KR102348003B1 - Target biomarker detection method using a Dual antibody-linked immuno sandwich assay - Google Patents

Abstract

The present invention relates to a method for detecting a target biomarker using a double antibody-linked immune sandwich assay and provides a composition for detecting a target biomarker comprising: superparamagnetic nanoparticles immobilized with antibodies; and a polymersome on which a signal amplifying material is supported and an antibody is bound, and a method for detecting a target biomarker using the same.

Description

Target biomarker detection method using a Dual antibody-linked immuno sandwich assay

The present invention relates to a composition and a detection method for detecting a target biomarker using a double antibody-linked immune sandwich assay.

Immunoassay refers to a technique that uses an antibody in the process of analyzing a sample, and is one of very important techniques for analyzing a biomarker because of its high selectivity and affinity for a target antigen. In the early days, it was mainly used for the detection or quantification of proteins, and according to the detection principle and method, radioimmunoassay (RIA) that detects signals using radioactive isotopes, enzymes that use signal amplification by enzymes It can be divided into an enzyme-linked immunoassay (ELISA) and a chemiluminescence immunoassay (CLIA) using chemiluminescence. Among them, the most widely used method is ELISA.

ELISA is a method of measuring the concentration of a specific biomarker in various types of samples and has the advantage of excellent sensitivity and specificity. It greatly affects the precision. In addition, there is a possibility that the enzyme bound to the antibody inhibits the antigen-antibody reaction, and thus the detectable concentration of the biomarker is very irregular.

In order to detect diseases early and prevent the spread of infectious diseases, low-concentration biomarker detection technology is required. In order to construct such a system, it is essential to develop an ultra-sensitive detection technology that can separate and detect only biomarkers from non-specific proteins in a sample.

In a related prior study (Non-Patent Document 1), there is a method of colorimetric discrimination through aggregation of gold nanoparticles that appear after the liposome collapse by binding a liposome carrying a reducing agent instead of an enzyme to the end of the secondary antibody (detection antibody). Due to the nature of the sandwich method, the analysis process is complicated and may cause loss of the target protein. In addition, there is a limit in that it cannot escape the difficulties of the antibody immobilization method.

A study on a biosensor using magnetic nanoparticles includes a nanocomposite in which magnetic nanoparticles and gold nanoclusters are combined and a method for manufacturing the same [Patent Document 1], a biosensor containing reusable magnetic nanoparticles, and a target using the same Detection method [Patent Document 2], magnetic silica nanoparticles immobilized with protein G multimer, a manufacturing method thereof, an immune sensor for detection using the same, and an immunodetection method using the same [Patent Document 3] have been reported, but the existing In biosensors using magnetic nanoparticles, signals are induced by combining a target material with a signal material or a signal control material one-to-one. In this case, there is a problem in that it takes about 2 to 3 hours due to the time required for antigen-antibody binding, the process of treating a material that prevents adsorption between the antibody and the antibody, and the washing process in several steps.

In addition, the concentration of biomarkers is relatively low compared to other body fluids, such as blood, because non-hematologic fluids such as tears contain less serum proteins, immune response-derived proteins, and cells. Therefore, there is an urgent need for an analytical method for isolating and purifying a small amount of biomarkers.

Domestic Patent Registration No. 2074316 (20.01.31) Domestic Patent Registration No. 2105263 (20.04.21) Domestic Patent Registration No. 1273964 (2013.06.03)

Magnetically Enhanced Fluorescence: Signal Amplification by Magnetic Force on Polydiacetylene Supramolecules for Detection of Prostate Cancer, small 2012, 8, No. 2, 209-213 Protein detection using biobarcodes, Mol. BioSyst., 2006, 2, 470-476 Analysis of nonspecific reactions in enzyme-linked immunosorbent assay testing for human rotavirus, J Clin Microbiol., 1979 Nov;10(5):703-707 Determination of Human Tumor Necrosis Factor α by a Highly Sensitive Enzyme Immunoassay, Biochemical and Biophysical Research Communications 289, 295-298 (2001) Single-Digit Pathogen and Attomolar Detection with the Naked Eye Using Liposome-Amplified Plasmonic Immunoassay, Nano Lett. 2015, 15, 9, 6239-6246

Accordingly, the present inventors have studied to solve the problems of the prior art. As a result, superparamagnetic nanoparticles immobilized with the first antibody and polymer nanoparticles loaded with a signal amplifying material and bound with a second antibody were used in a double antibody-linked immune sandwich assay. Thus, the present invention was completed by developing a target biomaterial detection platform.

Accordingly, an object of the present invention is to provide a composition for detecting a target biomaterial and a method for detecting a target biomaterial using a double antibody-linked immune sandwich assay.

As a means for solving the above problems, the present invention

superparamagnetic nanoparticles immobilized with a first antibody; and

Polymer nanoparticles (polymersome) to which a signal amplifying material is supported and a second antibody is bound;

It provides a composition for detecting a target biomaterial comprising a.

In addition, as another means for solving the above problems, the present invention

Immobilizing the first antibody to the superparamagnetic nanoparticles;

preparing a polymer nanoparticle (polymersome) on which a signal amplifying material is supported;

binding a second antibody to the polymersome;

adding the first antibody immobilized superparamagnetic nanoparticles to the sample in which the target biomaterial is present;

preparing a sandwich complex by adding polymer nanoparticles (polymersomes) to which the second antibody is bound to the sample to induce binding of superparamagnetic nanoparticles and polymersomes with a target biomaterial interposed therebetween;

disrupting the polymersome to release a signal amplifying material in the polymersome; and

Separating from the superparamagnetic nanoparticles and analyzing the emitted signal

It provides a method for detecting a target biomaterial comprising a.

The present invention relates to a method for detecting a target biomaterial by using superparamagnetic nanoparticles and polymersomes in a double antibody-linked immune sandwich assay. By binding to the cotton, it can be detected selectively and sensitively. In addition, the separation rate can be controlled by controlling the size of the superparamagnetic nanoparticles, and the loading amount of the signal amplifying material can also be controlled by adjusting the size of the polymersome. In particular, further, the detection time of the target material is less than 1 hour.

1 shows the superparamagnetic nanoparticle modification step.
Figure 2 shows the size and distribution of each particle after the modification of the superparamagnetic nanoparticles as a result of DLS.
3 is a TEM result of each particle after modification of superparamagnetic nanoparticles.
4 is a VSM graph showing the magnetization degree of each modified particle.
5 shows the minimum amount of MNP required for the target material to be separated.
6 shows the polymer cotton used in the present invention.
7 shows a process of emitting a fluorescent material by disintegration by an external stimulus after synthesis of the polymersome.
8 shows the structural formulas and NMR results of each of the block copolymer polymers used for synthesizing the polymersome.
9 is a result of DLS showing a polymersome (black) on which a signal amplifying material is supported and a polymersome (blue) on which a signal amplifying material is supported and bound to an antibody.
10 is a result showing the polymersome bound to the antibody by TEM.
11 is a graph showing the effect of emitting a fluorescent material over time after polymersome decay.
12 is a result showing particle disintegration patterns under various concentration treatments of surfactants.
13 is a fluorescence signal graph showing a polymersome decay pattern under various buffers.
14 shows a mechanism of action of a platform for detecting a target biomarker according to an embodiment of the present invention.
15 is a graph showing a graph of fluorescence signal versus biomarker concentration as a result of the process of FIG. 14 .
16 shows the detection time required for the detection sensing platform of the target biomarker according to the present invention.

The present invention relates to superparamagnetic nanoparticles immobilized with a first antibody; and

Polymer nanoparticles (polymersome) to which a signal amplifying material is supported and a second antibody is bound;

It relates to a composition for detecting a target biomaterial comprising a.

In the present invention, superparamagnetic nanoparticles are materials in the form of nano-sized particles having superparamagnetism, which usually do not have magnetism, but are instantaneously magnetized by an external magnetic field to have magnetism.

The superparamagnetic nanoparticles may be used without limitation as long as they are made of a magnetic metal material, a magnetic material, or a magnetic alloy.

The superparamagnetic metal nanoparticles may be at least one selected from the group consisting of iron oxide, cobalt oxide, nickel oxide, and iron oxide doped with a transition metal.

For example, the metal material may be at least one selected from the group consisting of Pt, Pd, Ag, Cu and Au, and the magnetic material is Co, Mn, Fe, Ni, Gd, Mo, MM' ₂ O ₄ , and MpOq (M and M' each independently represent Co, Fe, Ni, Mn, Zn, Gd or Cr, and p and q are the formulas 0 < p ≤ 3 and 0 < q ≤ 5, respectively). It may be at least one selected from, and the magnetic alloy may be at least one selected from the group consisting of CoCu, CoPt, FePt, CoSm, NiFe, NiFeCo and MnFe ₂ O _{4 As} it may vary depending on the purpose, it is not limited thereto. does not

In the present invention, the method for preparing the superparamagnetic nanoparticles is not particularly limited. For example, the co-precipitation method, the sol-gel method, the thermal decomposition method or the emulsion method may be used without limitation, but the thermal decomposition method may be preferably used. The thermal decomposition method can easily control the size of the superparamagnetic nanoparticles compared to the co-precipitation method, and can prepare nanoparticles having excellent dispersibility, crystallinity, and paramagnetic properties. In addition, the growth of the superparamagnetic nanoparticles can be manufactured to a desired size by reapplying a seed mediated growth method. In addition, the average particle size of the superparamagnetic nanoparticles (including the particle size after modification) may be 100 to 500 nm, preferably 100 to 300 nm.

In one embodiment of the present invention, the first antibody was immobilized and used by modifying the superparamagnetic nanoparticles with a functional group (eg, a carboxyl group) after silica coating.

A method of coating the superparamagnetic nanoparticles with silica is not particularly limited, but a nano-emulsion method may be preferably used. The silica-coated superparamagnetic nanoparticles may have a thickness of 1 to 100 nm, preferably 5 to 30 nm, of a silica layer, and by coating silica, hydrophilicity is imparted and can be easily modified into various functional groups.

The first antibody is an arbitrarily named antibody used for immobilizing superparamagnetic nanoparticles, and refers to a targeting antibody that specifically binds to a biomarker to be detected. Specifically, the first antibody may vary depending on the target biomarker, and since the binding site of the target biomarker (eg, antigen) is different, it should be used as different from the second antibody.

Since the superparamagnetic nanoparticles exhibit an average magnetizing force that can be sufficiently attractive to a general magnet, the biomarker-polymer nanoparticles and the sandwich complex can be formed and separated simply by using a magnet.

The composition for detecting biomarkers according to the present invention can target biomarkers by immobilizing an antibody on the surface of superparamagnetic nanoparticles, and can be easily collected and separated using magnetic separation.

As used herein, the term "polymer nanoparticles" refers to nanoparticles formed in a double-layer structure by self-assembly of an amphiphilic polymer, and is called a polymersome (Psome).

The "amphiphilic polymer" refers to a polymer having a region exhibiting hydrophilicity and a region exhibiting hydrophobicity. When synthesizing polymer cotton, the amphiphilic polymer can be used without limitation as long as it is a polymer having a hydrophilic region and a hydrophobic region. Amphiphilic polymers include hydrophilic polymers, hydrophobic polymers, and combinations thereof. The polymer may be included in the form of a copolymer.

Specifically, the amphiphilic polymer is polyphosphazene, polylactide (PLA), polylactide-co-glycolide, poly(lactic-co-glycolic acid) (PLGA), polycaprolactone, polyanhydride , polymalic acid or its derivatives, polyalkylcyanoacrylate, polyhydrooxybutylate, polycarbonate and polyorthoester, polypropylene glycol (PPG), polyoxypropylene (PPO) poly-L-lysine, polyglycol A hydrophobic region selected from the group consisting of ride, polyalkyl (meth)acrylate and polyvinylpyrrolidone, polyethylene glycol (PEG), polyoxyethylene (PEO), polyetherimide (PEI), polyvinylpyrrolidone ( PVP) and N-isopropylacrylamide (NIPAM) may include a hydrophilic region selected from the group consisting of, but is not limited thereto.

In one embodiment of the present invention, the amphiphilic polymer is a hydrophilic polymer, PEG, as a hydrophobic polymer, lactide (Lactide, 3,6-Dimethyl-1,4-dioxane-2,5-dione) containing PEG and poly lactide copolymer (PEG)m-(b-PLA)n. In this case, m may be 10 to 50, and n may be 30 to 100.

In one embodiment, the amphiphilic polymer (CH ₃ PEG)m-(b-PLA)n [1] can be mixed with the amphipathic polymer [2] having a functional group (eg, COOH) for later binding to the antibody. have. The synthesis of particles is possible without mixing, and in order to control the amount of antibody conjugated to the surface, an appropriate ratio of [1] and [2], for example, CH ₃ :COOH=7.5:2.5, is used by mixing. It is preferred, but not limited thereto. (m = 45, n = 97 of the amphiphilic polymer used in the Examples.)

The signal amplifying material may be supported on the hydrophilic portion or the hydrophobic portion of the amphiphilic polymer nanoparticles.

The signal amplification material is a material capable of easily detecting a target biomarker through signal amplification, and a fluorescent material, a light absorbing material, gold nanoparticles, or the like may be used.

The fluorescent material is not particularly limited, but specifically, Dylight 488 NHE-ester dye, Vybrant™ DiI, Vybrant™ DiO, quantum dots nanoparticles, fluorescein, rhodamine, lucifer yellow, B-phytoerythrin, 9 -acridineisothiocyanate, lucifer yellow VS, 4-acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid, 7-diethylamino-3-(4' -Isothiocyatophenyl)-4-methylcoumarin, succinimidyl-pyrenebutyrate, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid derivative, LC™- Red 640, LC™-Red 705, Cy5, Cy5.5, lysamine, isothiocyanate, erythrosine isothiocyanate, diethylenetriamine pentaacetate, 1-dimethylaminonaphthyl5-sulfonate , 1-anilino-8-naphthalene sulfonate, 2-sotitouidinyl-6-naphthalenesulfonate, 3-phenyl-7-isocyanatocoumarin, 9-isothiocyanatoacridine, creidine orange 9 -I(sothi(2-benzoxazolyl)phenyl)melimide sadiazole, stilbene, pyrene, even dot, silica containing fluorescent material, group II/IV semiconductor quantum dot, group III/V semiconductor quantum dot, IV Group semiconductor quantum dots, or may include a multi-component hybrid structure, preferably the fluorescent material is included in the hydrophobic film of the amphipathic polymer nanoparticles, quantum dots (quantum dots) nanoparticles, Cy3.5, Cy5, Cy5. 5, Cy7, ICG (indocyanine green), Cypate, ITCC, NIR820, NIR2, IRDye78, IRDye80, IRDye82, Cresy Violet, Nile Blue, Oxazine 750, Rhodamine800, fluorescence resonance from the group consisting of lanthanide series and Texas Red (FRET) Energy transfer, fluorescence resonance energy transfer) can be used by selecting two or more species.

In the present specification, the FRET phenomenon refers to a phenomenon in which, when two fluorescent materials of different wavelength regions are adjacent to each other, energy that excites one fluorescent molecule (donor) does not emit light, but is transferred to the other fluorescent molecule (acceptor) to generate fluorescence. A phenomenon, that is, a phenomenon in which the energy (donor) causing one fluorescence is transmitted to another fluorescence material and resonance occurs to generate another fluorescence (acceptor). Since FRET efficiency is determined as the distance between two fluorescent molecules, FRET appears when the donor and acceptor are close to each other, and FRET weakens when they move away from each other.

In one embodiment of the present invention, two types of DiO (3,3'-Dioctadecyloxacarbocyanine Perchlorate) and DiI (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate) in the preparation of the polymer nanoparticles It was synthesized using a FRET pair fluorescent substance of DiO excites at a wavelength of 488 nm as a donor to generate energy including a wavelength of 501 nm, and DiI as an acceptor excites at a wavelength of 549 nm to generate energy corresponding to a wavelength of 565 nm.

When the structure of the FRET pair fluorescent material-supported polymer nanoparticles is stable, energy corresponding to a wavelength of 565 nm is generated high because the distance between the two pairs of fluorescent materials is close. As this is emitted and the distance increases, the energy transfer is weakened, and energy corresponding to a wavelength of 501 nm is generated high.

A second antibody is bound to the amphiphilic polymer nanoparticles on which the signal amplifying material is supported (antibody binding is made by a polymersome modified with a functional group (eg, COOH). The reaction used at this time is an EDC/NHS coupling reaction, CH3 7.5 and COOH 2.5 are exposed on the surface of the polymersome, and the antibody binds to the COOH present in the proportion of 2.5.)

The second antibody is a detection antibody that is immobilized on a polymersome that specifically binds to a target biomarker, and refers to an antibody different from the first antibody (when the first antibody and the second antibody use the same antibody, the target biomarker It is not preferable because it is difficult to combine the superparamagnetic nanoparticles and the polymer cotton in a sandwich form between them).

In particular, the antibody is exposed to the surface of the polymer nanoparticle through a covalent bond between the carboxyl group exposed at the end of the polymer nanoparticle and the amine group of the antibody, thereby specifically complexing with various biomarkers according to the type of antibody. can be formed.

The biomarker may be a protein, specifically a membrane protein of a virus or exosome, an antigen, or the like.

The average size of the polymer cotton may be 35 to 200 nm.

In the present invention, specific biomarkers can be detected by a sandwich method by using superparamagnetic nanoparticles and a polymersome loaded with a signal amplifying material. Since it is possible to obtain an amplified detection signal from the superparamagnetic nanoparticle-biomarker-polymersome complex, it is possible to minimize the error in detecting trace amounts of biomarkers, and it is possible to absolutely quantify disease biomarkers through the detection signal according to the concentration. .

the present invention

Immobilizing the first antibody to the superparamagnetic nanoparticles;

preparing a polymersome on which a signal amplifying material is supported;

binding a second antibody to the polymersome;

adding the first antibody immobilized superparamagnetic nanoparticles to the sample in which the target biomarker is present;

preparing a sandwich complex by adding the polymersome to which the second antibody is bound to the sample to induce binding of the superparamagnetic nanoparticles to the polymersome;

disrupting the polymersome to release a signal amplifying material in the polymersome; and

Separating from the superparamagnetic nanoparticles and analyzing the emitted signal

It includes a method for detecting a target biomarker comprising a.

All of the contents described above with respect to the composition for detecting the target biomarker may be directly applied or applied mutatis mutandis to the method for detecting the target biomarker.

First, the first antibody is immobilized on the superparamagnetic nanoparticles.

The superparamagnetic nanoparticles are preferably modified with functional groups. The functional group may be, for example, a carboxyl group, an amine group, NHS, a thiol group, etc. for antibody binding, but is not limited thereto.

A first antibody capable of binding to a target biomarker is immobilized on the surface of the superparamagnetic nanoparticles.

Separately, a polymer cotton on which a signal amplifying material is supported is prepared. That is, an amphiphilic polymer and a signal amplifying material are self-assembled to synthesize a polymersome having a signal amplifying material supported inside the double membrane.

A second antibody is bound to the thus-prepared polymersome.

Superparamagnetic nanoparticles immobilized with the first antibody are added to the sample sample in which the target biomarker is present.

The second antibody-bound polymersome is further added to the sample, and a sandwich complex is prepared by sandwiching the polymersome with the superparamagnetic nanoparticles by double-antibody sandwich bonding.

With the target biomarker in between, the superparamagnetic nanoparticles and the polymersome form a double antibody sandwich bond. Thereafter, only the sandwich complex is separated using a magnet, and other non-target proteins and unbound polymer nanoparticles are discarded [see FIG. 14].

The polymer cotton of the sandwich complex is treated with a surfactant to disintegrate the polymer cotton so that the signal amplifying material in the polymer cotton is released.

The surfactant inducing the disintegration of the polymer cotton is not particularly limited as long as it has an effect of dissolving the polymer membrane. For example, the surfactant may be any one selected from Triton X-100, TWEEN 20, TWEEN 80, and the like.

Finally, when the polymersome is disintegrated by treatment with a surfactant, the signal amplifying material is dissolved and the signal is amplified. In this case, a signal appears in the presence of a target biomarker, and it is possible to quantify the amplified signal according to the concentration of the marker. That is, the signal emitted by separating from the superparamagnetic nanoparticles is analyzed.

When the surfactant is treated, the fluorescent material supported on the polymer cotton is freely circulating in the solution as a single molecule. Thereafter, only the fluorescent material of the supernatant is obtained and the fluorescent signal is measured.

In the sandwich targeted biomarker, one part is bound to a targeting antibody (first antibody) bound to the surface of a superparamagnetic nanoparticle, and a detection antibody ( By binding by the second antibody), different parts of the biomarker may be detected and detected by the superparamagnetic nanoparticle-biomarker and the biomarker-polymersome. In addition, in the present invention, only the sandwich-bonded complex is obtained and the polymersome is disrupted to induce the emission of a fluorescent substance or quantum dot.

In one embodiment of the present invention, the biomarker in the sample may be quantified by comparing the absorbance or intensity of fluorescence of the emitted fluorescent material or quantum dots.

Hereinafter, the present invention will be described in more detail through Examples according to the present invention, but the scope of the present invention is not limited by the Examples presented below.

[Example]

Preparation Example 1: Preparation of biomarker-specific superparamagnetic nanoparticles

As shown in FIG. 1 , the preparation of biomarker-specific superparamagnetic nanoparticles is as follows.

1-1: superparamagnetic nanoparticles (Fe ₃ O ₄ ) synthesis

In a 150 ml beaker, 260 mg of iron trichloride (Iron(III) chloride, FeCl ₃ ), polyvinylpyrrolidone (PVP ) 2 g, sodium acetate 1.5 g, sonication was performed for 1 hour, and then transferred to a Teflen bottle and heated at 200° C. for 12 hours.

After pouring out all the supernatant from the bottle, the remaining lower layer material (synthesized superparamagnetism and nanoparticles) was transferred into a 70 ml vial. Here, 20 ml of anhydrous ethyl alcohol were added, and 70 watts of sonication was added for 5 minutes. 70　ml　vial　Attached a magnet to the side 　to obtain only superparamagnetic nanoparticles 　and the supernatant was poured away. This 　 process is called 　 washing process, and this process was repeated 3 times. Finally, only the synthesized particles were transferred to a 20 ml vial, treated with anhydrous ethyl alcohol, treated with anhydrous ethyl alcohol, and finally dispersed well and stored at room temperature.

1-2: Silica coating on superparamagnetic nanoparticles

A 150 ml beaker was _{treated with 50 mg of superparamagnetic nanoparticles (Fe 3} O ₄ ), 40 ml of ethyl alcohol, and 10 ml of distilled water, and ultrasonication (bath sonicator, 70 watt) was performed for 5 minutes. 2 ml of aqueous ammonia and 200 ul of tetraethyl silicate mineral (Tetraethylorthosilicate, TEOS) were sequentially treated, followed by sonication (70 watt) for 3 hours. Then, only the supernatant was discarded using a magnet. After washing 3 times using anhydrous ethyl alcohol, it was finally dispersed in alcohol to coat the silica on the surface of the superparamagnetic nanoparticles.

The superparamagnetic nanoparticles remaining in the 150 ml beaker were coated with a silica shell. To remove unreacted silica material, 20 ml of anhydrous ethyl alcohol was added, and ultrasonic waves (70 watt) were applied for 5 minutes. Similarly, a magnet was applied to separate only silica-coated magnetic nanoparticles, and the supernatant was discarded. This process was repeated 3 times, and finally, only the coated magnetic nanoparticles were transferred to a 20ml vial, and 15ml of anhydrous ethyl alcohol was injected, dispersed well, and stored.

1-3: Modification of superparamagnetic nanoparticles into functional groups on the surface

In a 20 ml vial, 10 mg of silica-coated superparamagnetic nanoparticles SiMNP and 9.5 ml of a pegylation solution (EtOH 95%, DW 5%) were added. Specifically, 9.025 ml of EtOH and 0.475 ml of DW were added here. After ultrasonic treatment for 5 minutes, it was well dispersed in the PEGylation solution. After that, 0.5 ml of silane PEG solution was treated in the 20 ml vial. (The silane PEG solution was prepared as follows. Add 0.5 ml of PEGylation solution, 23.75 mg of mPEG-Silane powder, and 1.25 mg of COOHPEG-Silane powder to a 1.5 ml EP tube and disperse by pipetting 20 times.) In order to evenly coat the COOH functional group, the above solution mixed in a 20 ml vial was fixed on a shaker and mixed at a strength of 3 for 30 minutes. After the reaction was completed, only CMNP was obtained using a magnet and the supernatant was discarded. After 10 ml of DW was injected and sonicated for 3 minutes, only washed CMNP was obtained by applying a magnet again. This process was repeated 3 times. Finally, 15 ml of anhydrous ethyl alcohol was injected into the CMNP obtained in a 20 ml vial and well dispersed and stored (manufactured by CMNP).

1-4: Binding of Targeting Antibodies to Superparamagnetic Nanoparticles

After dispersing the superparamagnetic nanoparticles having COOH on the surface prepared in Preparation Example 1-3 in PBS buffer, 1 mg/ml of EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl) and NHS (N-hydroxysuccinimide) at a concentration of 1 mg/ml, first antibody LS-C20749 at a concentration of 1 mg/ml, and Apolipoprotein E antibody (monoclonal) were treated at a molar ratio of 1:10:10:1, respectively. After reacting for 2 hours with strong stirring, it was washed with PBS buffer using a magnet.

Preparation Example 2: Analysis of physical properties of superparamagnetic nanoparticles

2 is a graph measuring the particle size and distribution of superparamagnetic nanoparticles (MNP, SiMNP, CMNP, AbMNP) prepared in each step of Preparation Example 1 by DLS.

Samples were prepared by injecting 950 ul of anhydrous ethyl alcohol + 50 ul each of the synthesized MNP, SiMNP, CMNP, and AbMNP solutions into a 1.5 ml EP tube and adding sonication for 1 minute. When measuring DLS, it was measured by injecting 1 ml into a disposable UV cell.

The diameter measured by DLS can be slightly larger than electron microscopy because it reads the area up to the electrostatic layer of the particle.

3 is a result of observation with a transmission electron microscope (TEM: Transmission electron microscope, JEM-F200 model manufactured by JEOL). The average silica coating thickness was observed to be 10 nm.

Transmission electron microscopy was measured in the same way as the sample prepared for DLS measurement, and 20 ul was dropped on the grid and dried for one day.

The accompanying drawings show MNP, SiMNP (silica-coated nanoparticles on MNP surface), CMNP (nanoparticles functionalized with COOH on SiMNP surface) and AbMNP from the left diagram of FIG.

Table 1 below shows the size and distribution of each superparamagnetic nanoparticles prepared in each step of Preparation Example 1, which can be seen in FIGS. 2 and 3 .

[Table 1]

Therefore, it can be seen that the superparamagnetic nanoparticles prepared and modified according to Preparation Example 1 have a very uniform size and are evenly dispersed without agglomeration.

10 mg of each superparamagnetic nanoparticles (MNP, SiMNP, CMNP, AbMNP) were lyophilized to prepare a sample, and 10 mg each was loaded in a sample holder for VSM measurement to measure a vibrating sample magnetometer (VSM).

4 shows the results of measuring the magnetizing force using VSM (7407-s model manufactured by LakeShore), MNP, SiMNP, and CMNP, respectively, the average magnetizing force was 80, 50, 30 emu / g. Since it exhibits an average magnetizing force that can be sufficiently attractive to a general magnet, the superparamagnetic nanoparticles can be easily separated using a magnet by forming a sandwich complex with the biomarker-polymersome.

Therefore, the superparamagnetic nanoparticles surface-modified with a functional group prepared according to Preparation Example 1 of the present invention exhibit an average magnetizing force capable of sufficiently engaging a general magnet and attractive force without a significant difference from before that even if the modification steps are sequentially performed. It was confirmed that biomarkers can be easily separated using a magnet.

Preparation Example 3: Confirmation of resolution of superparamagnetic nanoparticles

A preliminary test was performed by binding biotin instead of antibody to the superparamagnetic nanoparticles of Preparation Example 1-4 and replacing avidin as a target biomarker.

[Synthesis of superparamagnetic nanoparticles with biotin conjugated to the surface]

3.3 mg of superparamagnetic nanoparticles were dispersed in 2.64 ml of PBS, and 330 nmol of biotin was dispersed in 0.66 ml. The two were mixed in a 1.5 ml EP tube and reacted for 3 hours at RT (room temperature) with a rotate shaker. And it was washed 3 times to remove unreacted biotin with PBS using magnetic separation.

[Avidin separation]

Avidin 500 nM and biotin-PEG1k-MNP solution for each concentration condition were prepared in 1.5 ml EP tube. After reaction for 6 hours, it was washed twice. The concentration of avidin captured by the biotin-modified MNP was inversely calculated using the BCA assay kit.

The results are shown in FIG. 5, and it was confirmed that 50% or more of the target material could be separated only by the presence of 100 ug of superparamagnetic nanoparticles.

Preparation Example 4: Preparation and analysis of physical properties of a polymer cotton carrying a fluorescent material for capturing a target biomarker

7 is a sequence in which a polymersome is synthesized from an amphiphilic polymer, a second antibody LS-C124977 that captures a target biomarker (ApoE), an Apolipoprotein E antibody (polyclonal) is bound, and then the particles are disintegrated by artificially treating with a surfactant. represents the steps of In the last process, after the polymersome collapses, the process in which the fluorescent material supported in the membrane is dissolved and the signal is amplified is to be explained.

4-1. Synthesis of fluorescent material-supported polymersome

The polymer cotton on which the fluorescent material was supported was prepared as follows.

Like MNP, mPEG-b-PLA polymer (Nanosoft Polymer, 9511-7000-2000-1g) and COOHPEG-b-PLA so that the second antibody is conjugated to the surface of polymer nanoparticles through COOH and EDC/NHS coupling reaction A polymer (Nanosoft polymer, 24027-7000-2000-1g) was mixed and used to have a concentration of 1 mg/ml in a chloroform solvent at a mass ratio of 7.5:2.5. 10 ml of the solution was added to a round flask, and 100 ul each of DiO and DiI fluorescent substances having a concentration of 1 mg/ml were treated. After vortexing so as to be evenly dispersed in the flask, the polymer mixture and fluorescent materials were self-assembled to prepare a polymer nanoparticle (polymersome, Psome) on which the fluorescent material was supported [FIG. 7].

8 is an NMR result of mPEG-b-PLA amphiphilic polymer used for polymersome synthesis. A hydrophilic fraction (f) can be obtained by comparing the integral values of the peaks b and c, and the f value of the polymer used in the present invention is 0.22. (mPEG-b-PLA is expressed in blue and COOHPEG-b-PLA in pink. Since both polymers have the same hydrophilic fraction, it is appropriate that b and c appear at the same position)

4-2. Binding of the second antibody to the surface of the polymersome

An EDC/NHS coupling reaction was performed for binding the second antibody. Polymersome Psome was dispersed in MES buffer, and EDC and NHS were also dispersed in MES buffer at a concentration of 1 mg/ml. The second antibody LS-C124977 dispersed at 1 mg/ml in PBS after strong vortexing was performed for 15 minutes by treatment with a molar ratio of 1:10:10 in the order of COOH functional groups exposed on the polymersome surface, EDC, and NHS; Apolipoprotein E antibody (polyclonal) was treated and reacted for 2 hours.

Figure 9 shows the results of DLS measurement of the fluorescent material-supported polymersome Psome (left) and the antibody-bound polymersome AbPsome (right) prepared in Preparation Example 4-2 (right) prepared in Preparation Example 4-1. It can be seen that the difference is small and has a size of 50-120 nm.

FIG. 10 is a result of analyzing the antibody-bound polymersome prepared by the above-mentioned method using a transmission electron microscope, and it was observed that the average particle diameter was about 50 nm.

Preparation 5: Confirmation of signal change due to fluorescence emission of polymersome

5-1: Method for inducing and analyzing polymersome degradation

The polymersome includes a fluorescent material inside the hydrophobic film and may include two types of dyes in which a fluorescence resonance energy transfer (FRET) phenomenon occurs.

In the preparation of the polymersome, DiO and DiI were synthesized using two types of FRET pair fluorescent materials. DiO was excited at a wavelength of 488 nm as a donor to generate energy including a wavelength of 501 nm, and DiI was excited at a wavelength of 549 nm as an acceptor to generate energy corresponding to a wavelength of 565 nm.

When the structure of the FRET pair fluorescent material-supported polymersome is stable, energy corresponding to a wavelength of 565 nm is generated high because the distance between the two pairs of fluorescent materials is close. As it is emitted and the distance increases, the energy transfer is weakened, and the energy corresponding to the wavelength of 501 nm is high.

5-2: Measure the time required for polymer cotton disintegration

[sample preparation]

1. AbPsome solution 80 ul + distilled water 20 ul

2. AbPsome solution 80 ul + Tx-100 (10 wt%) solution 20 ul

Each was treated in a 96-well microplate.

[Fluorescent signal measurement]

After inserting the plate into a multi-mode microplate reader and setting the exit wavelength 475 nm, emission wavelength 501 - 599 nm, and interval 5 nm, the fluorescence value was measured by pressing the start button. 0 minutes is the value measured as soon as the plate is put in, and it took about 4 minutes in total to be measured. After every 5 to 30 minutes, the plate was reinserted into the device and the start button was pressed according to the desired time. The set value to be measured was kept the same.

11 shows a comparison of fluorescence signals over time after each polymer cotton was treated with a surfactant and distilled water. The red graph treated with surfactant showed the same shape as the condition treated with distilled water at 0 min, but the intensity of the wavelength of 565 nm decreased and the intensity of 501 nm increased over time. This means that the FRET phenomenon occurs due to particle disintegration, which means that it takes 20 minutes for the particle to completely disintegrate.

12 is a result showing the signal by treating different concentrations of surfactant on the polymersome. It was confirmed that the effect of particle disintegration was not significantly different as the concentration of the surfactant was diluted.

5-3: Effect of decay in various buffers

13 is a result of confirming the polymersome degradation in various buffers. Here, the FRET ratio was used for signal analysis. The FRET ratio has the formula (fluorescence signal intensity at 565 nm wavelength)/(fluorescence signal at 501 nm wavelength + fluorescence signal intensity at 565 nm wavelength). It can be confirmed that the polymersome disintegration effect is maintained because there is no change in the FRET ratio even on various buffers.

Example 1: Target biomarker magnetic separation and fluorescence signal amplification sensing platform fabrication

In a 1.5 ml EP tube, 100 ul of the sample having the target biomarker (ApoE) was treated, 100 ul of the antibody-bound superparamagnetic nanoparticle solution prepared in Preparation Example 1-4, and the antibody prepared in Preparation Example 4-2 100 ul of the bound polymersome solution was sequentially treated and reacted at 37° C. for 30 minutes. Using a magnet, a sandwich composite of superparamagnetic nanoparticles and polymersome bonded to the target biomaterial was separated. By injecting 200 ul of PBS again, the separated complex was dispersed in the solution. The above process was repeated 3 times to remove unreacted polymer nanoparticles or non-specific proteins in the sample. Finally, 80 ul of PBS and 20 ul of a TX-100 (10 wt%) solution were injected and reacted at 37° C. for 20 minutes. The superparamagnetic nanoparticles and the emitted fluorescent material were separated using a magnet, and only the supernatant was obtained, and 100 ul was injected into an amber 96 well plate. An amber 96 well plate was inserted into a multi-mode microplate reader and the fluorescence signal was measured.

14 is a process of performing a sensing platform in which superparamagnetic nanoparticles and polymersomes are introduced. The sample was treated with a superparamagnetic nanoparticle solution surface-modified with an antibody, followed by treatment with a polymersome solution. Sandwich bonding between the superparamagnetic nanoparticles and the polymersome with the antigen interposed therebetween. After that, only the sandwich complex was separated using a magnet, and other non-target proteins and unbound polymer particles were discarded. Finally, as the polymersome was disintegrated by treatment with a surfactant, the fluorescent material was dissolved and the signal was amplified. In this case, a signal appears in the presence of a target biomarker, and the fluorescence signal can be quantified according to the concentration of the marker.

15 is a graph showing a fluorescence signal according to a concentration of a biomarker. It was confirmed that the signal was quantitatively decreased according to the concentration change from 100 pM to 1.56 pM of the target antigen.

Example 2: Sensing Platform Verification

16 is result data regarding the required time of the sensing platform, and the experimental process is as follows. Considering the washing process and measurement after particle disintegration, the total inspection time was about 1 hour.

[Experimental process]

1. A solution of 1 nM concentration of target biomarker (ApoE) protein was prepared.

2. In a 1.5 ml EP tube, 100 ul of the antibody-binding superparamagnetic nanoparticles AbMNP prepared in Preparation Example 1-4 + 100 ul of the target biomarker + 100 ul of the antibody-binding polymersome AbPsome prepared in Preparation Example 4-2 were treated with Thermo The reaction was carried out in a -shaker at 37 °C for different time conditions from 0 to 80 minutes. (Complex formation was confirmed from 30 minutes.)

3. In order to separate only the sandwich complex, the EP tube was fixed on a magnetic magnetic separator, and the separated supernatant was discarded. Washed 3 times with 300 ul of PBS (this process takes about 2 minutes)

4. Finally, 180 ul of PBS + 20 ul of surfactant TX-100 was put in and reacted in a thermo-shaker at 37 ℃ for 20 minutes.

5. After separating the superparamagnetic nanoparticles with a magnetic separator, only the supernatant was obtained and sampled in a 96 well plate.

6. Fluorescence values were measured with a multi reader. (takes 26 seconds)

Claims (19)

superparamagnetic nanoparticles having an average particle size of 100 nm to 151.8 nm and immobilized with a first antibody; and
a polymersome having an average particle size of 35 nm to 200 nm, on which a signal amplifying material is supported, and to which a second antibody is bound;
including,
The polymer cotton is a polymer nanoparticle formed by self-assembly of an amphiphilic polymer, and the amphipathic polymer includes a hydrophobic region of polylactide and a hydrophilic region of polyethylene glycol, and the polyethylene glycol is a methyl group-modified polyethylene glycol and A composition for detecting a target biomaterial comprising polyethylene glycol modified with a carboxyl group.
The method of claim 1,
The composition of the superparamagnetic nanoparticles is a superparamagnetic metal oxide nanoparticles.
3. The method of claim 2,
The superparamagnetic metal oxide nanoparticles are at least one composition selected from the group consisting of iron oxide, cobalt oxide, nickel oxide, and iron oxide doped with a transition metal.
The method of claim 1,
The signal amplifying material is a composition that exhibits a fluorescence resonance energy transfer (FRET) phenomenon.
The method of claim 1,
The superparamagnetic nanoparticles are functional group-modified composition.
delete delete delete delete The method of claim 1,
The biomaterial is a protein or antigen composition.
Immobilizing the first antibody to superparamagnetic nanoparticles having an average particle size of 100 nm to 151.8 nm;
Preparing a polymer cotton having an average particle size of 35 nm to 200 nm and carrying a signal amplifying material;
binding a second antibody to the polymersome;
adding the first antibody immobilized superparamagnetic nanoparticles to the sample in which the target biomaterial is present;
preparing a sandwich complex by adding the polymersome to which the second antibody is bound to the sample to induce binding of the superparamagnetic nanoparticles and the polymersome with a target biomaterial interposed therebetween;
disrupting the polymersome to release a signal amplifying material in the polymersome; and
Separating from the superparamagnetic nanoparticles and analyzing the emitted signal
including,
The polymer cotton is a polymer nanoparticle formed by self-assembly of an amphiphilic polymer, and the amphipathic polymer includes a hydrophobic region of polylactide and a hydrophilic region of polyethylene glycol, and the polyethylene glycol is a methyl group-modified polyethylene glycol and A method for detecting a target biomaterial comprising polyethylene glycol modified with a carboxyl group.
12. The method of claim 11,
A detection method in which impurities are removed with a magnet after the sandwich composite is manufactured.
12. The method of claim 11,
A detection method for disintegrating polymer cotton by treatment with a surfactant.
12. The method of claim 11,
The detection method wherein the superparamagnetic nanoparticles are superparamagnetic metal oxide nanoparticles.
15. The method of claim 14,
The superparamagnetic metal oxide nanoparticles are at least one selected from the group consisting of iron oxide, cobalt oxide, nickel oxide, and iron oxide doped with a transition metal.
12. The method of claim 11,
The signal amplifying material is a material exhibiting a fluorescence resonance energy transfer (FRET) phenomenon.
12. The method of claim 11,
The detection method of the superparamagnetic nanoparticles modified with a functional group.

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