Retroreflective signal analysis-type biosensor chip for bacterial detection, food pathogenic bacteria detection chip, retroreflective signal analysis-type bacterial detection method, and food pathogenic bacteria detection method

Abstract

Disclosed are a retroreflective signal analysis-type biosensor chip for bacterial detection, a food pathogenic bacteria detection chip, a retroreflective signal analysis-type bacterial detection method, and a food pathogenic bacteria detection method. The retroreflective signal analysis-type biosensor chip for bacterial detection may comprise: a fluid chip including a flow path therein; an injection unit which is connected to the flow path and injects a sample to be detected including retroreflective particles modified with a first antibody capable of binding to bacteria to be detected; and a detection unit in which a second antibody capable of binding to the bacteria to be detected is immobilized on the inner surface of the flow path.

Description

Retroreflective signal analysis type bacteria detection biosensor chip, food pathogen detection chip, retroreflective signal analysis type bacteria detection method and food pathogen detection method

[0001] The present invention relates to a retroreflective signal analysis-based bacteria detection biosensor chip, a food pathogen detection chip, a retroreflective signal analysis-based bacteria detection method, and a food pathogen detection method.

[0002] Specifically, the present invention relates to a field diagnostic pathogenic bacteria detection system using a biosensor chip for retroreflective signal analysis and a method for quantitatively analyzing the number of pathogenic bacteria using the same. Specifically, the present invention relates to the development of a detection system capable of detecting and quantitatively analyzing bacteria using a simple optical device by introducing a newly developed biosensor chip for signal detection and a retroreflective Janus Particle (RJP) as an optical marker to overcome the limitations of conventional bacteria detection methods (selective culture medium method, PCR, ELISA) in field diagnosis.

[0003] Conventional techniques for detecting pathogenic bacteria include selective medium culture, which cultures bacteria on selective media and visually observes the number of colonies formed on the media; enzyme-linked immunosorbent assay (ELISA), which analyzes antigen concentration through an antigen-antibody reaction using an enzyme bound to an antibody; and polymerase chain reaction (PCR), which amplifies a specific DNA sequence to analyze the bacterial concentration in a sample. While these methods can detect and quantitatively analyze pathogenic bacteria, they are time-consuming and require complex optical measurement equipment to ensure high sensitivity and accuracy. To overcome these limitations, simpler methods such as lateral flow immunoassay have been developed. However, these methods require additional optical analysis equipment to quantitatively analyze bacterial counts and have relatively low detection sensitivity. Therefore, a novel method for quantitatively analyzing pathogenic bacterial counts that enables rapid and accurate testing in the field is needed.

[0004] One object of the present invention is to provide a retroreflective signal analysis-based bacterial detection biosensor chip capable of qualitatively and quantitatively analyzing whether or not a pathogenic bacteria is infected in a space-limited environment, and a retroreflective signal analysis-based bacterial detection method using the same.

[0005] Another object of the present invention is to provide a food pathogen detection chip capable of determining whether food poisoning bacteria are present in food and detecting them with high sensitivity, and a method for detecting food pathogens using the chip.

[0006] Specifically, the novel biosensor chip for retroreflective signal analysis proposed in the present invention has a detection unit capable of capturing pathogenic bacteria-particle complexes and a control unit capable of capturing residual particles that have not reacted with pathogenic bacteria, similar to a lateral flow immunoassay. At the same time, the biosensor chip is composed of a transparent material, so that signals from retroreflective particles captured in each unit can be obtained using a simple, non-spectral optical system. Because retroreflective particles have a special mirror structure that reflects light with a wavelength smaller than the particle size toward the light source, unlike conventional fluorescent / colorimetric particles, they exhibit strong signals without attenuation even under non-spectral conditions. By utilizing this characteristic, a transparent biosensor chip that can transmit light can be fabricated, thereby constructing a simple measurement system that can be used in the field. In addition, since the optical signal of retroreflective particles appears in the form of bright light spots on the image, these can be counted and quantitatively analyzed. In the case of the above-mentioned various pathogenic bacteria detection methods, highly sensitive and complex equipment is required for signal detection, whereas the proposed system uses white light as a light source and retroreflective particles as signal markers, enabling the implementation of a non-spectroscopic signal detection system, and thus it is expected to be easy to enter the diagnostic testing market.

[0007] In one aspect, the present invention provides a retroreflective signal analysis type bacteria detection biosensor chip, comprising: a fluid chip including a flow path therein; an injection unit connected to the flow path and injecting a detection target sample including a retroreflective particle modified with a first antibody capable of binding to a detection target bacteria; and a detection unit in which a second antibody capable of binding to the detection target bacteria is fixed to the inner surface of the flow path.

[0008] In one embodiment, the retroreflective signal analysis type bacterial detection biosensor chip may further include a control portion in which a third antibody capable of binding to the retroreflective particle is fixed to the inner surface of the channel.

[0009] In one embodiment, the first antibody and the second antibody may comprise the same antibody.

[0010] In one embodiment, the first antibody and the second antibody may be fixed to a surface facing the injection portion within the euro.

[0011] In another aspect, the present invention provides a food pathogen detection chip including a retroreflective signal analysis type bacteria detection biosensor chip.

[0012] In one embodiment, the target bacteria may include Salmonella bacteria.

[0013] In another aspect, the present invention provides a method for detecting bacteria using a retroreflective signal analysis method, comprising: a fluid chip including a channel therein; an injection unit connected to the channel and injecting a sample to be detected, the sample including a retroreflective particle modified with a first antibody capable of binding to a target bacteria; and a detection unit having a second antibody capable of binding to the target bacteria fixed to the inner surface of the channel; and a sample injection step of injecting the sample to be detected into the injection unit of the biosensor chip and causing it to flow within the channel.

[0014] In one embodiment, the biosensor chip may be prepared by further including a control portion in which a third antibody capable of binding to the retroreflective particle is fixed to the inner surface of the euro.

[0015] In one embodiment, the first antibody and the second antibody may be prepared to include the same antibody.

[0016] In one embodiment, the first antibody and the second antibody may be prepared to be fixed to a surface facing the injection portion within the euro.

[0017] In one embodiment, the retroreflective signal analysis method for detecting bacteria may further include a light analysis step of irradiating light onto the detection unit or the control unit, and then detecting and analyzing light reflected from the retroreflective particles coupled to the detection unit or the control unit to quantitatively analyze the target bacteria.

[0018] In one embodiment, the light can be irradiated from the outside of the surface on which the first antibody and the second antibody are fixed.

[0019] In one embodiment, the direction of irradiating the light and the direction of detecting the light can be irradiated and detected at an angle of 60° or less.

[0020] In another aspect, the present invention provides a method for detecting pathogenic bacteria in food, including a food pathogenic bacteria detection step for detecting pathogenic bacteria in food using the retroreflective signal analysis method.

[0021] In one embodiment, the target bacteria may include Salmonella bacteria.

[0022] Specifically, a sandwich-type sensing technique was applied to enable antigen-antibody-based bacterial detection by immobilizing antibodies that recognize bacteria as antigens on retroreflective particles and modifying the chip surface, which corresponds to the detection area, with antibodies that recognize the same bacteria as antigens. In addition, a secondary antibody that recognizes the antibodies modified with retroreflective particles as antigens was immobilized on the chip surface, which corresponds to the control area, so that bacterial detection and the effectiveness of the chip can be confirmed simultaneously. When a sample is injected into the microfluidic channel of the biosensor chip, a retroreflective signal appears in the shape of a circle based on the contrast between the detection area and the surrounding area depending on the presence or absence of bacteria. By counting retroreflective particles, quantitative analysis according to the bacterial concentration is possible. Unlike conventional pathogenic bacteria detection methods that require complex measuring and analyzing equipment, this system uses retroreflective particles as optical probes, enabling highly sensitive quantitative bacterial analysis using only an optical device consisting of white light and a CMOS camera without the need for a spectral filter.

[0023] A retroreflective signal analysis biosensor chip according to an embodiment of the present invention can perform qualitative and quantitative analysis of pathogenic bacterial infection in a space-limited environment.

[0024] A food pathogen detection chip according to an embodiment of the present invention can determine whether food poisoning bacteria are present in food and detect them with high sensitivity.

[0025] The retroreflective signal analysis method according to an embodiment of the present invention can perform qualitative and quantitative analysis of pathogenic bacteria infection in a space-limited environment by using the retroreflective signal analysis method bacteria detection biosensor chip.

[0026] The method for detecting food pathogens according to an embodiment of the present invention can determine whether food poisoning bacteria are present in food and detect them with high sensitivity using the food pathogen detection chip.

[0027] According to the present invention, quantitative analysis of bacteria can be performed using a simple biosensor chip and retroreflective particles, unlike conventional pathogenic bacteria detection methods that require complex measuring equipment and long detection times.

[0028] FIG. 1 is a schematic diagram illustrating a retroreflective signal analysis type bacterial detection biosensor chip according to an embodiment of the present invention.

[0029] Figure 2 is a flowchart schematically illustrating a method for detecting bacteria using a retroreflective signal analysis method according to an embodiment of the present invention.

[0030] Figure 3 is a flowchart illustrating a method for detecting food pathogens according to an embodiment of the present invention.

[0031] Figure 4 is about the development of a biosensor chip for analyzing retroreflection signals and a method for quantitative analysis of pathogenic bacteria using the same.

[0032] Figure 5 is a schematic diagram of an example of constructing a biosensor chip by modifying an antibody on a chip substrate made of glass.

[0033] Figure 6 is a schematic diagram of a transparent microfluidic chip driving method for retroreflection signal analysis.

[0034] Figure 7 illustrates a method for analyzing a retroreflection signal using an image analysis program.

[0035] Figure 8 shows a schematic diagram of a method for detecting retroreflection signals according to the concentration of Salmonella typhimurium.

[0036] Figure 9 shows the results of retroreflection signal analysis according to the concentration of Salmonella bacteria.

[0037] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. The present invention may be modified in various ways and may take various forms. Therefore, specific embodiments are illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to a specific disclosed form, but should be understood to include all modifications, equivalents, and substitutes included in the spirit and technical scope of the present invention. In describing each drawing, similar reference numerals are used to indicate similar components. In the attached drawings, the dimensions of structures are shown larger than actual size to ensure clarity of the present invention.

[0038] The terminology used in this application is only used to describe specific embodiments and is not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this application, the terms "comprise" or "have" should be understood to indicate the presence of a feature, number, step, operation, component, or combination thereof described in the specification, but do not preclude the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, or combinations thereof.

[0039] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and will not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0040] FIG. 1 is a schematic diagram illustrating a retroreflective signal analysis type bacterial detection biosensor chip according to an embodiment of the present invention.

[0041] Referring to FIG. 1, a retroreflective signal analysis biosensor chip (100) according to an embodiment of the present invention may include a fluid chip (110) including a flow path (110') therein; an injection unit (120) connected to the flow path (110') and injecting a detection target sample (S) including a retroreflective particle (P) modified with a first antibody (not shown) capable of binding to a detection target bacteria (G); and a detection unit (130) in which a second antibody (130A) capable of binding to the detection target bacteria (G) is fixed to the inner surface of the flow path (110').

[0042] The fluid chip (110) includes a flow path (110') therein, and is a component that constitutes the main body of a retroreflective signal analysis type bacteria detection biosensor chip (100) according to an embodiment of the present invention. By including the fluid chip (110), the retroreflective signal analysis type bacteria detection biosensor chip (100) according to an embodiment of the present invention can define a surface on which an injection portion (120) into which a sample to be detected is injected, a flow path (110') through which the sample flows, and a detection portion (130) or control portion (140) to which a component of the sample can be combined are formed. The fluid chip (110) is not particularly limited in its structure or material as long as it performs the above-described function, but may have a hardness of a significant level or higher in order to ensure the stability of the component, and / or may be formed of a material having a significant level or higher transparency in whole or in part for optical analysis.

[0043] The above-described path (110') is a channel or passage-like member through which a sample to be analyzed by the retroreflective signal analysis type bacteria detection biosensor chip (100) according to an embodiment of the present invention flows. The shape or structure of the path (110') is not particularly limited as long as the sample can flow. In one embodiment, the fluid chip (110) may be extended in the longitudinal direction, and the path (110') may be extended in the same direction as the fluid chip (110). In one embodiment, the injection portion (120) may be formed on one side of the fluid chip (110) and / or may be formed at the beginning of the path (110'). The retroreflective signal analysis type bacteria detection biosensor chip (100) according to an embodiment of the present invention may include one or more paths (110'). In one embodiment, the one or more paths (110') may have different functions, for example, may detect different pathogens.

[0044] The above injection unit (120) is a member into which a sample for detecting bacteria can be injected by the retroreflective signal analysis type bacteria detection biosensor chip (100) according to an embodiment of the present invention. The structure or shape of the injection unit (120) is not particularly limited as long as it can inject a sample into the flow path (110').

[0045] The sample injected into the injection unit (120) is not an essential component defining the retroreflective signal analysis type bacteria detection biosensor chip (100) according to an embodiment of the present invention, but may include a retroreflective particle (P) in order to fully express the function of the retroreflective signal analysis type bacteria detection biosensor chip (100) according to an embodiment of the present invention. The retroreflective particle (P) may be modified with a first antibody (not shown), and the first antibody (not shown) may be combined with a detection target bacteria (G). The sample may or may not include the detection target bacteria (G), but since the retroreflective particle (P) modified with the first antibody (not shown) is included, when the detection target bacteria (G) are included in the sample, the retroreflective particle (P) and the detection target bacteria (G) may be combined.

[0046] The above detection unit (130) is a part of a retroreflective signal analysis type bacteria detection biosensor chip (100) according to an embodiment of the present invention to which a detection target bacteria (G) can bind in order to qualitatively and / or quantitatively detect the detection target bacteria (G) to be detected by the retroreflective signal analysis type bacteria detection biosensor chip (100) according to an embodiment of the present invention. The detection unit (130) may include a second antibody (130A). The second antibody (130A) may bind to the detection target bacteria (G).

[0047] Referring to the description of the first antibody (not shown) and the second antibody (130A), when the second antibody (130A) is fixed in the fluid chip (110) and exposed to a sample containing both the retroreflective particle (P) on which the first antibody (not shown) is modified and the target bacteria (G), since both the first antibody (not shown) and the second antibody (130A) bind to the target bacteria (G), a binding structure having the order of the second antibody (130A), the target bacteria (G), the first antibody (not shown), and the retroreflective particle (P) can be formed from the second antibody (130A) fixed to the fluid chip (110). The present invention is at least based on the discovery that qualitative and / or quantitative analysis of the target bacteria (G) is possible through the structure as described above.

[0048] The retroreflective signal analysis type bacteria detection biosensor chip (100) according to an embodiment of the present invention does not exclude the addition of additional components. In one embodiment, the retroreflective signal analysis type bacteria detection biosensor chip (100) may further include a control unit (140) in which a third antibody (140A) capable of binding to the retroreflective particle (P) is fixed to the inner surface of the passage (110').

[0049] The control unit (140) may include a third antibody (140A) capable of binding to the retroreflective particle (P). Therefore, when a sample containing the retroreflective particle (P) is normally injected through the injection unit (120), the excess retroreflective particle (P) that is not bound to the detection unit (130) binds to the control unit (140), thereby confirming that the sample containing the retroreflective particle (P) was normally injected through the injection unit (120).

[0050] The types of the first antibody (not shown) and the second antibody (130A) are not particularly limited, as long as they functionally bind to the target bacteria (G). In one embodiment, the first antibody (not shown) and the second antibody (130A) may comprise the same antibody. In one embodiment, the first antibody (not shown) and the second antibody (130A) may comprise different antibodies.

[0051] In one embodiment, the first antibody (not shown) and the second antibody (130A) may be fixed to the surface facing the injection portion (120) within the flow path (110'). By being arranged as described above, when the retroreflective signal analysis type bacterial detection biosensor chip (100) according to the embodiment of the present invention is performed with the injection portion (120) as the upper surface, the first antibody (not shown) and the second antibody (130A) may be fixed to the lower surface inside, thereby ensuring stability in the reaction, and the lower surface may be used for subsequent optical analysis.

[0052]

[0053] Meanwhile, a food pathogen detection chip according to an embodiment of the present invention may include the retroreflective signal analysis type bacteria detection biosensor chip described above.

[0054] The description of the food pathogen detection chip according to the embodiment of the present invention may be applied identically or similarly to the same or similar terms or configurations in the description of the retroreflective signal analysis type bacteria detection biosensor chip described above.

[0055] In one embodiment, the target bacteria may include Salmonella bacteria. That is, the food pathogen detection chip according to the embodiment of the present invention can detect Salmonella bacteria.

[0056] Figure 2 is a flowchart schematically illustrating a method for detecting bacteria using a retroreflective signal analysis method according to an embodiment of the present invention.

[0057] Referring to FIG. 2, the retroreflection signal analysis method (200) according to an embodiment of the present invention may include a sample injection step (S210) of injecting a detection target sample into an injection unit of a biosensor chip and causing it to flow within a flow path.

[0058] The description of the retroreflective signal analysis type bacteria detection method (200) according to the embodiment of the present invention may be applied identically or similarly to the same or similar terms or configurations in the description of the food pathogen detection chip described above and / or the description of the retroreflective signal analysis type bacteria detection biosensor chip described above. Accordingly, the biosensor chip may be a retroreflective signal analysis type bacteria detection biosensor chip according to the embodiment of the present invention described above and / or a food pathogen detection chip according to the embodiment of the present invention described above.

[0059] Accordingly, the method (200) for detecting bacteria using a retroreflective signal analysis method according to the embodiment of the present invention may include a sample injection step (S210) of injecting the detection target sample into the injection unit of a biosensor chip, which includes a fluid chip having a flow path therein; an injection unit connected to the flow path and injecting a detection target sample including a retroreflective particle modified with a first antibody capable of binding to the detection target bacteria; and a detection unit in which a second antibody capable of binding to the detection target bacteria is fixed to the inner surface of the flow path; and causing the detection target sample to flow within the flow path.

[0060] The above sample injection step (S210) is a step in which a detection target sample that may include a detection target bacteria is injected into an injection portion of a biosensor chip, and it is possible to determine whether the detection target bacteria exist in the detection target sample.

[0061] Since the biosensor chip may be a retroreflective signal analysis type bacteria detection biosensor chip according to the embodiment of the present invention described above and / or a food pathogen detection chip, in one embodiment, the biosensor chip may be prepared to further include a control portion in which a third antibody capable of binding to the retroreflective particle is fixed to the inner surface of the channel. In addition, in one embodiment, the first antibody and the second antibody may be prepared to include the same antibody. In addition, in one embodiment, the first antibody and the second antibody may be prepared to be fixed to a surface facing the injection portion within the channel.

[0062] The method (200) for detecting bacteria using a retroreflective signal analysis method according to an embodiment of the present invention does not exclude the addition of additional steps. In one embodiment, after the sample injection step (S210), the method (200) for detecting bacteria using a retroreflective signal analysis method according to an embodiment of the present invention may further include a washing step for washing away unbound retroreflective particles and target bacteria by flowing a washing solution in the flow path of the fluid chip.

[0063] In addition, in one embodiment, the retroreflective signal analysis method (200) for detecting bacteria may further include a light analysis step of quantitatively analyzing the target bacteria by detecting and analyzing light reflected from the retroreflective particles coupled to the detection unit or the control unit after irradiating light to the detection unit or the control unit.

[0064] The above-mentioned photoanalysis step is a step of at least quantitatively analyzing the target bacteria by irradiating light. The present invention is based at least on the discovery that quantitative analysis of target bacteria can be performed quickly, accurately, and at low cost without a separate spectroscopic device using retroreflective particles.

[0065] In the above-described light analysis step, as long as light analysis is possible, the direction of the light irradiation is not particularly limited. In one embodiment, the light may be irradiated from the outside of the surface on which the first antibody and the second antibody are fixed. In one embodiment, the direction of irradiating the light and the direction of detecting the light may be irradiated and detected at an angle of 60° or less.

[0066] Figure 3 is a flowchart illustrating a method for detecting food pathogens according to an embodiment of the present invention.

[0067] Referring to FIG. 3, a food pathogen detection method (300) according to an embodiment of the present invention may include a food pathogen detection step (S310) of detecting a pathogen detection target bacteria in food through a retroreflection signal analysis method (300).

[0068] The description of the food pathogen detection method (300) according to the embodiment of the present invention may be applied identically or similarly to the description of the retroreflective signal analysis type bacteria detection method (300) described above and / or the description of the food pathogen detection chip described above and / or the description of the retroreflective signal analysis type bacteria detection biosensor chip described above for the same or similar terms or configurations.

[0069] Therefore, the retroreflection signal analysis method for detecting bacteria (300) may be the retroreflection signal analysis method for detecting bacteria (300) according to the embodiment of the present invention described above.

[0070] In one embodiment, the target bacteria may include Salmonella bacteria. Therefore, Salmonella bacteria can be detected by the food pathogen detection method (300) according to an embodiment of the present invention.

[0071] Hereinafter, embodiments of the present invention will be described in detail. However, the embodiments described below are merely some embodiments of the present invention, and the scope of the present invention is not limited to the embodiments described below.

[0072] Figure 4 describes the development of a biosensor chip for analyzing retroreflective signals and a method for quantitatively analyzing pathogenic bacteria using the same. Existing methods such as selective media culture, ELISA, and PCR require long detection times and complex optical measurement equipment, limiting their use in fields where pathogenic bacteria are present. While simple lateral flow immunoassay-based bacterial detection methods exist that can detect pathogenic bacteria in the field, they are difficult to quantitatively analyze bacterial counts. Therefore, for rapid and accurate field diagnosis, the development of a method capable of confirming the presence of bacteria and quantitatively analyzing bacterial counts using simple equipment is necessary. In the present invention, retroreflective particles are introduced as markers, enabling the use of a simple non-spectral optical system as the analytical equipment. Furthermore, a microfluidic biosensor chip with a structure similar to that of lateral flow immunoassays is developed to enable simple and rapid measurement. The developed biosensor chip for analyzing retroreflective signals and the method for quantitatively analyzing pathogenic bacteria using the same are discussed, including the fabrication method of the biosensor chip and the quantitative analysis method for bacterial counts using retroreflective signal analysis.

[0073] Manufacturing example

[0074] To fabricate a biosensor chip for analyzing retroreflective signals for detecting target pathogenic bacteria, transparent glass or polymers (e.g., polycarbonate, poly(methyl methacrylate), polystyrene, polydimethylsiloxane) were used as the lower substrate of the chip. Reactive moieties are introduced into the detection and control regions of the lower substrate of the chip to immobilize antibodies that specifically bind to each target. Antibodies are immobilized by reacting the reactive moieties (e.g., amine, aldehyde, carboxylic acid) on the lower substrate with the reactive moieties on the antibody surface. When immobilizing antibodies, cross-linking agents such as bis(sulfosuccinimidyl)suberate (BS3), glutaraldehyde (GA), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) are introduced as a medium to enable antibody immobilization. In this process, the detection unit immobilizes an antibody targeting the target bacteria as an antigen to enable capture of retroreflective particle-pathogenic bacteria complexes, and the control unit immobilizes a secondary antibody capable of capturing antibodies on the surface of retroreflective particles to enable capture of any remaining single retroreflective particles. After antibody immobilization, the substrate is treated with ethanolamine, L-lysine, bovine serum albumin (BSA), etc. to prevent nonspecific binding due to reactive residues remaining on the substrate. A biosensor chip is manufactured by combining a chip lower plate where the detection unit and the control unit are formed and a chip upper plate where microfluidic channels are formed. At this time, transparent glass or a polymer (polycarbonate, poly(methyl methacrylate), polystyrene, polydimethylsiloxane, etc.) with high light transmittance, similar to the lower plate, can be used as the chip upper plate. Figure 5 is a schematic diagram of an example of constructing a biosensor chip by modifying an antibody on a chip lower plate made of glass.

[0075] Example

[0076] Chip operation method for quantitative analysis of bacterial counts

[0077] Figure 6 is a schematic diagram of the operating method of a transparent microfluidic chip for retroreflective signal analysis. Referring to Figure 6, a mixture of retroreflective particles and bacteria is injected into a transparent biosensor chip manufactured through the above method to induce an antigen-antibody reaction in the detection unit and control unit within the chip. The operating method is similar to the lateral flow immunoassay. The analyte and marker in the sample pass through the detection unit to the control unit, and finally exit through the outlet. During this process, if there are no bacteria in the sample, the retroreflective particles are captured only in the control unit and a signal is generated, and no signal is generated in the detection unit. Conversely, if bacteria are present in the sample, the retroreflective particle-pathogenic bacteria complex is first captured in the detection unit, and the remaining particles are captured in the control unit, so that signals are generated in both capture units. The signals are converted into light spots on an image using a CMOS camera, and the number of light spots can be analyzed to quantitatively analyze the number of bacteria in the sample.

[0078] Image processing for retroreflection signal analysis

[0079] Figure 7 illustrates a method for analyzing a retroreflection signal using an image analysis program.

[0080] Referring to Fig. 7, the retroreflection signal obtained through the biosensor chip for retroreflection signal analysis implemented in the present invention and the method for quantitative analysis of pathogenic bacteria using the same appears on the image in the form of a bright light spot due to the white light reflected by the retroreflection particles. The retroreflection signal is a light signal that is reflected and returned by a special mirror structure, and has a larger size than fluorescent / chromogenic molecules, so it shows a signal in the form of a light spot that can be counted, and thus concentration analysis of target bacteria is possible by counting the number of these. At the same time, retroreflection images can be obtained using a white LED as a light source and a CMOS camera as a light receiver without the need for a specific spectral filter, so quantitative analysis of bacteria is possible by analyzing the image obtained with a simple optical system in the field.

[0081] A basic retroreflective optical system is configured as shown in Figure 7. The chip to be measured is positioned parallel to the light receiver, and the angle of the white LED light source is positioned within 60° relative to the CMOS camera, which is the light receiver. In the optical system configured as described above, light from the light source is retroreflected by particles within the chip, reaches the CMOS camera, and is converted into an image signal. The area to be measured is enlarged to obtain a retroreflective image, which is then processed using an image analysis program.

[0082] Check the number of retroreflective signals

[0083] 1) Convert the original image acquired with a CMOS camera to a 32-bit image.

[0084] 2) Set the background threshold to 10 to reduce noise.

[0085] 3) Retroreflective particle count (A) with a threshold of 10 or more pixels appearing in the entire area of ​​the image converted to 32 bits.

[0086] 4) Set a circle with a diameter of 2800 pixels in each of the detection and contrast areas of the converted image.

[0087] 5) Retroreflective particle count (B) with a threshold of 10 or more pixels within the selection area.

[0088] 6) Quantitative analysis of Salmonella bacteria using a formula using the ratio of A and B in the detection area and A and B in the control area

[0089] Quantitative analysis of the number of Salmonella bacteria in a sample using a biosensor chip for retroreflective signal analysis

[0090] In an embodiment of the present invention, a field diagnostic detection system based on the retroreflective phenomenon was designed to detect food poisoning bacteria in food, and a detection unit and a control unit were configured inside the biosensor chip similar to a lateral flow immunoassay so that the results can be intuitively confirmed on a transparent microfluidic biosensor chip. In order to confirm the target pathogenic bacteria count analysis ability of the designed biosensor chip, an analysis was performed on Salmonella typhimurium, a food poisoning bacteria in food, in an embodiment. An antibody that recognizes an antigen (LPS, flagella, etc.) present on the surface of Salmonella as an antigen was immobilized on the detection unit of the retroreflective particle surface, which is an optical marker, and a secondary antibody that recognizes the antibody on the surface of the retroreflective particle as an antigen was immobilized on the control unit, thereby configuring the chip.

[0091] Figure 8 shows a schematic diagram of a method for detecting retroreflection signals according to the concentration of Salmonella typhimurium.

[0092] Referring to Fig. 8, the operating method begins with injecting a mixture of a sample and retroreflective particles into the inlet, similar to the lateral flow immunoassay. The analytes and markers in the sample pass through the detection unit to the control unit, and finally exit through the outlet. During this process, if there is no Salmonella in the sample, the retroreflective particles are captured only in the control unit and a signal is generated, and no signal is generated in the detection unit. Conversely, if there is Salmonella in the sample, the retroreflective particle-Salmonella complex is first captured in the detection unit, and the remaining particles are captured in the control unit, so that signals are generated in both capture units. The signals are converted into light spots on an image using a CMOS camera, and the number of Salmonella in the sample can be quantitatively analyzed through the above analysis method.

[0093] Detection and quantitative analysis of Salmonella using retroreflective signal analysis

[0094] In order to verify the practical feasibility of the Salmonella detection system developed through the present invention, retroreflective particles were introduced as optical signal markers, and the signal difference according to the Salmonella concentration was analyzed through the retroreflective signal count. The measurement range was from 0 CFU / mL, where no Salmonella was present, to 10 3 The concentration was measured up to CFU / mL. In order to detect the target bacteria, Salmonella typhimurium, the detection area of ​​the glass sensing surface was modified with an antibody that specifically reacts with Salmonella, and the control area was modified with a secondary antibody that reacts with the antibody modified with the retroreflective particle, thereby verifying the effectiveness of the detection chip through control and simultaneously configuring the chip to detect the target bacteria.

[0095] Figure 9 shows the results of retroreflective signal analysis according to Salmonella concentration. The left figure of Figure 9 shows an image of the analysis results using retroreflective particles, and the right figure shows a calibration curve resulting from the analysis using ImageJ. The data in the right figure are presented in the table below.

[0096] Salmonella concentration (CFU / mL) Retroreflective signal count ratio 0 17.8 ± 2.2 10 21.2 ± 2.3 10 2 33.2±3.610 3 40.5±3.6

[0097] Retroreflection images were acquired using an optical system consisting of a CMOS camera and a white LED light source, and retroreflection signals appearing as light spots were counted using an image analysis program. As a result, as the concentration of Salmonella increased, the number of particles observed in the detection area increased, and in the control area, the number of particles observed was constant regardless of the concentration of Salmonella. On the other hand, referring to Fig. 9, as the concentration of Salmonella present in the sample increased, it reacted with more retroreflective particles during pre-mixing, so the retroreflection signal measured by the overall non-specific reaction around the detection area and the control area in the measured image decreased. In order to reduce the signal deviation according to the Salmonella concentration, the formula (control B / control A)*(detection area B / detection area A)*100 was applied to perform quantitative analysis of Salmonella. Finally, a calibration curve according to the concentration of Salmonella was obtained. The graph on the right side of Fig. 9 shows the average value of the results obtained through three repeated experiments of the retroreflection signals appearing in the control and detection sections according to the concentration of Salmonella. Through the invented biosensor chip for analyzing retroreflection signals and the method for quantitative analysis of pathogenic bacteria using the same, bacteria could be detected up to a concentration of 10 CFU / mL. The biosensor chip developed in the present invention is expected to be advantageously applied to field diagnosis in that it can measure with high sensitivity in a short time without requiring complicated equipment or procedures used in existing bacteria detection methods through PCR or ELISA.

[0098] Although the present invention has been described above with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and changes may be made to the present invention without departing from the spirit and scope of the present invention as set forth in the claims below.

[0099] [Explanation of symbols]

[0100] 100: Retroreflective signal analysis type fungal detection biosensor chip

[0101] 110: Fluid chip

[0102] 110': Euro

[0103] 120: Injection

[0104] 130: Detection unit

[0105] 130A: Second antibody

[0106] 140: Contrast

[0107] 140A: Third antibody

[0108] P: Retroreflective particle

[0109] G: Target bacteria for detection

[0110] S: Detection target sample

[0111] 200: Retroreflective signal analysis method for detecting bacteria

[0112] 300: Method for detecting food pathogenic bacteria

Claims

1. Fluid chip containing a euro inside; An injection unit for injecting a detection target sample, which is connected to the above-mentioned euro and includes a retroreflective particle modified with a first antibody capable of binding to a detection target bacteria; A detection unit including a second antibody capable of binding to the target bacteria fixed to the inner surface of the urea; Retroreflective signal analysis biosensor chip for bacterial detection.

2. In paragraph 1, The above retroreflective signal analysis type bacterial detection biosensor chip further includes a control part in which a third antibody capable of binding to the retroreflective particle is fixed to the inner surface of the euro; Retroreflective signal analysis biosensor chip for bacterial detection.

3. In paragraph 1, The first antibody and the second antibody comprise the same antibody, Retroreflective signal analysis biosensor chip for bacterial detection.

4. In paragraph 1, The first antibody and the second antibody are fixed to the surface facing the injection portion in the euro. Retroreflective signal analysis biosensor chip for bacterial detection.

5. A biosensor chip comprising a retroreflective signal analysis method for detecting bacteria according to any one of claims 1 to 4. Food pathogen detection chip.

6. In paragraph 5, The above detection target bacteria include Salmonella bacteria. Food pathogen detection chip.

7. A biosensor chip comprising a fluid chip including a flow path therein; an injection unit for injecting a detection target sample including a retroreflective particle modified with a first antibody capable of binding to a detection target bacterium, which is connected to the flow path; and a detection unit in which a second antibody capable of binding to the detection target bacterium is fixed to the inner surface of the flow path; a sample injection step for injecting the detection target sample into the injection unit and causing it to flow within the flow path; Retroreflective signal analysis method for detecting bacteria.

8. In paragraph 7, The above biosensor chip is prepared by further including a control portion in which a third antibody capable of binding to the retroreflective particle is fixed to the inner surface of the euro; Retroreflective signal analysis method for detecting bacteria.

9. In paragraph 7, The first antibody and the second antibody are prepared to contain the same antibody, Retroreflective signal analysis method for detecting bacteria.

10. In paragraph 7, The first antibody and the second antibody are prepared to be fixed to the surface facing the injection portion in the euro. Retroreflective signal analysis method for detecting bacteria.

11. In paragraph 7, The above retroreflective signal analysis method for detecting bacteria further includes a light analysis step of irradiating light to the detection unit or the control unit, and then detecting and analyzing light reflected from the retroreflective particles coupled to the detection unit or the control unit to quantitatively analyze the target bacteria. Retroreflective signal analysis method for detecting bacteria.

12. In paragraph 11, The above light is irradiated from the outside of the surface on which the first antibody and the second antibody are fixed. Retroreflective signal analysis method for detecting bacteria.

13. In paragraph 11, The direction of irradiating the light and the direction of detecting the light are irradiated and detected so as to have an angle of 60° or less. Retroreflective signal analysis method for detecting bacteria.

14. A food pathogen detection step for detecting pathogenic bacteria in food using a retroreflective signal analysis method according to any one of Articles 7 to 13; including; Method for detecting food pathogens.

15. In paragraph 14, The above detection target bacteria include Salmonella bacteria. Method for detecting food pathogens.

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