KR102291650B1 - Quantum dot-polymer complex and diagnosis kit using the same - Google Patents

Abstract

The present invention relates to a quantum bead of a three-dimensional self-assembly structure consisting of one or more quantum dots and one or more polymer ligands; and at least one antibody (antibody); and a quantum dot-polymer complex comprising the same, and a diagnostic kit using the same.

Description

Quantum dot-polymer complex and diagnostic kit using same {Quantum dot-polymer complex and diagnosis kit using the same}

The present invention relates to a quantum dot-polymer complex and a diagnostic kit using the same.

In the field of biotechnology, semiconductors and metal nanoparticles have brought significant advances in diagnosis and treatment. If the pathogenesis of disease can be observed at the molecular or protein level, it will provide important information on the mechanism of disease development.

In particular, semiconductor nanoparticles exhibiting optical properties have very high application potential in bio-imaging. Quantum dots (QDs) exhibit fluorescence with high quantum yield, and since the band gap can be adjusted by adjusting the size, there is an advantage in that the wavelength band of the emitted light can be easily adjusted. Because these optical properties show very bright imaging and are stable, it is expected to be a much more effective material than the conventionally used dyes or protein fluorophores for cell imaging.

Conventionally, gold nanoparticles have been used for diagnosing diseases, but since the color change of gold nanoparticles is not large, there is a limit to increasing the sensitivity of diagnosis through the color of gold nanoparticles.

In addition, attempts have been made to increase the diagnostic sensitivity by changing the size and shape of the gold nanoparticles, but there is a limit to its application to diseases requiring high sensitivity.

Accordingly, there is a high need for developing a nanomaterial as a highly efficient labeling material capable of in vitro diagnosis while increasing sensitivity and a diagnostic kit using the same.

One aspect is a quantum bead of a 3D self-assembly structure consisting of one or more quantum dots and one or more polymer ligands; and

Quantum dots comprising one or more antibodies (antibody); to provide a polymer complex.

Another aspect is to provide a diagnostic kit using such a quantum dot-polymer complex.

In one aspect, the present invention, a quantum bead of a three-dimensional self-assembly structure (3D self-assembly structure) consisting of one or more quantum dots (quantum dot) and one or more polymer ligands; and

Quantum dots comprising one or more antibodies (antibody) - provides a polymer complex.

For example, the quantum dots are cadmium selenide (CdSe), cadmium sulfide (CdS), cadmium telorium (CdTe), zinc telorium (ZnTe), zinc selenide (ZnSe), zinc sulfide (ZnS), zinc Oxide (ZnO), zinc nitride (ZnN), lead sulfide (PbS), indium phosphide (InP), indium arsenide (InAs), indium nitride (InN), gallium nitride (GaN), mercury HgTe) and mercury selenide (HgSe); and

Cadmium Selenide (CdSe), Cadmium Sulfide (CdS), Cadmium Tellurium (CdTe), Zinc Telorium (ZnTe), Zinc Selenide (ZnSe), Zinc Sulfide (ZnS), Zinc Oxide (ZnO), Zincite Ride (ZnN), lead sulfide (PbS), indium phosphide (InP), indium arsenide (InAs), indium nitride (InN), gallium nitride (GaN), mercury telorium (HgTe) and mercury selenide ( HgSe) on a core containing at least one of cadmium oxide (CdO), cadmium selenide (CdSe), cadmium sulfide (CdS), cadmium telorium (CdTe), zinc telorium (ZnTe), cadmium Zinc sulfide (CdZnS), zinc sulfide (ZnS), zinc oxide (ZnO), zinc selenide (ZnSe), zinc selenium sulfide (ZnSeS), lead selenium sulfide (PbSeS), indium gallium nitride (InGaN), indium gallium phosphorus Pide (InGaP), Indium Zinc Phosphide (InZnP), Capaselenium Tellurium (CuSeTe), Capaindium Selenium (CuInSe ₂ ), Capaindium Sulfide (CuInS ₂ ), Silver Indium Sulfide (AgInS ₂ ), and Tintellorium a core-shell coated with a shell comprising at least one of (SnTe); It may be one or more selected from among.

For example, when there are two or more quantum dots, the two or more quantum dots may be the same or different.

For example, the polymer ligand may be selected from a random copolymer, a block copolymer, a graft copolymer, or a combination thereof.

For example, the polymer ligand may include a hydrophobic moiety and a hydrophilic moiety.

For example, a lipophilic portion of the polymer ligand may be arranged in a central direction of the proton bead, and a hydrophilic portion of the polymer ligand may be arranged in an outer direction of the proton bead.

For example, the hydrophilic portion of the polymer ligand may be exposed to the outside of the proton bead.

For example, the hydrophilic portion may be in the form of a needle or a brush.

For example, the polymer ligand may include at least one of an amine group, an amide group, a hydroxy group, a maleimide group, a thiol group, and a carboxylic acid group at the terminal end.

For example, the polymeric ligand may include polystyrene, polyolefin, polyvinylacetal, polyester, polyketone, polyether, polyvinylpyridine, polycarbonate, polydiene, polyacrylic acid, polyamide, and polyimide; random copolymers thereof, and block copolymers; polymers containing polymaleic anhydride groups; and a graft copolymer consisting of a polymaleic anhydride group and an alkylamine; It may be one or more selected from among.

For example, the polymer including the polymaleic anhydride group may be represented by the following Chemical Formula 1, and the graft copolymer composed of the polymaleic anhydride group and an alkylamine may be represented by the following Chemical Formula 1A or 1B:

<Formula 1>

<Formula 1A>

<Formula 1B>

In Formulas 1, 1A and 1B,

L ₁ and L ₂ are each independently selected from a C ₁ -C ₂₀ alkyl group or a C ₁ -C ₂₀ alkylene group; and deuterium, -F, -Cl, -Br, -I, a cyano group, a nitro group, a C ₁ -C ₁₀ alkyl group, a C ₆ -C ₂₀ aryl group, and a C ₁ -C ₁₀ alkoxy group substituted with at least one selected from the group consisting of selected from C ₁ -C ₂₀ alkylene groups,

a1 is an integer selected from 1 to 5,

a2 is an integer selected from 0 to 5;

R ₂ is a C ₁ -C ₂₀ alkyl group; and deuterium, -F, -Cl, -Br, -I, a cyano group, a nitro group, a C ₁ -C ₁₀ alkyl group, a C ₆ -C ₂₀ aryl group, and a C ₁ -C ₁₀ alkoxy group substituted with at least one selected from the group consisting of selected from C ₁ -C ₂₀ alkyl groups,

n is 1 or more and 1000 or less.

For example, when there are two or more polymer ligands, the two or more polymer ligands may be the same or different.

For example, a hydrodynamic diameter (HD) of the quantum bead may be 50 to 1000 nm.

For example, the antibody may be bound to the surface of the polymer ligand.

For example, the bond may be an amide bond.

In another aspect, the present invention provides a kit for diagnosis using a quantum dot-polymer complex.

For example, the diagnostic kit may detect an antigen in vitro.

For example, the diagnostic kit may detect fertility by detecting HCG hormone.

The diagnostic kit is not particularly limited as long as it is an antigen to which an antigen-antibody system can be applied, but may detect, for example, a malaria antigen.

For example, the diagnostic kit may be applied to all in vitro diagnostic kits according to changes in the antigen-antibody system.

For example, the diagnostic kit may use a LFA (Lateral Flow Assay) system.

For example, in the LFA system, the quantum dot-polymer complex may act as a labeling material.

For example, the diagnostic kit may use an enzyme-linked immunosorbent assay.

Unlike the conventional technique of diagnosing the occurrence of disease by color change, the quantum dot-polymer complex according to the present invention has excellent light stability and quantum efficiency, and has excellent luminescence intensity and antigen-antibody binding force, so that the Diagnosis using light is possible.

In addition, by varying the type of antibody bound to the quantum dot-polymer complex according to the disease to be detected, early diagnosis of various diseases is possible and in vitro diagnosis is possible, so the diagnosis method is easier and faster than the conventional analysis method .

In addition, the process cost can be reduced by using quantum dots that are cheaper than gold nanoparticles.

1 is a schematic diagram for explaining an LFA system.
2A is a schematic diagram of a quantum dot-polymer composite including a brush-type polymer ligand, FIG. 2B is a schematic diagram of a quantum dot-polymer composite including a needle-type polymer ligand, and FIG. 2C is a quantum dot including a brush-type polymer ligand. - It is a schematic diagram showing the high sensitivity of the polymer composite.
3A is a schematic diagram showing the structures of a block copolymer and a random copolymer, which are one embodiment of a polymer ligand, and FIG. 3B is a schematic diagram showing the structure of a graft copolymer, which is another embodiment of a polymer ligand.
4a to 4f are TEM images of quantum beads having different shapes and sizes, respectively, and FIG. 4g is a hydrodynamic diameter (HD) value measurement result of quantum beads containing a polymer ligand having a brush shape and having different sizes. , FIG. 4H is a measurement result of surface zeta potential of quantum beads having different sizes and containing a polymer ligand in a brush form, and FIG. 4I is a PL emission peak of quantum beads containing a polymer ligand having a brush shape and having different sizes. Measurement results, FIGS. 4j and 4k are TEM images of quantum beads according to Synthesis Example 4.
5a to 5d show the LFA results corresponding to the schematic diagram of the quantum dot-polymer complexes having different types of polymer ligands and sizes, respectively.
6a to 6d are graphs showing the LFA results of the quantum dot-polymer complexes including the polymer ligands in the form of brushes and needles, respectively, for each concentration of HCG antigen.
7a to 7b are graphs showing the antibody binding efficiency of quantum beads containing a polymer ligand in brush form and needle form, respectively, and FIGS. 7c to 7d are quantum dots containing a polymer ligand in brush form and needle form, respectively - Polymer complexes This is a graph showing the HD value measurement result.
8a to 8b are stability evaluation results according to the pH change and NaCl concentration change of the proton beads containing the polymer ligand in the form of a brush, respectively, and FIGS. 8c to 8d are the pH changes and Stability evaluation results according to changes in NaCl concentration, FIGS. 8e to 8f are results of HD value measurement according to pH change and NaCl concentration change of quantum dot beads including brush-type polymer ligands, respectively, and FIGS. 8g to 8h are needle-type polymers These are the HD value measurement results according to the pH change and NaCl concentration change of the proton beads including the ligand, respectively.
Figure 9a is a photographic image under UV-lamp showing the LFA test results of EPCAM, Tnl, CRP, HRP II, and HCG antibodies, Figure 9b is a graph showing the T / C ratio of each antibody of Figure 9a, Figure 9c is a photographic image of R, G, and B quantum beads, and FIG. 9D is a graph showing the quantum beads of FIG. 9C and corresponding PL emission data of quantum dots forming the same.

Hereinafter, with reference to the accompanying drawings, various embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily implement them. The present invention may be embodied in many different forms and is not limited to the embodiments described herein.

In order to clearly explain the present invention, parts irrelevant to the description are omitted, and the same reference numerals are given to the same or similar elements throughout the specification.

In addition, since the size and thickness of each component shown in the drawings are arbitrarily indicated for convenience of description, the present invention is not necessarily limited to the illustrated bar.

In addition, the terms used below are only used to describe specific embodiments, and are not intended to limit the present inventive concept. The singular expression includes the plural expression unless the context clearly dictates otherwise. Hereinafter, terms such as “comprises” or “have” are intended to indicate that a feature, number, step, action, component, part, component, material, or combination thereof described in the specification is present, but one or the It should be understood that the above does not preclude the possibility of the presence or addition of other features, numbers, steps, operations, components, parts, components, materials, or combinations thereof. "/" used below may be interpreted as "and" or "or" depending on the situation.

In one aspect, the present invention, a quantum bead of a three-dimensional self-assembly structure (3D self-assembly structure) consisting of one or more quantum dots (quantum dot) and one or more polymer ligands; And one or more antibodies (antibody); quantum dots comprising a - provides a polymer complex.

As described above, in the case of gold nanoparticles, which have been mainly used as a material for a conventional in vitro diagnostic kit, the diagnostic sensitivity is low, so that it is difficult to diagnose a disease in the initial stage.

The quantum dot-polymer complex according to the present invention, having the above-described structure, can exhibit excellent photostability and quantum efficiency, thereby maximizing luminescence intensity and antigen-antibody binding force, thereby improving diagnostic sensitivity.

On the other hand, Figure 1 is a schematic diagram for explaining the LFA system.

Referring to FIG. 1 , in the LFA system, the color or light observed in the test line and the control line according to the presence of an antigen in the analyte during the process of the analyte added to the diagnostic kit being developed by capillary action. It is a system for diagnosing a specific antigen or disease by analyzing the change in intensity.

Specifically, when a signal is generated in the test line, the antigen is present in the analyte, and the control line is to verify whether the diagnostic kit operates normally, and a signal must be generated.

In this case, the test line and the control line may be coupled with a labeling material. In order to develop a high-sensitivity diagnostic kit that has not been reached in the prior art, it is essential to develop a labeling material having excellent properties.

The quantum dot-polymer composite according to the present invention has high diagnostic sensitivity as described above, and thus can be used as a labeling material in an in vitro diagnostic kit utilizing a Lateral Flow Assay (LFA) system.

As described above, the quantum dot-polymer composite is a three-dimensional self-assembled structure of the quantum dot and the polymer ligand. Specifically, the quantum bead is not a separate magnetic bead, latex bead, or quantum dot bonded to an inorganic or organic support, but has a structure in which quantum dots are uniformly distributed at a certain distance in a polymer ligand structure, so that in the prior art Compared to the developed material, the number of quantum dots that can be included in the same volume is large, and through this, the effect of maximizing the emission intensity can be exhibited.

2a to 2b is a schematic diagram of the quantum dot-polymer composite of the present invention. Hereinafter, the structure of the quantum dot-polymer composite of the present invention will be described in detail with reference to FIGS. 2a to 2b.

2A to 2B , the quantum dot-polymer composites 10 and 20 may include quantum beads of a three-dimensional self-assembly structure composed of one or more quantum dots and one or more polymer ligands; and one or more antibodies (13, 23).

Specifically, the quantum bead includes core portions 11 and 21 of the quantum bead formed by the quantum dots and the lipophilic portion of the polymer ligand and the hydrophilic portions 12 and 22 of the polymer ligand to be described later.

For example, the quantum bead may include a core portion 11 and 21 of a quantum bead formed by a quantum dot and a lipophilic portion of a polymer ligand and a shell portion including a hydrophilic portion 12 and 22 of a polymer ligand. can be a structure.

For example, the quantum dots are not particularly limited as long as they are nanoparticles used in the industry,

Cadmium Selenide (CdSe), Cadmium Sulfide (CdS), Cadmium Tellurium (CdTe), Zinc Telorium (ZnTe), Zinc Selenide (ZnSe), Zinc Sulfide (ZnS), Zinc Oxide (ZnO), Zincite Ride (ZnN), lead sulfide (PbS), indium phosphide (InP), indium arsenide (InAs), indium nitride (InN), gallium nitride (GaN), mercury telorium (HgTe) and mercury selenide ( HgSe); and

Cadmium Selenide (CdSe), Cadmium Sulfide (CdS), Cadmium Tellurium (CdTe), Zinc Telorium (ZnTe), Zinc Selenide (ZnSe), Zinc Sulfide (ZnS), Zinc Oxide (ZnO), Zincite Ride (ZnN), lead sulfide (PbS), indium phosphide (InP), indium arsenide (InAs), indium nitride (InN), gallium nitride (GaN), mercury telorium (HgTe) and mercury selenide ( HgSe) on a core comprising at least one of, cadmium oxide (CdO), cadmium selenide (CdSe), cadmium sulfide (CdS), cadmium telorium (CdTe), zinc telorium (ZnTe), cadmium Zinc sulfide (CdZnS), zinc sulfide (ZnS), zinc oxide (ZnO), zinc selenide (ZnSe), zinc selenium sulfide (ZnSeS), lead selenium sulfide (PbSeS), indium gallium nitride (InGaN), indium gallium phosphorus Pide (InGaP), Indium Zinc Phosphide (InZnP), Capaselenium Tellurium (CuSeTe), Capaindium Selenium (CuInSe ₂ ), Capaindium Sulfide (CuInS ₂ ), Silver Indium Sulfide (AgInS ₂ ), and Tintellorium a core-shell coated with a shell comprising at least one of (SnTe); It may be one or more selected from among.

In this case, when there are two or more quantum dots, the two or more quantum dots may be the same or different.

In addition, the polymer ligand interacts with the quantum dots to help with the lipophilic moiety (not specifically shown in the figure) forming the central portion of the quantum bead and aqueous dispersion, and is used for binding to the antibodies (13, 23). It may include hydrophilic portions 12 , 22 .

In this case, the polymer ligand may be a random copolymer, a block copolymer, a graft copolymer, or a combination thereof. Here, the random copolymer refers to a copolymer in which two or more different types of units are randomly arranged. In addition, the block copolymer refers to a copolymer in which two or more different types of polymer chains are connected through a chemical bond.

3A is a schematic diagram showing the structures of a block copolymer and a random copolymer, which are one embodiment of a polymer ligand, and FIG. 3B is a schematic diagram showing the structure of a graft copolymer, which is another embodiment of a polymer ligand.

Referring to FIG. 3A first, the block copolymer has a polymer chain bonded to hydrophilic units and a polymer chain bonded to lipophilic units, and the random copolymer is polymerized in which hydrophilic units and lipophilic units are randomly arranged. is in the form

Also, referring to FIG. 3B , the graft copolymer is a form in which another unit monomer is polymerized on a previously formed polymer.

On the other hand, referring again to FIGS. 2A to 2B , the quantum bead may be formed by a hydrophobic interaction between the quantum dot and the lipophilic portion of the polymer ligand, and formed by the quantum dot and the lipophilic portion of the polymer ligand. The aqueous dispersion and binding force with the antibody are ensured by the hydrophilic portions 12 and 22 of the polymer ligand extending to the surface of the core portions 11 and 21 of the quantum bead.

According to one embodiment, the lipophilic portion (not specifically shown) of the polymer ligand is arranged in the central direction of the proton bead, and the hydrophilic portion (12, 22) of the polymer ligand is in the outer direction of the proton bead. can be arranged.

According to an embodiment, the hydrophilic portions 12 and 22 of the polymer ligand may have a needle shape 22 or a brush shape 12 .

Here, "needle shape" means a shape in which the hydrophilic portion 22 of the polymer ligand is generally short and has a single functional group as shown in FIG. 2B, and "brush shape" is shown in FIG. 2A As shown, the hydrophilic portion 12 of the polymer ligand is generally long and refers to a form having several or more functional groups.

For example, the hydrophilic portion of the polymer ligand is not particularly limited, but may be in the form of a brush. As described above, when the hydrophilic part of the polymer ligand is in the form of a brush, compared to when it is in the form of a needle, when the antibody binds as will be described later, the surface area where the hydrophilic part of the polymer ligand and the antibody can bind increases because the surface area is wider. It can bind to a large amount of antibody.

For example, the polymer ligand is not particularly limited, but may be in the form of a brush in the case of a block copolymer, and may be in the form of a needle in the case of a random copolymer and a graft copolymer.

Specifically, referring to FIGS. 2A to 2B , when the content of the antibodies 13 and 23 is increased (the antibody content increases from left to right), each quantum dot-polymer complex 10, 20 of the antibody 13 , 23) can be confirmed. In the case of the quantum dot-polymer composite 10 of FIG. 2A including the hydrophilic portion 12 of the polymer ligand in the form of a brush, even if the content of the antibody 13 is increased, as described above, the hydrophilic portion 12 of the polymer ligand and Since the surface area to which the antibody 13 can bind is large, the antibodies 13 bind well to the surface of the hydrophilic portion 12 of the polymer ligand.

On the other hand, in the case of the quantum dot-polymer complex 20 of FIG. 2b including the hydrophilic portion 22 of the polymer ligand in the form of a needle, when the content of the antibody 23 is increased, the hydrophilic portion 22 of the polymer ligand and the antibody Since the surface area on which (23) can bind is not sufficient, the antibody binding efficiency is reduced because the antibodies (23) aggregate between the antibodies (23) rather than on the surface of the hydrophilic portion (22) of the polymer ligand. It may be somewhat inferior to the hydrophilic portion of the polymer ligand.

In addition, referring to Figure 2c, when the quantum dot-polymer ligand complex of the present invention is applied to the LFA system, the case where the hydrophilic part of the polymer ligand is in the form of a brush (top) and when the hydrophilic part of the polymer ligand is in the form of a needle (bottom) ), it can be seen that the antigen 31 binds more to the antibodies 13 and 23.

For example, the polymer ligand may include at least one of an amine group, an amide group, a hydroxy group, a maleimide group, a thiol group, and a carboxylic acid group at the terminal end.

For example, the polymeric ligand may include polystyrene, polyolefin, polyvinylacetal, polyester, polyketone, polyether, polyvinylpyridine, polycarbonate, polydiene, polyacrylic acid, polyamide, and polyimide; random copolymers thereof, and block copolymers; polymers containing polymaleic anhydride groups; And it may be at least one selected from a graft copolymer consisting of a polymaleic anhydride group and an alkylamine.

For example, the polymer containing the polymaleic anhydride group and a block copolymer; polymers containing polymaleic anhydride groups; And it may be at least one selected from a graft copolymer consisting of a polymaleic anhydride group and an alkylamine.

For example, the polymer including the polymaleic anhydride group may be represented by the following Chemical Formula 1, and the graft copolymer composed of the polymaleic anhydride group and an alkylamine may be represented by the following Chemical Formula 1A or 1B:

<Formula 1>

<Formula 1A>

<Formula 1B>

In Formulas 1, 1A and 1B,

L ₁ and L ₂ are each independently, a C ₁ -C ₂₀ alkylene group; and deuterium, -F, -Cl, -Br, -I, a cyano group, a nitro group, a C ₁ -C ₁₀ alkyl group, a C ₆ -C ₂₀ aryl group, and a C ₁ -C ₁₀ alkoxy group substituted with at least one selected from the group consisting of selected from C ₁ -C ₂₀ alkylene groups,

a1 is an integer selected from 1 to 5,

a2 is an integer selected from 0 to 5;

R ₂ is a C ₁ -C ₂₀ alkyl group; and deuterium, -F, -Cl, -Br, -I, a cyano group, a nitro group, a C ₁ -C ₁₀ alkyl group, a C ₆ -C ₂₀ aryl group, and a C ₁ -C ₁₀ alkoxy group substituted with at least one selected from the group consisting of selected from C ₁ -C ₂₀ alkyl groups,

n is 1 or more and 1000 or less.

For example, in Formulas 1A and 1B,

L ₁ and L ₂ are each independently selected from a methylene group, an ethylene group, a propylene group, an isopropylene group, a butylene group, an isobutylene group, a pentylene group, a hexylene group, a heptylene group, an octylene group, a nonylene group, and a decylene group; and

A methylene group, ethylene group, propylene group, isopropylene group, butylene group, isobutyl group, substituted with one or more selected from a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a phenyl group, a methoxy group, and an ethoxy group It may be selected from a lene group, a pentylene group, a hexylene group, a heptylene group, an octylene group, a nonylene group, and a decylene group.

For example, in Formulas 1A and 1B,

R ₂ may be selected from a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, and a decyl group.

For example, the polymer ligand may be a random copolymer of an alkylene maleic anhydride monomer and an alkylamine monomer.

For example, the polymer ligand may be a graft copolymer of a polyalkylene maleic anhydride polymer and an alkylamine monomer.

For example, the polymer ligand may be a poly(isobutylene-alt-maleic anhydride)-hexadecylamine graft copolymer.

In this case, when there are two or more polymer ligands, the two or more polymer ligands 12 and 22 may be the same or different.

According to one embodiment, a hydrodynamic diameter (HD) of the quantum bead may be 50 to 1000 nm.

At this time, out of the above range, when the hydrodynamic diameter of the quantum bead is less than 50 nm, there is a problem that the luminescence intensity is weak due to the small number of quantum dots included therein, whereas the hydrodynamic diameter of the quantum bead is 1000 nm When it exceeds, there is a problem in that the dispersibility of the particles is deteriorated.

For example, the number of quantum dots constituting the core portions 11 and 21 of the quantum bead may be appropriately adjusted according to the size of the quantum bead, and is not particularly limited, but may be, for example, 50,000 or less, for example For example, it may be 50 to 50,000. For example, the number of quantum dots may be 40,000 or less, for example, 30,000 or less, for example, 20,000 or less, for example, 10,000 or less, for example, 5,000 or less, for example For example, it may be 4,000 or less, for example, 3,000 or less, for example, 1500 or less, for example 50 to 1500, for example 50 to 1450, for example 60 to 1450, for example, 60 to 1400, for example, 70 to 1400, for example, 70 to 1350, for example, 80 to 1350.

At this time, out of the above range, when the number of quantum dots constituting the core portions 11 and 21 of the quantum bead exceeds 1500, fluorescence quenching between quantum dots located nearby may occur, whereas When the number of the quantum dots is less than 50, there is a problem in that the light emission intensity is weak.

The emission wavelength of the quantum bead is preferably 450 to 700 nm, but is not limited thereto.

According to one embodiment, the antibodies 13 and 23 may be bound to the surface of the polymer ligand.

Since the antibody binds to the functional groups of the hydrophilic portions 12 and 22 of the polymer ligand present on the surface of the proton bead, the degree of binding of the antibodies may be increased by controlling the relative ratio of the proton bead to the antibody.

For example, the bond may be an amide bond.

Here, the antibodies 13 and 23 are specific immunoglobulins directed against an antigenic site as a term known in the art. The form of the antibody includes all of polyclonal antibodies, monoclonal antibodies, and recombinant antibodies, and may include all immunoglobulin antibodies as well as special antibodies such as humanized antibodies. In addition, the antibody includes functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule means a fragment having at least an antigen-binding function, and may be Fab, F(ab'), F(ab')2 and Fv.

In the present invention, the antibody is not particularly limited as long as it can bind directly to a polymer ligand or indirectly using a linker, and any type of antibody may be used. For example, the antibody may be directly bound to the polymer ligand by reacting the reactive amino group of the antibody with the carboxylic acid group of the polymer ligand to form an amide bond. There is a problem that there may be. In order to solve this problem, an antibody and a polymer ligand can be indirectly linked using a linker such as peptide, glutaraldehyde, succinic anhydride, etc. as long as it is not particularly limited.

According to another aspect of the present invention, a diagnostic kit using the above-described quantum dot-polymer complex is provided. In this case, the use of the diagnostic kit may vary without limitation depending on the type of antibody included in the complex.

According to one embodiment, the diagnostic kit may detect an antigen in vitro. That is, by simply detecting an antigen through urine, blood, etc. outside the body, the speed of diagnosis is increased and diagnosis is made easier, and at the same time, it is possible to diagnose a number of samples to be analyzed.

For example, the diagnostic kit may be applied to all in vitro diagnostic kits according to changes in the antigen-antibody system.

For example, the diagnostic kit is not particularly limited as long as it is an antigen to which the antigen-antibody system can be applied, but may be for diagnosing pregnancy potential by detecting HCG hormone. it could be However, it is not limited to the above uses, and as described above, by regulating the antibody included in the complex as necessary, a diagnosable disease can be changed.

According to one embodiment, the diagnostic kit may use a LFA (Lateral Flow Assay) system.

In this case, in the LFA system, the quantum dot-polymer complex may act as a labeling material.

According to one embodiment, the diagnostic kit may use an enzyme-linked immunosorbent assay.

The present invention will be described in more detail through the following synthesis examples and evaluation examples. However, the synthesis examples are for illustrating the present invention and do not limit the scope of the present invention only to these.

Synthesis Example 1 (Synthesis of small sized brush type QBs)

CdSe@CdZnS (quantum dots with CdSe core coated with CdZnS shell) (0.6 mg) and polystyrene-polyacrylic acid block copolymer (2.2 mg) (polymer ligand) were dispersed in tetrahydrofuran and stirred After the addition of water in the purified, proton beads were prepared. At this time, the HD of the quantum bead was about 120 nm.

The TEM images of the quantum beads are shown in FIGS. 4A and 4D, and the HD values, surface zeta potential, and PL emission peaks of the quantum beads were measured and shown in FIGS. 4G, 4H and 4I, respectively.

Synthesis Example 2 (Synthesis of middle sized brush type QBs)

CdSe@CdZnS (quantum dots) (0.9 mg) and polystyrene-polyacrylic acid block copolymer (3.3 mg) (polymer ligand) were dispersed in tetrahydrofuran, stirred, and water was added thereto, followed by purification. Quantum beads were prepared. At this time, the HD of the quantum bead was about 210 nm.

The TEM images of the quantum beads are shown in FIGS. 4b and 4e, and the HD values, surface zeta potential, and PL emission peaks of the quantum beads were measured and shown in FIGS. 4g, 4h and 4i, respectively.

Synthesis Example 3 (Synthesis of large sized brush type QBs)

CdSe@CdZnS (quantum dots) (1.8 mg) and polystyrene-polyacrylic acid block copolymer (6.6 mg) (polymer ligand) were dispersed in tetrahydrofuran, stirred, and water was added thereto, followed by purification. Quantum beads were prepared. At this time, the HD of the quantum bead was about 290 nm.

The TEM images of the quantum beads are shown in FIGS. 4c and 4f, and the HD values, surface zeta potential, and PL emission peaks of the quantum beads were measured and shown in FIGS. 4g, 4h and 4i, respectively.

Synthesis Example 4 (Synthesis of large sized needle type QBs)

CdSe@CdZnS (quantum dots) (0.5 mg) and poly(isobutylene-alt-maleic anhydride)-hexadecylamine random copolymer (1.6 mg) (polymer ligand) were mixed with tetrahydrofurene ( After dispersion in tetrahydrofuran), water was added under stirring, and then purified to prepare quantum beads, in which case, the HD of the quantum beads was about 300 nm.

TEM images of the quantum beads are shown in FIGS. 4j and 4k.

Preparation Example 1 (Quantum Dot-Polymer Composite Synthesis)

Quantum beads (0.1 mg) synthesized in Synthesis Example 1, ethyldimethylaminopropylcarbodiamide (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) (13.32 ug), hydroxysulfosuccimide (N-hydroxysulfosuccimide) ) (30.18 ug) was dispersed in pH 6 morpholinoethanesulfonic acid (2-(N-morpholino)ethanesulfonic acid) buffer for activation. After that, only the proton beads are submerged with a centrifuge, and then redispersed in phosphate buffered saline, and then, anti-beta hCG antibody (anti-Beta Human chorionic gonadotropin) (25 ug) is added, and quantum dots- A polymer composite was prepared.

Preparation Examples 2 to 4 (quantum dots-synthesis of polymer composite)

A quantum dot-polymer composite was prepared in the same manner as in Preparation Example 1, except that the quantum beads synthesized in Synthesis Examples 2 to 4 were used instead of the quantum beads synthesized in Synthesis Example 1.

Evaluation Example 1 (Measurement of Size)

For the quantum beads according to Synthesis Examples 1 to 4 and the quantum dots used for synthesizing them, average particle diameter through TEM analysis, average particle diameter through dynamic laser scattering (DLS) analysis, zeta potential, PL emission peak range, photoluminescence quantum yield ( PLQY), quantum dots-the number of quantum dots per polymer composite was analyzed, and is shown in Table 1 below.

Sample TEM
(nm) DLS
(nm) ζ-potential
(mV) PL luminescence
(nm) PLQY(%) number of quantum dots quantum dots 8.037±0.666 - - 613.1 84.3 - Synthesis Example 1 91.0 ±5.8 124.7 -37.9 614.3 39.18 80 Synthesis Example 2 139.1 ±12.0 212.6 -27.1 614.3 41.11 344 Synthesis Example 3 217.8 ±15.6 289.2 -25.9 614.4 59.52 1,326 Synthesis Example 4 269.7 ±25.9 300.2 -33.5 614.0 60.74 1,262

Referring to Table 1, it can be seen that in the case of a large-sized quantum bead, the number of quantum dots included therein increases. Also, it can be seen that PLQY is much better.

Evaluation Example 2 (LFA test)

The results of the LFA test for the HCG hormone using the quantum dot-polymer complexes according to Preparation Examples 1 to 4 are shown in FIGS. 5A to 5D .

From left to right in FIGS. 5A-5D , the HCG hormone concentration becomes lower.

A portion marked with * in FIGS. 5A to 5D indicates a limit of detection and means a sample concentration at a level that is difficult to confirm with the naked eye.

Referring to FIGS. 5A to 5D , it can be seen that the quantum dot-polymer composite ( FIG. 5C ) having a brush-type polymer ligand and having a large size has higher diagnostic sensitivity.

In particular, compared with the needle-type polymer ligand (FIG. 5d), when the brush-type polymer ligand is used, even if the size of the quantum dot-polymer complex is smaller, the diagnostic sensitivity is better (Figure 5b) or a similar level ( 5a) can be confirmed.

Evaluation Example 3 (LFA Test)

Using the quantum dot-polymer complex according to Preparation Examples 3 to 4, the LFA results according to the change in HCG hormone concentration were measured, and are shown in FIGS. 6a to 6d.

Specifically, LFA results at an antigen concentration in the range of 50 pg/mL to 30 ng/mL are shown in FIGS. 6A (Preparation Example 3) and 6B (Preparation Example 4), respectively, and the antigen concentration is 50 pg/mL to 3 The LFA results in the ng/mL range are shown in FIG. 6c (Preparation Example 3), and the LFA results in the antigen concentration range of 100 pg/mL to 3 ng/mL are shown in FIG. 6c (Preparation Example 4).

Referring to FIGS. 6A to 6D , it can be seen that when the same concentration of HCG is used, the diagnostic sensitivity of the complex including the brush-type polymer ligand is much superior.

Evaluation Example 4 (antibody binding force test)

Using the quantum beads according to Synthesis Examples 3 and 4, the antibody binding efficiency (7a, 7b) according to the change in the antibody content and the quantum dot-polymer complexes prepared in Preparation Examples 3 and 4 HD values (7c, 7d) were measured. , are shown in FIGS. 7A to 7D.

Referring to FIGS. 7A and 7B , it can be seen that the antibody binding efficiency is excellent at ~95% or more regardless of the amount of antibody added in both the brush form and the needle form. The portion indicated by the red dotted line is the amount of antibody capable of binding to the quantum bead surface as a monolayer.

7c and 7d, in the case of the quantum dot polymer complex including the polymer ligand in the form of a brush, the hydrodynamic radius has a constant value regardless of the increase in the amount of antibody added, but in the case of the quantum dot polymer complex including the polymer ligand in the form of a needle It can be seen that it is continuously increasing. This is because, in the case of a brush-type polymer ligand, the volume of the ligand itself is large and there are many functional groups, so the bound antibody exists between the ligands, and the increase in the hydrodynamic radius is minimized. The blue dotted line indicates the hydrodynamic radius of the quantum bead before antibody binding.

Evaluation Example 5 (pH and salt concentration stability test)

Using the quantum beads according to Synthesis Examples 3 and 4, the stability evaluation results according to the pH change and the NaCl concentration change were measured and shown in FIGS. 8a to 8d, and the HD value according to the pH change and NaCl concentration change was measured. , are shown in FIGS. 8E to 8H.

8A to 8D , the quantum beads according to Synthesis Example 3 did not generate a separate precipitate even when the pH or NaCl concentration was changed, whereas the quantum beads according to Synthesis Example 4 had a low pH or a high NaCl concentration. As a result, it can be confirmed that the precipitate is generated.

That is, the quantum beads according to Synthesis Example 3 may be more useful as a labeling material in the LFA system, since stability may be maintained regardless of the pH or salt concentration of the analyte sample.

On the other hand, referring to FIGS. 8e to 8h , the quantum beads according to Synthesis Example 3 did not have a large HD value change even when the pH or NaCl concentration was changed, whereas the quantum beads according to Synthesis Example 4 had a low pH or a high NaCl concentration. It can be seen that the HD value increases accordingly.

That is, the quantum beads according to Synthesis Example 3 can maintain the HD value regardless of the pH or salt concentration of the analyte sample, and thus can be more useful as a labeling material in the LFA system.

Evaluation Example 6 (Antigen Antibody Specificity Test)

In the LFA system in which the quantum dot-polymer complex using the HCG hormone antibody as the quantum bead and antibody according to Synthesis Example 3 was applied as a label, the antigen among the analytes was measured while different with EPCAM, Tnl, CRP, HRP II, and HCG hormone. is shown in Figures 9a to 9b.

Referring to FIGS. 9A and 9B , it can be confirmed that a signal is generated in the test line only for the HCG hormone, and the T/C ratio is also high. Through this, it can be confirmed that the quantum dot-polymer complex of the present invention can be applied as a labeling material in the LFA system for HCG hormone detection.

Also, referring to FIGS. 9C and 9D , it can be seen that the quantum bead according to Synthesis Example 3 can change the color by adjusting the emission wavelength by adjusting the quantum dots.

Through this, it can be confirmed that the quantum dot-polymer complex of the present invention can be applied to a multiplexing system that can detect various diseases with one LFA diagnostic kit.

Although the preferred embodiment of the present invention has been described above, the present invention is not limited to the above embodiment, and it is easily changed by a person skilled in the art from the embodiment of the present invention to equivalent It includes all changes to the extent recognized as such.

Claims (20)

a quantum bead of a three-dimensional self-assembly structure comprising one or more quantum dots and one or more polymer ligands; and
one or more antibodies;
The quantum dots are cadmium selenide (CdSe), cadmium sulfide (CdS), cadmium telorium (CdTe), zinc telorium (ZnTe), zinc selenide (ZnSe), zinc sulfide (ZnS), zinc oxide (ZnO) , zinc nitride (ZnN), lead sulfide (PbS), indium phosphide (InP), indium arsenide (InAs), indium nitride (InN), gallium nitride (GaN), mercury telorium (HgTe) and mercury selenide (HgSe); and
Cadmium Selenide (CdSe), Cadmium Sulfide (CdS), Cadmium Tellurium (CdTe), Zinc Telorium (ZnTe), Zinc Selenide (ZnSe), Zinc Sulfide (ZnS), Zinc Oxide (ZnO), Zincite Ride (ZnN), lead sulfide (PbS), indium phosphide (InP), indium arsenide (InAs), indium nitride (InN), gallium nitride (GaN), mercury telorium (HgTe) and mercury selenide ( HgSe) on a core containing at least one of cadmium oxide (CdO), cadmium selenide (CdSe), cadmium sulfide (CdS), cadmium telorium (CdTe), zinc telorium (ZnTe), cadmium Zinc sulfide (CdZnS), zinc sulfide (ZnS), zinc oxide (ZnO), zinc selenide (ZnSe), zinc selenium sulfide (ZnSeS), lead selenium sulfide (PbSeS), indium gallium nitride (InGaN), indium gallium phosphorus Phosphide (InGaP), Indium Zinc Phosphide (InZnP), Capaselenium Tellurium (CuSeTe), Carpaindium Selenium (CuInSe2), Carpaindium Sulfide (CuInS2), Silver Indium Sulfide (AgInS2), and Tintellorium (SnTe) a core-shell coated with a shell comprising at least one of; at least one selected from
The polymer ligand comprises a hydrophobic moiety and a hydrophilic moiety,
The hydrophilic portion of the polymer ligand is in the form of a brush,
wherein the antibody is bound to the polymer ligand,
The hydrodynamic diameter of the quantum bead (Hydrodynamic Diameter: HD) is 50 to 1000 nm, quantum dots-polymer composite. delete According to claim 1,
When there are two or more quantum dots, the two or more quantum dots are the same or different, quantum dots-polymer composite. According to claim 1,
The polymer ligand is a random copolymer (random copolymer), block copolymer (block copolymer), graft copolymer (graft copolymer), or a combination thereof selected from, quantum dots-polymer composite. delete According to claim 1,
The lipophilic portion of the polymer ligand is arranged in the central direction of the quantum bead, and the hydrophilic portion of the polymer ligand is arranged in the outer direction of the quantum dot-polymer composite. delete According to claim 1,
wherein the polymer ligand comprises at least one of an amine group, an amide group, a hydroxy group, a maleimide group, a thiol group, and a carboxylic acid group at a terminal end. According to claim 1,
The polymer ligands include polystyrene, polyolefin, polyvinylacetal, polyester, polyketone, polyether, polyvinylpyridine, polycarbonate, polydiene, polyacrylic acid, polyamide, and polyimide; random copolymers thereof, and block copolymers; polymers containing polymaleic anhydride groups; and a graft copolymer consisting of a polymaleic anhydride group and an alkylamine; At least one selected from among, quantum dots-polymer composite. 10. The method of claim 9,
The polymer including the polymaleic anhydride group is represented by the following Chemical Formula 1, and the graft copolymer consisting of the polymaleic anhydride group and the alkylamine is represented by the following Chemical Formula 1A or 1B, quantum dot-polymer composite:
<Formula 1>

<Formula 1A>

<Formula 1B>

In Formulas 1, 1A and 1B,
L ₁ and L ₂ are each independently, a C ₁ -C ₂₀ alkylene group; and deuterium, -F, -Cl, -Br, -I, a cyano group, a nitro group, a C ₁ -C ₁₀ alkyl group, a C ₆ -C ₂₀ aryl group, and a C ₁ -C ₁₀ alkoxy group substituted with at least one selected from the group consisting of selected from C ₁ -C ₂₀ alkylene groups,
a1 is an integer selected from 1 to 5,
a2 is an integer selected from 0 to 5;
R ₂ is a C ₁ -C ₂₀ alkyl group; and deuterium, -F, -Cl, -Br, -I, a cyano group, a nitro group, a C ₁ -C ₁₀ alkyl group, a C ₆ -C ₂₀ aryl group, and a C ₁ -C ₁₀ alkoxy group substituted with at least one selected from the group consisting of selected from C ₁ -C ₂₀ alkyl groups,
n is 1 or more and 1000 or less. delete delete According to claim 1,
The bond is an amide bond, quantum dots-polymer composite. A diagnostic kit using the quantum dot-polymer complex according to any one of claims 1, 3, 4, 6, 8 to 10, and 13. 15. The method of claim 14,
The diagnostic kit is a diagnostic kit for detecting an antigen in vitro. 15. The method of claim 14,
The diagnostic kit is a diagnostic kit for diagnosing fertility by detecting HCG hormone. 15. The method of claim 14,
The diagnostic kit is a diagnostic kit for detecting a malaria antigen. 15. The method of claim 14,
The diagnostic kit is a diagnostic kit using a LFA (Lateral Flow Assay) system. 19. The method of claim 18,
In the LFA system, the quantum dot-polymer complex acts as a labeling material, diagnostic kit. 15. The method of claim 14,
The diagnostic kit is a diagnostic kit using an enzyme-linked immunosorbent assay.

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