KR20190083192A - System and Method for Capturing and Assaying Hazardous Biomaterials Using Hybrid Nano-structures - Google Patents

Abstract

One embodiment of the present disclosure describes a system and method for conveniently and accurately collecting and diagnosing biomaterials in a sample, by detecting fluorescence signals by cleavage of probes that are responsive to nucleases released by the cell lysis after efficiently capturing biomaterials such as various pathogens and infectious microorganisms on a hybrid nanostructure, wherein a nanoweb of a second polymer material is formed on a surface of a nanopillar array of a first polymer material.

Description

TECHNICAL FIELD [0001] The present invention relates to a system and a method for capturing and diagnosing harmful biomaterials using a composite nano structure,

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a system and a method for collecting and diagnosing harmful biomaterials using a composite nanostructure. More particularly, the present disclosure relates to a method for efficiently and efficiently removing harmful biomaterials such as various pathogens and infectious microorganisms on a composite nano structure having nanoparticles of a second polymer material formed on the surface of a nanopillar array of a first polymer material Capturing, lysing and releasing nuclease, detecting fluorescence signal by cutting of probe having responsiveness to released nuclease, and collecting and diagnosing biomaterial in sample easily and accurately And more particularly,

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, it can be performed only by an expert. For example, the time required for detection is long as it is analyzed by visual observation through a culturing process of about 3 to 5 days, Have defects

On the other hand, in order to diagnose an environmental sample containing harmful bio-substances such as E. coli , ATP measurement method using the principle that ATP (adenosine triphosphate) and luciferin / luciferase react and emit is applied .

Luciferin, which is a luminescent substance, is activated by reacting with ATP and then oxidized by oxygen under the action of luciferase, which is a luminescent enzyme, as shown in Reaction Scheme 1 below, converting chemical energy into light energy, Release.

[Reaction Scheme 1]

That is, luciferin, which is a luminescent substance, forms two inorganic phosphoric acid (H ₃ PO ₄ ) molecules while binding to ATP to form a complex of luciferin-ATP, in which luciferin is in a reduced form. The reduced form of luciferin-AMP produced in the reaction is oxidized and reacted with oxygen to be in an unstable energy state. Therefore, the oxidized product in an unstable state is rapidly decomposed to generate light ( hv ) while producing oxidized luciferin and AMP do. At this time, the quantity of light emitted can be measured and quantified, and ATP corresponds to an indicator indicating the presence of an organism. That is, when microorganisms are present, ATP, which is essentially contained in the cells, is also present, so that light can be emitted. Through the ATP measurement method, it is possible to perform a rapid diagnosis work by reducing a series of processes from ATP erase, ATP extraction and luminescence measurement.

The ATP method has the advantage of detecting not only microorganisms but also other organic substances. However, since a small amount of ATP is contained in a sample such as food, it is necessary to remove ATP derived from food or the like for accurate diagnosis, - Only the derived ATP should be measured. As such, the APT measurement method itself has a fundamental limitation in collecting only harmful bio-materials and performing accurate diagnosis. Particularly, when the concentration of the target biomaterial to be diagnosed in the sample is low, the difficulty of accurate detection and diagnosis can be further exacerbated.

Therefore, in order to accurately diagnose the environmental sample, it is required to effectively recover the nucleic acid of the sample in the sample and to develop a diagnostic platform that can accurately diagnose the nucleic acid.

Generally, nucleic acids of biomaterials are chemically extracted from cell lysates using phenol, chloroform, guanidinium thiocyanate, and the like. However, chemical methods are difficult to apply because they require complicated steps requiring the use of toxic chemicals and skilled techniques. In order to recover the nucleic acid, a solid phase extraction (SPE) has been used in the past, but a large number of complicated processes such as centrifugation, voltexing and heating in an SPE-based nucleic acid extraction process And thus can not meet the demand for point-of-care testing of infectious diseases.

Recently, research is underway to develop polymers capable of interacting with nucleic acids by ion exchange mechanism or affinity. For example, there has been proposed a method of collecting DNA using charge interaction based on chitosan microparticles, but there are still limitations due to the use of uneven particles and expensive equipment. In addition, a technique for synthesizing and coating a catechol-based polymer on a variety of substrates to fix the DNA was proposed, and a multi-layer structure of poly (2-oxazoline) was formed on a plastic substrate to efficiently extract DNA from the cell lysate And performing genetic diagnosis using RT-PCR (real-time PCR) techniques. However, in these methods, a nucleic acid can be extracted to a certain extent using a synthetic polymer, but it takes a comparatively long time to synthesize a polymer, and also involves complicated procedures, resulting in problems in terms of reproducibility and efficiency. Further, in order to carry out the PCR, it is required to use various reagents used for the controlled temperature regulation and amplification reaction, so that the cost for diagnosis is relatively high.

In recent years, various kinds of infectious microorganisms are propagated and contaminated from human hands, cooking utensils, food surfaces due to infectious harmful bio-materials, and secondary contamination occurs in the process of consuming or consuming food Sometimes happens. Considering the above points, various micro or nanostructures have been applied to collect the infectious microorganisms contained in the surface or the solution, and studies are underway to analyze them. However, in the process of pressing and rubbing various types of surfaces, Is broken or damaged, making analysis difficult.

Accordingly, it is an object of the present invention to solve the problems of the prior art, and to easily and effectively collect target biomaterials even for samples containing harmful biomaterials (for example, pathogens) present in low concentrations such as environmental samples, There is a need to develop a method that can do this.

In an embodiment according to the present disclosure, a method for collecting and diagnosing a bio-material (for example, a harmful bio-material such as various pathogens) by a direct physicochemical method (or fixing) is provided.

Also, in accordance with embodiments of the present disclosure, a composite nanostructure platform having an effective binding (immobilization or trapping) property to a biomaterial can be used to provide a biomaterial with a biomaterial without additional equipment, subsequent amplification processes or reagents And a smart diagnostic method capable of easily detecting a smart diagnostic method.

In addition, it is intended to provide a collection and diagnosis platform capable of collecting harmful biomaterials such as food poisoning bacteria with high efficiency from various foods, food ingredients, cooking utensils and the like requiring rapid on-the-spot detection and quickly and accurately diagnosing them.

According to a first aspect of the present disclosure,

a) contacting a sample containing a biomaterial with a composite nanostructure having fixability to the biomaterial to collect an increased density of the biomaterial on the composite nanostructure relative to the sample;

b) lysing the captured biomaterial;

c) contacting a cell lysate of the biomaterial with a probe in a quenching state and cleaving the chain in the probe by a nuclease released by the lysis of the cell, A nucleic acid probe having responsiveness to a clase, comprising a phosphor and a quencher at each end connected by the chain, wherein color development of the phosphor is suppressed by the action of a quencher prior to cleavage of the chain;

d) performing at least one of quantitative analysis and qualitative analysis on the fluorescence generated from the liberated fluorescent material according to cleavage of the probe;

/ RTI >

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 of collecting and diagnosing a bio-material in a sample including a nano-web structure of a second polymer material is provided.

According to a second aspect of the present disclosure,

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

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) a nucleic acid probe having responsiveness to a nuclease derived from the biomaterial, wherein the probe comprises a fluorescent substance and a quencher at each end connected by a chain, and before the chain is cleaved by a nuclease, The coloring of the phosphor is suppressed by the action of the optical body; And

c) an apparatus for detecting at least one of fluorescence and fluorescence intensity (or fluorescence amount) of a phosphor liberated from the probe by cutting the chain;

A collection and diagnosis system is provided.

According to an exemplary embodiment, the probe may be a DNA strand of a double strand.

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.

The system and method for collecting and diagnosing bio-materials provided in accordance with embodiments of the present disclosure provide a three-dimensional fiber-like structure to provide topographical interactions with surfaces of biomaterials such as bacteria To provide an effective selective fixing force against a target biomaterial contained in a sample (specifically, an environmental sample) without special surface treatment, and to use a probe having responsiveness to nuclease released from cell dissolution of a target biomaterial It is possible to realize a simple and accurate quantitative and / or qualitative diagnostic system with good signal characteristics for background noise.

In particular, the present invention proposes a method for easily and accurately detecting biomaterials without requiring additional steps (e.g., washing or purification steps) or devices and / or chemicals in the diagnosis process for biomaterials. In particular, can be applied effectively with respect to the sample, and further the harmful bio-material containing the biomaterial with a low concentration (e. G., Such as various pathogens causing food poisoning), such as environmental samples - in-situ (in-situ) And provides a new platform for detection. Therefore, it is expected to be widely used in various fields such as clinical diagnosis, environmental monitoring, and pharmacogenetics analysis in the future.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view showing a principle of trapping and diagnosing a biomaterial using a nuclease-responsive probe derived from a composite nanostructure-based film and a biomaterial produced according to one embodiment of the present disclosure,
Fig. 2 is a diagram showing an example of a combinable range of a phosphor and a quencher to be attached to the end of a probe for detecting a bio material in an exemplary embodiment,
3 is a conceptual view illustrating a process of fabricating a composite nanostructure formed with a nanowire according to one embodiment of the present disclosure;
4 is a view showing a series of processes for fabricating a plurality of nanopillar-shaped nanopattern structures of a first polymer material according to an exemplary embodiment of the present disclosure;
FIG. 5 is a graph showing the principle of nanofiller nanofilaments formed on a nanopattern structure of a first polymer material to which a surface modifying metal layer is attached according to one embodiment of the present invention, Fig.
FIG. 6 is a view schematically showing a sequence of steps for fabricating a composite nanostructure according to Embodiment 1; FIG.
7 is a graph showing the appearance of each of the nanopattern structure of (a) PUNO material, (b) the nanopattern structure having Au / Ti metal layer attached, and (c) the composite nanostructure- Photos, diagrams and SEM pictures;
8 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;
9 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.
FIGS. 10A to 10C are diagrams showing resistance test results of the composite nanostructure fabricated in Example 1 against the horizontal external force and the vertical external force, respectively, and the changes before and after rubbing the composite nanostructure on the surface of various objects ,
11 is a SEM photograph of a composite nanostructure produced by directly forming a nanoweb on a nanostructured structure without a surface modification metal layer according to Comparative Example 1 and a composite nanostructure produced according to Example 1 ;
12 is an 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; FIG.
13 is a SEM photograph (a) of E. coli O157: H7 collected on the composite nanostructure-based film produced according to Example 1, and E. coli O157: H7 collected on the composite nanostructure- The enlarged SEM photograph (b, c).
14 is a SEM photograph of E. coli O157: H7 collected on a bare film produced according to Comparative Example 3
15 is a graph showing fluorescence spectrum measurement results obtained by dissolving E. coli O157: H7 collected in each of the composite nanostructure-based film (Example 1) and the bare film (Comparative Example 3) by cell lysis and reacting with DNA probe of double strand Graph,
Fig. 16 shows the results obtained by collecting the Gram-negative bacteria E. coli O157: H7, S. enteritidis, and Y. enterocolitica into a composite nanostructure-based film by concentration (each bacterial concentration range is 0 to 10 ⁷ CFU) Fluorescence intensity (ac), fluorescence intensity (df (nm)) obtained by capturing with bare film by concentration (each bacterial concentration range is 0 to 10 ⁷ CFU) for E. coli O157: H7, S. enteritidis and Y. enterocolitica bacteria ) And the fluorescence intensity (gi) of the live cells and dead cells collected on the composite nanostructure-based film for each bacterial (1 x 10 ⁷ CFU / rxn) Lt; / RTI >
Fig. 17 shows the fluorescence intensity (Fig. 17) obtained by capturing the Gram-positive bacteria S. aureus, B. cereus and L. monocytogenes by concentration (each bacterial concentration range is 0 to 10 ⁷ CFU) The fluorescence intensity (df) obtained by collecting the bacillus strains of S. aureus, B. cereus and L. monocytogenes by their concentration (each bacterial concentration range 0 to 10 ⁷ CFU) on a bare film, (Gi) of viable cells and dead cells collected in a composite nanostructure-based film for each of the bacteria (each number of bacteria is 1 x 10 ⁷ CFU / rxn) for identification of specificity, and
Fig. 18 shows the results of an artificial ( E. coli O157: H7 (a, b, e and f; concentration: 10 ⁷ CFU / rxn) and S. aureus (c, d, g and h; concentration: 10 ⁷ CFU / Is a graph showing fluorescence intensities measured after contamination of knife, chopping board, salad, and drinking water, and then directly collecting bacteria from the surface of the sample by using a composite nano-structured film, a bare film and wet wipes.

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 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" 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.

"Nuclease" may mean an enzyme capable of cleaving a phosphodiester bond between nucleotide subunits of a nucleic acid (eg, DNA or RNA), wherein the nucleotide endonuclease (exo-nuclease) While the cleavage is mainly an enzyme that cleaves the nucleotide phosphodiester bond, the nucleic acid internal hydrolyzing enzyme (endo-nuclease) is an enzyme that cleaves phosphodiester bonds in the nucleotide bond.

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.

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

FIG. 1 schematically shows the principle of capturing and diagnosing biomaterials using nuclease-responsive probes derived from composite nanostructure-based films and biomaterials fabricated according to one embodiment of the present disclosure.

(A) First, a film (film structure) based on a composite nanostructure having specific morphological characteristics is prepared and used as a means for collecting harmful bio-materials such as biomaterials, particularly bacteria.

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. These biomaterials may include Gram-positive bacteria and Gram-negative bacteria. Specific examples thereof include E. coli O157: H7 , Salmonella choleraesuis , Salmonella bongori , Salmonella typhimurium , Staphylococcus aureus, Listeria monocytogenes , Listeria denitrificans , Listeria grayi , Listeria murrayi , cholera, red germ, pertussis, diphtheria, typhoid, pest, hemolytic streptococcus or staphylococcus aureus, and more specifically, 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. monocytognes . In addition, virus types 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, Viruses, leukocyte or West Nile virus, Rift Valley or Tuscan virus, Chichungnia virus, Respiratory syncytial virus, Rossiovirus, Measles virus, Murray encephalitis virus, Vesicular swine virus, Zika virus, a virus selected from the group consisting of lymphocytic choreomeningitis virus, Ebola virus, Marburg virus, Crimea-Congo hemorrhagic fever virus, Lisa virus, Junin virus, Machupo virus, Sabia virus, Guanarito virus , Mumps virus, rabies bar (Eg, rubella virus, rubella virus, varicella zoster virus, herpes simplex 1 and 2 virus, more typically alphavirus, adenovirus, eco virus, rotavirus, flavivirus, linovirus, orthobunyavirus ), Poliovirus, human parvovirus, enterovirus, coronavirus, human papillomavirus, human cytomegalovirus, Epstein-Barr virus, parainfluenza viruses of type 1, 2 and 3, or any confirmed virus . ≪ / RTI > It should be understood that the types listed above are illustrative, and are not necessarily limited to specific types.

Such bio-materials can be propagated through various ways, i.e., via various vapors, liquid and / or solid media or medium, and in some cases, through human contact (e.g., hand contact).

Next, the sample containing the biomaterial is brought into contact with the film based on the composite nanostructure, wherein the composite nanostructure is configured to utilize the physical or non-specific fixing (or trapping) principle by the nanoweb.

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. When hydrophilic properties of the solid surface are high, the hydrophobic interaction is dominant, and biomaterials (specifically, gram-negative and gram-positive bacteria) can be adsorbed and fixed on the hydrophilic solid surface relatively easily. Particularly, in a high concentration sample, 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. As described later, the composite nanostructure can provide an effective collecting effect.

Specifically, in this embodiment, instead of using a chemical method or a biochemical method of an antigen-antibody, the nano-ciliol structure of the surface of the nano-web structure and the physicochemical method using the interaction of the ciliated structure of the surface of the noxious bio- The principle of fixing biomaterials is used. Furthermore, since the nanopile length, diameter and spacing between the pillars can be freely controlled during the fabrication of the composite nanostructure, it is possible to apply not only bacteria but also viruses of a smaller size to the collection and detection.

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 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.

In this way, a cell lysis step is performed on the biomaterial (specifically, bacteria) immobilized on the composite nanostructure, thereby releasing the nucleases from the target biomaterial. Typically, cell lysis involves, for example, (i) heating a biomass-containing sample under controlled temperature conditions at a temperature of about 70-95 ° C (specifically about 80-90 ° C). (ii) a method in which pulse on and pulse off are alternately performed using an ultrasonic sonicator, and (3) a lysis buffer.

In one embodiment, a cell lysis step using a cell lysis buffer can be performed. At this time, the cell lysis buffer may have a composition that functions to minimize the damage of the protein in the biomaterial during cell lysis and at the same time release the nucleases from the biomaterial. According to an exemplary embodiment, the cell lysis buffer may contain components capable of uniformly lysing Gram-negative and Gram-positive bacteria, such as, for example, lysozyme, lysostaphin, and the like, (3 - ((3-cholamidopropyl) dimethylammonio) -1-propanesulfonate) and Triton x-100. In this regard, resource tapin is widely known as an antimicrobial enzyme that cleaves the pentaglycine bridge from the cell wall of staphylococci. In addition, vancomycin, HEPES, SDS and the like may be additionally contained alone or in combination.

Exemplary compositions of cytolytic buffers usable in this embodiment are shown in Table 1 below.

ingredient density Lysozyme About 0.1 to 2 mg / ml (specifically about 0.5 to 1.5 mg / ml, more specifically about 0.8 to 1.2 mg / ml) Resource Tapin About 0.05 to 1 mg / ml (specifically about 0.2 to 0.8 mg / ml, more specifically about 0.3 to 0.6 mg / ml) EDTA About 0.05 to 0.2 mM (specifically about 0.07 to 0.15 mM, more specifically about 0.09 to 0.12 mM) Protease inhibitor cocktail About 0.1 to 1X (specifically about 0.2 to 0.8X, more specifically about 0.3 to 0.6X) Tris-HCl (pH 7.4) About 0.3 to 1.5 M (specifically about 0.5 to 1.3 M, more specifically about 0.8 to 1.2 M) NaCl About 3 to 8 M (specifically about 4 to 7 M, more specifically about 4.5 to 6 M) Triton X-100 About 10 to 25 weight percent (specifically about 15 to 22 weight percent, more specifically about 18 to 20 weight percent) CHAPS About 0.5 to 1.5% by weight (specifically about 0.7 to 1.3% by weight, more specifically about 0.9 to 1.2% by weight)

As described above, nuclease is released from the biomaterial through cell lysis using a cell lysis buffer. In one embodiment, the temperature during the cell lysis process of the biomaterial can be adjusted to an appropriate range in consideration of the type of enzyme used, for example, about 30 to 95 캜 (typically about 40 to 80 캜, more typical To about 55-72 < 0 > C). The temperature range is described for illustrative purposes only, and the present disclosure is not limited thereto.

Separately, in the diagnosis system according to the present embodiment, a nucleic acid probe showing responsiveness to the released nucleases, specifically, a DNA probe having a double strand is provided. As a result of contacting a target biomaterial-derived nuclease with a probe, a chain (bond) in the probe is cleaved by a nuclease. As described above, the contact between the probe and the nuclease can be performed by adding a probe (specifically, a DNA probe having a double strand) to at least a portion of the buffered solution after cell lysis.

The probe includes a fluorophore or a dye and a quencher at each end connected by a chain. As the phosphor and the quencher are positioned adjacent to each other before the chain in the probe is cleaved by the nucleases, fluorescence or fluorescence signals of the phosphor are minimized by fluorescence resonance energy transfer (FLET) phenomenon. That is, it is in a state where the coloring of the phosphor is suppressed by the action of the quencher (quenching state). According to an exemplary embodiment, a fluorescent substance is attached to the 5'-end of the probe and a quencher is attached to the 3'-end. This small housing may be a typically light component-free molecules, and the like Specifically, DABCYL, ROX, Black Hole Quencher ^ㄾ (BHQ ^ㄾ), Eclipse, TAMRA, QSY-6, and the gold nanoparticles (Gold nano-particle). More specifically, BHQ, particularly BHQ-1, can be used as a light extender.

On the other hand, a kind known in the art such as FAM (fluorecein amidite), HEX, JOE, TET, MAX, VIC and the like is attached to the probe end (specifically, 5'-end) as a phosphor. The distance between the fluorophores and the quencher (i. E., Base sequence or chain length) in the probe is, for example, about 25 to 60, specifically about 31 to 50, more specifically about 35 to 40 bases . In this regard, it may be desirable for the absorption spectrum of the quencher to match as closely as possible to the emission spectrum of the phosphor in order to obtain optimal performance. An example of a possible combination of a fluorescent substance and a quencher to be attached to the probe tip for detection of a biomaterial is shown in Fig. 2, and an exemplary combination of the selected fluorescent substance and quencher is shown in Table 2 below.

Mold and sieve (dye) Maximum excitation wavelength (nm) Maximum emission wavelength (nm) Small extinction body FAM 494 515 BHQ-1 JOE 520 548 BHQ-1 TET 521 536 BHQ-1 HEX 535 555 BHQ-1

The phosphor-quencher combinations described in the above table are illustrative and the present invention should not be construed as being limited thereto. However, the combination of FAM / BHQ-1 may be advantageous in terms of detection accuracy.

As described above, the probe usable in the present specific example should exhibit responsiveness to a nuclease (specifically, a nuclease derived from a target biomaterial). Specifically, the probe reacts with a nuclease to form a fluorescent substance and a small molecule It may be one having a base sequence capable of cleaving a chain (bond) between the light bodies. For this purpose, the base sequence in the probe can be calibrated so as to have at least one mismatch or mismatch in the chain (base sequence).

When the probe is cleaved by using a nuclease derived from a bio-material immobilized on the complex nano-complex, the fluorescent substance in the probe in the extinction state is recovered and developed, and the biomaterial in the sample, specifically, The substance can be detected qualitatively and / or quantitatively. That is, the diagnosis can be performed by measuring the presence or intensity of the fluorescent signal according to the concentration of the bio-material in the sample. According to an illustrative embodiment, the amount of probe used may range, for example, from about 5 to 30 [mu] l, specifically about 8 to 25 [mu] l, more specifically about 10 to 20 [ .

In an exemplary embodiment, the fluorescence emitted upon probe cleavage can be detected using, for example, a confocal microscope, a fluorescence microscope, a fluorometer, a fluorescence scanner, a flow cytometer (or apparatus) known in the art, such as a flow cytometer, and the presence or concentration of the target biomaterial can be analyzed in consideration of the presence or absence of fluorescence and the intensity of fluorescence. According to an exemplary embodiment, a laser-induced confocal fluorescence microscope can be used. In addition, it can be used without any limitation, for example, QuantaMaster PTI spectrofluorimeter (PTI) and SPEX Fluorolog 3 (ISA), as long as it can confirm the presence or absence of fluorescence and the change of fluorescence intensity according to the presence of the target biomaterial. have.

The detection limit (LOD) of the diagnostic system according to the exemplary embodiment may be, for example, in the range of about 10 ³ to 10 ⁷ CFU, specifically about 10 ⁴ to 10 ⁵ CFU, but is not limited thereto.

Fabrication of composite nanostructures

According to one embodiment, as described above, the composite nanostructure of the capture (fixation) of the biomaterial in the sample can be fabricated as shown in FIG.

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 using a master mold of a metallic material as described later, when the nano-pattern 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. 4 is a view showing a series of processes for manufacturing a plurality of nanopillar-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 1500 nm. The present invention is not limited to the numerical range of the above-described nanoscale holes 12 but may be appropriately adjusted in consideration of the material of the polymer (the first polymer and a second polymer to be described later), the use of the composite nano structure, 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 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 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. 5 shows a principle of forming a web structure by growing a nanofilament structure of a nanopillar second polymer 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 basis of the nano pillar side, for example, about 10 to 170 degrees, specifically about 30 to 150 degrees, (E.g., the end of the growing fiber strand has a wedge or wedge shape) of the second polymer grown in the direction of about 60 to about 120 [deg.], More specifically about 90 [ The nanofibers of the second polymer grown in the transverse direction from at least one point (specifically, a plurality of points) of the nanofibers are interconnected to form a network similar to a web or a 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;  , Polyaniline (PANI), specifically polyaniline, and the like, which can be selected singly or in combination of two or more selected from the group consisting of polyaniline, polyaniline, polyaniline, have.

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 certain embodiments, the bar to use the polyaniline as the second polymer, wherein if the available aniline monomer, for example a substituted or (e. _{G., P-CH 3, p-} OCH 3, o-CF 3, It may be a _{m-CF 3, p-COOH} , o-NH 2, p-NH 2 such as substituted acceptable), unsubstituted aniline, aniline unsubstituted in detail.

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 about 1: 0.1, specifically from about 1: 0.01 to about 1: 0.05, 0.0 &gt; 1: 0.02. &Lt; / RTI &gt; 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 .

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, a 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 (HClO ₄ ): 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, 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.

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 having an Au / Ti metal layer attached thereto, and (c) a composite nanostructure-based film having PANI nanobubbles formed in this embodiment, And SEM photographs are shown in Fig.

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 is confirmed that polyaniline fibers (or strands) are grown from the side of the nanopillar in the composite nanostructure-based film, and the polyaniline fibers (or strands) grown from the side of the adjacent nanopillar form a network similar to the mesh structure have.

In addition, the diameter of the nanofiller itself before formation of the polyaniline nanofiber structure was about 500 nm, and the diameter of the nanofiller coated with polyaniline was about 550 nm. After the formation of the polyaniline, the depth of the nanopillar (the depth of the polyaniline grown on the bottom surface between the plurality of nanopillars) was about 800 nm. The thickness of the polyaniline nanoweb formed between the nanopillars was about 150 nm.

Analysis of Composite Nanostructured Film

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

(B) a nanopattern structure with an Au / Ti metal layer, and (c) a composite nanostructure-based film formed with a PANI nanowire by using EDS equipment, The results of the EDS analysis are shown in FIG. 8 together with the SEM photograph.

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-based film 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 based film formed with PANI nanoweb were measured using a contact angle meter, The results are shown in Fig.

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 for horizontal external force of composite nano structure based film

In order to evaluate the stiffness of the composite nanostructure-based film produced in this example, the deformation degree of the nanostructure generated by applying a force in the horizontal direction was observed by SEM, and the angle of the nanopillar in the composite nanostructure with respect to the applied horizontal pressure The degree of change 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 for vertical external force of composite nanostructure-based film

In order to evaluate the rigidity of the composite nanostructure-based film produced in this example, 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. 10B.

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.

- Test for external force against various object surfaces

The external force was applied by rubbing the composite nanostructure-based film on the chopping board by using a hand, and the degree of the induced deformation was observed by SEM, and the results before (rubbing) and after rubbing (rubbing) Respectively.

As shown in the figure, it can be seen that a part of the upper part of the composite nanostructure is damaged, but most of the structure is maintained.

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, 12 kPa), the shape changes of the nanopile array in the nanopattern structure are shown in FIG. 12, 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, after the modification of the distance between the nanopillars, it was judged that it is difficult to maintain the original structure due to the van der Waals force generated between the nanopillars, despite the elastic characteristics of the PUNO polymer.

Comparative Example 3

A film-like structure without nanopatterns was fabricated. Specifically, a PET film (Mitsubishi) sold on the market was used to cut the film using a cutter in accordance with the size (2 x 2 cm) to be applied.

Example 2

Capture and detection test procedures for biomaterials (bacteria)

Hereinafter, the composite nanostructure-based film produced according to Example 1 is referred to as a " b-SNAP film ", and the film according to Comparative Example 3 is referred to as a "bare film ".

- Preparation of probe

A nuclease responsive DNA probe with responsiveness to nuclease was custom-made on GenoTech Corp. (Korea). The sequence of the probe used is shown in Table 3 (blue color indicates mismatch).

According to the above table, the phosphor and quencher were placed in pairs at both ends of the double strand probe. In addition, a mismatch occurred at the 3 'end of the sticky-end to cause the degradation of the 3' → 5 'nucleic acid as a part of the correcting operation by DNA polymerase. However, when the complementary base sequence is separated by 3 or more from the 5 'end, the effect of the quencher is reduced, so only one mismatch is given.

Three mismatched bases were constructed in the probe so that it could be decomposed by nucleic acid internal hydrolytic enzymes in addition to the nucleic acid terminator.

Each oligonucleotide probe was dissolved in distilled water (DW) at 100 pmol. Each of the complementary oligonucleotides was mixed with 20 μl of each of the oligonucleotides in 50 μL of an annealing buffer (10 mM Tris (pH 8.0), 50 mM NaCl, 1 mM EDTA, and 2.5 mM MgCl ₂ ) 2 &lt; / RTI &gt;

Followed by denaturation at 95 ° C for 10 min and hybridization at 45 ° C for 70 min. In the double stranded DNA (dsDNA) thus formed, the fluorescent substance and the quencher are adjacent to each other to minimize the fluorescence signal.

- Bacteria preparation

Escherichia coli O157: H7, Salmonella enteritidis and Yersinia enterocolitica , which are gram-negative bacteria, and Y. enterocolitica , and Staphylococcus aureus , a gram-positive bacterium, S. aureus, B. cereus and Listeria monocytogenes were prepared. Each of the bacteria was cultivated in a shaking incubator for 12-24 hours in Luria-Bertani broth (LB broth).

Optical density was measured at 600 nm (Beckman Coulter DU-730, USA) to adjust the concentration of each bacterial to 10 ⁸ CFU / mL (colony-forming units per milliliter).

Bacterial samples (108 CFU / mL) were centrifuged at 12,000 rpm for 10-20 minutes. Then, the LB supernatant was discarded and the bacteria were dispersed in PBS (phosphate buffered saline solution) to prepare bacterial samples at a concentration of 10 ³ CFU / mL to 10 ¹⁰ CFU / mL. Bacterial bacterial samples (107 CFU) were each prepared by heating at 100 ° C for 1 hour or by treating with 70% EtOH for 3 hours.

- Collection of bacteria

A b-SNAP film was prepared in a size of 5 × 5 mm 2, and 10 μL of the prepared bacterial sample was added dropwise. The complex nanostructure formed on the film was allowed to react at room temperature for about 1 hour so that the bacteria could react sufficiently. Then, in order to accurately evaluate the trapping ability of b-SNAP film, bacteria were added dropwise by PBS using PBS and then washed to a severe level. For comparative experiments with or without composite nanostructures, the bare films were treated in the same manner.

- SEM sampling and measurement

E. coli O157: H7 and S. aureus were prepared for each concentration and added dropwise to the b-SNAP film by 10 μL. After reacting at room temperature for about 1 hour, it was washed with PBS. The thus prepared sample was observed by SEM using Nova 230 (FEI company).

- Cell lysis and fluorescence measurement of captured bacteria

Bacteria harvested with b-SNAP film were lysed with optimized cell lysis buffer. The buffer for cell lysis is lysozyme, lysostaphin, EDTA, protease inhibitor cocktail, Tris-HCl (pH 7.4), NaCl, Triton X-100 and CHAPS (3- -cholamidopropyl) dimethylammonio) -1-propanesulfonate.

Then, the cell-lysed bacterial solution was diluted 10-fold with a reaction buffer (50 mM Tris-HCl pH 8.0, 5 mM CaCl ₂ ) to protect the prepared probe and increase nuclease activity. The cell lysate was also subjected to the same method for the bacterial cell sample.

Before measuring fluorescence for each sample, 10 μL dsDNA probe (20 μM) and 100 μL of reaction sample were mixed and then loaded onto a 96-well black microwell plate (Greiner bioone, Germany) Respectively. Fluorescence intensity of individual bacterial samples ranging from 10 &^lt; ¹ &gt; to 10 &^lt; ^{7 & gt} ; CFU / rxn was measured using a fluorescence spectrophotometer (Infinite TM M200 PRO, Tecan Trading AG, Switzerland). The excitation wavelength was 470 nm and the emission wavelength was 520 nm. All data were acquired and analyzed using i-control ™ software (Tecan Trading AG, Switzerland).

- Preparation of actual sample

For evaluation of the actual samples, cutting boards, knives, salads (cabbage and drop tomatoes) and drinking water were prepared. The surfaces of chopping boards, knives and salads were artificially contaminated with E. coli O157: H7 (10 ⁷ CFU / rxn) and S. aureus (10 ⁷ CFU / rxn). In addition, the final concentration of E. coli O157: H7 and S. aureus in drinking water was 10 ⁷ CFU / rxn.

Analyze test results

- Capture analysis of biomaterials (bacteria)

The result of the collection test of bacteria on the b-SNAP film is shown in Figs. 13A to 13C.

Referring to the figure, it can be seen that E. coli O157: H7 was successfully captured on the surface of the b-SNAP film without any surface treatment. 13b and 13c, it was clearly observed that nano-scale protruding regions were formed between the E. coli O157: H7 and the complex nanostructure captured on the b-SNAP film, indicating that the bacterial surface and the complex nanostructure And that there is a strong topographical interaction between the three-dimensional fiber-like structures of

On the other hand, E. coli O157: H7 was also deposited on the bare film in the same manner and then observed with SEM. The results are shown in Fig. According to the figure, it can be seen that the number of bacteria trapped on the flat bare film is significantly lower than that of the b-SNAP film. This suggests that the three-dimensional fiber-like structure of b-SNAP film plays an important role in trapping bacteria.

- Fluorescence Spectrum Analysis

15 is a fluorescence spectrum obtained by lysing E. coli O157: H7 collected in each of a b-SNAP film (Example 1) and a bare film (Comparative Example 3) and then reacting with DNA probe of double strand. As can be seen from the figure, each spectrum shows a main signal at 520 nm, which means that the double stranded DNA probe was cleaved by the nucleases from E. coli . In addition, the area (size) of the fluorescence spectrum is consistent with the number of E. coli captured on each film.

- Diagnostic reliability assessment of biomaterials (bacteria)

Quantitative analysis of six representative bacterial species causing food poisoning was performed using the capture ability of b-SNAP film. The bacteria to be analyzed were Escherichia coli O157: H7, Salmonella enteritidis and Yersinia enterocolitica as Gram-negative bacteria and Staphylococcus aureus (S. aureus) as Gram- Bacillus cereus (B. cereus) and Listeria monocytogenes (L. monocytogenes) . These bacteria are typical pathogenic bacteria causing food poisoning.

Each of the bacteria was prepared by concentration (from 10 ⁷ CFU / rxn to 0 CFU / rxn), and b-SNAP film was used to collect the bacteria. In order to compare the capture performance of b-SNAP films, experiments were conducted under the same conditions using a bare film. Each of the bacteria captured on the b-SNAP film and the bare film was cell-lysed using a cell lysis buffer. At this time, the cell lysis buffer used contains lysozyme and resourcetapine and is applicable to both Gram-negative and Gram-positive bacteria.

Bacteria captured on each film through cell lysis will release bacterial nuclease, the released bacterial nuclease will include the exo-nuclease and the nucleic acid endo-nuclease, and It is DNAase (DNase).

The double stranded DNA probe chain was cleaved by nuclease from Escherichia coli O157: H7, Salmonella enteritidis (S. enteritidis) and Yersinia enterocolitica (Y. enterocolitica) (Fluorescent dye) was measured. In this connection, FIGS. 16A to 16C show cell colonies obtained by collecting E. coli O157: H7, S. enteritidis and Y. enterocolitica by concentration using a b-SNAP film, respectively , followed by reaction with double strand DNA probes Indicating fluorescence intensity. 16D to 16F show fluorescence intensities obtained in the same manner except for using a bare film.

Referring to the figure, b-SNAP For film 10 ⁷ CFU / rxn in the concentration range of 10 ⁴ CFU / rxn to observe the difference in the fluorescence signal of the bacterial concentration iteotneun bars, E. coli O157: H7, S The enteritidis and Y. enterocolitica bacteria have a detection limit of approximately 10 ⁴ CFU. On the other hand, in the case of the bare film, fluorescence signal difference according to the bacterial concentration could not be observed. This means that the b-SNAP film can directly detect and accurately detect bacteria by concentration.

Simple sterilization methods such as dry heat and chemical disinfectants were applied to each bacteria to confirm the specificity for detection of live bacteria.

70% EtOH is a well-known chemical disinfectant which is mainly used in hospitals and kitchen processing plants. To prepare bacteria samples, each bacteria is reacted at 100 ° C for 1 hour or in 70% ethanol for 2 hours Lt; / RTI &gt; After the inactivation process, the b-SNAP film was used to collect.

Each of Figures 16g-16i shows fluorescence intensities of live cells and dead cells captured using b-SNAP films for three Gram-negative bacteria. The fluorescence intensities of heat - treated E. coli O157: H7, S. enteritidis and Y. enterocolitica bacteria were significantly reduced compared to live bacteria. Unlike E. coli O157: H , the fluorescence signal of S. enteritidis and Y. enterocolitica was not significantly reduced by 70% ethanol compared with dry heat treatment but could be clearly distinguished from that of living bacteria.

Thus, live cells and dead cells could be clearly distinguished through the capture and detection system using b-SNAP film.

In the same manner as described above, S. aureus, B. cereus and L. monocytognes , which are Gram-positive bacteria, were collected by b-SNAP film and bare film for each concentration and then lysed and reacted with a double strand DNA probe Fluorescence intensity was measured. The results are shown in Fig.

Referring to the figure, similar to the gram-negative bacteria, the signal difference according to the bacterial concentration was observed in the b-SNAP film, but it was difficult to distinguish it from the bare SNP film. In the case of S. aureus and B. cereus , the difference of fluorescence signal from 10 ⁷ CFU / rxn to 10 ⁵ CFU / rxn was observed, and the detection limit was 10 ⁵ CFU. On the other hand, L. monocytogenes was difficult to differentiate between fluorescence signals in the range of 10 ⁶ CFU / rxn to 0 CFU / rxn, and signal intensity was remarkably high only at the concentration of 10 ⁷ CFU / rxn.

For the bacteria, a bacterial sample was prepared using dry heat and 70% ethanol to evaluate the false positives. Figures 17g-17i each show the fluorescence intensities of live and dead bacteria captured with b-SNAP film for three Gram-positive bacteria. Both S. aureus, B. cereus and L. monocytognes treated with dry heat and 70% ethanol showed a significant decrease in fluorescence signal compared to live bacteria.

Through the above-mentioned results, it is possible to directly collect pathogenic bacteria such as food poisoning bacteria, which are harmful biomaterials, with high efficiency through a detection method using a DNA probe having responsiveness to b-SNAP film and bacteria, And 10 ⁴ CFU and 10 ⁵ CFU for Gram-positive bacteria, respectively. The detection limits of b-SNAP film-based detection systems exhibit similar sensitivities when compared to the sensitivity of conventional detection equipment commonly used.

Furthermore, for the purpose of accurately assessing the ability of the b-SNAP film to capture bacteria, this detection limit is significant in light of bacterial losses occurring during the course of a severe washing procedure after collection. In addition, indirect detection of food poisoning bacteria through the swab sample ring and ATP measurement method, which is the most widely used method commercially, can not distinguish between live cells and dead cells, and the detection limit due to a false signal due to non-bacterial-derived ATP , But the b-SNAP film-based system clearly distinguishes live cells from dead cells and has excellent detection of false positives.

- Diagnostic testing and evaluation of real samples

In order to evaluate the field applicability of the b-SNAP film-based system, food poisoning bacteria were detected from actual cookware and food ingredients. Tomato, salad and drinking water were artificially contaminated with representative Gram-negative and Gram-positive bacteria, E. coli O157: H7 (10 ⁷ CFU / rxn) and S. aureus (10 ⁷ CFU / rxn). In order to evaluate the bacterial capture ability in actual samples, the bacteria were directly collected from the surface of the sample using b-SNAP film, a bare film and a wet wipe, and cytolysis and fluorescence detection were performed according to the above-described method.

18, fluorescence intensities of E. coli O157: H7 (a, b, e and f) and S. aureus (c, d, g and h) As a result of analysis, b-SNAP film successfully detected E. coli O157: H7 and S. aureus in knife, cutting board, salad and drinking water unlike in bare film, and most b-SNAP The intensity of fluorescence in the film was remarkable, which means that the b-SNAP film was excellent in the collection efficiency.

Thus, it has been confirmed that the detection platform using the b-SNAP film and the bacterial nuclease-responsive DNA probe can be effectively applied to prevention of food poisoning and the like in actual sites (for example, a foodservice, a restaurant, etc.).

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) contacting a sample containing a biomaterial with a composite nanostructure having fixability to the biomaterial to collect an increased density of the biomaterial on the composite nanostructure relative to the sample;
b) lysing the captured biomaterial;
c) contacting a cell lysate of the biomaterial with a probe in a quenching state and cleaving the chain in the probe by a nuclease released by the lysis of the cell, A nucleic acid probe having responsiveness to a clase, comprising a phosphor and a quencher at each end connected by the chain, wherein color development of the phosphor is suppressed by the action of a quencher prior to cleavage of the chain;
d) performing at least one of quantitative analysis and qualitative analysis on the fluorescence generated from the liberated fluorescent material according to cleavage of the probe;
/ RTI &gt;
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 for collecting and diagnosing a bio-material in a sample comprising a nano-web structure of a second polymer material formed on a substrate; 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. 3. The method of claim 2, wherein the first polymer is a blend of polyurethane acrylate (PU) and a NOA-based adhesive,
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%. 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 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. 7. The method of claim 6, 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, . 8. The method according to claim 7, wherein the material of each of the surface modifying metal layer and the intermediate layer is Au and Ti. The method of claim 3, wherein the second polymer is polyaniline. 4. The method of claim 3, wherein the diameter of the fibers (strands) in the nano-web structure of the second polymeric material is in the range of 10 to 200 nm,
Wherein the average mesh size of the nano-web structure of the second polymeric material ranges from 1 to 1,500 nm. 2. The method of claim 1, wherein the probe is a double stranded DNA probe. The method of claim 1, wherein the bio-material is at least one of a bacteria, a gram-positive bacterium, and a gram-negative bacterium. The method according to claim 1, wherein step b) is carried out using a cell lysis buffer,
Wherein the cell lysis buffer comprises lysozyme and lysostaphin. 3. The phosphor according to claim 1, wherein the phosphor is selected from the group consisting of FAM, HEX, JOE, TET, MAX and VIC, and the quencher is DABCYL, ROX, Black Hole and Quencher (BHQ) And gold nanoparticles. &Lt; RTI ID = 0.0 &gt; 8. &lt; / RTI &gt; 15. The method according to claim 14, wherein the phosphor is FAM and the quencher is BHQ-1. 12. The method according to claim 11, wherein the distance between the fluorophores and the quencher in the probe is 25 to 60 bases,
Wherein the probe comprises a mismatch in the base sequence such that the chain between the fluorophore and the quencher is cleaved in response to the released nucleases. The method of claim 1, wherein the generated fluorescence is generated by a confocal microscope, a fluorescence microscope, a fluorometer, a fluorescence scanner or a flow cytometer, a photo multiplier tube, and a photo diode. As a system for collecting and diagnosing bio-materials in a sample,
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) a nucleic acid probe having responsiveness to a nuclease derived from the biomaterial, wherein the probe comprises a fluorescent substance and a quencher at each end connected by a chain, and before the chain is cleaved by a nuclease, The coloring of the phosphor is suppressed by the action of the optical body; And
c) detecting at least one of fluorescence and fluorescence intensity of the fluorescent material released from the probe by cutting the chain;
&Lt; / RTI &gt; The method of claim 18 wherein the limit of detection (LOD) of 10 ³ to 10 ⁷ CFU of collection and diagnostic system, characterized in that in the collecting and diagnostic system. The probe according to claim 18, wherein the probe is a double-stranded DNA probe, wherein FAM is attached as a fluorescent substance to the 5'-end of the nucleotide sequence shown in Table 3 below, and BHQ-1 is attached as a quencher to the 3'- Collecting and diagnosing system characterized by:
[Table 3]

In the above table, blue indicates a mismatch.

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