KR101639862B1 - A high efficient device to capture magnetic particles for a rapid detection of food poisoning bacteria - Google Patents
A high efficient device to capture magnetic particles for a rapid detection of food poisoning bacteria Download PDFInfo
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- KR101639862B1 KR101639862B1 KR1020140036873A KR20140036873A KR101639862B1 KR 101639862 B1 KR101639862 B1 KR 101639862B1 KR 1020140036873 A KR1020140036873 A KR 1020140036873A KR 20140036873 A KR20140036873 A KR 20140036873A KR 101639862 B1 KR101639862 B1 KR 101639862B1
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Abstract
The present invention relates to a method and apparatus for isolating food poisoning bacteria, and more particularly, to a method for isolating and detecting large amount of food poisoning bacteria using magnetic nanoparticles and a magnet collecting apparatus.
The collecting device according to the present invention includes a double tubular body portion having a magnetic body accommodating portion in its center portion and having a micro unit interval between an outer wall and an inner wall; A plurality of disk-shaped magnets stacked at predetermined intervals in the magnetic body accommodating portion; And magnetic nanoparticles to which a pathogenic substance immobilized by the magnet can be bonded to the inner wall of the body part.
Description
The present invention relates to a method and apparatus for isolating food poisoning bacteria, and more particularly, to a method for isolating and detecting large amount of food poisoning bacteria using magnetic nanoparticles and a magnet collecting apparatus.
Pollution of agricultural products and frequent food poisoning accidents. It has been increasing worldwide in recent 10 years. Accordingly, there is a growing demand for diagnostic methods that can quickly and quickly diagnose food poisoning bacteria contamination during pre-distribution processing of agricultural products, prevent the occurrence of food poisoning, and reduce social costs associated with the occurrence of food poisoning. There has been a problem in that it is not suitable for prevention of food poisoning accidents due to pre-circulation measurement and diagnosis in advance as a method which takes about 3-5 days, although there is a method through conventional culture and biochemical inspection.
In order to solve this problem, Jun et al. Proposed a "multicomponent recognition and separation system established via fluorescent, magnetic, dual-coded multifunctional bioprobes" of Biomaterials 32 (2011) A method of binding particles and an antibody to bind to a specific bacterium, attaching magnets to the outer wall of the container, separating the particles, and analyzing the particles.
However, such a separation method requires a considerable time to bond the magnetic particles with specific bacteria, and also has a problem in that it takes a long time to separate the magnetic nanoparticles from the sample with increasing capacity. In particular, there is a time-consuming problem in large-volume samples larger than 50 ml, which are useful for detection and quantification.
Accordingly, there is a continuing need for a novel method for effectively and rapidly separating bacteria from large-volume samples.
A problem to be solved by the present invention is to provide a new method for quickly separating bacteria from a large-volume sample.
Another object of the present invention is to provide a new apparatus capable of rapidly separating bacteria from a large-volume sample.
In order to solve the above problems,
Applying a magnetic field outside the flow path through which a sample containing a pathogenic substance flows, and fixing magnetic nanoparticles capable of binding to a pathogenic substance in the flow path;
Introducing a sample containing a pathogenic substance through the channel, separating the pathogenic substance into a sample bound to the magnetic nanoparticle; And
Removing the magnetic field and discharging the magnetic nanoparticles coupled with a pathogenic substance from the flow path
And a control unit.
In the present invention, the pathogenic substance refers to a pathogenic microorganism or pathogenic protein including food poisoning bacteria.
In the present invention, the pathogenic substance refers to a pathogenic microorganism or pathogenic protein including food poisoning bacteria.
In the present invention, the pathogenic microorganism may be food poisoning bacteria which may be included in food or the like and cause food poisoning. Examples include Salmonella, Staphylococcus aureus, Enterobacteriaceae, Listeria monocytogenes, Bacillus cereus, Welsh onion, Botuluris.
In the present invention, the pathogenic proteins are understood to be proteins that can cause diseases or diseases in the human body. For example, alpha-fetoprotein (AFP), prostate-specific antigen (PSA), interleukin- 6, carcinoembryonic antigen (CEA).
In the present invention, the magnetic nanoparticles are understood as magnetic fine particles which react with magnetic force.
In the present invention, the magnetic nanoparticles may be microparticles smaller than pathogenic substances so that a pathogenic substance can be attached to one or more nanoparticles. In the practice of the present invention, it is preferred to have a size of 100 to 500 nanometers, such as 1 to 10000 nanometers, preferably 50 to 1000 nanometers, and even more preferably 100 to 500 nanometers, . In the practice of the present invention, the magnetic nanoparticles may be manufactured through a known method and commercially available. For example, γ-Fe 2 O 3 may be used as the magnetic nanoparticles. have.
In the present invention, the pathogenic substance, for example, a magnetic nanoparticle capable of binding bacteria can be bound to bacteria by forming a functional group capable of binding to bacteria on the surface or inside thereof. In the practice of the present invention, the functional groups capable of binding to the bacteria are antibodies capable of binding to bacteria, and various antibodies capable of binding to specific bacteria are known. As a method for forming an antibody capable of binding bacteria on the surface of nanoparticles, Jun ha et al., Which is incorporated herein by reference in its entirety, discloses "A multicomponent recognition and separation system established via fluorescent of Biomaterials 32 (2011) magnetic, dualencoded multifunctional bioprobes ". In one embodiment of the present invention, the antibodies can use known antibodies capable of binding to bacteria. Binding of the bacterium and the magnetic nanoparticles can be made by antigen-antibody binding, and one bacterium can be bound to a plurality of magnetic particles.
In the present invention, 'large capacity' is understood to mean a sample of 50 mL or more, preferably a sample of 100 mL or more, and most preferably a sample of 200 mL or more.
In the present invention, the term 'rapid separation' refers to separation in an amount of not less than 1 hour, preferably not more than 50 minutes, and most preferably not more than 30 minutes in a sample of 50 mL or more.
In the present invention, it is preferable that the channel is narrow so that the magnetic nanoparticles fixed in the channel by the pathogenic substance included in the sample and the magnetism can efficiently react. The width may mean the inner diameter in the case of a tubular flow path and the shortest distance between the flat plates in the case of a flat flow path. In the practice of the present invention, it is preferable that the flow path has a width of micro flow path, preferably 100 to 1000 micrometers, and more preferably 200 to 500 micrometers. If the width of the flow path is excessively narrow, the passage time of the sample becomes long and rapid separation may become difficult.
In the present invention, the magnetic field may be fixed by a magnet located outside the flow path, and preferably, the permanent magnet may be used to continuously apply a magnetic field without a separate external power source.
In the present invention, in order to quickly separate a sample of a large capacity, a strong magnetic force is required so that the magnetic nanoparticles do not flow away by a sample flowing at a high speed, and accordingly, a plurality of magnets having edges than one magnet are used . When a plurality of magnets having edges are used, it is preferable that they are spaced apart from each other by a predetermined distance so as to prevent magnetic fields from being canceled out between adjacent magnets. In the practice of the present invention, the magnet having the corner may take various forms such as a rectangular parallelepiped, a rod, or a disk.
According to one aspect of the present invention, there is provided a flow pathway in which a sample containing a pathogenic substance flows, a magnetic substance which is located outside the flow channel and applies a magnetic field to the flow channel, and a pathological substance contained in the sample, The present invention also provides a pathogenic material separating apparatus comprising magnetic nanoparticles fixed to the inner wall of a substrate.
In the preferred embodiment of the present invention, the flow path is a double-tube type flow path having microchannels, and the magnetic body is formed by disc-shaped magnets stacked along the flow direction of the flow path at a predetermined interval in the center of the flow path, And includes magnetic nanoparticles capable of binding food-borne bacteria fixed therein.
The present invention can examine the presence of pathogenic substances in a sample without passing through a complicated and time-consuming production process of binding magnetic nanoparticles capable of binding with a pathogenic substance to a sample and binding the same to a pathogenic substance.
Further, in the present invention, a method in which a sample is actively moved to a device including a magnetic body is used instead of a passive method in which the magnetic nanoparticles are moved by the magnetic force by keeping the magnet around the sample, Lt; / RTI >
Fig. 1 is a schematic view of a device for testing the efficiency of magnetic nanoparticle separation according to the number of corners of a magnet in a collecting device. (a) Number of
2 is a photograph of a device for testing the efficiency of magnetic nanoparticle separation according to the number of corners of a magnet in a collecting device. (a) Number of
3 is a graph showing the difference in separation efficiency according to the number of corners.
4 is a schematic diagram of a large-capacity food poisoning bacteria separation system using a magnet based on an edge effect.
5 is a photograph of magnetic particle agglomerates at the corners of the magnets.
6 is a graph showing the separation efficiency by the flow velocity.
7 is a simulation result showing the change of the magnetic field distribution according to the distance between magnets. Here, the distance between the magnets is (a) none (b) 1/4 (c) 2/4 (d) 3/4 (e) 4/4
FIG. 8 is a photograph showing magnetic nanoparticle separation photographs and magnetic field distributions (a) when there is no gap between magnets and FIG. 8 (b)
Fig. 9 is a photograph (b) of a magnet collector; Fig.
10 shows a process of collecting magnetic nanoparticles using a magnet collecting apparatus.
FIG. 11 shows the case of (a) photographs of magnetic nanoparticles separated by using a magnet collecting apparatus, (b) absorbance measurement results (black), and absences (blue)
12 is a side cross-sectional view showing a state in which a double pipe type flow path a and a disk type magnet are stacked according to an embodiment of the present invention.
FIG. 13 shows (a) the result of the food poisoning bacteria detection process and (b) results using the magnet collection device in which the magnetic nanoparticles immobilized with the antibody are preliminarily contained.
Hereinafter, the present invention will be described in detail with reference to examples. Hereinafter, the present invention will be described in detail with reference to examples. The following examples illustrate the present invention in detail, but are for the purpose of illustrating the present invention and are intended to limit the scope of the present invention, and it is to be noted that the scope of the present invention is defined by the claims.
Reagents and equipment
Iron (III) chloride, FeCl 3 , Urea, sodium citrate, polyacrylamide, [3-Aminopropyl] triethoxysilane , APTES), glutaraldehyde and bovine serum albumin (BSA) were purchased from Sigma-Aldrich. Polyethylene glycol was purchased from Fluka. Antibodies against food poisoning bacteria were obtained from Abcam Inc. .
The deionized distilled water was obtained by using a commercial ultrapure water producing apparatus (Human Science, Korea) and used to synthesize magnetic nanoparticle clusters and to make a phosphate buffer solution. The neodymium magnet used in the collecting device was purchased from Seoul Magnetics Co., Ltd. The magnitude of the magnet measured using a gauge meter is 30 milli tesla.
Fe 3 O 4 Magnetic nanoparticle clusters (hereinafter referred to as magnetic nanoparticles) synthesis and antibody immobilization
Fe 3 O 4 magnetic nanoparticles were synthesized using a one-pot solvothermal method. First, 4 millimoles of iron chloride, 12 millimoles of element and 8 millimoles of sodium citrate are dissolved in 80 milliliters of water. Thereafter, 0.6 g of polyacrylamide is added while stirring continuously using a magnet rod. The reaction solution thus prepared is transferred to a 100 ml volume Teflon autoclave vessel and maintained at a temperature of 200 ° C for 12 hours to cause a synthesis reaction. After cooling the solution to room temperature, the synthesized particles are collected using a magnet, and the remaining reactants are discarded and washed several times with ethanol and water to finally obtain Fe 3 O 4 magnetic nanoparticles. The size of the nanoparticles thus obtained is about 200 nanometers.
The synthesized magnetic nanoparticles react with APTES and glutaraldehyde, respectively, so that amine groups on the surface can be activated and bind to the antibody. Thereafter, the antibody is immobilized on the particle surface by mixing the antibody and the magnetic nanoparticles, and then the BSA treatment is performed to prevent non-specific binding.
Comparison of the separation efficiency of food poisoning bacteria by the number of permanent magnet corners
This experiment was conducted on Salmonella typhimurium. The experiment was conducted by mixing magnetic nanoparticles immobilized with antibodies and a solution containing food poisoning bacteria to bind food poisoning bacteria with magnetic nanoparticles. The sample was filled into a AL-1000 syringe pump from World Precision Instrument and discharged through a 1/8 inch tube at a flow rate of 0.1 mL / min for 10 minutes. 1 and 2 are schematic and photographs of the device showing the efficiency of magnetic nanoparticle separation by the number of corners of the magnet in the collecting device. As can be seen, it can be seen that more magnetic nanoparticles are collected at the corners than the surface of the magnet And the absorbance of the solution passed through the collecting device as shown in FIG. 2 was measured. The results are shown in FIG. As the number of corners of the magnet in the collecting device increases, the measured absorbance decreases, indicating that the number of collected magnetic nanoparticles increases. Therefore, it can be seen that the collection efficiency increases as the number of corners of the magnetic nanoparticles on the same area magnet increases. At this time, the tube through which the magnetic nanoparticle solution flows is perpendicular to the edge of the magnet.
Manufacture of a collection device using magnets and separation of food poisoning bacteria
FIG. 4 is a schematic diagram of a collecting apparatus designed to maximize separation efficiency based on the above results. Since the magnetic field affecting the magnetic nanoparticles is inversely proportional to the cube of the distance from the magnet to the particle, the smaller the conduit through which the solution containing the magnetic nanoparticles or the magnetic nanoparticle- It becomes stronger. Therefore, when a very small fluid conduit is used as in conventional microfluidic devices, the intensity of the magnetic field on the entire magnetic nanoparticles can be increased. In this case, however, the volume of the sample to be treated is reduced. / 8 inch tube. The tube then meets the edge of the magnet horizontally and a larger volume of the solution meets the edges of the magnet, enabling higher separation efficiency to be achieved.
FIG. 5 is a photograph showing that more magnetic nanoparticles are gathered in the corner portion of the magnet. FIG. Since the thickness of the magnet is 1 cm, it can be seen that the collection of the magnetic nanoparticles is more smooth in the tube passing the edge portion than the tube passing the surface portion of the magnet.
FIG. 6 shows the results of measuring the absorbance of the magnetic nanoparticle solution passing through the manufactured collecting device and comparing the separation efficiency according to the flow velocity. The equipment used is the same as that of the third embodiment except for the collecting device. At a flow rate of 25 mL / min, a separation efficiency of 99% or more can be achieved, and 250 mL samples can be treated in 10 minutes.
Magnet collecting device maximizing the strength of magnetic field
7 is a simulation result of the magnetic field distribution of the magnet for optimizing the manufactured collecting device. The magnetic field of the N pole is represented by a dark red color and the magnetic field of the S pole is represented by a light yellow color. When the magnets are closely contacted as shown in FIG. 4, a magnetic field is canceled at the corners between the magnets. It can be seen that the offset of the magnetic field decreases as the distance between the magnets increases.
FIG. 8 is a photograph showing that the trapping efficiency for the magnetic nanoparticles is remarkably increased by setting a gap between the actual magnets. As the gap increases, the magnetic field canceling phenomenon decreases. However, since the size of the collecting device increases, the distance between the magnets is optimized to 1/4 of the magnet thickness.
9 is a schematic view and a photograph of a collecting device suitable for collecting magnetic nanoparticles than the tube in Example 4; The fluid conduit in which the magnetic nanoparticles or the magnetic nanoparticle-food poisoning bacteria conjugate passes through the device is more preferably a form in which the fluid conduit surrounds the magnet rather than a tube wound around the magnet as in FIGS. 4, 5 and 6. This is because such a fluid conduit has the effect of reducing the volume in the collecting device and consequently reducing the volume of the collecting solution. A solution containing magnetic nanoparticles is injected into the lower part of the device, and a solution other than the magnetic nanoparticles is discharged to the upper part of the device, and a magnet is disposed in the center of the device.
10 is a photograph showing a process of collecting magnetic nanoparticles using a magnet collecting device. The collection process of magnetic nanoparticles consists of 1) injection of a solution containing magnetic nanoparticles, 2) collection of magnetic nanoparticles, 3) removal of the magnet inside the device, and precipitation of magnetic nanoparticles.
FIG. 11 shows the results of absorbance measurement showing the separation efficiency of magnetic nanoparticles depending on the presence of the gap between the magnet and the magnet nanoparticle separation photograph using the magnet collector. The photomicrographs show the difference in concentration between the magnetic nanoparticles before and after passage through the device. The magnetic nanoparticles were collected from a 250 mL solution into a collecting device having an internal volume of 1.8 mL. . The solution passing through the collecting device can be seen that the magnetic nanoparticles are completely collected and transparent. In the absorbance results, the absorbance of each magnetic nanoparticle concentration is indicated (black). In the magnet collector, about 70% of the collecting efficiency is obtained when there is no gap between the magnets, and 100% You can see what you see.
Magnet trapping apparatus with magnetic immobilized magnetic nanoparticles fixed and isolation of bacteria
12, the magnet collecting apparatus according to the present invention has a double
The inner
The magnetic nanoparticles are fixed between the inner walls of the
FIG. 13 shows the results of the detection and accumulation of food poisoning bacteria more rapidly by injecting the food poisoning bacteria solution after embedding the magnetic nanoparticles immobilized with the antibody into the apparatus in advance. Detection of food poisoning bacteria was accomplished within about 3 minutes by collecting and detecting the food poisoning bacteria captured in the device after injecting milk (10 mL) containing food poisoning bacteria at 25 mL / min.
Claims (14)
Moving a sample containing a pathogenic substance through the flow path to bond the pathogenic substance contained in the sample to the magnetic nanoparticles and separating the pathogenic substance; And
Removing the magnetic field and discharging the magnetic nanoparticles coupled with a pathogenic substance from the flow path
A method for isolating a pathogenic material,
Wherein the magnetic field is applied by a plurality of magnets having corners spaced apart at predetermined intervals.
A magnetic body positioned outside the flow path and applying a magnetic field to the flow path, and
A plurality of magnetic nanoparticles capable of binding pathogenic substances contained in the sample and fixed to the inner wall of the flow path by the magnetic material,
/ RTI >
Wherein the magnetic field is applied by a plurality of magnets having corners spaced apart at predetermined intervals.
A plurality of disk-shaped magnets stacked at predetermined intervals in the magnetic body accommodating portion; And
A magnetic nanoparticle capable of binding a pathogenic substance fixed by the magnet to the inner wall of the body;
Wherein the pathological substance collecting device comprises:
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| KR20200078219A (en) | 2018-12-21 | 2020-07-01 | 경북대학교 산학협력단 | Detection device for foodborne pathogen and its method for foodborne pathogen detection |
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| KR102170259B1 (en) | 2019-07-15 | 2020-10-28 | 주식회사 어큐노스 | Solution Substitution Type Magnetic Particle Concentration Device and Method for Rapid Detection of Bacteria Based on Fluorescence Imaging |
| KR102300213B1 (en) * | 2019-09-11 | 2021-09-09 | 포항공과대학교 산학협력단 | Lenz's law-based virtual net for rapid capture of pathogenic microorganisms |
| CN115254068B (en) * | 2022-05-30 | 2024-01-26 | 西北农林科技大学 | Magnetic nanometer bacterial catching agent containing phytic acid and preparation method and application thereof |
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| KR20110025975A (en) * | 2008-06-17 | 2011-03-14 | 조지아 테크 리서치 코오포레이션 | Superparamagnetic nanoparticles for removal of cells, pathogens or viruses |
| KR101080045B1 (en) * | 2009-02-27 | 2011-11-04 | 동아대학교 산학협력단 | Fluid mixing system by microchannel and mixing method thereof |
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