KR101871572B1 - Hybrid nanoparticles for detection of pathogenic bacteria and Manufacturing methods thereof - Google Patents

Abstract

The present invention relates to magnetic composite nanoparticles for pathogen detection, and more particularly, to a magnetic composite nanoparticle for pathogen detection which has excellent antioxidation and saturation magnetization and is capable of effectively detecting pathogens and a method for producing the same .
The present invention provides magnetic composite nanoparticles for pathogen detection which have excellent antioxidation and saturation magnetization and can effectively detect pathogens and a method for producing the same.

Description

TECHNICAL FIELD [0001] The present invention relates to magnetic nanoparticles for detecting pathogens,

The present invention relates to magnetic composite nanoparticles for pathogen detection, and more particularly, to a magnetic composite nanoparticle for pathogen detection which has excellent antioxidation and saturation magnetization and is capable of effectively detecting pathogens and a method for producing the same .

Magnetic nanoparticles having super magnetic properties have been used as various functional materials because of their ease of trapping and redispersion of particles in a solution depending on the presence or absence of a magnetic field. Among them, Fe ₃ O ₄ magnetic nanoparticles have inherent superparamagnetism and biocompatibility and are known to be suitable for application in the biotechnology field. In particular, the functionalized magnetic nanoparticles to which antibodies are bound can be selectively captured with DNA, biomarker proteins, bacteria, and the like, and then collected using a magnetic field. However, Fe ₃ O ₄ has a decisive disadvantage of losing superparamagnetism due to oxidation when moisture is present.

As a general method for preventing the oxidation of Fe ₃ O ₄ , a protective film such as silica (SiO ₂ ) is deposited. However, if the reaction conditions can not be precisely controlled, the formation of silica film tends to solidify the particles in a lump state, and the saturation magnetization of the magnetic particles is remarkably reduced due to the high density of silica (2.195 g / cm ³ ). In addition, when a silane reaction is used to bond the antibody to the silica on the particle surface, the orientation of the antibody is deteriorated and the binding efficiency with the detection substance is decreased.

A problem to be solved by the present invention is to provide a magnetic composite nanoparticle for pathogen detection which has excellent antioxidation and saturation magnetization and is capable of effectively detecting pathogens.

Another problem to be solved by the present invention is to provide a method for producing magnetic composite nanoparticles for pathogen detection, which has excellent antioxidation and saturation magnetization and is capable of effectively detecting pathogens.

Another object of the present invention is to provide a method for detecting pathogens using magnetic composite nanoparticles for pathogen detection having excellent oxidation resistance and saturation magnetization.

In order to solve the above problems,

The present invention relates to magnetic nanoparticles;

A metal organic structure coating film surrounding the magnetic nanoparticles; And

Characterized in that the metal atom present in the coating and the sulfur atom of the biological functional molecular sieve are fixed by coordinate bonding.

The present invention relates to magnetic nanoparticles;

A metal organic structure coating film surrounding the magnetic nanoparticles; And

Characterized in that the sulfur atom of the biological functional molecular sieve is fixed by coordination bonding to the metal atom in an unsaturated coordination bond state present in the coating.

In one aspect, the present invention provides magnetic nanoparticles;

A metal organic structure coating film surrounding the magnetic nanoparticles; And

There is provided a composite nanoparticle for trapping pathogenic bacteria comprising an antibody immobilized through a bond between a metal atom present in the coating and a thiol group.

In another aspect, the present invention provides magnetic nanoparticles;

Zeolitic imidazolate frameworks (ZIFs) coatings surrounding the magnetic nanoparticles; And

Characterized in that sulfur atoms of the biological functional molecular sieve are fixed by coordination bonding to metal atoms coordinated in an unsaturated state present in the coating.

In another aspect, the present invention provides magnetic nanoparticles;

A ZIF-8 coating surrounding the magnetic nanoparticles; And

To provide a cleavage antibody that coordinates with the coating.

According to another aspect of the present invention, there is provided a magnetic nanoparticle comprising a magnetic nanoparticle, a metal organic structure film surrounding the magnetic nanoparticle, and a complex nanoparticle in which a sulfur atom of a metal atom existing in the film and a biological functional molecular sieve is fixed by coordinate bonding, Into a sample comprising a material that binds to the functional molecular sieve;

Separating the composite nanoparticles using magnetic force; And

Detecting the substance bound to the biological functional molecular sieve

And a detection method.

The present invention, in one aspect,

Surrounding the magnetic nanoparticles with a metal organic structure; And

And fixing the sulfur atom of the biological functional molecular sieve to the metal atom of the metal organic structure by coordination bonding.

In one aspect, the present invention relates to a method of reducing a disulfide bond between heavy chains of an antibody and separating the antibody into a half-antibody having a thiol group; And

Forming a zinc-sulfur bond between the zinc of the unsaturated coordination state and the thiol group of the anti-fragment antibody present in the coating of the metal organic structure of the magnetic nanoparticles, and immobilizing the antibody; to provide.

In the present invention, the magnetic nanoparticles are understood to be nanoparticles composed of one or more magnetic particles that are magnetically responsive to a magnetic field.

In the present invention, the size of the magnetic nanoparticles is 1 to 1000 nanometers. When the antigen is a pathogenic bacterium, the size of the magnetic nanoparticles is preferably 100 to 500 nanometers so that one antigen can bind to several particles . In the practice of the present invention, the magnetic nanoparticles can be manufactured and used by known methods, and commercially available.

In the present invention, the metal organic structure is understood as a porous structure in which a metal ion and an organic molecule undergo coordination bonding. Preferably, the metal organic structure is a crystalline organic / inorganic hybrid polymer formed through coordination bonding with an organic ligand Lt; / RTI >

The metal ion of the metal organic structure is a metal to which the sulfur atom of the biological functional molecular sieve can coordinate and includes, but is not limited to, iron, copper, manganese, cadmium, palladium,

The organic molecules of the metal organic structure may be various organic molecules capable of forming a crystalline porous structure by coordinating with metal ions. Organic ligands which form a crystalline porous structure in the form of zeolite may be used so as to have hydrothermal stability, preferably. The organic ligand is not limited to 2-methylimidazole but may be an organic compound having two or more carboxyl groups including oxalic acid, malonic acid, succinic acid, glutaric acid, phthalic acid, terephthalic acid, And the like, and further, the present invention is not particularly limited to the above description.

In the practice of the present invention, the metal-organic structure are possible to use to manufacture by known methods where.

In the present invention, the metal organic structure may be zeolitic imidazole frameworks-8 (ZIF-8). In the case of ZIF-8, it can be synthesized without particle agglomeration at a lower temperature than silica and within a short time, and has chemical and thermal stability. In addition, the zinc atom present on the outer surface of ZIF-8 does not form a complete coordination bond and can react with the thiol group of the antibody to form a zinc-sulfur bond. Unlike the silane reaction in which an antibody is immobilized using an amine group randomly present in the antibody using such a characteristic, it is possible to immobilize an orientable antibody using a thiol group present in the heavy chain of the antibody. In addition, the low density of ZIF-8 (0.35 g / cm ³ ) does not significantly reduce the saturation magnetization of magnetic nanoparticles when used as a coating material.

In the present invention, the functional group bound to the surface of the metal organic structure is not limited to an antibody but includes a protein ligand, nucleic acid (DNA or RNA) molecule sequence capable of binding to the detection substance. The detection substance includes not only pathogenic bacteria but also biomolecules and chemical substances including proteins, virus phage, DNP, and aptamer in the nucleic acid field that can bind to the functional group And is not particularly limited to the above description.

The present invention provides magnetic composite nanoparticles for pathogen detection which have excellent antioxidation and saturation magnetization and can effectively detect pathogens and a method for producing the same.

The present invention proposes a method of immobilizing an antibody so as to have orientation in a ZIF-8 coating of magnetic nanoparticles, thereby improving the efficiency of collecting pathogens and showing the possibility of application of the metal organic structure to immunoassay.

Further, in the present invention, ZIF-8 coating is formed on Fe ₃ O ₄ magnetic nanoparticles to delay the oxidation of particles due to moisture, which is useful in increasing the chemical stability and storage stability of the particles.

Fig. 1 is a schematic diagram of an enlarged surface of a ZIF-8 coating synthesized by the method of the present invention
Figure 2 is a schematic diagram showing the preparation of an antibody having a thiol group by the method of the present invention. (a) shows reduction of the disulfide bond between the heavy chains of the antibody to separate the antibody into half pieces, and (b) shows the half-fragment antibody showing the thiol group.
Figure 3 is a schematic diagram showing the capture and detection of Staphylococcus aureus by using an antibody-immobilized Fe ₃ O ₄ @ ZIF-8 composite nanoparticle and an ATP measurement kit. (1) is Fe ₃ O ₄ (2) is ZIF-8, (3) is an antibody having a thiol group, and (4) is staphylococcus aureus.
Figure ₄ shows the detection of Staphylococcus aureus by using Fe ₃ O ₄ @ ZIF-8 complex nanoparticles immobilized with antibodies. (a) is a photograph of Fe ₃ O ₄ @ ZIF-8 composite nanoparticles selectively bound to the surface of Staphylococcus aureus. (b) is the result of collecting Staphylococcus aureus using Fe ₃ O ₄ @ ZIF-8 composite nanoparticles and then detecting bacteria using ATP detection kit.
Figure 5. Selectivity test results of Fe ₃ O ₄ @ ZIF-8 composite nanoparticles immobilized with Staphylococcus aureus antibody.

Hereinafter, the present invention will be described in detail with reference to examples. It is to be understood that the following examples are illustrative of the present invention but are not intended to limit the scope of the present invention and that the scope of the present invention is defined by the appended claims.

1. Reagents and equipment

6H₂O), 구연산나트륨(Sodium citrate), 요소(urea), 폴리아크릴아마이드(Polyacrylamide, PAM, Mw 5,000,000-6,000,000 g/mol), 질산아연(6수화물)(Zinc nitrate hexahydrate, Zn(NO₃)₂

6H₂O), 2-메틸이미다졸(2-Methylimidazole, 2-MIM), 메탄올(methanol), 2-(N-모르폴리노)에테인설폰산(2-(N-morpholino)ethanesulfonic acid, MES), 염화나트륨(sodium chloride, NaCl), 인산이수소칼륨(potassium dihydrogen phosphate, H₂KPO₄), 제2인산칼륨(potassium phosphate dibasic, HK₂PO₄), 트리스(2-카복시에틸)-포스핀(Tris(2-carboxyethyl)-phosphine, TECP), 트윈 20(Tween 20)은 시그마-알드리치 社에서 구매하였다.Iron (III) chloride (hexahydrate, FeCl ₃₎

6H ₂ O), sodium citrate, urea, polyacrylamide (PAM, Mw 5,000,000-6,000,000 g / mol), zinc nitrate hexahydrate, Zn (NO ₃ ) ₂

6H ₂ O), 2- methylimidazole (2-Methylimidazole, 2-MIM ), methanol (methanol), 2- (N - morpholino) ethanesulfonic acid (2- (N -morpholino) ethanesulfonic acid , MES ), Sodium chloride (NaCl), potassium dihydrogen phosphate (H ₂ KPO ₄ ), potassium phosphate dibasic (HK ₂ PO ₄ ), tris (2-carboxyethyl) (Tris (2-carboxyethyl) -phosphine, TECP) and Tween 20 were purchased from Sigma-Aldrich.

Staphylococcus antibodies were obtained from Abcam Inc. .

Casein hydrolyzate is MP Biomedicals. .

The Amicon centrifugal 10K filter was purchased from Millipore.

Portable photometers and ATP detection kits were purchased from Kikkoman.

2. Synthesis of Fe ₃ O ₄ magnetic nanoparticle clusters (hereinafter referred to as magnetic nanoparticles)

F ₃ O ₄ magnetic nanoparticles were synthesized using the one-pot solvothermal method. First, 8 millimoles of iron (III) chloride (hexahydrate), 24 millimoles of urea and 16 millimoles of sodium citrate are dissolved in 30 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.

3. Synthesis of ZIF-8 film on magnetic nanoparticle surface

The ZIF-8 coating was synthesized by an ultrasonic chemical method. First, 0.8 millimoles of zinc nitrate (hexahydrate) and 0.8 millimoles of 2-methylimidazole are dissolved in 30 milliliters of methanol. To this solution, 0.1 g of the magnetic nanoparticles synthesized in 2 are mixed, sonicated, and reacted at 60 ° C. After the solution is cooled to room temperature, the synthesized particles are collected using a magnet, and washed several times with methanol and water to finally obtain Fe ₃ O ₄ @ ZIF-8 composite nanoparticles. FIG. 1 is a schematic view showing an enlarged surface of a synthesized ZIF-8 coating. Zinc on the outermost surface of ZIF-8 is in a state of unsaturated coordination with nitrogen of 2-methylimidazole to achieve additional coordination with another electron pair.

4. Antibody immobilization to Fe ₃ O ₄ @ ZIF-8 composite nanoparticles

In order to immobilize the antibody on the surface of Fe ₃ O ₄ @ ZIF-8 complex nanoparticles, the disulfide bond of the antibody was reduced and separated into a semi-antibody having a thiol group. First, 9.76 grams of MES and 4.5 grams of sodium chloride are dissolved in 500 milliliters of water to make a MES buffer solution. Then, 14 micrograms of TCEP and 10 micrograms of antibody are dissolved in 400 milliliter of MES buffer, mixed well, and reacted. The reacted antibody is purified with an MES buffer solution using an amiconic centrifugal filter and reacted with 1.5 milligrams of Fe ₃ O ₄ @ ZIF-8 nanoparticles. Thereafter, the immobilized particles are mixed with 0.07 grams of casein and 0.01 gram of tween 20 to prevent non-specific binding and wash several times with phosphate buffer. Phosphate buffers are prepared by dissolving 0.272 grams of potassium dihydrogenphosphate and 1.714 grams of potassium diphosphate in 1 liter of water.

Figure 2 shows a schematic diagram in which TCEP reduces the disulfide bond between the heavy chains of the antibody to separate the antibody into half-pieces and reveal the thiol group. Zinc, which forms an unsaturated coordination bond on the ZIF-8 outermost surface, has a higher energy than zinc having a saturated coordination bond and can react with the thiol group of the antibody to lower the energy. Antibody immobilization on the particle surface through the thiol group is performed in a single step, and the antibody is immobilized with the orientation, increasing the binding efficiency with the detection substance.

5. Detection of pathogens using Fe ₃ O ₄ @ ZIF-8 complex nanoparticles

This experiment was conducted on Staphylococcus aureus. FIG. 3 is a schematic diagram for collecting and detecting Staphylococcus aureus using an antibody-immobilized Fe ₃ O ₄ @ ZIF-8 composite nanoparticle and an ATP measurement kit. After mixing 0.1 ml of immobilized Fe ₃ O ₄ @ ZIF-8 complex nanoparticles in 10 ml of milk containing bacteria, separate them with magnets. The bacterial-particle complex is washed several times with phosphate buffer and concentrated to 100 microliters. The concentrated complex solution is added to the ATP detection kit and shaken, and the light intensity is measured using a portable photometer. FIG. 4 shows the results of detection of Staphylococcus aureus using Fe ₃ O ₄ @ ZIF-8 complex nanoparticles immobilized with antibodies. As shown in FIG. 4 (a), the Fe ₃ O ₄ @ ZIF-8 complex nanoparticles selectively bound to the surface of the microorganism in the milk. As a result of measuring the luminosity through the ATP detection kit, the luminosity was increased as the concentration of the bacteria increased in FIG. 4 (b), and the detection sensitivity was lower than 300 CFU per milliliter.

6. Selectivity test of Fe ₃ O ₄ @ ZIF-8 composite nanoparticles

In order to test the selectivity of antibody-immobilized Fe ₃ O ₄ @ ZIF-8 complex nanoparticles, four kinds of pathogenic bacteria other than Staphylococcus aureus were tested for detection. The pathogenic bacteria used in the test were Escherichia coli, T. typhi, Enterobacteriaceae, and Listeria. And separated into each of the 0.1 mg antibody four strains of milk in 10 ⁶ CFU fixed Fe ₃ O ₄ @ ZIF-8 gave after mixing a composite magnetic nanoparticles. The bacterial-particle complex is washed several times with phosphate buffer and concentrated to 100 microliters. The concentrated complex solution is added to the ATP detection kit and shaken, and the light intensity is measured using a portable photometer. FIG. 5 shows the selectivity test results of Fe ₃ O ₄ @ ZIF-8 composite nanoparticles immobilized with Staphylococcus aureus antibody. ATP emission from other species is lower than that of Staphylococcus aureus. Therefore, antibodies to Staphylococcus aureus were immobilized on Fe ₃ O ₄ @ ZIF-8 complex nanoparticles, and these particles can selectively detect target bacteria.

Claims (15)

Magnetic nanoparticles;
A metal organic structure coating film surrounding the magnetic nanoparticles; And
Wherein the zinc atom present in the metal organic structure coating and the sulfur atom of the biological functional molecular sieve are fixed by coordinate bonding. delete delete The functional magnetic nanoparticle according to claim 1, wherein the functional molecular sieve is an antibody. 5. The functional magnetic nanoparticle according to claim 4, wherein the antibody is a half-fragment antibody having a thiol group. The functional magnetic nanoparticle according to claim 1, wherein the metal organic structure is a zeolite-like structure. The functional magnetic nanoparticle of claim 1, wherein the metal organic structure is ZIF-8. delete A magnetic nanoparticle, a metal organic structure film surrounding the magnetic nanoparticle, and a substance that binds to the biological functional molecular sieve, complex nano-particles in which the sulfur atoms of the metal atom and the biological functional molecular sieve are fixed by coordination bonding Into a sample containing the sample;
Separating the composite nanoparticles using magnetic force; And
Detecting the substance bound to the biological functional molecular sieve
≪ / RTI > 10. The detection method according to claim 9, wherein the biological functional molecular sieve is a half-fragment antibody having a thiol group. 11. The detection method according to claim 10, wherein the substance binding to the biological functional molecular sieve is a pathogenic bacteria. 12. The detection method according to claim 11, wherein the pathogenic bacterium is Staphylococcus aureus. Surrounding the magnetic nanoparticles with a metal organic structure; And
And fixing the sulfur atom of the biological functional molecular sieve to the zinc metal atom of the metal organic structure by coordinate bonding. 14. The method of claim 13, wherein the biological functional molecular sieve is a semi-assembled antibody having a thiol group by reducing a disulfide bond between heavy chains of the antibody.

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