JP4654414B2 - Immunochemical detection method and reagent kit for the detection - Google Patents

Description

  The present invention relates to a detection method for immunochemically detecting a test substance with high sensitivity and ease using a biotin-labeled quantum dot. Furthermore, the present invention relates to a detection kit used in the detection method.

  Conventionally, as a method for detecting a test substance using an immunochemical technique, a method using a primary antibody that specifically binds to a test substance and a labeled secondary antibody that binds to the primary antibody It has been known. In general, fluorescent substances, enzymes (for example, peroxidase), biotin, and the like are used for labeling the secondary antibody, and a coloration method or a detection method corresponding to the type of each labeling substance is employed. ing. However, in the conventional method, the binding reaction of the secondary antibody with respect to the primary antibody is essential, and there is a disadvantage that not only the measurement time is long but also the detection sensitivity is low. Against the background of such conventional techniques, development of a detection method for immunochemically detecting a test substance with high sensitivity and simplicity has been desired.

On the other hand, the technology related to quantum dots has also made remarkable progress year by year, and various types of quantum dots have been developed. For example, Patent Document 1 proposes a quantum dot that has excellent quantum efficiency and does not exhibit a blinking phenomenon. However, as an application example of a quantum dot relating to detection of a test substance by an immunochemical technique, there has been no specific information conventionally except that it is used as a labeling substance bound to the secondary antibody.
JP 2005-187314 A

  An object of the present invention is to provide a detection method for immunochemically detecting a test substance with high sensitivity and simplicity, and a detection kit used in the detection method.

  The present inventor has intensively studied to solve the above problems, and in a detection method for immunochemically detecting a test substance, after binding a biotin-labeled antibody that specifically binds to the test substance to the test substance, It was found that the test substance can be detected with high sensitivity and ease by reacting with avidin or streptavidin and further reacting with biotin-labeled quantum dots. The present invention has been completed by making further improvements based on these findings.

That is, the present invention provides the following detection methods and detection kits:
Item 1. A method for detecting a test substance by an immunochemical method,
(1) reacting a solid-phased test substance obtained by immobilizing a test substance on a solid phase and a biotin-labeled substance that specifically binds to the test substance,
(2) a step of reacting the biotin-labeled substance captured by the immobilized test substance in the step (1) with avidin or streptavidin,
(3) reacting avidin or streptavidin captured by biotin in step (2) with a biotin-labeled quantum dot; and
(4) A detection method comprising a step of measuring the fluorescence intensity of the biotin-labeled quantum dots captured by avidin or streptavidin in the step (3).
Item 2. Item 2. The detection method according to Item 1, wherein the biotin-labeled quantum dots are biotin-labeled CdSe nanoparticles.
Item 3. Item 3. The detection method according to Item 1 or 2, wherein Western blotting is used as an immunochemical method.
Item 4. A detection reagent kit for carrying out the detection method according to any one of Items 1 to 3, comprising a reagent containing avidin or streptavidin, and a reagent containing biotin-labeled quantum dots, kit.

Hereinafter, the present invention will be described in detail.
1. Hereinafter, the detection method of the present invention will be described step by step.
Process (1)
First, a solid-phased test substance obtained by immobilizing a test substance on a solid phase is reacted with a biotin-labeled substance that specifically binds to the test substance.

  Here, the test substance is a substance to be detected, and is not particularly limited as long as it is a substance that can be measured by an immunochemical method. Specific examples of the test substance include in vivo proteins, viral surface antigens, tumor markers, haptens, and antibodies.

The term “biotin-labeled substance that specifically binds to the test substance” used in step (1) means a substance that binds to the test substance immunochemically and is labeled with biotin. To do. Specific examples of the substance that immunochemically binds to the test substance include an antibody when the test substance is an antigen substance, and an antigen substance when the test substance is an antibody. When an antibody is used as a substance that immunochemically binds to a test substance, the antibody may be a fragment such as F (ab) ₂ or Fab ′, regardless of whether it is a polyclonal antibody or a monoclonal antibody. May be. Further, biotin labeling for a substance that immunochemically binds to a test substance can be performed according to a known method. As an embodiment of the present invention, a method using a biotin-labeled antibody as a “biotin-labeled substance that specifically binds to a test substance” is preferably exemplified.

  In the detection method of the present invention, the solid phase is an insoluble carrier for trapping a test substance. As the solid phase, those generally used in immunochemical methods can be used. Specific examples of the constituent material of the solid phase include glass, cellulose powder, Sephadex, Sepharose, polystyrene, polyacryl, polycarbonate, polymethacrylate, filter paper, carboxymethylcellulose, ion exchange resin, dextran, plastic film, plastic. Examples thereof include tubes, nylon, glass beads, silk, polyamine-methyl vinyl ether-maleic acid copolymers, agarose, amino acid copolymers, ethylene-maleic acid copolymers, and polybilinidene difluoride (PVDF). The shape of the solid phase is not particularly limited, and examples thereof include beads, plates, sticks, gels, column filling resins, sheets, capsules, test tubes and the like.

  There is no particular limitation on the method for immobilizing the test substance on the solid phase, and either physical bonding or chemical bonding can be used. As the immobilization method, solid phase immobilization by physical adsorption is the most common and preferred.

  Moreover, it is desirable that the solid phase on which the test substance is immobilized is subjected to a blocking treatment in order to suppress adsorption of the reagent to be added. For this blocking treatment, a method generally used in immunochemical methods can be adopted, and examples of substances used for the blocking treatment include bovine serum albumin, gelatin, skim milk, and the like.

  In this step (1), the conditions for reacting the “immobilized test substance” and the “biotin-labeled substance that specifically binds to the test substance” are not particularly limited, and the conditions for general immune reaction are as follows. Adopted. Generally, the above reaction is carried out by immobilizing an appropriate amount of a solvent containing “biotin-labeled substance that specifically binds to a test substance” at a concentration of 0.05 to 0.2% by weight, preferably 0.075 to 0.1% by weight. By adding to the "test substance". The above reaction is usually performed at a temperature of about 20 to 35 ° C., preferably about 25 to 30 ° C., for about 0.5 to 1.5 hours, preferably about 0.75 to 1 hour, with gentle stirring as necessary. , By incubating.

  In addition, as a solvent used in the said reaction, what is used by a normal immunochemical method (sandwich method) is used. Specifically, a buffer solution having a pH of about 5 to 9 such as a citrate buffer solution, a phosphate buffer solution, a tris salt buffer solution, and an acetate buffer solution can be exemplified.

  Thus, a “biotin-labeled substance that specifically binds to the test substance” is bound to the solid-phased test substance immobilized on the solid phase, and a complex (test substance-biotin label is bound on the solid phase. Material) is formed. The obtained complex on the solid phase is washed and subjected to the next step (2). As for the washing conditions, the same conditions as those used in the usual immunochemical method (sandwich method) or conditions based thereon can be adopted. Specifically, as a washing solution used for the washing, a buffer solution having a pH of about 5 to 9 (for example, a citrate buffer solution, a phosphate buffer solution, a Tris salt buffer solution, an acetate buffer solution, etc.) Examples include those obtained by adding an appropriate amount of a surfactant such as Tween20.

Process (2)
Next, the biotin-labeled substance captured on the solid phase in the step (1) is reacted with avidin or streptavidin.

  In this step (2), the conditions for reacting “biotin-labeled substance captured on the solid phase” with “avidin or streptavidin” are not particularly limited, and are employed in general immunochemical methods. The conditions for the formation reaction of the biotin-avidin (or streptavidin) complex are employed. Generally, the above reaction is carried out by adding an appropriate amount of a solvent containing “avidin or streptavidin” at a concentration of 0.1 to 1% by weight, preferably 0.3 to 0.5% by weight, “biotin-labeled substance captured on a solid phase”. It is performed by adding to. The above reaction is usually performed at a temperature of about 20 to 35 ° C., preferably about 25 to 30 ° C., for about 0.1 to 0.6 hours, preferably about 0.3 to 0.5 hours, with gentle stirring as necessary. This is done by incubating.

  The solvent used in the reaction is the same as that used in the step (1).

  Thus, “avidin or streptavidin” binds to “biotin-labeled substance captured on the solid phase” and a complex [test substance-biotin-labeled substance-avidin (or streptavidin)] on the solid phase. Is formed. The obtained complex on the solid phase is washed and subjected to the next step (3). The cleaning conditions are the same as in the case of the step (1).

Process (3)
Next, avidin or streptavidin captured by biotin in the step (2) is reacted with biotin-labeled quantum dots.

  The biotin-labeled quantum dot used in this step is a quantum dot in which biotin is bound by a chemical bond. For reference, an example of a model structure of biotin-labeled quantum dots is shown in FIG.

  The quantum dot used for the biotin-labeled quantum dot is not particularly limited as long as it emits light that can be detected by a commonly used detection method, but as an example, a quantum dot composed of a metal atom and a group 5B or 6B atom Preferably at least selected from the group consisting of Cd, Zn, Hg, Cu, Ag, Ti, V, Cr, Mn, Fe, Co, Ni, Zr, Mo, Ta, W, Ir, Eu, Sm and Mg; Quantum dots consisting of one metal atom and at least one 5B group or 6B group atom selected from the group consisting of S, Se and Te; more preferably CdSe.

  The average particle diameter of the quantum dots used for the biotin-labeled quantum dots is usually 2 to 6 nm, preferably 2.5 to 5 nm, and more preferably 2.5 to 3 nm.

  The quantum dots can be produced according to a conventionally known method (preferably, the method described in Patent Document 1). In addition, biotin-labeled quantum dots are generally produced by binding biotin after the hydrophilization of the quantum dots. In addition, the method for hydrophilizing the quantum dots and the method for binding biotin to the hydrophilized quantum dots are as described later.

  In this step (3), the conditions for reacting “avidin or streptavidin captured on the solid phase” with “biotin-labeled quantum dots” are not particularly limited, and are employed in general immunochemical methods. The biotin-avidin (or streptavidin) complex formation reaction conditions are employed. In general, the above reaction is performed by adding an appropriate amount of a reagent containing “biotin-labeled quantum dots” at a concentration of 50 to 100% by weight, preferably 95 to 100% by weight, “avidin or streptavidin captured on a solid phase. Is added to the above. The above reaction is usually performed at a temperature of about 20 to 35 ° C., preferably about 25 to 30 ° C., for about 0.1 to 0.3 hours, preferably about 0.2 to 0.25 hours, with gentle stirring as necessary. This is done by incubating.

  The solvent used in the reaction is the same as that used in the step (1).

  Thus, “biotin-labeled quantum dots” bind to “avidin or streptavidin captured on the solid phase”, and a complex [test substance-biotin-labeled substance-avidin (or streptavidin) -Biotin labeled quantum dots] are formed. The obtained complex on the solid phase is washed and subjected to the next step (4). The cleaning conditions are the same as in the case of the step (1).

  In addition, after performing this process (3), you may repeat the said process (2) and (3) again. Thus, it becomes possible to further improve the detection sensitivity of the test substance by repeating the steps (2) and (3) again. In the detection method of the present invention, the operations of steps (2) and (3) are desirably performed twice or more, preferably 2 to 4 times.

  For reference, FIG. 2 shows an example of the model structure of the complex (test substance-biotin-labeled substance-avidin-biotin-labeled quantum dot) formed on the solid phase when this step (3) is completed. .

Process (4)
Next, the fluorescence intensity of the biotin-labeled quantum dots captured by avidin or streptavidin in the step (3) is measured.

  In this step (4), the fluorescence intensity can be measured by a conventionally known method according to the emission spectrum of the quantum dots used. For example, when CdSe nanoparticles are used as the quantum dots, the fluorescence intensity in the wavelength region of 475 to 625 nm may be measured.

  Thus, a test substance can be detected qualitatively or quantitatively by measuring the fluorescence intensity of a biotin-labeled quantum dot.

Embodiments The detection method of the present invention may be carried out in any embodiment such as, for example, Western blotting or ELISA, as long as it is a method usually employed as an immunochemical method. When the test substance is a protein, the Western blotting method is preferably employed.

2. Detection reagent kit The present invention further provides a detection reagent kit for carrying out the detection method described above.

  The detection reagent kit contains a reagent containing avidin or streptavidin and a reagent containing biotin-labeled quantum dots. The reagent kit may further contain an appropriate reaction solution, dilution solution, washing solution and the like for the convenience of measurement.

<Hydrophilic treatment method of quantum dots>
Hereinafter, a suitable example is described regarding the hydrophilic treatment method of a quantum dot.

  The hydrophilization of the quantum dots is performed by coating the surface of the quantum dots using a compound having a mercapto group and / or a selenomercapto group as a hydrophilic compound.

  Here, the hydrophilic compound having a mercapto group is a compound having a hydrophilic group such as a carboxyl group, an amino group and a hydroxyl group together with a mercapto group (-SH), and imparts hydrophilicity to quantum dots. There is no particular limitation as far as possible. Specific examples of the hydrophilic compound having a mercapto group include monomercaptosuccinic acid, dimercaptosuccinic acid, mercaptopropionic acid, D, L-cysteine, cysteamine, 2-mercaptocysteamine, mercaptoacetic acid, and salts thereof (for example, Examples thereof include organic acid salts such as hydrochloride.

  The hydrophilic compound having a selenomercapto group is a compound having a selenomercapto group (-SeH) and a hydrophilic group such as a carboxyl group, an amino group, a hydroxyl group, and the like obtained in the step (1). There is no particular limitation as long as hydrophilicity can be imparted to the nanoparticles. Specific examples of such compounds include seleno-L-methionine.

  Said hydrophilic compound may be used individually by 1 type, and may be used in combination of 2 or more type.

  Among the above hydrophilic compounds, a hydrophilic compound having a mercapto group is preferred, and monomercaptosuccinic acid is more preferred. By using these hydrophilic compounds, the reaction can be easily carried out, and the resulting reaction product can be made difficult to aggregate.

  For coating the surface of the quantum dot with the hydrophilic compound, for example, the quantum dot is dispersed in a hydrophobic solvent, and the hydrophilic compound is added to and mixed with the quantum dot.

  Here, specific examples of the hydrophobic solvent used for dispersing the quantum dots include chloroform, hexane, methanol, butanol and the like.

  The quantum dots may be adjusted to a concentration of usually 30 to 40 g / l, preferably 20 to 30 g / l, and more preferably 15 to 20 g / l in the hydrophobic solvent.

  In order to add the hydrophilic compound to the hydrophobic solvent in which the quantum dots are dispersed, the hydrophilic compound is usually added in an amount of 300 to 400 parts by weight with respect to 100 parts by weight of the hydrophobic solvent in which the quantum dots are dispersed. The addition ratio is preferably 200 to 300 parts by weight, more preferably 100 to 150 parts by weight.

  The mixing of the hydrophobic solvent in which quantum dots are dispersed and the hydrophilic compound is performed at a temperature of 30 to 40 ° C., preferably 25 to 35 ° C., more preferably 20 to 30 ° C. It is carried out by stirring for about -1.0 hour, preferably about 0.2-0.5 hour, more preferably about 0.1-0.2 hour.

  Thus, by adding and mixing the hydrophobic solvent in which quantum dots are dispersed and the hydrophilic compound, the mercapto group and / or the selenomercapto group of the hydrophilic compound are bonded to the surface of the quantum dot. Water-soluble quantum dots (quantized dots subjected to hydrophilic treatment) are generated. Since the target hydrophilic quantum dot is present in the hydrophilic fraction in the mixed liquid, it can be obtained by separating and collecting the hydrophilic quantum dot from the hydrophilic fraction by a known means.

<Binding of biotin to hydrophilic quantum dots>
Hereinafter, a suitable example will be described regarding a method of binding biotin to hydrophilic quantum dots.

  Examples of the method of binding biotin to the quantum dots include a method of reacting hydrazine biotin (biotin hydrazide) with hydrophilic quantum dots. Specifically, the hydrazine biotin is hydrophilic to 1 mol of hydrazine biotin in a solvent adjusted to 0.025 to 0.1 M, preferably 0.035 to 0.075 M, more preferably 0.045 to 0.05 M. An example is a method in which quantum dots are added at 0.00005 to 0.0002 mol, preferably 0.000075 to 0.00015 mol, more preferably 0.000095 to 0.0001 mol, and both are reacted.

  In addition, the reaction between the hydrophilic quantum dots and biotin is performed in the presence of a crosslinking accelerator such as EDC (1-Ethyl-3- [3-Dimethylaminopropyl] carbodiimide Hydrochloride) in order to accelerate the dehydration condensation reaction. It is desirable. When the reaction is carried out in the presence of such a crosslinking accelerator, the addition ratio of the crosslinking agent is, for example, 0.005 to 0.02 mol, preferably 0.0075 to 0.015 mol, more preferably 1 mol of hydrazin biotin. Is a ratio of 0.010 to 0.0104 mol.

  Specific examples of the solvent used in the reaction include 0.08 to 0.1 M MES [(2-N-morpholino) ethanesulfonic acid] buffer.

  In the reaction, in order to efficiently perform the reaction between the hydrophilic quantum dots and biotin, the pH in the reaction solution is appropriately adjusted within the range of 3.8 to 5.7, preferably 4 to 5.5 during the reaction. It is desirable to keep control.

  The reaction is performed, for example, under a temperature condition of 30 to 40 ° C., preferably 25 to 35 ° C., more preferably 20 to 30 ° C., for 0.5 to 1 hour, preferably 0.2 to 0.5 hour, Preferably it is carried out for 0.1 to 0.2 hours. In addition, the reaction is desirably performed under light-shielding conditions.

  After the reaction, the synthesized biotin-labeled quantum dots can be purified by a known purification means such as centrifugal filtration.

  According to the detection method of the present invention, the primary antibody bound to the test substance can be highly amplified and detected by a biotinylated quantum dot in which a plurality of avidin or streptavidin is bound as a cross-linking agent. There is an advantage that the test substance can be detected with high sensitivity. In addition, quantum dots (especially CdSe nanoparticles) are not easily decomposed or quenched by light irradiation, so the results can be observed stably for a long time, and observation and quantification using an image analyzer can be performed accurately. Become.

  In addition, as described above, according to the detection method of the present invention, the test substance can be detected with high sensitivity. Therefore, pre-treatment such as purification of the test substance (test substance (Concentration treatment) is not necessary. Also in this respect, it can be said that the detection method of the present invention is more advantageous than conventional immunochemical methods.

  Furthermore, according to the detection method of the present invention, since the immunochemical method is carried out using only the primary antibody without using the secondary antibody, the test can be performed inexpensively, easily and in a short time. There is also an advantage that a substance can be detected.

EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited at all by these examples.
Example 1
Using the reagents shown below, the proteins (TRF1 (Telomeric Binding Factor 1), Tin2 (TRF1-interacting nuclear protein 2), and β-Actin) contained in the lysate of leukemia cell line K562 cells by the following method Detection was performed. TRF1 and Tin2 are proteins that are known to exist in trace amounts in K562 cells.
<Main reagents used>
Lysate: Composition 10 mM Tris, pH 7.8, 1 % Nonidet P-40, 0.15 M NaCl, 1 mM EDTA, and protease inhibitor cocktail
2 x Laemmly buffer : Composition 1.1 M Tris-HCl, pH 6.0, 3.3% SDS, 22% glycerol, 10% b-mercaptoethanol, 0.001% bromphenol blue
Tris-Glycine-SDS solution : Composition 3 g / L Tris, 14.4 g / L Glycine, 1 g / L SDS, remaining distilled water
Protein transfer buffer : Composition 3.03 g / L Trizma-base, 14.4 g / L Glycine, 200 ml / L Methanol, distilled water remaining
Blocking solution : Composition 100 mM phosphate-buffered saline, 0.1% Tween-20, 2-5% dry skimmed milk
Wash solution: 10xPBS-T (100 mM phosphate-buffered saline, containing 1% Tween-20)
PVDF membrane : Trade name Hybond-P PVDF transfer membranes, Amersham Bioscience catalog # 25006203
NeutrAvidin : Pierce catalog # 31000
Biotin-labeled quantum dots : produced by the method shown in Reference Example 1 described later.

<Experiment method>
Leukemia cell line K562 cells (1 × 10 ⁶ cells) were incubated in a lysis solution at 4 ° C. for 1 hour to obtain a cell lysate. Next, the obtained cell lysate was centrifuged at 14,000 × g for 10 minutes, and the supernatant was collected. The obtained supernatant was mixed with 2 × Laemmly buffer at a ratio of 1: 1 to obtain a suspension. The resulting suspension was warmed at 95 ° C for 10 minutes, applied to 4-12% SDS-polyacrylamide gel electrophoresis, run in Tris-Glycine-SDS solution at room temperature for 15 minutes at 80V, and then at 120V. Run for 2-4 hours. After gel electrophoresis, the gel was taken out and immersed in protein transfer buffer at low temperature.

  The PVDF membrane was then cut to the desired gel size and the membrane was immersed in methanol for 10 seconds. After washing with distilled water for 5 minutes, the mixture was equilibrated with protein transfer buffer for 10 minutes. In all the operations at this time, the film was not dried.

  Next, in order to perform protein transfer from the gel to the PVDF membrane, the following operation was performed. First, an electroblot cassette (XCell II Blot Module) was assembled as a sandwich from top to bottom in the following order 1) to 10): 1) negatively charged plate, 2) cold protein transfer 2 filter papers soaked in buffer, 3) Sponge pad soaked in protein transfer buffer under cold temperature, 4) 3 filter papers soaked in protein transfer buffer under cold temperature, 5) Activated PVDF membrane, 6) Gel, 7) Three filter papers soaked in protein transfer buffer under cold temperature, 8) Sponge pad soaked in protein transfer buffer under cold temperature, 9) Two filter papers soaked in protein transfer buffer under cold temperature, 10) Positively charged plate. Here, all the bubbles in the filter paper, the PVDF membrane, and the gel were removed. The protein was transferred from the gel to the PVDF membrane by electrophoresis at 4 ° C. and 35 V for 18 hours.

After the protein transfer operation from the gel to the PVDF membrane, the PVDF membrane was washed with methanol for 10 seconds, and then washed with distilled water for 5 minutes. The PVDF membrane was then incubated in the blocking solution for 1 hour at room temperature with agitation. At this time, the amount of the blocking solution was> 4 mL / cm ² (PVDF membrane area). Thereafter, each of the PVDF membranes was labeled with a biotin-labeled antibody (anti-TRF-1-rabbit, Calbiochem, 1 mg / ml; anti-Tin2-mouse, Calbiochem, 1 mg / ml; or anti-b-actin-mouse, Calbiochem; Incubated for 1 hour at room temperature. After incubation, the PVDF membrane was washed a total of 3 times with a washing solution (5 minutes per time). Then 5 μg / ml NeutrAvidin was added and incubated for 30 minutes at room temperature with agitation. Then, it was washed a total of 2 times with the washing solution (5 minutes per time). Next, 0.5 mg / ml of biotin-labeled quantum dots were added, incubated for 15 minutes at room temperature with stirring, and then washed twice with a washing solution (5 minutes per time). This NeutrAvidin addition operation and biotin-labeled quantum dot addition operation were performed twice in total. The PVDF membrane was finally washed 3 times with a washing solution (5 minutes per time), 3 mM Glutathione was added, and the mixture was incubated for 10 minutes. As a result of analyzing the PVDF membrane with several types of fluorescence detection systems (Fluor-S ^™ MultiImager, BioRad; FluorChem ^™ , AlphaInnothech), it was observed that fluorescence was emitted regardless of which antibody was used (FIG. 3). (See (a)). The use of an emission filter with a wavelength of 475-625 nm was the most suitable for both fluorescence images. It was also confirmed that the PVDF membrane used could be kept for 2 weeks in PBS at 4 ° C.

  For comparison, TRF1 and Tin2 in the lysate of leukemia cell line K562 cells were compared with the usual Western blotting method (Bakalova, R., Ohba, H., Zhelev, Z., Kubo, T., Fujii, M ., Ishikawa, M., Shinohara, Y., Baba, Y. Antisense inhibition of bcr-abl / c-abl synthesis promotes telomerase activity and upregulates tankyrase in human leukemia cells., FEBS Letters 2004, 564 (1-2), 73-84, and Ohba, H., Zhelev, Z., Bakalova, R., Ewis, A., Omori, T., Ishikawa, M., Shinohara, Y., Baba, Y. Inhibition of bcr-abl and / or c-abl gene expression by small interfering, double-stranded RNAs: cross-talk with cell proliferation factors and other oncogenes., Cancer. 2004 Sep 15; 101 (6): 1390-403. Detection was performed. As a result, although TRF1 and Tin2 could be detected under some conditions, it was confirmed that the sensitivity was significantly lower than that of the method of the present invention (see FIG. 3B).

Reference Example 1 Production of biotin-labeled quantum dots <Synthesis of quantum dots>
1. Preparation of tri-n-octylphosphine selenide (TOP-Se, Lancaster) solution
By dissolving Se powder (0.7896 g, 10 mM, Aldrich) in TOP (7.413 g, 20 mM) solution and incubating at 100 ° C. under argon atmosphere for 2 hours, the temperature of the reaction solution is lowered to room temperature. A trioctylphosphine selenide solution was prepared.

2. Preparation of Cd dispersion Cadmium acetate (1 g, 3.75 mM, Wako) was dispersed in 2 mL of Tri-n-octylphosphine (TOP) solution in a round bottom flask under an argon atmosphere at 100 ° C. for 20 minutes. After reacting with vigorous stirring, a Cd dispersion was prepared by immediately lowering the temperature to room temperature.

3. Preparation of CdSe nanocrystals (quantum dots)
Using a 100 mL flask, 4 mL of trioctylphosphine selenide solution and 4 mL of Cd dispersion were mixed and reacted strongly with stirring at room temperature (25 ° C.) for (1/6) hour. During the reaction, formation of CdSe nanocrystals was confirmed by monitoring with UV / VIS absorption spectrum and fluorescence spectrum. The reaction was stopped by adding 30 mL of chloroform. The crude solution thus obtained was slightly turbid because unreacted cadmium acetate present in chloroform was insoluble. This crude solution was centrifuged (16,000 rpm × 20 min), and the supernatant was transferred to a new flask to remove insoluble precipitates. The supernatant in which pure CdSe nanocrystals were dispersed was extremely transparent.

  About the obtained CdSe nanocrystal (quantum dot), when the fluorescence spectrum was measured by Hitachi F-4500 fluorescence spectrometer (excitation wavelength 420nm), it was confirmed that an emission spectrum is shown in the wavelength range of 475-625nm. In addition, the surface morphology of the obtained CdSe nanocrystals (quantum dots) was evaluated using a high resolution transmission electron microscope (JEOL (model JEM 3010, Japan) transmission electron microscope operating at 300 kV). It was also confirmed that it was about 2 nm. The observation with a high resolution transmission electron microscope was prepared by dispersing a sample (CdSe nanocrystal) in methanol by sonication and then dropping it onto a carbon-coated copper grid and drying it.

<Hydrophilic treatment of quantum dots>
In order to impart water solubility to the CdSe nanocrystals (quantum dots) obtained above, the reaction was performed by the following method using monomercaptosuccinic acid as a surface modifier.

The CdSe nanocrystals (quantum dots) obtained above were dispersed in chloroform and adjusted so that the absorbance at _{430 nm} (OD _{430 nm} ) was 0.5. 3 mL of monomercaptosuccinic acid (Sigma-Aldrich, dissolved in 100 mM PBS, pH 7.3 at concentration 30 mg / mL) was added to 3 mL of this solution. Shake vigorously for 5-10 minutes until the mixture is yellow. After mixing, the organic solvent layer and the aqueous layer were separated by centrifugation (80 × g for 10 min). The aqueous layer portion containing water-soluble CdSe nanocrystals (water-soluble quantum dots) was carefully collected and immediately purified by centrifugal filtration to purify the water-soluble CdSe nanocrystals. All operations for purification of water-soluble CdSe nanocrystals (water-soluble quantum dots) were performed at 4 ° C. using a Vivaspin concentrator (Vivascience, Sartorius). Specifically, first, by centrifugal filtration concentration (3,000 xg for 15 min) using Vivaspin-20 (filtered molecular weight: 3,000 MW, manufactured by Sartorius), those having a molecular weight of 3,000 or less (unreacted monomercaptosuccinol). After removing the acid, the filter was washed three times with 6 mL of 100 mM PBS (Phosphate buffered saline; pH 7.3). Collect the supernatant of the filter, and concentrate it by centrifugal filtration (3,000 xg for 10 min) using Vivaspin-20 (filtered molecular weight: 10,000 MW, manufactured by Sartorius). The solution was further concentrated with a filter having a molecular weight cut-off of 5,000, and adjusted to a target concentration (OD _{430 nm} = 0.1; about 7.6 μM) in a PBS (−) solution (pH 7.3). The composition of PBS (−) is 100 mM Na ₂ HPO ₄ × 12H ₂ O, 100 mM KH ₂ PO ₄ , 136 mM NaCl, 2 mM KCl.

  Eventually, about 90% of the hydrophilic nanocrystals (water-soluble quantum dots) pass through a filter with a molecular weight cut-off of 10,000, and 95% of the hydrophilic nanocrystals (water-soluble quantum dots) have a molecular weight cut-off of 5,000. Could be recovered above. Considering the size of the filter, this result indicates that the purified crystal size is 1.8-2.1 nm.

  In this way, hydrophilic CdSe nanocrystals (water-soluble quantum dots) having a carboxyl group as a functional group were obtained, and this was evaluated for fluorescence spectrum and surface morphology in the same manner as the above CdSe nanocrystals (quantum dots). . As a result, it was confirmed that the hydrophilic CdSe nanocrystal (water-soluble quantum dot) has the same fluorescence spectrum and surface morphology as the CdSe nanocrystal (quantum dot) before the hydrophilic treatment.

<Binding of biotin to quantum dots>
Hydrazinated biotin (D-biotin hydrazide; manufactured by Pierce) was added to anhydrous dimethyl sulfoxide (DMSO) immediately before use to prepare a 50 mM hydrazinated biotin solution. Separately, the hydrophilic CdSe nanocrystals (water-soluble quantum dots) obtained above were dissolved in 0.1 M MES buffer (pH 5.5) to prepare a 5 μM quantum dot-containing solution. Separately, 100 mg of EDC was dissolved in 0.1 M MES buffer (pH 5.5) immediately before use to prepare an EDC-containing solution.

  Next, 25 μL of a hydrazinated biotin solution and 25 μL of an EDC-containing solution were added to 950 μL of the quantum dot-containing solution and incubated at room temperature for 2 hours under light shielding. During the incubation, the pH of the reaction solution was maintained between 4 and 5.5.

  The biotin-labeled quantum dots synthesized in this way were purified by centrifugal filtration using Vivaspin filter (5,000 MW), and the MES buffer used for the reaction was also PBS (50-100 mM, pH 7.3). Replaced with

It is a figure which shows an example of the model structure of a biotin label quantum dot in the detection method of this invention in FIG. In the detection method of the present invention, an example of a model structure of a complex (test substance-biotin-labeled substance-avidin-biotin-labeled quantum dot) formed on the solid phase when this step (3) is completed is shown. FIG. In the figure, (a) shows the results of the detection of proteins (TRF1, Tin2 and β-Actin) contained in the lysate of leukemia cell line K562 cells by the method of the present invention (Example 1). (B) shows the results of detecting TRF1 and Tin2 in the lysate of leukemia cell line K562 cells by the usual Western blotting method. In the figure, in (a) and (b), the K562 cell lysate applied to lanes 1-5 is as follows: lane 1: K562 cell lysate in an amount corresponding to 10 μg of protein lane 2: protein amount K562 cell lysate in an amount equivalent to 20 μg Lane 3: K562 cell lysate in an amount equivalent to 30 μg protein Lane 4: K562 cell lysate in an amount equivalent to protein amount 40 μg Lane 5: An amount equivalent to an amount of protein 50 μg K562 cell lysate.

Claims (4)

A method for detecting a test substance by an immunochemical method,
(1) reacting a solid-phased test substance obtained by immobilizing a test substance on a solid phase with a biotin-labeled substance that specifically binds to the test substance;
(2) reacting the biotin-labeled substance captured by the immobilized test substance in the step (1) with avidin or streptavidin,
(3) a step of reacting avidin or streptavidin captured by biotin in step (2) with a biotin-labeled quantum dot; and (4) a biotin label captured by avidin or streptavidin in step (3). Including the step of measuring the fluorescence intensity of the quantum dots,
A detection method in which the steps (2) and (3) are sequentially performed as a series of steps, and this step is performed twice or more. The detection method according to claim 1, wherein the biotin-labeled quantum dots are biotin-labeled CdSe nanoparticles. The detection method according to claim 1 or 2, wherein Western blotting is used as an immunochemical method. A detection reagent kit for carrying out the detection method according to any one of claims 1 to 3,
A reagent comprising avidin or streptavidin for use in step (2) , and
A kit comprising a reagent containing a biotin-labeled quantum dot for use in the step (3) .

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