KR20150114037A - Biomarker protein and bacteria detection method based on pipette using magnetic nanoparticle and viscous solution - Google Patents

Abstract

The present invention relates to a method for detecting pathogenic proteins and microorganisms. More specifically, the present invention relates to a method for rapidly detecting and quantifying microorganisms or pathogenic proteins included in foods or the like by using a pipette, a high viscous solution, and magnetic nanoparticles. To solve the problem, the method of the present invention comprises the steps of: feeding and coupling magnetic nanoparticles capable of being coupled with a pathogenic material in a sample including the pathogenic material; separating the magnetic nanoparticles; separating magnetic nanoparticles coupled with the pathogenic material by making the separated magnetic nanoparticles penetrate through a high viscous liquid by using magnetism; and analyzing the separated magnetic nanoparticles coupled with the pathogenic material. According to the present invention, disclosed is a method for selectively separating the pathogenic material coupled with separately existing magnetic nanoparticles from the high viscous fluid by using a pipette. Accordingly, error occurring in a step of separating the pathogenic material coupled with magnetic nanoparticles from the high viscous fluid can be reduced. Moreover, in the present invention, disclosed is a method for improving accuracy in a quantitative analysis through signal amplification by using magnetic nanoparticles coupled with the pathogenic material from the high viscous fluid, through signal amplification.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for detecting a pipette-based biomarker protein and a microorganism using magnetic nanoparticles and a high viscosity solution,

The present invention relates to a method for detecting pathogenic proteins and microorganisms, and more particularly, to a method for rapidly detecting and quantifying microorganisms or pathogenic proteins contained in foods and the like using pipettes, high viscosity solutions and magnetic nanoparticles will be.

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, Korean Patent Registration No. 1149418 granted to the Republic of Korea discloses that a fluorescent nanoparticle conjugate containing an antibody is put into a bacterial population, adhered to the fluorescent nanoparticle conjugate, Discloses a method for quantifying the amount of a specific bacterium by removing the nanoparticle binding substance and measuring the intensity of fluorescence. To this end, a method has been disclosed in which an avidin layer, which is a biomolecule, is formed on the surface of a fluorescent nanoparticle (QD), and the avidin is reacted with an antibody conjugated with biotin to allow the specific bacteria to react with the antibody .

In addition, Junha et al. Reported the use of fluorescent particles and antibodies to 3-micrometer-sized carboxyl-terminal magnetic particles in a paper entitled "A multicomponent recognition and separation system established via fluorescent, magnetic, and dual-coded multifunctional bioprobes" in Biomaterials 32 (2011) And then binding them to specific bacteria and then separating them using magnetic force.

However, this method has a problem in that it is difficult to efficiently separate bacteria-bound particles and non-bacteria-bound particles from the antibody, and that separate fluorescence particles are required for quantification. Accordingly, there is a continuing need for a new method for effectively separating and easily quantifying bacteria-bound particles and non-bacteria-bound particles in an antibody.

In order to solve this problem, the present inventors have proposed a method for detecting microbial cells by injecting magnetic nanoparticles capable of binding to bacteria into a sample for measuring bacteria in Korean Patent Application No. 2012-0134544, Separating the magnetic nanoparticles; Separating the magnetic nanoparticles to which the bacteria are bound and the magnetic nanoparticles to which the bacteria are not bound by passing the separated magnetic nanoparticles through the high viscosity liquid using magnetic force; And quantifying the magnetic nanoparticles to which the bacteria are bound.

This method is advantageous in that the magnetic nanoparticles to which the bacteria are not bonded are clearly separated from the magnetic nanoparticles to which the bacteria are bound while having a band shape in the microtube containing the high viscosity liquid. However, Because of the separation of the conjugate, the unbound magnetic nanoparticles remained in the upper part had to be removed in order to quantitatively measure the binding agent, which resulted in a large measurement error between the concentrations of food poisoning bacteria. In addition, there is a problem that the concentration of the magnetic nanoparticles themselves bound to the separated food poisoning bacteria is very small, so that quantification by absorbance analysis is not easy. On the other hand, there is a continuing demand for analysis methods for pathogenic substances other than food poisoning bacteria.

A problem to be solved by the present invention is to provide a new method for isolating and quantifying pathogenic substances.

Another problem to be solved by the present invention is to provide a new method for accurately and easily separating magnetic nanoparticles bound with separated pathogenic substances at the bottom of a high viscosity liquid.

Another problem to be solved by the present invention is to provide a new method for precisely quantifying the magnetic nanoparticles to which a pathogenic substance is bound.

In order to solve the above problems,

Introducing and binding magnetic nanoparticles capable of binding to a pathogenic substance into a sample containing a pathogenic substance;

Separating the magnetic nanoparticles;

Separating and separating the separated magnetic nanoparticles through a high-viscosity liquid by using a magnetic force; And

And quantifying the magnetic nanoparticles to which the pathogenic substance is bound.

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 step of permeating and separating the high-viscosity liquid may be performed on the basis of a pipette. In the practice of the present invention, after the high-viscosity liquid is filled in the pipette tip, the separated magnetic nanoparticles are placed on the top of the high-viscosity liquid, and then the magnetic nanoparticles bound with the pathogenic substance are applied to the pipette It can be collected at the bottom of the tip and then discharged out of the pipette tip using a pipette. When using a micropipette tip, it is possible to observe qualitatively with a small amount of the naked eye.

In the present invention, the quantification step can quantitatively analyze the magnetic nanoparticles to which a pathogenic substance is bound without performing a signal amplification process. In the practice of the present invention, the quantitative analysis can be performed through ultraviolet-visible light absorbance analysis.

In addition, the quantification step can increase the amount more accurately through signal amplification. In the practice of the present invention, the magnetic nanoparticles used in the present invention can act as artificial enzymes in addition to magnetic separation. When hydrogen peroxide (H ₂ O ₂ ) is present, 3,3 ', 5,5'-Tetramethylbenzidine ) To make it blue. This coloration can increase the absorbance signal for the conjugate, thus obtaining an absorbance signal for very low AFP concentrations.

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 the pathogenic material so that one or more nanoparticles can be attached to the pathogenic material. In the practice of the present invention, one having a size of 100 to 500 nanometers, preferably 1 to 10000 nanometers, preferably 50 to 1000 nanometers, and more preferably a size of 100 to 500 nanometers, desirable. 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, magnetic nanoparticles capable of binding to bacteria can be bound to bacteria by forming functional groups 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. The binding of the bacteria and the magnetic nanoparticles can be made by antigen-antibody binding, and a plurality of magnetic particles can be bound to one bacterium.

In the present invention, the magnetic nanoparticles are separated by magnetic force, and the magnetic nanoparticles to which pathogenic substances are coupled are separated from the non-bound magnetic nanoparticles to which the pathogenic substances are not combined.

In the present invention, when a mixture of a magnetic nanoparticle to which a pathogenic substance is bound and a magnetic nanoparticle to which a pathogenic substance is not bound is passed through a high viscosity liquid by using a magnetic force, a different behavior for the same magnetic force, The distance that they pass through will be different and separate from each other. For example, a plurality of magnetic nanoparticles can be bound to one food poisoning bacteria, and the resulting food poisoning bacteria-magnetic nanoparticle binding body (hereinafter referred to as a binding body) may be a single particle having a relatively large size relative to unbound magnetic nanoparticles Behavior. Therefore, the binding body has a larger magnetic force due to the external magnetic field than the non-binding magnetic nanoparticles. When the magnetic nanoparticles are induced to pass through the high-viscosity solution, only the binding bodies pass through the high-viscosity liquid.

In the present invention, the high-viscosity liquid means a liquid having a viscosity higher than that of water, and the viscosity of a liquid suitable for separation of magnetic nanoparticles can be determined by magnetic force, moving distance, or the like. Preferably 10 to 1000 times the viscosity of water, and preferably 20 to 200 times the viscosity so that the magnetic nanoparticles can be separated using a short distance. For example, in the range of about 20 to 100 milli-pascals-sec (mPa-s) at room temperature.

In the present invention, Chuanbin Mao et al ., Which is incorporated herein by reference in its entirety as the high-viscosity liquid, Mater. An aqueous solution of polyvinylpyrrolidone can be used as disclosed in the "Viscosity Gradient as a Novel Mechanism for Centrifugation-Based Separation of Nanoparticles", 2011 , 23 , 4880-4885, wherein the molecular weight of the polyvinylpyrrolidone is 5,000 To 30,000, preferably from 6,000 to 20,000, for example, polyvinylpyrrolidone having a molecular weight of about 10,000 may be used in a concentration of 10 to 60% by weight.

According to an aspect of the present invention, there is provided a method of analyzing a sample, comprising: injecting magnetic nanoparticles capable of binding to the analyte into a sample containing the analyte to bind and separating the magnetic nanoparticles; And

Separating the separated magnetic nanoparticles using a magnet to penetrate the high viscosity liquid filled in the pipette tip and selectively separating the combined samples of the magnetic nanoparticles into the bottom of the pipette tip. ≪ / RTI >

According to another aspect of the present invention, there is provided a method of separating a magnetic nanoparticle to which a pathogenic substance is bound by passing a high viscosity liquid through a sample containing a magnetic nanoparticle to which a pathogenic substance is bound, The present invention provides a method of analyzing a sample using magnetic nanoparticles.

According to another aspect of the present invention, there is provided a method for quantifying the content of a pathogenic substance, which comprises quantifying a chromophore-containing compound using magnetic nanoparticles having a pathogenic substance bound thereto.

In the present invention, a method for selectively separating a pathogenic material having magnetic nanoparticles bound in a separated phase from a high-viscosity fluid using a pipette has been proposed. As a result, a pathogenic material having a magnetic nanoparticle- It is possible to reduce an error occurring in the process of separating from the display device.

Also, in the present invention, a method of improving the accuracy of quantitative analysis by signal amplification using magnetic nanoparticles having a pathogenic substance bound thereto from a viscous fluid has been proposed.

The present invention also provides a general method for separating and quantifying analytes from a sample containing the analyte using magnetic nanoparticles.

The analytical method according to the present invention has an effect that relatively accurate qualitative analysis is possible through basic equipment such as magnetic nano-particles, pipette, and magnet without any expensive equipment such as a centrifuge or an absorber.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view showing separation of food poisoning bacteria-magnetic nanoparticle aggregates using a conventional microtube (a) separating food poisoning bacteria using magnetic nanoparticles (b) magnets;
FIG. 2 is a schematic diagram showing a comparison between a separation method using a conventional microtube and a high-viscosity solution and a detection method using a pipette.
3 is a photograph of a pipette-based food-borne pathogen detection apparatus using a high-viscosity solution.
FIG. 4 is a schematic diagram of a pipette-based food poisoning bacteria detection method using a high-viscosity solution.
Fig. 5 is a photograph showing actual separation according to the concentration of food poisoning bacteria.
Fig. 6 shows the absorbance of food poisoning bacteria-magnetic nanoparticles separated by food poisoning concentration (A: lowest 15L, B: lower 15L, C: next 15L, D: upper 45L)
7 is a schematic diagram of a pipette-based AFP detection method using a high-viscosity solution.
Fig. 8 is a photograph showing actual separation according to AFP concentration.
FIG. 9 is a photograph of a result of color development of the separated AFP-magnetic nanoparticles immediately after mixing with TMB and H ₂ O ₂ solution (left) for 10 minutes (right).
FIG. 10 is a graph showing the absorbance (AFP: 100 ng / mL, red: 10 ng / mL, green: 1 ng / mL, blue: 0.1 ng / mL, light blue: 0 ng / mL, ) (Left) Absorbance at 652 nm for AFP concentration (right).
11 is a photograph showing a state after centrifugal separation and separation using a magnet according to a comparative example.

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.

Example 1: Analysis of food poisoning bacteria

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. .

Deionized distilled water was obtained by using a commercial ultrapure water production system (Human Science, Korea) and was used to synthesize magnetic nanoparticle clusters and to make phosphate buffer solutions.

The neodymium magnet used for the separation of unbonded food poisoning bacteria and non-bonded magnetic nanoparticles was purchased from Seoul Magnetics Co., and the intensity of the magnet measured using a gauge meter was 30 milli tesla.

Quantitative determination of the separated food poisoning bacteria-magnetic nanoparticle conjugate was carried out by absorbance measurement using an ultraviolet-visible spectrophotometer of Ocean Optics.

Synthesis of Fe ₃ O ₄ magnetic nanoparticle clusters (hereinafter referred to as magnetic nanoparticles) and immobilization of antibodies

Fe3O4 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 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.

Preparation of high viscosity solution

Polyethylene glycol was used in this experiment, and a high viscosity solution can be prepared by dissolving the water in water at a concentration of 30 wt% (weight percent) at a number average molecular weight of 8,000. The viscosity of this high viscosity solution is about 50 millipascal seconds (mPa · s) at room temperature and about 50 times higher than the viscosity of ordinary water (0.89 mPa · s).

Food poisoning bacteria detection

This experiment was carried out on Salmonella typhimurium, and the magnetic nanoparticles immobilized with antibody were mixed with a solution containing food poisoning bacteria to bind the food poisoning bacteria with the magnetic nanoparticles. Then, the magnetic nanoparticles were separated using a magnet I'll do it.

Thereafter, 10 g of the separated particles were dispersed in 50 L of a phosphate buffer solution, 50 L was sucked into the pipette tip using a micropipette, and 80 L of the prepared high-viscosity liquid was sucked, And a high-viscous fluid layer is present in the lower part of the magnetic particle layer.

The micropipette was placed on a neodymium magnet for 10 minutes to qualitatively analyze the presence of food poisoning bacteria by collecting it in the lower part of the viscous fluid, that is, the narrow lower part of the pipette tip, as shown in Figs. 4 and 5, . Quantitative analysis was carried out using this.

Quantitative Analysis 1

The lower 15L of the pipette tip was drained using a pipette volume control wheel, diluted to 70 L in a plastic cuvette (BRAND), and absorbance was measured using a UV-visible spectrometer from Oceanoptics. 70L is the minimum volume for absorbance measurement. As the concentration of food poisoning bacteria increases, the amount of discharged magnetic nanoparticles increases, resulting in an increase in the absorbance measured.

Quantitative Analysis 2

The released Fe ₃ O ₄ magnetic nanoparticles act as an artificial enzyme to oxidize 3,3 ', 5,5'-Tetramethylbenzidine (TMB) when present in the presence of hydrogen peroxide, resulting in colorless to blue color. The 15 L discharged from the above was mixed with 25 L of 0.1 MH ₂ O ₂ and 30 L of 5 mM TMB solution to cause color development for 10 minutes, and the absorbance was measured. The measured absorbance showed a much better signal than when it was measured without color, and the degree of improvement increased with increasing concentration of the detection target.

Example 2: Analysis of pathogenic proteins

This experiment was conducted on the pathogenic protein alpha-fetoprotein (AFP), and the AFP and the magnetic nanoparticles were combined by mixing the immobilized magnetic nanoparticles with the AFP-containing solution, and then the magnetic nanoparticles .

Thereafter, 10 g of the separated particles were dispersed in 50 L of a phosphate buffer solution, 50 L was sucked into the pipette tip using a micropipette, and 80 L of the prepared high-viscosity liquid was sucked, And a high-viscous fluid layer is present in the lower part of the magnetic particle layer.

The micropipette was placed on a neodymium magnet for 10 minutes to qualitatively analyze the presence of AFP such that it was collected at the bottom of the high viscosity fluid, i.e. the narrow bottom of the pipette tip, as in Figures 4 and 5, . Quantitative analysis was carried out using this.

Comparative Example

The procedure of Example 1 was repeated except that the pipette container was separated by using a centrifuge. As shown in FIG. 11, since the magnetic nanoparticles are continuously banded from the upper layer to the lower layer, it is difficult to separate the magnetic nanoparticles without bacteria from the magnetic nanoparticles to which the bacteria are attached.

Claims (16)

Introducing and binding magnetic nanoparticles capable of binding to a pathogenic substance into a sample containing a pathogenic substance;
Separating the magnetic nanoparticles;
Separating the magnetic nanoparticles coupled with the pathogenic substance by permeating the separated magnetic nanoparticles through the high viscosity liquid using magnetic force; And
Analysis of the magnetic nanoparticles bound to the separated pathogenic material
Of the pathogenic agent. The method according to claim 1, wherein the pathogenic agent is a pathogenic microorganism or a pathogenic protein. The method according to claim 1, wherein the separated magnetic nanoparticles collect the magnetic nanoparticles coupled with a pathogenic substance at the lower part of the pipette tip while passing through the high-viscosity liquid filled in the pipette tip. [4] The method of claim 3, wherein the magnetic nanoparticles coupled with the pathogenic material collected at the lower portion of the pipette tip are discharged by pressing the pipette. The method according to claim 1, wherein the pathogenic substance-bound magnetic nanoparticles are quantified using ultraviolet-visible light absorption. The method according to claim 1, wherein the pathogenic substance is quantitated by analyzing a chromogenic compound by coloring the compound using the magnetic nanoparticles coupled with the pathogenic substance. The method of claim 1, wherein the pathogenic agent is selected from the group consisting of Salmonella, Staphylococcus aureus, Vibrio parahaemolyticus, Listeria monocytogenes, Escherichia coli E. coli O157, Campylobacterium, Bacillus cereus, Wherein the microorganism is selected from one or more microorganisms. The pathogenic agent according to claim 1, wherein the pathogenic agent is a pathogenic protein selected from the group consisting of alpha-fetoprotein (AFP), prostate-specific antigen (PSA), IL-5, IL-6, carcinoembryonic antigen A method for measuring a pathogenic material characterized by: The method of claim 1, wherein the magnetic nanoparticles are bound to an antibody capable of binding to a pathogenic substance.         The method according to claim 1, wherein the viscous liquid has a viscosity of 20 to 100 times that of water. The method according to claim 1, wherein the high viscosity liquid is a polymer solution. [7] The method according to claim 6, wherein the magnetic nanoparticles to which pathogenic substances are bound are added, and 3,3 ', 5,5'-tetramethylbenzidine (TMB) is discolored in the presence of hydrogen peroxide to measure and measure the absorbance signal. Method of measurement of material. Introducing magnetic nanoparticles capable of binding to the analyte into a sample containing the analyte to bind and separating the magnetic nanoparticles; And
Separating the separated magnetic nanoparticles by passing the high viscosity liquid filled in the pipette tip using a magnet and selectively separating the combined samples of the magnetic nanoparticles into the bottom of the pipette tip
A method for analyzing a sample using magnetic nanoparticles comprising the magnetic nanoparticles. 14. The method according to claim 13, further comprising discharging magnetic nanoparticles coupled with a separated analyte at a lower portion by pressing a pipette. A method for separating a magnetic nanoparticle having a pathogenic substance bound thereto by separating a pathogenic substance-bound magnetic nanoparticle by permeating a high viscosity liquid through a sample containing a magnetic nanoparticle coupled with a pathogenic substance and a magnetic nanoparticle not bound to the pathological substance, Analysis method. A method for analyzing the content of a pathogenic substance characterized by coloring and quantifying a compound having a chromophore by using magnetic nanoparticles having a pathogenic substance bound thereto

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