Chip for detecting bacteria and manufacturing method therefor

Abstract

The present invention provides a chip for detecting bacteria, a manufacturing method therefor, and a method for detecting bacteria, the chip comprising: a substrate; a plurality of pillar structures on the substrate; and a reaction layer formed of a thermo-responsive polymer on the plurality of pillar structures, wherein the thermo-responsive polymer exhibits hydrophilicity or hydrophobicity depending on temperature.

Description

A chip for detecting bacteria and a method for manufacturing the same

[0001] The present invention relates to a chip for detecting bacteria and a method for manufacturing the same.

[0002] Pathogenic bacterial infections cause millions of deaths annually and have a significant impact on human health, life, and society as a whole. In particular, bacterial contamination in food is a major threat to public health and food safety, leading to continuous development of bacterial detection technologies to address this issue.

[0003] Currently, various techniques such as microbial culture methods, protein-based detection methods (e.g., enzyme-linked immunosorbent assays, rapid test kits, or mass spectrometry), and nucleic acid-based detection methods (e.g., PCR, qPCR, or DNA microarray analysis) are used for the detection of pathogenic bacteria. However, each of these techniques has limitations, such as analysis errors in samples during the pretreatment step, reduced detection sensitivity, and time consumption.

[0004] In particular, for detecting trace amounts of pathogenic bacteria in food and kitchen utensils, accuracy in the bacterial capture and detection process is crucial. However, existing sampling devices and techniques cannot guarantee the efficient release of captured bacteria within the samples, which can reduce the reliability of the analysis results.

[0005] There is a need for a new system that can solve these problems and improve the sensitivity and accuracy of bacterial detection.

[0006] The background description of the invention is provided to facilitate a better understanding of the present invention. The matters described in the background description should not be construed as an acknowledgment that they exist as prior art.

[0007] The inventors of the present invention recognized that the main problem in the bacterial detection process occurs during the sample collection and release stages.

[0008] Although recent studies have reported that 3D structured chips can improve bacterial capture performance, it is difficult to completely release the captured bacteria, requiring additional lysis treatment. This leads to reduced detection sensitivity and accuracy, and there is a high possibility of false negatives, especially in samples containing small amounts of pathogenic bacteria.

[0009] To overcome the aforementioned limitations, the inventors of the present invention sought to develop a new bacterial detection system capable of simultaneously achieving efficient bacterial capture and easy release during the sample pretreatment process.

[0010] The inventors of the present invention focused on stimulus-responsive polymer technology capable of transforming physical properties in response to external stimuli. In particular, they recognized that by introducing a heat-responsive polymer capable of reversibly switching between hydrophilicity and hydrophobicity solely through temperature changes, it is possible to replace conventional chemical solvent treatment and implement a more simplified bacterial detection process.

[0011] More specifically, the inventors of the present invention noted that the heat-reactive polymer provides higher flexibility and rapid recovery characteristics, and possesses the advantage of effectively capturing bacteria under specific temperature conditions and releasing the attached bacteria solely through temperature changes.

[0012] At this time, the inventors of the present invention sought to improve bacterial capture performance by coating a thermoreactive polymer onto a nano-filament structure, taking into account that it exhibits hydrophobicity below a certain temperature, thereby stably attaching bacteria to the surface of the nano-filament.

[0013] Furthermore, the inventors of the present invention sought to implement a system capable of performing a bacterial detection process more stably without additional chemical solvents or enzyme treatment by easily releasing bacteria captured by a heat-reactive polymer from the surface at temperatures above a certain level.

[0014] Accordingly, the inventors of the present invention recognized that by providing a new bacterial detection system, they could minimize errors in sample analysis that occurred during the existing bacterial detection pretreatment process and significantly improve the sensitivity and accuracy of pathogenic bacteria.

[0015] Furthermore, the inventors of the present invention recognized that by providing a new bacterial detection system, it can be effectively utilized in various application fields, such as bacterial capture in the air and detection of multidrug-resistant bacteria, in addition to the detection of bacteria in food and kitchen utensils.

[0016] Accordingly, the problem to be solved by the present invention is to provide a chip for detecting bacteria comprising a substrate, a pillar structure formed on the substrate, and a heat-reactive polymer-based reaction layer disposed on the pillar structure, and a method for manufacturing the same.

[0017] Another problem that the present invention aims to solve is to provide a method for detecting bacteria using a chip for bacteria detection.

[0018] The problems of the present invention are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0019] To solve the problem described above, a chip for detecting bacteria comprising a substrate according to an embodiment of the present invention is provided. The chip for detecting bacteria comprises a substrate, a plurality of pillar structures on the substrate, and a reaction layer composed of a heat-reactive polymer on the plurality of pillar structures, wherein the heat-reactive polymer exhibits hydrophilicity or hydrophobicity depending on the temperature.

[0020] According to a feature of the present invention, the heat-reactive polymer may be a copolymer composed of an MNAGA monomer (methacryloyl glycinamide monomer) and a hydrophobic acrylate monomer.

[0021] According to another feature of the present invention, in the copolymer, the proportion of hydrophobic acrylate monomer may be 1 mol % to 25 mol %.

[0022] According to another feature of the present invention, the hydrophobic acrylate monomer may be one of ethyl acrylate, butyl acrylate, and benzyl acrylate.

[0023] According to another feature of the present invention, the chip for bacterial analysis further comprises a mediating layer on a pillar structure, and a reaction layer may be disposed on the mediating layer.

[0024] According to another feature of the present invention, the mediating layer may be composed of a fibrous polymer.

[0025] According to another feature of the present invention, the mediating layer may be composed of at least one of polyaniline (PANI), polypyrrole (PPy), polythiophene (Poly(3,4-ethylenedioxythiophene), including PEDOT), nylon, polyvinyl alcohol (PVA), polyethyleneimine (PEI), polyurethane (PU), polyimide (PI), polysiloxane, and conductive nanocomposites.

[0026] According to another feature of the present invention, the diameter of the pillar structure may be 0.5 μm to 2 μm.

[0027] According to another feature of the present invention, the height of the pillar structure may be 0.5 μm to 2 μm.

[0028] According to another feature of the present invention, the heat-reactive polymer may be a material that exhibits hydrophobicity below a predetermined temperature level and hydrophilicity above that temperature.

[0029] According to another feature of the present invention, the predetermined temperature level may be 20 ℃ to 40 ℃.

[0030] To solve the problems described above, a method for manufacturing a bacterial analysis chip according to another embodiment of the present invention is provided. The manufacturing method comprises the steps of providing a substrate, arranging a plurality of pillar structures on the substrate, preparing a heat-reactive polymer, and arranging a reaction layer composed of the heat-reactive polymer on the plurality of pillar structures. At this time, the heat-reactive polymer exhibits hydrophilicity or hydrophobicity depending on the temperature.

[0031] According to a feature of the present invention, the step of preparing a heat-reactive polymer may include the step of obtaining a copolymer of an MNAGA monomer and a hydrophobic acrylate monomer.

[0032] According to another feature of the present invention, the step of obtaining a copolymer may include the step of polymerizing an MNAGA monomer and a hydrophobic acrylate monomer into a copolymer such that the ratio of the hydrophobic acrylate monomer is 1 mol % to 25 mol %.

[0033] According to another feature of the present invention, the method may further include the step of synthesizing an MNAGA monomer (Methacryloyl glycinamide monomer) based on an amidation reaction of glycinamide and methacryloyl chloride.

[0034] According to another feature of the present invention, the manufacturing method further comprises the step of placing a mediating layer made of a fibrous polymer on a plurality of pillar structures, and the step of placing a reaction layer may include the step of placing a reaction layer on the mediating layer.

[0035] According to another feature of the present invention, the method may further include the steps of placing an auxiliary layer made of metal on a pillar structure and placing a mediating layer on the auxiliary layer.

[0036] According to another feature of the present invention, the step of arranging an auxiliary layer comprises the step of sequentially arranging a titanium layer and a gold layer through vacuum deposition, wherein the thickness of the titanium layer is 10 nm to 50 nm and the thickness of the gold layer is 100 nm to 300 nm, a method for manufacturing a chip for detecting bacteria.

[0037] According to another feature of the present invention, the step of placing a mediating layer may include the step of polymerizing polyaniline (PANI) to place the mediating layer on an auxiliary layer.

[0038] According to another feature of the present invention, the step of arranging a reaction layer may include the step of functionalizing a plurality of pillar structures with an amine, the step of immobilizing BiBB (2-bromoisobutryl bromide) on the plurality of functionalized pillar structures, and the step of polymerizing an MNAGA monomer and a hydrophobic acrylate monomer so that a thermally reactive polymer of a thermally reactive copolymer is arranged on the plurality of pillar structures on which BiBB is immobilized.

[0039] To solve the problems described above, a method for detecting bacteria using a bacterial detection chip according to another embodiment of the present invention is provided. The detection method comprises the steps of: preparing a sample containing target bacteria; placing the sample on a bacterial detection chip according to any one of claims 1 to 12; adjusting the temperature to below a predetermined level so that the bacterial detection chip captures the target bacteria; and adjusting the temperature to above a predetermined level so that the bacterial detection chip releases the target bacteria.

[0040] The present invention will be explained in more detail below through examples. However, since these examples are merely illustrative of the present invention, the scope of the present invention should not be interpreted as being limited by these examples.

[0041] The present invention can provide a novel bacterial detection system that efficiently implements the capture and release of bacteria by utilizing a heat-reactive polymer capable of reversibly switching between hydrophilicity and hydrophobicity according to a specific temperature change.

[0042] Accordingly, the present invention can minimize analysis errors of samples that occurred during the conventional bacterial detection pretreatment process and can contribute to significantly improving the sensitivity and accuracy of pathogenic bacteria detection.

[0043] In addition, the present invention provides a bacteria detection system based on a heat-reactive polymer, which enables the release of bacteria through simple temperature control without the need for additional chemical solvents or enzyme treatment, thereby contributing to further simplifying the bacteria detection process and increasing reliability.

[0044] Furthermore, the present invention can be effectively utilized in various application fields, such as the detection of bacteria in food and kitchen utensils, as well as the capture of bacteria in the air and the detection of multidrug-resistant bacteria, and thereby can be applied to various fields including public health, food safety, and environmental hygiene management.

[0045] The effects according to the present invention are not limited to those exemplified above, and various other effects are included in this specification.

[0046] FIGS. 1a to 1c, FIGS. 2a and 2b illustrate exemplary configurations of a bacterial detection chip comprising a substrate, a plurality of pillar structures, and a reaction layer according to various embodiments of the present invention.

[0047] FIGS. 3a and 3b illustrate, exemplarily, a chip for detecting bacteria including a mediating layer and its configurations according to various embodiments of the present invention.

[0048] FIGS. 4a to 4f illustrate, exemplarily, the manufacturing procedure of a chip for detecting bacteria according to various embodiments of the present invention.

[0049] FIGS. 5a and 5b illustrate, exemplarily, a procedure for analyzing target bacteria using a bacterial detection chip according to various embodiments of the present invention.

[0050] FIGS. 6a to 6h illustrate the evaluation results of a bacterial detection chip according to various embodiments of the present invention.

[0051] The advantages of the invention and the methods for achieving them will become clear by referring to the embodiments described below in detail together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below but may be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0052] The shapes, sizes, ratios, angles, numbers, etc. disclosed in the drawings for explaining embodiments of the present invention are exemplary, and therefore the present invention is not limited to the depicted details. Furthermore, in describing the present invention, if it is determined that a detailed description of related known technology may unnecessarily obscure the essence of the present invention, such detailed description is omitted. Where terms such as "comprising," "having," or "consisting of" are used in this specification, other parts may be added unless "only" is used. Where a component is expressed in the singular, it includes cases where it includes the plural unless specifically stated otherwise.

[0053] In interpreting the components, they are interpreted to include a margin of error even in the absence of a separate explicit description.

[0054] The features of each of the various embodiments of the present invention may be combined or combined with one another, either partially or wholly, and as will be fully understood by those skilled in the art, various technical interlocking and operation are possible, and each embodiment may be implemented independently of one another or together in an interlocking relationship.

[0055] For clarity in the interpretation of this specification, the terms used in this specification are defined below.

[0056] As used in this specification, the term "chip for bacterial detection" refers to a chip designed for the purpose of capturing and analyzing bacteria, comprising a substrate, a pillar structure, a mediating layer, and a heat-reactive polymer reaction layer, and may mean a chip that operates in response to temperature changes.

[0057] For example, chips for bacterial detection include Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Salmonella spp., Pseudomonas aeruginosa (P. aeruginosa), Listeria monocytogenes (L. monocytogenes), Klebsiella pneumoniae (K. pneumoniae), Enterococcus faecalis (E. faecalis), Enterococcus faecium (E. faecium), Vibrio cholerae (V. cholerae), Shigella spp., Bacillus cereus (B. cereus), Clostridium difficile (C. difficile), Helicobacter pylori (H. pylori), Campylobacter jejuni (C. jejuni), Legionella pneumophila (L. pneumophila), Mycobacterium tuberculosis (M. It can target and detect pathogenic bacteria such as tuberculosis, Acinetobacter baumannii (A. baumannii), Yersinia pestis (Y. pestis), and Haemophilus influenzae (H. influenzae). However, it is not limited to this, and the bacterial detection chip may also be used for the analysis of fungi.

[0058] As used in this specification, the term "substrate" refers to a material that serves as a support for forming a pillar structure, and may refer to a material capable of supporting a structure through various physical and chemical treatments.

[0059] In various embodiments of the present invention, the substrate may be glass, a silicon wafer, or a polymer (PET (Polyethylene Terephthalate), PDMS (Polydimethylsiloxane), PU (Polyurethane), etc.). However, it is not limited thereto, and the substrate may be a ceramic, a metal thin film, or other synthetic material.

[0060] In various embodiments of the present invention, the substrate may be made of a material such as a plurality of pillar structures.

[0061] As used in this specification, the term "multiple pillar structures" refers to pillar-shaped nano or microstructures formed on a substrate and may mean structures having the function of facilitating the capture and analysis of target substances.

[0062] In various embodiments of the present invention, a plurality of pillar structures may be nano-pillars having a diameter of about 0.5 μm to 2 μm and a height of about 0.5 μm to 2 μm.

[0063] However, its shape and size are not limited to those described above, and the pillar structure may be a structure with a conical, pyramidal, or polygonal cross-section, and its diameter, height, and spacing may be selected within a wide range depending on the type of target bacteria, the volume of the sample, etc.

[0064] As used in this specification, the term "thermally reactive polymer" refers to a polymeric material whose physical properties change from hydrophilic to hydrophobic, or from hydrophobic to hydrophilic, depending on a specific temperature change. Based on these properties, the capture and release of bacteria can be controlled.

[0065] More specifically, the heat-reactive polymer may be a material that exhibits hydrophobicity below a predetermined temperature level and hydrophilicity above a predetermined temperature level.

[0066] Here, the predetermined temperature level may be the Upper Critical Solution Temperature point (UCST point), which is an indicator indicating that the interaction between polymers weakens above a certain temperature and transitions to an insoluble state below a certain temperature, but is not limited thereto.

[0067] In various embodiments of the present invention, a predetermined temperature level may be 20°C to 40°C.

[0068] For example, a nanopillar structure coated with a reaction layer made of a heat-reactive polymer can exhibit hydrophobicity at temperatures below 25°C, with a captive bubble contact angle of about 129°, which indicates the property of the material to repel interactions with water.

[0069] Furthermore, a nanopillar structure coated with a reaction layer made of a heat-reactive polymer can be converted to a hydrophilic state at a temperature of 37°C or higher, with a capture bubble contact angle of approximately 139.0°.

[0070] That is, in more diverse embodiments, nanopillar structures coated with a reaction layer made of a heat-reactive polymer can be enabled to release for the capture and analysis of target bacteria by controlling the critical temperature.

[0071] In various embodiments of the present invention, the heat-reactive polymer may be a copolymer of an MNAGA monomer (methacryloyl glycinamide monomer) and a hydrophobic acrylate monomer. It is not limited thereto.

[0072] As used in this specification, the term "copolymer" refers to a polymer formed by the combination of two or more different monomers.

[0073] In various embodiments of the present invention, the copolymer may refer to a polymer composed of an MNAGA monomer and a hydrophobic acrylate monomer to impart thermal reactive properties.

[0074] For example, in a copolymer composed of an MNAGA monomer and a hydrophobic acrylate monomer, the hydrophobic acrylate monomer may be included in an amount of 1 mol% to 25 mol%. However, it is not limited thereto.

[0075] The term "MNAGA monomer" as used in this specification may refer to a substance synthesized through the amidation reaction of glycinamide and methacryloyl chloride.

[0076] In this case, the MNAGA monomer may be a compound that imparts hydrophilic properties to the copolymer.

[0077] The term "hydrophobic acrylate monomer" as used in this specification may refer to an acrylate-based compound having a low affinity for water.

[0078] In various embodiments of the present invention, the hydrophobic acrylate monomer may be ethyl acrylate, butyl acrylate, or benzyl acrylate. Preferably, the hydrophobic acrylate monomer may be benzyl acrylate, but is not limited thereto.

[0079] More preferably, the copolymer is a copolymer of MNAGA monomer and benzyl acrylate (Bn) used as a hydrophobic acrylate monomer, and the ratio of benzyl acrylate may be 5 mol%, but is not limited thereto.

[0080] The term "medium layer" as used in this specification may refer to a layer that can perform the role of increasing adhesion with the reaction layer or promoting interaction with the sample through specific chemical properties.

[0081] In one embodiment of the present invention, the mediating layer may serve as a layer to more stably fix the reaction layer to the pillar structure. However, it is not limited thereto, and the mediating layer may also be used as a mediating layer to maximize the interaction between the substance in the sample (e.g., target bacteria) and the reaction layer.

[0082] The term "medium layer" as used in this specification refers to a layer composed of a fibrous polymer among the layers constituting the medium layer, which can serve to enhance interaction and adhesion with the reaction layer or adjust the physical structure of the surface.

[0083] In one embodiment of the present invention, the mediating layer is composed of polyaniline (PANI) and can serve to enhance the chemical bonding strength with the reaction layer. That is, the mediating layer may be a functional layer in the form of fibers that improves bacterial capture performance.

[0084] In various embodiments of the present invention, the mediating layer may be a single mediating layer, but is not limited thereto.

[0085] In this case, polyaniline is hydrophobic, but it can be hydrophilic depending on the shape of the nanofilament structure.

[0086] Meanwhile, the mediating layer is not limited to polyaniline and may be composed of at least one of polypyrrole (PPy), polythiophene (Poly(3,4-ethylenedioxythiophene), including PEDOT), nylon, polyvinyl alcohol (PVA), polyethyleneimine (PEI), polyurethane (PU), polyimide (PI), polysiloxane, and conductive nanocomposites.

[0087] The term "auxiliary layer" as used in this specification refers to a layer made of metal and can provide electrical and chemical stability to the surface.

[0088] In one embodiment of the present invention, a mediating layer may be disposed on an auxiliary layer. At this time, the auxiliary layer may be composed of gold (Au) and / or titanium (Ti). However, it is not limited thereto, and the auxiliary layer may be composed of at least one of silver (Ag), copper (Cu), platinum (Pt), palladium (Pd), aluminum (Al), nickel (Ni), chromium (Cr), and tungsten (W).

[0089] The term "vacuum deposition" as used in this specification refers to a process of forming a thin layer by evaporating a metal or other material in a vacuum environment and depositing it onto the surface of a substrate, and may refer to a technology capable of forming a layer of precise thickness.

[0090] For example, an auxiliary layer can be formed on a pillar structure by sequentially arranging a titanium layer and a gold layer through vacuum deposition. In this case, the thickness of the titanium layer may be 10 to 50 nm, and the thickness of the gold layer may be 100 nm to 300 nm.

[0091] As used in this specification, the term "functionalized with amine" refers to a specific compound or surface having an amine functional group (-NH 2) It can refer to the process of introducing to impart chemical reactivity or to impart the property of being able to bind to specific molecules.

[0092] At this time, functionalization with amine may be a treatment that increases the reactivity of the pillar structure.

[0093] In one embodiment of the present invention, functionalization with an amine may be achieved through treatment using glycine or ethylenediamine. However, it is not limited thereto, and may be functionalized in various ways using other amine compounds.

[0094] As used in this specification, the term "immobilization" may refer to a process of fixing a specific material to a surface by chemical or physical means so that it does not move.

[0095] In this case, immobilization may be a treatment that maintains the function of a specific functional group or enhances reactivity.

[0096] In one embodiment of the present invention, immobilization may be performed by attaching 2-bromoisobutryl bromide (BiBB) to the surface of a pillar structure. However, it is not limited thereto, and immobilization may also be used to attach proteins, enzymes, or other polymers to the surface of a pillar structure.

[0097] Hereinafter, with reference to FIGS. 1a to 1c, FIGS. 2a and 2b, and FIGS. 3a and 3b, a chip for detecting bacteria according to various embodiments of the present invention will be described in detail.

[0098] FIGS. 1a to 1c and FIGS. 2a and 2b illustrate exemplary configurations of a bacterial detection chip and the same, comprising a substrate, a plurality of pillar structures, and a reaction layer according to various embodiments of the present invention.

[0099] First, referring to FIGS. 1a and 1b, a chip (100) for detecting bacteria may be composed of a substrate (110), a pillar structure (120), and a reaction layer (130) on the pillar structure (120).

[0100] In various embodiments of the present invention, the substrate (110) may be made of a material such as the pillar structure (120). However, it is not limited thereto, and the substrate may be glass, a silicon wafer, or a polymer (PET (Polyethylene Terephthalate), PDMS (Polydimethylsiloxane), PU (Polyurethane), etc.) that supports the pillar structure.

[0101] In various embodiments of the present invention, each of the plurality of pillar structures (120) may be a nanopillar having a diameter of about 0.5 μm to 2 μm and a height of about 0.5 μm to 2 μm. However, the shape and size thereof are not limited to those described above, and the pillar structures may be structures having a conical, pyramidal, or polygonal cross-section, and the diameter, height, and spacing thereof may be selected within a variety of ranges depending on the type of target bacteria, the volume of the sample, etc.

[0102] Meanwhile, the reaction layer (130) made of a heat-reactive polymer may be a material that exhibits hydrophobicity at temperatures below a predetermined level and hydrophilicity at temperatures above a predetermined level.

[0103] In various embodiments of the present invention, the reaction layer (130) may be composed of a copolymer of MNAGA monomer (Methacryloyl glycinamide monomer) and hydrophobic acrylate monomer.

[0104] For example, the nano-pillar structure (120) coated with the reaction layer (130) may exhibit hydrophobicity at a temperature of 25°C or lower, with a capture bubble contact angle of about 129°C, which indicates the properties of the material to promote interaction with water. Furthermore, the nano-pillar structure (120) coated with the reaction layer (130) may be converted to a hydrophilic state at a temperature of 37°C or higher, with a capture bubble contact angle of about 139.0°C.

[0105] More specifically, referring to FIG. 1c, when an analysis sample containing target bacteria is processed on a bacterial analysis chip (100), the nano-pillar structure (120) coated with a reaction layer (130) at 25°C or lower may have hydrophobicity, allowing the capture of target bacteria in the pillar structure (120). Then, when heated to a temperature of 37°C or higher, the nano-pillar structure (120) coated with a reaction layer (130) may have hydrophilicity, allowing for the effective release of target bacteria from the pillar structure (120).

[0106] At this time, the target bacteria are Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Salmonella spp., Pseudomonas aeruginosa (P. aeruginosa), Listeria monocytogenes (L. monocytogenes), Klebsiella pneumoniae (K. pneumoniae), Enterococcus faecalis (E. faecalis), Enterococcus faecium (E. faecium), Vibrio cholerae (V. cholerae), Shigella spp., Bacillus cereus (B. cereus), Clostridium difficile (C. difficile), Helicobacter pylori (H. pylori), Campylobacter jejuni (C. jejuni), Legionella pneumophila (L. pneumophila), Mycobacterium tuberculosis (M. It may be at least one of pathogenic bacteria such as tuberculosis), Acinetobacter baumannii (A. baumannii), Yersinia pestis (Y. pestis), and Haemophilus influenzae (H. influenzae), but is not limited thereto.

[0107] That is, a bacterial analysis chip (100) according to one embodiment of the present invention may be configured to perform capture, release, and analysis of target bacteria by temperature control.

[0108] Meanwhile, referring further to FIGS. 2a and 2b, in various embodiments of the present invention, a reaction layer (130) on a chip (100) for detecting bacteria may be disposed on a substrate (110) as well as on a plurality of pillar structures (120).

[0109] Accordingly, it is possible to capture and release bacteria with higher efficiency.

[0110] Referring to FIGS. 3b and 3c, according to various embodiments of the present invention, a reaction layer (130) may be disposed on a mediating layer (140), and the mediating layer (140) may serve as a layer to more stably fix the reaction layer (130) on a pillar structure (120).

[0111] At this time, the mediating layer (140) may be a polyaniline (PANI) layer in the form of fibers, but is not limited thereto, and may be composed of at least one of polypyrrole (PPy), polythiophene (Poly(3,4-ethylenedioxythiophene), including PEDOT), nylon, polyvinyl alcohol (PVA), polyethyleneimine (PEI), polyurethane (PU), polyimide (PI), polysiloxane, and conductive nanocomposites.

[0112] That is, a thermally reactive reaction layer (130) layer can be more stably disposed on a pillar structure (120) on which a mediating layer (140) is formed.

[0113] In various embodiments of the present invention, the mediating layer (140) may be disposed alone on the pillar structure (120), but is not limited thereto, and may also be disposed on an auxiliary layer (not shown) made of metal.

[0114] In various embodiments of the present invention, the auxiliary layer (not shown) may be composed of gold (Au) and / or titanium (Ti) to impart hydrophilicity to the pillar structure. However, it is not limited thereto and may be composed of at least one of silver (Ag), copper (Cu), platinum (Pt), palladium (Pd), aluminum (Al), nickel (Ni), chromium (Cr), and tungsten (W).

[0115] In more diverse embodiments of the present invention, an auxiliary layer (not shown) may be formed on a pillar structure by sequentially arranging a titanium layer and a gold layer through vacuum deposition. However, it is not limited thereto.

[0116] As described above, a chip (100) for detecting bacteria according to various embodiments of the present invention is provided, so that the process of capturing and releasing bacteria can be performed simply and efficiently.

[0117] In particular, the bacterial detection chip of the present invention detects target bacteria with high sensitivity and specificity and can rapidly release bacteria according to temperature changes.

[0118] Accordingly, the present invention provides a chip for bacterial detection, thereby replacing the existing complex bacterial analysis process and enabling faster and simpler bacterial detection and analysis.

[0119] Hereinafter, the manufacturing procedure of a chip for detecting bacteria will be explained with reference to FIGS. 4a to 4f.

[0120] FIGS. 4a to 4f illustrate, by way of example, a procedure for manufacturing a chip for bacterial detection according to various embodiments of the present invention.

[0121] First, referring to FIG. 4a, for the manufacture of a chip for detecting bacteria, a substrate is first provided (S410), a plurality of pillar structures are arranged on the substrate (S420), a heat-reactive polymer is prepared (S430), and a reaction layer made of the heat-reactive polymer is arranged on the plurality of pillar structures (S440).

[0122] According to a feature of the present invention, in the step (S410) where a substrate is provided, the substrate may be glass, a silicon wafer, a polymer (PET (Polyethylene Terephthalate), PDMS (Polydimethylsiloxane), PU (Polyurethane), etc.). However, it is not limited thereto.

[0123] Referring further to FIG. 4b, a mediating layer is first disposed on a plurality of pillar structures on a substrate (S450), and in the step (S440) where a reaction layer is disposed, a reaction layer can be disposed on the mediating layer.

[0124] In one embodiment of the present invention, in the step (S450) where the mediating layer is disposed, a mediating layer made of a fibrous polymer may be disposed.

[0125] At this time, the mediating layer may be formed on the pillar structure by polymerizing polyaniline (PANI). However, it is not limited thereto, and the mediating layer may be composed of at least one of polypyrrole (PPy), polythiophene (Poly(3,4-ethylenedioxythiophene), including PEDOT), nylon, polyvinyl alcohol (PVA), polyethyleneimine (PEI), polyurethane (PU), polyimide (PI), polysiloxane, and conductive nanocomposites.

[0126] In another embodiment of the present invention, in the step (S450) where the mediating layer is disposed, an auxiliary layer made of metal may be disposed on the pillar structure, and a mediating layer may be disposed on the auxiliary layer.

[0127] At this time, the auxiliary layer can be formed by sequentially arranging a titanium layer and a gold layer through vacuum deposition, and the thickness of the titanium layer may be 10 to 50 nm, and the thickness of the gold layer may be 100 nm to 300 nm. However, it is not limited thereto, and the auxiliary layer may be composed of at least one of silver (Ag), copper (Copper, Cu), platinum (Pt), palladium (Pd), aluminum (Al), nickel (Ni), chromium (Cr), and tungsten (W).

[0128] For example, referring to FIG. 4c, the manufacturing process of a chip for detecting bacteria may include the following steps.

[0129] First, in the step of providing a substrate (S410) and the step of placing a pillar structure (S420), a hole-patterned silicon master mold is prepared and used as a mold for forming the pillar structure. At this time, the size and arrangement of the pillar structure can be selected according to the shape of the mold.

[0130] Next, polyurethane (PU) is cast onto a mold to form the initial shape of the pillar structure, and after coating the polyurethane onto a polyethylene terephthalate (PET) film, a UV curing process is performed. That is, the cured polyurethane pillar structure is separated from the mold, forming multiple pillar structures on a polyethylene terephthalate substrate.

[0131] Next, in the step (S450) where the mediating layer is placed, a mediating layer made of a fiberized polymer is formed.

[0132] At this time, an auxiliary layer may be selectively formed, and a medium layer may be formed thereon. For example, a titanium (Ti) layer and a gold (Au) layer may be formed sequentially through vacuum deposition, wherein the thickness of the titanium layer may be 10 nm to 50 nm and the thickness of the gold layer may be 100 nm to 300 nm. Subsequently, a fibrous medium layer is formed by polymerizing polyaniline (PANI) on the auxiliary layer.

[0133] Subsequently, a reaction layer composed of a heat-reactive polymer is placed on the medium layer through the step (S440) in which the reaction layer is placed.

[0134] Referring further to FIGS. 4a and 4d, a copolymer of MNAGA monomer and hydrophobic acrylate monomer is obtained for the preparation of a heat-reactive polymer (S432), a plurality of pillar structures are functionalized with an amine for the arrangement of a reaction layer (S442), BiBB (2-bromoisobutryl bromide) is immobilized on the functionalized plurality of pillar structures (S444), and the MNAGA monomer and hydrophobic acrylate monomer are polymerized on the plurality of pillar structures immobilized with BiBB to form a reaction layer (S446).

[0135] In this case, BiBB can act as an initiator for polymerization.

[0136] According to various embodiments of the present invention, in the step (S432) where the copolymer is obtained, the MNAGA monomer and the hydrophobic acrylate monomer can be polymerized into a copolymer such that the ratio of the hydrophobic acrylate monomer is 1 mol % to 25 mol %.

[0137] According to a more diverse embodiment of the present invention, in the step (S432) where a copolymer is obtained, an MNAGA monomer (Methacryloyl glycinamide monomer) can be synthesized based on the amidation reaction of glycinamide and methacryloyl chloride.

[0138] For example, referring to FIGS. 4e and 4f, a process for forming a heat-reactive polymer may include the following steps.

[0139] First, referring to FIG. 4e, in the step (S432) where the copolymer is obtained, glycinamide and methacryloyl chloride are synthesized into MNAGA monomer (methacryloyl glycinamide monomer) through an amidation reaction. At this time, glycinamide is deprotonated in the presence of K2CO3, and amide bonding can be induced by dropping methacryloyl chloride using diethyl ether in an ice bath environment. Next, the MNAGA monomer can be combined with a hydrophobic acrylate monomer through copolymerization to form a heat-reactive copolymer. At this time, the proportion of the hydrophobic acrylate monomer can be adjusted from 1 mol% to 25 mol%.

[0140] Next, referring to FIG. 4f, in the step (S442) where a plurality of pillar structures are functionalized with amines, hydroxyl groups (-OH) are introduced to the surface of the pillar structures through oxygen plasma treatment, and APTES (Aminopropyltriethoxysilane) treatment is performed so that the surface of the pillar structures is functionalized with amine groups (-NH2).

[0141] Next, through the step (S444) in which BiBB (2-bromoisobutryl bromide) is immobilized on a plurality of functionalized pillar structures, BiBB is attached to the surface of the amine-functionalized pillar structures by vacuum deposition, at which time BiBB can act as an initiator for polymer polymerization.

[0142] Finally, through the step (S446) in which a thermally reactive polymer is formed on a plurality of pillar structures on which BiBB is immobilized, a polymerization reaction is induced with respect to the MNAGA monomer and the hydrophobic acrylate monomer with copper(II) bromide (CuBr2) and ascorbic acid as a reducing agent to obtain a thermally reactive copolymer. At this time, the reaction temperature is maintained at approximately 45°C, and as a result, a reaction layer based on the thermally reactive copolymer can be formed on the pillar structures.

[0143] The thermoreactive functionalized pillar structures obtained through this process exhibit hydrophilicity or hydrophobicity depending on temperature changes, thereby providing properties optimized for bacterial capture and release.

[0144] In other words, the present invention can provide a novel bacterial detection system that efficiently implements the capture and release of bacteria by utilizing a heat-reactive polymer capable of reversibly switching between hydrophilicity and hydrophobicity according to temperature changes.

[0145] Accordingly, the present invention can minimize analysis errors of samples that occurred during the conventional bacterial detection pretreatment process and can contribute to significantly improving the sensitivity and accuracy of pathogenic bacteria detection.

[0146] In addition, the present invention provides a bacteria detection system based on a heat-reactive polymer, which enables the release of bacteria through simple temperature control without the need for additional chemical solvents or enzyme treatment, thereby contributing to further simplifying the bacteria detection process and increasing reliability.

[0147] Hereinafter, a method for detecting bacteria using a bacterial detection chip according to various embodiments of the present invention will be described with reference to FIGS. 5a and 5b.

[0148] FIGS. 5a and 5b illustrate, exemplarily, a procedure for detecting target bacteria using a bacterial detection chip according to various embodiments of the present invention.

[0149] First, referring to FIG. 5a, a sample containing target bacteria is prepared to detect target bacteria (S510), and the sample is placed on a bacterial detection chip (S520). Then, the temperature is adjusted below a predetermined level so that the bacterial detection chip captures the target bacteria (S530), and the temperature is adjusted above a predetermined level so that the bacterial detection chip releases the target bacteria (S540).

[0150] Referring further to FIG. 5b, the bacterial detection chip of the present invention can perform the process of capturing and releasing target bacteria by utilizing the phase transition characteristics of a heat-reactive polymer.

[0151] First, in the sample preparation step (S510), a sample containing target bacteria is prepared. This sample may be a sample extracted from food, the surface of kitchen utensils, particles in the air, or other objects of analysis.

[0152] Next, in the sample placement step (S520), the prepared sample is placed on the bacterial detection chip. In this step, the sample is made to sufficiently contact the reaction layer to enable bacterial capture.

[0153] Next, in the step (S530) where bacteria are captured, the temperature is controlled to a level below a predetermined level so that the bacterial detection chip can capture target bacteria. At this time, when the temperature is set to about 25°C, the heat-reactive polymer exhibits hydrophobicity, so that the surface of the reaction layer forms a strong interaction with the target bacteria and can effectively capture the target bacteria. In this state, the reaction layer can maintain a dehydrated state.

[0154] Finally, in the step where bacteria are released (S540), the temperature is adjusted above a predetermined level so that the bacterial detection chip can release target bacteria. At this time, when the temperature is set to approximately 37°C, the heat-reactive polymer is converted to a hydrophilic state, and the surface of the reaction layer changes to a hydrated state, so that the captured target bacteria are effectively released from the chip surface and analysis is performed (S550).

[0155] According to various embodiments of the present invention, the predetermined temperature level may be 20°C to 40°C, but is not limited thereto.

[0156] Through this, the bacterial detection chip can rapidly and efficiently capture and release target bacteria in response to temperature changes without the need for separate solvents or treatments.

[0157] That is, the present invention can provide a novel bacterial detection system that efficiently implements the capture and release of bacteria by utilizing a heat-reactive polymer capable of reversibly switching between hydrophilicity and hydrophobicity according to temperature changes.

[0158] Accordingly, the present invention can minimize analysis errors of samples that occurred during the conventional bacterial detection pretreatment process and can contribute to significantly improving the sensitivity and accuracy of pathogenic bacteria detection.

[0159]

[0160] Evaluation: Evaluation of a bacterial detection chip according to various embodiments of the present invention

[0161] Hereinafter, with reference to FIGS. 6a to 6h, the evaluation results of a chip for detecting bacteria according to various embodiments of the present invention will be described.

[0162] First, referring to Fig. 6a, data showing the change in water contact angle according to surface modification of the pillar structure is illustrated. The water contact angle is an indicator used to evaluate the hydrophilicity or hydrophobicity of a surface and plays an important role in determining surface characteristics by measuring the angle at which a water droplet forms on a solid surface.

[0163] More specifically, referring to the top, the water contact angle of the uncoated pillar structure was measured to be 31.2° ± 2.2°, and in the amine-functionalized pillar structure, it showed a decreasing trend to 28.8° ± 2.7°. Subsequently, in the pillar structure with BiBB fixed thereon, the water contact angle increased significantly to 115.1° ± 1.2°.

[0164] Referring to the bottom, the water contact angle of the thermally reactive functionalized pillar structure changes with temperature, and is measured at 19.2° ± 0.1° at 25 ℃, then decreases to 14.0° ± 1.8° at 37 ℃.

[0165] In this case, the significant increase in the contact angle when BiBB is fixed indicates that hydrophobicity has been enhanced, while the decrease in the contact angle with temperature change after the introduction of the heat-reactive polymer may mean that it has been converted to hydrophilicity.

[0166] Referring further to FIG. 6b, the results of analyzing the surface characteristics of the pillar structure through changes in water contact angle according to various coating conditions and temperature changes, and the type and ratio of hydrophobic acrylate monomers are shown.

[0167] First, the initial water contact angle of the Au substrate was measured to be 110.9° ± 5.3°, and after functionalization with amine (-NH2), the water contact angle decreased significantly to 16.9° ± 2.4°, making the surface hydrophilic. Subsequently, after immobilizing the BiBB (2-bromoisobutryl bromide) initiator, the water contact angle increased again to 127.0° ± 1.8°, enhancing hydrophobicity. After MNAGA sole coating, the water contact angle at 25°C decreased to 12.0° ± 0.2°, indicating that the thermally reactive polymer imparted hydrophilicity.

[0168] Next, before examining the change in water contact angle according to the type and ratio of hydrophobic acrylate monomers, notations such as "MNAGA-X YY %" clearly indicate the constituent components and molar ratios of the polymer. Here, "MNAGA" refers to the methacryloyl glycinamide monomer, which acts as the hydrophilic element of the polymer. X stands for "Bn," "Et," and "Bu," representing benzyl acrylate, ethyl acrylate, and butyl acrylate, respectively, distinguishing the types of hydrophobic acrylate monomers within the polymer. YY represents "5%," "10%," and "20%," indicating the molar ratio of hydrophobic acrylate monomers within the copolymer.

[0169] Under room temperature (RT) conditions, MNAGA-Bn 5% exhibits a low water contact angle, which implies that the surface maintains relative hydrophilicity due to the low proportion of hydrophobic acrylates. On the other hand, for MNAGA-Bn 10% and MNAGA-Bn 20%, the water contact angle gradually increases as the molar ratio increases, indicating that the hydrophobicity of the surface is enhanced. The same trend is observed in MNAGA-Et and MNAGA-Bu, where the water contact angle increases as the molar ratio of hydrophobic acrylate monomers increases.

[0170] Under conditions of 37 ℃, the hydrophobicity of the heat-reactive polymer decreases and the surface transitions to hydrophilicity. MNAGA-Bn 5% shows a decrease in water contact angle compared to 25 ℃, indicating that the hydrophilicity of the surface is enhanced with increasing temperature.

[0171] Under conditions of 45 ℃, the water contact angle decreases further, indicating that at high temperatures, the hydrophobic properties of the thermally reactive polymer weaken and hydrophilic properties become prominent. As the proportion of hydrophobic acrylate increases, the increase in the contact angle becomes greater, and MNAGA-Bu 20% exhibits the highest hydrophobicity.

[0172] In other words, the above results demonstrate that the type and molar ratio of hydrophobic acrylate monomers have a significant influence on the surface properties of the thermoreactive polymer, particularly the water contact angle. It appears that surface hydrophobicity is enhanced as the proportion of hydrophobic acrylate increases. The water contact angle is controlled by changes in temperature (25°C, 37°C, and 45°C), and as the temperature increases, the thermoreactive polymer exhibits a characteristic of switching from hydrophobic to hydrophilic. This suggests that a filament structure utilizing a thermoreactive polymer is an important factor in effectively controlling bacterial capture and release in a bacterial detection chip.

[0173] Referring further to FIG. 6c, the change in surface characteristics of a heat-reactive functionalized pillar structure in a bacterial detection chip of the present invention according to temperature is illustrated.

[0174] At this time, the heat-reactive polymer is a copolymer of MNAGA monomer and benzyl acrylate (Bn) used as a hydrophobic acrylate monomer, and the ratio of benzyl acrylate may be 5 mol%, but is not limited thereto.

[0175] More specifically, referring to Fig. 6b (a), the capture bubble contact angle of the heat-reactive functionalized pillar structure was measured to be 129.9° ± 0.8° at 25°C, which means that the surface is relatively hydrophobic. On the other hand, in Fig. 6b (b), when the temperature was raised to 37°C, the capture bubble contact angle increased to 139.0° ± 3.1°, which indicates that the surface was converted to hydrophilic.

[0176] These changes are attributed to the properties of MNAGA-Bn 5%, a heat-reactive polymer, and may mean that the structure of the polymer changes near the highest critical temperature, causing the surface properties to switch from hydrophobic to hydrophilic.

[0177] In other words, the above results may imply that the thermally reactive functionalized pillar structure can precisely control surface characteristics according to temperature changes and possesses thermally reactive properties capable of efficiently capturing and releasing target bacteria on a bacterial detection chip.

[0178] Next, referring to Fig. 6d, the results of a step-by-step analysis of the surface chemical properties of a thermally reactive functionalized pillar structure based on FT-IR (Fourier-transform infrared) spectra are shown.

[0179] More specifically, FT-IR data include spectral changes of uncoated nanopillar structures, amine-functionalized nanopillar structures, nanopillar structures with BiBB initiator immobilized, and nanopillar structures coated with MNAGA-Bn 5%.

[0180] The FT-IR spectrum of the uncoated nanopillar structure shows a C=C stretching signal at 1560 cm¹ and a CN stretching signal at 1240 cm¹, which indicates the basic chemical properties of the nanopillar structure.

[0181] The amine-functionalized nanopillar structures clearly exhibit a CN stretching signal at 1297 cm⁻¹, which may indicate the successful introduction of amine groups (-NH₂) to the surface. Additionally, a CH₄ aromatic stretching signal is observed at 1146 cm⁻¹, demonstrating chemical changes on the surface.

[0182] The nanopillar structure immobilized with BiBB initiator shows an additional C=O stretching signal at 1718 cm¹, indicating that the carbonyl group of BiBB is immobilized on the surface.

[0183] The nano-pillar structure (Thermo-URCHANO) coated with 5% MNAGA-Bn shows an amide signal at 1681 cm⁻¹, which may indicate that amide bonds, the main component of 5% MNAGA-Bn, have been successfully formed on the surface. Additionally, a CO signal appears at 1088 cm⁻¹, which may indicate that a copolymer between MNAGA and a hydrophobic acrylate monomer has been formed.

[0184] In other words, the above FT-IR analysis results indicate that the surface chemical properties of the nanopillar structure were successfully transformed at each stage, and in particular, suggest that the 5% MNAGA-Bn polymer can be effectively coated on the surface to provide optimized functionality in the process of capturing and releasing target bacteria on a bacterial detection chip.

[0185] Next, referring to FIG. 6e, the results of comparing the surface and cross-sectional shapes of nanopillar structures before and after coating using scanning electron microscope images are shown. At this time, the shape differences between uncoated nanopillar structures ((a) to (c)) and nanopillar structures coated with 5% of the heat-reactive copolymer MNAGA-Bn ((d) to (f)) were compared.

[0186] More specifically, referring to Fig. 6e (a), a top view of an uncoated nanopillar structure is shown, revealing a distinct nanopillar structure. Referring to Fig. 6e (d), a top view of a nanopillar structure coated with 5% MNAGA-Bn is shown, revealing a relatively smooth surface of the nanopillar. This may indicate that the copolymer has been coated on the surface.

[0187] Referring to Figures 6e (b) and (c), the vertical structure of the nanopillar structure is shown in the cross-sectional image of the uncoated nanopillar structure. Referring to Figures 6e (e) and (f), in the cross-section of the nanopillar structure coated with MNAGA-Bn 5%, it is shown that the copolymer is uniformly coated on the surface of the nanopillar structure, and the thickness and location of the MNAGA-Bn 5% coating layer, which is about 50 nm thick, are clearly indicated by the arrow.

[0188] The above results suggest that a 5% MNAGA-Bn reactive layer was successfully deposited on the surface of the nanopillar structure, forming a thermally reactive functionalized nanopillar structure. In particular, the smooth surface after coating implies that the thermally reactive polymer uniformly covers the surface of the pillar structure, which can optimize bacterial detection and release functions.

[0189]

[0190] Next, referring to Fig. 6f, the process of bacteria being captured and released from a heat-reactive functionalized nanopillar structure is visually illustrated through scanning electron microscope images.

[0191] More specifically, referring to (a) of Fig. 6f, it appears that the nanopillar structure effectively captures bacteria at 25°C, and in particular, the structure of the surface of the pillar structure and the heat-reactive polymer form a strong interaction with the target bacteria, thereby stably capturing the bacteria.

[0192] Referring to (b) of Fig. 6f, the number of bacteria remaining on the surface of the nanopillar structure is shown to be significantly reduced after the temperature was raised to 37°C. This may mean that the thermally reactive polymer on the surface was converted to hydrophilic at high temperatures, weakening the interaction with bacteria and accelerating the release of bacteria.

[0193] The above results suggest that a bacterial detection chip based on a heat-reactive functionalized nanofilament structure is an effective platform capable of precisely controlling the capture and release of target bacteria according to temperature changes. In particular, the characteristic of stably capturing bacteria at 25°C and rapidly releasing them at 37°C can serve as a factor in increasing the efficiency of the bacterial detection and analysis process.

[0194] Next, referring to Fig. 6g, the results of comparing changes in bacterial concentration and Ct values ​​via RT-PCR analysis after capturing and releasing the target bacterium S. aureus using heat-reactive functionalized nanofilament structures and uncoated nanofilament structures are shown. At this time, this evaluation was conducted at various bacterial concentrations (10 2 inside 10 5 It was performed to verify bacterial release efficiency with and without heat treatment in CFU / mL.

[0195] More specifically, when heat-treated at 37°C in a heat-reactive functionalized nanopillar structure, the Ct value at all bacterial concentrations was found to be similar to the initial bacterial concentration. This may indicate that bacterial release was promoted as the 5% coating of the heat-reactive polymer MNAGA-Bn became hydrophilic due to the increase in temperature. In particular, when heat treatment was not performed at 25°C, the Ct value was found to be almost undetectable, which may mean that the heat-reactive polymer stably captured bacteria while maintaining hydrophobicity at low temperatures.

[0196] In contrast, the Ct value remained constant in the uncoated nanopillar structures regardless of heat treatment, indicating that bacterial release from the nanopillar surface is not regulated by temperature changes.

[0197] The above results may imply that a heat-reactive polymer-based reaction layer plays an important role in effectively controlling bacterial release. In particular, at temperatures above 37°C, the MNAGA-Bn 5% coating undergoes hydration as the hydrogen bonds of the amide bonds are broken, and this change can help to push bacteria away from the coating surface.

[0198] Referring to Fig. 6h, the results of capturing and analyzing bacteria from actual kitchen utensils and food using a heat-reactive functionalized filament structure are shown.

[0199] More specifically, referring to (a) of FIG. 6h, it is shown that target bacteria are captured on kitchenware such as gloves and aprons, and on food surfaces such as eggs and sausages, which may be designed to evaluate the performance of a bacterial detection chip by simulating a real-life environment.

[0200] Referring to Fig. 6h (b), the bacterial composition used in the experiment and the bacterial mixture applied to each sample are shown. In particular, Staphylococcus aureus, Salmonella enteritidis, Listeria monocytogenes, and Bacillus cereus, which are pathogenic bacteria that cause food poisoning, were used, and various conditions were reproduced by varying the bacterial composition for each sample.

[0201] Referring to Fig. 6h (c), the results of evaluating the Ct values ​​of bacteria captured in a sample by comparing the heat-reactive functionalized filament structure and the uncoated nanofilament structure as RT-PCR analysis results are shown. More specifically, it appears that the captured bacteria were effectively released as the heat-reactive polymer was activated by a change in temperature.

[0202] Furthermore, it was observed that bacteria were effectively released from all samples when using heat-reactive functionalized filament structures. This implies that heat-reactive functionalized filament structures enhance the efficiency of bacterial capture and release from kitchenware and food surfaces, and can be effectively utilized for the analysis of foodborne pathogens.

[0203] In various embodiments of the present invention, the heat-reactive polymer-based reaction layer may be composed of 5% MNAGA-Bn, but is not limited thereto.

[0204] According to the above evaluation results, the present invention can provide a novel bacterial detection system that efficiently implements the capture and release of bacteria by utilizing a heat-reactive polymer capable of reversibly switching between hydrophilicity and hydrophobicity according to a specific temperature change.

[0205] Accordingly, the present invention can minimize analysis errors of samples that occurred during the conventional bacterial detection pretreatment process and can contribute to significantly improving the sensitivity and accuracy of pathogenic bacteria detection.

[0206] In addition, the present invention provides a bacteria detection system based on a heat-reactive polymer, which enables the release of bacteria through simple temperature control without the need for additional chemical solvents or enzyme treatment, thereby contributing to further simplifying the bacteria detection process and increasing reliability.

[0207] Furthermore, the present invention can be effectively utilized in various application fields, such as the detection of bacteria in food and kitchen utensils, as well as the capture of bacteria in the air and the detection of multidrug-resistant bacteria, and thereby can be applied to various fields including public health, food safety, and environmental hygiene management.

[0208] [Explanation of the symbol]

[0209] 100: Chip for detecting bacteria

[0210] 110: Substrate

[0211] 120: Pillar structure

[0212] 130: Reaction layer

[0213] 140: Mediating layer

[0214]

[0215] [National R&D projects that supported this invention]

[0216] [Project ID] 2540000269

[0217] [Project No.] RS-2024-00401639

[0218] [Ministry Name] Ministry of Agriculture, Food and Rural Affairs

[0219] [Project Management (Specialized) Agency Name] Korea Institute of Planning and Evaluation for Food, Agriculture and Forestry Technology

[0220] [Research Project Name] Technology Commercialization Support

[0221] [Project Title] Development of Modality-Based Nano-Drugs for the Treatment of Allergic Diseases in Companion Animals

[0222] [Name of Project Performing Organization] Korea Research Institute of Bioscience and Biotechnology

[0223] [Research Period] April 1, 2024 ~ December 31, 2024

[0224]

[0225] [Project ID] 2480000170

[0226] [Assignment No.] 2021003370003

[0227] [Ministry Name] Ministry of Environment

[0228] [Project Management (Specialized) Agency Name] Korea Environmental Industry & Technology Institute

[0229] [Research Project Name] Development of Indoor Air Biological Hazardous Factor Management Technology Project

[0230] [Research Project Title] Development of Field-Portable Real-Time Detection Technology

[0231] [Name of Project Performing Organization] Korea Research Institute of Bioscience and Biotechnology

[0232] [Research Period] 2024.01.01 ~ 2024.12.31

[0233]

[0234] [Project ID] 2710018584

[0235] [Project No.] CSM2105M101

[0236] [Ministry Name] Ministry of Science and ICT

[0237] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0238] [Research Project Name] Support for Operating Expenses of National Nanotechnology Center (Major Project Expenses)

[0239] [Project Title] Development of Contactless Digital PCR for Next Wave (Post-COVID) Response

[0240] [Name of Project Performing Organization] Korea Research Institute of Bioscience and Biotechnology

[0241] [Research Period] 2024.01.01 ~ 2024.12.31

[0242]

[0243] [Project ID] 2710018250

[0244] [Project No.] RS-2024-00459749

[0245] [Ministry Name] Ministry of Science and ICT

[0246] [Project Management (Specialized) Agency Name] Korea Institute of Information & Communications Technology Planning & Evaluation

[0247] [Research Project Name] Development of Core Technologies for Immersive Content

[0248] [Research Project Title] Development of an AI-based Non-contact Intelligent Multi-complex Drug Detection Solution

[0249] [Name of Project Performing Organization] Korea Research Institute of Bioscience and Biotechnology

[0250] [Research Period] July 1, 2024 ~ March 31, 2025

[0251]

[0252] [Project ID] 2710008896

[0253] [Assignment No.] KGM5472413

[0254] [Ministry Name] Ministry of Science and ICT

[0255] [Name of Project Management (Specialized) Agency] National Science and Technology Research Council

[0256] [Research Project Name] Korea Research Institute of Bioscience and Biotechnology Research Operational Expense Support (Major Project Expenses)

[0257] [Project Title] Development of Diagnostic-Therapy Platform Technology Based on Innovative Bio-nano Materials

[0258] [Name of Project Performing Organization] Korea Research Institute of Bioscience and Biotechnology

[0259] [Research Period] 2024.01.01 ~ 2024.12.31

[0260]

[0261] [Project ID] 2710018369

[0262] [Project No.] RS-2024-00438316

[0263] [Ministry Name] Ministry of Science and ICT

[0264] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0265] [Research Project Name] Biomedical Technology Development

[0266] [Project Title] Development of Full-Cycle Diagnostic Platform Technology for Diffuse Molecular Subtype Gastric Cancer

[0267] [Name of Project Performing Organization] Korea Research Institute of Bioscience and Biotechnology

[0268] [Research Period] July 1, 2024 ~ December 31, 2024

[0269]

[0270] [Project ID] 2710012794

[0271] [Assignment No.] 2022R1C1C1008815

[0272] [Ministry Name] Ministry of Science and ICT

[0273] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0274] [Research Project Name] Individual Basic Research (Ministry of Science and ICT)

[0275] [Project Title] Development of High-Precision Detection Technology for Antibiotic-Resistant Bacteria Based on Non-Enzymatic Nucleic Acid Cross-Chain Reaction

[0276] [Name of Project Performing Organization] Korea Research Institute of Bioscience and Biotechnology

[0277] [Research Period] 2024.03.01 ~ 2025.02.28

[0278]

[0279] [Project ID] 2710012823

[0280] [Assignment No.] 2023R1A2C2005185

[0281] [Ministry Name] Ministry of Science and ICT

[0282] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0283] [Research Project Name] Individual Basic Research (Ministry of Science and ICT)

[0284] [Project Title] Development of Source Molecular Diagnostic Technology Using Novel Protein / Gene Complexes

[0285] [Name of Project Performing Organization] Korea Research Institute of Bioscience and Biotechnology

[0286] [Research Period] 2024.03.01 ~ 2025.02.28

[0287]

[0288] [Project ID] 2710004962

[0289] [Project No.] RS-2024-00348576

[0290] [Ministry Name] Ministry of Science and ICT

[0291] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0292] [Research Project Name] Individual Basic Research (Ministry of Science and ICT)

[0293] [Project Title] Development of a Nanodrug Platform Based on Cancer-Associated Fibroblast (CAF) Removal Modality

[0294] [Name of Project Performing Organization] Korea Research Institute of Bioscience and Biotechnology

[0295] [Research Period] May 1, 2024 – April 30, 2025

[0296]

[0297] [Project ID] 2710016592

[0298] [Assignment No.] 2021M3H4A1A02051048

[0299] [Ministry Name] Ministry of Science and ICT

[0300] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0301] [Research Project Name] Nanomaterial Technology Development

[0302] [Project Title] Development of CRISPR Materials for Cancer Diagnosis and Application of 3D Plasmonic Nanostructures

[0303] [Name of Project Performing Organization] Korea Research Institute of Bioscience and Biotechnology

[0304] [Research Period] 2024.01.01 ~ 2024.12.31

[0305]

[0306] [Project ID] 2710015183

[0307] [Assignment No.] 2021M3E5E3080844

[0308] [Ministry Name] Ministry of Science and ICT

[0309] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0310] [Research Project Name] Development of Core Technologies for Novel and Emerging Infectious Disease Response Platform

[0311] [Project Title] Development of Biocontent Production and Application Technologies for Establishing a NiRAN Inhibitor Therapeutic Discovery Platform

[0312] [Name of Project Performing Organization] Korea Research Institute of Bioscience and Biotechnology

[0313] [Research Period] 2024.01.01 ~ 2024.12.31

[0314]

[0315] [Project ID] 2340001540

[0316] [Project No.] RS-2023-00275869

[0317] [Ministry Name] Ministry of Education

[0318] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0319] [Research Project Name] Establishment of Academic Research Infrastructure in Science and Engineering

[0320] [Research Project Title] Development of High-Efficiency Bacteria Detection Technology Based on Intelligent Nanostructures

[0321] [Name of Project Performing Organization] Korea Research Institute of Bioscience and Biotechnology

[0322] [Research Period] 2024.03.01 ~ 2025.02.28

[0323]

[0324] [Project ID] 2410000042

[0325] [Project No.] RS-2022-00154853

[0326] [Ministry Name] Ministry of Trade, Industry and Energy

[0327] [Project Management (Specialized) Agency Name] Korea Institute of Industrial Technology Planning and Evaluation

[0328] [Research Project Name] Market-Driven K-Sensor Technology Development Project

[0329] [Project Title] Development of High-Sensitivity Nano-Optical Biosensor Technology for Rapid On-Site Virus Diagnosis

[0330] [Name of Project Performing Organization] Korea Research Institute of Bioscience and Biotechnology

[0331] [Research Period] 2024.01.01 ~ 2024.12.31

[0332]

[0333] [Project ID] 2410000525

[0334] [Project No.] RS-2024-00403563

[0335] [Ministry Name] Ministry of Trade, Industry and Energy

[0336] [Project Management (Specialized) Agency Name] Korea Institute of Industrial Technology Planning and Evaluation

[0337] [Research Project Name] Market-Driven K-Sensor Technology Development Project

[0338] [Project Title] Development of a Rapid Sepsis Diagnostic System Based on Multi-Biomarkers and Ultra-High Sensitivity Sensors

[0339] [Name of Project Performing Organization] Korea Research Institute of Bioscience and Biotechnology

[0340] [Research Period] April 1, 2024 ~ December 31, 2024

[0341]

[0342] [Project ID] 2710018588

[0343] [Project No.] CP24007M

[0344] [Ministry Name] Ministry of Science and ICT

[0345] [Project Management (Specialized) Agency Name] National Nanotechnology Center

[0346] [Research Project Name] Support for Operating Expenses of National Nanotechnology Center (Major Project Expenses)

[0347] [Project Title] Development of an i-Lab-on-Human-based In-vivo Biosignal Monitoring System

[0348] [Name of Project Performing Organization] National Nanotechnology Center

[0349] [Research Period] 2022.01.01 ~ 2025.12.31

[0350]

[0351] [Project ID] 2710006283

[0352] [Assignment No.] PSD24010M

[0353] [Ministry Name] Ministry of Science and ICT

[0354] [Name of Project Management (Specialized) Agency] Korea Institute for Science and Technology Commercialization

[0355] [Research Project Name] Strengthening the Technological Competitiveness of Domestic Research Equipment

[0356] [Project Title] Development of Gene-Loaded Nanoparticle Formulation System for Cell Delivery

[0357] [Name of Project Performing Organization] National Nanotechnology Center

[0358] [Research Period] April 1, 2024 ~ December 31, 2025

Claims

1. Substrate; A plurality of pillar structures on the above substrate; and It includes a reaction layer composed of a thermally reactive polymer on the plurality of pillar structures above, and The above-mentioned heat-reactive polymer is a chip for detecting bacteria, which exhibits hydrophilicity or hydrophobicity depending on temperature.

2. In Paragraph 1, The above-mentioned heat-reactive polymer is, A chip for detecting bacteria, which is a copolymer composed of MNAGA monomer (methacryloyl glycinamide monomer) and hydrophobic acrylate monomer.

3. In Paragraph 2, A chip for detecting bacteria, wherein the ratio of the hydrophobic acrylate monomer in the copolymer is 1 mol % to 25 mol %.

4. In Paragraph 2, The above hydrophobic acrylate monomer is one of ethyl acrylate, butyl acrylate, and benzyl acrylate, a chip for bacterial detection.

5. In Paragraph 1 It further comprises a mediating layer composed of a fibrous polymer on the above-mentioned pillar structure, and The above reaction layer is a chip for detecting bacteria, disposed on a mediating layer.

6. In Paragraph 5 It further includes an auxiliary layer made of metal, and The above mediating layer is disposed on the above auxiliary layer, a chip for detecting bacteria.

7. In Paragraph 5 A chip for detecting bacteria, wherein the above-mentioned mediating layer is composed of at least one of polyaniline (PANI), polypyrrole (PPy), polythiophene (Poly(3,4-ethylenedioxythiophene), including PEDOT), nylon, polyvinyl alcohol (PVA), polyethyleneimine (PEI), polyurethane (PU), polyimide (PI), polysiloxane, and conductive nanocomposites.

8. In Paragraph 1 A chip for detecting bacteria, wherein the diameter of the above-mentioned pillar structure is 0.5 μm to 2 μm.

9. In Paragraph 1 A chip for detecting bacteria, wherein the height of the above-mentioned pillar structure is 0.5 μm to 2 μm.

10. In paragraph 1 The above-mentioned heat-reactive polymer is, A chip for detecting bacteria, which is a material that exhibits hydrophobicity below a predetermined temperature level and hydrophilicity above the temperature level.

11. In Paragraph 10 The above-mentioned predetermined level of temperature is, A chip for detecting bacteria, with a temperature of 20°C to 40°C.

12. Step of providing a substrate; A step of arranging a plurality of pillar structures on the above substrate; Step of preparing a heat-reactive polymer; and The method includes the step of placing a reaction layer composed of a heat-reactive polymer on the plurality of pillar structures above. A method for manufacturing a chip for bacterial detection, wherein the above-mentioned heat-reactive polymer exhibits hydrophilicity or hydrophobicity depending on the temperature.

13. In Paragraph 12, The step of preparing the above-mentioned heat-reactive polymer is, A method for manufacturing a chip for bacterial detection, comprising the step of obtaining a copolymer of MNAGA monomer and hydrophobic acrylate monomer.

14. In Paragraph 13, The step of obtaining the above copolymer is, A method for manufacturing a chip for detecting bacteria, comprising the step of polymerizing the MNAGA monomer and the hydrophobic acrylate monomer into a copolymer such that the ratio of the hydrophobic acrylate monomer is 1 mol % to 25 mol %.

15. In Paragraph 13, A method for manufacturing a chip for bacterial detection, further comprising the step of synthesizing an MNAGA monomer (Methacryloyl glycinamide monomer) based on an amidation reaction of glycinamide and methacryloyl chloride.

16. In Paragraph 12, The method further includes the step of placing a mediating layer composed of a fibrous polymer on the plurality of pillar structures above. The step of placing the above reaction layer is, A method for manufacturing a chip for detecting bacteria, comprising the step of placing the reaction layer on the above mediating layer.

17. In Paragraph 16, A step of placing an auxiliary layer made of metal on the above pillar structure; and A method for manufacturing a chip for bacterial detection, further comprising the step of arranging the above-mentioned mediating layer.

18. In Paragraph 17, The step of placing the above auxiliary layer is, A method for manufacturing a chip for detecting bacteria, comprising the step of sequentially arranging a titanium layer and a gold layer through vacuum deposition, wherein the thickness of the titanium layer is 10 nm to 50 nm and the thickness of the gold layer is 100 nm to 300 nm.

19. In Paragraph 17, The step of arranging the mediating layer is, A method for manufacturing a chip for detecting bacteria, comprising the step of polymerizing polyaniline (PANI) and placing a mediating layer on the auxiliary layer.

20. In Paragraph 12, The step of placing the above reaction layer is, A step of functionalizing a plurality of pillar structures with an amine; A step of immobilizing BiBB (2-bromoisobutryl bromide) on the functionalized plurality of pillar structures; A method for manufacturing a bacterial detection chip, comprising the step of polymerizing an MNAGA monomer and a hydrophobic acrylate monomer so that a thermally reactive polymer of the thermally reactive copolymer is disposed on a plurality of pillar structures on which BiBB is immobilized.

21. A method for detecting bacteria using a chip for bacterial detection, Step of preparing a sample containing target bacteria; A step of placing the above sample on a bacterial detection chip according to any one of claims 1 to 12; A step of adjusting the temperature below a predetermined level so that the above-mentioned bacterial detection chip captures target bacteria, A method for detecting bacteria, comprising the step of adjusting the temperature above a predetermined level so that the bacterial detection chip releases target bacteria.

22. In Paragraph 21, A method for detecting bacteria, wherein the above-determined level is 20 ℃ to 40 ℃.

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