KR20190083136A - System and Method for Capturing and Assaying Biomaterials Using Hybrid Nano-structures Coated with Zinc Oxide - Google Patents

Abstract

According to an embodiment of the present disclosure, described are a system and a method that can be used to provide a diagnostic platform, capable of easily and efficiently collecting and detecting biomaterials such as bacteria and viruses, by manufacturing and using hybrid nanostructures with improved capturing ability for biomaterials by developing wrinkled morphological topology on the surface thereof and by forming a zinc oxide layer on top to increase chemical interaction with harmful biomaterials such as bacteria while attaching at least two metal layers onto the surface of a nano-pattern structure (specifically, nanopillar array) made of a polymer material.

Description

TECHNICAL FIELD [0001] The present invention relates to a system and a method for capturing and diagnosing biomaterials using a composite nano-structure coated with zinc oxide,

The present disclosure relates to a system and a method for collecting / diagnosing biomaterial using a composite nano structure coated with zinc oxide. More particularly, the present disclosure relates to a method of depositing at least two metal layers on the surface of a polymeric nanopatterned structure (specifically, a nanofiller array), wherein a zinc oxide layer is formed as an upper layer, By increasing the interaction and developing the morphological topology of the wrinkles on the surface, it is possible to manufacture composite nanostructures with improved ability to collect bio-materials, and furthermore, to use harmful bio-materials such as bacteria and viruses And more particularly, to a system and method for providing a diagnostic platform capable of collecting and detecting at high efficiency.

Within the intestines of all warm-blooded animals, including humans, there is a very good environment in which various pathogens such as bacteria and viruses are inhabited. Pathogens are widely distributed in the surrounding environment. Specifically, bacterial pathogens are found in objects such as water kitchens, kitchen tools, desks, tableware, etc. that are contaminated by soil, animal organs and animal stools. 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 , the Escherichia coli (or E. coli ) family does not cause major problems when consumed, but some are known as pathogens that produce toxins that cause diarrhea.

In particular, E. coli O157: H7, a pathogenic Escherichia coli , has been widely known as a causative organism of food poisoning as enterohemorrhagic Escherichia coli and has become a worldwide problem. 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 paratyphoid, and can be contaminated with various livestock and animals. In addition, if adequate cleaning, processing temperatures and storage conditions are not maintained, Salmonella may multiply and cross contaminate with other foods through such contaminated drinking water, food, and the like.

Pathogens can easily infect humans through contaminated soil, water, food, and cooking environment. Under normal conditions, the propagation rate of pathogens is very fast, so that even a small number of pathogens can quickly grow in a field that is well suited to their growth environment and can threaten human health once they enter the human body. Thus, there is a need for a diagnostic technique that can quickly and easily detect the presence or absence of pathogens (particularly E. coli ) from a contaminated environment. Thus, clinical diagnostic techniques based on genetic analysis typically involve the recovery of nucleic acids from bacteria, fungi or viruses to perform amplification reactions, and then various detection means (e.g., optical , An electrochemical or mechanical biosensor device) is used to analyze the amplicon.

On the other hand, naturally occurring environmental samples can be collected from various pollutants such as drinking water, food (e.g. milk, food, etc.), drinking water, blood and the like, Is present in a trace amount. In order to analyze such a harmful bio-material, a method of measuring the concentration or amount of the harmful bio-material after the food or the like is washed and cultured in water is performed. However, since this method involves a complicated process, only an expert can conduct the analysis. For example, the time required for detection is long as the analysis is carried out with the naked eye through a culturing process of about 3 to 5 days, Is low.

Recently, H1N1 influenza virus, which is a problem with influenza virus, is contained in air and propagated. In order to collect such a hazardous bio-material in the vapor, an attachment method using a filter is generally used. In this regard, techniques have been developed (e. G., In Korean Patent No. 1243631) using a microstructure fabricated through a photomask pattern or a digital micro-mirror device process to collect bacteria and viruses. Also,

In order to capture bacteria, a soft lithography process is used and an antibody having a specific reaction characteristic is attached to the upper part of the cell, followed by ozone plasma treatment (J. Nanosci., Nanotechnol. 2017, Vol. ) Or a planar polyamide reverse osmosis (PARO) membrane is prepared in a mLbL manner, followed by performing a nanoimprinting process using a sol-gel method to produce a TiO2 pillar (ACS Appl. Mater. Interfaces 2016 , 8, 31433- 31441).

However, in the case of the above-described technique, microorganisms or viruses can be separated / collected at a certain level, but there are technical limitations in quickly analyzing or diagnosing genes. Therefore, relatively long time is required for collection and diagnosis, and complicated procedures are involved, thereby causing problems in terms of reproducibility and efficiency, and in particular, the cost of diagnosis is high.

As described above, it is possible to easily and effectively collect target biomaterials on samples containing harmful bio-materials (for example, pathogens) present in low concentrations such as environmental samples obtained in various phases, There is a need to develop a method that can do this.

In the embodiments according to the present disclosure, a method for easily and effectively collecting a biomaterial (for example, various pathogens, etc.) is provided.

In addition, a collection and diagnosis platform capable of collecting biomaterials such as food poisoning bacteria with high efficiency from various foods, food ingredients, cooking utensils and the like requiring rapid on-site detection and capable of accurately diagnosing the collected bio materials at low cost .

According to a first aspect of the present disclosure,

A method for collecting a biomaterial contained in a sample,

a) contacting a sample containing a biomaterial with a composite nanostructure having fixability to the biomaterial, and collecting the biomaterial in the sample on the composite nanostructure,

Here, the composite nano-

(i) a nanopattern structure of a polymer material having a plurality of nanopillars formed on its upper side, (ii) a surface-modifying metal layer adhered to the surface of the nanopattern structure, and (iii) a nanopattern structure There is provided a method comprising forming a zinc oxide layer of positively and corrugated morphological character.

According to a second aspect of the present disclosure,

a) contacting a sample containing a biomaterial with a composite nanostructure having a fixability with respect to the biomaterial, and collecting the biomaterial in the sample on the composite nanostructure;

b) lysing the captured biomaterial to extract nucleic acid; And

c) amplifying and detecting the extracted nucleic acid;

A method for diagnosing a biomaterial comprising:

Here, the composite nano-

(i) a nanopattern structure of a polymer material having a plurality of nanopillars formed on its upper side, (ii) a surface-modifying metal layer adhered to the surface of the nanopattern structure, and (iii) a nanopattern structure There is provided a method comprising forming a zinc oxide layer of positively and corrugated morphological character.

According to a third aspect of the present disclosure,

As a system for collecting and diagnosing bio-materials in a sample,

(i) a nanopattern structure of a polymer material having a plurality of nanopillars formed on its upper side, (ii) a surface-modifying metal layer adhered to the surface of the nanopattern structure, and (iii) a nanopattern structure A composite nanostructure comprising a zinc oxide layer of positively and corrugated morphology formed;

An amplification means for a nucleic acid derived from a bio-material in the sample captured in the complex nano structure; And

Means for detecting the amplified nucleic acid;

Is provided.

According to an exemplary embodiment, the zinc oxide layer may be formed by an ultrasonic synthesis method.

According to exemplary embodiments, the surface roughness of the zinc oxide layer may be, R _z range of 205 to 260 nm of R _q, 170 to 220 nm of the R _a, and 970 to 1100 nm.

According to an exemplary embodiment, the ultrasonic synthesis method comprises:

Providing a zinc precursor-containing solution; And

Converting the zinc precursor into zinc oxide by contacting the nanopattern structure having the surface-modifying metal layer with the zinc precursor-containing solution and irradiating the nanopattern structure with ultrasonic waves;

. ≪ / RTI >

According to an exemplary embodiment, the zinc precursor-containing solution may include hexamethylenetetramine, ethylenediamine, ammonia, polyethyleneimine, or a combination thereof as the reducing agent.

According to an exemplary embodiment, the reaction under ultrasonic irradiation may include irradiating ultrasound with an output of 20 to 200 W for 3 to 120 minutes.

According to an embodiment of the present disclosure, a system and a method for collecting and diagnosing a bio material are provided by depositing at least two metal layers on the surface of a nanoscale structure of a polymer material, that is, a nanopillar array, A zinc oxide layer is formed on the surface of the zinc oxide layer to increase the chemical interaction with a biomaterial such as bacteria by forming a zinc oxide layer on the surface of the zinc oxide layer to synthesize a zinc oxide layer by ultrasonic irradiation, By improving the spatial or morphological characteristics of the shape, it is possible to remarkably improve the ability to collect the biomolecule and secure the physico-chemical stability resulting from the underlying polymeric nanostructure.

In addition, in another embodiment of the present disclosure, sonication that is converted to zinc oxide in an aqueous phase over a short reaction time has been applied, resulting in a cost effective and cost-effective way to quickly capture biomaterials on the nanopattern structure at low cost. It is possible to form a zinc oxide coating layer having a large area, which is advantageous in terms of economy and environmental friendliness.

Therefore, it is possible to diagnose harmful pathogens in various foods and drinking water by effectively collecting or fixing biomaterials such as pathogens using a complex nano-structure, and to diagnose various direct and indirect propagation or infection pathways such as human skin contact, coughing, Can provide a flexible diagnosis method, and thus it is expected to be widely used in various fields such as clinical diagnosis, environmental monitoring, and pharmacogenetics analysis in the future.

FIG. 1A is a view showing a series of processes for manufacturing a composite nanostructure according to one embodiment of the present disclosure; FIG.
FIG. 1B is a view showing a series of processes for fabricating a nanopattern structure having a plurality of nanopiles of a polymer material; FIG.
2A to 2C are scanning electron microscope (SEM) photographs showing the surface state of the ZnO / Au-coated nanopattern structure according to the reaction time (60 minutes, 90 minutes and 120 minutes) under ultrasonic irradiation in the embodiment,
FIG. 3 is a schematic diagram of a nanopattern structure (PUNO) of a polymer material, (b) a nanopattern structure (G-PUNO) of an Au coated polymer material and a probe type ultrasonic irradiation apparatus, (ZG-PUNO) coated with a zinc oxide layer while changing the thickness of the nanopattern structure (5 minutes);
4 is a graph showing zeta potential values of a zinc oxide layer formed in an embodiment;
FIG. 5 is a cross-sectional view of a nanopattern structure (PUNO), a Au-coated nanopattern structure (G-PUNO), and a ZnO / Au-coated nanopattern structure (ZG-PUNO), respectively;
FIG. 6 is a view showing a mapping analysis result for a ZnO / Au-coated nanopattern structure (ZG-PUNO); FIG.
FIG. 7 is a cross-sectional view of a nanopattern structure (PUNO), a Au-coated nanopattern structure (G-PUNO), and a ZnO / Au-coated nanopattern structure (ZG-PUNO), respectively; Fig.
FIG. 8 is a cross-sectional view of a nanopattern structure (PUNO), a Au-coated nanopattern structure (G-PUNO), and a ZnO / Au-coated nanopattern structure (E) a PUNO structure, (f) a ZG-PUNO structure, and (g) a ZG-PUNO structure in which bacteria are captured. SEM photograph (10 ⁷ CFU / ml) of the PUNO structure;
Figure 9 is a FIB (focused ion beam) photograph showing the capture state of bacteria on the ZG-PUNO structure;
Figure 10 is a SEM image (10-10 ⁶ CFU / ml) of bacteria captured on the ZG-PUNO structure;
11 is an FT-IR spectrum for each of (a) a PUNO structure, (b) a ZG-PUNO structure and (c) a ZG-PUNO structure in which bacteria are captured;
Figure 12 is an XRD spectrum for each of (a) a PUNO structure, (b) a ZG-PUNO structure, and (c) a ZG-PUNO structure in which bacteria are captured;
FIG. 13 is a graph showing AFM analysis results and surface roughness measured from (a) PUNO structure, (b) ZG-PUNO structure and (c) ZG-PUNO structure in which bacteria are collected;
Each of Figs. 14A to 14D shows the collection state of the bacteria of the ZnO / Au-coated nanopattern structure (ZG-PUNO) formed while varying the reaction time (10 minutes, 30 minutes, 40 minutes and 50 minutes) SEM picture;
15 is an SEM photograph showing (a) S. enteritidis and (b) S. aureus as food poisoning bacteria on the ZG-PUNO structure;
16 is a graph showing the result of analysis of agarose gel electrophoresis after PCR amplification according to bacterial concentration in a sample (Lane M: Molecular Weight Marker 50 bp (Amersham, USA). Lane N: Negative Control. Lane G: G-PUNO Number: ZG-PUNO; bacterial concentration in sample: 10 ⁷ -10 ¹ CFU / ml); And
FIG. 17 is a graph showing the results of agarose gel electrophoresis analysis after PCR amplification in which a sample containing 10 ⁶ CFU / ml of bacteria was sprayed into each of a transparent vinyl glove and a rubber glove, and then contacted with the ZG-PUNO structure.

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 other layers in order to facilitate understanding, and the meaning thereof may 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 1,000 nm, specifically 1 to 100 nm) May refer to any nanoscale object, including nanopiles, nano-rods, nano-walls, nanowires, nano-webs, and the like.

"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.

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

"Biomaterial" may broadly refer to a material derived from any type of biological organism, and may specifically be comprised of a plurality of cells and need not be nucleic acids, for example, pathogens Or bacteria).

The "sample" is not limited to a particular kind or form as long as it can contain the biomaterial to be detected. Illustratively, the sample can be a biological sample, such as a biological fluid or biological tissue. Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, feces, sputum, cerebrospinal fluid, tears, mucus, amniotic fluid and the like. Biological tissues are a collection of cells, which are a collection of intracellular substances and certain types of aggregates that typically form one of the structural components of human, animal, plant, bacterial, fungal or viral constructs, connective tissue, epithelial tissue, Organizations, and so on. In addition, examples of biological tissues may include organs, tumors, lymph nodes, arteries, and individual cell (s). In addition, the sample may include an environmental sample containing a low concentration of the biomaterial, and may include various types and types such as drinking water, food, and the like.

"Cell lysis" may be a phenomenon in which cell membranes are ruptured and cell contents are exposed by decomposition of cells. Generally, they are used to separate DNA or RNA in the previous step of amplification such as PCR have.

&Quot; Nucleic acid " may mean a plurality of nucleotides consisting of a sugar linked to an organic base (a substituted pyrimidine such as cytosine, thymidine or uracil, or a substituted purine such as ardenine or guanine). That is, examples thereof include ribonucleic acid (RNA), deoxyribonucleic acid (DNA), methylphosphonate nucleic acid, S-oligo, c-DNA, cRNA, miRNA, siRNA, and aptamer, Or artificially synthesized. More typically, however, it may be DNA, RNA, or the like.

The term "probe ", as a matter of reference, may refer to a component or a biomolecule that hybridizes under appropriate conditions, including a specific base sequence and a complementary base sequence contained in the target nucleic acid, Can refer to components that interact with the target component in an optional, predetermined manner.

"Dissolution buffer or lysis buffer" means a buffer having a pH of, for example, from about 6 to about 9 at 25 DEG C and a pH value of, for example, from 6 to 9 (specifically about 7.5 to 9, ≪ / RTI > about 7.8 to about 8.5). At this time, the buffer may typically refer to a physiologically compatible buffer that is compatible with the function of the enzyme activity and can maintain the normal physiological and biochemical functions of the biological molecule.

"Bacteria" refers to the fact that the bacterial envelope is distinguished from the Gram staining reaction by its structure and is characterized by gram-negative bacteria (having a thin murein or peptidoglycan layer and a lipid bilayer of the outer membrane) Gram-positive bacteria (having a thick muraane or peptidoglycan layer and thus having a crystal violet). The membranes of gram-negative bacteria are composed of phospholipids and other glycoproteins, and most of the phospholipids are (-) negatively charged. Examples thereof include salmonella, meningococcus, spirocheta cholera, pest, typhoid, heterozygous, E. coli, and gonorrhea. On the other hand, in the case of Gram-positive bacteria, the cell wall is composed mainly of peptidoglycan and teichoic acid. The surface of the cell wall has charge characteristics close to neutrality. Staphylococcus, streptococcus, , Diphtheria bacteria, tetanus bacteria, and pneumococci.

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

"PCR" means 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 are alternately made), and the PCR mixture usually contains (i) (Iii) a mixture of dATP, dTTP, dGTP, and dCTP (in a PCR amplification process), a double stranded DNA molecule, which is a template having a double stranded DNA molecule, which is capable of binding to a complementary DNA base sequence in the template DNA, A nucleotide subunit that is joined to form a new DNA molecule), and (iv) an enzyme that synthesizes a new DNA molecule using Taq DNA polymerase (dNTP).

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.

The term "fluorescence" may mean a type of luminescence that is generated by electromagnetic excitation for a short time. That is, fluorescence means a phenomenon that occurs when a specific substance absorbs light energy at a short wavelength and then emits light energy at a longer wavelength. Illustratively, the length of time between absorption and release is typically relatively short, e.g., about 10 ^-9 to 10 ^-8 seconds.

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.

Overall disclosure

According to one embodiment, there is provided a method for collecting a zinc oxide-coated composite nano-structure by contacting the zinc oxide with a biomaterial. At this time, the coated composite nanostructure includes (i) a nanopattern structure of a polymer material having a plurality of nanopillars formed on its upper side, (ii) a surface-modifying metal layer attached to the surface of the nanopattern structure, and (iii) May comprise a positively charged, corrugated, morphological zinc oxide layer formed on the attached nanostructured structure.

Generally, the interaction between a biomaterial such as a bacteria and a solid surface involves charge-induced interaction (electrostatic bonding), hydrogen bonding, and hydrophobic interaction. On the other hand, these three forces also act on the interaction between biomaterials.

In high concentration samples, they can easily aggregate with each other by hydrophobic bonding regardless of the surface charge of the biomaterial. On the other hand, hydrogen bonding and hydrophobic interactions are reduced in low concentration samples due to the large distance between biomaterials. Therefore, in a low-concentration sample, it is necessary to increase not only the interaction between the solid surface and the biomaterial, but also the force related to the interaction between the biomaterials. In this regard, the morphological characteristics of the solid surface to which the bio material is attached can also be enhanced if the contact area, etc., increases as a physical factor affecting the attachment of the bio material. In this regard, as described below, the composite nanostructure according to one embodiment provides an effective collecting effect.

Specifically, when a sample containing a biomaterial is brought into contact with a composite nano-structure coated with zinc oxide, the biomaterial in the sample is exposed to a positive electrostatic attraction or chemical interaction with a positively charged zinc oxide layer as a surface layer of the nano- Can be increased and easily attached. Further, as a result of forming a zinc oxide layer by a method of synthesizing while interacting with a lower surface-modifying metal layer under ultrasonic irradiation, the surface topography or morphological characteristics of the wrinkles are developed on the surface thereof, Thereby providing an effect that the material can be attached more firmly. Furthermore, the physicochemical stability due to the physical properties of the nano-pillar array of the polymeric material, which is the base structure of the composite nano structure, can be secured at the same time. In addition, since the length, diameter, and spacing between the pillars of the nanopile can be freely adjusted during the fabrication of the composite nanostructure, it is possible to capture not only bacteria but also viruses of a smaller size.

Applicable biomaterials herein may typically include pathogens or pathogenic bacteria. Pathogens may include all microorganisms that enter the living organisms of animals and plants, parasitize them, cause disease, or cause harm.

According to exemplary embodiments, the biomaterials, such as pathogens, can be administered in a variety of ways or via a variety of media, including, for example, human (e.g., skin) contact, liquid (e.g. saliva during cough) For example, air), and can be collected by contact with the composite nanostructure. Particularly, in the case of a pathogen, propagation by skin transmission can be easily carried out even by hand contact.

According to the exemplary embodiment, the biosubstance, specifically the pathogen, can be detected through the subsequent amplification reaction. As such an amplification method, for example, PCR technique (DNA amplification), NASBA technique (RNA amplification), and the like, and can be detected more accurately through the amplification technique.

Hereinafter, the production and characteristics of the composite nanostructure and the collection (and detection) of the biomaterial will be described in detail.

Fabrication of nanopattern structure

FIG. 1A is a view conceptually showing a manufacturing process of a composite nano-structure coated with zinc oxide according to one embodiment of the present disclosure.

In the illustrated embodiment, the composite nanostructure has a structure in which a surface-modifying metal layer is formed on a nanopattern structure of a polymer material on which a plurality of nanopillars are formed, and a zinc oxide layer is coated on the nanopattern structure on which the surface- .

The nanopattern structure may have a plurality of nanopillars (pillar structure having nanoscale) projected on a plane, and may be made of a polymer material. At this time, the polymer can be appropriately selected in consideration of the application field of the complex 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. In addition, it may be advantageous to select and use a polymer having good physical properties such as flexibility, photosensitivity, mechanical stability, thermal stability, and chemical stability.

For this purpose, the polymer preferably has a viscosity in an uncured (fluid) state at room temperature, for example, about 300 to 5000 cps, specifically about 500 to 3000 cps, more specifically about 800 to 2000 cps Lt; / RTI > 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 polymer layer from the master mold.

According to an exemplary embodiment, at least one polymer is selected from the group consisting of polyurethane (PU), polydimethylsiloxane (PDMS), NOA (Noland Optical Adhesive), and epoxy . 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. 1B is a view showing a series of processes for manufacturing a nanopatterned structure in which a plurality of nano-pillar-shaped polymeric materials are formed 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 and polymer of the master mold 11 will typically be different, but may be the same type in some cases.

In this regard, the nanoscale hole or nano hole 12 formed in the master mold 11 has a shape and dimensions corresponding to the nanopile of a polymer material to be formed later, and may have various shapes such as circular, elliptical, And more specifically 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 1500 nm. It should be understood that the present invention is not limited to the numerical range of the nanoscale hole 12 described above and can be suitably adjusted in consideration of the material of the polymer and the use of the composite nano structure.

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 polymer (or a 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 application of the polymer layer 13, a flexible support film layer of a polymeric material having a thickness of about 1 to 500 m (specifically about 5 to 300 m, more specifically about 20 to 200 m) It may be advantageous to attach the polymeric material 14 and roll it so that the 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.

Further, considering that the rolling process performed after the application (application) of the support film layer 14 can be performed in a temperature range of typically from room temperature to 90 ^° C, it is possible to maintain the durability in this temperature range It may be advantageous to select a category. In addition, it may be advantageous to show the chemical resistance to various compounds during the formation of the zinc oxide layer.

Alternatively, in order to facilitate separation of the polymer layer 13 from the master mold 11 in the subsequent process, the release agent (for example, 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 can 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 a polymer, a polymer solution is prepared using a curing agent together with a polymer. The weight ratio of the polymer (PDMS) to the curing agent may be in a 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 (e.g., NOA 68) as the polymer is used, the master mold is spin-coated under a condition of, for example, about 1000 to 2000 rpm, As the film, for example, a PET film is attached 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 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 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, a surface-modifying metal layer is deposited on the nanopatterned structure 15 fabricated as described above.

Of course, a zinc oxide layer may be formed on the nanopattern structure to some extent without attaching such a surface-modifying metal layer, but it is more preferable that a surface-modified metal layer is interposed between the zinc oxide layer and the zinc oxide layer, Do. Although the present invention is not limited to a specific theory, such a surface-modifying metal layer not only improves adhesion to a polymer, but also improves the adhesion of the zinc oxide layer to the surface of the zinc oxide layer (for example, It is believed that it is advantageous to give a spatial characteristic or morphological characteristic, particularly a corrugated surface characteristic, favorable for attachment of a bio material.

More specifically, examples of the surface-modifying metal layer include Ni, Pd, Ag, Cd, Pt, Ga, In and Au. More specifically, Au and Ag are exemplified. (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 for use 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 nanopatterned structure using methods known in the art, thermal vapor deposition, sputtering, E-beam deposition, and the like. The thickness of the surface modifying 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 with respect to the nanopatterned structure may be at least about 70%, specifically at least about 80%, more specifically at least about 90%, and even 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 the lower nanopattern structure to the surface, an intermediate layer can be selectively interposed between the nanopattern structure and the surface-modifying metal layer in a specific embodiment (for example, a surface Modified metal layer / 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. It is considered that such a metal can form a chemical bond with polar atoms on the surface of the nanopattern structure so that the nanopattern structure of the polymer material on the lower surface and the surface-modified metal layer 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 an 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, And then heated to a glass transition temperature of the target to deposit on the nanopattern structure to be deposited. At this time, the deposition thickness may be adjusted according to the deposition time.

Formation of zinc oxide layer

According to one embodiment, a surface-modifying metal layer is formed on the nanopattern structure as described above, and then a zinc oxide layer is formed on the nanopattern structure having the surface-modifying metal layer, Fixed) surface.

As described above, the ability of the composite nanostructure to capture the bio-material in the sample is affected by the surface contacting with the biomaterial. In one embodiment, the surface contacting the bio-material has a corrugated morphology (a kind of texture characteristic) A zinc oxide layer can be formed. To this end, in one embodiment, a zinc oxide layer (or a coating layer) is formed in such a manner that a zinc precursor-containing solution (liquid medium) is converted into zinc oxide by contact with a nanopattern structure in which the surface-modifying metal layer is formed under ultrasonic irradiation. At this time, dipping, ultrasonic treatment, precipitation, hydrothermal synthesis and the like can be exemplified as a contact method, but the present invention is not limited thereto as long as it provides sufficient contact between the liquid medium and the structure.

According to an exemplary embodiment, the zinc precursor solution may contain a zinc precursor and a reducing agent in a liquid medium (solvent).

Here, the liquid medium may be an aqueous medium, for example, water, an alcohol having 1 to 3 carbon atoms (e.g., methanol, ethanol, isopropanol, etc.), or a mixture thereof. Specifically, water can be used as a liquid medium. Since water serves as a dispersion medium of a zinc precursor and also serves as a mother liquor in which zinc oxide crystal grains formed through synthesis are grown, impurities capable of affecting crystal growth are maximally Removed purified water, more specifically deionized water, can be used.

In the case of a zinc precursor, it may be a precursor soluble in a water-based medium, that is, a water-soluble zinc compound (or salt), and specifically includes zinc nitrate, zinc sulfate, zinc acetate, zinc formate, zinc (II) chloride, zinc iodide, Natrium, hydrate thereof and the like, which may be used alone or in combination. More specifically, it may be zinc acetate dihydrate, zinc nitrate hexahydrate, or a combination thereof. The concentration of the zinc precursor in the liquid medium is a factor that may affect the density of the zinc oxide layer. For example, as the concentration of zinc ions increases, the nucleation point generated on the surface of the surface-modified metal- It will increase. In view of the above, in accordance with exemplary embodiments, the concentration of the zinc precursor in the liquid medium is, for example, in the range of about 0.0005 to 1 M, specifically about 0.0025 to 0.1 M, more specifically about 0.005 to 0.01 M As shown in FIG.

As the reducing agent, for example, hexamethylenetetraamine, ethylenediamine, ammonia, polyethyleneimine, sodium hydroxide or a combination thereof may be used. More specifically, hexamethylenetetramine, ammonia, or a combination thereof may be used. By way of example, the concentration of reducing agent in the liquid medium may range, for example, from about 0.5 to 5 M, specifically about 1 to 3 M, and more specifically about 1.5 to 2.5 M.

In this regard, the molar ratio of reducing agent / zinc precursor in the zinc precursor-containing solution may range, for example, from about 1000 to 3000, specifically from about 1200 to 2000, more specifically from about 1500 to 1800.

According to an exemplary embodiment, when applying a zinc precursor-containing solution, the pH of the medium in the synthesis reaction of zinc oxide is, for example, about 9 to 12, typically about 9 to 11, more typically about 9.5 to 10, 10.5, but it can vary depending on the kind of zinc precursor and reducing agent, and the relative ratio between them.

In this regard, since the synthesis reaction mechanism of zinc oxide under irradiation with ultrasonic waves can be performed through a plurality of steps, when ammonia is used as a reducing agent, it can be converted into zinc oxide after forming an ammonium complex, have.

[Reaction Scheme 1]

On the other hand, when hexamethylenetetraamine (HMTA) is used as a reducing agent, it can be converted to zinc oxide through zinc hydroxide as shown in the following reaction formula (2).

[Reaction Scheme 2]

According to another exemplary embodiment, the base component may optionally be added, and it may act as a catalyst (in particular, a hydrolysis catalyst) during the reaction under ultrasonic irradiation. The base component that can be selectively added can be, for example, sodium hydroxide, potassium hydroxide, and the like.

In the zinc oxide synthesis reaction using the ultrasonic irradiation method, the reaction temperature may be, for example, about 20 to 50 ° C, specifically about 25 to 45 ° C, more specifically about 30 to 35 ° C. . This is distinguished from the fact that it is heated at a high temperature in the conventional hydrothermal synthesis method. While the present invention is not bound by any particular theory, cavitation effects are induced by ultrasound energy as ultrasound is applied to the precursor-containing solution, and as a result microthermal heating by the resulting cavitation hot spots leads to zinc oxide It can be converted. Specifically, cavitation is formed by repetitive processes of compression and expansion of a liquid phase when an ultrasonic wave passes through a liquid phase. When cavitation occurs, it grows under the influence of negative pressure upon expansion. When cavitation reaches a critical value in the compression process, it collapses. As the micro-bubble collapses through the formation, growth, and decay of cavitation by the ultrasonic waves, instantaneous high temperature and high pressure are formed. Therefore, even at a low temperature compared with the conventional hydrothermal synthesis method, As shown in Fig. As described above, it is possible to form a zinc oxide layer under mild system reaction conditions (especially at normal temperature and atmospheric pressure) by using an ultrasonic irradiation method.

According to exemplary embodiments, the ultrasound irradiated with the zinc precursor solution may have an output in the range of about 20 to 200 W, specifically about 30 to 170 W, more specifically about 50 to 150 W. The characteristics of the ultrasonic waves to be irradiated affect the formation of the zinc oxide layer to be synthesized. For example, when the frequency or its output is too low, the nucleation is insufficient and the reaction time is increased while the frequency or its output is excessively high It may be advantageous to properly control within the aforementioned range as long as the nucleation can exhibit a tendency to occur in a liquid medium other than on the surface of the surface-modified metal coated nanostructure. Further, in the exemplary embodiment, the reaction time under ultrasonic irradiation may be, for example, in the range of about 3 to 120 minutes, specifically about 4 to 60 minutes, more specifically about 5 to 40 minutes. In this connection, in the case where the reaction time is too long, it may be advantageous to adjust it appropriately so as to cause damage to the substrate surface.

However, the above-described reaction conditions are described for exemplary purposes. The reaction conditions include the kind of the surface-modified metal coated on the nanopattern structure, the type of the ultrasonic irradiation apparatus, the kind of the reactant (for example, zinc precursor, And the like.

On the other hand, the device required to deliver ultrasonic energy to a liquid system consists largely of three components: (i) a generator that drives the transducer assembly by converting electrical energy into high frequency alternating current, (ii) a high frequency alternating current To a mechanical vibration, and (iii) a transmission system for transmitting vibration to the liquid.

In this connection, the ultrasonic irradiation may be a vessel type (bath type) or a probe type (small note rod type).

In the case of a container type of ultrasonic irradiation, for example, an ultrasonic wave irradiation device can be fixedly disposed on one side portion of the container, for example, the lower surface, and the irradiated ultrasonic waves can be directly transmitted to the liquid medium in the container.

In the case of the container type ultrasonic irradiation apparatus, since the ultrasonic intensity applied to the surface of the ultrasonic wave irradiation apparatus and the surface-modified metal-coated nanopattern structure is relatively weak, it is possible to suppress surface damage caused by excessive ultrasonic irradiation, Will require a longer reaction time.

 On the other hand, in the case of the probe type, a relatively short reaction time is required because the emitter surface can generate a relatively high output as it is in direct contact with the liquid medium, whereas when the irradiation time is excessively long, - May damage the surface of the coated nanopattern structure.

In this regard, in the case of the container type ultrasonic irradiation method, the ultrasonic output is, for example, about 20 to 100 W, specifically about 30 to 60 W, and the zinc oxide synthesis reaction time is, for example, about 20 to 120 Min, specifically about 25 to 60 minutes. On the other hand, in the case of the probe-type ultrasonic irradiation method, the ultrasonic output is, for example, about 100 to 200 W, specifically about 120 to 170 W. The zinc oxide synthesis reaction time is, for example, about 3 to 20 minutes, Specifically about 4 to 10 minutes.

 According to exemplary embodiments, the ultrasonic wave irradiation device may be made of a flexible material such as titanium or aluminum alloy to enhance the ultrasonic wave transmission effect, and typically about 65 to 80%, specifically about 70 to 75% And the average efficiency of the ultrasonic generator may be about 80 to 90%.

According to an exemplary embodiment, the thickness of the zinc oxide layer formed as described above may be in the range of, for example, about 10 to 100 nm, specifically about 15 to 50 nm, more specifically about 20 to 30 nm. At this time, the zinc oxide layer may exhibit a surface roughness range as shown in Table 1 below:

division Surface roughness value R _q (nm) For example about 205-260, specifically about 210-250,
More specifically from about 220 to 240 R _a (nm) For example, about 170-220, specifically about 180-215,
More specifically about 190 to 210 &lt; RTI ID = 0.0 &gt; R _z (nm) For example, about 970-1100, specifically about 980-1070,
More specifically from about 990 to 1050

The above-described surface roughness is remarkably increased compared with that before each of the surface-modifying metal layer formation (the nanopattern structure of the polymeric material) and the surface-modified metal-coated nanopattern structure. This is because zinc oxide is synthesized on the surface of the surface modification layer by ultrasonic irradiation to form a layer having corrugated morphological characteristics.

Collecting bio-materials in the sample

According to one embodiment of the present disclosure, a biomaterial (such as bacteria) can be collected using the zinc oxide-coated composite nanostructure produced as described above.

These biomaterials may include Gram-positive bacteria and Gram-negative bacteria, and specifically, E. coli O157: H7 , Salmonella choleraesuis , Salmonella bongori , Salmonella typhimurium , Staphylococcus aureus, Listeria monocytogenes , Listeria denitrificans , Listeria grayi , Listeria murrayi ), cholera, erythritis , pertussis, diphtheria, typhoid, pest, hemolytic streptococcus or staphylococcus aureus, and more particularly E. coli O157 : H7 , Salmonella choleraesuis , Salmonella bongori , Salmonella typhimurium , Listeria monocytogenes , Listeria denitrificans , Listeria grayi , Listeria murrayi, S. enteritidis, Y. enterocolitica, S. aureus, B. cereus , L. monocytogenes and the like.

Examples of the virus include influenza virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, hepatitis G virus, HIV virus, yellow fever virus, dengue virus, Japanese encephalitis virus, tick- Leukocyte or West Nile virus, Rift Valley or Tuscan virus, Chichungnia virus, respiratory syncytial virus, rossiovirus, measles virus, Murray encephalitis virus, Besselsbron virus, Zika virus, lymphocytic mucositis choreomeningitis virus, Ebola virus, Marburg virus, Crimea-Congo hemorrhagic fever virus, Lisa virus, Junin virus, Machupo virus, Sabia virus, Guanarito virus, Mumps virus, rabies virus, Lu Rubella virus, varicella zoster virus, herpes simplex 1 and 2 virus, more typically alphavirus, adenovirus, eco virus, rotavirus, flavivirus, linovirus, orthobunyavirus, A virus, a human parvovirus, an enterovirus, a coronavirus, a human papillomavirus, a human cytomegalovirus, an Epstein-Barr virus, a parainfluenic virus of type 1, 2 and 3, or any confirmed virus . It should be understood that the types listed above are illustrative, and are not necessarily limited to specific types.

In particular, in this embodiment, instead of using the biochemical method of the antigen-antibody, the electrostatic attraction between the biomaterial and the zinc oxide layer and / or the corrugated morphological characteristics of the surface of the composite nanostructure (i.e., the surface of the zinc oxide layer) The principle of physically fixing the biomaterial using the interaction with the ciliary structure on the surface of the biomaterial such as a pathogen can be used. Furthermore, since the nanopillar length, diameter and spacing can be controlled during the fabrication of composite nanostructures, not only bacteria but also smaller viruses can be trapped and detected.

Diagnosis of captured biomaterials

In this way, a lysis step can be performed for nucleic acid extraction of the biomaterial (specifically, bacteria) captured on the composite nanostructure. In this connection, various methods of melting known in the art can be applied so that the collected biomaterial under controlled temperature conditions is heated at about 70 to 95 캜 (specifically about 80 to 90 캜). A method of alternately performing pulse on and pulse off using an ultrasonic wave sonicator or a method of using a lysis buffer can be applied.

According to a specific embodiment, a nucleic acid extraction method using a lysis buffer can be used. Examples of buffer components that can be added to such a dissolution buffer include HEPES ((4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid), MOPS (3- (N-morpholino) Tris (hydroxymethyl) methylamine acid (Tris), piperazine-N, N'-bis (2-ethanesulfonic acid) (PIPES) And an acetate or phosphate-containing buffer (K ₂ HPO ₄ , KH ₂ PO ₄ , Na ₂ HPO ₄ , and / or NaH ₂ PO ₄ ), or combinations thereof, but the present invention is not necessarily limited thereto. The concentration of the buffer component in the lysis buffer may be, for example, in the range of about 3 to 30 mM, specifically about 5 to 20 mM, more specifically about 6 to 10 mM, Alternatively, the lysis buffer can be a protease K, trypsin, chymotrypsin, elastase, streptogel The may further contain a protease to be used for nucleic acid extraction in the art, such as new. Illustratively according to the embodiment, to the nucleic acid extract, a commercially available G-spin ^™ nucleic acid extraction kit, easy-spin ^™ nucleic acid extraction kit, A Pathogen-spin ^™ nucleic acid extraction kit, and the like.

In one embodiment, the dissolution temperature of the collected biomaterial can be adjusted to an appropriate range in consideration of the kind of enzyme used, for example, about 30 to 95 캜 (typically about 40 to 80 캜, more typically About 55-72 &lt; 0 &gt; C). The temperature range is described for illustrative purposes only, and the present disclosure is not limited thereto.

After the dissolution process of the captured biomaterial, an amplification reaction is performed on the nucleic acid extracted according to a method known in the art. For the amplification reaction, any amplification technique known in the art can be utilized such as PCR (DNA amplification method), NASBA (Nucleic Acid Sequence Based Amplification (amplification method using isothermal conditions using RNA), etc.).

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, . Examples of PCR methods include 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 of 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 can form a competitive relationship with the probe in binding with the nucleic acid. That is, hybridization of the probe and the target nucleic acid may increase 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 exemplary embodiment, an insertion dye may be added to the amplification mixture prior to or during the amplification step. At this time, when the DNA is amplified as a nucleic acid, the insertion dye is inserted between the double strand DNA (dsDNA), and the degree of amplification can be confirmed by measuring the intensity according to the detection of fluorescence later. In this regard, the intercalating dye is a dye that can be inserted into the double strand in the amplification process, as SYBR Green, PicoGreen, Hoechst series, BOBO, TOTO, YOYO, JOJO, POPO, LOLO, PO- -PRO, TO-PRO, JO-PRO, LO-PRO, SYTO series, etc., and they can be used singly or in combination. Typically, SYBR Green, TOTO-3 of the TOTO series, POPO-3 of the POPO series, and BOBO-3 of the BOBO series can be used. The present invention is not necessarily limited to the above-described kind, and various types may be used as long as they can be inserted into the double strand during the amplification reaction and the fluorescence characteristic derived therefrom can be detected. The amount of the intercalating dye to be added can be appropriately adjusted in consideration of the characteristics of the intercalating dye and the like, and may be, for example, in the range of about 0.1 to 20 μM% by weight (specifically, about 1 to 5 μM). However, the content of the present disclosure is not limited to the above numerical range.

As described above, fluorescence derived from an amplification product can be detected by, for example, a confocal microscope, a fluorescence microscope, a fluorometer, a fluorescence scanner, a flow cytometer ). The presence and concentration of the target nucleic acid can be analyzed in consideration of the presence or absence of fluorescence and the intensity of the fluorescence. According to an exemplary embodiment, for fluorescence detection, for example, a laser-induced confocal fluorescence microscope can be used. Examples of such a device include QuantaMaster PTI spectrofluorimeter (PTI), SPEX Fluorolog 3 (ISA), and the like, as long as it is capable of confirming the presence or absence of fluorescence and the change in fluorescence intensity according to the presence of nucleic acid (target nucleic acid) can do.

The detection limit (LOD) of the diagnostic system according to the exemplary embodiment may, for example, be in the range of about 10 to 10 ⁶ CFU / ml, but is not limited thereto.

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 example, a composite nanostructure was prepared according to the following procedure (see FIG. 8). 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

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

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

- Zinc acetate dihydrate: Sigma-Aldrich (99%)

- Sputtering equipment: Custom made by Ultech.

- Scanning electron microscope: Nova230 (FEI company)

In addition, the samples used in the examples were prepared as follows.

- Bacterial Containing Samples

E. coli O157: H7 (ATCC 43895: American Type Culture Collection (Washington, DC, USA)) was selected and the pathogen was placed in a tube and stirred at 37 ° C with 15 mL of Luria-Bertani 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.

A. Fabrication of zinc oxide coated nanopattern structures

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, SKKA-8670, a product of Dongjin Semichem Co., Ltd., was applied as a photoresist corresponding to a thickness of 0.7 탆 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 (Cl ₂ , HBr and O ₂ ). As a result, a plurality of nanoholes were formed on the Si wafer, wherein the diameter, depth, and spacing between the nanoholes were 500 nm, 1000 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.

As a result of analysis, the diameter, height, and spacing between the nanopillars formed on the nanopattern structure (i.e., the nanopatterned film-like structure) were 500 nm, 1000 nm, and 500 nm, respectively.

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 formed as described above, and then Au was sequentially deposited to a thickness of 200 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 (G-PUNO structure).

Formation (coating) of zinc oxide layer

- Formation of zinc oxide layer using ultrasonic irradiation device of vessel type

Using a container-type ultrasonic irradiation apparatus (Bandelin), a precursor aqueous solution containing 0.01 M of zinc acetate dihydrate as a precursor and 1.57 M of ammonia as a reducing agent, based on a nanopattern structure (G-PUNO) And the reaction was carried out by dipping under an ultrasonic condition of 50 W.

At this time, a zinc oxide (ZnO) layer was formed by changing the reaction time as shown in Table 2, and then washed with deionized water to obtain a zinc oxide-coated nanopattern structure (ZG-PUNO structure).

Ultrasonic output Reaction time 50 W 10 minutes 50 W 30 minutes 50 W 40 minutes 50 W 50 minutes 50 W 60 minutes 50 W 90 minutes 50 W 120 minutes

As a result of observation under an optical microscope, the surface of the substrate was hardly damaged within the reaction time range shown in the above table. As a result of analysis using AFM (Atomic Force Microscope), the thickness of the zinc oxide layer formed through various reaction times (30 minutes, 40 minutes, 50 minutes, and 60 minutes) was in the range of 20 to 30 nm.

In this connection, a scanning electron microscope (SEM) photograph showing the surface state of the ZnO / Au-coated nanopattern structure according to the reaction time (60 min, 90 min and 120 min) under ultrasonic irradiation is shown in each of Figs. Respectively.

According to the figure, a smooth curved zinc oxide layer was formed by wrinkling over the surface of the base structure by the reaction under ultrasonic irradiation, and the corrugated characteristics of the zinc oxide layer increased as the reaction time was longer.

- Formation of zinc oxide layer using probe type ultrasonic irradiation device

A zinc oxide layer was formed using a probe-type ultrasonic wave irradiation apparatus (VC-750 ultrasonic processor (Sonics @ materials, USA)) using the same precursor aqueous solution as described above, and ZG-PUNO structures were obtained Respectively. The nano-pattern structure (PUNO) of the polymer material, the nanopattern structure of the Au coated polymer (G-PUNO) and the reaction time (1 minute, 2 minutes and 5 minutes) SEM photographs of each of the pattern structures (ZG-PUNO) are shown in FIG.

As shown in the figure, when the reaction time was 1 minute and 2 minutes, the zinc oxide layer was not formed on the entire top surface of the nanopile (nano pillar), but when the reaction time was 5 minutes, the zinc oxide layer was formed as a whole.

B. Characterization of zinc oxide-coated nanopattern structure (ZG-PUNO)

The zinc oxide-coated nanopattern structure (ZG-PUNO) prepared by reacting with a container-type ultrasonic irradiation apparatus for 30 minutes was evaluated as follows.

- zeta potential measurement

The zeta potential of the zinc oxide-coated nanopattern structure (ZG-PUNO) prepared by reacting with a vessel type ultrasonic irradiation apparatus for 30 minutes was measured. The results are shown in FIG. According to the figure, it can be seen that the surface of the zinc oxide layer shows positive charge.

- XRD analysis

(A) polymer nanopattern structure (PUNO), (b) Au-coated nanopattern structure (G-PUNO) and (c) ZnO / Au-coated nanopattern structure (ZG-PUNO) The results are shown in Fig.

According to the figure, in the case of G-PUNO, a characteristic peak for gold (Au) was observed together with PUNO. In the case of ZG-PUNO, characteristic peaks for PUNO, gold (Au) and zinc (Zn) were all observed, but the peak intensity for gold (Au) Is coated.

- Mapping analysis

Mapping analysis was performed on the ZG-PUNO structure, and the results are shown in FIG.

According to the figure, it can be seen that gold (Au) and zinc (Zn) components are uniformly distributed on the surface of the nanopillar in the PUNO structure.

- FT-IR analysis

PUNO structure, G-PUNO structure, and ZG-PUNO structure, respectively. The results are shown in FIG.

- Surface roughness measurement

Surface roughness was measured for each of PUNO structure, G-PUNO structure and ZG-PUNO structure using AFM (atomic force microscopy). The results are shown in Table 3 below.

division PUNO G-PUNO ZG-PUNO R _q (nm) 182.3 203.7 227.1 R _a (nm) 157.6 170.5 194.4 R _z (nm) 384.4 967.3 1035

According to the table, the surface roughness of the ZG-PUNO structure was highest, which is attributable to the result that the zinc oxide was coated in a corrugated form on the nanopattern structure to enhance the texture property.

C. Collection of Bacteria

- Collection of E. Coli

Bacteria-containing samples prepared at a distance of 5 cm from the ZG-PUNO structure were sprayed back and forth over 3 seconds each (6 seconds total) and then dried at room temperature for 1 hour to collect the bacteria in the sample ( E. coli ) (10 ⁷ CFU / ml). In this regard, FIG. 8 is a drawing showing a series of processes for collecting bacteria using (a) PUNO structure, (b) G-PUNO structure, (c) ZG-PUNO structure, and (d) ZG-PUNO structure , And (e) PUNO structure, (f) ZG-PUNO structure, and (g) ZG-PUNO structure in which bacteria are captured.

FIB analysis was performed on whether or not the bacteria were collected, and the results are shown in FIG. In addition, a SEM image of food poisoning bacteria on the ZG-PUNO structure at various bacterial concentrations (10-10 ⁶ CFU / ml) in various samples is shown in FIG.

In addition, the FT-IR spectrum, XRD spectrum, and AFM analysis results of the PUNO structure, the ZG-PUNO structure and the ZG-PUNO structure in which the bacteria were collected, and the surface roughness measured therefrom are shown in FIGS. 11 to 13, respectively .

Considering the results according to the above figures, it can be seen that the bacteria in the sample are effectively trapped on the surface of the ZG-PUNO structure. In particular, referring to Figure 13, increased surface roughness after formation of the zinc oxide layer was somewhat reduced after bacterial entrapment.

The collection state of bacteria of ZnO / Au-coated nanopattern structure (ZG-PUNO) formed by varying the reaction time (10 min, 30 min, 40 min and 50 min) under ultrasonic irradiation was analyzed by SEM, The results are shown in Figs. 14A to 14D. According to the figure, ZG-PUNO structure formed by reacting under ultrasonic irradiation for 30 minutes is relatively advantageous in terms of nano pillar shape retention and bacterial trapping.

- Collection of S. enteritidis and S. aureus

A bacterial capture test was performed in the same manner as in E. coli except that S. enteritidis and S. aureus were used as food poisoning bacteria, and the results are shown in FIG.

According to the figure, the ZG-PUNO structure exhibited a trapping ability for all two bacteria to be trapped, and the trapping effect for S. aureus was particularly good.

D. Amplification and Detection of Collected Bacteria

Bacteria were captured on the ZG-PUNO structure with varying bacterial concentrations in the bacterial sample (bacterial concentration in sample: 10 ⁷ -10 ¹ CFU / ml). The collected bacteria were subjected to cell lysis using a G-spin ^™ nucleic acid extraction kit to extract nucleic acids. Each of these was subjected to amplification reaction by PCR using i-StarMAX ^™ , and electrophoresis analysis was performed on the amplified products. To prepare agarose gel (2%) for electrophoresis analysis, 100 mL of 1X TAE buffer and 2 g of Agarose LE (intron, Cat No. 32034) were mixed and dissolved in a microwave oven. The fluorescent dye (red safe) was added, mixed and then poured into a gel plate to solidify.

The electrophoresis gel experiment was carried out by mixing 6 μl of the amplified product sample by PCR and 1 μl of the 6 × loading buffer, loading 5 μl into the 2% agarose gel prepared above, and applying the gel for 20 minutes on the electrophoresis apparatus And then UV was measured. The results are shown in Figure 16 (Lane M: Molecular Weight Marker 50 bp (Amersham, USA). Lane N: Negative Control. Lane P: PUNO Lane G: G-PUNO Number: ZG-PUNO).

According to the figure, when the detection platform according to the present embodiment was applied, bacteria could be effectively detected even at a low concentration of bacteria-containing sample (10 CFU / ml).

On the other hand, bacterial-containing samples (concentration of bacteria: 10 ⁶ CFU / mL) were sprayed on each of the transparent vinyl gloves and rubber gloves, contacted with the ZG-PUNO structure, and analyzed by agarose gel electrophoresis after PCR amplification. The results are shown in Fig.

According to this figure, ZG-PUNO structures can effectively detect bacteria even when carrying out direct contact with two kinds of gloves.

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 collecting a biomaterial contained in a sample,
a) contacting a sample containing a biomaterial with a composite nanostructure having fixability to the biomaterial, and collecting the biomaterial in the sample on the composite nanostructure,
Here, the composite nano-
(i) a nanopattern structure of a polymer material having a plurality of nanopillars formed on its upper side, (ii) a surface-modifying metal layer adhered to the surface of the nanopattern structure, and (iii) a nanopattern structure &Lt; / RTI &gt; wherein the zinc oxide layer comprises a positively, corrugated morphological zinc oxide layer. a) contacting a sample containing a biomaterial with a composite nanostructure having a fixability with respect to the biomaterial, and collecting the biomaterial in the sample on the composite nanostructure;
b) lysing the captured biomaterial to extract nucleic acid; And
c) amplifying and detecting the extracted nucleic acid;
A method for diagnosing a biomaterial comprising:
Here, the composite nano-
(i) a nanopattern structure of a polymer material having a plurality of nanopillars formed on its upper side, (ii) a surface-modifying metal layer adhered to the surface of the nanopattern structure, and (iii) a nanopattern structure &Lt; / RTI &gt; wherein the zinc oxide layer comprises a positively, corrugated morphological zinc oxide layer. According to claim 1 or 2, wherein the surface roughness of the zinc oxide layer is characterized in that R _z range of 205 to 260 nm of R _q, 170 to 220 nm of the R _a, and 970 to 1100 nm. The method according to claim 1 or 2, wherein the zinc oxide layer is formed by an ultrasonic synthesis method. 4. The method according to claim 3,
Providing a zinc precursor-containing solution; And
Converting the zinc precursor into zinc oxide by contacting the nanopattern structure having the surface-modifying metal layer with the zinc precursor-containing solution and irradiating the nanopattern structure with ultrasonic waves;
&Lt; / RTI &gt; 6. The method of claim 5, wherein the zinc precursor-
As the solvent, water, an alcohol having 1 to 3 carbon atoms or a mixture thereof,
As the zinc precursor, zinc nitrate, zinc sulfate, zinc acetate, zinc formate, zinc (II) chloride, zinc iodide, zinc acetylacetonate, its hydrate or a combination thereof, and
Wherein the reducing agent comprises hexamethylenetetramine, ethylenediamine, ammonia, polyethyleneimine, or a combination thereof. 7. The method of claim 6, wherein the concentration of the zinc precursor in the zinc precursor-containing solution is in the range of 0.0005 to 1 M and the concentration of the reducing agent is in the range of 0.5 to 5 M. 8. The method of claim 7, wherein the molar ratio of reducing agent / zinc precursor in the zinc precursor-containing solution is in the range of 1000 to 3000. The method according to claim 5, wherein the reaction under ultrasonic irradiation is performed at 20 to 50 ° C. 6. The method according to claim 5, wherein the reaction under ultrasonic irradiation comprises irradiating ultrasonic waves having an output of 20 to 200 W for 3 to 120 minutes. 11. The method according to claim 10, wherein the reaction under ultrasonic irradiation comprises:
Is performed for 20 to 120 minutes under ultrasound irradiation with a power of 20 to 100 W using a container type ultrasonic irradiation apparatus, or
Characterized in that the method is performed for 3 to 20 minutes under ultrasonic irradiation at a power of 100 to 200 W using an ultrasonic irradiation apparatus of probe type. The method according to claim 1 or 2, wherein the thickness of the zinc oxide layer is in the range of 10 to 100 nm. The method of claim 1, wherein the 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 diameter and height of the plurality of nanofillers are in the range of 100 to 1000 nm and 100 to 1500 nm, respectively, and the spacing between the plurality of nanofillers is in the range of 100 to 3500 nm. The method of claim 1, wherein the surface-modifying metal layer is at least one selected from the group consisting of Ni, Pd, Ag, Cd, Pt, Ga, In and Au. 16. The method of claim 15, 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, Mo and W is selected. As a system for collecting and diagnosing bio-materials in a sample,
(i) a nanopattern structure of a polymer material having a plurality of nanopillars formed on its upper side, (ii) a surface-modifying metal layer adhered to the surface of the nanopattern structure, and (iii) a nanopattern structure A composite nanostructure comprising a zinc oxide layer of positively and corrugated morphology formed;
An amplification means for a nucleic acid derived from a bio-material in the sample captured in the complex nano structure; And
Means for detecting the amplified nucleic acid;
&Lt; / RTI &gt; 18. The collection and diagnostic system of claim 17, wherein the detection limit (LOD) of the capture and diagnostic system is between 10 and 10 &lt; ⁶ &gt; CFU / ml. (i) a nanopattern structure of a polymer material having a plurality of nanopillars formed on its upper side,
(ii) a surface-modified metal layer attached to the surface of the nanopattern structure, and
(iii) a zinc oxide layer formed on the nanopattern structure with the surface-modifying metal layer attached thereto, the zinc oxide layer having a positively charged and corrugated morphology;
&Lt; / RTI &gt; 20. The method of claim 19, wherein the surface roughness of the zinc oxide layer, a composite nano-structure, characterized in that R _z range of 205 to 260 nm of R _q, 170 to 220 nm of the R _a, and 970 to 1100 nm.

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