WO2022158437A1

WO2022158437A1 - Detection kit and detection method

Info

Publication number: WO2022158437A1
Application number: PCT/JP2022/001528
Authority: WO
Inventors: 康郎新留
Original assignee: 国立大学法人鹿児島大学
Priority date: 2021-01-19
Filing date: 2022-01-18
Publication date: 2022-07-28
Also published as: JPWO2022158437A1

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

Detection kit and detection method

The present invention relates to detection kits and detection methods.

Antibody-modified gold nanoparticles are widely used as probes (markers) for immunodetection such as immunoaffinity chromatography and ELISA (Enzyme-Linked Immuno Sorbent Assay). Since gold nanoparticles can be ionized without using matrix molecules, gold nanoparticles are useful for detecting large-molecular-weight proteins that are difficult to ionize by MALDI (Matrix Assisted Laser Desorption/Ionization).

Gold nanoparticles also function as ultra-sensitive mass probes that efficiently emit gold ions by MALDI-MS (Mass Spectrometry). Furthermore, even in the case of the immunodetection described above, it can be detected with ultrahigh sensitivity by mass spectrometry. In Patent Document 1, gold ions or gold clusters are obtained by fixing an immunoaffinity chromatogram membrane using antibody-modified gold nanoparticles as probes on a substrate for MALDI-MS measurement and irradiating with laser light. A target substance detection method for detecting desorption of ions is disclosed. According to this detection method, even very small amounts of gold nanoparticles that cannot be observed with the naked eye can be detected, thereby greatly improving analytical sensitivity.

JP 2015-1453 A

Some of the gold nanoparticles are non-specifically adsorbed to the membrane or the like. Signals over 1000 times the detection limit of the mass spectrometer are detected from non-specific adsorption alone. In the case of immunodetection by normal fluorescence or color development, nonspecific adsorption can be reduced to a level that does not interfere with detection by performing blocking treatment according to a standard method. However, for gold nanoparticles, even if optimized blocking treatment is applied, it is considered difficult to reduce non-specific adsorption to the level required for mass spectrometry. For this reason, it has not yet been possible to fully utilize the ultrahigh sensitivity of gold nanoparticle probes reaching 10 ⁻¹⁸ mol/mm ² .

The present invention has been made in view of the above circumstances, and aims to provide a detection kit and a detection method that can detect a target to be detected with high sensitivity without being hindered by nonspecific adsorption.

The detection kit according to the first aspect of the present invention comprises
a first carrier carrying a first probe that binds to a detection target and containing a first metal;
a second carrier carrying a second probe that binds to the detection target and containing a second metal different from the first metal;
with
When energy is supplied to the first carrier and the second carrier that are close to each other by binding to the detection target through the first probe and the second probe, specific ions are desorbed.

The first carrier is a gold-silver alloy nanoparticle,
wherein the second carrier is a gold-palladium alloy nanoparticle;
You can do it.

Further, the first carrier is a gold nanoparticle,
The second carrier is platinum nanoparticles,
You can do it.

Further, the first probe and the second probe are
An antibody that specifically binds to the detection target,
You can do it.

A detection method according to a second aspect of the present invention comprises:
carrying a first probe that binds to the target of detection, carrying a first carrier containing a first metal, and carrying a second probe that binds to the target of detection, a second metal different from the first metal a binding step of binding a second carrier containing a metal to the detection target 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 detection target;
Detection of unique ions that are desorbed by supplying the energy to the first carrier and the second carrier that are adjacent to each other by binding to the detection target through the first probe and the second probe. a detection step for
including.

According to the present invention, the detection target can be detected with high sensitivity without being interfered with by non-specific adsorption.

FIG. 3 is a diagram showing 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 isotope abundance ratio. FIG. 4 is a diagram showing absorption spectra of gold nanoparticles; FIG. 4 shows absorption spectra of palladium-shell gold nanoparticles; FIG. 4 shows absorption spectra of AuPd nanoparticles; FIG. 4 shows absorption spectra of silver-shelled gold nanoparticles; FIG. 4 shows absorption spectra of AuAg nanoparticles; FIG. 3 shows absorption spectra of goat normal immunoglobulin G (IgG)-AuAg nanoparticles. FIG. 4 shows absorption spectra of anti-goat IgG-AuPd nanoparticles. FIG. 4 shows the ionization behavior of complexes of anti-goat IgG-AuPd nanoparticles and normal goat IgG-AuAg nanoparticles. FIG. 2 shows the ionization behavior of AuAg nanoparticles and AuPd nanoparticles cast on a substrate. It is a figure which shows the absorption spectrum of platinum nanoparticles. FIG. 4 is a diagram showing absorption spectra of grown platinum nanoparticles; FIG. 4 shows absorption spectra of anti-goat IgG-platinum nanoparticles. FIG. 3 shows absorption spectra of goat normal IgG-gold nanoparticles. FIG. 10 shows the intensity of signals on membranes cast with normal goat IgG. FIG. 4 shows the ionization behavior of complexes of anti-goat IgG-platinum nanoparticles and normal goat IgG-gold nanoparticles. FIG. 4 shows absorption spectra of AuPd nanoparticles; FIG. 3 shows the ionization behavior of a sample according to Example 3 that does not contain prostate-specific antigen. FIG. 3 shows the ionization behavior of a sample according to Example 3 containing prostate specific antigen. FIG. 10 is a diagram showing the relationship between the concentration of PSA solution and the signal intensity of AgPd ⁺ ions in a sample containing no whole blood according to Example 3; FIG. 10 is a diagram showing the relationship between the concentration of a PSA solution and the signal intensity of AgPd ⁺ ions in a sample containing whole blood according to Example 3; FIG. 10 is a diagram showing ionization behavior after mixing of a sample according to Example 4; FIG. 10 is a diagram showing the relationship between the elapsed time after mixing the sample and the signal intensity according to Example 4; FIG. 10 is a diagram showing platinum nanoparticles according to Example 5 imaged with a transmission electron microscope (TEM). FIG. 10 is a diagram showing ionization behavior of platinum nanoparticles according to Example 5 cast on a substrate.

An embodiment according to the present invention will be described with reference to the drawings. The present invention is not limited by the following embodiments and drawings. In the following embodiments, expressions such as "have", "include" and "contain" also include the meaning of "consisting of" or "consisting of".

The detection kit according to the present embodiment includes carrier 1 (first carrier) and carrier 2 (second carrier). A carrier 1 carries a probe 10 (first probe) that binds to a detection target. Objects to be detected include peptides, proteins, nucleic acids, sugar chains, lipids, and the like. The carrier 2 carries probes 20 (second probes) that bind to the target of detection independently of the carrier 1 . The probe 20 may be the same as or different from the probe 10 as long as it binds to the target of detection. Hereinafter, the

probes

10 and 20 are also collectively referred to as "probes".

The probe is not particularly limited as long as it binds to the detection target. Modes of binding with the detection target include interaction due to van der Waals forces, hydrogen bonding, coordinate bonding, alloying, and the like. For the binding between the probe and the target to be detected, for example, binding between antigen and antibody fragment or antibody, hybridization of nucleic acid or peptide nucleic acid, binding of aptamer, binding between receptor and ligand, redox reaction, and the like can be used. be. Preferably, the probe specifically binds to the target of detection. "Specific" means that the probe does not show any significant binding to a substance other than the substance to be detected.

Antibodies and antibody fragments include all types of antibodies that specifically bind to detection targets. For example, the antibody is a monoclonal antibody, polyclonal antibody, single chain antibody, chimeric antibody and any fragment or derivative of such antibody capable of binding to a detection target. 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 what is to be detected.

Nucleic acids include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and chemically modified derivatives thereof. Nucleic acids can be used particularly when the object to be detected is a nucleic acid. In this case, the nucleic acid is partially or completely complementary to the nucleic acid to be detected or part thereof.

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

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

The receptor as a probe specifically interacts with the ligand to be detected. A ligand as a probe specifically interacts with the receptor to be detected.

The carrier 1 contains metal 11 (first metal). Carrier 2 contains a metal 21 (second metal) different from metal 11 . Hereinafter, the

carriers

1 and 2 are also collectively referred to as "carriers", and the

metals

11 and 21 are also collectively referred to as "metals". A carrier is, for example, a particle containing a metal. The particle size of the particles is not particularly limited, and may be on the order of micrometers or on the order of nanometers. The metal may be contained in the carrier not only as a single metal element but also as an alloy in which metal elements are combined. Note that when the metal 11 and the metal 21 are an alloy, the metal 21 different from the metal 11 means that the metal 21 contains at least one metal atom that is not contained in the metal 11 .

Preferably, the carrier is metal nanoparticles or alloy nanoparticles. Several preparation methods are known for metal nanoparticles, and different sizes and various surface modifications are possible. Gold nanoparticles are particularly preferred because they are chemically stable and methods for immobilizing various physiologically active substances on their surfaces are known. In addition, gold nanoparticles are preferable as carriers because of the high desorption efficiency of gold ions and gold cluster ions when irradiated with pulsed laser light. Silver nanoparticles are also suitable for carriers because their preparation methods are widely known, and the efficiency of desorption of silver ions and silver cluster ions when irradiated with pulsed laser light is high. Other metal nanoparticles include nanoparticles of platinum, palladium, iridium, copper, nickel, manganese, and the like. Preferably, carrier 1 is gold nanoparticles and carrier 2 is platinum nanoparticles.

As the alloy nanoparticles, alloy nanoparticles obtained by combining two or more metals related to the above metal nanoparticles are preferable. For example, alloy nanoparticles include AuAg nanoparticles and AuPd nanoparticles. Alloy nanoparticles can be prepared by known methods. For example, alloy nanoparticles are obtained by alloying nanoparticles containing a core containing a metal and a shell containing a metal different from the metal contained in the core and covering the core with a pulsed laser. The alloy nanoparticles are not limited to binary alloys containing two kinds of metal elements, and may be ternary alloys, quaternary alloys, or the like.

The carrier may also be a silicate such as silicon dioxide (silica) containing metals as metal ions. When the carrier comprises a silicate, embodiments of the carrier may be microparticles, polymers, and the like. For example, the carrier is obtained by ionically bonding a manganese ion to an anion of silicate.

The method for supporting the probe on the carrier is not particularly limited. For example, probes are adsorbed or covalently attached to a support. When the carrier is a metal nanoparticle and the probe is a protein (immunoglobulin) such as an antibody, the metal nanoparticles prepared by reduction with a reducing agent such as citric acid or tannic acid are treated with the protein under predetermined conditions. Just mix. As a result, the protein binds to the carrier via electrostatic interactions, hydrogen bonds, free thiol groups with high affinity contained in the protein, and the like.

Alternatively, the protein used as the probe can be chemically modified and immobilized on the carrier through covalent bonding with a thiol group or the like. As a method for efficiently immobilizing an antibody on a carrier, an antibody immobilization technique using a protein (protein A, protein G, protein A/G or protein L) that specifically binds to the antibody may be used. When the carrier contains gold, peptides with gold-reducing and gold-binding ability may be used.

When energy is supplied to the carrier 1 and the carrier 2 which are close to each other by binding to the detection target through the probe 10 and the probe 20, specific ions are desorbed. "Intrinsic" means an ion that is desorbed only when carrier 1 and carrier 2 are brought into close proximity due to binding to the target of detection. For example, when the carrier 1 is a gold nanoparticle and the carrier 2 is a platinum nanoparticle, energy is supplied to the adjacent carrier 1 and the carrier 2, so that gold platinum ions as ions specific to the adjacent carrier 1 and carrier 2 (AuPt ⁺ ) is eliminated. In addition, when the carrier 1 is AuAg nanoparticles and the carrier 2 is AuPd nanoparticles, energy is supplied to the

carriers

1 and 2 that are in close proximity to fuse the two types of nanoparticles to form a ternary alloy. . Silver palladium ions (AgPd ⁺ ) are released as unique ions from the ternary alloy.

The carrier 1 may be silica or protein that occludes metal ions, and the carrier 2 may be metal nanoparticles. Metal ions include manganese ions, copper ions, cobalt ions, tin ions, and the like, which do not exist in large amounts in nature. When the carrier 1 is silica that absorbs manganese ions, and the carrier 2 is platinum nanoparticles, manganese ions are desorbed from the carrier 1 by supplying energy to the carrier 1 and the carrier 2 that are in close proximity due to binding to the detection target. do. Further, when the carrier 1 is silica that occludes manganese ions and the carrier 2 is a gold nanoparticle, energy is supplied to the

carriers

1 and 2 that are close to each other due to binding to the detection target, so that alloy ions (AuMn ²⁺ ) desorbs.

Energy can be supplied using known methods such as the electron impact method, the chemical ionization method, the field desorption method, the ion beam method, and the particle impact method. Preferably, the energy is supplied by electromagnetic radiation that is absorbed by the nanoparticles. Preferably, the electromagnetic waves are optimized for desorption ionization of the material forming the carrier rather than from the detection target itself.

An electromagnetic wave is, for example, a pulsed electromagnetic wave of 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 to near-infrared range and having a pulse width of 100 nanoseconds to 1 picosecond. The use of a pulsed laser is efficient in selective desorption and ionization from the detection target. In particular, a device that combines a pulse laser and a time-of-flight mass spectrometer is a suitable analytical instrument for the detection kit according to this embodiment. A combination of a pulsed laser and a time-of-flight mass spectrometer is generally widely used as MALDI-MS using matrix molecules. A widely used laser is an ultraviolet pulsed laser, but visible light or near-infrared light pulsed laser light may also be used.

More specifically, when the carrier 1 contains gold nanoparticles and the carrier 2 contains platinum nanoparticles, the ultraviolet pulsed laser light of the MALDI-MS device is applied to the

carriers

1 and 2 that are in close proximity due to binding to the detection target. Upon irradiation, desorption of AuPt ⁺ occurs with high efficiency. The presence of carrier 1 and carrier 2 isolated without proximity gives no competing signal to AuPt ⁺ . Therefore, when AuPt ⁺ is detected, it indicates that the target of detection is contained in the sample.

Further, when the carrier 1 contains AuAg nanoparticles and the carrier 2 contains AuPd nanoparticles, when the ultraviolet pulsed laser light of the MALDI-MS apparatus is irradiated to the

carriers

1 and 2 that are close to each other due to binding to the detection target, AgPd ⁺ elimination occurs with high efficiency. ^Specifically , the ^seven ^types of ^AgPd ⁺ ^shown in ^FIG ^. AgPd ⁺ can be detected at the corresponding m/z of 219 signal magnitude. Since palladium is less likely to be desorbed and ionized than gold and silver, AgPd ⁺ can be detected even at a signal magnitude of m/z 215 corresponding to ¹⁰⁹ Ag ¹⁰⁶ Pd and ¹⁰⁷ Ag ¹⁰⁸ Pd. On the other hand, carrier 1 and carrier 2, which are not in close proximity and isolated, do not give a signal that competes with AgPd ⁺ . Therefore, the detection of AgPd ⁺ indicates that the sample contains a factor that brings carrier 1 and carrier 2 closer to each other, that is, the detection target.

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 detection target via probe 10 and probe 20, respectively. The binding of the carrier to the detection target is performed by a known method, and for example, the sample may be exposed to carrier 1 and carrier 2. In the binding step, the sample may be exposed to the carrier 1 and then to the carrier 2, or, for example, by immersing the sample in a dispersion containing the carrier 1 and the carrier 2, the sample may be combined with the carrier 1 and the carrier 2. It may be exposed to carrier 2 simultaneously.

In the supply step, energy is supplied as described above to the carrier 1 and carrier 2 bound to the detection target. In the detection step, the unique ions desorbed by binding to the detection target via the

probes

10 and 20 and supplying energy to the

adjacent carriers

1 and 2 are detected.

It is possible to detect viruses, biomolecules, and disease markers as detection targets in specimens by immunodetection using this desorption ionization property. For example, in the case of immunoaffinity chromatograms, the above probes may be antibodies. By fixing a membrane for chromatogram on a substrate for MALDI-MS measurement and irradiating a pulsed laser, ions specific to carrier 1 and carrier 2 that are close to each other can be detected. The detection kit can also be applied to a blotting method in which DNA, RNA, and protein are transferred to a membrane and analyzed. The carrier may be exposed to the specimen transferred to the blotting membrane, and the blotting membrane may be irradiated with a pulse laser. In the application to ELISA, the carrier may be exposed to the sample immobilized on the ELISA plate and irradiated with a pulsed laser.

In addition, by condensing the pulsed laser, detecting desorbed ions from a narrow area, and scanning the laser irradiation position of the specimen, it is possible to map the distribution of the detection target in the specimen two-dimensionally.

Detection is also possible using other mass spectrometry methods, such as quadrupole mass spectrometers, ion trap mass spectrometers, and Fourier transform mass spectrometers.

According to the detection kit according to the present embodiment, it is possible to identify ions bound to the detection target, that is, ions that are desorbed only when the carrier 1 and the carrier 2 are in close proximity. Therefore, competing signals (background signals) due to ions derived from carrier 1 and carrier 2 non-specifically adsorbed on the membrane or the like and not bound to the detection target can be excluded from the analysis. As a result, the detection target can be detected with high sensitivity without being interfered with by non-specific adsorption.

The detection kit according to the present embodiment uses mass spectrometry to detect the presence of a target to be detected in a sample having a complex composition such as a tissue section and an insulating sample having a certain thickness such as a membrane with high sensitivity. has the advantage of being able to detect Furthermore, there is an advantage that the distribution of the detection target can be visualized using the technique of imaging mass spectrometry.

It should be noted that the detection kit according to the present embodiment may further include a substrate on the surface of which a capturing substance that binds to at least one of

probes

10 and 20 is immobilized. The capture substance is not particularly limited as long as it binds to the probe. For example, the capture agent is an antibody that specifically binds to the probe. In the detection method using the detection kit comprising the substrate, the target to be detected to which the carrier 1 and the carrier 2 are bound is preferably transferred to the substrate via at least one of the probe 10 and the probe 20 between the binding step and the supply step described above. A capture step is included which captures the capture material above.

More preferably, the detection method further includes, between the capturing step and the supplying step, a removing step of washing the substrate to remove substances not captured on the substrate from the substrate. By removing substances other than the target to be detected, carrier 1 and carrier 2 using a capture substance, it is possible to exclude signals due to organic fragments generated by photoheating such as metal nanoparticles. As a result, for example, even if a biological sample such as whole blood containing various contaminants is analyzed as it is, the detection target can be detected with high sensitivity without being interfered with by non-specific adsorption.

The present invention will be explained more specifically by the following examples, but the present invention is not limited by the examples.

(Example 1: AuAg-AuPd)
[Preparation of gold nanoparticles]
100 mL of pure water and 0.7 mL of HAuCl ₄ (42 mM) were added to a 500 mL three-necked flask and heated under reflux. After boiling, 2 mL of 1% by weight trisodium citrate was added to the reaction solution and heating was continued for 60 minutes. The spectrum of the reaction solution was measured using an absorption photometer (V-670, manufactured by JASCO Corporation) (see FIG. 2) to confirm the progress of the reaction.

The reflux tube was removed from the three-necked flask, and the reaction solution was concentrated by heating for 10 minutes. The reaction solution was cooled to room temperature, divided into 2 mL aliquots, and centrifuged at 5000×G and 30° C. for 10 minutes. The supernatant was removed, and 1.3 mM trisodium citrate was added to the obtained gold nanoparticles to redisperse them, and the total volume was adjusted to 10 mL. The spectrum was measured by diluting the gold nanoparticle dispersion 10-fold (see FIG. 2).

[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 HPdCl ₄ and 330 μL of the gold nanoparticle dispersion prepared above were added to a 50 mL vial. The amount of the gold nanoparticle dispersion liquid was calculated from the absorbance at a spectral wavelength of 355 nm. Further, 500 μL of 10 mL of 100 mM L-ascorbic acid was added to each vial and stirred at 4° C. for 30 minutes. The progress of the reaction was confirmed while measuring the spectrum (see FIG. 3).

While evaluating the spectral change over time, the reaction solution was irradiated with laser light from an Nd-YAG laser (manufactured by NEWWAVE Research) (532 nm, 10 ns, 20 Hz, 250 mW). Each 8 mL of the reaction solution was transferred to a 15 mL tube and centrifuged at 5000×G and 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 tube and the spectrum was measured (see Figure 4).

[Preparation of silver-shelled gold nanoparticles and alloying by pulsed laser irradiation]
30 mL of 1.3 mM trisodium citrate, 100 μL of 10 mM AgNO ₃ and 330 μL of the gold nanoparticle dispersion prepared above were added to a 50 mL vial. The amount of the gold nanoparticle dispersion liquid was calculated from the absorbance at a spectral wavelength of 355 nm. Subsequently, the mixture was stirred for 30 minutes in a constant temperature bath. Further, 500 μL of 10 mL of 100 mM L-ascorbic acid was added to each vial, and the progress of the reaction was confirmed while measuring the spectrum (see FIG. 5).

While evaluating the spectral change over time, the reaction solution was irradiated with laser light from an Nd-YAG laser (manufactured by NEWWAVE Research) (532 nm, 10 ns, 20 Hz, 200 mW). Each 8 mL of the reaction solution was transferred to a 15 mL tube and centrifuged at 5000×G and 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 tube and the spectrum was measured (see Figure 6).

[Antibody Modification of Alloy Nanoparticles]
Using low adsorption nanotubes, AuPd nanoparticles and AuAg nanoparticles were diluted with 10 mM phosphate buffer and each prepared to OD ₃₅₅ =0.3 cm ⁻¹ (total volume 450 μL). 50 μL of 10 mM phosphate buffer was added to each of 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.

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 each of the AuPd nanoparticle dispersion and the AuAg nanoparticle dispersion, and allowed to stand at 25° C. for 30 minutes.

50 μL of 1% by weight polyethylene glycol (PEG) was added to each of the AuPd nanoparticle dispersion and the AuAg nanoparticle dispersion, and centrifuged at 1000×G and 15° C. for 10 minutes. The supernatant was transferred to another tube and centrifuged at 6000×G, 15° C. for 10 minutes. Tris-Buffered-Solution (pH 8.2) was added to the precipitate, and the total volume of each was 200 μL. The spectra of the obtained goat normal IgG-AuAg nanoparticle dispersion and the anti-goat IgG-AuPd nanoparticle dispersion were measured (see FIGS. 7 and 8).

[Mass spectrometry of antibody-modified alloy nanoparticles]
Anti-goat IgG-AuPd nanoparticles were prepared in a total volume of 100 μL such that their OD ₃₅₅ values were those of normal goat IgG- _AuAg nanoparticles. 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. Dispersions were prepared by further diluting the prepared dispersion with phosphate buffered saline (1×PBS) 10-fold, 100-fold and 1000-fold. The resulting dispersion was cast onto a transparent conductive film (ITO) substrate.

Mass spectrometry was performed using Autoflex Speed (manufactured by Bruker) (number of laser shots: 500, laser power: 90%, reflector voltage: 9.9, random walk: OFF)

(result)
As shown in Figure 9, the signal intensity of AgPd ⁺ ions (m/z 217, 219) could be discerned. No AgPd ⁺ ions were detected even when the AuPd nanoparticles and AuAg nanoparticles not modified with antibodies were cast on the substrate. When the nanoparticles are cast onto a substrate and dried, they can clump together in small areas as the water evaporates. As shown in FIG. 10, it was confirmed that aggregates that give a ternary alloy (AuAgPd) were not generated even under conditions of a relatively high concentration of nanoparticles with an absorbance of 0.3.

(Example 2: Au-Pt)
[Preparation of platinum nanoparticles]
50 mL of pure water and 144 μL of H ₂ PtCl ₆ (96.5 mM) were added to a 100 mL three-necked flask and heated under reflux. After boiling, add 1.1 mL of citrate buffer (1 wt% trisodium citrate and 0.05 wt% citric acid), 30 seconds later add 0.8 wt% NaBH4 buffer ₍ 0.8 wt% per mL of citrate buffer). 550 μL of 0.08 w/v % NaBH ₄ buffer obtained by 10-fold dilution of NaBH ₄ (0.044 g) was added. Continue heating for 10 minutes and cool to room temperature. The spectrum of the resulting product was measured (see Figure 11).

[Growth of platinum nanoparticles]
29 mL of pure water, 5 mL of the product prepared above, 144 μL of H ₂ PtCl ₆ (96.5 mM), and 500 μL of 1 wt % trisodium citrate-1.25 wt % L-ascorbic acid were added to a 100 mL three-necked flask and refluxed. heated below. After boiling, heating was continued for 30 minutes, cooled to room temperature, and the spectrum of the product was measured (see FIG. 12).

The product was centrifuged at 5000 x G and 30°C for 10 minutes. The supernatant was discarded, and 1.3 mM trisodium citrate was used to adjust the total amount of the platinum nanoparticle dispersion to 10 mL.

[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 to prepare OD ₃₅₅ =0.3 cm ⁻¹ (total volume: 450 μL ). 50 μL of 10 mM phosphate buffer was added to each of 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 allowed to stand at 25° C. for 30 minutes.

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% by weight BSA was added to each of the gold nanoparticle dispersion and the platinum nanoparticle dispersion, and allowed to stand at 25° C. for 30 minutes.

50 μL of 1% by weight PEG was added to each of the gold nanoparticle dispersion and the platinum nanoparticle dispersion, and centrifuged at 1000×G and 15° C. for 10 minutes. The supernatant was transferred to another tube and centrifuged at 6000×G, 15° C. for 10 minutes. Tris-Buffered-Solution (pH 8.2) was added to the precipitate, and the total volume of each was adjusted to 2 mL. The spectra of the obtained anti-goat IgG-platinum nanoparticle dispersion and normal goat IgG-gold nanoparticle dispersion were measured (see FIGS. 13 and 14).

[Dot blotting]
Various concentrations of normal goat IgG were cast on hydrophobic membranes, dried, and then immersed in an anti-goat IgG-platinum nanoparticle dispersion. The membrane was washed, and mass spectrometry was performed under the same conditions as in Example 1 except for non-specifically adsorbed anti-goat IgG-platinum nanoparticles.

[Mass spectrometry of antibody-modified gold nanoparticles and antibody-modified platinum nanoparticles]
Anti-goat IgG-platinum nanoparticles were prepared in a total volume of 100 μL such that their OD ₃₅₅ values were the OD ₃₅₅ values of normal goat IgG-gold nanoparticles prepared in Example 1. 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, and the resulting dispersion was cast on an ITO substrate. Mass spectrometry was performed under the same conditions as in Example 1.

(result)
Signals (m/

z

194, 195, 196 and 198) derived from platinum ions (Pd ⁺ ) were confirmed in the mass spectrum obtained by dot blotting. Summation of m/

z

194, 195 and 196 signals versus dot position is shown in FIG. A platinum ion signal was observed in the portion cast with normal goat IgG serving as an antigen, confirming that the antibody could be immobilized on platinum.

FIG. 16 shows a mass spectrum obtained by measuring the ITO substrate. AuPt ⁺ signals specific to the antigen-antibody reaction were observed at m/z 392 and 395.

(Example 3: PSA sandwich assay)
Two types of antibody-modified alloy nanoparticles (AuAg nanoparticles and AuPd nanoparticles) are used to sandwich the prostate-specific antigen (PSA) as the target substance, and the two types of alloy nanoparticles are close to each other. Immunodetection was performed using the resulting cluster ions (AgPd ⁺ ions) as reporter ions.

[Preparation of Palladium Shell Gold Nanoparticles and Alloying by Pulsed Laser Irradiation]
To 30 mL of 1.3 mM trisodium citrate solution, 100 μL of 10 mM palladium chloride solution and 2 mL of 0.5 mM gold nanoparticle dispersion prepared in Example 1 were added. 500 μL of 0.1 M ascorbic acid solution was added at room temperature and reacted for 30 minutes. The reaction solution was irradiated with a laser beam (532 nm, 10 ns, 20 Hz, 250 mW) for 15 minutes using an Nd-YAG laser (manufactured by NEWWAVE Research). After centrifugation at 5000×G for 10 minutes, the precipitate was dispersed in 16 mL of 1.3 mM trisodium citrate solution. When the resulting AuPd nanoparticle dispersion was transferred to a screw tube and the spectrum was measured (see FIG. 17), almost no gold surface plasmon band around 520 nm was observed. Thus, AuPd (alloy) nanoparticles with a high palladium ratio were obtained.

[Antibody Modification of Alloy Nanoparticles]
The AuPd nanoparticle dispersion prepared in this example and the AuAg nanoparticle dispersion prepared in Example 1 were diluted with 10 mM phosphate buffer, and the absorbance at 355 nm was adjusted to 0.6 cm ⁻¹ . . As an antibody, the antibody of the PSA detection ELISA kit (Human PSA ELISA Kit, manufactured by Abcam, ab264615) was used.

To 450 μL of the AuPd nanoparticle dispersion, 50 μL of 10 mM phosphate buffer was added, 50 μL was added to the anti-PSA antibody solution (capture), and left standing at 25° C. for 30 minutes. 50 μL of 10 mM phosphate buffer was added to 450 μL of the AuAg nanoparticle dispersion, 50 μL was added to the anti-PSA antibody solution (detective), and the mixture was allowed to stand at 25° C. for 30 minutes.

50 μL of 1 wt % bovine serum albumin (BSA) was added to each alloy nanoparticle dispersion and allowed to stand for 30 minutes. Add 1 wt% poly(ethylene glycol) (molecular weight 5000), allow to stand for 5 minutes, centrifuge at 1000 x G for 10 minutes, centrifuge the supernatant twice at 5000 x G, and combine the three precipitates. Dispersed in 200 μL of 10 mM phosphate buffer.

[Immune detection]
20 μL each of the two types of alloy nanoparticle dispersions were mixed, 10 μL of solutions containing PSA with different concentrations were added, and 10 μL of carp blood was added as necessary, and allowed to stand for 1 hour. The sample solution was diluted 10-fold (500 μL) with water, cast on an ITO substrate, and subjected to mass spectrometry.

Mass spectrometry was performed using Autoflex Speed (manufactured by Bruker) (number of laser shots: 100, laser power: 90%, reflector voltage: 9.9)

(result)
FIG. 18 shows the mass spectrum of the sample without PSA. Three strong signals from Ag ²⁺ (m/

z

214, 216, 218) were observed from samples without PSA, and only weak noise was observed at the other m/z. FIG. 19 shows the mass spectrum of a sample to which 10 μL of 4 ng/mL PSA solution was added. When PSA was added to the sample solution, the peak intensities of m/

z

212, 213, 215, 217 and 219 increased, and signals of AgPd ⁺ ions could be observed.

FIG. 20 shows the signal intensity of AgPd ⁺ ions (m/z 215) versus the concentration of added PSA solution. The signal intensity at m/z 215 increased depending on the amount of PSA added. AgPd ⁺ ions were found to function as reporter ions for antigen-antibody reactions. As shown in FIG. 21, the sample to which 10 ^μL of carp whole blood was added decreased the signal intensity, but the tendency of the signal intensity to increase depending on the PSA concentration did not change. It was found that immunodetection using reporter ions is possible.

(Example 4: Examination of acceleration of analysis)
The sample solution is concentrated on the substrate and dried. Without waiting for the antigen-antibody reaction to proceed in the solution, the reaction proceeds during concentration and drying, enabling rapid analysis.

As in Example 1, AuPd nanoparticles and AuAg nanoparticles were modified with goat IgG and normal goat IgG, respectively. The sample mixed with the modified nanoparticle dispersion was subjected to mass spectrometry at predetermined time intervals under the same conditions as in Example 1.

(result)
FIG. 22 shows mass spectra for each elapsed time after mixing. FIG. 23 shows signal intensity (m/z 217) versus time elapsed after mixing. Even when casting and measuring immediately after mixing, AgPd ⁺ ions (m/z 217) could be detected with about half the intensity when waiting for 60 minutes. The concentration of nanoparticles is 4.4×10 ⁻¹² M. Given sufficient signal intensity, little waiting time is required after sample mixing. In order to obtain the ultimate detection sensitivity, it is better to wait until the antigen-antibody reaction occurs sufficiently in the solution. signal can be detected even

(Example 5: Preparation of platinum nanoparticles and ionization behavior)
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, 550 μL of 0.08 wt % NaBH ₄ was added, 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 a dispersion. The dispersion liquid was cast on an ITO substrate and subjected to mass spectrometry under the same conditions as in Example 1.

(result)
FIG. 24 shows platinum nanoparticles imaged by TEM. The particle size of the platinum nanoparticles obtained from the TEM image was 4.9±1 nm. FIG. 25 shows a mass spectrum obtained by measuring the 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 it has been shown that if the laser light irradiation intensity is sufficient, ions are desorbed at a level at which cluster ions with silver or palladium can be detected. rice field.

Various embodiments and modifications of the present invention are possible without departing from the broad spirit and scope of the present invention. Moreover, the embodiment described above is for explaining the present invention, and does not limit the scope of the present invention. That is, the scope of the present invention is indicated by the claims rather than the embodiments. Various modifications made within the scope of the claims and within the meaning of equivalent inventions are considered to be within the scope of the present invention.

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

The present invention is suitable for detecting trace components.

1, 2 carrier, 10, 20 probe, 11, 21 metal

Claims

a first carrier carrying a first probe that binds to a detection target and containing a first metal;
a second carrier carrying a second probe that binds to the detection target and containing a second metal different from the first metal;
with
When energy is supplied to the first carrier and the second carrier that are close to each other by binding to the detection target through the first probe and the second probe, unique ions are desorbed.
detection kit.
The first carrier is a gold-silver alloy nanoparticle,
wherein the second carrier is a gold-palladium alloy nanoparticle;
The detection kit according to claim 1.
The first carrier is a gold nanoparticle,
The second carrier is platinum nanoparticles,
The detection kit according to claim 1.
The first probe and the second probe are
An antibody that specifically binds to the detection target,
A detection kit according to any one of claims 1 to 3.
carrying a first probe that binds to the target of detection, carrying a first carrier containing a first metal, and carrying a second probe that binds to the target of detection, a second metal different from the first metal a binding step of binding a second carrier containing a metal to the detection target 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 detection target;
Detection of unique ions that are desorbed by supplying the energy to the first carrier and the second carrier that are adjacent to each other by binding to the detection target through the first probe and the second probe. a detection step for
A method of detection, including