KR20200121496A - Complex for separation and detection of pathogens comprising magnetic nanoparticles immobilized with antibody and method for separation and detection of pathogens using the same - Google Patents

Abstract

The present invention relates to a complex for separation and detection of pathogenic microorganisms, and a method for pre-treatment, separation, and detection of pathogenic microorganisms using the same. More specifically, by rapidly separating and accurately detecting pathogenic microorganisms, the complex can be used for hygiene management of food and livestock products, diagnosis of causative bacteria, and disease diagnosis.

Description

Complex for separation and detection of pathogens comprising magnetic nanoparticles immobilized with antibody and method for separation and detection of pathogens using the same}

The present invention relates to a complex for separating and detecting pathogenic microorganisms and a method for pretreatment, separation and detection of pathogenic microorganisms using the same, and more particularly, by rapidly separating and accurately detecting pathogenic microorganisms, hygiene management of food, livestock products, etc., diagnosis of causative bacteria, and / Or can be used to diagnose disease.

Despite advances in science and technology, it is still considered difficult to completely prevent diseases caused by pathogenic microorganisms. For example, food poisoning is caused by natural poisoning, spoilage of food, and the incorporation of bacteria in food. Of these, it is easy to prevent natural poison or spoilage of food, whereas contamination by bacteria is not observed with the naked eye. However, it is most difficult to prevent because it is very difficult to detect without relying on a specialized method.

In recent years, as the number of facilities that supply food in large quantities, such as restaurants, order delivery businesses, catering centers, and fast food stores, and the number of consumers who use them increases, it is more difficult to manage the level of contamination of pathogenic microorganisms in food.

If food poisoning occurs due to infection with pathogenic microorganisms, symptoms are severe, such as severe vomiting, diarrhea, abdominal pain, and high fever, and there are many group infections through facilities, and long-term treatment is required even after infection, and treatment is often difficult. Early detection and prevention is urgently needed.

Existing methods for detecting and confirming pathogenic microorganisms include culturing the collected sample using a selective medium, observing the colonies formed in the medium with the naked eye, or confirming using other means. Recently, polymerase A test method using a chain reaction (PCR) has been developed and used. Polymerase chain reaction has a simple and convenient advantage of being able to amplify the DNA sequence of a specific site exponentially with only a very small amount of DNA. Compared to the general test method, culture using medium (takes more than 96 hours) or enzyme-linked immunoassay (enzyme-linked ImmumoSorbent Assay: takes 48 hours), the accuracy and speed (takes 24 hours) of omitting the step of the confirmation experiment. However, since the process of proliferating pathogenic microorganisms infected with food must necessarily be accompanied in order to secure sufficient gene concentration, there is a disadvantage in that 16 to 20 hours of enrichment culture time may be consumed. Therefore, there is an urgent need to develop a new technology that enables accurate testing in a short time by quickly separating only pathogenic microorganisms into detectable concentrations with only a simple process from actual samples in which several components are mixed.

The matters described as the above-described background art are only for enhancing understanding of the background of the present invention, and should not be taken as acknowledging that they correspond to the prior art already known to those of ordinary skill in the art.

Japanese Patent Application Publication No. 2008-530993

The present inventors have made diligent efforts to invent a technology that can quickly isolate and accurately detect pathogenic microorganisms for hygiene management and disease diagnosis of food and livestock products. As a result, a complex for separation and detection of pathogenic microorganisms in magnetic nanoparticles that can be easily immobilized without additional chemical reactions with antibodies that form a specific bond with pathogenic microorganisms was devised, and by using this, pathogenic microorganisms present in the sample were quickly separated. Thus, the present invention was completed by accurately detecting.

Accordingly, an object of the present invention is to provide a complex for separating and detecting pathogenic microorganisms capable of rapidly separating and accurately detecting pathogenic microorganisms from a sample.

Another object of the present invention is to provide a manufacturing method capable of mass-producing the complex for separating and detecting pathogenic microorganisms.

Another object of the present invention is to provide a method for rapidly separating and detecting pathogenic microorganisms from a sample using the complex for isolation and detection of pathogenic microorganisms.

Other objects and advantages of the present invention will become more apparent by the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention is a pathogenic microorganism capable of quickly separating and accurately detecting pathogenic microorganisms from food or livestock products by easily immobilizing antibodies that bind pathogenic microorganisms on the surface of magnetic nanoparticles without additional chemical reactions. It provides a separation and detection complex or a method for separation and detection of pathogenic microorganisms using the same.

The present inventors have tried to discover new materials and methods for separating and detecting pathogenic microorganisms from samples. As a result, using copper-iron oxide magnetic nanoparticles and antibodies specific for pathogenic microorganisms, immobilization efficiency does not require additional chemical reactions. It was confirmed that this remarkably excellent complex for isolation and detection of pathogenic microorganisms was provided, and that pathogenic microorganisms could be effectively separated and detected from a sample by using this.

An aspect of the present invention is a copper-iron oxide magnetic nanoparticles; And an antibody that is immobilized on the surface of the magnetic nanoparticles and specifically binds to pathogenic microorganisms. It relates to a complex for isolation and detection of pathogenic microorganisms.

In the present invention, "magnetic nanoparticles" are particles having ferromagnetic properties and generally mean particles having a size of several nanometers to hundreds of nanometers. In order to impart biological functionality to such'magnetic nanoparticles', it is essential to immobilize biomolecules on the surface. Since this requires an additional chemical reaction, additional cost, time, and effort must be consumed. If this can be omitted, it has a great advantage in the use of magnetic nanoparticles. In the present invention, copper-iron oxide, cobalt-iron oxide, nickel-iron oxide magnetic nanoparticles produced by adding cationic metal ions such as copper, cobalt, and nickel to iron oxide, which are commonly used magnetic nanoparticles, and proteins such as antibodies. It was confirmed that immobilization without additional chemical reactions was possible by using the interaction of existing anionic amino acids such as histidine. As a result of confirming the immobilization efficiency of the antibody or linker and the magnetic nanoparticles using this, copper-iron oxide magnetic nanoparticles are 3 to 3 than those of other magnetic nanoparticles in order to efficiently immobilize the antibody or linker as in the experiment described below. 6 times more preferable. The copper-iron oxide magnetic nanoparticles may be most preferably represented by CuFe ₂ O ₄ .

The complex for separating and detecting pathogenic microorganisms of the present invention has a remarkable advantage in that it can omit the process of using a harmful solvent such as modifying or coating the surface, and can immobilize more antibodies or linkers than conventional magnetic nanoparticles. Have.

The magnetic nanoparticles may be in the form of clusters, and the average diameter is not particularly limited, but is preferably 10 to 2000 nm, and is preferably 50 nm or more in order to be separated by magnetism applied from the outside.

Because the pathogen or biomarker of the disease is determined by the antibody include, but not particularly limited to, Escherichia coli (E. coli), Salmonella genus (Salmonella), a break in Gela (Shigella), Enterobacter bacteria in three (Enterobacteriaceae), Pseudomonas species (Pseudomonas), morak Cellar in (Moraxella), Helicobacter genus (Helicobacter) and a stereo notes Pomona's in (Stenotrophomonas), Listeria genus (Listeria), Staphylococcus Cocos genus (Staphylococcus), Vibrio genus (Vibrio) and viruses , It may include disease biomarkers, such as cancer or immune disease, preferably E. coli ( E. coli O157:H7), Salmonella enteritidis , Salmonella typhi , and Shigella D. Centeriae ( Shigella dysenteriae ), Klebsiella pneumoniae , Pseudomonas aeruginosa , Moraxella catarrhalis , Helicobacter pylori, and Helicobacter pylori stenotrobacter pylori and pilriah (Stenotrophomonas maltophilia), L. monocytogenes Zen (Listeria monocytogene), Staphylococcus Aru Leu (Staphylococcus aureus), Vibrio cholera (Vibrio cholerae), Vibrio Burley blood kusu (Vibrio vulnificus), norovirus (norovirus), Human Immunodeficiency virus , Human Epithelial Growth Factor Receptor 2, interferon (Interferons) and interleukins (Interleukins) may be any one or more selected from the group consisting of And, most preferably, it may be Salmonella enteritidis .

That is, the complex for separating and detecting pathogenic microorganisms of the present invention specifically binds to an antigen located on the surface of Salmonella enteritidis, a pathogenic microorganism that causes food poisoning, to separate the presence of pathogenic microorganisms present in a liquid or solid sample, or Not only can it be concentrated, it can be detected.

Therefore, the antibody is not particularly limited as long as it specifically binds to the pathogenic microorganism, but most preferably may be an anti-Salmonella H antibody or an anti-Salmonella O antibody.

The copper-iron oxide magnetic nanoparticles can be directly bonded to the antibody, and the efficiency of immobilization is 3 to 6 times higher than when using other magnetic nanoparticles without additional processes such as adding a linker or additional surface modification. It is preferable in that it can be improved.

A linker may be further included between the copper-iron oxide magnetic nanoparticles and the antibody, but since there is no difference in immobilization efficiency compared to the added process step, the copper-iron oxide magnetic nanoparticles directly bind the antibody in terms of process convenience and cost-effectiveness. It is desirable to make it.

The linker is not particularly limited as long as it can be immobilized on the surface of the nanoparticles known in the art.

The complex for separating and detecting pathogenic microorganisms according to the present invention is a multifunctional particle having a nanometer-sized level with a property capable of specifically separating and detecting pathogenic microorganisms while having magnetic properties affected by magnets. Using this, it is possible to qualitatively/quantitatively detect the presence or absence of pathogenic microorganisms from various food and clinical samples, such as liquids and solids, in which several different components are mixed, and can be applied simultaneously to separate pathogenic microorganisms. The present invention can separate and detect pathogenic microorganisms from a sample at high speed within 1 to 3 hours. This simplifies the entire process of detecting existing pathogenic microorganisms, thereby improving the accuracy, reproducibility and reliability of sample preparation, and has a remarkable advantage in being able to detect trace amounts of pathogenic microorganisms at high speed.

The magnetism may be applied through a permanent magnet or an electromagnet.

In the present invention,'quantitative' means that the amount of pathogenic microorganisms bound to the complex for isolation and detection of pathogenic microorganisms of the present invention can be numerically detected. Specifically, the complex for separating and detecting pathogenic microorganisms combined with pathogenic microorganisms is separated under magnetism, and the pathogenic microorganisms are separated from the complex through heat treatment and lysed, and then DNA is extracted to determine the Ct value through Real-tim PCR. It can be confirmed by converting it to the concentration of microorganisms.

According to an embodiment of the present invention, it was confirmed that the quantitative detection range of pathogenic microorganisms detected by the complex for isolation and detection of pathogenic microorganisms of the present invention was 4.0 × 10 ³ to 1.6 × 10 ⁹ cells.

Another aspect of the present invention relates to a method for producing a complex for isolation and detection of pathogenic microorganisms comprising the following steps.

1) ultrasonically treating the copper-iron oxide magnetic nanoparticles;

2) obtaining a complex by immobilizing the antibody on the sonicated copper-iron oxide magnetic nanoparticles; And

3) removing unreacted material from the complex.

Step 1) is a step of ultrasonically treating the copper-iron oxide magnetic nanoparticles. Specifically, this is a step of forming a colloid of magnetic nanoparticle clusters by applying ultrasonic waves to the copper-iron oxide magnetic nanoparticles.

Step 2) is a step of obtaining a complex by immobilizing the antibody on the clustered copper-iron oxide magnetic nanoparticles through the ultrasonic wave. That is, an antibody capable of specifically recognizing and binding pathogenic microorganisms is immobilized on the copper-iron oxide magnetic nanoparticles clustered by the ultrasonic treatment, so that pathogenic microorganisms are separated and detected.

The step 2) may be performed for 10 to 40° C., 1 to 60 minutes, preferably 15 to 30° C., may be performed for 5 to 15 minutes, and magnetic nanoparticles are performed within the lower and upper limits. Antibodies can be most efficiently immobilized on particles. The amount of antibody immobilized on the surface of magnetic nanoparticles in the industry is highly related to the detection range and detection efficiency, so even 1% of the antibody can be further immobilized without surface modification or substitution and coating processes using harmful solvents. Has a meaningful meaning.

After performing the step 1) and before performing the step 2), the step of immobilizing the linker on the surface of the ultrasonically treated copper-iron oxide magnetic nanoparticles may be further included. In the case of including a linker, the orientation of the antibody may be improved, but despite the need for an additional chemical process step, there is no significant difference in the immobilization ratio and when directly binding the antibody, so the copper- It is most preferable to directly bind the iron oxide magnetic nanoparticles to the antibody.

The linker is not particularly limited as long as it can be immobilized on the surface of the nanoparticles known in the art.

Finally, step 3) is a step of removing the unreacted material from the complex, and if it is a method commonly used in the art, it is not particularly limited, but for example, after separating the complex by applying magnetism from the outside, the solution is removed and After adding a PBS buffer solution, magnetism is applied again to remove only the solution.

The complex for separating and detecting pathogenic microorganisms of the present invention is preferably prepared by immobilizing an antibody that specifically binds to a pathogenic microorganism on copper-iron oxide magnetic nanoparticles.

The complex for isolation and detection of pathogenic microorganisms of the present invention directly immobilizes an antibody that specifically binds to a pathogenic microorganism on copper-iron oxide magnetic nanoparticles, or after fixing the BC domain of the modified protein A represented by SEQ ID NO: 1 By immobilization, the amount of immobilization of the antibody immobilized on the copper-iron oxide magnetic nanoparticles is greatly increased while maintaining the activity of the antibody, so that pathogenic microorganisms can be removed from the actual food or livestock products using the magnetic nanoparticles to which the antibody according to the present invention is immobilized. It has the effect of being able to accurately separate and detect.

Another aspect of the present invention relates to a method for isolation and detection of pathogenic microorganisms comprising the following steps.

Ⅰ) contacting the complex for isolation and detection of pathogenic microorganisms with a sample to be analyzed;

Ⅱ) separating the complex for separating and detecting the pathogenic microorganism by applying magnetism; And

Ⅲ) detecting pathogenic microorganisms specifically bound to the complex for isolation and detection of pathogenic microorganisms.

First, as in step I), the complex for separating and detecting the pathogenic microorganism is brought into contact with the sample to be analyzed.

The sample to be analyzed includes all subjects that may be suspected of inhabiting pathogenic microorganisms, and may preferably be food or livestock products.

It was confirmed that the quantitative detection range of pathogenic microorganisms by the detection method of the present invention was 4.0 × 10 ³ to 1.6 × 10 ⁹ cells.

Step I) may be performed for 10 to 120 minutes, preferably 50 to 120 minutes, and most preferably 60 to 80 minutes in terms of cost and time.

Next, as in step II), the complex for separating and detecting the pathogenic microorganism is separated by applying magnetism. Specifically, when magnetism is applied to a part of the result obtained from step Ⅰ), the complex for separating and detecting pathogenic microorganisms according to the present invention is collected at the corresponding site, thereby enabling separation.

Because the pathogen or biomarker of the disease is determined by the antibody include, but not particularly limited to, Escherichia coli (E. coli), Salmonella genus (Salmonella), a break in Gela (Shigella), Enterobacter bacteria in three (Enterobacteriaceae), Pseudomonas species (Pseudomonas), morak Cellar in (Moraxella), Helicobacter genus (Helicobacter) and a stereo notes Pomona's in (Stenotrophomonas), Listeria genus (Listeria), Staphylococcus Cocos genus (Staphylococcus), Vibrio genus (Vibrio) and viruses , It may include disease biomarkers, such as cancer or immune disease, preferably E. coli ( E. coli O157:H7), Salmonella enteritidis , Salmonella typhi , and Shigella D. Centeriae ( Shigella dysenteriae ), Klebsiella pneumoniae , Pseudomonas aeruginosa , Moraxella catarrhalis , Helicobacter pylori, and Helicobacter pylori stenotrobacter pylori and pilriah (Stenotrophomonas maltophilia), L. monocytogenes Zen (Listeria monocytogene), Staphylococcus Aru Leu (Staphylococcus aureus), Vibrio cholera (Vibrio cholerae), Vibrio Burley blood kusu (Vibrio vulnificus), norovirus (norovirus), Human Immunodeficiency virus , Human Epithelial Growth Factor Receptor 2, interferon (Interferons) and interleukins (Interleukins) may be any one or more selected from the group consisting of And, most preferably, it may be Salmonella enteritidis .

That is, the complex for separating and detecting pathogenic microorganisms of the present invention specifically binds to an antigen located on the surface of Salmonella enteritidis, a pathogenic microorganism that causes food poisoning, to separate the presence of pathogenic microorganisms present in a liquid or solid sample, or Not only can it be concentrated, it can be detected.

Therefore, the antibody is not particularly limited as long as it specifically binds to the pathogenic microorganism, but most preferably may be an anti-Salmonella H antibody or an anti-Salmonella O antibody.

In the step (II), the magnetism may be applied through a permanent magnet or an electromagnet.

The step (III) includes (i) dissolving (Lysis) pathogenic microorganisms bound to the complex for isolation and detection of pathogenic microorganisms; And (ii) centrifuging the dissolved solution obtained from (i), obtaining a supernatant, and analyzing pathogenic microorganisms using Real time-PCR.

The dissolution in step (i) may be performed through heating, and the temperature may be 50 to 150°C.

The features and advantages of the present invention are summarized as follows:

(I) The present invention provides a complex for separating and detecting pathogenic microorganisms capable of rapidly separating and accurately detecting pathogenic microorganisms from a sample, and a method for manufacturing the same.

(Ii) In addition, the present invention provides a method for separating and detecting pathogenic microorganisms from a sample using the complex for isolation and detection of pathogenic microorganisms.

(Iii) The complex for separating and detecting pathogenic microorganisms of the present invention and the method for separating and detecting pathogenic microorganisms using the same can be practically used in liquid or solid foods or clinical samples in which a large amount of components are mixed. It shows the function of quickly and accurately separating and detecting pathogenic microorganisms from food through a simple process, and by using this function, the degree of contamination of food is quickly checked to prevent food poisoning infection or in case of disease caused by pathogenic microorganisms, pathogenic microorganisms can be detected. It can be quickly separated and detected and used for precise treatment.

1 is a diagram schematically showing the structure and principle of action of a magnetic nanoparticle-antibody complex according to the present invention.
2 is a result of quantitative analysis of BCA protein in order to confirm the concentration of the antibody immobilized on the magnetic nanoparticle-antibody complex prepared from Example 1 and Comparative Examples 1 to 3.
FIG. 3 is a photograph taken by time of a process of separating the magnetic nanoparticle-antibody complex and the solution from the solution by applying magnetism to the mixture prepared in Example 1. FIG.
4A is a graph showing the measurement of the amount of antibody immobilized in the magnetic nanoparticle-antibody complex prepared from Examples 1_4, 1_8, Example 1_12, Example 1_16, Example 1_20, and Example 1_24 (N = 4).
4B is a magnetic nanoparticle-antibody complex prepared from Examples 1_4, 1_8, Example 1_12, Example 1_16, Example 1_20, and Example 1_24 by calculating the immobilization efficiency (%) of the antibody. It is a graph (N = 4).
5 is a result of analyzing the detection efficiency of pathogenic microorganisms of the magnetic nanoparticle-antibody complex according to the immobilization time of the magnetic nanoparticle and the antibody in Example 1_8 (N = 4).
6 is a result of analyzing the detection efficiency of pathogenic microorganisms of the magnetic nanoparticle-antibody complex prepared from Example 1_8 under various temperature conditions (N = 4).
7 is a result of analyzing the detection efficiency of pathogenic microorganisms of the magnetic nanoparticle-antibody complex according to the immobilization time of the magnetic nanoparticle and the antibody in Example 1_8 under various time conditions (N = 4).
8 is a result of measuring the linearity and detection limit of the magnetic nanoparticle-antibody complex prepared in Example 1.
9 is a result of treatment of the magnetic nanoparticle-antibody complex prepared in Example 1 on 10 random samples and measuring pathogenic microorganisms by Real-time PCR analysis.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Experiment method

<Measurement of protein concentration>

The protein and antibody concentrations eluted from the complex according to the present invention were measured by Bradford assay. As a standard solution, a Bovine Serum Albumin solution at concentrations of 10 μg/ml, 5 μg/ml, 2.5 μg/ml, 1.25 μg/ml, and 0 μg/ml was used. 100 [mu]l sample and Bradford's reagent (Sigma-Aldrich, Inc., St. Louis, MO, USA) were each placed in a 96-well plate. The reaction was performed at room temperature for 5 minutes, and the result was confirmed by measuring OD (optical density) at 595 nm.

<nucleic acid analysis by real-time PCR>

The complex according to the present invention reacted with the pathogenic microorganism was washed twice with 500 µl of PBS buffer, and 200 µl of distilled water (DW) was added. The mixture was heated to lyse cells at 100° C. for 10 minutes. It was left on ice for 15 minutes. After centrifugation at 12,000 rpm for 10 minutes, the supernatant was used as a DNA sample. Real-time PCR was performed on the DNA sample, and the Ct value was continuously calculated.

Specifically, Real-time PCR and data analysis were performed using the QuantStudio 3 Real-Time PCR System (Thermo-Fisher Scientific, Inc., Walthan, MA, USA). The cycle threshold (Ct) value was automatically calculated by the QuentStudio 3 system. PCR mixture 18 in 2 μl of pathogenic microorganism DNA extracted with hot water After adding μl, the mixture was mixed so that no air bubbles were generated using a pipette. The PCR mixture includes Thunderbird SYBR qPCR mix (Toyobo, Osaka, Japan), two primers (Bioneer Co., Daejeon, Korea), ROX reference dye, and distilled water, and the specific composition is shown in Table 1 below. For real-time PCR, the 5'-CTCACCAGGAGATTACAACATGG-3' base sequence was used as the forward primer, and the 5'-AGCTCAGACCAAAAGTGACCATC-3' base sequence was used as the reverse primer. The cycling sequence proceeded as follows. First, it was maintained at 95°C for 10 minutes, and then 15 seconds at 95°C and 60 seconds at 60°C were repeated.

reagent Reaction volume Final concentration SYBR qPCR Mix 10 μl 1 x Forward primer 6 pmol 0.3 μM Reverse primer 6 pmol 0.3 μM ROX reference dye 0.4 μl 0.1 x DNA 2 μl Distilled water Remaining amount (5.6 μl) gun 20 μl

Example

<Example 1> Preparation of magnetic nanoparticle-antibody complex

Copper-iron oxide magnetic nanoparticles (CuFe ₂ O ₄ , <100 nm, Cu-FMNP) (Sigma-Aldrich, Inc., St. Louis, MO, USA) in 50 mM PBS (pH 7.0; Genomicbase, Inc., Seoul) , Korea) After mixing to a concentration of 1 mg/ml in a buffer solution, it was reacted in an ultrasonic water bath for about 3 minutes. The ultrasonically applied mixture was subdivided into 500 µl, and a PBS buffer solution excluding the copper-iron oxide magnetic nanoparticles was removed using a magnet. Here, 4 μl of antibodies of various concentrations (1 μg/ml, 2 μg/ml, 3 μg/ml, 4 μg/ml, 5 μg/ml and 6 μg/ml) were added, respectively, and 496 μl of PBS buffer was added. After that, by incubating for 10 minutes at 25 ℃ using a rotary shaker to prepare a complex for detecting microorganisms of magnetic nanoparticles and antibodies. In order to remove the unreacted material from the complex, it was washed twice with PBS buffer using a magnet. Anti-salmonella Omnivalent antibody (ab78688, abcam) was used as the antibody.

When the actual antibody concentration in the microbial detection complex solution was calculated, 4 ng/ml, 8 ng/ml, 12 ng/ml, 16 ng/ml, 20 ng/ml, and 24 ng/ml were used, respectively. Example 1_4, Example 1_8, Example 1_12, Example 1_16, Example 1_20, Example 1_24.

At this time, antibodies that did not bind to magnetic nanoparticles were measured by Bradford assay (Sigma-Aldrich, Inc., St. Louis, MO, USA).

<Comparative Example 1> Preparation of cobalt-iron oxide magnetic nanoparticle-antibody complex

Copper-iron oxide magnetic nanoparticles (CuFe ₂ O ₄ , <100 nm, Cu-FMNP) (Sigma-Aldrich, Inc., St. Louis, MO, USA) instead of cobalt-iron oxide magnetic nanoparticles (CoFe ₂ O ₄ , <100 nm, Co-FMNP) (Sigma-Aldrich, Inc., St. Louis, MO, USA) was prepared in the same manner as in Example 1 except for using a cobalt-iron oxide magnetic nanoparticle-antibody complex. .

<Comparative Example 2> Preparation of nickel-iron oxide magnetic nanoparticle-antibody composite

Copper-iron oxide magnetic nanoparticles (CuFe ₂ O ₄ , <100 nm, Cu-FMNP) (Sigma-Aldrich, Inc., St. Louis, MO, USA) instead of nickel-iron oxide magnetic nanoparticles (LiFe ₂ O ₄ , <100 nm, Li-FMNP) (Sigma-Aldrich, Inc., St. Louis, MO, USA) was prepared in the same manner as in Example 1 except for using a nickel-iron oxide magnetic nanoparticle-antibody complex. .

<Comparative Example 3> Preparation of iron oxide magnetic nanoparticle-antibody complex

Copper-iron oxide magnetic nanoparticles (CuFe ₂ O ₄ , <100 nm, Cu-FMNP) (Sigma-Aldrich, Inc., St. Louis, MO, USA) instead of iron oxide magnetic nanoparticles (Fe ₂ O ₄ , <100) nm, Li-FMNP) (Sigma-Aldrich, Inc., St. Louis, MO, USA) was used to prepare an iron oxide magnetic nanoparticle-antibody complex in the same manner as in Example 1.

<Test Example 1> Analysis of antibody immobilization efficiency by magnetic nanoparticle type

In order to confirm the antibody immobilization efficiency of the magnetic nanoparticle-antibody complex prepared according to Example 1 and the complexes prepared from Comparative Examples 1 to 3, quantitative analysis of BCA protein was performed.

2 is a result of quantitative analysis of BCA protein in order to confirm the protein concentration immobilized on the magnetic nanoparticle-antibody complex prepared from Example 1 and Comparative Examples 1 to 3. According to this, it can be seen that among the various magnetic nanoparticles, the copper-iron oxide magnetic nanoparticles most efficiently immobilize the antibody (3 mg of antibody is immobilized per 1 g of iron oxide). Through these results, it can be confirmed that the copper-iron oxide magnetic nanoparticles have a high fixed capacity of the antibody by 3 to 6 times or more by other magnetic nanoparticles.

<Test Example 2> Time required for separation of magnetic nanoparticle-antibody complex and solution

When the magnetic nanoparticle-antibody complex prepared in Example 1 was mixed with the solution, the time required to separate from the solution by applying magnetism was measured, and the process was photographed.

3 is a photograph taken by time of a process of separating from the solution by applying magnetism to the mixture of the magnetic nanoparticle-antibody complex and the solution prepared in Example 1. FIG. According to this, it was confirmed that the copper-iron oxide magnetic nanoparticles can be separated from the solution within about 1 minute with magnetism applied through a magnet.

<Test Example 3> Analysis of optimal conditions of the magnetic nanoparticle-antibody complex prepared in Example 1

1) Analysis of the amount of antibody to be immobilized

In the magnetic nanoparticle-antibody complex prepared from Example 1_4, Example 1_8, Example 1_12, Example 1_16, Example 1_20, and Example 1_24, it was attempted to confirm the optimal amount of antibody immobilized. In addition, the immobilization efficiency (%) attached to the copper-iron oxide magnetic nanoparticles was also analyzed. The concentration of the antibody was calculated by subtracting the amount of unimmobilized antibody from the total amount of antibody using the Bradford assay (Sigma-Aldrich, Inc., St. Louis, MO, USA) and dividing the total amount of antibody.

4A is a graph showing the measurement of the amount of antibody immobilized in the magnetic nanoparticle-antibody complex prepared from Examples 1_4, 1_8, Example 1_12, Example 1_16, Example 1_20, and Example 1_24 (N = 4). 4B is a magnetic nanoparticle-antibody complex prepared from Examples 1_4, 1_8, Example 1_12, Example 1_16, Example 1_20, and Example 1_24 by calculating the immobilization efficiency (%) of the antibody. It is a graph (N = 4).

4A, it was confirmed that the copper-iron oxide magnetic nanoparticle-antibody complex according to Examples 1_12, 1_16, Example 1_20, and Example 1_24 was saturated and thus the antibody was no longer immobilized. Therefore, in the process of preparing the copper-iron oxide magnetic nanoparticle-antibody complex according to the present invention, it is preferable to use an antibody at a concentration of 1 to 10 ng/ml, and an antibody at a concentration of 4 to 8 ng/ml is used in terms of immobilization efficiency. It was confirmed that it is more preferable.

Referring to FIG. 4B, the immobilization efficiency of the antibody is a percentage of the amount of the antibody (Immobilized Ab) immobilized on the magnetic nanoparticles relative to the total amount of the added antibody (Added Ab), and the copper-iron oxide magnetic nanoparticles prepared from Example 1_8- It was confirmed that the antibody complex was the best.

2) Analysis of optimal time to be fixed

In accordance with Example 1_8, an optimal time required for the copper-iron oxide magnetic nanoparticles and the antibody to be immobilized was analyzed. The experiment was carried out as follows.

A copper-iron oxide magnetic nanoparticle-antibody complex was prepared in the same manner as in Example 1_8, except that the immobilization time of the magnetic nanoparticles and the antibody was changed to 10, 20, 30, 40, 50, and 60 minutes, respectively.

Salmonella enteritidis , which had been lyophilized at -80°C, was inoculated into 10 mL Luria-Bertani (LB) medium in a 15 mL tube using a pipette from the cell stock. The inoculated LB medium was cultured in a 37°C incubator at a speed of 125 rpm, and when the optical density at 200 nm was 2.295, the obtained sample was diluted 1.6 × 10 ⁴ times to prepare a pathogenic microorganism sample. Next, as described above, the magnetic nanoparticle-antibody complex prepared by varying the immobilization time was added. By reacting for 40 minutes at various temperatures (4° C., 25° C., 37° C.) using a vortex at room temperature, the antibody-antigen reaction between the magnetic nanoparticle-antibody complex and the pathogenic microorganism was induced. After 40 minutes, if the reaction is completed and the sample is left on the magnet for 1 minute, pathogenic microorganisms that reacted with the magnetic nanoparticles are attracted toward the magnet. In order to separate the reacted pathogenic microorganisms from the magnetic nanoparticle-antibody complex, the supernatant containing the unreacted material was carefully removed twice with a pipette. As described above, the ct value was calculated for the complex reacted with each pathogenic microorganism obtained according to the detection temperature using real-time PCR.

5 is a result of analyzing the detection efficiency of pathogenic microorganisms of the magnetic nanoparticle-antibody complex according to the immobilization time of the magnetic nanoparticle and the antibody in Example 1_8 (N = 4). According to this, it was confirmed that the time to immobilize the magnetic nanoparticles and the antibody was sufficient in 10 minutes.

<Test Example 4> Optimal detection temperature and time of pathogenic microorganisms of the magnetic nanoparticle-antibody complex prepared in Example 1

1) Optimal detection temperature for pathogenic microorganisms

First, Salmonella enteritidis , which had been lyophilized at -80°C, was inoculated into 10 mL Luria-Bertani (LB) medium in a 15 mL tube using a pipette from the cell stock. The inoculated LB medium was cultured in a 37°C incubator at a speed of 125 rpm, and when the optical density at 200 nm was 2.295, the obtained sample was diluted 1.6 × 10 ⁴ times to prepare a pathogenic microorganism sample. After adding the magnetic nanoparticle-antibody complex prepared in Example 1_8, the magnetic nanoparticle-antibody complex and the pathogenic microorganism were reacted for 40 minutes at various temperatures (4°C, 25°C, 37°C) using a vortex at room temperature. The liver antibody-antigen reaction was induced. After 40 minutes, if the reaction is completed and the sample is left on the magnet for 1 minute, pathogenic microorganisms that reacted with the magnetic nanoparticles are attracted toward the magnet. In order to separate the reacted pathogenic microorganisms from the magnetic nanoparticle-antibody complex, the supernatant containing the unreacted material was carefully removed twice with a pipette. As described above, the ct value was calculated for the complex reacted with each pathogenic microorganism obtained according to the detection temperature using real-time PCR.

6 is a result of analyzing the detection efficiency of pathogenic microorganisms of the magnetic nanoparticle-antibody complex prepared from Example 1_8 under various temperature conditions (N = 4). According to this, it was confirmed that when the detection temperature was 25°C, the binding of the pathogenic microorganism and the antibody was efficiently activated. That is, 25°C is considered to be the temperature at which antibodies that specifically bind to pathogenic microorganisms are efficiently activated.

2) Optimal detection time for pathogenic microorganisms

First, Salmonella enteritidis , which had been lyophilized at -80°C, was inoculated into 10 mL Luria-Bertani (LB) medium in a 15 mL tube using a pipette from the cell stock. The inoculated LB medium was cultured in a 37°C incubator at a speed of 125 rpm, and when the optical density at 600 nm was 1.979, the obtained sample was diluted 1.6×10 ⁴ times to prepare a pathogenic microorganism sample. After adding the magnetic nanoparticle-antibody complex prepared in Example 1_8, the magnetic nanoparticle-antibody complex and the magnetic nanoparticle-antibody complex were reacted at 25° C. for various times (20, 40, 60, 80 and 120 minutes) using a vortex at room temperature. Antibody-antigen reactions between pathogenic microorganisms were induced. When the reaction is completed sample is left on the magnet for 1 minute, pathogenic microorganisms that reacted with the magnetic nanoparticles are attracted toward the magnet. In order to separate the reacted pathogenic microorganisms from the magnetic nanoparticle-antibody complex, the supernatant containing the unreacted material was carefully removed twice with a pipette. As described above, the Ct value was calculated for the complex reacted with each pathogenic microorganism obtained according to the detection temperature using real-time PCR.

7 is a result of analyzing the detection efficiency of pathogenic microorganisms of the magnetic nanoparticle-antibody complex according to the immobilization time of the magnetic nanoparticle and the antibody in Example 1_8 under various time conditions (N = 4). According to this, it was confirmed that the Ct value gradually decreased as time increased. That is, it can be seen that the copper-iron oxide magnetic nanoparticle-antibody complex prepared in Example 1_8 takes 60 minutes to form a reaction with the pathogenic microorganism.

<Test Example 5> Analysis of the detection limit of the magnetic nanoparticle-antibody complex prepared from Example 1 according to the concentration of pathogenic microorganisms

Samples of pathogenic microorganisms of various concentrations were prepared. The preparation process of the pathogenic microorganism sample was as follows. Salmonella enteritidis , which had been lyophilized at -80°C, was inoculated into 10 mL Luria-Bertani (LB) medium in a 15 mL tube using a pipette from the cell stock. The inoculated LB medium was incubated overnight at a speed of 125 rpm in a 37°C incubator. The number of Salmonella enteritidis bacteria was measured using the optical density at 600 nm and multiplied by 10 ⁹ . Centrifugation at 4,000 g for 20 minutes to remove all of the supernatant except for the precipitated cells, and suspend the cells in PBS buffer. Each of these was subjected to a 4-fold dilution rate to prepare pathogenic microbial samples at concentrations of 2288000, 572000, 143000, 35750, 8937.5, 2234.375 cells/ml, and the magnetic nanoparticle-antibody complex prepared from Example 1 above. After the addition of, the reaction was carried out at room temperature using a vortex for 1 hour to induce an antibody-antigen reaction between the magnetic nanoparticle-antibody complex and the pathogenic microorganism. After an hour, when the reaction is completed and the sample is left on the magnet for 1 minute, pathogenic microorganisms that react with the magnetic nanoparticles are attracted toward the magnet. In order to separate the reacted pathogenic microorganisms from the magnetic nanoparticle-antibody complex, the supernatant containing the unreacted material was carefully removed twice with a pipette. After DNA extraction was performed on each sample, real-time PCR was performed, and Ct values were continuously calculated. Through the previous experiment, an example having the best antibody immobilization efficiency was confirmed, and since the amounts of the final immobilized antibodies were all the same, it was collectively referred to as Example 1, but the magnetic nanoparticle-antibody complex used in this experiment was Example 1_8.

8 is a result of measuring the linearity and detection limit of the magnetic nanoparticle-antibody complex prepared in Example 1. According to this, it can be determined that the detection limit for pathogenic microorganisms can be measured up to about 13,000 cells. Based on this, the detection range of pathogenic microorganisms of the magnetic nanoparticle-antibody complex prepared in Example 1 could be determined to be 1.3 × 10 ⁴ to 2.3 × 10 ⁶ cells.

<Test Example 6> Analysis using real samples

From actual food (milk (liquid formulation), confectionery (solid formulation)), the prepared complex for detecting pathogenic microorganisms of the present invention was applied to compare the accuracy. Specifically, 25 ml of milk in a liquid formulation and 25 g of confectionery (cookies) in a solid formulation were prepared, and each of them was mixed in 100 ml LB medium and incubated at 37°C and 125 rpm for 6 hours to prepare 5 milk and snack samples. Ready.

Next, Salmonella enteritidis , which had been lyophilized at -80°C, was inoculated into 10 mL Luria-Bertani (LB) medium in a 15 mL tube using a pipette from the cell stock. The inoculated LB medium was incubated overnight at a speed of 125 rpm in a 37°C incubator. At this time, the number of Salmonella enteritidis bacteria was measured using the optical density at 600 nm, and measured by multiplying by 10 ⁹ , a pathogenic microbial sample (1.6 * 10 ⁶ Cells/ml) were prepared.

The pathogenic microorganism samples were added to only 5 samples randomly among the milk samples (No 1-5) and confectionery samples (No 6-10). Here, in the case of confectionery samples, after centrifugation, only the supernatant was collected, and pathogenic microorganism samples were added to prepare a total of 10 samples. After DNA extraction was performed for each sample, real-time PCR was performed, and Ct values were continuously calculated.

Through the previous experiment, an example having the best antibody immobilization efficiency was confirmed, and since the amounts of the final immobilized antibodies were all the same, it was collectively referred to as Example 1, but the magnetic nanoparticle-antibody complex used in this experiment was Example 1_8.

9 is a result of treatment of the magnetic nanoparticle-antibody complex prepared in Example 1 on 10 random samples and measuring pathogenic microorganisms by Real-time PCR analysis. According to this, it was quickly and accurately detected that pathogenic microorganisms were added to 1, 2, 3, 9, and 10 samples and no pathogenic microorganisms were added to 4, 5, 6, 7, and 8 samples. It was confirmed that the sample with pathogenic microorganism and the sample without pathogenic microorganism clearly showed statistically significant difference. In addition, it can be seen that the same amount of pathogenic microorganisms were added to the samples 1, 2, 3, 9, and 10.

Therefore, the magnetic nanoparticle-antibody complex prepared from Example 1 according to the present invention detected pathogenic microorganisms in 10 food samples with an accuracy of 100%.

As described above, specific parts of the present invention have been described in detail, and it is clear that these specific techniques are merely preferred embodiments, and the scope of the present invention is not limited thereto for those of ordinary skill in the art. Therefore, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (8)

Copper-iron oxide magnetic nanoparticles; And
An antibody that is immobilized on the surface of the magnetic nanoparticles and specifically binds to a pathogenic microorganism; a complex for isolation and detection of pathogenic microorganisms. The method of claim 1,
The magnetic nanoparticles are CuFe ₂ O ₄ A complex for separation and detection of pathogenic microorganisms, characterized in that. The method of claim 1,
The antibody is an anti-Salmonella H antibody or an anti-Salmonella O antibody, characterized in that the complex for isolation and detection of pathogenic microorganisms. 1) ultrasonically treating the copper-iron oxide magnetic nanoparticles;
2) obtaining a complex by immobilizing the antibody on the sonicated copper-iron oxide magnetic nanoparticles; And
3) removing the unreacted material from the complex; method for producing a complex for separating and detecting pathogenic microorganisms comprising. Ⅰ) contacting the complex for isolation and detection of pathogenic microorganisms according to claim 1 with a sample to be analyzed;
Ⅱ) separating the complex for separating and detecting the pathogenic microorganism by applying magnetism; And
Ⅲ) Separating and detecting pathogenic microorganisms specifically bound to the complex for isolation and detection of pathogenic microorganisms; pathogenic microorganism isolation and detection method comprising a. The method of claim 5,
The step I) is a pathogenic microorganism separation and detection method, characterized in that performed for 60 to 80 minutes. The method of claim 5,
In the step (II), the magnetism is applied through a permanent magnet or an electromagnet. The method of claim 5,
The step (III) includes (i) dissolving (Lysis) pathogenic microorganisms bound to the complex for isolation and detection of pathogenic microorganisms; And
(Ii) centrifuging the dissolved solution obtained from the above (i), obtaining a supernatant, and analyzing the pathogenic microorganism using Real time-PCR.

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