KR20190010001A - Nanoparticle assembly structure and immunoassay method using the same - Google Patents

Abstract

Provided are a nanoparticle assembly structure for facilitating immunoassay and an immunoassay method using the same. The nanoparticle assembly structure includes a first nanoparticle assembly structure including a first assembly particle formed by a plurality of first nanoparticles, wherein the first assembly particle has magnetism. According to embodiments of the present invention, the immunoassay method includes: a step of providing an analyte, the first nanoparticle assembly structure having magnetism, and a second nanoparticle assembly structure having luminescence in a buffer solution; and a step of providing a magnetic force to the buffer solution. The first nanoparticle assembly structure comprises: the first assembly particle comprising the plurality of first nanoparticles; and a first antibody linked to the first assembly particle. The second nanoparticle assembly structure comprises: a second assembly particle comprising a plurality of second nanoparticles; and a second antibody linked to the second assembly particle. The first and second antibodies may be combined with the analyte.

Description

TECHNICAL FIELD The present invention relates to a nanoparticle assembly structure and an immunoassay method using the nanoparticle assembly structure.

The present invention relates to a nanoparticle assembly structure and immunoassay using the same.

Recently, various immunoassay methods have been developed and proposed according to advances in nanotechnology. However, there are still a variety of technical difficulties and limitations such as suboptimal reproductibility, unevenness of the substrate, lack of multiplex detection capability, and the need for custom designed devices.

In order to solve the above problems, the present invention provides a nanoparticle assembly structure that facilitates immunoassay.

The present invention provides an immunoassay method using the nanoparticle assembly structure.

Other objects of the present invention will become apparent from the following detailed description and the accompanying drawings.

The nanoparticle assembly structure according to embodiments of the present invention includes a first nanoparticle assembly structure including a first assembly particle formed by gathering a plurality of first nanoparticles, and the first assembly particle has magnetism.

The first nanoparticles may include iron oxide nanoparticles. The first nanoparticle assembly structure may further include a first antibody coupled to the first assembly particle.

The nanoparticle assembly structure may further include a second nanoparticle assembly structure including a second assembly particle formed by gathering a plurality of second nanoparticles, and the second assembly particle has a luminescent property.

The second nanoparticle assembly structure may further comprise a second antibody coupled to the second assembly particle.

At least one of the first assembly particle and the second assembly particle may include streptavidin and at least one of the first antibody and the second antibody may include biotin, At least one of the particle assembly structure and the second nanoparticle assembly structure may include a streptavidin-biotin bond.

The second nanoparticles may include at least one of ZnS: Mn nanoparticles and Cd _{1 - x} Zn _x S / ZnS nanoparticles.

The second nanoparticle assembly structure may further include a second antibody linked to the ZnS: Mn nanoparticles and a third antibody coupled to the Cd _{1 - x} Zn _x S / ZnS nanoparticles. The second antibody and the third antibody may be different from each other.

An immunoassay method according to embodiments of the present invention includes: providing an analyte, a first nanoparticle assembly structure having magnetism, and a second nanoparticle assembly structure having fluorescence property in a buffer solution; And providing a magnetic force to the buffer solution. Wherein the first nanoparticle assembly structure comprises a first assembly particle formed by gathering a plurality of first nanoparticles and a first antibody connected to the first assembly particle, wherein the second nanoparticle assembly structure comprises a plurality of 2 nanoparticles gathered and a second antibody linked to the second assembly particle, wherein the first antibody and the second antibody are capable of binding to the analyte.

The first nanoparticles may include iron oxide nanoparticles, and the second nanoparticles may include at least one of ZnS: Mn nanoparticles and Cd _{1 - x} Zn _x S / ZnS nanoparticles.

The immunoassay method may further include the step of providing a third nanoparticle assembly structure having luminescence in the buffer solution. Wherein the third nanoparticle assembly structure comprises a third assembly particle comprising a plurality of third nanoparticles gathered and a third antibody linked to the third assembly particle, wherein the third antibody binds to the analyte . The first nanoparticles may include iron oxide nanoparticles, the second nanoparticles may include ZnS: Mn nanoparticles, and the third nanoparticles may include Cd _{1 - x} Zn _x S / ZnS nanoparticles .

At least one of the first assembly particle and the second assembly particle may include streptavidin and at least one of the first antibody and the second antibody may include biotin, At least one of the particle assembly structure and the second nanoparticle assembly structure may include a streptavidin-biotin bond.

The immunoassay can be easily performed using the nanoparticle assembly structure according to the embodiments of the present invention. The nanoparticle assembly structure can overcome the limitations of conventional immunoassay methods by using the physical properties and chemical characteristics resulting from the intrinsic properties of the nanoparticles and the interaction of the nanoparticles.

1 shows a TEM image of iron oxide nanoparticles according to an embodiment of the present invention.
Fig. 2 shows the size distribution of the iron oxide nanoparticles of Fig.
Figure 3 shows a magnetic hysteresis loop of the iron oxide nanoparticles of Figure 1;
4 shows a TEM image of ZnS: Mn nanoparticles according to an embodiment of the present invention.
Fig. 5 shows the size distribution of the ZnS: Mn nanoparticles of Fig.
FIG. 6 shows the absorption spectrum (blue) and the optical luminescence spectrum (red) of the ZnS: Mn nanoparticles of FIG. 4.
7 schematically illustrates a process of forming a nanoparticle assembly structure according to an embodiment of the present invention.
Figure 8 shows the change in zeta potential according to PAA coating of a nanoparticle assembly structure according to an embodiment of the present invention.
9 shows a TEM image of an iron oxide nanoparticle assembly structure according to an embodiment of the present invention.
Figure 10 shows the size distribution of the iron oxide nanoparticle assembly structure of Figure 9;
Figure 11 shows a magnetic hysteresis loop of the iron oxide nanoparticle assembly structure of Figure 9;
12 shows a TEM image of a ZnS: Mn nanoparticle assembly structure according to an embodiment of the present invention.
13 shows the size distribution of the ZnS: Mn nanoparticle assembly structure of FIG.
Figure 14 shows a standardized one-photon PLE spectrum (blue) and a photoluminance spectrum (red) of the ZnS: Mn nanoparticle assembly structure of Figure 12.
15 shows the hydrodynamic diameter change after binding of streptavidin and antibody of the iron oxide nanoparticle assembly structure according to an embodiment of the present invention.
16 shows the change in hydrodynamic diameter of the ZnS: Mn nanoparticle assembly structure after antibody binding according to an embodiment of the present invention.
17 is a view for explaining the immunoassay according to an embodiment of the present invention.
18 shows the PL intensity of a ZnS: Mn nanoparticle assembly structure according to an embodiment of the present invention.
FIG. 19 is an enlarged view of the low density region of FIG.
20 shows optical luminescence for CRP concentration obtained using the iron oxide nanoparticle assembly structure and the ZnS: Mn nanoparticle assembly structure according to an embodiment of the present invention.
21 is a TEM image of a colloidal nanoparticle assembly structure obtained after self-separation of ZnS: Mn nanoparticle assembly structure and iron oxide nanoparticle assembly structure bound to a target protein.
Figure 22 shows the detection limit of immunoassay.
23 shows the optical luminescence for the CRP concentration obtained by varying the amount of use of the iron oxide nanoparticle assembly structure and the ZnS: Mn nanoparticle assembly structure.
Figure 24 shows the correlation of CRP concentrations in human serum samples measured using immunoassay and ELISA according to one embodiment of the present invention.
25 shows a TEM image of Cd _{1 - x} Zn _x S / ZnS nanoparticles according to an embodiment of the present invention.
26 shows the size distribution of Cd _{1 - x} Zn _x S / ZnS nanoparticles of Fig.
27 shows a TEM image of a Cd _{1 - x} Zn _x S / ZnS nanoparticle assembly structure according to an embodiment of the present invention.
28 shows the size distribution of the Cd _{1 - x} Zn _x S / ZnS nanoparticle assembly structure.
29 shows the comparison of the optical luminescence of the Cd _{1 - x} Zn _x S / ZnS nanoparticles and the Cd _{1 - x} Zn _x S / ZnS nanoparticle assembly structure.
30 shows optical luminescence for EpCAM obtained using a Cd _{1 - x} Zn _x S / ZnS nanoparticle assembly structure and an iron oxide nanoparticle assembly structure.
Figure 31 shows photoluminescence obtained for various concentrations of EpCAM in the presence of various concentrations of CRP.
Figure 32 shows photoluminescence obtained for various concentrations of CRP in the presence of various concentrations of EpCAM.

Hereinafter, the present invention will be described in detail with reference to examples. The objects, features and advantages of the present invention will be easily understood by the following embodiments. The present invention is not limited to the embodiments described herein, but may be embodied in other forms. The embodiments disclosed herein are provided so that the disclosure may be thorough and complete, and that those skilled in the art will be able to convey the spirit of the invention to those skilled in the art. Therefore, the present invention should not be limited by the following examples.

Although the terms first, second, etc. are used herein to describe various elements, the elements should not be limited by such terms. These terms are only used to distinguish the elements from each other. In addition, when an element is referred to as being on another element, it may be directly formed on the other element, or a third element may be interposed therebetween.

The sizes of the elements in the figures, or the relative sizes between the elements, may be exaggerated somewhat for a clearer understanding of the present invention. In addition, the shape of the elements shown in the drawings may be somewhat modified by variations in the manufacturing process or the like. Accordingly, the embodiments disclosed herein should not be construed as limited to the shapes shown in the drawings unless specifically stated, and should be understood to include some modifications.

As used herein, the term ZnS: Mn nanoparticles refers to ZnS nanoparticles doped with Mn, Cd _{1 - x} Zn _x S / ZnS nanoparticles have a Cd _{1 - x} Zn _x S core and ZnS is a shell Core / shell nanoparticles, and in Cd _{1 - x} Zn _x S / ZnS, x represents a real number between 0 and 1. Assembly particles mean that nanoparticles are gathered and formed in the form of particles, and the nanoparticle assembly structure means a structure containing assembly particles. For example, the iron oxide assembly particles are aggregates of iron oxide nanoparticles formed in the form of particles, and the iron oxide nanoparticle assembly structure is a structure containing the iron oxide assembly particles. IONA means an iron oxide nanoparticle assembly structure, MZSNA means a ZnS: Mn nanoparticle assembly structure, and CZSNA means a Cd _{1 - x} Zn _x S / ZnS nanoparticle assembly structure.

The nanoparticle assembly structure according to an embodiment of the present invention may include a first nanoparticle assembly structure and a second nanoparticle assembly structure.

The first nanoparticle assembly structure may include a first assembly particle formed by gathering a plurality of first nanoparticles, and a first antibody coupled to the first assembly particle, wherein the first assembly particle has magnetic properties have. The first nanoparticles may include iron oxide nanoparticles.

The second nanoparticle assembly structure may include a second assembly particle formed by gathering a plurality of second nanoparticles, and a second antibody linked to the second assembly particle, wherein the second assembly particle has a luminescent property have. The second nanoparticles may comprise ZnS: Mn nanoparticles.

The nanoparticle assembly structure according to another embodiment of the present invention may further include a third nanoparticle assembly structure. The third nanoparticle assembly structure may include a third assembly particle formed by gathering a plurality of third nanoparticles, and a third antibody linked to the third assembly particle, wherein the third assembly particle has a luminescent property have. The third nanoparticles may include Cd _1-x Zn _x S / ZnS nanoparticles.

The first assembly particle, the second assembly particle, and / or the third assembly particle may comprise streptavidin, and the first antibody, the second antibody, and / or the third antibody may comprise biotin And the antibody may be linked to the assembly particle by binding of the biotin with the streptavidin.

An immunoassay method according to an embodiment of the present invention includes the steps of: providing an analyte, a first nanoparticle assembly structure having magnetism, and a second nanoparticle assembly structure having luminescence in a buffer solution; And providing a magnetic force to the buffer solution. Wherein the first nanoparticle assembly structure comprises a first assembly particle formed by gathering a plurality of first nanoparticles and a first antibody connected to the first assembly particle, wherein the second nanoparticle assembly structure comprises a plurality of 2 nanoparticles gathered and a second antibody linked to the second assembly particle, wherein the first antibody and the second antibody are capable of binding to the analyte.

The immunoassay method may further include the step of providing a third nanoparticle assembly structure having luminescence in the buffer solution. Wherein the third nanoparticle assembly structure comprises a third assembly particle comprising a plurality of third nanoparticles gathered and a third antibody linked to the third assembly particle, wherein the third antibody binds to the analyte .

The first nanoparticles may include iron oxide nanoparticles, the second nanoparticles may include ZnS: Mn nanoparticles, and the third nanoparticles may include Cd _{1 - x} Zn _x S / ZnS nanoparticles .

The first assembly particle comprises streptavidin, the first antibody comprises biotin, and the first antibody may be linked to the first nanoparticle by binding of the biotin with the streptavidin.

[ Example ]

Iron oxide nanoparticles, ZnS: Mn  Nanoparticles, and CD _{One - x} Zn _x S / ZnS  Synthesis of nanoparticles

Iron oleate (1.8 g) and oleic acid (0.28 g) are added to 1-octadecene (10 g) to form a mixed solution. The mixed solution is heated to 320 DEG C and maintained at this temperature for 30 minutes. The mixed solution is cooled to room temperature and washed with ethanol. The mixed solution is centrifuged to obtain iron oxide nanoparticles.

0.4 g of zinc chloride and 20 mg of manganese chloride are dissolved in 55 g of dibenzylamine to form a mixed solution. After adding sulfur (0.6 g), the mixed solution is heated to 260 ° C and maintained at this temperature for 15 minutes. The mixed solution was cooled to 150 캜 and zinc chloride (0.4 g) dissolved in dibenzylamine (5 g) was added. The mixed solution is heated to 260 DEG C and maintained at this temperature for 15 minutes. After cooling the mixed solution, 1-propanol is added to wash the reaction mixture. The mixed solution is centrifuged to obtain ZnS: Mn nanoparticles.

0.13 g of cadmium oxide, 1.8 g of zinc acetate, and 6.3 g of oleic acid are mixed with 1-octadecene (11.8 g) to form a mixed solution. The mixed solution was heated to 320 DEG C and sulfur (48 mg) dissolved in 1-octadecene (1.9 g) was added. After 10 minutes, sulfur (256 mg) dissolved in tributylphosphine (2.4 g) is added. The mixed solution is kept at 320 DEG C for 30 minutes. The mixed solution is cooled to room temperature and washed with ethanol. The mixed solution is centrifuged to obtain Cd _1-x Zn _x S / ZnS nanoparticles.

Iron oxide nanoparticle assembly structure, ZnS: Mn  Nanoparticle assembly structure, and Cd _1-x Zn _x Synthesis of S / ZnS nanoparticle assembly structure

The colloidal nanoparticle assembly structure is formed by a self-assembly process in an oil-in-water microemulsion system.

For the synthesis of the iron oxide nanoparticle assembly structure, iron oxide nanoparticles in chloroform (150 mg) in chloroform (4.5 g) were dissolved in 150 mg of dodecyltrimethylammonium bromide in deionized water ) To form a mixed solution. The mixed solution is stirred at room temperature overnight. Polyacrylic acid (0.9 g) (poly (acrylic acid) ethylene glycol) in ethylene glycol (11.1 g) was added to the mixed solution and mixed well. The mixed solution is washed with deionized water and then centrifuged to obtain an iron oxide nanoparticle assembly structure.

The ZnS: Mn nanoparticle assembly structure and the Cd _{1 - x} Zn _x S / ZnS nanoparticle assembly structure are also obtained in the same manner.

Iron oxide nanoparticle assembly structure, ZnS: Mn  Nanoparticle assembly structure, and Cd _1-x Zn _x Antibody binding to S / ZnS nanoparticle assembly structure

EDC (1 mg) and NHS (1 mg) (EDC and NHS in 2- (N-morpholino) ethanesulfonic acid buffer) were dissolved in deionized water (500 μl) in a 2- (N-morpholino) ethanesulfonic acid buffer Is added to the iron oxide nanoparticle assembly structure (1 mg [Fe]) (IONAs in deionized water) and incubated for 30 minutes. The mixed solution is centrifuged to separate the iron oxide nanoparticle assembly structure. Streptavidin in phosphate-buffered saline (100 μg) in PBS (1 ml) was added to the iron oxide nanoparticle assembly structure and allowed to react for 1 hour. The streptavidin-bonded iron oxide nanoparticle assembly structure was washed with a dilution buffer solution (for example, a borate buffer solution containing 0.1% bovine serum albumin and 0.05% Tween 20) and centrifuged. Then, a dilution buffer solution ) With biotin-conjugated CRP antibody or EpCAM antibody and reacted for 2 hours. The antibody is bound to the iron oxide nanoparticle assembly structure by streptavidin-biotin bonding, and an antibody-bound iron oxide nanoparticle assembly structure is formed. The antibody-bound iron oxide nanoparticle assembly structure is washed, centrifuged and dispersed in a dilution buffer solution (500 μl).

Such EDC / NHS activation is performed on the ZnS: Mn nanoparticle assembly structure (1 mg [Zn]). The ZnS: Mn nanoparticle assembly structure is separated by centrifugation and mixed with a CRP antibody in a dilution buffer (500 [mu] l) to form an antibody-bound ZnS: Mn nanoparticle assembly structure. Antibodies that do not adhere to the biotin are bound directly to the ZnS: Mn nanoparticle assembly structure. In this way, an antibody-bound Cd _{1 - x} Zn _x S / ZnS nanoparticle assembly structure is formed.

The prepared antibody-bound nanoparticle assembly structure is stored at 4 캜.

Immunoassay using colloidal nanoparticle assembly structure

A sample CRP solution (100 μl) was added to a CRP antibody-bound iron oxide nanoparticle assembly structure (80ng [Fe]) and a CRP antibody-bound ZnS: Mn nanoparticle assembly structure (80ng [Zn]) in a dilution buffer And then reacted. 100 占 퐇 of the mixed solution is provided in a magnet-equipped 96-well plate and incubated for 30 minutes. The plate is loaded into the plate reading instrument and the PL intensity is measured using 340 +/- 30 nm excitation and 579 +/- 12 nm emission filters. The obtained PL intensity is normalized to the PL intensity of the zero-analyte.

Same as CRP analysis except that EpCAM antibody-bound iron oxide nanoparticle assembly (20 μg [Fe]) and EpCAM antibody-bound Cd _{1 -x} Zn _x S / ZnS nanoparticle assembly (20 μg [Cd + Zn]) were used Gt; EpCAM < / RTI > analysis. The PL intensity is measured using a 355 20 nm excitation and a 460 12 nm emission filter. The obtained PL intensity is normalized to the PL intensity of the zero-analyte.

Two duplex assays were performed with mixed solutions containing antibody-bound iron oxide nanoparticle assembly structures, antibody-bound ZnS: Mn nanoparticle assembly structures, and antibody-bound Cd _{1 - x} Zn _x S / ZnS nanoparticle assemblies . The rest of the procedure is the same as the singleplex analysis.

FIG. 1 shows a TEM image of iron oxide nanoparticles according to an embodiment of the present invention, FIG. 2 shows the size distribution of the iron oxide nanoparticles of FIG. 1, and FIG. 3 shows a magnetic hysteresis loop of the iron oxide nanoparticle of FIG. .

1 to 3, iron oxide nanoparticles can be synthesized by thermal decomposition of iron oleate. The iron oxide nanoparticles have a spherical shape with an average size of about 18 nm, and the size distribution is narrow. The magnetic (magnetization) behavior of the iron oxide nanoparticles measured at 300 K does not show a hysteresis loop, and iron oxide nanoparticles exhibit superparamagnetic properties at room temperature.

FIG. 4 shows TEM images of ZnS: Mn nanoparticles according to an embodiment of the present invention, FIG. 5 shows a size distribution of ZnS: Mn nanoparticles of FIG. (Blue) and a photo-luminescence spectrum (red) of the sample.

Referring to FIGS. 4 to 6, ZnS: Mn nanoparticles can be formed by reacting zinc chloride and manganese chloride with sulfur in dibenzylamine. The ZnS: Mn nanoparticles may have a nearly spherical shape with an average size of about 4.8 nm. According to the UV-Vis absorption and PL spectra of ZnS: Ms nanoparticles, a strong emission peak at 575 nm derived from the transition from ⁴ T ₁ to ⁶ A ₁ of Mn ^{2 +} ions results in a high-quality ZnS: Mn nanoparticles were synthesized. The Mn doping level determined by ICP-AES (inductively coupled plasma atomic emission spectroscopy) is 1%, and the quantum yield of PL in ZnS: Mn nanoparticles (QY) is about 40%.

7 schematically illustrates a process of forming a nanoparticle assembly structure according to an embodiment of the present invention.

Referring to FIG. 7, the iron oxide nanoparticle assembly structure and the ZnS: Mn nanoparticle assembly structure are prepared by mixing an iron oxide nanoparticle and a ZnS: Mn nanoparticle assembly using an oil-in-water microemulsion system Is formed from self-assembly.

The nanoparticles dispersed in chloroform are mixed with deionized water in the presence of DTAB (dodecyltrimethylammonium bromide), and the self-assembled nanoparticles form colloid assembly particles after the chloroform evaporation. The positively charged colloid assembly particles are coated with PAA (poly (acrylic acid)), a polymer electrolyte that is negatively charged by electrostatic interaction. Iron oxide assembly particles and the PAA coating on the ZnS: Mn assembly particle surface provide useful carboxylic acid functionalities for further functionalization.

Although not shown in the figure, the iron oxide nanoparticle assembly structure is functionalized with streptavidin through an N-hydroxysuccinimide (NHS) coupling reaction with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, The biotinylated antibodies are immobilized on the iron oxide nanoparticle assembly structure via high-affinity streptavidin-biotin linkage. Antibodies without biotin attachment are attached directly to the ZnS: Mn nanoparticle assembly surface using EDC / NHS coupling. The conjugation of the antibody increases the hydrodynamic diameter.

Figure 8 shows the change in zeta potential according to PAA coating of a nanoparticle assembly structure according to an embodiment of the present invention.

Referring to FIG. 8, the zeta potential of the nanoparticle assembly structure is shown to change from initial positive charge to negative after PAA coating.

FIG. 9 shows TEM images of the iron oxide nanoparticle assembly structure according to an embodiment of the present invention, FIG. 10 shows a size distribution of the iron oxide nanoparticle assembly structure of FIG. 9, and FIG. 11 shows the iron oxide nanoparticle assembly structure Lt; RTI ID = 0.0 > hysteresis loop.

9 to 11, the iron oxide nanoparticle assembly structure has a polyhedral shape with an average size of about 190 nm. The iron oxide nanoparticles behave like artificial atoms in the iron oxide nanoparticle assembly structure and form a high order superlattice structure. Considering the close-packing of iron oxide nanoparticles, one iron oxide nanoparticle assembly structure contains about 700 iron oxide nanoparticles. The size of the iron oxide nanoparticle assembly structure measured in a DLS (dynamic light scatter) analysis shows a hydrodynamic diameter of about 200 nm, which is slightly larger than the size measured by TEM.

The iron oxide nanoparticle assembly structure exhibits a superparamagnetic behavior at room temperature. The iron oxide nanoparticle assembly structure has a much larger volume than individual iron oxide nanoparticles and can generate a larger magnetic force. In addition, the larger surface area of the iron oxide nanoparticle assembly structure has advantages in applications involving surface reactions and magnetic separation.

12 is a TEM image of a ZnS: Mn nanoparticle assembly structure according to an embodiment of the present invention, FIG. 13 shows a size distribution of the ZnS: Mn nanoparticle assembly structure of FIG. 12, and FIG. : Represents the standardized one-photon PLE spectrum (blue) and the optical luminescence spectrum (red) of the Mn nanoparticle assembly structure.

Referring to Figures 12-14, a ZnS: Mn nanoparticle assembly structure is formed similar to an iron oxide nanoparticle assembly structure except that ZnS: Mn nanoparticles are replaced by iron oxide nanoparticles. The ZnS: Mn nanoparticle assembly structure has a spherical shape with an average size of about 320 nm and lacks the superlattice structure observed in the iron oxide nanoparticle assembly structure due to the non-spherical and less uniform size of the ZnS: Mn nanoparticles. One ZnS: Mn nanoparticle assembly structure (ZnS: Mn assembly particles) contains about 100,000 ZnS: Mn nanoparticles. The hydrodynamic diameter of the ZnS: Mn nanoparticle assembly structure measured by DLS is about 390 nm. The ZnS: Mn nanoparticle assembly structure does not show a red shift in the PL spectrum due to a large Stokes shift, while the PL QY decreases to 9%. Despite reduced PL QY, the ZnS: Mn nanoparticle assembly structure is much brighter than individual ZnS: Mn nanoparticles because of the large number of nanoparticles included.

FIG. 15 shows hydrothermal diameter changes after binding of streptavidin and an antibody in the iron oxide nanoparticle assembly structure according to an embodiment of the present invention, and FIG. 16 is a graph showing the hydrodynamic diameter change of ZnS: Mn nanoparticle assembly structure Of the hydrodynamic diameters after antibody binding.

15 and 16, the iron oxide nanoparticle assembly structure has a hydrodynamic diameter increased after binding of streptavidin and antibody, and a ZnS: Mn nanoparticle assembly structure has a larger hydrodynamic diameter after antibody binding.

17 is a view for explaining the immunoassay according to an embodiment of the present invention.

17, in the immunoassay according to the embodiments of the present invention, the first nanoparticle assembly structure, the second nanoparticle assembly structure, the antigen, etc. are reacted at once, and after the reaction, the second nanoparticle assembly Since only the PL signal of the structure can be selectively measured through magnetic separation, it does not participate in the reaction and does not require washing of the remaining material (wash-free).

First, a sample solution is added to the mixed solution of antibody-bound iron oxide nanoparticle assembly structure and antibody-bound ZnS: Mn nanoparticle assembly structure. Since the antibody of the iron oxide nanoparticle assembly structure and the antibody of the ZnS: Mn nanoparticle assembly structure selectively bind to other epitopes of the target protein, the target protein is bound to the iron oxide nanoparticle assembly structure and the ZnS: Mn A sandwich structure is formed between the nanoparticle assembly structures.

The ZnS: Mn nanoparticle assembly structure, coupled with the target protein, is separated with the iron oxide nanoparticle assembly structure by magnetic isolation and then the PL intensity of the remaining ZnS: Mn nanoparticle assembly is measured and used for quantitative analysis do. Because the ZnS: Mn nanoparticle assembly structure is brighter than the ZnS: Mn nanoparticles, the number of ZnS: Mn nanoparticle assembly structures required for detectable changes in PL intensity is greatly reduced and the sensitivity of the assay is increased.

FIG. 18 shows the PL intensity of the ZnS: Mn nanoparticle assembly structure according to an embodiment of the present invention, and FIG. 19 shows an enlarged view of the low concentration region of FIG.

Referring to Figures 18 and 19, only a few hundred ZnS: Mn nanoparticle assembly structures are required for reliable detection of small numbers of target protein molecules. If a similar number of individual ZnS: Mn nanoparticles is used for immunoassay, the total PL intensity will not be strong enough for detection. Conversely, when a much larger number of ZnS: Mn nanoparticles are used to obtain PL intensity as much as the ZnS: Mn nanoparticle assembly structure, PL reduction induced by a small amount of target protein is not detected due to strong background PL signals .

20 shows optical luminescence for CRP concentration obtained using the iron oxide nanoparticle assembly structure and the ZnS: Mn nanoparticle assembly structure according to an embodiment of the present invention.

20, C-reactive protein (CRP), which is widely used as an inflammatory biomarker, was used as a target protein to evaluate the immunoassay performance of the colloid nanoparticle assembly core. As the CRP concentration increases, the slope becomes negative and the dynamic range is about 10 to 1000 pg / ml.

21 is a TEM image of a colloidal nanoparticle assembly structure obtained after self-separation of ZnS: Mn nanoparticle assembly structure and iron oxide nanoparticle assembly structure bound to a target protein.

Referring to FIG. 21, in the self-isolated colloidal nanoparticle assembly structure obtained after reaction with 1000 pg / ml CRP, the existence of a ZnS: Mn nanoparticle assembly structure embedded between iron oxide nanoparticle assembly structures can be confirmed.

Because the excess iron oxide nanoparticle assembly structure is used with fewer ZnS: Mn nanoparticle assembly structures, each ZnS: Mn nanoparticle assembly structure is surrounded by several iron oxide nanoparticle assembly structures.

Since the decrease in PL intensity is proportional to the ratio of the ZnS: Mn nanoparticle assembly structure to the total ZnS: Mn nanoparticle assembly structure bound to the target protein, a smaller amount of the ZnS: Mn nanoparticle assembly structure is more sensitive . However, the amount of ZnS: Mn nanoparticle assembly structure should not be reduced below the threshold due to measurement noise.

Figure 22 shows the detection limit of immunoassay.

22, in the immunoassay according to the embodiments of the present invention, when using the same amount of antibody, a detection limit of 0.7 pg / ml, which is lower than the detection limit of ELISA (9.1 pg / ml) The amount of antibody used can be reduced to one tenth. The detection limit is defined as the concentration corresponding to the PL intensity, which is three times lower than the PL intensity standard deviation of the zero-analyte (blank sample) at the PL intensity of the zero-analyte control (blank sample).

23 shows the optical luminescence for the CRP concentration obtained by varying the amount of use of the iron oxide nanoparticle assembly structure and the ZnS: Mn nanoparticle assembly structure. The blue graph shows the case where the amount of iron and zinc in the iron oxide nanoparticle assembly structure and the amount of iron and zinc in the ZnS: Mn nanoparticle assembly structure are respectively 160ng, and the red graph shows the case where the amounts of iron and zinc are respectively 1280ng.

Referring to FIG. 23, another feature of the immunoassay using the colloidal nanoparticle assembly structure is that the dynamic range can be adjusted by controlling the amount of the iron oxide nanoparticle assembly structure and the ZnS: Mn nanoparticle assembly structure. For example, by increasing the amount of the iron oxide nanoparticle assembly structure and the amount of the ZnS: Mn nanoparticle assembly structure by 8 times, a standard curve covering an 8-fold higher CRP concentration can be obtained.

Figure 24 shows the correlation of CRP concentrations in human serum samples measured using immunoassay and ELISA according to one embodiment of the present invention.

Referring to FIG. 24, immunoassay using the colloidal nanoparticle assembly structure shows a good correlation with ELISA for human serum and CRP-spiked PBS samples. In addition, the immunoassay has a reproducibility of less than 5% in inter-assay and intra-assay coefficients of variation.

25 shows a TEM image of Cd _{1 - x} Zn _x S / ZnS nanoparticles according to an embodiment of the present invention, and Fig. 26 shows a size distribution of Cd _{1 - x} Zn _x S / ZnS nanoparticles of Fig. 25 . Referring to FIGS. 25 and 26, Cd _{1 - x} Zn _x S / ZnS nanoparticles have an average size of about 6.8 nm.

FIG. 27 is a TEM image of a Cd _{1 - x} Zn _x S / ZnS nanoparticle assembly structure according to an embodiment of the present invention, and FIG. 28 is a TEM image of a Cd _{1 - x} Zn _x S / ZnS nanoparticle assembly structure .

Referring to FIGS. 27 and 28, the Cd _{1 - x} Zn _x S / ZnS nanoparticle assembly structure is a ZnS: Mn nanoparticle assembly except for using Cd _{1 - x} Zn _x S / ZnS nanoparticles of about 6.8 nm in size . ≪ / RTI > The size of the Cd _{1 - x} Zn _x S / ZnS nanoparticle assembly structure measured by TEM analysis is about 350 nm and the hydrodynamic diameter is about 440 nm.

29 shows the comparison of the optical luminescence of the Cd _{1 - x} Zn _x S / ZnS nanoparticles and the Cd _{1 - x} Zn _x S / ZnS nanoparticle assembly structure.

29, the Cd _{1 - x} Zn _x S / ZnS nanoparticles exhibit an emission peak with a PL QY of 60% at 450 nm, whereas the emission peak of the Cd _{1 - x} Zn _x S / ZnS nanoparticle assembly structure exhibits a Due to the absorption, the red shift to 458 nm and the PL QY decrease to 4%.

30 shows optical luminescence for EpCAM obtained using a Cd _{1 - x} Zn _x S / ZnS nanoparticle assembly structure and an iron oxide nanoparticle assembly structure.

30, the Cd _{1 - x} Zn _x S / ZnS nanoparticle assembly structure and the iron oxide nanoparticle assembly structure are matched against an epithelial cell adhesion molecule (EpCAM), which is a diagnostic biomarker expressed in various cancers. , A standard curve covering about 1-100 ng / ml is obtained. Because the Cd _{1 -x} Zn _x S / ZnS nanoparticle assembly structure and the ZnS: Mn nanoparticle assembly structure do not overlap in the PL spectrum, the two colloidal nanoparticle assembly structures can be used simultaneously in two duplex assays.

Figure 31 shows photoluminescence obtained for various concentrations of EpCAM in the presence of various concentrations of CRP and Figure 32 shows photoluminescence obtained for various concentrations of CRP in the presence of various concentrations of EpCAM.

31 and 32, combinations of various concentrations of CRP (0, 16, 125, and 1000 pg / ml) and EpCAM (0, 2, 15, and 120 ng / ml) were evaluated. The PL intensity is not affected by the coexistence of other types of target proteins or colloidal nanoparticle assembly structures. The adjustable dynamic range of immunoassays using colloidal nanoparticle assembly structures can have advantages in multiplex analysis. While the dynamic range of individual analytes can be easily controlled by adjusting the amount of colloidal nanoparticle assembly structure, conventional immunoassays using two-dimensional solid substrates make it difficult to alter the amount of antibody and its dynamic range. That is, wash-free immunoassays using colloidal nanoparticle assembly structures enable multiplex detection with high sensitivity, high specificity, and good reproducibility. Adjustable dynamic range and no signal amplification cleanup procedures provide additional advantages. The colloidal nanoparticle assembly immunoassay can generalize the methods and reagents used and is useful for protein genetic information or disease diagnosis.

In the above embodiments, the iron oxide nanoparticle assembly structure comprises a streptavidin-biotin bond, and the ZnS: Mn nanoparticle assembly structure and the Cd _1-x Zn _x S / ZnS nanoparticle assembly structure comprise streptavidin- But are not limited to, direct binding of the assembly particle and antibody without the streptavidin-biotin linkage. The iron oxide nanoparticle assembly structure may include a direct bond between the assembly particle and the antibody and the ZnS: Mn nanoparticle assembly structure and the Cd _{1 - x} Zn _x S / ZnS nanoparticle assembly structure may contain streptavidin-biotin ) Bonds.

Hereinafter, specific embodiments of the present invention have been described. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (13)

A first nanoparticle assembly structure including first assembly particles formed by gathering a plurality of first nanoparticles,
Wherein the first assembly particles have magnetic properties. The method according to claim 1,
Wherein the first nanoparticles comprise iron oxide nanoparticles. 3. The method of claim 2,
Wherein the first nanoparticle assembly structure further comprises a first antibody coupled to the first assembly particle. The method according to claim 1,
Further comprising a second nanoparticle assembly structure comprising second assembly particles comprising a plurality of second nanoparticles gathered,
Wherein the second assembly particles have a luminescent property. 5. The method of claim 4,
Wherein the second nanoparticle assembly structure further comprises a second antibody coupled to the second assembly particle. 6. The method of claim 5,
Wherein at least one of the first assembly particle and the second assembly particle comprises streptavidin,
Wherein at least one of the first antibody and the second antibody comprises biotin,
Wherein at least one of the first nanoparticle assembly structure and the second nanoparticle assembly structure comprises a streptavidin-biotin bond. 5. The method of claim 4,
Wherein the second nanoparticles comprise at least one of ZnS: Mn nanoparticles and Cd _{1 - x} Zn _x S / ZnS nanoparticles. 8. The method of claim 7,
Wherein the second nanoparticle assembly structure comprises:
A second antibody linked to the ZnS: Mn nanoparticles and a third antibody linked to the Cd _{1 - x} Zn _x S / ZnS nanoparticles. 9. The method of claim 8,
Wherein the second antibody and the third antibody are different. Providing an analyte, a first nanoparticle assembly structure having magnetism, and a second nanoparticle assembly structure having luminescence in a buffer solution; And
Providing a magnetic force to the buffer solution,
Wherein the first nanoparticle assembly structure comprises:
A first assembly particle in which a plurality of first nanoparticles are gathered, and
A first antibody coupled to the first assembly particle,
Wherein the second nanoparticle assembly structure comprises:
A second assembly particle formed by gathering a plurality of second nanoparticles, and
And a second antibody linked to the second assembly particle,
Wherein the first antibody and the second antibody bind to the analyte. 11. The method of claim 10,
Wherein the first nanoparticles comprise iron oxide nanoparticles,
Wherein the second nanoparticles comprise at least one of ZnS: Mn nanoparticles and Cd _{1 - x} Zn _x S / ZnS nanoparticles. 11. The method of claim 10,
Further comprising providing a third nanoparticle assembly structure having luminescence in the buffer solution,
Wherein the third nanoparticle assembly structure comprises:
A third assembly particle formed by gathering a plurality of third nanoparticles, and
A third antibody linked to said third assembly particle,
The third antibody binds to the analyte,
Wherein the first nanoparticles comprise iron oxide nanoparticles,
Wherein the second nanoparticles comprise ZnS: Mn nanoparticles,
Wherein the third nanoparticle comprises Cd _{1 - x} Zn _x S / ZnS nanoparticles. 11. The method of claim 10,
Wherein at least one of the first assembly particle and the second assembly particle comprises streptavidin,
Wherein at least one of the first antibody and the second antibody comprises biotin,
Wherein at least one of the first nanoparticle assembly structure and the second nanoparticle assembly structure comprises a streptavidin-biotin bond.

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