Simultaneous analysis method for multiple targets using multiple metal nano-tags

Abstract

The present invention relates to a simultaneous analysis method for a target using a plurality of metal nano-tags and, more particularly, to a simultaneous analysis method for a target using a plurality of metal nano-tags, wherein the method fuses a nano-particle technology on the basis of an antigen-antibody reaction, which is a conventional biological immune response, and simultaneously diagnoses a plurality of target materials by using a plurality of antigen-antibody reactions and a plurality of metal nano-tags, thereby enhancing diagnostic effect.

Claims

1. A method for simultaneous analysis of a plurality of targets in a blood sample using a plurality of metals, comprising: (i) preparing a first analysis platform to which a first antibody, that specifically binds to a target, is bound; (ii) reacting the first analysis platform comprising the first antibody with a blood sample comprising a plurality of targets and thereby forming a second analysis platform to which target materials are bound; (iii) reacting a second antibody, that specifically binds to a target, with the second analysis platform in which the first antibody and targets are bound; and (iv) performing a quantitative analysis of the target material to which the second antibody is bound; wherein the first analysis platform to which the first antibody is bound is silica nanoparticles, and comprises a plurality of types of first antibodies, wherein the silica nanoparticles have a metal-comprising core and silica that coats the surface of the core, wherein the targets analyzed are targets in blood samples and comprise at least two selected from the group consisting of hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV), wherein the first analysis platform to which the first antibody is bound comprises at least two types of silica nanoparticles containing different types of metals, wherein the silica nanoparticle comprises Au (gold) when the target to be analyzed is HBV, the silica nanoparticle comprises Gd (gadolinium) when the target to be analyzed is HIV, and the silica nanoparticle comprises Eu (europium) when the target to be analyzed is HCV.

2. The method of claim 1, wherein the first antibody and the second antibody are the same.

3. The method of claim 1, wherein the first antibody is a monoclonal antibody and the second antibody is a polyclonal antibody.

4. The method of claim 1, wherein step (iv) of performing a quantitative analysis of the target material to which the second antibody is bound comprises: (iv-1) capturing the target material to which the second antibody is bound by applying an external magnetic force; and (iv-2) analyzing the captured target material to which the second antibody is bound using a spectrophotometer.

5. The method of claim 4, wherein step (iv-2) of analyzing the captured target material to which the second antibody is bound using a spectrophotometer comprises analyzing by using an inductively coupled plasma mass spectrometry (ICP-MS) or graphite furnace atomic absorption spectrophotometer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1 and 2 show schematic diagrams illustrating a method for simultaneous analysis of targets using a plurality of metal nano-tags by the present invention.

(2) FIGS. 3 and 4 show the analysis results by ICP-MS in the blood according to an exemplary embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

(3) The method for simultaneous analysis of a plurality of targets using a plurality of metal nano-tags according to the present invention may include:

(4) (i) preparing an analysis platform to which a first antibody, that specifically binds to a target, is bound;

(5) (ii) reacting the analysis platform including the first antibody with a sample containing a plurality of targets and thereby forming an analysis platform to which target materials are bound;

(6) (iii) reacting a second antibody, that specifically binds to a target, with the analysis platform in which the first antibody and targets are bound; and

(7) (iv) performing a quantitative analysis of the material to which the second antibody is bound.

(8) In the method for simultaneous analysis of a plurality of targets using a plurality of metal nano-tags according to the present invention, the analysis platform to which the first antibody is bound may include a plurality of types of antibodies.

(9) In the method for simultaneous analysis of a plurality of targets using a plurality of metal nano-tags according to the present invention, the analysis platform to which the first antibody is bound may be a silica nanoparticle which contains a magnetic metal-containing core and a silica that coats the surface of the core. The silica nanoparticle contains a single type of metal; and the analysis platform to which the antibody is bound contains at least two types of silica nanoparticles containing different types of metals. The metals contained in the silica nanoparticle may be selected from the group consisting of Au, Ag, Pt, Pd, Ir, Rh, Ru, Al, Cu, Te, Bi, Pb, Fe, Ce, Mo, Nb, W, Sb, Sn, V, Mn, Ni, Co, Zn, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Sc, Y, and Ti. The analysis platform to which the first antibody is bound may include at least two types of a plurality of types of silica nanoparticles selected from the group consisting of a silica nanoparticle containing a silica nanoparticle containing Au, a silica nanoparticle containing Gd, a silica nanoparticle containing Y, and a silica nanoparticle containing Eu. The second antibody may be bound to a silica nanoparticle, which contains a magnetic metal-containing core and a silica that coats the surface of the core.

(10) In the method for simultaneous analysis of a plurality of targets using a plurality of metal nano-tags according to the present invention, the analysis platform to which the first antibody is bound may be a plate to which a plurality of types of the first antibody is bound.

(11) In the method for simultaneous analysis of a plurality of targets using a plurality of metal nano-tags according to the present invention, the second antibody may be bound to a silica nanoparticle which contains a magnetic metal-containing core and a silica that coats the surface of the core.

(12) In the method for simultaneous analysis of a plurality of targets using a plurality of metal nano-tags according to the present invention, the core of the silica nanoparticle bound to the second antibody may contain at least two metals selected from the group consisting of Au, Ag, Pt, Pd, Ir, Rh, Ru, Al, Cu, Te, Bi, Pb, Fe, Ce, Mo, Nb, W, Sb, Sn, V, Mn, Ni, Co, Zn, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Sc, Y, and Ti.

(13) In the method for simultaneous analysis of a plurality of targets using a plurality of metal nano-tags according to the present invention, the core of the silica nanoparticle bound to the second antibody may contain Au.

(14) In the method for simultaneous analysis of a plurality of targets using a plurality of metal nano-tags according to the present invention, the first antibody and the second antibody may be the same.

(15) In the method for simultaneous analysis of a plurality of targets using a plurality of metal nano-tags according to the present invention, the first antibody may be a monoclonal antibody and the second antibody may be a polyclonal antibody.

(16) In the method for simultaneous analysis of a plurality of targets using a plurality of metal nano-tags according to the present invention,

(17) step (iv) of performing a quantitative analysis of the material to which the second antibody is bound may include:

(18) (iv-1) capturing the target material to which the second antibody is bound by applying an external magnetic force; and

(19) (iv-2) analyzing the captured target material to which the second antibody is bound using a spectrophotometer.

(20) In the method for simultaneous analysis of a plurality of targets using a plurality of metal nano-tags according to the present invention,

(21) step (iv) of performing a quantitative analysis of the material to which the second antibody is bound may include:

(22) (iv-1) separating the material, which is bound to the first antibody of the plate, to which the second antibody is bound; and

(23) (iv-2) analyzing only the material, which is bound to the first antibody of the plate, to which the second antibody is bound using a spectrophotometer.

(24) In the method for simultaneous analysis of a plurality of targets using a plurality of metal nano-tags according to the present invention,

(25) step (iv-2) of analyzing the captured target material to which the second antibody is bound using a spectrophotometer may be to analyze using an inductively coupled plasma mass spectrometry (ICP-MS) or graphite furnace atomic absorption spectrophotometer.

EXAMPLES

(26) Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not further limited by the following examples.

<Example 1> Case Using Plate as Analysis Platform Containing First Antibody

(27) After attaching a Human anti-p24 monoclonal antibody as a first antibody to a plate, HBsAg was attached as a second antibody to the plate, and silica nanoparticles containing an Au particle were prepared using the Gold Nanoparticle Conjugation kit

(28) A blood sample was allowed to flow through the plate to induce a reaction between the first antibody and a target in the blood sample and unreacted impurities were removed by washing. The HBsAg was attached the resultant and allowed to react with a second antibody containing an Au particle.

(29) Then, the conjugate bound to the second antibody was separated and recovered by a reaction with nitric acid and the weight of the conjugate was measured using an ICP-MS. The results are shown in FIG. 3.

<Example 2> Case Using Silica Nanoparticle as Analysis Platform Containing First Antibody

(30) Gadolinium-doped silica nanoparticles, yttrium-doped silica nanoparticles, and europium-doped silica nanoparticles were synthesized as an analysis platform containing the first antibody, respectively.

(31) Human anti-p24 monoclonal antibody was attached to each of the synthesized silica nanoparticles as a first antibody and mixed, and thereby an analysis platform containing silica nanoparticles was prepared.

(32) Iron nanoparticles were prepared as magnetic nanoparticles and by attaching human anti-p24 monoclonal antibody thereto as a second antibody.

(33) Silica nanoparticles, in which Gadolinium-doped silica nanoparticles, yttrium-doped silica nanoparticles, and europium-doped silica nanoparticles were mixed, were reacted with a sample containing target materials. After removing the unreacted materials, the conjugate bound to the second antibody was separated and recovered by a reaction with nitric acid, and the weight of the resultant was measured by ICP-MS. The results are shown in FIG. 4.

(34) It was confirmed that a plurality of targets can be quantitatively analyzed when a method for simultaneous analysis of a plurality of targets using a plurality of metal nano-tags according to the present invention.

ADVANTAGEOUS EFFECTS OF INVENTION

(35) The analysis method for a target material using metal nano-tags according to the present invention fuses a nanotechnology to a conventional biological immune response, and the method thereby makes it possible to accurately detect even a trace amount of virus without the burden of inspection cost, in the business of verification/diagnosis of blood preparations, viruses, and other biomedicines as well as in the blood management business which deals with a large amount of blood samples.