WO2015122391A1

WO2015122391A1 - Quantum dot fluorescence enhanced immunoassay

Info

Publication number: WO2015122391A1
Application number: PCT/JP2015/053572
Authority: WO
Inventors: 龍洙朴; 在郁李; 在範李
Original assignee: 国立大学法人静岡大学; 釜山大学校
Priority date: 2014-02-13
Filing date: 2015-02-10
Publication date: 2015-08-20
Also published as: JPWO2015122391A1; JP6593840B2

Abstract

Provided is a method for detecting a target substance in a sample, including: an incubation step in which a first probe and a second probe are added to the sample and to a negative contrast that does not include the target substance and incubation is performed, said first probe being bonded to metal nanoparticles fixed in a carbon nanotube and said second probe being bonded to quantum dots; a fluorescent light measurement step in which the fluorescent light intensity of the sample and the quantum dots in the negative contrast, after the incubation step, is measured; and a determination step in which a determination is made that the target substance is present in the sample if the fluorescent light intensity of the quantum dots in the sample is greater than the fluorescent light intensity of the quantum dots in the negative contrast. The first and second probes bond to the target substance but do not bond to each other. The metal nanoparticles and the quantum dots move closer as a result of the bonding of the first and second probes to the target substance, and the fluorescent light intensity of the quantum dots increases.

Description

Quantum dot fluorescence enhanced immunoassay

The present invention relates to a quantum dot fluorescence enhanced immunoassay.

Recently, many studies on the detection and diagnosis of infectious diseases using nanomaterials have been conducted. In particular, research examples for detecting or diagnosing target cells using the optical properties, electrical conductivity, fluorescence properties, and magnetic properties of nanoparticles have been reported. For example, a method of detecting an antigen by modifying an antibody to gold nanoparticles and changing the degree of aggregation of the gold nanoparticles according to the amount of the antigen, and the accompanying change in optical properties, and a magnetic nanoparticle modified with an antibody and gold Magnetophoresis using nanoparticles, the detection method of tuberculosis by the change in the optical properties of gold nanoparticles accompanying this, the DNA complementary to the disease is modified on the surface of the nanoparticles to induce hybridization with the target DNA, electrons A method for detecting or diagnosing a disease using nanoparticles has been reported, such as a method for diagnosing a disease caused by a change in a physical signal. For example, in Non-Patent Document 1, detection of methyl parathion is enhanced using a device in which carbon nanotubes are supported on a glass-like carbon electrode and methyl parathionase is immobilized on the carbon nanotubes via CdTe quantum dots. It is reported that.

On the other hand, the present inventors have reported the technique which coats a metal dot with a quantum dot until now. Specifically, in Non-Patent Document 2, imaging of colon cancer is performed by binding fluorescent magnetic nanoparticles having a surface of iron oxide Fe ₃ O ₄ coated with CdTe quantum dots to an antibody using electrostatic interaction. Have reported to do. Non-Patent Document 3 reports that the fluorescence of quantum dots is enhanced when the surface of a film having silver nanoneedles is coated with CdSe / ZnS quantum dots. Furthermore, Non-Patent Document 4 reports that an anti-Neospora antibody can be detected using a nanocomposite in which the surface of a gold nanoparticle having a protrusion is coated with a CdTe quantum dot on which an antibody is immobilized.

If the target molecule in the sample can be detected or quantified by more various methods, there are possibilities that advantages such as improved detection sensitivity, improved detection accuracy, and measurement that has been impossible in the past can be obtained. Accordingly, an object of the present invention is to provide a new method and kit for detecting or quantifying a target substance in a sample.

The present invention is a method for detecting a target substance in a sample, comprising an incubation step of adding a first probe and a second probe to each of a sample and a negative control not containing the target substance and incubating. , The first probe is bound to the metal nanoparticles immobilized on the carbon nanotubes, and the second probe is bound to the quantum dots, the step, the sample after the incubation step and the quantum dots in the negative control A fluorescence measurement step for measuring the fluorescence intensity, and a determination step for determining that the target substance is present in the sample when the fluorescence intensity of the quantum dot in the sample is stronger than the fluorescence intensity of the quantum dot in the negative control, and The first probe and the second probe bind to the target substance but do not bind to each other, and the first probe and the second probe By binding to the target substance, the metal nanoparticles and the quantum dots are close, thereby providing a method of fluorescence intensity of the quantum dots is enhanced.

According to the present invention, a new method for detecting a target substance in a sample can be provided. This method utilizes the phenomenon that the fluorescence intensity of the quantum dot increases when the metal nanoparticles and the quantum dot come close to each other. By combining the target substance in the sample with the metal nanoparticles to which the first probe is immobilized and the quantum dots to which the second probe is immobilized, the metal nanoparticles and the quantum dots are close to each other, The fluorescence intensity is enhanced. Based on this increase in fluorescence intensity, the target substance in the sample can be detected. In addition, according to the method of the present invention, a target substance in a sample can be detected without performing a washing step, which is a necessary step in the conventional ELISA method. For this reason, a target substance can be detected simply. In addition, it is possible to prevent a decrease in detection sensitivity of the target substance due to the washing process.

The present invention is also a method for quantifying a target substance in a sample, wherein a first probe and a second probe are added to each of a sample and a plurality of standard samples containing a target substance at a known concentration and incubated. An incubation step, wherein the first probe is bound to the metal nanoparticles immobilized on the carbon nanotubes, and the second probe is bound to the quantum dot, the sample after the incubation step and a plurality of samples Quantify the target substance in the sample by comparing the fluorescence intensity of the quantum dot in the sample with the fluorescence measurement process that measures the fluorescence intensity of the quantum dot in the standard sample, and the fluorescence intensity of the quantum dot in multiple standard samples The first probe and the second probe bind to the target substance but do not bind to each other, and the first probe and the second probe By lobe binds to the target substance, near the metal nanoparticles and the quantum dots, thereby providing a method of fluorescence intensity of the quantum dots is enhanced.

According to the present invention, a new method for quantifying a target substance in a sample can be provided. Further, according to the method of the present invention, the target substance in the sample can be quantified without requiring a washing step, and therefore the target substance can be quantified easily.

The present invention is also a kit for detecting or quantifying a target substance in a sample, which includes a first probe and a second probe, and the first probe is bound to metal nanoparticles fixed to carbon nanotubes. The second probe is coupled to the quantum dot, the first probe and the second probe bind to the target substance but not to each other, and the first probe and the second probe bind to the target substance. This provides a kit in which the metal nanoparticles and quantum dots are in close proximity, thereby enhancing the fluorescence intensity of the quantum dots.

According to the kit of the present invention, a target substance can be easily detected based on a new principle.

It is preferable that the metal nanoparticles are confetti sugar metal nanoparticles.

By using the gold-peeled metal nanoparticles, the detection sensitivity of the target substance can be increased, and the target substance can be quantified more accurately.

The metal nanoparticles are preferably gold nanoparticles. Gold nanoparticles can efficiently enhance the fluorescence intensity of quantum dots.

The quantum dots may emit fluorescence in the visible light region. Thereby, the enhancement of the fluorescence intensity of the quantum dots can be confirmed with the naked eye, and the target substance can be easily detected or quantified.

The first probe and the second probe are preferably antigens, antibodies, lectins, sugars, receptors, ligands, aptamers or nucleic acids.

If the first probe and the second probe are such, a target substance that can bind to an antigen, antibody, lectin, sugar, receptor, ligand, aptamer or nucleic acid can be detected or quantified.

According to the present invention, it is possible to provide a new method and kit for detecting or quantifying a target substance in a sample that does not require a washing step.

In addition, according to the method and kit of the present invention, the metal nanoparticle having the first probe immobilized thereon is immobilized on the carbon nanotube, and the metal nanoparticle has the second probe via the target substance. By combining with the fixed quantum dots, a composite is formed that is three-dimensionally arranged around the carbon nanotubes. By forming the composite, the metal nanoparticles and the quantum dots can be brought closer to each other, and the fluorescence intensity of the quantum dots is further enhanced. In addition, the fluorescence intensity of the quantum dots is further enhanced by bringing the plurality of metal nanoparticles and quantum dots close to each other on the surface of the carbon nanotube. That is, the difference in fluorescence intensity caused by the difference in the presence or absence of the target substance becomes more prominent, and the target substance can be detected more easily and with higher sensitivity than conventional detection methods.

It is a schematic diagram which shows one Embodiment of the production method of the 1st probe fixed carbon nanotube. It is a mimetic diagram explaining one embodiment of a method of detecting a target substance in a sample. 2 is an ultraviolet / visible light spectrum of AuCNT and carbon nanotube obtained in Example 1. 2 is an X-ray diffraction pattern of AuCNT and carbon nanotubes obtained in Example 1. FIG. It is a photograph which shows the transmission electron microscope image of AuCNT (FIG. 5B and FIG. 5C) and carbon nanotube (FIG. 5A) which were obtained in Example 1. FIG. It is an infrared absorption spectrum of AuCNT processed with cysteamine. It is a graph which shows the result of the ELISA analysis of anti- HA antibody fixed AuCNT and anti-NA antibody fixed AuCNT. It is the photograph which shows the confocal laser microscope image (FIG. 8A) and differential interference microscope image (FIG. 8B) of the composite_body | complex obtained in Example 1, FIG. 8C is the photograph which accumulated these images. 2 is a graph showing fluorescence intensity of a complex using anti-HA antibody-immobilized AuCNT and anti-HA antibody-immobilized CdTe obtained in Example 1. 6 is a graph showing the fluorescence intensity of a complex using anti-NA antibody-immobilized AuCNT and anti-HA antibody-immobilized CdTe obtained in Example 2.

(principle)
A quantum dot is a semiconductor crystal of several tens of nm or less, and emits fluorescence when irradiated with excitation light. The detection method and quantification method of the present invention utilize the phenomenon that the fluorescence intensity of a quantum dot increases when the metal nanoparticles and the quantum dot come close to each other.

In the method for detecting a target substance in a sample according to the present embodiment, the first probe and the second probe immobilized on the quantum dot are added to each of the sample and the negative control not containing the target substance and incubated. The incubation step, the fluorescence measurement step that measures the fluorescence intensity of the quantum dots in the sample and the negative control after the incubation step, and the fluorescence intensity of the quantum dots in the sample is compared with the fluorescence intensity of the quantum dots in the negative control. And a determination step of determining that the target substance is present in the sample when it is strong.

(Production method of first probe-immobilized carbon nanotube)
FIG. 1 is a schematic view showing an embodiment of a method for immobilizing metal nanoparticles on carbon nanotubes and further immobilizing a first probe on the metal nanoparticles. It is a mimetic diagram explaining one embodiment of a method of detecting a target substance in a sample. In the present embodiment, after the carbon nanotubes 10 are treated with the metal ions 20 to obtain the metal ion-treated carbon nanotubes 100, the metal nanoparticles 30 are formed on the surfaces of the carbon nanotubes 10 by performing a treatment with a reducing agent. Then, the carbon nanotube 200 on which the metal nanoparticles 30 are immobilized is obtained. Further, the first probe 40 is immobilized on the surface of the metal nanoparticle 30 to obtain the first probe-immobilized carbon nanotube 300. Further, the second probe 61 is immobilized on the surface of the quantum dot 60 by the same operation.

(Method of detecting target substance in sample)
FIG. 2 is a schematic diagram for explaining an embodiment of a method for detecting a target substance in a sample. By adding the quantum dots 60 on which the first probe-immobilized carbon nanotubes 300 and the second probes 61 obtained by the above method are immobilized to a sample containing the target substance 50, the first probe 40, the target substance A complex 400 in which 50 and the second probe 61 are combined in this order is formed. By forming the composite 400, the metal nanoparticles 30 bonded to the first probe 40 and the quantum dots 60 bonded to the second probe 61 are close to each other, and the fluorescence intensity of the quantum dots 60 is enhanced. . On the other hand, the presence of the target substance 50 in the sample can be detected more easily by comparing with the fluorescence intensity of the quantum dot 60 when a negative control not containing the target substance 50 is used instead of the sample.

(Target substances and probes)
In the method of the present embodiment, one of a pair of substances that specifically bind to each other, such as an antigen-antibody, a lectin-sugar, a receptor-ligand, an aptamer-aptamer target substance, and a nucleic acid-nucleic acid, is used as a target substance. The other can be used as a probe. More specifically, proteins, peptides, DNA, RNA, chemical substances, hormones, viruses, sugars and the like can be used as target substances or probes.

Here, the first probe and the second probe are selected to bind to the target substance but not to each other. When the first probe and the second probe are directly bonded, the metal nanoparticles and the quantum dots are brought close to each other regardless of the presence or absence of the target substance, which causes erroneous detection and determination. For example, when the target substance is an antibody that specifically binds to a certain antigen, examples of the first probe and the second probe include an antigen to which the target substance (antibody) binds and a target substance (antibody). Secondary antibodies that bind can be used in combination. Any of these may be the first probe or the second probe. For example, when the target substance is an antigen, two types of antibodies that bind to different epitopes on the target substance (antigen) may be used as the first probe and the second probe. In addition, for example, when the target substance is an antigen and a plurality of antigens are present in the vicinity, antibodies that bind to the same epitope on the target substance (antigen) are used as the first probe and the second probe. May be. Here, the case where a plurality of antigens are present in close proximity includes, for example, the case where the antigen forms a multimer, and the case where the target substance is an antigen present on the surface of a virus, microorganism, cell or the like. It is done. That is, the combination of the first probe and the second probe is not limited as long as the target substance, the first probe, and the second probe can be combined to allow the metal nanoparticles and the quantum dots to approach each other.

The target substance to be detected may be present in a liquid, or may be present in a sample such as a solid, powder, fluid, or gas. The method for detecting a target substance in a sample according to this embodiment is preferably performed in a liquid. For this reason, when the sample is other than a liquid, it is preferable to dissolve or suspend the sample in an appropriate buffer or the like to obtain a liquid.

(carbon nanotube)
The first probe of the present invention is immobilized on the surface of metal nanoparticles immobilized on carbon nanotubes. As the carbon nanotube used in the present invention, a single-walled carbon nanotube, a double-walled carbon nanotube, a multi-walled carbon nanotube, or the like can be used. By immobilizing the metal nanoparticles on the carbon nanotube, fluorescence is further enhanced by the effect of π electrons existing in the benzene ring constituting the carbon nanotube.

(Metal nanoparticles)
Examples of the metal nanoparticles include metal particles having a plasmon phenomenon such as gold or silver having a nano-order particle size. Especially, since the effect which enhances the fluorescence of a quantum dot is large, it is preferable that a metal nanoparticle is a gold particle. The plasmon phenomenon means a phenomenon in which free electrons in a metal collectively vibrate and behave as pseudo particles.

The method for preparing the metal nanoparticles is not particularly limited. For example, gold nanoparticles can be prepared by reducing chloroauric acid (HAuCl ₄ ) with citric acid and tannic acid, and reducing chloroauric acid (HAuCl ₄ ) with gallic acid and isoflavone. It can also be prepared. Silver nanoparticles can be prepared by reducing an aqueous silver nitrate solution with citric acid or the like.

(Kinpira sugar-like metal nanoparticles)
The gold-peeled metal nanoparticle is a metal nanoparticle having a gold-peel-like unevenness on the surface and is also referred to as a urchin-like metal nanoparticle. Whether or not the surface has irregularities can be confirmed, for example, by observing with a transmission electron microscope (TEM). Ordinary metal nanoparticles (metal nanoparticles with a smooth surface) are almost spherical, whereas gold-peeled metal nanoparticles are shaped like gold-peel sugar or sea gall. For example, after adding 250 μL of 20 mM chloroauric acid solution to 10 mL of 10 mM HEPES buffer (pH 7.4), the gold-peeled metal nanoparticles are allowed to stand still for 30 minutes at room temperature until the color of the solution changes from yellow to cloudy blue. Can be prepared.

(Quantum dot)
A quantum dot is a nanocrystal having a quantum well structure with a diameter of about 2 to 10 nm. Quantum dots are known to have only a core structure and a core / shell structure. Known examples of the former include Cd-based quantum dots such as CdS, CdSe, CdTe, and CdSeTe; Pb-based quantum dots such as PbS and PbSe; and Zn-based quantum dots such as ZnSe and ZnTe. As examples of the latter, quantum dots such as CdSe / ZnS, CdSe / CdS / ZnS, CdSe / ZnSe / ZnS, and GaAs / AlGaAs are known. In the present embodiment, any of these quantum dots can be used.

Quantum dot fluorescence wavelength depends on quantum dot particle size. For example, in CdSe quantum dots, fluorescence from blue-green to red (wavelength: 500 to 650 nm) can be generated by changing the particle size from 3 to 5 nm. In general, the particle size of the quantum dots can be controlled by the reaction time of the synthesis reaction, the temperature of the thermal decomposition reaction of the organometallic compound used for the synthesis, and the like.

Also, the fluorescence wavelength of the quantum dot depends on the type of semiconductor of the quantum dot material. Quantum dots that emit fluorescence from visible to near-infrared (wavelength 400 to 2000 nm) can be synthesized using semiconductors such as ZnSe, CdS, CdSe, CdSeTe, PbS, and PbSe.

There are two main types of quantum dot synthesis methods: top-down and bottom-up methods. In the top-down method, quantum dots are synthesized on a semiconductor substrate using electron beam lithography, molecular beam epitaxy, or the like. In the bottom-up method, chemical synthesis is performed in the liquid phase. In addition, there are two types of chemical synthesis in the liquid phase, mainly synthesized in an aqueous solution and synthesized in an organic solvent.

In the synthesis in an aqueous solution, for example, CdTe quantum dots can be synthesized by reacting sodium hydride or the like with an aqueous solution of a cadmium salt using a thiol compound as a protective agent.

In the synthesis in an organic solvent, for example, trioctylphosphine (TOP) or trioctylphosphine oxide (TOPO), which is a coordinating organic compound, is used as a solvent, and TOP complexes or organic compounds of dimethylcadmium and S, Se, Te. A metal compound can be thermally decomposed at about 300 ° C. to synthesize quantum dots.

In this embodiment, quantum dots synthesized by any of these methods can be used.

(Method of fixing probes to metal nanoparticles and quantum dots)
The method for immobilizing the probe to the metal nanoparticles and quantum dots is not particularly limited. For example, a functional group such as an amino group, a carboxy group, or a thiol group is introduced on the surface of the metal nanoparticles or quantum dots, and Using chemical crosslinking agents such as-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) The probe can be fixed. Examples of the method for introducing a functional group on the surface of metal nanoparticles or quantum dots include a method of reacting metal nanoparticles or quantum dots with an amphiphilic thiol compound such as 8-mercaptooctanoic acid or cysteamine. It is done. When introducing a functional group on the surface of a metal nanoparticle or quantum dot, a carboxy group can be introduced when 8-mercaptooctanoic acid is used, and an amino group can be introduced when cysteamine is used. Based on the type of probe used in the present invention, a functional group such as a carboxy group, an amino group, or a thiol group can be appropriately selected.

(Incubation process)
In the incubation step, the quantum dots 60 on which the first probe-immobilized carbon nanotubes 300 and the second probes 61 are immobilized are added to each of the sample and the negative control not containing the target substance, and incubated. By this step, when the target substance is present in the sample, the target substance, the first probe, and the second probe are bound to bring the metal nanoparticles and the quantum dots close to each other. What is necessary is just to set the quantity of a metal nanoparticle and a quantum dot suitably. The incubation temperature can be appropriately selected depending on the target substance and the probe. The incubation time may be set to a time required for the binding of the target substance, the first probe, and the second probe to reach equilibrium, for example, 1 hour.

(Fluorescence measurement process)
In the fluorescence measurement step, the fluorescence intensity of the quantum dot in the sample after the incubation step and the negative control is measured. For the measurement, a general fluorescence measuring instrument such as a spectrofluorometer can be used. More specifically, the sample after the incubation step and the negative control may be irradiated with excitation light of quantum dots and the generated fluorescence intensity may be measured.

For example, when a quantum dot that emits fluorescence in the visible light region is used, if there is an excitation light irradiation device, it is possible to detect fluorescence by observing with the naked eye without using a fluorescence measurement device. is there. As a result, it becomes easy to detect the presence of the target substance on the spot at a site where the target substance needs to be detected.

(Judgment process)
In the determination step, it is determined whether or not the target substance is present in the sample based on the fluorescence intensity measured in the fluorescence measurement step. Specifically, when the fluorescence intensity of the quantum dots in the sample is stronger than the fluorescence intensity of the quantum dots in the negative control, it is determined that the target substance is present in the sample. As described above, when a quantum dot that emits fluorescence in the visible light region is used, it is possible to detect fluorescence by observing with the naked eye without using a fluorescence measuring device. The presence or absence of the target substance can be determined based on the enhancement of the fluorescence intensity with the naked eye.

(Method of quantifying the target substance in the sample)
The method for quantifying the target substance in the sample is basically the same as the method for detecting the target substance 50 in the sample, except that a plurality of standard samples including the target substance 50 at a known concentration are used together with the sample. Specifically, the quantum dots 60 on which the first probe-immobilized carbon nanotubes 300 and the second probes 61 are immobilized are added to each of a plurality of standard samples including the sample and the target substance 50 having a known concentration, The same incubation step and fluorescence measurement step are performed. Subsequently, the quantitative process described below is performed.

(Quantitative process)
In the quantification step, the abundance of the target substance in the sample is determined by comparing the fluorescence intensity of the quantum dots in the sample with the fluorescence intensity of the quantum dots in the plurality of standard samples. For example, by creating a calibration curve based on the fluorescence intensity of quantum dots in multiple standard samples, and applying the fluorescence intensity of quantum dots in the sample to this calibration curve, the concentration of the target substance in the sample is determined. Can do.

(kit)
In one embodiment, a kit for detecting or quantifying a target substance in a sample includes a first probe-immobilized carbon nanotube 300 and a quantum dot 60 on which a second probe 61 is immobilized. The first probe-immobilized carbon nanotube 300 and the quantum dot 60 on which the second probe 61 is immobilized may be supplied in a solution or suspension state, or supplied in a dry state and used. Sometimes it may be dissolved or suspended in a buffer. By performing the incubation step, the fluorescence detection step, the determination step, and the quantification step using the quantum dots 60 on which the first probe-immobilized carbon nanotube 300 and the second probe 61 are immobilized, in the sample Target substances can be detected or quantified.

In the kit for detecting or quantifying a target substance in a sample according to another embodiment, the quantum dots 60 on which the first probe-immobilized carbon nanotube 300 and the second probe 61 are immobilized are, for example, filter paper. Or the like soaked in a medium such as. The medium may be appropriately adjusted to a shape and size suitable for use, and may be, for example, a strip shape. When the kit is used, a negative control that does not contain the sample and the target substance is dropped onto a medium in which the first probe-immobilized carbon nanotube 300 and the second probe 61 are immobilized. Thereby, when the target substance 50 exists in a sample, a target substance, a 1st probe, and a 2nd probe couple | bond together in a medium, and a metal nanoparticle and a quantum dot adjoin. In the fluorescence detection step, fluorescence is generated from the quantum dots by irradiating the medium with excitation light. The kit of this embodiment is particularly suitable for simple detection in the field that requires rapid detection of a target substance.

Hereinafter, the present invention will be described using examples, but the present invention is not limited to the examples.

In the examples, as an example of the method of the present invention, a method for detecting influenza virus as a target substance using carbon nanotubes (AuCNT) surface-treated with gold nanoparticles and CdTe fluorescent quantum dots (CdTe) will be described.

(Production of AuCNT)
HAuCl ₄ .3H ₂ O (0.01 mmol, manufactured by Sigma-Aldrich) and acid-treated multi-walled carbon nanotubes (2 mg, manufactured by Sigma-Aldrich) were placed in distilled water (30 mL) and dispersed by sonication for 30 minutes. A CNT solution was obtained. The obtained CNT solution (600 μL) was vigorously stirred for 1 hour in a mixed solution (10 mL) of gallic acid (0.01 M, manufactured by Sigma-Aldrich) and isoflavone (10 mg, extracted directly from Korean beans). did. After stirring, the mixture was centrifuged at 13,000 rpm for 10 minutes, and the supernatant was removed to obtain powdered AuCNT.

Example 1
1. Preparation of anti-HA antibody-immobilized AuCNT The obtained AuCNT (1 mg) was placed in distilled water (10 mL) and dispersed by sonication for 5 minutes to obtain an AuCNT solution. In order to introduce amino groups on the surface of the gold nanoparticles, cysteamine (0.01 M, 1 mL) is added to the AuCNT solution, stirred for 30 minutes, then centrifuged at 13,000 rpm for 10 minutes, and the supernatant is removed. An amine-treated AuCNT was obtained. On the other hand, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC: 100 μL, 10 mM, manufactured by Sigma-Aldrich) and N-hydroxysuccinimide (NHS: 100 μL, 10 mM, manufactured by Sigma-Aldrich) A hemagglutinin (HA) antibody (1 μL, Ab66189, manufactured by Abcam) was added and reacted for 30 minutes to obtain an antibody solution. The obtained antibody solution was mixed with amine-treated AuCNT (1 mg / mL, 30 μL) and reacted for 3 hours.

2. Preparation of anti-HA antibody-immobilized CdTe An anti-HA antibody (Ab66189, manufactured by Abcam) was immobilized on the surface of CdTe by the same operation as the preparation of anti-HA antibody-immobilized AuCNT.

3. Detection of influenza virus by antigen-antibody reaction After mixing anti-HA antibody-immobilized AuCNT and anti-HA antibody-immobilized CdTe, it was added to a 96-well plate, and influenza virus (A / Beijing / 262/95, H1N1, type Sino Biological Inc. The reaction was carried out for 1 hour. The fluorescence intensity was measured at an excitation wavelength of 380 nm and an emission wavelength of 518 nm.

(Example 2)
1. Preparation of anti-NA antibody-immobilized AuCNT The obtained AuCNT (1 mg) was placed in distilled water (10 mL) and dispersed by sonication for 5 minutes to obtain an AuCNT solution. In order to introduce amino groups on the surface of the gold nanoparticles, cysteamine (0.01 M, 1 mL) is added to the AuCNT solution, stirred for 30 minutes, then centrifuged at 13,000 rpm for 10 minutes, and the supernatant is removed. An amine-treated AuCNT was obtained. On the other hand, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC: 100 μL, 10 mM, manufactured by Sigma-Aldrich) and N-hydroxysuccinimide (NHS: 100 μL, 10 mM, manufactured by Sigma-Aldrich) A neuraminidase (NA) antibody (1 μL, A / New Caledonia / 20/1999, H1N1 type, manufactured by Cosmo bio) was added and reacted for 30 minutes to obtain an antibody solution. The obtained antibody solution was mixed with amine-treated AuCNT (1 mg / mL, 30 μL) and reacted for 3 hours.

2. Preparation of anti-HA antibody-immobilized CdTe An anti-HA antibody (Ab66189, manufactured by Abcam) was immobilized on the surface of CdTe by the same operation as the preparation of anti-NA antibody-immobilized AuCNT.

3. Detection of influenza virus by antigen-antibody reaction After mixing anti-NA antibody-immobilized AuCNT and anti-HA antibody-immobilized CdTe, it was added to a 96-well plate, and influenza virus (A / New Caledonia / 20 / 99IvR116, H1N1 type, Sino Biological) The reaction was carried out for 1 hour after the addition of Inc.). The fluorescence intensity was measured at an excitation wavelength of 380 nm and an emission wavelength of 518 nm.

(Ultraviolet-visible spectrum analysis)
FIG. 3 shows the result of measuring the absorbance of AuCNT using an ultraviolet-visible light spectrophotometer (infinite F500, manufactured by TECAN). In FIG. 3, the horizontal axis indicates the wavelength (nm) and the vertical axis indicates the absorbance. As shown in FIG. 3, in the AuCNT obtained in Example 1, an absorption peak due to surface plasmon resonance of gold nanoparticles was observed at a wavelength of about 550 nm. Further, when the absorbance was similarly measured using carbon nanotubes (CNT), no absorption peak at a wavelength of about 550 nm was observed.

(Powder X-ray diffraction pattern)
The result of having measured the X-ray-diffraction pattern of AuCNT using the powder X-ray-diffraction apparatus (RINT ULTIMA, Rigaku company make) is shown in FIG. In FIG. 4, the horizontal axis represents 2θ, and the vertical axis represents intensity. As shown in FIG. 4, in the carbon nanotube (CNT), a pattern of only the (002) plane was observed, but in the AuCNT obtained in Example 1, various diffraction patterns due to gold nanoparticles were observed. In particular, since the crystallinity of carbon is lower than that of gold, it was confirmed that the intensity of the diffraction pattern was weak.

(Observation with transmission electron microscope)
The results of observation of carbon nanotubes (CNT) and AuCNT using a transmission electron microscope (JEM-2100F, manufactured by JEOL) are shown in FIGS. 5A, 5B, and 5C. In the case of carbon nanotubes, as shown in FIG. 5A, an image showing a transparent tube structure having a diameter of 180 to 200 nm and a length of several micrometers was obtained. On the other hand, in the case of AuCNT, as shown in FIGS. 5B and 5C, an image in which black particles having a diameter of 10 to 100 nm are fixed to a transparent tube was obtained. According to an image at a higher magnification, the black particles shown in FIG. 5B are aggregates of a plurality of gold nanoparticles, and the particle diameter of each gold nanoparticle is 10 to 20 nm. In the AuCNT shown in FIG. 5C, many smaller gold nanoparticles were immobilized on the carbon nanotubes than in the AuCNT shown in FIG. 5B.

(Infrared absorption spectrum analysis)
The results of measuring the infrared absorption spectrum of the amine-treated AuCNT obtained in Example 1 are shown in FIG. In FIG. 6, the horizontal axis indicates the absorption rate (%), and the vertical axis indicates the wave number (cm ⁻¹ ). As shown in FIG. 6, an infrared absorption peak indicating the stretching vibration energy of the carbon-carbon double bond of the benzene ring (C ₆ H ₆ ) constituting the carbon nanotube was observed in the vicinity of the wave number of 1450 to 1580 cm ⁻¹ . . In addition, an infrared absorption peak indicating NH vibrational energy was observed in the vicinity of a wave number of 3400 to 3500 cm ⁻¹ . In Example 1, the thiol group of cysteamine is bonded to the surface of the gold nanoparticle by the reaction between the carbon nanotube on which the gold nanoparticle is immobilized and cysteamine, and the amino group is introduced into the carbon nanotube via the gold nanoparticle. The

(ELISA analysis)
ELISA analysis was performed using the anti-HA antibody-immobilized AuCNT obtained in Example 1 and AuCNT to which the anti-HA antibody was not immobilized, respectively. The results are shown in FIG. 7A. The absorbance of the anti-HA antibody-immobilized AuCNT (HA Ab / AuCNT) was about 4 times that of AuCNT not immobilized with the anti-HA antibody, confirming the antigen specificity of the anti-HA antibody. Absorbance could not be detected when no antigen was added. In addition, ELISA analysis was similarly performed using the anti-NA antibody-immobilized AuCNT obtained in Example 2. The result is shown in FIG. 7B. In the case of anti-NA antibody-immobilized AuCNT (NA Ab / AuCNT), the antigen specificity of the anti-NA antibody could be confirmed.

In addition, an influenza virus (A / Beijing / 262/95, H1N1 type, manufactured by Sino Biological Inc) was added as a target substance to the anti-HA antibody-immobilized AuCNT and anti-HA antibody-immobilized CdTe obtained in Example 1. Then, the anti-HA antibody-immobilized AuCNT was observed using a confocal laser microscope (LSM700, Carl Zeiss Microscopy GmbH) and a differential interference microscope (DIC). FIG. 8A shows an image obtained with a confocal laser microscope, FIG. 8B shows an image obtained with a differential interference microscope, and FIG. 8C shows a superposition of these images. As shown in FIG. 8C, the anti-HA antibody immobilized on AuCNT and the influenza virus antigen-antibody reaction, and the anti-HA antibody immobilized on CdTe and the influenza virus antigen-antibody reaction proceed, CdTe accumulated on the surface of AuCNT, and CdTe exhibited fluorescence.

Next, according to the above method, the final concentration (virus concentration) of influenza virus (A / Beijing / 262/95, H1N1 type, manufactured by Sino Biological Inc) is 10 ⁻³ ng / mL (1 pg / mL) to 10 ^3. The CdTe fluorescence intensity (PL intensity) of the complex obtained in Example 1 was measured by changing the concentration in the range of ng / mL (1 μg / mL). When the virus concentration is plotted on the horizontal axis and the fluorescence intensity is plotted on the vertical axis, as shown in FIG. 9, the fluorescence intensity of CdTe increases with increasing virus concentration when the influenza virus concentration is in the range of 1 ng / mL to 1 μg / mL. did. At this time, the fluorescence detection limit (lower limit) was 1 ng / mL.

Furthermore, anti-NA antibody-immobilized AuCNT and anti-HA antibody-immobilized CdTE obtained in Example 2 and influenza virus (A / New Caledonia / 20 / 99IvR116, H1N1 type, manufactured by Sino Biological Inc) as a target substance Similarly, the fluorescence concentration (PL intensity) of CdTe of the complex obtained in Example 2 was varied by changing the virus concentration in the range of 10 ⁻⁴ ng / mL (0.1 pg / mL) to 10 ng / mL. Was measured. When the virus concentration was plotted on the horizontal axis and the fluorescence intensity was plotted on the vertical axis, a calibration curve with a correlation coefficient of 0.98 or more was obtained as shown in FIG. In Example 2, since the antibody immobilized on AuCNT was different from the antibody immobilized on CdTe, the virus could be detected with higher sensitivity. At this time, the fluorescence detection limit (lower limit) was 0.1 pg / mL.

Therefore, according to the detection method of the present invention, the target substance can be detected easily and with very high sensitivity. Further, the fluorescence intensity of the quantum dots increases depending on the concentration of the target substance, and the target substance can be detected even if the concentration of the target substance is 0.1 pg / mL. Furthermore, compared with the conventional detection method using a metal film, the detection method of the present invention forms a complex centered on carbon nanotubes, thereby increasing the difference in fluorescence intensity depending on the presence or absence of the target substance. It is very easy to determine the presence or absence of a substance.

DESCRIPTION OF SYMBOLS 10 ... Carbon nanotube, 20 ... Metal ion, 30 ... Metal nanoparticle, 40 ... 1st probe, 50 ... Target substance, 60 ... Quantum dot, 61 ... 2nd probe, 100 ... Metal ion processing carbon nanotube, 200 ... Carbon nanotubes on which metal nanoparticles are immobilized, 300 ... first probe-immobilized carbon nanotubes, 400 ... composite.

Claims

A method for detecting a target substance in a sample,
An incubation step in which a first probe and a second probe are added to each of a sample and a negative control not containing a target substance and incubated, wherein the first probe is attached to metal nanoparticles immobilized on carbon nanotubes. The second probe is coupled to the quantum dot; and
A fluorescence measurement step for measuring the fluorescence intensity of the quantum dots in the sample and the negative control after the incubation step;
A determination step of determining that the target substance is present in the sample when the fluorescence intensity of the quantum dot in the sample is stronger than the fluorescence intensity of the quantum dot in the negative control;
Including
The first probe and the second probe bind to the target substance but not to each other, and the first probe and the second probe bind to the target substance, whereby the metal nanoparticle and the quantum dot In proximity, thereby increasing the fluorescence intensity of the quantum dots.
A method for quantifying a target substance in a sample, comprising:
An incubation step in which a first probe and a second probe are added to each of a sample and a plurality of standard samples containing a known concentration of a target substance, and the first probe is immobilized on the carbon nanotube. A second probe coupled to the metal nanoparticle and the second probe coupled to the quantum dot; and
A fluorescence measurement step for measuring the fluorescence intensity of the quantum dots in the sample after the incubation step and the plurality of standard samples;
A quantitative process for quantifying the target substance in the sample by comparing the fluorescence intensity of the quantum dots in the sample with the fluorescence intensity of the quantum dots in a plurality of standard samples;
Including
The first probe and the second probe bind to the target substance but not to each other, and the first probe and the second probe bind to the target substance, whereby the metal nanoparticle and the quantum dot In proximity, thereby increasing the fluorescence intensity of the quantum dots.
The method according to claim 1 or 2, wherein the metal nanoparticles are confetti sugar metal nanoparticles.
The method according to any one of claims 1 to 3, wherein the metal nanoparticles are gold nanoparticles.
The method according to any one of claims 1 to 4, wherein the quantum dots emit fluorescence in the visible light region.
The method according to any one of claims 1 to 5, wherein the first probe and the second probe are antigens, antibodies, lectins, sugars, receptors, ligands, aptamers or nucleic acids.
A kit for detecting or quantifying a target substance in a sample,
Including a first probe and a second probe;
The first probe is bound to metal nanoparticles fixed to the carbon nanotube,
The second probe is coupled to the quantum dot,
The first probe and the second probe bind to the target substance but not to each other, and the first probe and the second probe bind to the target substance, whereby the metal nanoparticle and the quantum dot , Which increases the fluorescence intensity of the quantum dots.
The kit according to claim 7, wherein the metal nanoparticles are confetti sugar metal nanoparticles.
The kit according to claim 7 or 8, wherein the metal nanoparticles are gold nanoparticles.
The kit according to any one of claims 7 to 9, wherein the quantum dots emit fluorescence in the visible light region.
The kit according to any one of claims 7 to 10, wherein the first probe and the second probe are antigens, antibodies, lectins, sugars, receptors, ligands, aptamers or nucleic acids.