KR101990426B1 - Composition for quantifying target material comprising metal nanoparticle assembled template particle, and quantification method using the same - Google Patents

Abstract

The present invention provides a composition for detecting a target substance comprising template particles into which metal nanoparticles are introduced, and a method for detecting a target substance using the composition.
The composition for detecting a target substance containing the template particles into which the metal nanoparticles of the present invention are introduced and the method for detecting the target material using the composition can be usefully used as a high sensitivity immunoassay technique in the field of biomaterial detection.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for detecting a target substance containing template particles having metal nanoparticles incorporated therein, and a detection method using the composition,

The present invention relates to a composition for detecting a target substance, and more particularly, to a composition for detecting a target substance containing template particles into which metal nanoparticles are introduced and a detection method using the same.

Immunologic methods have become the most prominent analytical technique for monitoring the efficacy of disease treatment as well as early diagnosis. In order to improve the sensitivity, various analyzes related to surface plasmon resonance, quartz crystal microbalance, surface enhanced Raman spectroscopy, fluorescence, electrochemistry, chemiluminescence, colorimetric assay Much effort has been sought to combine technology with immunology. Among these methods, the enzyme-linked immunosorbent assay (ELISA) is widely used to measure clinically important biological target molecules because of its simplicity, practicality, low cost, and rapid / prompt discrimination due to visual observation (Non-patent documents 1-4). Accordingly, much attention has been focused on the development of an enzyme-mediated signal amplification method. Conventional ELISA methods showed detection limits at the picomole (pM) level and could not be applied to the detection of trace amounts of proteins in body fluids or tissues (Non-Patent Document 5), indicating a narrow assay range for performing quantitative detection Document 6).

In addition, the conventional polymerase chain reaction (PCR) is a DNA amplification method, which is useful for detecting a trace amount of DNA. However, since there is a problem in that a technique such as protein amplification can not be applied in protein detection, There was a limit to protein detection.

Recent nanomaterial-based signal amplification has received considerable attention due to the ultra-high sensitivity of these technologies. Nanomaterials can combine catalytic activity, conductivity, and biocompatibility to accelerate signal transduction as well as amplify signals, resulting in synergistic effects. Most importantly, nanoscale materials can be used as chemical and biological sensors at single molecule detection levels. Among them, gold nanoparticles (Au nanoparticles, Au NP) can be easily produced, are easily deformed, and are widely used in the field of biometrics due to their biocompatibility. To date, a number of highly sensitive immunoassays based on monodispersed or agglomerated Ag NPs have been reported (Non-Patent Documents 7-11). However, most of them have difficulty in quantitatively measuring the target molecule concentration due to the narrow linearity analysis range. There is still a need for the development of highly sensitive quantitative immunoassays using Au NP.

U.S. Published Patent Application No. 2013-0040292 (published on February 23, 2013). Korean Patent Publication No. 10-2010-0061603 (published on June 6, 2010). Korean Patent Publication No. 10-2014-0103875 (Disclosure Date: 2014.08.27.)

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We have focused on the bimetallic Au-Ag NP as an alternative to the nanoparticles used in the quantitative immunoassay. 2 Metallic Au-Ag alloy NP is characterized by high plasmon efficiency, high index of refraction index due to silver, and Au (Ag) NP because Au-Ag NP's localized surface plasmon resonance (LSPR) strengthens the electromagnetic field around the NP surface. Long-term stability and biocompatibility by < RTI ID = 0.0 > a < / RTI > In particular, the optical properties of Au-Ag NPs in Au and / or Ag NP-incorporating complexes on silica NP templates can be controlled by controlling the growth of the Ag shell thickness on Au NPs and using Au and / or Ag By controlling the density of the NP and the gap between the two NPs, it was pointed out that stronger LSPR characteristics can be obtained compared to a single NP.

Thus, the present inventors have further studied a detection method for amplifying an ELISA signal and improving the sensitivity of a colorimetric immunoassay, so that when a bio-target substance is detected using a composition containing template particles into which metal nanoparticles are introduced, And that the target substance can be detected in a wide range of analysis.

Accordingly, it is an object of the present invention to provide a composition for detecting a target substance comprising template particles into which metal nanoparticles are introduced, and a method for detecting a target substance using the composition.

According to an aspect of the present invention, there is provided a composition for detecting a target substance comprising template particles into which nanoparticles of a first metal are introduced, a cation of a second metal, and a precursor of a reducing agent.

In one embodiment, the template particles may be particles of a material selected from the group consisting of polystyrene, polymethylmethacrylate, proteins, viruses, silica, TiO ₂ , graphene, carbon nanotubes, and mixtures thereof.

In one embodiment, the first metal may be a metal selected from the group consisting of copper, palladium, silver, gold, and alloys thereof.

In one embodiment, the cation of the second metal may be a cation selected from the group consisting of copper ions, palladium ions, silver ions, gold ions, and mixtures thereof.

In one embodiment, the nanoparticles of the first metal may be nanoparticles of the first metal coated with the second metal.

In one embodiment, the precursor of the reducing agent may be 2-phospho- L -ascorbic acid.

According to another aspect of the present invention, there is provided a process for the preparation of a compound of formula I, comprising: reacting a first reactant comprising a precursor of a reducing agent; And a second reactant including template particles into which nanoparticles of the first metal are introduced and cations of the second metal.

In one embodiment, the kit may further comprise a capture antibody and an enzyme-bound detection antibody for the target material.

In one embodiment, the enzyme may be an alkaline phosphatase.

According to another aspect of the present invention there is provided a method of detecting a target substance comprising: (a) binding a target substance to a capture antibody immobilized on a platform; (b) binding the enzyme-bound detection antibody to the target material; (c) reacting the enzyme bound to the detection antibody with a first reactant comprising a reducing agent precursor, and a second reactant comprising template particles into which nanoparticles of the first metal are introduced and cations of the second metal, To obtain a resultant product; And (d) measuring the absorbance of the resultant.

In one embodiment, the method of detection comprises the steps of: (a) binding a target substance to a capture antibody immobilized on a platform; (b) binding the alkaline phosphatase-bound detection antibody to the target material; (c) reacting the alkaline phosphatase bound to the detection antibody with a first reactant comprising 2-phospho- L -ascorbic acid, and a second reactant comprising silver nanoparticle-introduced silica particles and silver nitrate, Obtaining the result; And (d) measuring the absorbance of the resultant at 430 nm.

According to the present invention, the plasmon immunoassay using a composition comprising template particles into which metal nanoparticles are introduced exhibits an absorbance linearly proportional to the IgG concentration in the range of 6.67 x 10 ^-13 M to 6.67 x 10 ^-8 M, It has been found that bio-target substances can be detected with a dynamic range 100 times wider than conventional enzyme-based colorimetric immunoassays.

Therefore, the composition for detecting a target substance including the template particles into which the metal nanoparticles of the present invention are introduced and the method for detecting the target material using the template particle can be usefully used as a high sensitivity immunoassay technique in the field of biomaterial detection.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of a plasma-based colorimetric immunoassay process.
2 is a transmission electron micrograph of SiO ₂ @ Au NP according to SiO ₂ modified with (a) SiO ₂ NP and (b) NH ₂ group (APTS) or (c) SH group (MPTS).
3 shows TEM images (A) and optical characteristics (B) of a SiO ₂ @ Au @ Ag solution at different concentrations of ascorbic acid [(A) 0 μM, (B) 10 μM and (C) 100 μM ; SiO ₂ @Au and silver nitrate (AgNO ₃₎ was 100 ㎍ and 1 nmol each Im.
4 is a TEM photograph of SiO ₂ @ Au @ Ag synthesized at different concentrations of ascorbic acid [(a) 0, (b) 2, (c) 4, 10, (g) 20, (h) 40, (i) 60, (k) 80, (1) 100 μM; SiO ₂ @Au and AgNO ₃ is 100 ㎍ and 1 nmol each Im.
FIG. 5 is a calibration curve of SiO ₂ @ Au @ Ag with different concentrations of ascorbic acid.
Figure 6 shows the color change (A) of the plasmon enzyme-linked immunosorbant assay according to different concentrations of IgG ranging from 6.67 x 10 ^-15 to 6.67 x 10 ^-8 M and (B) the corresponding UV-Vis absorbance plot. (Internal figure is a linear section of the 6.67 x 10 ^-13 to 6.67 x 10 ^-8 M IgG calibration curve).
Figure 7 is a UV-Vis spectrum of a plasmonzyme-linked immunosorbent assay according to different IgG concentrations ranging from 6.67 x 10 ^-15 to 6.67 x 10 ^-8 M.

The present invention provides a composition for detecting a target substance comprising a template particle into which nanoparticles of a first metal are introduced, a cation of a second metal and a precursor of a reducing agent.

In one embodiment, the template particles are not limited as long as they can be made into nano-sized particles by conventional nanoparticle preparation methods. For example, polystyrene (PS), polymethyl methacrylate methacrylate, and PMMA); Biopolymers such as proteins and viruses; Silica, TiO _2; Carbon nanotubes (CNTs), and other carbon-based materials (nanoparticles). In addition, the size of the template particles may be 50 nm - 250 nm, preferably 100 nm - 200 nm, and most preferably 150 nm.

The preparation of the template particles into which the nanoparticles of the first metal are introduced will be described below taking as an example the process of using the silica nanoparticles as template particles. When the process of introducing gold nanoparticles into the silica nanoparticles is carried out, it is possible to carry out after the surface of the silica is modified with an amine. Specifically, after silica NP is prepared, it is dispersed in a solvent (for example, anhydrous EtOH), and a commonly used amino group-modifying reagent [3-aminopropyltriethoxysilane (APTS) and NH ₄ OH] is added to the dispersion to modify the surface of the SiO ₂ to an amino group. The mixture is stirred and centrifuged to obtain aminated silica NP, which is then washed several times with EtOH to remove excess reagent.

In one embodiment, the first metal is not limited as long as it is a metal that can be made into nano-sized particles by conventional nanoparticle preparation methods, for example, copper, palladium, silver, gold, . The nanoparticle size of the first metal introduced into the template particles may be 0.1-10 nm, preferably 1-5 nm, more preferably 2-3 nm. Also, the number of nanoparticles of the first metal introduced per one template particle is 100 to 10,000, preferably 1,000 to 5,000, more preferably 2,000 to 3,000.

The process in which the nanoparticles of the first metal are introduced into the template particles is described below as the template particles of the silica nanoparticles and the gold as an example of the first metal. AuCl ₃ is reduced using a reducing agent (for example THPC [tetrakis (hydroxymethyl) phosphonium chloride]) to produce colloidal Au NP. The prepared Au NP and the aminated SiO ₂ solution were mixed, sonicated, incubated in a shaker, and then centrifuged to obtain Au NP-introduced silica NP, and a solvent (for example, EtOH) To remove unbound Au NP to obtain silica nanoparticle-incorporated silica particles.

The template particles into which the nanoparticles of the first metal are introduced are contained in the target substance detection composition at a concentration of 0.01 to 100 mg / mL, preferably 0.1 to 10 mg / mL, more preferably 1 mg / mL. The volume added to the reaction vessel is adjusted so that the template particles having the nanoparticles of the first metal introduced therein are added so as to correspond to 1 to 1000 μg, preferably 10 to 100 μg, more preferably 100 μg per reaction vessel, .

In one embodiment, for the cation of the second metal, the second metal is not limited as long as it is a metal that can be present in the ionic form of the cation, for example, copper, palladium, silver, gold, and the like. Therefore, the cation of the second metal may be copper ion, palladium ion, silver ion, gold ion, etc., preferably silver ion, more preferably silver nitrate silver ion. The concentration of the cation-containing material of the second metal (for example, silver nitrate) may be 0.1-1000 mM, preferably 1-100 mM, more preferably 10 mM. The amount of the cation-containing material of the second metal is adjusted so as to correspond to 0.01 to 100 nmole, preferably 0.1 to 10 nmole, and more preferably 1 nmole per reaction vessel.

In one embodiment, the nanoparticles of the first metal may be nanoparticles of the first metal coated with the second metal.

In one embodiment, the precursor of the reducing agent refers to a precursor of a substance that can act as a reducing agent, for example, but not limited to, 2-phospho- L -ascorbic acid. The 2-phospho- L -ascorbic acid is converted to ascorbic acid by the enzyme (i.e., alkaline phosphatase) bound to the detection antibody. The converted ascorbic acid reduces the cations of the second metal to form a shell of the second metal on the surface of the nanoparticles of the first metal.

The present invention also relates to a process for the preparation of a compound of formula I comprising: reacting a first reactant comprising a precursor of a reducing agent; And a second reactant including template particles into which nanoparticles of the first metal are introduced and cations of the second metal.

In one embodiment, the kit may further comprise a capture antibody and an enzyme-bound detection antibody for the target material. The capture antibody and the enzyme-bound detection antibody are antibodies comprising an epitope that specifically binds to a target substance to be detected.

In one embodiment, the enzyme is not limited as long as it is an enzyme capable of converting the precursor of the reducing agent to a reducing agent, for example, alkaline phosphatase. When the enzyme is an alkaline phosphatase, 2-phospho- L -ascorbic acid is converted to ascorbic acid.

In one embodiment, the target material is not limited as long as it is capable of acting as an antigen in an antigen-antibody reaction such as a peptide, a protein, a virus, a nucleic acid and the like. For example, a prostate specific antigen (PSA) Related antigens such as alpha-fetoprotein (AFP), which is an indicator for carcinoembryonic antigen (CEA), hepatocellular carcinoma, ovarian cyst, ovarian cyst in testis, fetal carcinoma, etc. for breast cancer, lung cancer and liver cancer; Nucleic acids such as circulating tumor DNA (ctDNA) and RNA; Cell organelles such as microcysts and exosomes; Cells such as circulating tumor cells (CTC); Liposomes.

The present invention also relates to a method of detecting a target substance comprising: (a) binding a target substance to a capture antibody immobilized on a platform; (b) binding the enzyme-bound detection antibody to the target material; (c) reacting the enzyme bound to the detection antibody with a first reactant comprising a reducing agent precursor, and a second reactant comprising template particles into which nanoparticles of the first metal are introduced and cations of the second metal, To obtain a resultant product; And (d) measuring the absorbance of the resultant.

In one embodiment, the method of detection comprises the steps of: (a) binding a target substance to a capture antibody immobilized on a platform; (b) binding the alkaline phosphatase-bound detection antibody to the target material; (c) reacting the alkaline phosphatase bound to the detection antibody with a first reactant comprising 2-phospho- L -ascorbic acid, and a second reactant comprising silver nanoparticle-introduced silica particles and silver nitrate, Obtaining the result; And (d) measuring the absorbance of the resultant at 430 nm.

Specifically, the detection method can be performed as follows.

Step (a) is the step of binding the target substance to the capture antibody immobilized on the platform. That is, an antibody (capture antibody) that specifically binds to a target substance is immobilized on a platform (for example, a 96-well microplate, a reaction container), and a platform surface (microplate surface) (Blocking) with a blocking agent (e.g. Thereafter, the detection target sample containing the target substance may be injected into the reaction container, and the antibody-antigen interaction may be advanced, followed by washing with a buffer (PBST).

Step (b) is the step of binding the enzyme-bound detection antibody to the target substance. That is, this can be performed by injecting an enzyme-bound detection antibody into a reaction container, binding the detection antibody to the target substance, and then removing the detection antibody not bound to the target substance by washing.

Wherein step (c) comprises adding to the enzyme bound to the detection antibody a first reactant comprising a precursor of a reducing agent and a second reactant comprising template particles into which nanoparticles of the first metal have been introduced and cations of the second metal, To obtain the final product. That is, a first reactant containing a precursor of a reducing agent is injected into a reaction vessel, followed by incubation. Then, a second reactant containing template particles into which nanoparticles of the first metal are introduced and cations of the second metal is added to the reaction vessel ≪ / RTI > incubation.

Step (d) is a step of measuring the absorbance of the resultant. That is, it can be obtained by measuring the absorbance (for example, the absorbance at 430 nm) of the result of the reaction, calculating the target substance in the sample and its concentration from the calibration curve.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.

< Example >

1. Experimental Method

(1) Material

Tetraethylorthosilicate (TEOS), 3-aminopropyltriethoxysilane (APTS), silver nitrate (AgNO ₃ ), tetrakis (hydroxymethyl) phosphonium chloride [tetrakis ), phosphonium chloride (THPC), gold (III) chloride trihydrate, HAuCl ₄ , ascorbic acid, polyvinylpyrrolidone (PVP), PBS tablet), tween 20 (tween 20), 2- phospho-L-ascorbic acid (2-phospho- L -ascorbic acid) , goat anti-rabbit IgG (goat anti-rabbit IgG) , rabbit IgG (rabbit IgG) and Alkaline phosphatase (AP) conjugated goat anti-rabbit IgG was purchased from Sigma-Aldrich (St. Louis, MO, USA) Respectively. Ethanol (EtOH) and aqueous ammonium hydroxide (aqueous NH ₄ OH, 27%) were purchased from Dae Jung (Siheung, Korea).

(2) Silica nanoparticles into which gold nanoparticles are introduced (SiO ₂ @Au NP)

Silica NP having a size of about 150 nm was prepared according to the method described in the prior art (Stober method, non-patent document 32). The prepared silica NP (50 mg mL ^{- 1} ) was dispersed in 4 mL of anhydrous EtOH, 250 μL of APTS and 40 μL of NH ₄ OH were added to the colloid solution to modify the SiO ₂ surface with an amino group. The mixture was vigorously stirred at 25 &lt; 0 &gt; C for 6 hours and then at 70 &lt; 0 &gt; C for 1 hour. After centrifugation, aminated silica NP was obtained, and excess reagent was removed by washing with EtOH several times (Non-Patent Documents 33 and 34).

AuCl ₃ was reduced using THPC [tetrakis (hydroxymethyl) phosphonium chloride] as a reducing agent to prepare colloidal Au NP. THPC capped Au NP was prepared according to the method described in the prior art (Non-Patent Documents 33, 35). The prepared solution was stored at 4 캜 for 3 days before use.

Au NP (1 mM, 10 mL) and aminated SiO ₂ solution (1 mg mL ^-1 , 1 mL) were mixed and sonicated for 30 minutes and then incubated overnight in the shaker. After centrifugation, Au NP-introduced silica NP was obtained and washed with EtOH several times to remove unbound Au NP. The obtained SiO ₂ @ Au NP was re-dispersed in anhydrous EtOH to obtain a 1 mg mL ^-1 SiO ₂ @ Au NP solution (Non-Patent Documents 33 and 34).

(3) SiO ₂ Manufacture of @ Au @ Ag NP

Au-Ag Core-Shell NP was prepared by reducing and depositing Ag source with ascorbic acid on gold NP in the presence of PVP in an aqueous medium. In summary, 100 μg of SiO ₂ @ Au NP (100 μL) was dispersed in 0.7 mL of PVP (1 mg / mL). Silver nitrate (10 mM, 100 μL) was added to the solution followed by 10 mM ascorbic acid (100 μL). This solution was incubated for 1 hour to reduce Ag ⁺ ions to Ag metal. The resulting SiO ₂ @ @ Au Ag NP is obtained by centrifugation for 15 minutes at 8,500 rpm, washed several times with EtOH to remove the excess reagent. SiO ₂ @ Au @ Ag NP was redispersed in 1.0 mL of anhydrous EtOH (non-patent documents 33 and 34).

(4) Detection of rabbit IgG

First, 100 占 퐇 of a rabbit anti-IgG solution (10 占 퐂 / ml) was fixed overnight in a 96-well microplate. After washing three times with PBS (PBST) containing 0.1% Tween 20, the microplate was blocked (blocked) with 300 μL of 1% BSA for 2 hours to prevent nonspecific adsorption. Subsequently, rabbit IgG (100 [mu] L) at various concentrations was injected into a microplate and incubated for 1 hour at 37 [deg.] C for antibody-antigen interaction, and then each well was washed 3 times with PBST. AP-conjugated anti-rabbit IgG (100 [mu] L) was then incubated at 37 [deg.] C for 1 hour to bind to the captured IgG and washed with PBST.

Finally, 100 μL of 2-phospho- L -ascorbic acid (substrate) in bicarbonate buffer (pH 8.5) was injected into the microplate and incubated at room temperature for 1 hour. 100 μL of 1 mg / mL SiO ₂ @ Au (100 μL) and 10 mM AgNO ₃ (100 μL) in PVP (1 mg / mL) were simultaneously added to the microplate and incubated for 1 hour. The UV-Vis absorbance of the prepared sample was measured at 430 nm.

2. Results

(1) Enzyme Catalyzed  SiO ₂ @Au Top Ag  On growth Based  Colorimetric immunoassay

Plasmon-based colorimetric immunoassay is shown in Fig. In this method, the amplified reduction of the Ag ⁺ ion by the enzyme and the Ag growth on the Au NP incorporated structure (SiO ₂ @ Au @ Ag) are combined, which causes a strong color change. As a model, rabbit IgG was used as a target material for immunoassay.

First, polyclonal goat anti-rabbit IgG (Ab ₁ ) was immobilized on a 96-well microplate and used as a platform to capture rabbit IgG (antigen). Was used as a rabbit IgG (Ab ₂₎ the tracking antibody (detection antibody), for the construction of the plasmon-based colorimetric immunoassay - Next, a polyclonal goat anti-AP-coupled. In the presence of the target IgG, the immunoconjugate of Ab ₁ -IgG-Ab ₂ containing AP can cause enzyme catalyzed conversion of 2-phospho- L -ascorbic acid to ascorbic acid. The resulting ascorbic acid reduces silver nitrate (AgNO ₃ ) to Ag metal, which is deposited on the surface of Au NP (SiO ₂ @ Au) on silica NP.

When Ag metal is deposited on the surface of SiO ₂ @ Au, the UV-Vis spectrum is shifted to 430 nm (absorbance peak of Ag). The change in absorbance was dependent on the IgG concentration in the sample. By monitoring the absorbance at 430 nm, the IgG concentration can be measured quantitatively.

(2) SiO ₂ On @Au Ag  growth

For the successful development of plasmon-based colorimetric immunoassays, one important prerequisite is the coating of Ag metal on the surface of SiO ₂ @ Au under an ascorbic acid reduction system. To this end, SiO ₂ @ Au was prepared by immobilizing Au NP on aminated silica NP (about 150 nm diameter). The method reported in the prior art was modified to produce colloidal Au NP (2-3 nm) by reducing HAuCl ₄ with tetrakis (hydroxymethyl) phosphonium chloride (THPC) (Non-Patent Document 35). The Au NP was immobilized on the aminated silica NP by shaking weakly.

Figure 2 shows that about 2300 Au NPs were introduced into the aminated silica NP surface. Thereafter, the Ag shell was selectively formed on the Au NP surface on silica when silver nitrate (AgNO ₃ ) was reduced in the presence of ascorbic acid and polyvinylpyrrolidone (PVP). The thickness of Ag NP was controlled by the concentration of ascorbic acid in the solution. The amount of SiO ₂ @ Au NP was 100 μg, the concentration of silver nitrate (AgNO ₃ ) was fixed to 1 nmole, and the concentration of ascorbic acid was in the range of 2 to 100 μM.

Representative transmission electron microscopy (TEM) images are shown in Figures 3A and 4. As ascorbic acid concentration increases from 2 to 100 μM, the size of Au @ Ag NP increases on the surface of silica NP. The UV-Vis spectrum of the resulting SiO ₂ @ Au @ Ag solution is shown in FIG. The UV-Vis absorbance of SiO ₂ @ Au @ Ag NP was consistent with their TEM image. Due to the low absorbance of 3 nm Au NP, SiO ₂ @ Au NP did not show typical UV peaks of Au NP at 500 - 520 nm. In general, Au NP exhibits red color (due to absorption of blue light due to plasmon effect), and Au NP of 3 nm size is too small for plasmon phenomenon to appear, unlike typical Au NP, The absorbance of the blue series was low because it could not absorb the light of the series.

After depositing Ag metal on SiO ₂ @ Au NP, a plasmon deposition peak was observed at about 430 nm, which is due to the formation of an Ag shell on the SiO ₂ @ Au NP surface. In addition, the peak intensity was proportional to ascorbic acid concentration over a wide range of 2-100 μM, which means that the Ag shell was grown proportionally on SiO ₂ @ Au @ Ag (FIG. 3B and FIG. 5). By the AP 2- phospho - L - ascorbic acid is produced from the ascorbic acid was able to promote the coating of the Ag in the shell surface @Au SiO _2, the new absorbance peak of SiO ₂ @ @ Au Ag was at 430 nm.

(3) colorimetric immunoassay Analytical ability

To investigate the analytical ability of plasmon-based colorimetric immunoassays, AP-conjugated goat anti-rabbit IgG was used as a detection antibody. After optimization of the assay conditions, UV-Vis absorbance spectra of the reaction solutions with various IgG concentrations were obtained. FIG. 6A shows the color of the SiO ₂ @ Au @ Ag solution after the color enhancement by the enzyme. As the IgG concentration increased from 6.67 x 10 ^-15 to 6.67 x 10 ^-7 M, the color of the solution changed sharply from light pink to brown and dark gray. This means that ascorbic acid was produced from 2-phospho- L -ascorbic acid in solution by AP coupled to anti-rabbit IgG. Ascorbic acid to the surface of the SiO ₂ @Au by reducing Ag ⁺ ions to Ag metal was generated to SiO ₂ @ Au @ Ag. The absorption intensity of the reaction sample containing SiO ₂ @ Au @ Ag is shown in FIG. 6B and FIG. When the IgG concentration was 6.67 x 10 ^-14 M or less, the maximum absorbance peak was hardly observed and the absorbance at 430 nm slightly changed. This is because there is little Ab ₂ and thus less ascorbic acid is formed in the solution and less Ag metal is coated on the SiO ₂ @ Au surface. When the concentration of IgG was 6.67 x 10 ^-13 M or more, the absorbance spectrum of SiO ₂ @ Au @ Ag was clearly observed at 430 nm. The absorbance spectrum of the solution showed a tendency to red shift. As the IgG concentration increased, the absorbance intensity was significantly increased by Ag growth on the surface of SiO ₂ @ Au (FIG. 6B).

Curve - fitting method was used for the calibration curve. A similar correlation can be fitted between the logarithmic values of absorbance and IgG concentration for experimental data measurements of 6.67 x 10 ^-13 M to 6.67 x 10 ^-8 M. Y = 0.183 x + 4.93 and R ² = 0.99 were obtained from the calibration curve. Compared with the existing ELISA, the scope of analysis of the present invention extended 100 times. The theoretical detection limit (LOD) was estimated to be 5 x 10 ^-13 M at 3 _Sblank standard.

3. Conclusion

In the present invention, a novel plasmonic immunoassay method for detecting a target substance with high sensitivity and a broad analysis range using an AP-bound detection antibody that catalyzes the conversion of 2-phospho- L -ascorbic acid to ascorbic acid has been developed . The presence of ascorbic acid induced the reduction of the Ag ⁺ ion to the Ag metal and coated onto the surface of SiO ₂ @ Au leading to an increase in the absorbance peak at 430 nm. As a result, the absorbance of SiO ₂ @ Au @ Ag was linearly proportional to the IgG concentration in the range of 6.67 × 10 ^-13 M to 6.67 × 10 ^-8 M. Compared with conventional enzymatic-based colorimetric immunoassays, plasmonic colorimetric assays are highly sensitive methods with detection limits of 5 x 10 ^-13 M over a 100-fold broader assay range.

Claims (11)

A silver cation, a precursor of a reducing agent, a capture antibody to a target substance, and an enzyme-bound detection antibody, wherein the enzyme of the enzyme-bound detection antibody binds a precursor of the reducing agent to a reducing agent To the target substance.
delete delete delete The composition according to claim 1, wherein the gold nanoparticles are silver-coated gold nanoparticles.
The composition according to claim 1, wherein the precursor of the reducing agent is 2-phospho- L -ascorbic acid.
A first reactant comprising a precursor of a reducing agent; And
A silica template particle into which gold nanoparticles are introduced and a second reactant containing silver cation
Characterized in that the kit further comprises a capture antibody for the target substance and an enzyme-bound detection antibody.
delete The kit for quantitative detection of a target substance according to claim 7, wherein the enzyme is an alkaline phosphatase.
(a) binding a target substance to a capture antibody immobilized on a platform;
(b) binding the enzyme-bound detection antibody to the target material;
(c) a step of sequentially reacting an enzyme bound to the detection antibody with a first reactant containing a reducing agent precursor and a second reactant containing silver template particles and silver cations in which gold nanoparticles are introduced, ; And
(d) measuring the absorbance of the resultant
And detecting the target substance.
11. The method of claim 10,
(a) binding a target substance to a capture antibody immobilized on a platform;
(b) binding the alkaline phosphatase-bound detection antibody to the target material;
(c) reacting the alkaline phosphatase bound to the detection antibody with a first reactant comprising 2-phospho- L -ascorbic acid, and a second reactant comprising silver nanoparticle-introduced silica particles and silver nitrate, Obtaining the result; And
(d) measuring the absorbance of the resultant at 430 nm
And detecting the target substance.

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