WO2022158437A1 - Trousse et procédé de détection - Google Patents

Trousse et procédé de détection Download PDF

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WO2022158437A1
WO2022158437A1 PCT/JP2022/001528 JP2022001528W WO2022158437A1 WO 2022158437 A1 WO2022158437 A1 WO 2022158437A1 JP 2022001528 W JP2022001528 W JP 2022001528W WO 2022158437 A1 WO2022158437 A1 WO 2022158437A1
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carrier
nanoparticles
detection
probe
metal
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PCT/JP2022/001528
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English (en)
Japanese (ja)
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康郎 新留
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国立大学法人 鹿児島大学
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Publication of WO2022158437A1 publication Critical patent/WO2022158437A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode

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  • 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).
  • MALDI Microdetection/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.
  • 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.
  • 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 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.
  • the first carrier is a gold nanoparticle
  • the second carrier is platinum nanoparticles, You can do it.
  • first probe and the second probe are An antibody that specifically binds to the detection target, You can do it.
  • a detection method 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.
  • 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.
  • 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.
  • 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).
  • TEM transmission electron microscope
  • the detection kit 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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 .
  • the carrier is metal nanoparticles or alloy nanoparticles.
  • metal 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.
  • 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.
  • carrier 1 is gold nanoparticles and carrier 2 is platinum nanoparticles.
  • alloy nanoparticles As the alloy nanoparticles, alloy nanoparticles obtained by combining two or more metals related to the above metal nanoparticles are preferable.
  • alloy nanoparticles include AuAg nanoparticles and AuPd nanoparticles.
  • Alloy nanoparticles can be prepared by known methods.
  • 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.
  • a silicate such as silicon dioxide (silica)
  • the carrier may be microparticles, polymers, and the like.
  • 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.
  • probes are adsorbed or covalently attached to a support.
  • 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.
  • the protein binds to the carrier via electrostatic interactions, hydrogen bonds, free thiol groups with high affinity contained in the protein, and the like.
  • 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.
  • an antibody immobilization technique using a protein protein A, protein G, protein A/G or protein L
  • the carrier contains gold
  • peptides with gold-reducing and gold-binding ability may be used.
  • ions 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.
  • 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.
  • 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.
  • the carrier 1 is silica that absorbs manganese ions
  • 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.
  • 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.
  • the energy is supplied by electromagnetic radiation that is absorbed by the nanoparticles.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • the carrier 1 contains AuAg nanoparticles and the carrier 2 contains AuPd nanoparticles
  • 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.
  • 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.
  • 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.
  • the detection method includes a binding step, a supply step, and a detection 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.
  • 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.
  • the supply step energy is supplied as described above to the carrier 1 and carrier 2 bound to the detection target.
  • 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.
  • the above probes may be antibodies.
  • 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.
  • the carrier may be exposed to the sample immobilized on the ELISA plate and irradiated with a pulsed laser.
  • Detection is also possible using other mass spectrometry methods, such as quadrupole mass spectrometers, ion trap mass spectrometers, and Fourier transform mass spectrometers.
  • the detection kit 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.
  • 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.
  • the capture agent is an antibody that specifically binds to the probe.
  • 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.
  • 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.
  • 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.
  • Example 1 AuAg-AuPd
  • 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).
  • 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).
  • Nd-YAG laser manufactured by NEWWAVE Research
  • 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).
  • Nd-YAG laser manufactured by NEWWAVE Research
  • Anti-goat IgG-AuPd nanoparticles were prepared in a total volume of 100 ⁇ L such that their OD 355 values were those of normal goat IgG- AuAg nanoparticles. 100 ⁇ L of the prepared anti-goat IgG-AuPd nanoparticle dispersion and 100 ⁇ L 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.
  • ITO transparent conductive film
  • 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)
  • Example 2 Au-Pt [Preparation of platinum nanoparticles] 50 mL of pure water and 144 ⁇ L 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 ⁇ L 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).
  • 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.
  • Anti-goat IgG-platinum nanoparticles were prepared in a total volume of 100 ⁇ L such that their OD 355 values were the OD 355 values of normal goat IgG-gold nanoparticles prepared in Example 1. 100 ⁇ L of the prepared anti-goat IgG-platinum nanoparticle dispersion and 100 ⁇ L 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.
  • 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.
  • 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 .
  • the antibody of the PSA detection ELISA kit Human PSA ELISA Kit, manufactured by Abcam, ab264615. was used.
  • BSA bovine serum albumin
  • Mass spectrometry was performed using Autoflex Speed (manufactured by Bruker) (number of laser shots: 100, laser power: 90%, reflector voltage: 9.9)
  • 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 ⁇ L 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.
  • FIG. 21 the sample to which 10 ⁇ L 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.
  • Example 2 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.
  • 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 ⁇ L 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 ⁇ L 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.
  • 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.
  • the present invention is suitable for detecting trace components.

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Abstract

La trousse de détection de l'invention comprend un premier support qui supporte une première sonde se liant à un objet à détecter et qui contient un premier métal, et un second support qui supporte une seconde sonde se liant à l'objet à détecter et qui contient un second métal différent du premier métal. Lorsque de l'énergie est fournie au premier support et au second support qui sont arrivés à proximité en se liant à l'objet détecté par le biais de la première sonde et de la seconde sonde, les ions intrinsèques sont désorbés.
PCT/JP2022/001528 2021-01-19 2022-01-18 Trousse et procédé de détection WO2022158437A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015001453A (ja) * 2013-06-15 2015-01-05 新留　康郎 ナノ粒子脱離イオンプローブ質量分析
CN106353394A (zh) * 2016-08-11 2017-01-25 厦门大学 一种电喷雾离子源金属团簇离子的价态分布调节方法
WO2019013464A1 (fr) * 2017-07-10 2019-01-17 재단법인대구경북과학기술원 Procédé de traitement de tissu biologique, dispositif de traitement laser et système d'imagerie par spectrométrie de masse à pression atmosphérique

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015001453A (ja) * 2013-06-15 2015-01-05 新留　康郎 ナノ粒子脱離イオンプローブ質量分析
CN106353394A (zh) * 2016-08-11 2017-01-25 厦门大学 一种电喷雾离子源金属团簇离子的价态分布调节方法
WO2019013464A1 (fr) * 2017-07-10 2019-01-17 재단법인대구경북과학기술원 Procédé de traitement de tissu biologique, dispositif de traitement laser et système d'imagerie par spectrométrie de masse à pression atmosphérique

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