KR20190089636A - Multiplexing Method Of Micro particle having theSuperparamagnetic Nanoparticle Pattern - Google Patents

Abstract

The present invention provides a diagnosing method for detecting bacteria in a short time using micro particles and detecting various bacteria simultaneously. According to an embodiment of the present invention, a multiplexing method for a super-paramagnetic nanoparticle pattern includes: a step of preparing a super-paramagnetic nanoparticle solution in which the super-paramagnetic nanoparticles are dispersed in a resin; a step of providing an alignment axis by aligning the super-paramagnetic nanoparticles in an outer magnetic field direction of encoding after injecting the super-paramagnetic nanoparticle solution into a micro-channel and applying an outer magnetic field of the encoding; a step of forming the super-paramagnetic nanoparticle pattern by locally hardening the super-paramagnetic nanoparticle solution with the alignment axis to have a predetermined pattern in the micro-channel; a step of preparing the super-paramagnetic nanoparticle patterns having the different alignment axes; and a step of individually aligning the alignment axis of the super-paramagnetic nanoparticle patterns in the outer magnetic field direction of decoding by exposing the super-paramagnetic nanoparticle patterns to an outer magnetic field of the decoding in a predetermined direction.

Description

TECHNICAL FIELD [0001] The present invention relates to a method of multiplexing microparticles having a magnetic nanoparticle pattern,

The present invention relates to a multiplex analysis method using microparticles having a specific direction within a magnetic field, and more particularly, to a method for analyzing multiplexed microparticles by attaching capture antibodies corresponding to microparticles, exposing the microparticles to pathogens, To a method for detecting pathogens at the same time.

Magnetic particles are used in various bio-studies because they can be adjusted to fix or move using only a magnetic field without any additional machine. Magnetic particles have been developed in various sizes from several nanometers to micrometers and are commonly used as analytical mediums for separating biosamples. Recently, they have been used in various ways such as cell counter or drug delivery system.

A Korean patent (KR 10-1196185 B1) discloses a method of manufacturing a structure having a controlled magnetic axis. In addition, a Korean patent (KR 10-1072187 B1) discloses a method of magnetically controlling a magnetic structure. However, a Korean patent (KR 10-1196185 B1) only discloses a structure having a controlled magnetic axis, and a Korean registered patent (KR 10-1072187 B1) discloses a color-coded magnetic structure.

However, the color-coded magnetic structure distinguishes the probe region from the code region. The color characteristic of the code region affects the color characteristic of the probe region by the code region and locally affects the fluorescence characteristic only in the probe region. An additional process for forming the sample is required.

Accordingly, there is a need for a new diagnostic method capable of labeling a sample without distinguishing between a code region and a probe region.

Disclosure of Invention Technical Problem [8] The present invention provides a diagnostic method for simultaneously detecting microorganisms in a short period of time using microparticles.

Disclosure of Invention Technical Problem [8] The present invention provides a labeling method or a multiplexing method using microparticles containing superparamagnetic nanoparticles aligned in a specific direction.

According to an embodiment of the present invention, there is provided a method of superimposing a superpowder nanoparticle pattern, comprising: preparing a superpowder nanoparticle solution in which superpowder nanoparticles are dispersed in a resin; Injecting the superpowder nanoparticle solution into a microchannel and applying an encoding external magnetic field to align the superpowder nanoparticles in the direction of the encoded external magnetic field to provide an alignment axis; Curing the superpowder nanoparticle solution having the alignment axis in the microchannel to prepare superparamagnetic nanoparticles patterns having different alignment axes; And exposing the superpowder nanoparticle patterns to a decoding external magnetic field in a specific direction to align the alignment axes of the superpowder nanoparticle patterns in the decoding external magnetic field direction.

In one embodiment of the present invention, each of the super-magnetic nanoparticle patterns may have the same shape and different alignment axes.

In one embodiment of the present invention, the superficial magnetic nanoparticle patterns are in the form of a film, the alignment axes of the superpowder nanoparticle patterns are disposed in the plane of the film, and the alignment axis of the superpowder nanoparticle patterns is And may have a geometric reference axis to determine the rotation angle of the alignment axis.

In one embodiment of the present invention, the step of curing the super-fine magnetic nanoparticle solution having the alignment axis in the microchannel to prepare super-magnetic nanoparticle patterns having different alignment axes may include changing the direction of the encoding magnetic field The superpowder magnetic nanoparticle solution may be cured to have a predetermined pattern so as to form the superpowder nanoparticle patterns corresponding to the direction of the encoding magnetic field.

In one embodiment of the present invention, the step of curing the super-fine magnetic nanoparticle solution with the alignment axis in the microchannel to prepare the super-magnetic nanoparticle patterns having different alignment axes comprises: And the super micro magnetic nano patterns corresponding to the rotation angle of the microchannel can be formed by curing the microchannel while rotating the microchannel in a predetermined pattern.

In one embodiment of the present invention, the step of curing the super-fine magnetic nanoparticle solution with the alignment axis in the microchannel to prepare the super-magnetic nanoparticle patterns having different alignment axes comprises: The super-high magnetic nano-particle solution may be cured and then cut to correspond to the rotation angle of the predetermined pattern to form super-strong magnetic nano-patterns.

In one embodiment of the present invention, the step of curing the super-fine magnetic nanoparticle solution having the alignment axis in the microchannel to prepare the super-magnetic nanoparticle patterns having different alignment axes comprises: The direction of the microchannel is fixed and the superficial magnetic nano patterns corresponding to the rotation angle of the predetermined pattern can be formed by curing by rotating the predetermined pattern.

According to an embodiment of the present invention, there is provided a pathogen multiplexing analysis method comprising: preparing capture antibodies and detection antibodies that respectively react with reference pathogenic fungi; Preparing superparamagnetic nanoparticles patterns having different alignment axes; Attaching capture antibodies corresponding to each of the superparamagnetic nanoparticle patterns having different alignment axes; Immersing the supernatant nanoparticle patterns having different alignment axes attached with different capture antibodies in an analytical sample containing at least one of the reference pathogenic fungi to deposit at least one of the reference pathogenic fungi Attaching; Attaching a detection antibody having a fluorescent substance to react with the reference pathogenic bacteria attached to the super-magnetic nanoparticle patterns; And exposing the superpowder nanoparticle patterns to a decoding external magnetic field in a specific direction to align the alignment axes of the superpowder nanoparticle patterns in the decoding external magnetic field direction.

In one embodiment of the present invention, each of the super-magnetic nanoparticle patterns may have the same shape and different alignment axes.

In one embodiment of the present invention, the superficial magnetic nanoparticle patterns are in the form of a film, the alignment axes of the superpowder nanoparticle patterns are disposed in the plane of the film, and the alignment axis of the superpowder nanoparticle patterns is And may have a geometric reference axis to determine the rotation angle of the alignment axis.

In one embodiment of the present invention, the intensity of light generated from the fluorescent material of the detection antibody is detected in a state in which the alignment axes of the super-magnetic nanoparticle patterns are aligned along the decoding external magnetic field, The method comprising the steps of:

In one embodiment of the present invention, the intensity of light generated from the fluorescent material of the detection antibody is detected in a state in which the alignment axes of the super-magnetic nanoparticle patterns are aligned along the decoding external magnetic field, The method comprising: obtaining a fluorescence image of the super-magnetic nanoparticle patterns using a microscope; Extracting a rotation angle between the alignment axis and the geometric reference axis for each of the super-strong magnetic nanoparticle patterns in the fluorescence image; Extracting intensity of light emitted from each of the super-magnetic nanoparticle patterns; And extracting the concentration of the pathogen using the relationship between the intensity of the light and the concentration of the pathogen depending on the rotation angle of the super-magnetic nanoparticle patterns.

In one embodiment of the present invention, the pathogenic bacteria may include at least one of Bacillus subtilis, Salmonella typhimurium, Staphylococcus aureus, Escherichia coli O157: H7. The capture antibodies may include at least one of anti-Bacillus subtilis, anti-Salmonella typhimurium, anti-Staphylococcus aureus, and anti-Escherichia coli O157: H7. The detection antibodies may include at least one of anti-Bacillus subtilis-FITC, anti-Salmonella typhimurium-FITC, anti-Staphylococcus aureus-FITC, and anti-Escherichia coli O157: H7-FITC.

According to an embodiment of the present invention, there is provided a method of preparing a nanoparticle nanoparticle solution, comprising: preparing a supernatant nanoparticle solution in which supernatant nanoparticles are dispersed in a resin;  Injecting the superpowder nanoparticle solution into a microchannel and applying an encoding external magnetic field to align the superpowder nanoparticles in the direction of the encoded external magnetic field to provide an alignment axis; And locally curing the supernatant nanocrystal solution having the alignment axis so as to have a specific pattern in the microchannel to form a supernatant nanoparticle pattern.

The multiplexing analysis set according to an embodiment of the present invention includes a plurality of super-magnetic nanoparticle patterns of the same shape. Each of the plurality of super-magnetic nanoparticle patterns has a different alignment axis. The alignment axes are axes in which the superparamagnetic nanoparticles are arranged in a chain structure in each of the superpowder nanoimpact patterns. Wherein the superparamagnetic nanoparticle patterns are in the form of a film and the alignment axes of the superpowder nanoparticle patterns are disposed in the plane of the film, and the alignment axes of the superpowder nanoparticle patterns rotate in accordance with the decoded external magnetic field, And has a geometric reference axis for discrimination.

According to one embodiment of the present invention, detection of pathogenic bacteria is possible with a small production cost. Microparticles containing super-magnetic nanoparticles aligned in a specific direction are easily fabricated using an external magnetic field and photopolymerization, and the microparticles are aligned with their alignment axis directions in a certain magnetic field without additional equipment It can serve as an encoder or label for storing information. When each microparticle is reacted with a sample containing several pathogenic bacteria by attaching the capture antibodies of different pathogenic bacteria to each other, the presence of pathogenic bacteria in one sample can be confirmed simultaneously.

FIG. 1 is a flowchart illustrating a method of superimposing a super-magnetic nanoparticle pattern according to an embodiment of the present invention.
FIG. 2 illustrates a method of fabricating a microchannel structure according to an embodiment of the present invention. Referring to FIG.
3 is a conceptual diagram illustrating a microfluidic device according to an embodiment of the present invention.
FIGS. 4A to 4D are views showing the alignment state of the super-magnetic nanoparticles according to the direction of the encoded external magnetic field and the case where no encoding external magnetic field is present.
FIG. 5 is a plan view illustrating superparamagnetic nanoparticle patterns according to another embodiment of the present invention. FIG.
Fig. 6 shows a state in which the microparticles (or super-magnetic nanoparticle patterns) of Fig. 5 are aligned according to the decoding external magnetic field Hdec.
7 is a view for explaining super-magnetic nanoparticle patterns according to another embodiment of the present invention.
8 is a conceptual diagram illustrating a method for analyzing pathogens according to another embodiment of the present invention.
FIG. 9 is a view showing a state after super-magnetic nanoparticle patterns are rotated under a decoded external magnetic field according to still another embodiment of the present invention. FIG.
10 is an experimental result showing the result of the advertisement purification process under various conditions.
Fig. 11 shows fluorescence images according to reaction time of pathogens.
12 is a graph showing the intensity of the fluorescence image of FIG. 11 according to the reaction time.
Figure 13 is fluorescence images according to the concentration of pathogens.
FIG. 14 is a graph showing the intensity of the fluorescence image of FIG. 13 according to the concentration of pathogens.
Figure 15 is a light microscope image showing cross-reactivity between a specific capture antibody and non-responding pathogen.
16 is the fluorescence intensity extracted from the glare microscope image of Fig.
17 is a fluorescence microscope image showing multiplex analysis according to another embodiment of the present invention.
FIG. 18 is a summary of the multiplex analysis result of FIG.

Conventional detection of pathogenic bacteria is carried out using a method of culturing bacteria, but this method is time-consuming and has a low detection limit and the disadvantage of diagnosing one bacterium in a single sample. Many biosensors have been developed for the purpose of diagnosis of pathogenic bacteria. Examples of typical diagnostic techniques include electrochemistry, surface-enhanced Raman scattering (SERS), and fluorescence resonance energy transfer (FRET).

In the detection technology using microparticles serving as a barcode among technologies to be developed, there is a need for a diagnostic technique for detecting bacteria simultaneously and for detecting various bacteria simultaneously.

The microparticles used in these techniques can now be manufactured in a variety of ways and are capable of simultaneous detection with low sensitivity detection limits, but they have drawbacks in that they require complex optical procedures for complex fabrication processes, high cost and detection confirmation. .

The present invention provides microparticles comprising superparamagnetic nanoparticles aligned in a particular direction. Each of the microparticles is labeled along the alignment axis of the supernatant nanoparticles. Attach different capture antibodies to each of the microparticles labeled with different alignment axes. The capture antibody attached to each microparticle reacts with a specific bacterium, and a specific bacterium can again bind with a detection antibody to exhibit fluorescence characteristics. In addition, each microparticle is aligned in the direction of the alignment axis labeled by applying a decoded external magnetic field. Thus, by detecting the rotation angle of the labeled alignment axis, it can be confirmed which capture antibody or detection antibody is attached.

According to an embodiment of the present invention, a differentiating method capable of performing a multiplexing assay using microparticles containing super-magnetic nanoparticles aligned in a specific direction is presented.

According to an embodiment of the present invention, the microparticles including the super-magnetic nanoparticles aligned in a specific direction can simultaneously detect a plurality of bacteria in one sample. In the case of bacterial diseases, the causative organism may be not only one kind of bacteria, but also various kinds of causative bacteria. When using the present invention, several bacteria can be detected simultaneously in one sample of a patient.

The pathogen analysis method according to an embodiment of the present invention can dramatically reduce inspection time and cost in a hospital diagnosing disease caused by pathogenic bacteria. The pathogen analysis method requires only a simple device capable of producing a fluorescent material of a wavelength and a constant magnetic field, so that it can be diagnosed simply without additional equipment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, exemplary embodiments of the present invention will be described with reference to the accompanying drawings. The following examples and results are provided so that the disclosure of the present invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Also, for convenience of explanation, the components may be exaggerated or reduced in size.

FIG. 1 is a flowchart illustrating a method of superimposing a super-magnetic nanoparticle pattern according to an embodiment of the present invention.

Referring to FIG. 1, a method of superimposing a superpowder nanoparticle pattern includes: preparing a superpowder nanoparticle solution in which superpowder nanoparticles are dispersed in a resin (S110); (S120) of injecting the supernatant magnetic nanoparticle solution into a microchannel and then applying an encoding external magnetic field to align the superpowder nanoparticles in the direction of the encoded external magnetic field to provide an alignment axis; (S130) forming the supercritical nanoparticle pattern by locally curing the supernatant nanocrystal solution having the alignment axis so as to have a specific pattern in the microchannel; Preparing (S140) superparamagnetic nanoparticles patterns having different alignment axes; And a step (S150) of exposing the super-magnetic nanoparticle patterns to a decoding external magnetic field in a specific direction to align the alignment axes of the super-magnetic nanoparticle patterns in the decoding external magnetic field direction.

Each of the super-magnetic nanoparticle patterns may have the same shape and different alignment axes. Wherein the superparamagnetic nanoparticle patterns are in the form of a film and the alignment axes of the superpowder nanoparticle patterns are disposed in the plane of the film, and the alignment axes of the superpowder nanoparticle patterns rotate in accordance with the decoded external magnetic field, You can have a geometric reference axis to identify it.

Prepare a supernatant magnetic nanoparticle solution in which the supernatant magnetic nanoparticles are dispersed in a resin. The resin is a monomer and may be a photocurable resin. The superpowder nanoparticles may have a diameter of 200 nm. The superparamagnetic nanoparticles may be composed of a magnetite core and a polyethylene glycol matrix.

The superpowder nanoparticle solution may be injected into a microchannel and then an encoding external magnetic field may be applied to align the superpowder nanoparticles in the direction of the encoded external magnetic field to provide an alignment axis.

Linear microchannels can be made using polydimethylsiloxane (PDMS). Specifically, microchannels are formed using PDMS having gas permeability. The inside of the flat microchannel is made of PDMS using the cover glass coated with PDMS.

A solution in which a monomer and superparamagnetic nanoparticles are mixed is prepared and the solution is injected into the microchannel and then the superposed magnetic nanoparticles are aligned with respect to the encoded external magnetic field using an encoded external magnetic field Thereby forming a structure having an alignment axis. The solution does not adhere to the microchannel surrounded by the PDMS constituting the microchannel.

The supernatant nanoparticle solution having the alignment axis is locally cured to have a specific pattern in the microchannel to form a supercritical nanoparticle pattern. After making a pattern of superparamagnetic nanoparticles aligned in a desired direction, a mask having the opening of the specific pattern is placed under the microchannel, and photopolymerization is performed using ultraviolet rays. As a result, the super-magnetic nanoparticle pattern having the alignment axis is fixed. Unexposed photocatalytic nanoparticle solutions are removed by wet etching.

Prepare superparamagnetic nanoparticle patterns with different alignment axes. For example, a plurality of super-magnetic nanoparticle patterns are generated while changing the direction of the encoding external magnetic field so as to have different alignment axes. The superficial magnetic nanoparticle patterns are labeled along the alignment axis.

And the super-magnetic nanoparticle patterns are exposed to a decoding external magnetic field in a specific direction to align the alignment axes of the super-magnetic nanoparticle patterns in the decoding external magnetic field direction. Labeled superparamagnetic nanoparticle patterns are classified by a decoding external magnetic field. That is, the alignment axis of the superpowder nanoparticle pattern is aligned in the direction of the decoded outer edge. The alignment axis of the super-fine magnetic nanoparticle patterns may have a geometric reference axis to rotate according to the decoded external magnetic field to determine the rotation angle of the alignment axis. The shapes of the super-magnetic nanoparticle patterns may not have a mirror-symmetric structure so that the rotation angle of the alignment axis can be discriminated even if they are inverted.

The alignment axis or directional microparticles (or superparamagnetic nanoparticle pattern) can be run through a sandwichimmuno assay to detect pathogenic bacteria. Capture antibodies of different microorganisms can be attached according to the labeling of the alignment axis of the superfine magnetic nanoparticle pattern.

In the present invention, the superparamagnetic nanoparticle pattern and the microparticles are mixed and used in the same constitution.

Different microbial capture antibodies are attached to microparticles (or superparamagnetic nanoparticle patterns) having directional or alignment axes at different angles using protein photoimmobilization. Microparticles (or superparamagnetic nanoparticle patterns) are all put into a sample containing several pathogenic bacteria. After a certain period of time, a detection antibody with a fluorescent substance is allowed to react with the microparticles. If a specific bacterium is present and attached to a capture antibody attached to the microparticle, the detection antibody is attached to the specific bacterium. Therefore, the microparticles exhibit fluorescence by the fluorescent substance of the detection antibody. On the contrary, if there is no specific bacterium in the sample, the specific bacterium does not react with the capture antibody and the detection antibody of microparticles, and thus does not show fluorescence. . Using this method, the presence or absence of bacteria can be known.

On the other hand, when the microparticles are placed under the decoding external magnetic field, the microparticles are aligned in the alignment axis direction. Each microparticle has a different alignment axis and a different capture antibody. Therefore, by detecting the rotation angle of the alignment axis of the microparticles, the microparticles are classified, and if the intensity of fluorescence of the microparticles is detected, presence or absence of a specific microbe and concentration can be confirmed.

This pathogen detection method has the alignment axis (or directionality) of the microparticles and detects the presence or absence of different microorganisms. These pathogen detection methods are as follows. Unlike conventional biosensors for detecting bacteria through microparticles, it is possible to detect fluorescence in one wavelength range without using various fluorescent materials. This pathogen detection method has a much lower cost and a simpler detection process.

Pathogenic or infectious diseases caused by bacteria, viruses, fungi, and parasites are important public health problems worldwide. Rapid, simple and accurate detection methods are urgent for screening several bacterial pathogens in situ. We present a new sensing method based on microparticles encoded by the alignment axis of aligned allparagraph superparamagnetic nanoparticles (SMNP) to detect multiple bacteria simultaneously. Encoded microparticles (or superparamagnetic nanoparticle patterns) with various magnetic axes (or alignment axes) are prepared using photo-curable polymers and superparamagnetic nanoparticles (SMNP) that can be aligned under an external magnetic field. The encoded microparticles (or superparamagnetic nanoparticle patterns) are arranged to rotate in the direction of the decoding external magnetic field because they have a particular magnetic axis (or alignment axis). Decoding By measuring the angle of rotation under each external magnetic field, the encoded microparticles can be decoded and used for multiplex analysis.

Specifically, four pathogens such as Bacillus subtillis, Salmonella typhimurium, Staphylococcus aureus and Escherichia coli O157: H7 were simultaneously analyzed. Using conjugated microparticles with corresponding capture antibodies, the concentration of each target pathogen was analyzed using fluorescent labels. As a result, we have successfully demonstrated multiplexing capabilities by quantitatively analyzing various pathogens simultaneously. This new pathogen detection method is a promising tool to detect various causes such as pathogens and prevent various diseases.

Foodborne diseases are a major concern for public health and are increasing worldwide. Foodborne diseases are generally inherently infectious, toxic and are caused by harmful factors such as pathogens, viruses and toxins in contaminated food or water. Foodborne pathogens can lead to severe diarrhea and fatal infections. Food safety depends on the rapid and sensitive detection of the risks that can enter food during food production, transportation, processing, storage and handling. Foodborne pathogens among a variety of causes, including chemicals, pathogenic bacteria, parasites, fungi, and viruses, are important tools in the serious development of food poisoning. Escherichia coli O157: H7, Campylobacter spp., Listeria monocytogenes, Salmonella enteritidis and the like are major global causes of food poisoning diseases.

Detection of foodborne pathogens is generally carried out using classical methods based on culture methods. Unfortunately, this method requires time-consuming procedures and several difficult steps, represents a limitation of rapid and sensitive detection, and is often used to detect a single type of bacteria in one sample. Above all, it is a problem that there is no ability to detect multiple food-borne pathogens at the same time, and it is necessary to solve them first. To overcome these limitations, various detection techniques such as electrochemistry, surface-enhanced Raman scattering (SERS), and quantum dot-based fluorescence resonance energy transfer (FRET) Many attempts have been made to detect pathogens as a single sample. However, since these methods mainly depend on the optical method for detection, there is a fundamental disadvantage in multiplexing capability or simultaneous detection due to technical constraints. Accordingly, there is an increasing need for alternative methods for improving multiplexing capabilities.

The present invention can simultaneously detect 2-4 bacterial pathogens showing sensitivity ranging from 10 < -10 > ⁵ CFU / ml.

Recently, encoded microparticles have been proposed as a diagnostic tool for rapid and multiplex assay platforms because of their advantages in detection and quantification. They can be processed in a variety of ways such as pectrometric, graphical, electronic and physical methods. Despite its excellent multiplexing capabilities, however, it has the disadvantage of requiring specific optical equipment for complex manufacturing processes and decoding. In order to overcome these disadvantages, encoded polymer microparticles including magnetic nanoparticles have been introduced into a multiplexed assay system.

In most cases the encoding capability is due to the optical properties of upconverting nanocrystals, quantum dots or color-barcoded magnetic microparticles. The problem remains that the number of encodings is limited by a combination of different materials.

In the present invention, polymeric microparticles encoded by aligned magnetic axes (or alignment axes) from aligned superparamagnetic nanoparticles (SMNP) are proposed. Encoded microparticles composed of superparamagnetic nanoparticles aligned with a UV curable polymer can be easily prepared by repeated exposure to the encoded external magnetic field and photopolymerization.

When aligned superparamagnetic nanoparticles are placed in the decoding external magnetic field, the encoded microparticles rotate and stop in the direction of the decoding external magnetic field because they have a particular magnetic axis (or alignment axis). The encoded microparticles can then be decoded simply by measuring their rotation angle. To demonstrate the feasibility of this new concept, we demonstrated multiplexing assays to detect pathogens using encoded microparticles. We successfully decoded the encoded microparticles at rotational angles, successfully classifying four types of pathogens concurrently, and quantitatively analyzed the concentration of the pathogen by the intensity of the fluorescent label.

According to an embodiment of the present invention, there is provided a pathogen multiplexing analysis method comprising: preparing capture antibodies and detection antibodies that respectively react with reference pathogenic fungi; Preparing a supernatant magnetic nanoparticle solution in which the supernatant magnetic nanoparticles are dispersed in a resin;  Injecting the superpowder nanoparticle solution into a microchannel and applying an encoding external magnetic field to align the superpowder nanoparticles in the direction of the encoded external magnetic field to provide an alignment axis; Locally curing the supernatant nanocrystal solution having the alignment axis so as to have a specific pattern in the microchannel to form a supernatant nanoparticle pattern; Attaching capture antibodies corresponding to each of the superparamagnetic nanoparticle patterns having different alignment axes; Immersing the supernatant nanoparticle patterns having different alignment axes, to which different capture antibodies are respectively attached, in a liquid sample containing a reference pathogenic bacterium; Attaching a detection antibody having a fluorescent substance to the liquid sample so as to react with the reference pathogenic bacteria; And And exposing the superpowder nanoparticle patterns to a decoding external magnetic field in a specific direction to align the alignment axes of the superpowder nanoparticle patterns in the direction of the decoding external magnetic field.

[matter]

The super-magnetic nanoparticle pattern used in the present invention is composed of superparamagnetic nanoparticles and a photocurable resin monomer solution. The superpowder magnetic nanoparticles may have a size of several to several hundred nanometers. The superparamagnetic nanoparticles may be composed of a magnetite core and a polyethylene glycol matrix.

 Photocurable resins may include poly (ethylene glycol) (700) diacrylate. The photocurable resin may comprise 10 weight percent (wt%) of photoinitiator. The photoinitiator may be 2,2-dimethoxy-2-phenylacetophenone (DMPA).

 4-Benzoyl benzylamine hydrochloride (Bz) was used as a photoinitiator to fix the protein to the surface of the microparticles. Streptavidin was used for identification of Bz function, and fluorescent biotin was used as detection label.

For the detection of pathogens, Bacillus subtilis, Salmonella typhimurium, Staphylococcus aureus, and Escherichia coli O157: H7 are prepared.

Anti-Bacillus subtilis, anti-Salmonella typhimurium, anti-Staphylococcus aureus, and anti-Escherichia coli O157: H7 are prepared as capture antibodies.

Anti-Bacillus subtilis-FITC, anti-Salmonella typhimurium-FITC, anti-Staphylococcus aureus-FITC and anti-Escherichia coli O157: H7-FITC are prepared as detection antibodies (or labeling substances).

[Microfluidic device for production of encoded microparticles]

The encoded microparticles according to the alignment of the superpowder magnetic nanoparticles are produced by a microfluidic device. The microfluidic device 100 may include a microchannel structure 110 and a UV curing device 120.

The microchannel structure 110 is manufactured by a molding process of polydimethylsiloxane (PDMS).

FIG. 2 illustrates a method of fabricating a microchannel structure according to an embodiment of the present invention. Referring to FIG.

3 is a conceptual diagram illustrating a microfluidic device according to an embodiment of the present invention.

Referring to Fig. 2 (a), a mold 10 is produced. The mold 10 may include a photoresist pattern 12 and a silicon wafer 11 formed using photolithography. The height of the photoresist pattern 12 may be about 100 micrometers and the length of the photoresist pattern 12 may be several hundred micrometers or more. The mold 10 can be formed in various ways.

Referring to FIG. 2 (b), a mixture of polydimethylsiloxane (PDMS) and a curing agent was poured into the mold 10 and thermally cured on a hot plate at 150 degrees Celsius for 15 minutes. The cured polydimethylsiloxane (PDMS) mixture can provide the patterned PDMS replica 13.

Referring to FIG. 2 (c), the patterned PDMS replica 13 is separated from the silicon wafer 11. The photoresist pattern 12 may be removed by oxygen plasma treatment.

Referring to Fig. 2 (d), the patterned PDMS replica 13 is bonded to the cover glass 15 including the PDMS coating layer 16 after oxygen plasma treatment.

The bonded microchannel structure 110 is heated for 10 minutes on a hot plate at 150 degrees Celsius for strong adhesion between the microchannel 14 and the PDMS coating layer 16.

Microchannel 14 is fabricated at about 100 [mu] m height for the production of the encoded microparticles. The microchannel 14 is filled with the supernatant nanoparticle solution 130 in which the supernatant nanoparticles 132 are dispersed in the resin. The microchannel 14 may include an inlet for receiving the supernatant nanoparticle solution 130 from the outside and an outlet for discharging the supernatant nanoparticle solution 130.

[Preparation of encoded microparticles]

 Superparamagnetic nanoparticles with a diameter of 200 nm are dispersed in a photocurable resin monomer solution at a concentration of 200 mg / ml.

The photocurable resin used was poly (ethylene glycol) (700) diacrylate (PEG-DA 700) and the photoinitiator was 10 wt% of 2,2-dimethoxy-2-phenylacetophenone (DMPA).

Under the encoding external magnetic field, the supercritical nanoparticles in the supernatant nanoparticle solution 130 may be aligned to form a chain-like structure along magnetic field lines. Based on this phenomenon, we produce microparticles encoded in the direction of the superparamagnetic nanoparticle chains.

Referring to FIG. 3, the microfluidic device 100 may include a microchannel structure 110 and a UV curing device 120.

The UV curing device 120 is composed of an ultraviolet source 121, a photomask 124, and an objective lens 122. Ultraviolet light passes through the photomask 124 and the stage 126 to the microchannel 14 to cause polymerization of the monomer solution. The microchannel 14 may include an inlet for receiving the supernatant nanoparticle solution 130 from the outside and an outlet for discharging the supernatant nanoparticle solution 130. The inlet may be supplied with the supernatant nanoparticle solution 130 from the outside through the hose 128.

The encoding magnetic field generators 142 and 144 may include two permanent magnets 142 and a permanent magnet alignment panel 144. The encoding magnetic field generators 142 and 144 can generate a uniform encoding magnetic field in the microchannel 14. [ The encoding magnetic field generators 142 and 144 may rotate at an arbitrary angle in the stage 124. [ The encoding magnetic field generators 142 and 144 may be modified to be comprised of electromagnets.

When the microchannel 14 is filled with the solution of super-magnetic nanoparticles 130 composed of the monomer solution and the super-magnetic nanoparticles, and then the microchannel 14 is exposed to the encoding external magnetic field, the super-magnetic nanoparticles are aligned in a predetermined direction . The superpowder magnetic nanoparticles are aligned in a linear chain in the direction of the encoded external magnetic field.

The supernatant nanoparticle solution 130 containing the superpowder magnetic nanoparticles may be cured to have a predetermined pattern by the UV curing apparatus 120. [ A photomask 124 is aligned with the microchannel 14 and exposed to UV light (? = 360 nm, 50 nm). The supernatant magnetic nanoparticle solution 130 is polymerized with a 1 second UV exposure at an intensity of 40 mW / cm ² .

FIGS. 4A to 4D are views showing the alignment state of the super-magnetic nanoparticles according to the direction of the encoded external magnetic field and the case where no encoding external magnetic field is present.

Referring to FIG. 4A, the supercritical nanoparticle solution 130 is filled in the microchannel, but the encoded external magnetic field is not applied. The superpowder magnetic nanoparticles 132 are randomly arranged without a specific alignment state.

Referring to FIG. 4B, an encoded external magnetic field Hx is applied in the longitudinal direction of the microchannel 14. The superpowder magnetic nanoparticles 132 are aligned in the direction of the encoded external magnetic field (Hx). The pattern of the photomask 124 is in an "L" shape, and the super-magnetic nanoparticle pattern cured by ultraviolet light has an "L" shape.

Referring to FIG. 4C, the encoded external magnetic field Hy is applied in a direction perpendicular to the longitudinal direction of the microchannel 14. The superpowder magnetic nanoparticles 132 are aligned in the direction of the encoded external magnetic field (Hy). The pattern of the photomask 124 is in an "L" shape, and the super-magnetic nanoparticle pattern cured by ultraviolet light has an "L" shape.

Referring to FIG. 4D, an encoded external magnetic field Hxy is applied in the direction of 45 degrees in the longitudinal direction of the microchannel 14. The superpowder magnetic nanoparticles 132 are aligned in the direction of the encoded external magnetic field (Hxy). The pattern of the photomask 124 is in an "L" shape, and the super-magnetic nanoparticle pattern cured by ultraviolet light has an "L" shape.

FIG. 5 is a plan view illustrating superparamagnetic nanoparticle patterns according to another embodiment of the present invention. FIG.

Referring to FIG. 5, the short exposure time of UV light ensures reproducible production of encoded microparticles (or super-magnetic nanoparticle patterns). The superparamagnetic nanoparticle patterns 200a to 200d having four kinds of individual magnetic axes (or alignment axes) have magnetic axis angles from 0 to 135 degrees at intervals of 45 degrees. Each of the super-magnetic nanoparticle patterns may have the same shape and different alignment axes. Wherein the superparamagnetic nanoparticle patterns are in the form of a film and the alignment axes of the superpowder nanoparticle patterns are disposed in a plane (xy plane) of the film, and the alignment axis of the superpowder nanoparticle patterns rotates in accordance with the decoded external magnetic field, You can have a geometric reference axis to determine the angle of rotation of the alignment axis. The reference axis may be x-axis.

Each of the superparamagnetic nanoparticle patterns 200a to 200d may have different angles between the alignment axis and the reference axis, respectively. Each of these superparamagnetic nanoparticle patterns 200a-200d may be implemented in various ways to adjust the angle between the encoding magnetic field and the geometric reference axis. Superparamagnetic nanoparticle solutions with alignment axes are cured in microchannels to produce superparamagnetic nanoparticle patterns with different alignment axes.

According to one embodiment, the direction of the encoding magnetic field is fixed, and the microchannel is rotated and cured in a predetermined pattern to form the super-magnetic nanoparticle patterns corresponding to the rotation angle of the microchannel. For example, a specific pattern of the microphone channel is cured in a specific pattern. Subsequently, the microchannel is rotated and the position to be cured is changed, and then the microchannel is cured in a predetermined pattern using ultraviolet rays at the changed position. Repeating this process produces superparamagnetic nanoparticle patterns with various alignment axes.

According to the modified embodiment, the direction of the encoding magnetic field is fixed, and the supernatant nano-particle pattern can be formed by curing the supernatant nanoparticle solution and then cutting to correspond to the rotation angle of the predetermined pattern. For example, all the supernatant nanoparticle solutions injected into the microchannel are cured simultaneously. The cured superporous nanoparticle film may then be subjected to a laser cutting process to provide the super-strong nano-particles patterns. The cured super high magnetic nanoparticle film is cut so that the angle between the geometrical reference axis and the alignment axis is different for each of the super high magnetic nano patterns.

According to another modified embodiment, the direction of the encoding magnetic field and the direction of the microchannel are fixed, and when the predetermined pattern is rotated, the super-magnetic nano patterns corresponding to the rotation angle of the predetermined pattern are formed by curing. For example, the rotation of the predetermined pattern may be performed by rotating a photomask or rotating an ultraviolet image forming a predetermined pattern. The rotation of the ultraviolet image can be accomplished by a scanning means such as a digital micromirror device without using a photomask.

Fig. 6 shows a state in which the microparticles (or super-magnetic nanoparticle patterns) of Fig. 5 are aligned according to the decoding external magnetic field Hdec.

The alignment axes of the superficial magnetic nanoparticle patterns 200a to 200d are rotated and aligned in the direction of the decoding external magnetic field Hdec.

7 is a view for explaining super-magnetic nanoparticle patterns according to another embodiment of the present invention.

Referring to FIG. 7 , each of the super-magnetic nanoparticle patterns (or microparticles) may have a size of 240 μm × 200 μm × 100 μm depending on the photomask and channel height. The encoded superparamagnetic nanoparticles patterns 300a-300d having magnetic axes (or alignment axes) of 0, 30, 60, and 90 degrees correspond to Bacillus subtillis, Salmonella typhimurium, Staphylococcus aureus, and Escherichia coli O157: H7 Respectively.

[Optical immobilization of proteins on microparticles]

4-Benzoyl benzylamine hydrochloride (Bz), which is capable of directly immobilizing the protein to bind the protein to the encoded superparamagnetic nanoparticle patterns 300a to 300d, is used as a linker A photoimmobilization process can be performed. To confirm the validity of this process, Streptavidin and fluorescent biotin were preferentially used. Benzoyl benzylamine hydrochloride (100 占 퐂 / ml) and Streptavidin (100 占 퐂 / ml) were mixed in phosphate buffered saline (PBS), and 200 占 퐇 of the mixed solution was added to the encoded super- .

They were irradiated with ultraviolet light (? = 365? 5 nm) for 20 minutes at an intensity of 18 mW / cm ² . The illuminated encoded microparticles were then washed with phosphate buffered saline and then reacted with fluorescent biotin (100 μg / ml) for 30 minutes at room temperature.

Next, the resulting microparticles were washed three times with phosphate buffered saline (PBS). The conjugated Streptavidin and fluorescent biotin were visualized by fluorescence microscopy to confirm the function of 4-benzoylbenzylamine hydrochloride (Bz).

For multiplexing analysis of pathogens, we applied this procedure to conjugate the captured microparticles and capture antibodies. At this time, 20 μg / ml of each capture antibody and 500 μg / ml of 4-benzoylbenzylamine hydrochloride (Bz) were used. As in the previous process, the mixed solution and each encoded microparticle were mixed and exposed to UV light (18 mW / cm ² ) for 20 minutes. Finally, to detect multiple pathogens, we kept them at 4 ° C. Each of the capture antibodies 310a to 310d may be anti-Bacillus subtilis, anti-Salmonella typhimurium, anti-Staphylococcus aureus, and anti-Escherichia coli O157: H7.

[Preparation of pathogens]

Bacillus subtilis, Salmonella typhimurium, Staphylococcus aureus and Escherichia coli O157: H7 were prepared for multiplex analysis. They were cultured in R2A medium for 24 hours at 25 ° C and then washed with sterile phosphate buffered saline (PBS).

To determine the concentration of pathogens, the absorbance was measured at a wavelength of 600 nm using a spectrophotometer. As a result, Bacillus subtilis, Salmonella typhimurium, Staphylococcus aureus and Escherichia coli O157: H7 in the absorbance is 1.80, 2.74, 2.71, and ^{2.06, 2.7ⅹ10 8, 1.2ⅹ10 8, 1.4ⅹ10} 8 and 2.6ⅹ10 ⁸ colony forming units (each colony -forming unit; CFU / ml), respectively.

[Multiplex analysis procedure for pathogen detection]

8 is a conceptual diagram illustrating a method for analyzing pathogens according to another embodiment of the present invention.

Referring to FIGS. 7 and 8, four types of pathogens 320a-320d of Bacillus subtillis, Salmonella typhimurium, Staphylococcus aureus, and Escherichia coli O157: H7 were grown on magnetic axes (or alignment) of 0, 30, 60, Axis) of each of the encoded super-magnetic nanoparticle patterns 300a-300d.

Reacted at a concentration of the pathogens corresponding to the encapsulated microparticles conjugated with each of the capture antibodies 310a to 310d at room temperature for 15 minutes and washed three times with phosphate buffered saline (PBS).

Each detection antibody (330a-330d) at 20 [mu] g / ml then reacted with the pathogen at the surface of the microparticles encoded at room temperature for 20 minutes. Each of the detection antibodies 330a to 330d is anti-Bacillus subtilis-FITC, anti-Salmonella typhimurium-FITC, anti-Staphylococcus aureus-FITC, and anti-Escherichia coli O157: H7-FITC.

 First, the standard curve of each pathogen was obtained by reacting the encoded microparticles with various concentrations of each pathogen through this process.

Next, we performed multiplex analysis using specific concentrations of each pathogen to assess reproducibility and multiplexing capabilities. The known concentration values were compared with the signals obtained from the standard curve.

[Decoding of encoded microparticles]

Encoded microparticles (or superparamagnetic nanoparticles patterns) having different magnetic axes exhibit specific motion along their magnetic axis. When randomly dispersed microparticles are exposed to a decoding external magnetic field in a specific direction, the microparticles rotate and stop so that the direction of magnetic field and magnetic field is the same.

Based on this principle, we determined the type of microparticles and performed multiple assays for the detection of pathogens.

Encoded microparticles 300a to 300d having different magnetic axes from 0 deg. To 90 deg. Are prepared at fourteen types of microparticles at intervals of 15 degrees.

Decoding When there is no external magnetic field, the microparticles do not move and have a random reference axis orientation.

FIG. 9 is a view showing a state after super-magnetic nanoparticle patterns are rotated under a decoded external magnetic field according to still another embodiment of the present invention. FIG.

Referring to Figure 9, the superparamagnetic nanoparticles pattern (300a ~ 300d) is in the decoded external magnetic field with a specific direction (H _dec), the superparamagnetic nanoparticles pattern (300a ~ 300d) arranged axis (or the magnetic axis) is And moved parallel to the direction of the magnetic field.

To avoid confusion in decoding, the shape of the microparticles may be in the "L" shape. The reference axis may be in a direction parallel to the geometric structure.

So we can easily find the direction of rotation using the reference axis. Thus, the direction of the reference axis acts as a kind of bar code so that the encoded microparticles can be used for multiple analysis.

Most existing bar code particles require additional detection programs or devices.

Meanwhile, the encoded microparticles according to an embodiment of the present invention can simultaneously perform multiplex analysis on 12 or more targets, and target analytes can be detected using only a magnetic field without a specific device. The system also has the clear advantage of using only one type of wavelength to detect multiple targets.

[Confirmation of protein advertising]

10 is an experimental result showing the result of the advertisement purification process under various conditions.

Referring to FIG. 10, in a photoimmobilization process, the target protein is conjugated to successfully encoded microparticles. However, since proteins need long-term exposure to ultraviolet light for a long time, the activity of conjugated proteins needs to be evaluated. Prior to application of the pathogen, we identified stable immobilization, target proteins and non-specific binding activity using streptavidin and fluorescent biotin.

Streptavidin reacted with microparticles using 4-benzoyl benzylamine hydrochloride (Bz) under ultraviolet light exposure for 18 minutes at a strength of 18 mW / cm ² .

Next, fluorescent biotin was reacted with streptavidin in microparticles for 30 minutes at room temperature. Referring to Fig. 10A, as a result, a clear fluorescence signal was measured.

Referring to Figure 10B, however, the fluorescence signal was also measured in the absence of Streptavidin. Based on these results, we found that 4-benzoyl benzylamine hydrochloride (Bz) is still activated by ultraviolet light and 4-benzoyl benzylamine hydrochloride (Bz) .

Thus, in order to block the activated 4-benzoyl benzylamine hydrochloride (Bz) after immobilizing the target protein (or capture antibody), we encode the encoded microparticles in 1% bovine serum Bovine serum albumin (BSA) was dissolved in phosphate buffered saline (PBS) for 1 hour.

Referring to FIG. 10C, after coating with bovine serum albumin (BSA), as a control, microparticles having no Streptavidin reacted with fluorescent biotin and no signal was detected.

 Since we confirmed that 4-Benzoyl benzylamine hydrochloride (Bz) was blocked, we performed a reaction between BSA and fluorescent biotin-coated Streptavidin and microparticles.

Referring to Figure 10D, when microparticles were reacted with streptavidin coated with BSA and fluorescent biotin, a clear fluorescence signal was measured. These experiments show that the activity of Streptavidin is retained despite the longer UV exposure time. Activated 4-Benzoyl benzylamine hydrochloride (Bz) is a bovine serum albumin ) Coating. ≪ / RTI >

[Detection of Pathogens]

Analysis was performed for each pathogen under optimized conditions. The capture antibodies of Bacillus subtillis, Salmonella typhimurium, Staphylococcus aureus and Escherichia coli O157: H7 were conjugated to encoded microparticles (300a to 300d) with 0, 30, 60 and 90 degree magnetic axes, respectively. The resulting encoded microparticles were incubated with a solution of the corresponding detection antibody-FITC (330a-330d) after reacting with each pathogen.

As a background test, four control experiments without pathogens were conducted. The fluorescence intensity of the control group was 52 au. Respectively. (Bacillus subtillis: 51.8 + - 2.78 au, Salmonella typhimurium: 46.8 + - 2.48 au, Staphylococcus aureus: 36.8 + 3.54 au, Escherichia coli O157: H7: 33.8 + 2.48 a.u).

Fig. 11 shows fluorescence images according to reaction time of pathogens.

12 is a graph showing the intensity of the fluorescence image of FIG. 11 according to the reaction time.

11, the concentration of the capture antibody is constant and the concentration of the detection antibody is constant. Also, the concentration of pathogenic bacteria is constant. On the other hand, the response times of the superparamagnetic nanoparticle patterns reacting with pathogens vary widely. The reaction time is 5 minutes, 10 minutes, 15 minutes, 20 minutes. 30 minutes, and 1 hour.

Referring to FIG. 12, the fluorescence intensity increases as the reaction time increases.

Figure 13 is fluorescence images according to the concentration of pathogens.

FIG. 14 is a graph showing the intensity of the fluorescence image of FIG. 13 according to the concentration of pathogens.

13 and 14, the concentration of Bacillus subtillis, Salmonella typhimurium, Staphylococcus aureus and Escherichia coli O157: H7 is increased by 10 times (10, 10, 2, 10 ^ 4, do. The standard curves for Bacillus subtillis, Salmonella typhimurium, Staphylococcus aureus and Escherichia coli O157: H7 are shown in the concentration range of 10 to 10 ⁸ CFU / ml.

The limit of detections (LOD) for each pathogen is 5.7 CFU / ml for Bacillus subtillis, 3.4 CFU / ml for Salmonella typhimurium, 12.3 CFU / ml for Staphylococcus aureus and Escherichia coli O157: H7 16.9 CFU / ml.

The detection limit (LOD) is defined as 3.3 x SD / b. Where SD and b represent the standard deviation of the blank measurement and the slope of the linear regression line, respectively.

The detection limit (LOD) value of our system showed better sensitivity than other systems for pathogen detection.

For Bacillus subtillis, y = 12.13 log (x) + 62.75.

For Salmonella typhimurium, y = 15.12 log (x) + 69.21.

For Staphylococcus aureus, y = 10.32 log (x) + 52.89.

For Escherichia coli O157: H7, y = 6.66 log (x) +25. These standard curves were used to convert the fluorescence signal measured at a known concentration of pathogen to the corresponding concentration in the multiplex analysis.

[Multiple analysis of pathogens]

To evaluate the reproducibility and multiplexing capabilities of this system, three analyzes based on encoded microparticles were performed.

Prior to multiplexed assays, we reacted four types of encoded microparticles to only one species of pathogen, cross-reacting between a particular capture antibody and non-corresponding pathogens -reactivity.

Figure 15 is a light microscope image showing cross-reactivity between a specific capture antibody and non-responding pathogen.

16 is the fluorescence intensity extracted from the glare microscope image of Fig.

Referring to Figures 15 and 16, when four types of encoded microparticles conjugated with the capture antibody of each pathogen are incubated with Bacillus subtillis, the fluorescence signal is encoded into an encoded micro- Only the particles were measured.

When we applied these investigations in all cases, the whole case showed similar results. As a result, it was confirmed that the specific antibody of a specific pathogen mainly reacted to the corresponding pathogen.

17 is a fluorescence microscope image showing multiplex analysis according to another embodiment of the present invention.

FIG. 18 is a summary of the multiplex analysis result of FIG.

17 and 18, the pathogen concentration of the first sample (Assay 1) is as follows. Bacillus subtilis: 10 CFU / ml, Salmonella typhimurium: 10 ⁸ CFU / ml, Staphylococcus aureus: 10 ² CFU / ml, Escherichia coli O157: H7: 10 ⁶ CFU / ml.

The concentration of the pathogenic bacteria extracted from the fluorescence intensity is as follows. Bacillus subtilis: 10 ^0.0149 CFU / ml, Salmonella typhimurium: 10 ^7.826 CFU / ml, Staphylococcus aureus: 10 ^2.2 CFU / ml, Escherichia coli O157: H7: 10 ^8.34 CFU / ml.

After evaluating the absence of cross-reactivity in this system, multiplexed assays using four types of encoded microparticles and four pathogens in an all-in-one reaction . The known fluorescence intensities were determined by analyzing known concentrations of the four types of pathogens. Based on these results, the concentration of the target pathogens was estimated from each standard curve.

Finally, known concentrations were compared to estimated values from the standard curve. As a result, the estimated concentrations from the three assays were in good agreement with known concentrations at acceptable tolerances.

In addition, the tilt angle of the microparticles arranged by the external magnetic field was easily analyzed and decoded without additional equipment.

However, in the case of Escherichia coli O157: H7, the fluorescence intensity as a whole was measured more strongly than the standard curve. This was particularly noticeable in samples 1 and 3 with high concentrations of Salmonella typhimurium.

Indeed, the system showed a slight overestimation at higher concentrations of Escherichia coli O157: H7 and Salmonella typhimurium.

However, in order to prevent foodborne or infectious diseases, it is more important to detect low-level pathogens and distinguish the safety of the object early. In this regard, the system has demonstrated excellent performance and convenient analysis in the critical concentration range of the target pathogen.

We have developed encoded microparticles with various magnetic axes for multiplexing analysis of pathogens. The corresponding target was easily decoded by measuring the tilt angle of each encoded microparticle under a parallel magnetic field, and the concentration of each target pathogen was successfully analyzed using fluorescent labels.

Although only four pathogens have been analyzed, these encoded microparticles have the ability to multiplex to expand the number of targets to be analyzed by the change of magnetic axis. This new sensing platform is expected to be a promising tool for the simultaneous diagnosis of various diseases including food poisoning.

While the invention has been shown and described with reference to certain preferred embodiments thereof, it will be understood by those skilled in the art that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. And all of the various forms of embodiments that can be practiced without departing from the spirit of the invention.

14: Microchannel
110: Microchannel structure
120: UV curing device
126: stage
132: Super magnetic nanoparticles
142: permanent magnet

Claims (15)

Preparing a supernatant magnetic nanoparticle solution in which the supernatant magnetic nanoparticles are dispersed in a resin;
Injecting the superpowder nanoparticle solution into a microchannel and applying an encoding external magnetic field to align the superpowder nanoparticles in the direction of the encoded external magnetic field to provide an alignment axis;
Curing the superpowder nanoparticle solution having the alignment axis in the microchannel to prepare superparamagnetic nanoparticles patterns having different alignment axes; And
And exposing the super-magnetic nanoparticle patterns to a decoding external magnetic field in a specific direction to align the alignment axes of the super-magnetic nanoparticle patterns in the direction of the decoding external magnetic field. . The method according to claim 1,
Wherein each of the super-magnetic nanoparticle patterns has the same shape and has different alignment axes. The method according to claim 1,
The super-magnetic nanoparticle patterns are in the form of a film,
Wherein the alignment axes of the superparamagnetic nanoparticle patterns are arranged in the plane of the film and the alignment axes of the superpowder nanoparticle patterns have geometrical reference axes so as to rotate according to the decoded external magnetic field to determine the rotation angle of the alignment axes A method of multiplexing a superfine magnetic nanoparticle pattern. The method according to claim 1,
Curing the superpowder nanoparticle solution with the alignment axis in the microchannel to prepare superparamagnetic nanoparticle patterns with different alignment axes comprising:
Wherein the superficial magnetic nanoparticle pattern is formed by curing the superficial magnetic nanoparticle solution so as to have a predetermined pattern while changing the direction of the encoding magnetic field to form the superficial magnetic nanoparticle pattern corresponding to the direction of the encoding magnetic field Way. The method according to claim 1,
Curing the superpowder nanoparticle solution with the alignment axis in the microchannel to prepare superparamagnetic nanoparticle patterns with different alignment axes comprising:
Wherein the direction of the encoding magnetic field is fixed and the microchannel is rotated and cured in a predetermined pattern to form super magnetic nano patterns corresponding to the rotation angle of the microchannel. . The method according to claim 1,
Curing the superpowder nanoparticle solution with the alignment axis in the microchannel to prepare superparamagnetic nanoparticle patterns with different alignment axes comprising:
Wherein the direction of the encoding magnetic field is fixed and the super high magnetic nanoparticle pattern is formed by curing the super high magnetic nanoparticle solution and cutting the super high magnetic nanoparticle pattern so as to correspond to the rotation angle of the predetermined pattern. Way. The method according to claim 1,
Curing the superpowder nanoparticle solution with the alignment axis in the microchannel to prepare superparamagnetic nanoparticle patterns with different alignment axes comprising:
Wherein the direction of the encoding magnetic field and the direction of the microchannel are fixed and the superficial magnetic nanoparticle pattern is formed by rotating the predetermined pattern and curing the superficial magnetic nano patterns corresponding to the rotation angle of the predetermined pattern. Multiplexing method. Preparing capture antibodies and detection antibodies that respectively react with the reference pathogenic fungi;
Preparing superparamagnetic nanoparticles patterns having different alignment axes;
Attaching capture antibodies corresponding to each of the superparamagnetic nanoparticle patterns having different alignment axes;
Immersing the supernatant nanoparticle patterns having different alignment axes attached with different capture antibodies in an analytical sample containing at least one of the reference pathogenic fungi to deposit at least one of the reference pathogenic fungi Attaching;
Attaching a detection antibody having a fluorescent substance to react with the reference pathogenic bacteria attached to the super-magnetic nanoparticle patterns; And
And exposing the super-magnetic nanoparticle patterns to a decoding external magnetic field in a specific direction to align the alignment axes of the super-magnetic nanoparticle patterns in the direction of the decoding external magnetic field. 9. The method of claim 8,
Wherein each of the super-magnetic nanoparticle patterns has the same shape and has different alignment axes. 9. The method of claim 8,
The super-magnetic nanoparticle patterns are in the form of a film,
Wherein the alignment axes of the superparamagnetic nanoparticle patterns are arranged in the plane of the film and the alignment axes of the superpowder nanoparticle patterns have geometrical reference axes so as to rotate according to the decoded external magnetic field to determine the rotation angle of the alignment axes Pathogen multiplexing analysis method. 11. The method of claim 10,
And extracting the concentration of the pathogen by detecting intensity of light generated from the fluorescent material of the detection antibody while aligning axes of the super-magnetic nanoparticle patterns are aligned along the decoding external magnetic field, Pathogen multiplexing analysis method. 12. The method of claim 11,
Detecting the intensity of light generated by the fluorescent material of the detection antibody in a state where the alignment axes of the super-magnetic nanoparticle patterns are aligned along the decoding external magnetic field to extract the concentration of the pathogen, comprising:
Obtaining a fluorescent image of the super-magnetic nanoparticle patterns using a microscope;
Extracting a rotation angle between the alignment axis and the geometric reference axis for each of the super-strong magnetic nanoparticle patterns in the fluorescence image;
Extracting intensity of light emitted from each of the super-magnetic nanoparticle patterns; And
And extracting the concentration of the pathogen using the relationship between the intensity of the light and the concentration of the pathogen in each rotation angle of the super-magnetic nanoparticle patterns. 9. The method of claim 8,
Wherein said pathogenic bacterium comprises at least one of Bacillus subtilis, Salmonella typhimurium, Staphylococcus aureus, Escherichia coli O157: H7,
Wherein said capture antibodies comprise at least one of anti-Bacillus subtilis, anti-Salmonella typhimurium, anti-Staphylococcus aureus, and anti-Escherichia coli O157: H7,
Wherein said detection antibodies comprise at least one of anti-Bacillus subtilis-FITC, anti-Salmonella typhimurium-FITC, anti-Staphylococcus aureus-FITC and anti-Escherichia coli O157: H7-FITC . 9. The method of claim 8,
Preparing a supernatant magnetic nanoparticle solution in which the supernatant magnetic nanoparticles are dispersed in a resin;
Injecting the superpowder nanoparticle solution into a microchannel and applying an encoding external magnetic field to align the superpowder nanoparticles in the direction of the encoded external magnetic field to provide an alignment axis; And
And locally curing the supernatant nanoparticle solution having the alignment axis so as to have a specific pattern in the microchannel to form a supernatant nanoparticle pattern. In the multiplexed analysis set,
A plurality of super-magnetic nanoparticle patterns of the same shape,
Each of the plurality of super-magnetic nanoparticle patterns has a different alignment axis,
Wherein the alignment axis is an axis in which the super-magnetic nanoparticles are arranged in a chain structure in each of the super-strong nano-particles patterns,
The super-magnetic nanoparticle patterns are in the form of a film,
Wherein the alignment axes of the superparamagnetic nanoparticle patterns are arranged in the plane of the film and the alignment axes of the superpowder nanoparticle patterns have geometrical reference axes so as to rotate according to the decoded external magnetic field to determine the rotation angle of the alignment axes Multiplexing analysis set.

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