclosure is intended to simulate the pathogen pathway of the human body and provide a fundamental way to more freely detect and analyze pathogens by non-specific / specific methods.

According to a first aspect of the present disclosure,

A method for immobilizing a pathogen in contact with a complex nanostructure,

The composite nano-

(i) a nanopattern structure of a first polymer material having a plurality of nanopillars formed on its upper side, (ii) a metal layer for surface modification attached to the surface of the nanopattern structure, and (iii) a nanopattern structure And a nano-web structure of a second polymeric material formed on the second polymeric material.

According to an exemplary embodiment, the immobilized pathogen can be detected nonspecifically, or specific detection can be performed by modifying the complex nanostructure with a specific chemical substance.

In an exemplary embodiment, the specific chemical for the specific detection may be an antibody corresponding to a specific antigen present on the surface of the pathogen cells to be detected.

According to a second aspect of the present disclosure,

As an analysis system of a pathogen,

a) a nanopattern structure of a first polymer material having (i) a plurality of nanopillars formed on an upper side thereof, (ii) a metal layer for surface modification attached to the surface of the nanopattern structure, and (iii) A composite nano structure including a nano-web structure of a second polymer material formed on a structure;

b) an amplification device of the pathogen immobilized on the complex nanostructure; And

c) a detection device for the amplified pathogen;

Is provided.

In an exemplary embodiment, the first polymer is at least one selected from the group consisting of polyurethane (PU), polydimethylsiloxane (PDMS), NOA (Noland Optical Adhesive), and epoxy Can be selected.

In an exemplary embodiment, the second polymer comprises polyether ketone (PEK, PEEK, PEEKK); Polysulfone; Polyether sulfone, polyphenyl ether sulfone, polyphenylene; Polyimidazole; Polyimide, polyamideimide; At least one selected from the group consisting of polyaniline, polyacetylene, polypyrrole, and polythiophene may be selected.

In an exemplary embodiment, an intermediate layer of a metal material is formed on the surface of the nanopattern structure and at least one selected from the group consisting of Ti, V, Cr, Sc, Nb, Mo and W is formed, A surface modifying metal layer may be formed on the intermediate layer, wherein at least one selected from the group consisting of Ni, Zn, Pd, Ag, Cd, Pt, Ga, In and Au is selected.

In an exemplary embodiment, a binary layer structure of Au / Ti may be formed on the nanopattern structure.

It is possible to provide flexible countermeasures against a variety of direct and indirect propagation or infection pathways such as human skin contact, coughing, air propagation, etc. through the method of immobilizing pathogens using the complex nanostructure provided in accordance with the specific embodiment of the present disclosure. Furthermore, since it can secure the good physicochemical stability of the composite nanostructure, it is not only a problem related to the stability of the immunoassay which is currently widely used but also applied to or attached to a soft material such as a fiber, and widely used in everyday life It is possible to build a biosensing platform as much as possible. Therefore, it is expected to be widely used in the future.

FIG. 1 is a view conceptually showing a manufacturing process of a composite nanostructure formed with a nanoweb according to one embodiment of the present disclosure; FIG.
2 is a view showing a series of processes for fabricating a plurality of nanopillar-formed nanopillar structures of a first polymer material according to an exemplary embodiment of the present disclosure;
FIG. 3 is a graph showing the relationship between the principle of nanofilament nanofibers in a nanofiller structure formed on a nanopattern structure of a first polymer to which a surface-modifying metal layer is attached, Fig.
4 is a view schematically showing a sequence of steps for fabricating a composite nanostructure according to Embodiment 1;
FIG. 5 is a SEM photograph showing the change of dimensions and surface state of the nanopile structure formed in Example 1; FIG.
FIG. 6A is a cross-sectional view of a Si master mold having a nanoscale hole formed in Example 1, (b) a nanopattern structure of a PUNO material, (c) a nanopattern structure having an Au / Ti metal layer adhered thereto, and (d) SEM image showing each of the composite nanostructures formed with the nanoweb;
6B is an SEM photograph showing a cross section of the composite nanostructure fabricated in Example 1;
7 is a graph showing SEM photographs and surface EDS analysis results after (a) a PUNO nanopattern structure, (b) after Au / Ti metal layer deposition, and (c) after nanofiber formation in Example 1, respectively;
8 is a graph showing the results of water contact angle test in (a) PUNO nanopattern structure, (b) Au / Ti metal layer-attached nanopattern structure, and (c) PANI nanoweb formed in Example 1, Fig.
FIG. 9A is a view showing a result of the immunity test on the horizontal direction external force of the composite nanostructure fabricated in Example 1; FIG.
FIG. 9B is a view showing a result of the immunity test for the vertical external force of the composite nanostructure fabricated in Example 1; FIG.
10 is a SEM photograph of a composite nanostructure produced by directly forming a nanoweb on a nanopattern structure without a surface modification metal layer according to Comparative Example 1 and a composite nanostructure produced according to Example 1 ;
11 is a SEM photograph showing the morphological change of the nanofiller according to the physical external force applied to the nanopattern structure having no nanofibers according to Comparative Example 2;
12 is a graph showing the result of simulation of the pathogen propagation through human contact using the composite nanostructure fabricated in Example 1 according to Example 2 and the results of PCR analysis of the pathogen immobilized or captured on the composite nanostructure Fig. And
FIG. 13 is a graph showing the results of a detection limit experiment to which various concentrations of a bacterial solution are applied to the composite nanostructure fabricated in Example 1 according to Example 3. FIG.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention can be achieved by the following description with reference to the accompanying drawings. It is to be understood that the following description is of a preferred embodiment of the present invention and that the present invention is not necessarily limited thereto.

The accompanying drawings may be exaggeratedly expressed relative to the actual layer thickness (or height) or the ratio with respect to the other layers for the sake of understanding, and the meaning thereof will be appropriately understood according to the concrete purpose of the related description to be described later .

The terms used in this specification can be defined as follows.

"Polymer" includes both homopolymers and copolymers, and the copolymer can be understood to include random copolymers and block copolymers.

"Nanostructure" or "nanostructure" refers to a feature or texture having a dimension or size of nanoscale (e.g., about 0.1 to 1000 nm, specifically 1 to 100 nm) May refer to any nanoscale object, including nanopiles, nano-rods, nano-walls, nanowires, nano-webs, and the like.

"Array" refers to fibers, columns, pillars, loops, tubes, cones, blocks, cubes, hemispheres, spares, or the like, which are derived from, or affixed (secured) to, a support, member or backing member , A wall, a grid, a hole, or a combination thereof, a textured surface, or a surface.

"Adhesion" may mean that a protein, cell, or other material is bound or linked to a surface in a covalent or non-covalent manner.

"Immobilization" may mean that any material or bioactive agent is attached to the substrate either directly or indirectly, either covalently or non-covalently.

The terms "on" and "on" can be understood to be used to refer to the relative position concept. Thus, there may be intervening or present other layers (interlayers) or components therebetween as well as where other elements or layers are directly present in the mentioned layer. Similarly, the expressions "under", "under" and "under" and "between" may also be understood as relative concepts of position. Also, the phrase "sequentially" can also be understood as a relative position concept.

"Nanopillar" may mean a rod shape having a diameter of less than about 1,000 nm, such as a few nanometers to a few hundred nanometers.

The "water contact angle" can be understood to refer to the angle at the three phase contact line to the moisture / air / dry surface.

By "contacting" is meant by direct contact between the two objects, it can be understood that, in the broadest sense, any additional component can be interposed.

"Polyaniline" is a polymeric cation with selective anion permeability, which is protonated in an oxidized state.

"Hydrophilic" means that the polymer, material or functional group has affinity for water.

"Amplification" may mean a reaction that occurs repeatedly to form multiple copies of at least one segment of the template molecule.

"PCR" refers to a reaction in which a large amount of the same DNA strand is formed from one initial template by a cycle process (heating and cooling alternately), wherein (i) a double helix (Iii) a mixture of dATP, dTTP, dGTP, and dCTP (which forms a new DNA molecule in the PCR amplification process), (ii) a primer (a single stranded DNA molecule capable of binding to the complementary DNA base sequence in the template DNA) , And (iv) an enzyme that synthesizes a new DNA molecule using Taq DNA polymerase (dNTP).

The term "complementary" means capable of hybridizing a nucleotide or base pair between nucleic acids, usually A and T (or U), or C and G.

Overall disclosure

According to one embodiment, there is provided a method of immobilizing a pathogen by contacting a composite nanostructure formed with a polyaniline nanoweb structure. The composite nanostructure may include (i) a nanopattern structure of a first polymer material having a plurality of nanopillars formed on its upper side, (ii) a metal layer for surface modification attached to a surface of the nanopattern structure, and (iii) And a nano-web structure of a second polymer material formed on the attached nanopattern structure.

The pathogens applicable in the present specification include all gram-positive and gram-negative microorganisms that enter the living organism of animals and plants to cause parasitic diseases or give harm, and specifically include E. coli O157: H7, Salmonella choleraesuis , Salmonella typhimurium , Salmonella typhimurium , Staphylococcus aureus, Listeria monocytogenes , Listeria denitrificans , Listeria grayi , Listeria murrayi , Cholera, Agrobacterium , Pertussis, Diphtheria, Typhoid, Pest, Hemolytic streptococcus, And more particularly E. coli O157: H7 , Salmonella choleraesuis , Salmonella bongori , Salmonella typhimurium , Listeria monocytogenes , Listeria denitrificans , Listeria grayi , Listeria murrayi , and the like.

These pathogens can be propagated in a variety of ways, i. E., Through various vapors, liquid and / or solid medium or medium, and in particular can propagate through human contact (e.g., hand contact).

According to an illustrative embodiment, when a pathogen is contacted with a complex nanostructure, it is captured in such a way that the pathogen is immobilized (or attached) in a nano-web structure (similar to a spider web structure) in the structure in a physical or non-specific manner. Such immobilized pathogens can be subsequently detected through an amplification reaction. As such amplification methods, amplification methods known in the art such as PCR technique (amplification of DNA), NASBA technique (RNA amplification) Technology can be used.

Hereinafter, the preparation and properties of complex nanostructures and the capture (and detection) of pathogens will be described in detail.

Fabrication of nanopattern structure

FIG. 1 is a view conceptually showing a manufacturing process of a composite nanostructure formed with a nanoweb according to one embodiment of the present disclosure.

In the illustrated embodiment, the composite nanostructure is fabricated by forming a surface-modifying metal layer on a nanopattern structure of a polymer material having a plurality of nanopillars and growing a web structure of a polymer material on the nanopattern structure.

The nanopattern structure has a plurality of nanopillars (pillar structure having nanoscale) projected on a plane, and may be made of a first polymer material. At this time, the first polymer can be appropriately selected in consideration of the application field of the composite nanostructure as the final object. Particularly, it is an object of the present invention to provide a polymer capable of easily coating (coating) during the process of manufacturing a nanopattern structure, capable of relatively easily separating (desorbing) from the mold and capable of easily controlling the dimension or arrangement of the formed nanofiller May be advantageous. Further, it may be advantageous to select and use a first polymer having good physical properties such as flexibility, photosensitivity, mechanical stability, thermal stability, and chemical stability.

For this purpose, the first polymer should have a viscosity in an uncured (fluid) state at room temperature for, for example, about 300 to 5000 cps, specifically about 500 to 3000 cps, more specifically about 800 to 800 cps, 2000 cps. Also, it may be advantageous that the first polymer typically has an adhesion to a plastic material equal to or greater than an adhesion to the metal. This is because when a nano pattern is formed by using a master mold of a metallic material as described later, when the nanopattern structure is separated (released) from the master mold after molding and curing (for example, UV curing) It is difficult to separate the first polymer layer from the master mold.

According to an exemplary embodiment, the first polymer is at least one selected from the group consisting of polyurethane (PU), polydimethylsiloxane (PDMS), NOA (Noland Optical Adhesive), and epoxy Can be selected. In this regard, NOA-based polymers (e.g., commercially available from Norland Products, Inc. under the trade names NOA 61, NOA 63, NOA 65, NOA 68 and the like) are liquid phases containing multifunctional thiols and multifunctional allyl monomers UV curable monomer mixture.

According to a particular embodiment, blends of NOA-based adhesives such as polyurethane acrylate (PU) and NOA 68 may be used. The content of each of the polyurethane acrylate (PU) and the NOA-based adhesive in the blend is, for example, about 20 to 80% by weight (specifically about 30 to 70% by weight, more specifically about 40 to 60% by weight) Can range from about 80 to 20 weight percent (specifically about 70 to 30 weight percent, more specifically about 60 to 40 weight percent). Hereinafter, the polymer produced from the blend of the PU and the NOA-based adhesive is referred to as "PUNO ".

In this regard, NOA 63 contains about 70 to 75 wt% of NOA 61 and about 25 to 30 wt% of urethane component, NOA 61 substantially comprises about 55 to 57 wt% of tetrathiol represented by the following formula I and triallyl About 43 to 45% by weight of isocyanurate.

[Chemical Formula 1]

The nanopattern structure described above is not limited to a specific manufacturing method but can be a method known in the art.

FIG. 2 is a view showing a series of processes for fabricating a plurality of nanofiller-formed nanopattern structures of a first polymer material according to an exemplary embodiment of the present disclosure.

First, a master mold 11 in which a plurality of nanoscale holes 12 are formed is provided. The master mold may be formed of a material such as silicon wafer, PDMS, glass, quartz, polyethylene terephthalate, polycarbonate, polyethylene (PE), polyurethane (PU) COC (cyclic olefin copolymer), and the like. At this time, the material of the master mold 11 and the first polymer will typically be different, but may be different depending on the case.

In this regard, the nanoscale hole or nano hole 12 formed in the master mold 11 has a shape and dimensions corresponding to the nano-pillar of the first polymer material to be formed later, and may be circular, elliptical, It may have various shapes, and more specifically, it may be circular. Such nanoscale holes 12 may be formed by processing techniques known in the art, such as photolithography, ion milling, e-beam lithography, and the like. Although the present invention is not limited to a specific processing technique, for example, a selective etching process using a photolithography method can be applied. As etching methods that can be used in the photolithography process, a dry etching method such as reactive ion etching (RIE), inductively coupled plasma reactive ion etching (ICP-RIE) Chemically assisted ion beam etching (CAIBE), or the like may be used, but the present invention is not limited thereto.

According to an exemplary embodiment, the diameter of the nanoscale hole 12 is about 100-1000 nm (specifically about 300-900 nm, more specifically about 400-700 nm), and the depth of the hole 12 is about May range from 100 to 1500 nm (specifically from about 300 to 1000 nm, more specifically from about 500 to 900 nm). In addition, the spacing between the plurality of holes 12 may range from about 100 to 3500 nm, specifically from about 300 to 2500 nm, and more specifically from about 500 to 150 nm. The present invention is not limited to the numerical range of the nanoscale hole 12 described above and can be appropriately adjusted in consideration of the material of the polymer (the first polymer and the second polymer to be described later), the use of the composite nanostructure, It should be understood that there is.

When the master mold 11 is provided, a cleaning step for selectively removing foreign matter may be performed. For this cleaning, for example, acetone, methanol, isopropanol or the like may be used, and in some cases, a plasma treatment may be performed to remove organic substances remaining on the surface.

A solution of a first polymer (or a first polymer) is applied on the master mold 11 to form a polymer layer 13 (which may be a kind of nanocasting or nanomolding). At this time, the coating method can be appropriately selected and applied by coating methods known in the art, for example, spin coating, spin coating, spray coating, and the like.

According to an exemplary embodiment, after the first polymer layer 13 is applied, a flexible polymeric support of a thickness of about 1 to 500 [mu] m (specifically about 5 to 300 [mu] m, more specifically about 20 to 200 [ It may be advantageous to attach and roll the film layer 14 so that the first polymer is sufficiently injected into the nano-scale holes in the master mold. The support film layer may be made of, for example, polyethylene terephthalate (PET), cycloolefin polymer (COC), polyethylene (PE), polycarbonate (PC), polyether ethyl ketone (PEEK) Polyamide (PA), polyurethane (PU), and the like. The support film layer 14 preferably has good flexibility, and it may also be desirable to have transparency so that UV irradiation can be smoothly performed in a subsequent step.

In addition, the rolling process performed after the application (application) of the support film layer 14 can be carried out typically in the temperature range of room temperature to 90 < ⁰ > C, and that the nanowire structure is relatively low (e.g., About 10 < ⁰ > C or less), it may be advantageous to select a species that can sustain durability within this temperature range. In addition, when the nano-web structure is formed in an acidic condition (for example, an acid solution), the chemical resistance against other chemical substances (for example, cleaning with acetone) It may be advantageous.

Alternatively, in order to facilitate separation of the polymer layer 13 from the master mold 11 in the subsequent process, a releasing agent (for example, a fluoroalkylsilane, specifically tridecafluoro- (1,1,2,2) -tetrahydrooctyl-trichlorosilane) may also be used.

The polymer layer 13 thus formed has a certain elasticity through, for example, a curing process. The curing process may be performed by various methods such as ultraviolet (UV) curing and thermal curing. For example, when PDMS, which is a silicone-based elastic polymer, is used as the first polymer, a polymer solution is prepared using a curing agent together with a polymer Wherein the weight ratio of polymer (PDMS): curing agent may be in the range commonly used in the art, for example in the range of about 10: 1 to about 11: 1. As a curing agent, a two-pack type Sylgard 184 kit (Sylgard A: Sylgard B = 10: 1) commercially available from Dow Corning is preferably used. The polymer solution thus prepared is coated on the master mold and then heated and cured, whereby the polymer layer 13 can be formed.

When a blend of a polyurethane acrylate (PU) and a NOA-based adhesive (for example, NOA 68) is used as the first polymer, the master mold is spin-coated under a condition of, for example, about 1000 to 2000 rpm, For example, a PET film as a support film, and then rolled using a roller. Subsequently, the intensity of ultraviolet light bar, at this time, for curing the PUNO polymer by UV irradiation, for example from about 100 to 600 mJ / cm ^2, specifically about 200 to 500 mJ / cm ^2, and more specifically about 400 To 480 mJ / cm < ² >.

The cured first polymer layer 13 is separated from the master mold 11 to obtain the nanopattern structure 15. [ As this separation method, various methods known in the art can be adopted, such as a method of using a solvent, a wet chemical etching method, a peeling method, and the like.

In an exemplary embodiment, the nanopatterned structure may typically exhibit hydrophobic (or superhydrophobic) and antibacterial properties.

In an exemplary embodiment, the aspect ratio of the nanopile in the nanopattern structure can be adjusted to an appropriate range in consideration of the use, material, and the like, for example, about 1 to 10, specifically about 2 to 7 , More specifically from about 3 to about 5. In this regard, the mechanical properties of PDMS (E <10 MPa; σ _ult <2.4 MPa) are lower than the mechanical properties of PUNO (E = 24 MPa; σ _ult = 11.5 MPa) It is possible to have a low aspect ratio as compared with the nano pillar of the PUNO material.

According to an alternative embodiment, the nanopattern structure can be fabricated using a polymer mold as described in Korean Patent Laid-Open Publication No. 2015-0053303 of the present applicant, which patent document is incorporated herein by reference do. In addition, as long as a nanopattern structure having a plurality of nanoscale filament (or rod) shapes can be provided, various methods can be adopted without being limited to a specific method.

Attachment of surface-modified metal layer

According to one embodiment, the surface modifying metal layer is attached on the nanopattern structure 15 manufactured as described above. The metal layer for surface modification serves as a catalyst for promoting the growth of the second polymer in the process of forming the nanowire structure described later. Of course, the nano-web structure can be formed to some extent on the nanopattern structure without attaching such a surface-modifying metal layer, but it is more advantageous when the surface-modifying metal layer is interposed in the formation degree and the formation time. Although the present invention is not limited to a particular theory, it is considered that this metal layer not only improves the adhesion to the polymer but also functions as a catalyst to promote the formation of a nanowire structure described later.

More specifically, examples of the metal for surface modification include Ni, Zn, Pd, Ag, Cd, Pt, Ga, In and Au. More specifically, Au and Ag are exemplified. Alone or in combination (or in the form of an alloy). In a specific embodiment, Au can be used as the material of the surface modifying metal layer, which can provide good oxidation and corrosion resistance, an inert surface when used in biological experiments, good electrical and thermal conductivity, optical reflectivity is high, and a smooth surface can be obtained. Utilizing these characteristics, for example, it is used as a useful substrate in the life science field or as a signal transducer in chemical and biochemical sensors. In addition, the Au thin film may also be used as a surface enhanced Raman scattering base material in a microfluidic device using an inactive property.

In certain embodiments, the surface modifying metal layer may be formed on the nanopattern structure using methods known in the art, thermal vapor deposition, sputtering, E-beam deposition, and the like. The thickness of the surface reforming metal layer may be, for example, in the range of about 1 to 500 nm, specifically about 5 to 300 nm, more specifically about 50 to 200 nm. The coverage of the surface modifying metal layer for the nanopatterned structure may be at least about 70%, specifically at least about 80%, more specifically at least about 90%, and even more substantially 100% .

The above-mentioned surface-modifying metal layer, particularly the Au layer, may have poor adhesion to the surface of the underlying polymer-based nanopattern structure, despite good physico-chemical properties. This is because the polymer or polymer-based surface has low wettability and bonding due to low surface energy, incompatibility, chemically inert or weak boundary layer present. For the purpose of alleviating the difficulty of adhering to the surface of the lower nanopattern structure, an intermediate layer may be selectively interposed between the nanopattern structure and the surface-modifying metal layer in a specific embodiment (for example, A two-layer structure of a metal layer for surface modification / an intermediate layer). As the intermediate layer, Ti, V, Cr, Sc, Nb, Mo, W or the like such as Ti, Cr or the like can be used singly or in combination. Since the metal can form a chemical bond with polar atoms on the surface of the nanopattern structure, it is considered that the nanopattern structure of the polymer material on the lower surface and the metal layer for surface modification on the upper surface can be firmly attached.

Of the metals for forming the intermediate layer listed above, Cr may improve the adhesion of the Au layer, but may diffuse to the Au surface and affect morphological or electrical properties of the Au layer. Accordingly, in a specific embodiment, a binary layer structure of Au / Ti using Ti as an intermediate layer and Au as a surface modification metal layer can be applied.

In the embodiments described above, the intermediate layer may also be deposited on the nanopattern structure using known methods such as thermal vapor deposition, sputtering, E-beam deposition, and the like. For example from about 1 to 500 nm, specifically from about 5 to 300 nm, more specifically from about 10 to 100 nm. In this regard, the thickness ratio of the surface modifying metal layer / intermediate layer can typically be adjusted to a range of about 1 to 50, specifically about 3 to 20, more specifically 5 to 10.

According to the exemplary embodiment, the above-described step of forming the surface-modifying metal layer (and the intermediate layer) can be performed at a chamber temperature of, for example, 50 ^o C and, for example, To the glass transition temperature of the target, and the deposited nanopattern structure may be deposited. The deposition thickness may be controlled according to the deposition time.

Formation of nano-web structure

According to one embodiment, when the surface-modifying metal layer is formed on the nanopattern structure as described above, the nanoparticle structure is formed by growing the second polymer on the nanopattern structure having the surface-modifying metal layer.

FIG. 3 shows a principle of forming a web structure by growing a nanofilament structure of a second polymer in a nanofiller of a nanopattern structure of a first polymer material to which a surface-modifying metal layer is attached according to an embodiment of the present disclosure FIG.

As shown in the figure, the nanopillar surface of the nanopattern structure having the metal layer attached thereto serves as the growth surface of the second polymer. Specifically, it is preferable that the distance from at least one point (specifically, a plurality of points) on the side of the nanofiller to the lateral direction (on the side of the nano pillar, for example, about 10 to 170 degrees, specifically about 30 to 150 degrees, The nanofibers of the second polymer (for example, the ends of the growing fiber strands have a wedge shape or a pile shape) grown in the direction of about 60 to 120 DEG, The nanofibers are combined with nanofibers of the second polymer grown transversely from at least one point (specifically, a plurality of points) of the nanofiller to form a network similar to a web or mesh structure. In addition, the fiber structure of the second polymer can also grow on the upper surface of the nanopillar, where the growth direction in the top face can be oriented or non-oriented (e.g., the fiber strands are arranged randomly or radially with respect to one another) have.

In an exemplary embodiment, the diameter of the second polymeric fibers (strands) growing to interconnect the plurality of nanofillers is about 10 to 200 nm, specifically about 30 to 100 nm, more specifically about 50 to 80 nm Lt; / RTI &gt; In addition, the average mesh size of the nanoweb can range from about 1 to 1,500 nm, specifically from about 50 to 1,200 nm, and more specifically from about 100 to 1,000 nm.

According to an exemplary embodiment, the second polymer is selected from the group consisting of polyether ketone (PEK, PEEK, PEEKK), polysulfone (PSU; e.g. Udel ^® , polyether sulfone, polyphenyl ether sulfone, polyphenylene, , Polyimide, polyamideimide, and polyaniline (PANI), specifically, polyaniline, which can be selected singly or in combination.

In an exemplary embodiment, the polymerization of the second polymer for nanoporous formation may be carried out using a template, or a template-free method such as an interfacial method, a seeding method, and a micellar method .

According to a specific embodiment, polyaniline can be used as the second polymer. In the case of the aniline monomer which can be used herein, for example, p-CH3, p-OCH3, o-CF3, , p-COOH, o-NH2, p-NH2, etc.), unsubstituted aniline, and specifically unsubstituted aniline.

In a specific embodiment, polyaniline having a repeating unit represented by the following general formula (1) may be used as the second polymer.

[Formula 1]

In this embodiment, polyaniline is a conductive polymer and is suitable for application to sensors, solar cells, displays, battery electrodes, corrosion-resistant coatings, and the like, for example, about 1,000 to 100,000 and it may specifically have an about 5,000 to 90,000, and more specifically a weight average molecular weight of from about 10,000 to about 6,5000 (M _w).

The polyaniline can typically be synthesized by a method of chemically polymerizing aniline monomers in an acidic medium (under agitation or comparative semi-conditions). Typically, a chemical oxidation method using an oxidant in an acidic aqueous medium containing an aniline monomer can be adopted. In this connection, as parameters affecting the polymerization reaction of the polyaniline and the properties of the final product, typically, the reaction medium, the amount of the oxidizing agent (concentration), the reaction time, and the temperature of the reaction medium can be exemplified.

According to an exemplary embodiment, the type of acid used in the acidic aqueous medium includes, for example, hydrochloric acid, sulfuric acid, nitric acid, perchloric acid,? -Naphtalenesulfonic acid, Various inorganic acids and / or organic acids (high molecular acids) such as poly (2-acrylamido-2-methyl-1-propanesulfonic acid) and poly (styrenesulfonic acid) can be used. The aniline monomer may be contained in the aqueous medium in the range of, for example, about 0.1 to 3% by weight, specifically about 0.9 to 3% by weight, more specifically about 0.9 to 1% by weight.

In certain embodiments, the molar ratio of acid: aniline monomer in the acid medium is, for example, from about 1: 0.001 to 1: 0.1, specifically from about 1: 0.01 to 1: 0.05, : 0.02. The pH range of the acidic medium may be, for example, in the range of about -1 to 7, specifically about -1 to 2, more specifically about 1 to 2. It should be noted that the numerical ranges set forth above are illustrative, and the present invention is not necessarily limited thereto.

As the oxidizing agent, there can be used, for example, ammonium persulfate, ammonium peroxydisulfate, sodium persulfate, potassium persulfate, FeCl ₃ , CuCl ₂ , hydrogen peroxide, potassium permanganate and the like. The amount of the above-mentioned oxidizing agent used may be, for example, about 1 to 100% by weight, specifically about 30 to 60% by weight, more specifically about 40 to 50% by weight, based on the weight of the aniline monomer. In addition, the polymerization reaction time may be in the range of, for example, about 1 to 60 hours, specifically about 12 to 60 hours, more specifically about 15 to 24 hours.

In the above-described embodiment, since polyaniline is insoluble in water, a precipitate of the polymer is formed as the polymerization proceeds. Although the invention is not bound to any particular theory, it can be seen that the monomer molecules first undergo nucleation and growth processes after accumulation to a certain supersaturation level, where the nuclei are instantaneously (homogeneous) in the parental phase Or in a non-uniform manner on a different kind of surface in the solution (specifically, a nanopatterned structure with a metal layer attached thereto).

In this connection, the polymerization reaction can be carried out at a temperature in the range of, for example, about 0 to 10 ° C, specifically about 2 to 7 ° C, more specifically about 3 to 5 ° C. As described above, the polyaniline produced by performing the polymerization reaction in a relatively low temperature range tends to nucleate in a heterogeneous manner, and since the interface energy between the nucleus and the substrate can be minimized, the nanopattern structure It is believed that a more rigid bond can be made on the surface.

In addition, when the polyaniline polymerization is carried out at a higher temperature, for example, at room temperature, the growth rate of the polyaniline fiber is increased, but the aggregation tendency of the formed polymer may also increase. Therefore, when the polymerization reaction is carried out at a relatively low temperature as described above, an effective polymerization reaction may occur throughout the nanostructure by slowing down the reaction rate. Particularly, the polyaniline fiber is formed between the nanofillers in the nanopattern structure Can grow uniformly.

As described above, the composite nanostructure in which the nanowire is formed in the nanopattern structure having the metal layer is different from the surface before formation of the nanowire. Specifically, when a nanoporous polyaniline nanoporous material is formed as a second polymer, the nanopattern structure with a metal layer exhibiting hydrophobicity changes in properties to exhibit hydrophilicity. For example, the water contact angle of the nanostructure before forming the nano-web is, for example, about 80 to 150 ° (specifically about 90 to 130 °), while it is about 5 to 50 ° To about 10 to 30 degrees).

In addition, the nanopillar of the nanopattern structure before the formation of the nanoparticle can be easily deformed by the physical force exerted by the external force due to a narrow space between the nanopiles. In the case of the flexible structure, And then they become entangled with each other by van der Waals force, it is difficult to maintain the original structure with stronger bonding force. However, in the case of a composite nanostructure in which a nanoweb is formed, the stiffness of the structure formed between the nanofillers increases, and as a kind of intermediate bumper layer, even if the magnitude of the external force (horizontal and / or vertical force) Even if a part of the structure of the pillar can be deformed, the entire structure can be supported at a certain level. Further, the shape or shape of the composite nanostructure formed in the middle can be kept constant.

The composite nanostructure produced as described above can be attached to the surface of an article having flexibility (for example, gloves, fibers, etc.). Particularly, it is possible to easily control the length, diameter, and spacing between the nanopillars of the nanopillar in the nanostructure during the manufacturing process, and ultimately control the shape and characteristics of the nanopillar by controlling the interval between the nanopillars .

Immobilization of pathogen using complex nano-structure

According to one embodiment, there is provided a method for contacting and fixing a pathogen to a composite nanostructure produced as described above, particularly a nanowire structure in the structure. Most of the prior art methods are methods for deriving an antimicrobial effect through a method of inhibiting the fixation or adherence of a pathogen on the surface, while in this specific example, by increasing the action of immobilizing the pathogen on the surface, the pathological agent can be qualitatively and / Way detection method.

Instead of using the physical or nonspecific fixation or capture principle, specifically the chemical method or the biochemical method of antigen-antibody by the nanobebs, the above-described method may be used for the mutual interaction of the nanosilm structure of the nanoweb structure surface and the ciliary structure of the pathogen surface By using the action, physiologists use the principle of fixing the pathogen. Furthermore, since the nanopile length, diameter and spacing between pillars can be freely controlled during the fabrication of composite nanostructures, it is possible to capture not only bacteria but also viruses of a smaller size.

According to exemplary embodiments, pathogens may be administered in a variety of ways or via a variety of mediators, e. G., Human (eg, skin) contact, liquid (eg saliva during cough) Air) to be able to be fixed in contact with the composite nanostructure. Particularly, in the case of pathogen, the propagation can be easily fixed by hand contact only because of skin spread.

Such immobilized pathogens can be detected through an amplification reaction, and can be detected using PCR techniques (DNA amplification method), NASBA (Nucleic Acid Sequence Based Amplification) technique (amplification method using isothermal conditions using RNA) Any amplification technique can be utilized. For example, the PCR method is described in Korean Patent No. 593687 and the NASBA method is exemplified in EP 0 329 822 B1 and US Patent No. 6,110,681, . In addition, as a PCR method, there are known PCR methods such as assembly-PCR, asymmetric PCR, digital PCR, endpoint PCR, inverse PCR, methylation-specific PCR, qualitative PCR, quantitative PCR Real-time PCR, reverse transcription (RT) -PCR, and the like.

The amplified products as described above can be detected by methods known in the art. For example, gel electrophoresis, enzyme-linked gel assay (ELGA), electrochromiluminescent (ECL) Can be used. However, in the above method, it is inconvenient to separately perform a detection step for each sample in order to confirm the result of the amplification reaction, so that a detection method using fluorescence may alternatively be used.

According to an exemplary embodiment, the amplified product can be detected using a labeled probe, specifically a fluorescence labeled probe. Representative examples of such fluorescently labeled probes include umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, TAMRA, Fluorescein including fluorescein, dichlorotriazinylamine fluorescein, dansyl chloride, quantum dots, phycoerythrin, FAM (fluorecein amidite) and the like, Fluorescent materials such as alexa fluor and cyanine including Cy3, Cy5, Cy7 and indocyanine green. One or more fluorescent materials can be used. However, the present invention is not limited to the above-exemplified fluorescent materials. In this regard, the fluorescent material is excited by light of a specific wavelength, and then emits light of another wavelength to emit surplus energy. Thus, a fluorescent material such as FITC emits light of a wavelength of 550 nm

In another embodiment, a detection method using fluorescence such as a molecular beacon probe or a TaqMan probe can be used. Particularly, in the case of using the biochip according to the present invention, fluorescence detection using a molecular beacon or a TaqMan probe is particularly advantageous because the sample can be concentrated, amplified and detected in a single biochip.

The molecular beacon probe is obtained by combining the 5 'end of the oligonucleotide with a fluorescent dye and the 3' end with a quencher. The oligonucleotide strand consists of a loop-type probe portion complementary to the target sequence and a self-complementary region of 5 to 6 nucleotides at both ends thereof. In the absence of a target, the complementary portion of the probe is in the form of an adjacent hairpin The phosphor and the quencher are adjacent to each other and exhibit a strong quenching effect. On the other hand, when reacting with the target, the loop-like arrangement is stretched and straightened, and the distance between the fluorescent substance and the quencher becomes distant and fluorescence is observed.

In the case of a TaqMan probe, a method of adding an oligonucleotide having a 5 'end modified with a fluorescent substance (FAM or the like) and a 3' end modified with a quencher (TAMRA or the like) to the amplification solution, wherein the TaqMan probe is specific to the template nucleic acid But fluorescence is inhibited by the quencher on the probe. During the extension reaction, the Taq DNA polymerase degrades the TaqMan probe hybridized to the template due to the activity of the 5 '→ 3' nucleic acid exonuclease. As a result, the fluorescent dye is liberated from the probe and the inhibition by the quencher is released Fluorescence. However, it may be more preferable to use molecular beacons in terms of detection specificity.

The above-mentioned probe is added to an amplification solution. When added before amplification in this way, the primer forms a competitive relationship with the probe in binding with the nucleic acid. That is, hybridization of the probe and the target nucleic acid increases non-specific binding. Therefore, when an excess amount is used, it can act as an inhibitor of the amplification reaction. On the other hand, when the amount of the probe is less than a certain level, it is difficult to expect a desired detection effect due to insufficient fluorescence characteristics as compared with the amplified nucleic acid. As a result, considering the amplification method, it is desirable to experimentally adjust the amount of probe used and other reaction conditions. In order to overcome this problem possibility, for example, by increasing the amplification reaction time, the maximum limit usage of the probe can be increased while suppressing the influence of the probe addition on the amplification reaction.

According to another embodiment of the present disclosure, there is provided a method of reducing or killing captured pathogens (i.e., removing a contact) by enhancing the action of immobilizing and trapping a pathogen on a complex nanostructure formed of a polyaniline nanoweb structure / RTI &gt; In this embodiment, when the pathogen is stably immobilized on the surface of the nanowire structure, a biochemically inducing mechanism is used. Although the present invention is not bound by any particular theory, it is believed that the positive charge of polyaniline permeates the cell membrane or induces cation exchange, disturbing the integrity of the cell membrane and inducing cell lysis.

According to a further embodiment, the pathogen can be specifically immobilized on the composite nanostructure by surface-modifying the nanoweb formed in the composite nanostructure with a specific chemical substance (specifically, a biochemical substance). As such chemical substances, (specific) antibodies corresponding to the antigens of a specific pathogen to be detected, genes derived from a specific pathogen, and the like can be exemplified. For example, when the surface of a nano-web is modified with an antibody, a specific pathogen can be selectively fixed through an antigen-antibody reaction, and subsequently it can be detected.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the present invention is not limited thereto.

Example 1

In this embodiment, the composite nanostructure was fabricated according to the procedure shown in FIG. The materials and devices used in this example are as follows:

- UV-curable adhesive: NOA 63 (Norland Optical Adhesives)

- PET film: Mitsubishi Co., Ltd. (thickness: 50 μm)

- Polyurethane acrylate (PU): MINS-311RM, product name of Minuta Tech, Japan

- Perchloric acid (HClO4): OCI, Korea (concentration: 70%)

- Aniline monomer: Sigma-Aldrich (99.5%)

- Ammonium Persulfate: Sigma-Aldrich (98%)

- Reactor: Neo 60 polypropylene box (KOMAX, Korea)

- Si wafer: product name of LG Siltron 8 inch wafer (thickness: 0.7 mm)

- E-beam evaporator: product name of Evatac Process System BAK641

Production of master mold

The Si wafer was placed in a furnace (Furnace E1200, a product of Centrotherm) to form a SiO ₂ layer with a thickness of 1500 nm. Thereafter, a photoresist (product name SKKA-8670 manufactured by Dongjin Semichem Co., Ltd.) in an amount corresponding to a thickness of 0.7 탆 was coated on a Si wafer at 3000 rpm for 60 seconds using a spin coater (product name: Kspin 8, manufactured by SEMES). Then, the surface of the Si wafer coated with the photoresist was exposed to UV light (intensity: 30 mJ / cm ² ) using a mask. Thereafter, the photoresist was removed using a developer, and then etched using ICP (product name TCP9400 SE, manufactured by Lam Research) and gas mixture (Cl2, HBr and O2). As a result, a plurality of nanoholes were formed on the Si wafer, and the diameters, depths, and nanoholes of the nanoholes were 500 nm, 1500 nm, and 500 nm, respectively.

Fabrication of nanopattern structure

A large-area nano-pillar array (nano-pattern structure) was fabricated using a Si wafer having a plurality of nano holes as a mold. Specifically, a liquid polymer blend (PUNO) was prepared by mixing polyurethane acrylate (PU) and NOA 68 at a ratio of 3: 7 (by weight) under stirring. The polymer blend was applied to the master mold by spin coating (condition: 1200 rpm for 30 seconds) to form a film having a thickness of about 100 mu m on the master mold, and a vacuum was applied to sufficiently remove the bubbles Respectively. The PET film was covered on the surface of the spin-coated PUNO polymer film and sufficiently rolled using a roller to remove air bubbles between the PET film and the PUNO film. The obtained film composite (PET film / PUNO film / master mold) was irradiated with UV of 480 mJ / cm ² intensity for 1 minute to cure the PUNO film. Then, the formed PUNO polymer layer was peeled off from the master mold to obtain a nanopattern structure, and ultraviolet light (UV) was further irradiated for 5 minutes. FIG. 5 shows a SEM photograph of the nanopattern structure manufactured as described above. As a result, the diameter, height, and spacing between the nanopillars of the nanopile formed on the nanopattern structure were 500 nm, 1500 nm, and 500 nm, respectively (aspect ratio: 1: 3).

Attachment of metal layer for surface modification

Ti was deposited to a thickness of 20 nm using an E-beam evaporator on the nanopattern structure fabricated as described above, and then Au was sequentially deposited to a thickness of 100 nm to form an Au / Ti metal layer on the nanopattern structure . The E-beam evaporator was operated in a chamber controlled at 50 ^° C and the target metal (Ti and Au) was irradiated ^specially with a laser and heated to the glass transition temperature of the corresponding metal (deposition condition: 2000 A, 1 nm thickness per 10 seconds). At this time, Ti was deposited for 200 seconds and Au was deposited for 1000 seconds.

Formation of Nano-web

A nano-web of polyaniline (PANI) was formed on the basis of a nanopattern structure having an Au / Ti metal layer attached thereto by the following procedure. The mixing and polymerization reactions involved in the formation of the nanobubbles were all performed in a refrigerator at 3 ° C.

940 mL of DI water and 60.16 mL of perchloric acid (1 M) were added to a 6 L capacity polypropylene box, and an additional 1.53 g of ammonium persulfate (0.0067 M) was added and mixed. Immediately prior to the polymerization reaction, 900 μL (0.001 M) of aniline monomer was added to the solution, and the nanopattern structure with the Au / Ti metal layer was impregnated to perform polymerization reaction for 24 hours. After the polymerization reaction was completed, the composite nanostructure was taken out and the remaining unreacted material was removed by washing with deionized water.

(B) a nanopattern structure of a PUNO material, (c) a nanopattern structure having an Au / Ti metal layer attached thereto, and (d) a nano-web of a PANI material, SEM photographs of each of the formed composite nanostructures are shown in FIG. 6A.

As shown in the figure, a nanopillar of a PUNO nanopattern structure is formed corresponding to a nanohole of a Si master mold, and an Au / Ti metal layer is attached to the surface of the nanopattern structure. Then, a polyaniline (PANI ) Nano-web structure was formed on the surface thereof. Particularly, it can be seen that polyaniline fibers (or strands) are grown from the side of the nanopillar in the composite nanostructure and are combined with the polyaniline fiber (or strand) grown from the side of the adjacent nanopillar to form a network similar to the mesh structure.

Meanwhile, an SEM photograph taken in the thickness direction of the composite nanostructure thus fabricated is shown in FIG. 6B. According to the figure, the diameter of the nanofiller itself before formation of the polyaniline nanofiller structure was about 500 nm, and the diameter of the nanofiller coated with polyaniline was about 800 nm. After the formation of the polyaniline, the depth of the nanofiller (the depth of the polyaniline grown on the bottom surface between the plurality of nanofillers) was about 1200 nm. The thickness of the polyaniline nanoweb formed between the nanopillars was about 150 nm.

Analysis of composite nanostructures

- Energy Dispersive X-ray Spectrometry (EDS) analysis on the surface

Using EDS equipment, surface EDS analysis was performed for each of (a) a nanopattern structure of PUNO material, (b) a nanopattern structure with an Au / Ti metal layer, and (c) a composite nanostructure formed of a PANI material nanoweb The results are shown in Fig. 7 together with SEM photographs.

As a result of analyzing the detected characteristic X-ray, (a) characteristic peaks of C, O and S constituting the PUNO polymer were mainly detected in the case of the nanopattern structure, and the characteristic peaks of Si originating from the master mold were detected . (b) In the case of a nanopattern structure having an Au / Ti metal layer, a characteristic peak corresponding to Au is remarkable, which means that Au is densely present on the surface of the structure. On the other hand, in the case of (c) a composite nanostructure formed with a PANI material nanoweb, characteristic peaks corresponding to nitrogen (N) originating from polyaniline constituting the nanoweb were detected in addition to Au. From the above SEM photographs and EDS analysis results, it can be confirmed that polyaniline having a nano-web structure was formed in the nanopattern structure having the surface-modifying metal layer.

- Measurement of change in water contact angle

The water contact angles of (a) the nanopattern structure, (b) the nanopattern structure with Au / Ti metal layer, and (c) the composite nanostructure with PANI nanoweb were measured using a contact angle meter, 8.

According to the figure, both the nanopattern structure made of PUNO and the nanopattern structure having the metal layer for surface modification have a water contact angle of 130 °, which means that the wettability to water is low (i.e., high hydrophobicity). On the other hand, as the polyaniline nanoweb structure is formed, the water contact angle is rapidly decreased to 20 °, which means that the wettability to water is remarkably increased (that is, the hydrophilicity is increased). From these results, it was confirmed that the surface characteristics of the nanopattern structure can be changed through the nanoweb.

- Immunity test of composite nanostructure against horizontal external force

In order to evaluate the stiffness of the composite nanostructure fabricated in this embodiment, the degree of deformation of the nanostructure generated by applying a force in the horizontal direction was observed by SEM, and the angle change of the nanopillar in the composite nanostructure with respect to the applied horizontal pressure Is shown in Fig.

As a result of the immunity test, the nano pillars arranged in the vertical direction gradually tilted as the magnitude of the horizontal external force increased. However, since the fiber structures forming the web connected to each other by growing at the side of a plurality of nano pillars mutually buffer, It can be confirmed that the basic structure is not easily collapsed even though an external force is applied.

- Immunity test of composite nanostructure against vertical external force

In order to evaluate the rigidity of the composite nanostructure fabricated in this embodiment, the degree of deformation of the nanostructure generated by applying a force in the vertical direction was observed by SEM, and the result is shown in FIG. 9B.

According to the figure, the shape of the basic nanopillar array in the composite nanostructure was maintained up to a vertical pressure of 588 kPa, and the structure of the composite nanostructure as a whole collapsed at a higher pressure of 1961 kPa. The reason why the nanofiller basic structure is maintained even at a high pressure (external force) up to 588 kPa in the vertical direction is that the nanofiber structures formed on the side of the nanofiller connect the nanofiller and the nanofiller This is due to the mutual buffering effect.

Comparative Example 1

After fabricating the nanopattern structure, a composite nanostructure was prepared in the same manner as in Example 1, except that a direct nanoweb was formed without interposing the Au / Ti metal layer.

For comparison, an SEM photograph of each of the composite nanostructure fabricated as described above and the composite nanostructure fabricated according to Example 1 is shown in FIG.

As shown in the figure, the composite nanostructure can be formed regardless of the presence or absence of a metal layer on the surface of the nanopattern structure. However, in the case where the surface modifying metal layer is attached, the metal layer plays a role as a kind of catalyst so that the formation speed of the web structure rapidly progresses over the same time, whereas when the metal layer is not attached, the growth rate of polyaniline remarkably decreases, It is expected that it will take considerable time to form the structure.

Comparative Example 2

The degree of deformation of the nanostructures generated by applying the force in the vertical and horizontal directions only to the nanopattern structure produced in Example 1 without formation of the nano-web was observed by SEM. As a result, 1200 kPa), the morphological changes of the nanopile array in the nanopattern structure are shown in FIG. 11, respectively.

According to the figure, a nanopillar arrayed in a vertical direction in a nanopattern structure having no nanoparticles is relatively easily deformed by external physical force. Especially, it was considered that it is difficult to maintain the original structure due to Van der Waals force despite the elastic characteristics of PUNO polymer after the nanopillar distance was modified to be close.

Example 2

Fixation test of complex nanostructures on pathogen

The complex nano structure fabricated in Example 1 was used to simulate pathogen propagation through human contact. For this, E. coli O157: H7 (ATCC 43895): American Type Culture Collection (Washington, DC, USA) was selected as the model pathogen and the pathogen was placed in a tube and mixed with 15 mL of Luria-Bertani (LB) 0.0 &gt; rpm &lt; / RTI &gt; for 24 hours. At this time, when the optical density (OD) value by the UV absorbance measurement method (600 nm) was 1, the culture was terminated and the culture was recovered.

Thereafter, the pathological culture was applied to the fingertips, and the contact was simulated by pressing the surface of the composite nano structure in the vertical direction, and rubbing was performed in the horizontal direction to simulate the rub. From this, SEM analysis was performed to determine whether the pathogen was immobilized and trapped on the surface of the composite nanostructure. The results are shown in FIG.

According to the figure, it can be confirmed that many cases of pathogens buried in the hand are fixed on the surface of the composite nano structure when the contact is simulated in the horizontal and vertical directions.

Amplification reaction and detection test for pathogen immobilized on complex nanostructure

As described above, the presence of a specific pathogen was confirmed by PCR analysis of the complex nano structure in which the pathogen was captured. At this time, PCR analysis was carried out through the following procedure.

The hybrid nanostructure, in which E. coli O157: H7 was immobilized as a pathogen, was cut into 3 mm- ² size and applied to PCR. Through the Primer3 program E.coli O157: PCR primer to target a gene of yliE H7 (forward primer: 5'-TCA AAT AAA TTG CTC TCA CTG AT-3 '; reverse primer: 5'-AC AGCC AGA TAC TGA TTA TTG for preparing a T-3 ') was carried out, and then, PCR using ^{SpeedSTAR TM HS DNA Polymerase (Takara Bio} INC., Shiga, Japan) products.

PCR was performed in a total volume of 25 μL and 10 μL of Fast Buffer I (20 mM Tris-HCl (pH 8.0), 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20, 0.5% Nonidet P- 2.0 μL of primer, 0.25 μL of Speed STAR HS (5 units / μL), and 1.0 μL of 0.7 mg / mL of bovine serum albumin (BSA) .

PCR temperature cycling conditions were first maintained for 5 minutes at 95 ° C for initial activation and for lysis of E. coli, followed by 5 seconds at 95 ° C, 15 seconds at 58 ° C, and 10 seconds at 72 ° C. The circulation was repeated 40 times and the final extension was performed at 72 ° C for 3 minutes to complete the gene amplification.

Next, electrophoresis patterns of PCR amplification products were analyzed using an electrophoresis apparatus (Mupid2plus), and the results are shown in FIG.

Considering that the band corresponding to the amplified E. coli O157: H7 is clearly present as shown in the figure, it can be confirmed that the complex nanostructure can be utilized as a diagnostic or detection platform of a pathogen in the future .

Example 3

One of the important considerations in the PCR analysis of a pathogen ( E. coli O157: H7 ) immobilized and captured on the complex nanostructure produced in Example 1 is the detection limit concentration. To verify this, a total of seven pathogen ( E. coli O157: H7 ) solutions were prepared by sequentially diluting 10 ⁷ cells to 10 ¹ cells 10 times, and each pathogen solution was mixed with 10 mm ² area After being placed on the surface of the nanostructure and left in the air for 30 minutes, the residue was washed with deionized water (DI water).

Thereafter, electrophoresis patterns after PCR amplification were analyzed in the same manner as in Example 2, and the results are shown in FIG.

As shown in the figure, a significant band was observed under the condition of 10 ⁷ to 10 ³ cell number / 3 mm ² . This means that signals can be detected by PCR until 10 ³ cells are present in a composite nanostructure of 3 mm ² size.

As described above, E. coli can be captured using the complex nanostructure produced in Example 1, and it can be directly applied to gene-based pathogen detection. Through the above results, it is possible to effectively fix (adhere) pathogens present on the surface of a substance that can be contacted with air or human by the composite nano structure, thereby overcoming the limitations of the conventional method of collecting pathogens However, it was confirmed that pathogen can be detected or diagnosed by directly using this in PCR.

Also, when a specific pathogen and / or virus antibody is immobilized on the surface of a composite nanostructure having a large specific surface area and brought into contact with the surface of a substance that can be exposed to air or a human body, And the possibility of diagnosing the presence or absence of specific microorganisms and the number of the microorganisms through direct gene diagnosis of the captured (fixed) pathogens or viruses can be selectively detected Respectively.

 It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (20)

A method for immobilizing a pathogen in contact with a complex nanostructure,
The composite nano-
(i) a nanopattern structure of a first polymer material having a plurality of nanopillars formed on its upper side, (ii) a metal layer for surface modification attached to the surface of the nanopattern structure, and (iii) a nanopattern structure A method comprising a nano-web structure of a second polymeric material The method of claim 1, wherein the first polymer is at least one selected from the group consisting of polyurethane (PU), polydimethylsiloxane (PDMS), NOA (Noland Optical Adhesive), and epoxy &Lt; / RTI &gt; The method of claim 1, wherein the second polymer comprises polyether ketone (PEK, PEEK, PEEKK); Polysulfone; Polyether sulfone, polyphenyl ether sulfone, polyphenylene; Polyimidazole; Polyimide, polyamideimide; Wherein at least one selected from the group consisting of polyaniline, polyacetylene, polypyrrole, and polythiophene is selected. The method of claim 2, wherein the first polymer is a blend of polyurethane acrylate (PU) and a NOA-based adhesive. 5. The method of claim 4, wherein the content of each of the polyurethane acrylate and the NOA-based adhesive in the blend is 20 to 80 wt% and 80 to 20 wt%. 2. The method of claim 1, wherein the diameters and heights of the plurality of nanofillers are in the range of 100 to 1000 nm and 100 to 1500 nm, respectively. 7. The method of claim 6, wherein the spacing between the plurality of nanopillars ranges from 100 to 3500 nm. The method according to claim 1, wherein the surface-modifying metal layer is at least one selected from the group consisting of Ni, Zn, Pd, Ag, Cd, Pt, Ga, In and Au. The method of claim 8, further comprising an intermediate layer between the surface modifying metal layer and the nanopattern structure, wherein at least one selected from the group consisting of Ti, V, Cr, Sc, Nb, . 10. The method according to claim 9, wherein the surface modifying metal layer and the intermediate layer are Au and Ti, respectively. The method of claim 1, wherein the second polymer is selected from the group consisting of polyether ketone, polysulfone, polyethersulfone, polyphenylether sulfone, polyphenylene, polyimidazole, polyimide, polyamideimide, and polyaniline (PANI) , Polypyrrole, and polythiophene. 12. The method of claim 11, wherein the second polymer is polyaniline. 13. The method of claim 12, wherein the diameter of the fibers (strands) in the nano-web structure of the second polymeric material ranges from 10 to 200 nm. 13. The method of claim 12, wherein the average mesh size of the nano-web structure of the second polymeric material ranges from 1 to 1,500 nm. The method according to claim 1, further comprising the step of specifically detecting a specific pathogen by nonspecifically detecting the fixed pathogen or modifying the complex nanostructure with a chemical substance. 16. The method according to claim 15, wherein the chemical substance is an antibody corresponding to a specific antigen present on a surface of a specific pathogen cell to be detected. A method for killing pathogens by contacting and fixing them with a composite nanostructure,
The composite nano-
(i) a nanopattern structure of a first polymer material having a plurality of nanopillars formed on its upper side, (ii) a metal layer for surface modification attached to the surface of the nanopattern structure, and (iii) a nanopattern structure And a second polymeric material nanowire structure formed on the second polymeric material. As an analysis system of a pathogen,
a) a nanopattern structure of a first polymer material having (i) a plurality of nanopillars formed on an upper side thereof, (ii) a metal layer for surface modification attached to the surface of the nanopattern structure, and (iii) A composite nano structure including a nano-web structure of a second polymer material formed on a structure;
b) an amplification device of the pathogen immobilized on the complex nanostructure; And
c) a detection device for the amplified pathogen;
/ RTI &gt; 19. The assay system of claim 18, wherein the amplification device is a PCR device. 19. The method of claim 18, wherein the detecting device is at least one of a gel electrophoresis device, an ELGA device (enzyme-linked gel assay), an ECL device (electrochromiluminescent device), and a fluorescence detecting device using a labeled probe .

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