Detection kit and detection method

Abstract

This detection kit comprises a first carrier that supports a first probe bonding to an object being detected and that contains a first metal, and a second carrier that supports a second probe bonding to the object being detected and that contains a second metal different from the first metal. When energy is supplied to the first carrier and the second carrier that have come into proximity by bonding to the object being detected via the first probe and the second probe, intrinsic ions are desorbed.

Description

[Technical Field]

[0001] The present invention relates to a detection kit and a detection method. [Background technology]

[0002] Antibody-modified gold nanoparticles are widely used as probes (markers) for immunoassays such as immunoaffinity chromatography and ELISA (Enzyme-Linked Immuno Sorbent Assay). Because gold nanoparticles can be ionized without the use of matrix molecules, they are useful for detecting large molecular weight proteins that are difficult to ionize using MALDI (Matrix-Assisted Laser Desorption / Ionization) methods.

[0003] Gold nanoparticles also function as ultra-sensitive mass probes that efficiently release gold ions by MALDI-MS (Mass Spectrometry). Furthermore, even in the case of immunodetection described above, they can be detected with ultra-high sensitivity by mass spectrometry. Patent Document 1 discloses a method for detecting a target substance by fixing an immunoaffinity chromatogram membrane using antibody-modified gold nanoparticles as probes onto a substrate for MALDI-MS measurement and irradiating it with laser light to detect the desorption of gold ions or gold cluster ions. According to this detection method, even extremely small amounts of gold nanoparticles that cannot be observed with the naked eye can be detected, thus significantly improving analytical sensitivity. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2015-1453 [Overview of the project] [Problems that the invention aims to solve]

[0005] Some of the gold nanoparticles are non-specifically adsorbed onto membranes or the like. Only due to non-specific adsorption, signals exceeding 1000 times the detection limit of mass spectrometry equipment are detected. In the case of ordinary immunodetection by fluorescence or color development, non-specific adsorption can be reduced to a level that does not interfere with detection by performing blocking treatment according to established methods. However, regarding gold nanoparticles, it is considered difficult to reduce non-specific adsorption to the level required for mass spectrometry even when an optimized blocking treatment is applied. Therefore, the ultra-high sensitivity of the gold nanoparticle probe reaching 10 -18 mol / mm 2 has not yet been fully utilized.

[0006] The present invention has been made in view of the above circumstances, and an object thereof is to provide a detection kit and a detection method capable of highly sensitively detecting a detection target without being hindered by non-specific adsorption.

Means for Solving the Problems

[0007] The detection kit according to the first aspect of the present invention carries a first probe that binds to a detection target, and a first carrier containing a first metal, carries a second probe that binds to the detection target, and a second carrier containing a second metal different from the first metal, and is provided with binding to the detection target via the first probe and the second probe Before When energy is supplied to the first carrier and the second carrier In a state of being in close proximity to each other. ions are desorbed. a cluster composed of both the constituent elements of the first metal and the constituent elements of the second metal.

[0008] The first carrier is gold-silver alloy nanoparticles, The second carrier is gold-palladium alloy nanoparticles, which may also be the case.

[0009] Also, the first carrier is gold nanoparticles, The second carrier is platinum nanoparticles, which may also be the case.​

[0010] Furthermore, the first probe and the second probe are The antibody is an antibody that specifically binds to the target of detection. It would be acceptable to do so.

[0011] A detection method according to a second aspect of the present invention is: A binding step involves binding a first carrier, which carries a first probe that binds to the target to be detected and contains a first metal, and a second carrier, which carries a second probe that binds to the target to be detected and contains a second metal different from the first metal, to the target to be detected via the first probe and the second probe, respectively. A supply step of supplying energy to the first carrier and the second carrier bound to the target to be detected, Binding to the detection target via the first probe and the second probe. Before Carrier No. 1 and The second carrier When the two are in close proximity The aforementioned energy but supply When Detachment a cluster composed of both the constituent elements of the first metal and the constituent elements of the second metal. A detection step for detecting ions, Includes. [Effects of the Invention]

[0012] According to the present invention, the target object can be detected with high sensitivity without interference from non-specific adsorption. [Brief explanation of the drawing]

[0013] [Figure 1] This figure shows the m / z of desorbed ions from gold-silver alloy (AuAg) nanoparticles and gold-palladium alloy (AuPd) nanoparticles, and the predicted intensity of each desorbed ion calculated from the isotopic abundance. [Figure 2] This figure shows the absorption spectrum of gold nanoparticles. [Figure 3] This figure shows the absorption spectrum of palladium-shelled gold nanoparticles. [Figure 4] This figure shows the absorption spectrum of AuPd nanoparticles. [Figure 5] This figure shows the absorption spectrum of silver-shelled gold nanoparticles. [Figure 6] This figure shows the absorption spectrum of AuAg nanoparticles. [Figure 7] This figure shows the absorption spectrum of goat normal immunoglobulin G (IgG)-AuAg nanoparticles. [Figure 8] This figure shows the absorption spectrum of anti-goat IgG-AuPd nanoparticles. [Figure 9] This figure shows the ionization behavior of a complex of anti-goat IgG-AuPd nanoparticles and normal goat IgG-AuAg nanoparticles. [Figure 10] This figure shows the ionization behavior of AuAg nanoparticles and AuPd nanoparticles cast on a substrate. [Figure 11] This figure shows the absorption spectrum of platinum nanoparticles. [Figure 12] This figure shows the absorption spectrum of grown platinum nanoparticles. [Figure 13] This figure shows the absorption spectrum of anti-goat IgG-platinum nanoparticles. [Figure 14] This figure shows the absorption spectrum of normal goat IgG-gold nanoparticles. [Figure 15] This figure shows the signal intensity in a membrane cast with normal goat IgG. [Figure 16] This figure shows the ionization behavior of a complex of anti-goat IgG-platinum nanoparticles and normal goat IgG-gold nanoparticles. [Figure 17] This figure shows the absorption spectrum of AuPd nanoparticles. [Figure 18] This figure shows the ionization behavior of the sample according to Example 3, which does not contain prostate-specific antigen. [Figure 19] This figure shows the ionization behavior of the sample according to Example 3, which contains prostate-specific antigen. [Figure 20] This figure shows the relationship between the concentration of the PSA solution and the signal intensity of AgPd+ ions in a sample that does not contain whole blood, according to Example 3. [Figure 21]This figure shows the relationship between the concentration of the PSA solution and the signal intensity of AgPd+ ions in a sample containing whole blood according to Example 3. [Figure 22] This figure shows the ionization behavior of the sample after mixing according to Example 4. [Figure 23] This figure shows the relationship between the elapsed time after mixing the sample in Example 4 and the signal intensity. [Figure 24] This figure shows platinum nanoparticles according to Example 5 as imaged using a transmission electron microscope (TEM). [Figure 25] This figure shows the ionization behavior of platinum nanoparticles according to Example 5, which were cast onto a substrate. [Modes for carrying out the invention]

[0014] Embodiments of the present invention will be described with reference to the drawings. However, the present invention is not limited to the embodiments and drawings described below. In the embodiments described below, expressions such as “having,” “including,” or “containing” also include the meaning of “consisting of” or “composed of.”

[0015] The detection kit according to this embodiment comprises a carrier 1 (first carrier) and a carrier 2 (second carrier). Carrier 1 carries a probe 10 (first probe) that binds to the target for detection. The target for detection includes peptides, proteins, nucleic acids, glycans, and lipids. Carrier 2 carries a probe 20 (second probe) that binds to the target for detection, independently of carrier 1. Probe 20 may be the same as probe 10 or different from probe 10, as long as it binds to the target for detection. Hereinafter, probe 10 and probe 20 will be collectively referred to as "probes".

[0016] The probe is not particularly limited as long as it binds to the target of detection. Examples of binding to the target of detection include van der Waals force interactions, hydrogen bonding, coordination bonding, and alloying. Binding between the probe and the target of detection can include, for example, binding of an antigen to an antibody fragment or antibody, hybridization of nucleic acids or peptide nucleic acids, aptamer binding, receptor-ligand binding, and redox reactions. Preferably, the probe binds specifically to the target of detection. "Specific" means that the probe does not show any significant binding to substances other than the target of detection.

[0017] Antibodies and antibody fragments encompass all types of antibodies that specifically bind to the target of detection. For example, antibodies include monoclonal antibodies, polyclonal antibodies, single-chain antibodies, chimeric antibodies, and any fragments or derivatives of such antibodies that can bind to the target of detection. Fragments and derivatives include bispecific antibodies, synthetic antibodies, Fab, F(ab)2, Fv, or scFv fragments, or any chemically modified derivatives of these antibodies. Antibodies and antibody fragments can be obtained by methods known in the art. Preferably, the probe is an antibody that specifically binds to the target of detection.

[0018] Nucleic acids encompass all deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and their chemically modified derivatives. Nucleic acids can be particularly used when the target of detection is a nucleic acid. In this case, the nucleic acid is partially or completely complementary to the nucleic acid being detected or a portion thereof.

[0019] Aptamers include nucleic acid and peptide aptamers. Nucleic acid aptamers, in addition to forming base pairs with other nucleic acids, specifically bind to targets such as small molecules, proteins, cells, tissues, and microorganisms.

[0020] Peptide nucleic acids are artificially synthesized polymers having a nucleic acid-like skeleton composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. Various purine and pyrimidine bases are linked to the skeleton by methylene crosslinks and carbonyl groups. Peptide nucleic acids have biological properties similar to nucleic acids and can bind to targets for detection.

[0021] The receptor acting as a probe specifically interacts with the ligand it is detecting.

[0022] Carrier 1 contains metal 11 (the first metal). Carrier 2 contains metal 21 (the second metal) that is different from metal 11. Hereinafter, carriers 1 and 2 will be collectively referred to as "carriers," and metals 11 and 21 will be collectively referred to as "metals." The carriers are, for example, particles containing metal. The particle size is not particularly limited and may be on the order of micrometers or nanometers. The metal may be contained in the carrier not only as a single metal element but also as an alloy of metal elements. When metals 11 and 21 are alloys, metal 21 that is different from metal 11 means that metal 21 contains at least one type of metal atom that is not contained in metal 11.

[0023] Preferably, the carrier is a metal nanoparticle or an alloy nanoparticle. Several preparation methods are known for metal nanoparticles, allowing for different sizes and various surface modifications. Gold nanoparticles are particularly preferred because they are chemically stable and various methods for immobilizing bioactive substances on their surface are known. Gold nanoparticles are also preferred as carriers because of their high desorption efficiency of gold ions and gold cluster ions when irradiated with pulsed laser light. Silver nanoparticles are also suitable as carriers because their preparation methods are widely known, and they exhibit high desorption efficiency of silver ions and silver cluster ions when irradiated with pulsed laser light. Other examples of metal nanoparticles include those of platinum, palladium, iridium, copper, nickel, and manganese. Preferably, carrier 1 is a gold nanoparticle, and carrier 2 is a platinum nanoparticle.

[0024] Preferably, alloy nanoparticles are alloy nanoparticles that combine two or more metals related to the above-mentioned metal nanoparticles. For example, alloy nanoparticles include AuAg nanoparticles and AuPd nanoparticles. Alloy nanoparticles can be prepared by known methods. For example, alloy nanoparticles can be obtained by alloying by irradiating nanoparticles, which include a core containing a metal and a shell containing a different metal from the metal in the core and covering the core, with a pulsed laser. Alloy nanoparticles are not limited to binary alloys containing two metal elements, but may also be ternary alloys or quaternary alloys, etc.

[0025] Furthermore, the support may be a silicate such as silicon dioxide (silica) containing a metal ion. When the support contains a silicate, the form of the support may be microparticles or polymers. For example, the support may be formed by ionically bonding a manganese ion to the anion of the silicate.

[0026] The method for supporting the probe on the carrier is not particularly limited. For example, the probe can be supported on the carrier by adsorption or covalent bonding. If the carrier is metal nanoparticles and the probe is a protein such as an antibody (immunoglobulin), the metal nanoparticles prepared by reduction with a reducing agent such as citrate or tannic acid can be mixed with the protein under predetermined conditions. This causes the protein to bind to the carrier via electrostatic interactions, hydrogen bonds, and free thiol groups with high affinity contained in the protein.

[0027] Furthermore, the protein used as a probe can be chemically modified and immobilized on a support via covalent bonds such as thiol groups. As an efficient method for immobilizing antibodies on a support, antibody immobilization techniques using proteins that specifically bind to antibodies (protein A, protein G, protein A / G, or protein L) may be used. If the support contains gold, peptides having gold-reducing and gold-binding capabilities may be used.

[0028] When energy is supplied to adjacent carriers 1 and 2 by binding to the target via probes 10 and 20, a specific ion is desorbed. "Specific" means an ion that is desorbed only when carriers 1 and 2 are in close proximity due to binding to the target. For example, if carrier 1 is gold nanoparticles and carrier 2 is platinum nanoparticles, when energy is supplied to adjacent carriers 1 and 2, a gold-platinum ion (AuPt) is desorbed as a specific ion for adjacent carriers 1 and 2. + ) is detached. Also, when support 1 is AuAg nanoparticles and support 2 is AuPd nanoparticles, energy is supplied to the adjacent support 1 and support 2, causing the two types of nanoparticles to fuse and a ternary alloy to be formed. From this ternary alloy, silver-palladium ions (AgPd) are released as unique ions. + ) detaches.

[0029] Support 1 may be silica or protein that absorbs metal ions, and Support 2 may be metal nanoparticles. Examples of metal ions include manganese ions, copper ions, cobalt ions, and tin ions, which do not exist in large quantities in nature. When Support 1 is silica that absorbs manganese ions and Support 2 is platinum nanoparticles, energy is supplied to adjacent Support 1 and Support 2 upon binding to the target object, thereby releasing manganese from Support 1. platinum Ions are released. Also, if support 1 is silica that absorbs manganese ions and support 2 is gold nanoparticles, energy is supplied to adjacent support 1 and support 2 by binding to the target of detection, resulting in alloy ions (AuMn 2+ ) detaches.

[0030] Energy can be supplied using known methods such as electron shock, chemical ionization, field desorption, ion beam, and particle shock. Preferably, energy is supplied by irradiation with electromagnetic waves absorbed by nanoparticles. Preferably, the electromagnetic waves are optimized for desorption ionization of the material forming the carrier rather than from the object being detected itself.

[0031] The electromagnetic wave is, for example, a pulsed electromagnetic wave from 10 milliseconds to 10 femtoseconds. Preferably, the energy is supplied to the carrier by irradiation with a pulsed laser having a wavelength in the ultraviolet region to near infrared and a pulse width from 100 nanoseconds to 1 picosecond. By using a pulsed laser, it is efficient in selective desorption and ionization from the detection target. In particular, an apparatus combining a pulsed laser and a time-of-flight mass spectrometer is a suitable analytical instrument for the detection kit according to the present embodiment. The combination of a pulsed laser and a time-of-flight mass spectrometer is generally widely used as MALDI-MS using matrix molecules. Although the widely used laser is an ultraviolet pulsed laser, visible light or near infrared pulsed laser light may be used.

[0032] More specifically, when carrier 1 contains gold nanoparticles and carrier 2 contains platinum nanoparticles, when the ultraviolet pulsed laser light of the MALDI-MS apparatus is irradiated onto carrier 1 and carrier 2 that are close to each other by binding to the detection target, the desorption of AuPt + occurs with high efficiency. Even if there are isolated carrier 1 and carrier 2 that are not close to each other, they do not give signals that compete with AuPt + . Therefore, when AuPt + is detected, it indicates that the detection target is contained in the sample.

[0033] Also, when carrier 1 contains AuAg nanoparticles and carrier 2 contains AuPd nanoparticles, when the ultraviolet pulsed laser light of the MALDI-MS apparatus is irradiated onto carrier 1 and carrier 2 that are close to each other by binding to the detection target, the desorption of AgPd + occurs with high efficiency. Specifically, the seven types of AgPd shown in FIG. 1 + are desorbed, but AgPd + containing only 109 [[ID=2२]]Ag 108 Pd and 107 Ag 110 Pd corresponding to the signal with m / z of {217}, and [[ID=2९]] 109 Ag 110 Pd corresponding to the signal with m / z of {219}, the magnitude of the signal can detect AgPd + . Since palladium is less likely to be desorbed and ionized compared to gold and silver,109 Ag 106 Pd and 107 Ag 108 Even with a signal magnitude of 215 m / z corresponding to Pd, AgPd + It can detect AgPd. On the other hand, carriers 1 and 2, which are isolated and not in close proximity, can detect AgPd. + It does not provide a competing signal. Therefore, AgPd + The detection of this factor indicates that a factor bringing carrier 1 and carrier 2 into close proximity, i.e., the target of detection, is present in the sample.

[0034] Next, a detection method using the detection kit according to this embodiment will be described. The detection method includes a binding step, a supply step, and a detection step. In the binding step, carrier 1 and carrier 2 are bound to the target to be detected via probe 10 and probe 20, respectively. The binding of the carriers to the target to be detected is performed by known methods, for example, by exposing the sample to carrier 1 and carrier 2. In the binding step, the sample may be exposed to carrier 1 first and then to carrier 2, or the sample may be exposed to carrier 1 and carrier 2 simultaneously by, for example, immersing the sample in a dispersion containing carrier 1 and carrier 2.

[0035] In the supply step, energy is supplied to carriers 1 and 2 bound to the target to be detected, as described above. In the detection step, specific ions that have been desorbed by supplying energy to carriers 1 and 2, which are adjacent to the target to be detected via probes 10 and 20, are detected.

[0036] By utilizing this desorption / ionization characteristic, immunodetection can detect viruses, biomolecules, and disease markers in a sample. For example, in the case of immunoaffinity chromatography, the above probe can be replaced with an antibody. By fixing the chromatogram membrane onto a substrate for MALDI-MS measurement and irradiating it with a pulsed laser, ions specific to adjacent carriers 1 and 2 can be detected. Furthermore, this detection kit can also be applied to blotting methods, which involve transferring DNA, RNA, and proteins onto a membrane for analysis. The sample transferred onto the blotting membrane is exposed to the carrier, and the blotting membrane is irradiated with a pulsed laser. For ELISA applications, the sample fixed on an ELISA plate is exposed to the carrier, and then irradiated with a pulsed laser.

[0037] Furthermore, by focusing a pulsed laser, detecting desorbed ions from a narrow area, and scanning the laser irradiation position of the sample, it is possible to create a two-dimensional map of the distribution of the target ions in the sample.

[0038] Furthermore, detection is also possible using other mass spectrometry methods, such as quadrupole mass spectrometers, ion trap mass spectrometers, and Fourier transform mass spectrometers.

[0039] The detection kit according to this embodiment can identify ions that are bound to the target, i.e., ions that desorb only when carrier 1 and carrier 2 are in close proximity. Therefore, competing signals (background signals) from ions derived from carrier 1 and carrier 2 that are not bound to the target on the membrane, etc., can be excluded from the analysis. As a result, the target can be detected with high sensitivity without interference from non-specific adsorption.

[0040] The detection kit according to this embodiment has the advantage of being able to detect the presence of a target substance in samples with complex compositions, such as tissue sections, and in insulating samples with a certain thickness, such as membranes, with high sensitivity by mass spectrometry. Furthermore, it has the advantage of being able to visualize the distribution of the target substance using imaging mass spectrometry technology.

[0041] The detection kit according to this embodiment may further include a substrate on which a capture substance that binds to at least one of probe 10 and probe 20 is immobilized. The capture substance is not particularly limited as long as it binds to the probe. For example, the capture substance is an antibody that specifically binds to the probe. A detection method using the detection kit comprising the substrate preferably includes a capture step between the binding step and the supply step described above, to which the target to be detected, to which carriers 1 and 2 are bound, is captured by the capture substance on the substrate via at least one of probe 10 and probe 20.

[0042] More preferably, the detection method further includes a removal step between the capture step and the supply step, in which the substrate is cleaned to remove any substances not captured on the substrate. By removing substances other than the target of detection, carrier 1, and carrier 2 using the captured substance, signals caused by organic fragments generated by photoheating, such as metal nanoparticles, can be excluded. As a result, for example, even if a biological sample such as whole blood containing various impurities is analyzed as is, the target of detection can be detected with high sensitivity without interference from non-specific adsorption.

[0043] The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples. [Examples]

[0044] (Example 1: AuAg-AuPd) [Preparation of gold nanoparticles] 100 mL of pure water and 0.7 mL of HAuCl4 (42 mM) were added to a 500 mL three-necked flask and heated under reflux. After boiling, 2 mL of 1 wt% trisodium citrate was added to the reaction mixture and heating was continued for 60 minutes. The spectrum of the reaction mixture was measured using a spectrophotometer (V-670, JASCO Corporation) (see Figure 2) to confirm the progress of the reaction.

[0045] The reflux condenser was removed from the three-necked flask, and the reaction mixture was concentrated by heating for 10 minutes. The reaction mixture was cooled to room temperature, dispensed into 2 mL portions, and centrifuged at 5000 × G at 30°C for 10 minutes. The supernatant was removed, and the resulting gold nanoparticles were redispersed with 1.3 mM trisodium citrate to a total volume of 10 mL. The gold nanoparticle dispersion was diluted 10-fold, and the spectrum was measured (see Figure 2).

[0046] [Preparation of palladium shell gold nanoparticles and alloying by pulsed laser irradiation] 30 mL of 1.3 mM trisodium citrate, 100 μL of 10 mM HPdCl4, and 330 μL of the gold nanoparticle dispersion prepared above were added to a 50 mL vial. The amount of gold nanoparticle dispersion was calculated from the absorbance at a spectral wavelength of 355 nm. Furthermore, 500 μL of 10 mL of 100 mM L-ascorbic acid was added to each vial, and the mixture was stirred at 4°C for 30 minutes. The reaction progress was monitored by measuring the spectrum (see Figure 3).

[0047] While evaluating the temporal changes in the spectrum, the reaction solution was irradiated with laser light from an Nd-YAG laser (NEWWAVE Research, Inc.) (532 nm, 10 ns, 20 Hz, 250 mW). 8 mL of the reaction solution was transferred to 15 mL tubes and centrifuged at 5000 × G, 30 °C for 10 minutes. The supernatant was removed, and 4 mL of 1.3 mM trisodium citrate was added to each tube to redisperse the resulting AuPd (alloy) nanoparticles. The AuPd nanoparticle dispersion was transferred to a screw-cap tube, and the spectrum was measured (see Figure 4).

[0048] [Preparation of silver-shell gold nanoparticles and alloying by pulsed laser irradiation] 30 mL of 1.3 mM trisodium citrate, 100 μL of 10 mM AgNO3, and 330 μL of the gold nanoparticle dispersion prepared above were added to a 50 mL vial. The amount of gold nanoparticle dispersion was calculated from the absorbance at a spectral wavelength of 355 nm. The mixture was then stirred in a constant temperature bath for 30 minutes. Furthermore, 500 μL of 10 mL of 100 mM L-ascorbic acid was added to the vial in batches, and the progress of the reaction was confirmed while measuring the spectrum (see Figure 5).

[0049] While evaluating the temporal changes in the spectrum, the reaction solution was irradiated with laser light from an Nd-YAG laser (NEWWAVE Research, Inc.) (532 nm, 10 ns, 20 Hz, 200 mW). 8 mL of the reaction solution was transferred to 15 mL tubes and centrifuged at 5000 × G, 30 °C for 10 minutes. The supernatant was removed, and 4 mL of 1.3 mM trisodium citrate was added to each tube to redisperse the resulting AuAg (alloy) nanoparticles. The AuAg nanoparticle dispersion was transferred to a screw-cap tube, and the spectrum was measured (see Figure 6).

[0050] [Antibody modification of alloy nanoparticles] Using low-adsorption nanotubes, AuPd nanoparticles and AuAg nanoparticles were diluted in 10 mM phosphate buffer, and each was OD 355 = 0.3cm -1 The mixture was prepared (total volume 450 μL). 50 μL of 10 mM phosphate buffer was added to each of the AuPd nanoparticles and AuAg nanoparticles. 50 μL of anti-goat IgG (30 μg / mL) was added to the AuPd nanoparticle dispersion and allowed to stand at 25°C for 30 minutes.

[0051] Similarly, 50 μL of normal goat IgG (30 μg / mL) was added to the AuAg nanoparticle dispersion and allowed to stand at 25°C for 30 minutes. 50 μL of 1 wt% bovine serum albumin (BSA) was added to both the AuPd nanoparticle dispersion and the AuAg nanoparticle dispersion, and allowed to stand at 25°C for 30 minutes.

[0052] 50 μL of 1 wt% polyethylene glycol (PEG) was added to both the AuPd nanoparticle dispersion and the AuAg nanoparticle dispersion, and the mixtures were centrifuged at 1000 × G at 15°C for 10 minutes. The supernatants were transferred to separate tubes and centrifuged at 6000 × G at 15°C for 10 minutes. Tris-Buffered Solution (pH 8.2) was added to the precipitates, and each mixture was combined into a single 200 μL tube. The spectra of the resulting normal goat IgG-AuAg nanoparticle dispersion and anti-goat IgG-AuPd nanoparticle dispersion were measured (see Figures 7 and 8).

[0053] [Mass spectrometry of antibody-modified alloy nanoparticles] Anti-goat IgG-AuPd nanoparticles, their OD 355 The value of the OD of normal goat IgG-AuAg nanoparticles 355 The solution was prepared in a total volume of 100 μL to achieve the specified value. 100 μL of the prepared anti-goat IgG-AuPd nanoparticle dispersion and 100 μL of normal goat IgG-AuAg nanoparticles were mixed and allowed to stand for 1 hour. The prepared dispersion was further diluted 10-fold, 100-fold, and 1000-fold with phosphate-buffered saline (1×PBS) to prepare new dispersions. The resulting dispersions were cast onto a transparent conductive film (ITO) substrate.

[0054] Mass spectrometry was performed using an Autoflex Speed ​​(Bruker) (Laser Shot Count: 500, Laser Power: 90%, Reflector Voltage: 9.9, Random Walk: OFF).

[0055] (result) As shown in Figure 9, AgPd + The signal intensity of ions (m / z 217,219) could be identified. Furthermore, even when unantibody-modified AuPd nanoparticles and AuAg nanoparticles were cast onto a substrate, AgPd + No ions were detected. When nanoparticles are cast onto a substrate and dried, the nanoparticles may aggregate into narrow regions as water evaporates. As shown in Figure 10, even under relatively high nanoparticle concentrations of 0.3 absorbance, we confirmed that aggregates that yield a ternary alloy (AuAgPd) were not formed.

[0056] (Example 2: Au-Pt) [Preparation of platinum nanoparticles] 50 mL of pure water and 144 μL of H2PtCl6 (96.5 mM) were added to a 100 mL three-necked flask and heated under reflux. After boiling, 1.1 mL of citrate buffer (1 wt% trisodium citrate and 0.05 wt% citric acid) was added, and 30 seconds later, 550 μL of 0.08 wt% NaBH4 buffer (0.8 wt% NaBH4 0.044 g per 1 mL of citrate buffer) diluted 10-fold was added. Heating was continued for 10 minutes and then allowed to cool to room temperature. The spectrum of the obtained product was measured (see Figure 11).

[0057] [Growth of platinum nanoparticles] In a 100 mL three-necked flask, 29 mL of pure water, 5 mL of the product prepared above, 144 μL of H2PtCl6 (96.5 mM), and 500 μL of 1 wt% trisodium citrate - 1.25 wt% L-ascorbic acid were added and heated under reflux. After boiling, heating was continued for 30 minutes, and the mixture was allowed to cool to room temperature. The spectrum of the product was then measured (see Figure 12).

[0058] The product was centrifuged at 5000×G at 30°C for 10 minutes. The supernatant was discarded, and the total volume of the platinum nanoparticle dispersion was reduced to 10 mL using 1.3 mM trisodium citrate.

[0059] [Antibody modification of gold nanoparticles and platinum nanoparticles] Using low-adsorption nanotubes, the gold nanoparticle dispersion and platinum nanoparticle suspension prepared in Example 1 were diluted with 10 mM phosphate buffer, and each was subjected to OD (Oxygen-Dose) analysis. 355 = 0.3cm -1 The following solutions were prepared (total volume 450 μL). 50 μL of 10 mM phosphate buffer was added to both the gold nanoparticle dispersion and the platinum nanoparticle dispersion. 50 μL of anti-goat IgG (30 μg / mL) was added to the platinum nanoparticle dispersion, and the mixture was allowed to stand at 25°C for 30 minutes.

[0060] Similarly, 50 μL of normal goat IgG (30 μg / mL) was added to the gold nanoparticle dispersion and allowed to stand at 25°C for 30 minutes. 50 μL of 1 wt% BSA was added to both the gold nanoparticle dispersion and the platinum nanoparticle dispersion, and allowed to stand at 25°C for 30 minutes.

[0061] 50 μL of 1 wt% PEG was added to both the gold nanoparticle dispersion and the platinum nanoparticle dispersion, and the mixtures were centrifuged at 1000 × G at 15°C for 10 minutes. The supernatants were transferred to separate tubes and centrifuged at 6000 × G at 15°C for 10 minutes. Tris-Buffered-Solution (pH 8.2) was added to the precipitates, and each mixture was brought to a total volume of 2 mL. The spectra of the resulting anti-goat IgG-platinum nanoparticle dispersion and normal goat IgG-gold nanoparticle dispersion were measured (see Figures 13 and 14).

[0062] [Dot blotting] The concentrations of normal goat IgG were varied and cast onto hydrophobic membranes. After drying, the membranes were immersed in a dispersion of anti-goat IgG-platinum nanoparticles. The membranes were washed to remove non-specifically adsorbed anti-goat IgG-platinum nanoparticles, and mass spectrometry was performed under the same conditions as in Example 1.

[0063] [Mass spectrometry of antibody-modified gold nanoparticles and antibody-modified platinum nanoparticles] Anti-goat IgG-platinum nanoparticles, their OD 355 The value of the OD of the normal goat IgG-gold nanoparticles prepared in Example 1 355 The solution was prepared in a total volume of 100 μL to achieve the specified value. 100 μL of the prepared anti-goat IgG-platinum nanoparticle dispersion and 100 μL of normal goat IgG-gold nanoparticle dispersion were mixed and allowed to stand for 1 hour. The resulting dispersion was then cast onto an ITO substrate. Mass spectrometry was performed under the same conditions as in Example 1.

[0064] (result) In the mass spectrum of dot blotting, platinum ions (P t + Signals originating from (m / z 194, 195, 196, and 198) were confirmed. Figure 15 shows the sum of the signals at m / z 194, 195, and 196 for the dot positions. A platinum ion signal was observed in the area where the normal goat IgG antigen was cast, confirming that the antibody could be immobilized on platinum.

[0065] The mass spectra measured on the ITO substrate are shown in Figure 16. AuPt, which is specific to the antigen-antibody reaction, is present at m / z 392 and 395. + The following signal was observed.

[0066] (Example 3: PSA sandwich assay) Prostate-specific antigen (PSA), the target substance, is sandwiched between two types of antibody-modified alloy nanoparticles (AuAg nanoparticles and AuPd nanoparticles), and cluster ions (AgPd) are generated by the proximity of the two types of alloy nanoparticles. + Immunodetection was performed using the ion (Ion) as the reporter ion.

[0067] [Preparation of palladium shell gold nanoparticles and alloying by pulsed laser irradiation] 30 mL of 1.3 mM trisodium citrate solution was mixed with 100 μL of 10 mM palladium chloride solution and 2 mL of the 0.5 mM gold nanoparticle dispersion prepared in Example 1. 500 μL of 0.1 M ascorbic acid solution was added at room temperature and the mixture was reacted for 30 minutes. The reaction mixture was irradiated with an Nd-YAG laser (NEWWAVE Research) for 15 minutes (532 nm, 10 ns, 20 Hz, 250 mW). The mixture was centrifuged at 5000 × G for 10 minutes, and the precipitate was dispersed in 16 mL of 1.3 mM trisodium citrate solution. The resulting AuPd nanoparticle dispersion was transferred to a screw-cap tube, and the spectrum was measured (see Figure 17). Almost no gold surface plasmon band was observed around 520 nm. Therefore, AuPd (alloy) nanoparticles with a high palladium ratio were obtained.

[0068] [Antibody modification of alloy nanoparticles] The AuPd nanoparticle dispersion prepared in this example and the AuAg nanoparticle dispersion prepared in Example 1 were diluted in 10 mM phosphate buffer, and the absorbance at 355 nm was 0.6 cm² for each. -1 The mixture was adjusted to achieve the desired result. The antibody used was from a PSA detection ELISA kit (Human PSA ELISA Kit, Abcam, ab264615).

[0069] 450 μL of AuPd nanoparticle dispersion was mixed with 50 μL of 10 mM phosphate buffer, and this mixture was added to 50 μL of anti-PSA antibody solution (capture). The mixture was then allowed to stand at 25°C for 30 minutes. 450 μL of AuAg nanoparticle dispersion was also mixed with 50 μL of 10 mM phosphate buffer, and this mixture was added to 50 μL of anti-PSA antibody solution (detective). The mixture was then allowed to stand at 25°C for 30 minutes.

[0070] 50 μL of 1 wt% bovine serum albumin (BSA) was added to each alloy nanoparticle dispersion and allowed to stand for 30 minutes. Then, 1 wt% poly(ethylene glycol) (molecular weight 5000) was added and allowed to stand for 5 minutes. After centrifugation at 1000 × G for 10 minutes, the supernatant was centrifuged twice more at 5000 × G, and the precipitates from all three centrifugations were dispersed together in 200 μL of 10 mM phosphate buffer.

[0071] [Immunodetection] Two types of alloy nanoparticle dispersions (20 μL each) were mixed, and 10 μL of solutions containing PSA at different concentrations were added. An additional 10 μL of carp blood was added as needed, and the mixture was allowed to stand for 1 hour. The sample solution was diluted with water to a volume of 10:1 (500 μL), cast onto an ITO substrate, and subjected to mass spectrometry.

[0072] Mass spectrometry was performed using an Autoflex Speed ​​(Bruker) (laser shot count: 100, laser power: 90%, reflector voltage: 9.9).

[0073] (result) Figure 18 shows the mass spectrum of a sample that does not contain PSA. Ag 2+ Three strong signals originating from (m / z 214, 216, 218) were observed, while only weak noise was observed at other m / z values. Figure 19 shows the mass spectrum of the sample to which 10 μL of 4 ng / mL PSA solution was added. When PSA was added to the sample solution, the peak intensities at m / z 212, 213, 215, 217, and 219 increased, indicating AgPd + Ion signals were observed.

[0074] Figure 20 shows the relationship between the concentration of AgPd added to the PSA solution. + The signal intensity of the ion (m / z 215) is shown. The signal intensity at m / z 215 increased depending on the amount of PSA added. AgPd + It was revealed that the ion functions as a reporter ion for antigen-antibody reactions. As shown in Figure 21, the signal intensity decreased in the sample to which 10 μL of carp whole blood was added, but the tendency for the signal intensity to increase depending on the PSA concentration remained unchanged, and AgPd was also detected in the sample solution containing whole blood. + It was found that immunoassay is possible using ions as reporter ions.

[0075] (Example 4: Examination of speeding up analysis) The sample solution is concentrated and dried on the substrate. Since the reaction proceeds during concentration and drying without waiting for the antigen-antibody reaction to take place in the solution, rapid analysis is possible.

[0076] Similar to Example 1, AuPd nanoparticles and AuAg nanoparticles were modified with goat IgG and normal goat IgG, respectively. The samples after mixing the modified nanoparticle dispersions were subjected to mass spectrometry at predetermined intervals under the same conditions as in Example 1.

[0077] (result) Figure 22 shows the mass spectra at different time intervals after mixing. Figure 23 shows the signal intensity (m / z 217) against the time interval after mixing. Even when cast and measured immediately after mixing, AgPd was detected at approximately half the intensity compared to when waiting 60 minutes. + Ions (m / z 217) were detected. The nanoparticle concentration was 4.4 × 10⁻⁶. -12 M is the value. If the signal intensity is sufficient, little to no waiting time is required after sample mixing. To obtain the ultimate detection sensitivity, it is better to wait until the antigen-antibody reaction has fully occurred in the solution, but since the antigen-antibody complex will be formed anyway during the solvent drying process, the signal can be detected even without waiting time.

[0078] (Example 5: Preparation and ionization behavior of platinum nanoparticles) 144 μL of 96.5 mM chloroplatinic acid was added to 50 mL of water and brought to a boil. 1.1 mL of 0.1 M citrate buffer was added, followed by 55 μL of 0.08 wt% NaBH, and the mixture was boiled for 10 minutes to prepare platinum nanoparticles. The prepared dispersion was diluted 10-fold with phosphate-buffered saline (1 × PBS) to prepare another dispersion. The dispersion was cast onto an ITO substrate and mass spectrometry was performed under the same conditions as in Example 1.

[0079] (result) Figure 24 shows platinum nanoparticles imaged by TEM. The particle size of the platinum nanoparticles obtained from the TEM images was 4.9 ± 1 nm. Figure 25 shows the mass spectrum measured on an ITO substrate. Platinum signals were observed at m / z 194, 195, and 196. It is generally understood that platinum has a high melting point and is difficult to ionize, but this study showed that if the laser irradiation intensity is sufficient, ions can be desorbed at a level that allows for detection of cluster ions with silver or palladium.

[0080] This invention allows for various embodiments and modifications without departing from the broad spirit and scope of the invention. Furthermore, the embodiments described above are for illustrative purposes only and do not limit the scope of the invention. In other words, the scope of the invention is indicated not by the embodiments, but by the claims. Various modifications made within the scope of the claims and the equivalent scope of the meaning of the invention are considered to be within the scope of the invention.

[0081] This application is based on Japanese Patent Application No. 2021-6270, filed on 19 January 2021. The entire specification, claims, and drawings of Japanese Patent Application No. 2021-6270 are incorporated herein by reference. [Industrial applicability]

[0082] This invention is suitable for detecting trace components. [Explanation of Symbols]

[0083] 1,2 Carrier, 10,20 Probe, 11,21 Metal

Claims

1. A first carrier containing a first metal supports a first probe that binds to the target to be detected, A second carrier supporting a second probe that binds to the target to be detected, and containing a second metal different from the first metal, Equipped with, When energy is supplied to the first and second carriers in close proximity by binding to the target of detection via the first and second probes, cluster ions composed of both the constituent elements of the first metal and the constituent elements of the second metal are desorbed. Detection kit.

2. The first carrier is a gold-silver alloy nanoparticle, The second carrier is a gold-palladium alloy nanoparticle. The detection kit according to claim 1.

3. The first support is gold nanoparticles, The second support is platinum nanoparticles. The detection kit according to claim 1.

4. The first probe and the second probe are, The antibody is an antibody that specifically binds to the target of detection. A detection kit according to any one of claims 1 to 3.

5. A binding step involves binding a first carrier, which carries a first probe to be bound to the target to be detected and contains a first metal, and a second carrier, which carries a second probe to be bound to the target to be detected and contains a second metal different from the first metal, to the target to be detected via the first probe and the second probe, respectively. A supply step of supplying energy to the first carrier and the second carrier bound to the target to be detected, A detection step for detecting cluster ions composed of constituent elements of both the first metal and the second metal, which dehiscence occurs when energy is supplied to the target for detection, bringing the first carrier and the second carrier into close proximity, by binding to the target via the first probe and the second probe; A detection method that includes this.

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