WO2019232797A1 - 磁珠-核酸适配体-多靶分子同时检测的方法及试剂盒 - Google Patents

磁珠-核酸适配体-多靶分子同时检测的方法及试剂盒 Download PDF

Info

Publication number
WO2019232797A1
WO2019232797A1 PCT/CN2018/090472 CN2018090472W WO2019232797A1 WO 2019232797 A1 WO2019232797 A1 WO 2019232797A1 CN 2018090472 W CN2018090472 W CN 2018090472W WO 2019232797 A1 WO2019232797 A1 WO 2019232797A1
Authority
WO
WIPO (PCT)
Prior art keywords
detection
nucleic acid
aptamer
magnetic bead
molecular
Prior art date
Application number
PCT/CN2018/090472
Other languages
English (en)
French (fr)
Inventor
廖世奇
曾家豫
廖正宇
栗怡
袁红霞
魏正丽
Original Assignee
Liao Shiqi
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Liao Shiqi filed Critical Liao Shiqi
Priority to DE112018007705.5T priority Critical patent/DE112018007705T5/de
Priority to AU2018426414A priority patent/AU2018426414A1/en
Priority to KR1020207034485A priority patent/KR20210013569A/ko
Priority to PCT/CN2018/090472 priority patent/WO2019232797A1/zh
Priority to CN201880003651.2A priority patent/CN110168376A/zh
Priority to US16/658,092 priority patent/US11293918B2/en
Publication of WO2019232797A1 publication Critical patent/WO2019232797A1/zh

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • G16B40/10—Signal processing, e.g. from mass spectrometry [MS] or from PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1048—SELEX
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2541/00—Reactions characterised by directed evolution
    • C12Q2541/10—Reactions characterised by directed evolution the purpose being the selection or design of target specific nucleic acid binding sequences
    • C12Q2541/101—Selex

Definitions

  • the invention relates to a method and a kit for simultaneous detection of magnetic beads-nucleic acid aptamers-multi-target molecules, in particular to a method for capturing marker molecules in a sample by using a marker nucleic acid aptamer group of a multi-molecular sample and using laser analysis to fly Qualitative and quantitative detection of detection markers by mass spectrometry, and then using the analysis and processing of the data platform to obtain the detection and analysis results of the comprehensive system of the sample, specifically related to the detection of the overall characteristic molecules of the sample and the analysis of the comprehensive system.
  • the antibody capture method is a simple and convenient screening method.
  • the antigen is coated on a solid support, then the antibody is used to bind the antigen, the plate is washed to remove the unbound antibody, and the labeled antibody specifically recognized by the bound antibody is used to detect the bound antibody.
  • Many antibody capture methods use Indirect method to detect antibodies.
  • the detection antibody is a murine antibody, and the detection molecule may be a rabbit anti-mouse antibody with a detection label.
  • Traditional detection labels include radioisotopes, dyes, and enzymes that act on substrates to produce detectable molecules such as chromogens.
  • Antigen capture test is to detect the presence or absence of antigen in the sample. First the antibody is bound to the support, then the antigen is added to react with the antibody to form a complex, and finally the complex is detected. It is also possible that the antigen-antibody reacts to form a complex, and then binds to the solid support, and then detects the complex.
  • ELISA is a well-known immunoassay. As soon as it came out in 1971, a revolution in diagnostic methods was initiated. The traditional ELISA technology is similar to the sandwich sandwich method, in which two antibodies bind to a certain antigen together. The capture antibody binds to the antigen in the sample, and then adds the detection antibody of the coupling enzyme that binds to the antigen to form a capture antibody-antigen-detection antibody "sandwich” complex. Finally, the activity of the coupling enzyme is measured to show the detection result.
  • the antibody detection method has great application value, but its detection range is limited by the Kd value of the capture antibody and antigen reaction. In practice, the detection bottom line is about 1% of the Kd value. When the concentration of the analyte decreases to this possible detection In a limited time, the percentage of antibody captured is not sufficient to produce a detectable signal relative to the signal-to-noise ratio. Therefore, the detection limit of the antibody detection method using a fluorescence or chemiluminescence detection system is about 1 pg / ml (10-4M versus a protein having an average molecular weight of 50,000 daltons).
  • nucleic acids and proteins interact in cells. Nucleic acids can fold to form secondary and tertiary structures, which is very important for their interaction with proteins. Methods for in vitro detection of nucleic acid-protein interactions have matured by diversifying nucleotide sequences. SELEX technology is used to isolate the nucleotide ligands of the selected target. These ligands are called ligands or aptamers, which means that the nucleic acid can form a certain structure and fit into the pocket of the target molecule. SELEX technology uses this Method for screening target molecule ligands in principle.
  • Gold et al. In 1995 (Gold L, et al. Annu Rev Biochem, 64: 763--797) applied the RNA and ssDNA ligands of systemic lupus erythematosus specific antibodies screened by SELEX to not only diagnose systemic lupus erythematosus, Moreover, condition monitoring and curative effect tests are performed. Gold et al. (1999) (Gold L, et al. Diagn Dec; 4 (4): 381-8) in the study of the molecular diagnostics of ligand microarrays, researched the resolution of the ligand microarrays, all showed nucleic acids Ligand detection has huge application prospects.
  • RNA molecules After the start of the SELEX technology, many target ligands have been screened.
  • proteins known to bind nucleic acids can be used as suitable targets for the SELEX technology, such as T4DNA polymerase, phage R17 envelope protein, and E. coli rho.
  • Factors E. coli ribosomal protein S1, phenylalanine-tRNA synthetase, autoimmune antibodies that recognize RNA, E2F transcription factors, different HIV-related proteins.
  • ligands can be obtained by SELEX technology in vitro screening. See US patents WO96 / 40991 and WD97 / 38134 for enzyme-linked oligonucleotide methods in which nucleotide ligands are used instead of the detection or capture antibodies in the sandwich method.
  • the sandwich method uses a conventional enzyme-linked detection antibody to detect the capture molecule-antigen complex, while labeling the oligonucleotide with a reporter enzyme molecule requires a chemical synthesis step and additional labor.
  • the above-mentioned method using an antibody reagent also has difficulties in itself.
  • MALDI-TOF MS is a new soft ionization organic mass spectrometer developed in recent years. It has become a powerful tool for the detection and identification of peptides, proteins, polysaccharides, nucleotides, glycoproteins, polymers and a variety of synthetic polymers. The principle is: when a laser beam of a certain intensity is irradiated to the co-crystal film formed by the sample and the substrate The matrix absorbs energy from the laser, and a charge transfer occurs between the matrix and the sample to ionize the sample molecules. The ionized sample accelerates through the flight tube under the action of the electric field, and is detected based on the time of flight to the detector.
  • the mass-to-charge ratio is proportional to the ion's flight time to detect the ion.
  • the central technology of MALDI-TOF-MS is to perform detection according to the mass-to-charge ratio (m / z) of the sample, and to determine the molecular weight of the sample molecule.
  • MALDI-TOF MS has the characteristics of high sensitivity, high accuracy, high resolution, concise map, wide mass range and fast speed. It is easy to prepare samples in operation, can be miniaturized, large-scale, Parallel and highly automated processing of biological samples to be tested, and has special advantages in the application of the determination of biological macromolecules and synthetic polymers. In recent years, it has become a powerful tool for the detection and identification of peptides, proteins, polysaccharides, nucleotides, glycoproteins, polymers, and many synthetic polymers.
  • MALDI-TOF MS can be used to determine the peptide mass fingerprint (PMF) of protein enzymatic hydrolysis, post-source fragmentation (PSD) fragment ion spectrum, and combined with mass spectrometry network database search to obtain the sequences of peptides and proteins.
  • PMF peptide mass fingerprint
  • PSD post-source fragmentation
  • MS mass spectrometry network database search
  • SNPs genomic single nucleotide polymorphisms
  • the detection of serum tumor markers can be used as a non-invasive diagnostic method, which plays an important role in the diagnosis of tumors. Therefore, a simple, fast, sensitive and convenient method for the detection of serum tumor marker groups is established. Is the key to early diagnosis and prevention of tumors.
  • the purpose of the present invention is to propose a method that involves using a nucleic acid ligand group to catch a marker group in a detection solution, and separating the marker group, and then qualitatively and quantifying the marker group through MALDI-TOF MS and bioinformatics Related protocols for omics analysis can be used for the detection of composite samples such as serum and body fluids.
  • a solution of the present invention is achieved by the following technical solution: a method for simultaneous detection of magnetic beads-aptamers-multitarget molecules for simultaneous detection and analysis of multiple markers, the method includes the following steps:
  • the specimen in step (1) includes at least one of serum, urine, body fluids, and cell and tissue suspensions.
  • the aptamer in step (1) is a specific nucleic acid aptamer group obtained by screening the specimen using an index-enriched ligand system evolution technology.
  • the elution efficiency of separating the aptamer and the target molecule by acid elution in step (2) is more than 98%.
  • the standard peptide in step (2) refers to a reference standard peptide carried in a mass spectrometry sample for accurate qualitative and quantitative determination of a specified protein peptide.
  • the target molecule in step (3) is a target molecule with a specific marker obtained through experimental screening.
  • step (4) infers a functional mechanism based on the dose-effect relationship of the specified protein detected, and makes an analysis report.
  • the specimen in step (1) is prepared by the following method:
  • the magnetic bead-aptamer-target molecule complex is formed by: incubating 100 ⁇ L of the specimen with 50 ⁇ L of the magnetic bead-aptamer at 37 ° C. for 30 minutes, After 3 ⁇ SSC washing once, 20 times volume of 0.4mM Binding Buffer washing 3 times, magnetic separation was obtained.
  • step (2) the following steps are included: adding 0.5 ml of elution buffer to the magnetic bead-aptamer-target molecule complex in step (1), shaking for 1 min, collecting the eluate, and immediately Add 250 ⁇ L of TrisHCL, pH 8.0 and 1M to neutralize, repeat the elution three times, and mix the eluted neutralization solution three times to obtain the mass spectrometer detection solution.
  • kits for simultaneous detection of magnetic beads-aptamers-multi-target molecules including:
  • Reagent 1 5-10mL, 0.01-0.1M phosphate buffer solution with pH value of 7.4, 50% streptavidin agar magnetic bead particles with a particle size of 5-50nm are dissolved therein;
  • Reagent 2 5-10 mL of 0.4 mM 1 ⁇ Binding Buffer buffer with a pH of 7.4, which contains 0.003-0.3ug / L biotinylated specific nucleic acid aptamer;
  • Reagent 3 Elution buffer.
  • the elution buffer includes glycine, sodium chloride and Tween20.
  • a magnetic bead-nucleic acid aptamer-mass spectrometry multi-molecule detection and analysis system includes:
  • At least one first reagent comprising a magnetic bead separation carrier having a specific nucleic acid aptamer group on the surface;
  • At least one second reagent comprising a molecular eluent
  • At least one instrument detection platform including a mass spectrometer
  • At least one data platform including data analysis instruments and corresponding software
  • the specific nucleic acid aptamer group is a specific nucleic acid aptamer group selected for a specific molecule of a complex sample, and the magnetic bead separation vector is coated by the specific nucleic acid aptamer group to a magnetic field. Bead formation and specific binding marker capacity;
  • the instrument detection platform is used to detect the content of specific molecules in the sample by washing the neutralization solution of the bound magnetic beads and analyzing the mass spectrometer by laser analysis;
  • the data analysis software is used to analyze the specific target molecular weight-effect relationship detected by mass spectrometry to determine molecular interrelationships and functional correlations.
  • the set of specific nucleic acid aptamers is obtained by screening the exponentially enriched ligand evolution system.
  • the mass spectrometer is used for qualitative and quantitative detection of multi-target molecules bound by the specific nucleic acid aptamer group.
  • the data analysis software is based on the molecular mechanism of the onset of disease in a certain disease test, the nature and content of specific markers, and the data analysis software is used to analyze and determine the probability of the onset of disease and the reference result of the pathogenesis.
  • the detection and analysis system is used for an analysis report system provided by an automatic analysis system for a certain disease and biological function based on the quantitative response of the qualitative and quantitative detection of characteristic molecules.
  • nucleic acid ligand signals Using magnetic beads as a carrier, specific nucleic acid aptamers on the magnetic beads are combined with target molecules (non-nucleic acid molecules), and then qualitative and quantitative analysis is performed by mass spectrometry, and data analysis is used to obtain omics analysis results.
  • the detection and / or bioinformatics analysis of nucleic acid ligand signals has the characteristics of fast, high sensitivity, strong specificity, complete multi-ligand group microarray detection, inductive analysis, simple human operation, and can be completed by mechanization.
  • the carrier Using magnetic beads (agar magnetic beads, etc.) as a carrier, the carrier has target molecules attached to it, and magnetically separates the detection molecules. Mechanical samples can be added, separated, eluted, incubated, etc., and the characteristic spectrum of related molecules can be obtained by qualitative and quantitative analysis by mass spectrometry Using the processing analysis of the data platform gives the corresponding results.
  • magnetic beads agar magnetic beads, etc.
  • Agar magnetic beads have the characteristics of low cost, good biocompatibility, and can maintain the excellent spatial results of binding molecules. Its structure is surrounded by agar around the paramagnetic magnet (iron tetroxide), closed by skimmed milk powder, plus active groups Active groups such as carboxyl, epoxy, and amino groups have a good ability to bind biomolecules.
  • the specific nucleic acid aptamer of the present invention is a specific nucleic acid aptamer molecule obtained by screening exponentially enriched ligand evolution system (SELEX) technology for different samples as required, and the nucleic acid aptamer can bind to the sample
  • the specific molecule in the molecule is formed by the abnormal function of the organism in the sample, so the qualitative and quantitative analysis of the molecule can indirectly determine the abnormal function (ie, the pathogenesis) produced by the organism.
  • washing solution is a strongly ion acidic eluent (0.1M glycine, 0.5M sodium chloride, pH 3.0, 0.05% Tween20), shake for 1min, and use magnetic binding to collect the eluate.
  • the data platform is based on the specific aptamers obtained by SELEX screening, the specific molecules obtained using aptamers are extracted, and pathogenesis functions such as diseases are obtained through research, and then specific marker proteomes in the functions are selected, and the protein properties in the marker proteome And quantity can determine the onset and / or physiological mechanism.
  • the data platform is an analysis platform established according to the relationship between the nature and quantity of each molecule measured by mass spectrometry and the pathogenesis and / or physiological mechanism.
  • the detection instrument used can complete all the detection processes mechanized, including: automatic sample loading, incubation, washing, relative qualitative and quantitative detection by mass spectrometer, data platform for data processing, and reporting. Adding samples, adding reagents, washing, mass spectrometry detection and multi-molecular data processing, result analysis, etc., can be fully mechanized and highly mechanized; through the multi-molecule detection of samples, a large amount of information can be stored, which can fully reflect the overall situation of the body and basic functions mechanism.
  • the specific nucleic acid aptamer of the present invention is a specific nucleic acid aptamer molecule obtained by screening exponentially enriched ligand evolution system (SELEX) technology for different samples as required; the nucleic acid aptamer can bind to a sample
  • the specific molecule in the molecule is formed by the abnormal function of the organism in the sample, so the qualitative and quantitative analysis of the molecule can indirectly determine the abnormal function (such as the pathogenesis) produced by the organism; mass spectrometry detection is very good
  • Molecular detection technology can systematically detect the specific target molecules of the sample caught by the aptamer, and maintain the dose-effect relationship between the molecules, which is very helpful to comprehensively understand the function of the biological body.
  • the invention has high sensitivity and strong specificity, and uses specific binding of ligand ligands to enhance the specificity of detection.
  • Mass spectrometry detection has a large amount of collected data and has an accurate information collection and processing system. Since the information acquisition and processing system of the instrument of the present invention is completed by mass spectrometry detection, accurate data reports are available.
  • the instrument used in the test is single, the operation process is simple, and the data collection and processing can be completed in a completely closed condition, which effectively ensures the safety and cleanness of the environment. Since the ligand and the ligand can be incubated for 45 minutes at room temperature during the reaction to complete the binding reaction between the ligand and the ligand, the operation is simple and convenient for general application in general laboratories or clinical laboratory departments; The kit or the constructed biochip can be widely used in basic research and clinical testing, and can bring certain economic and social benefits.
  • the kit for simultaneously detecting proteins (such as non-nucleic acid target molecules) and genes using the magnetic bead-nucleic acid aptamer-mass spectrometry multi-molecule detection method of the present invention utilizes nucleic acid beacon ligand molecules (the molecule has been applied for) "New Ligand Detection Method" (CN1521272 patent) ligands are combined with protein ligands to form ligand-nucleic acid beacon ligand molecular complexes, which converts target molecule ligand information into nucleic acid beacon information and enables target molecule information It is unified with the nucleic acid gene information, and then real-time quantitative-PCR is used to simultaneously detect the target molecule and the nucleic acid gene.
  • the kit of the present invention uses specific aptamer groups and magnetic separation technology to extract and separate target molecules. The difference is that the target molecule information is collected using mass spectrometry, and the multiple molecule information is collected against the composite sample to achieve multiple targets. Qualitative and quantitative quantification of molecules can better provide complete data information for understanding the function of organisms.
  • the kit not only has simple operation method and low cost, but also has the characteristics of fast detection, high sensitivity, strong specificity, large amount of molecular information, and mechanization. The study of the omics of pathogens, genes and proteins is of great significance.
  • FIG. 1 is a schematic diagram of Embodiment 1 of the present invention.
  • FIG. 2 is a schematic diagram of Embodiment 2 of the present invention.
  • FIG. 3 is a schematic diagram of Embodiment 3 of the present invention.
  • the method for simultaneous detection of multiple markers in this embodiment includes the following steps:
  • the magnetic beads are eluted with a specific marker bound by an alkaline buffer solution, and neutralized by adding 1/2 volume of an acidic buffer solution to form a target molecule extraction solution;
  • kit includes the following reagents:
  • Reagent 1 5-10mL, 0.01-0.1M phosphate buffer solution with pH value of 7.4, 50% streptavidin agar magnetic bead particles with a particle size of 5-50nm are dissolved therein;
  • Reagent 2 5-10 mL of 0.4 mM 1 ⁇ Binding Buffer buffer with a pH of 7.4, which contains 0.003-0.3ug / L biotinylated specific nucleic acid aptamer;
  • Reagent 3 Elution buffer.
  • the agar magnetic beads are connected to streptavidin through a carboxyl group (or an epoxy group), and then a target molecule binding carrier (preservative containing 0.01% sodium azide) formed by biotinylated aptamers is connected;
  • 0.4mM Banding Buffer with a pH value of 7.4 is a buffer solution containing aptamers and target molecules (preservative containing 0.01% sodium azide); (400000ml 1 ⁇ BB preparation: 138mmol / L NaCl, 2.7mmol / L KCl , 8.1mmol / L Na2HPO4, 1.1mmol / L KH2PO4, 1mmol / L MgCl2, adjust the pH of the solution to 7.4 with HCl, add water to volume to 1L, autoclave for 20min, and store at room temperature).
  • the magnetic bead-nucleic acid aptamer-mass spectrometry multi-molecule detection method of this embodiment uses magnetic beads coated with specific aptamers to separate and extract a target molecule group, and performs mass spectrometry and data platform analysis. Given the test results, the method includes the following steps:
  • Specimen preparation Take the required blood volume according to the venipuncture method, immediately remove the needle, inject the blood into the test tube containing the anticoagulant, and immediately shake it gently to mix the blood and the anticoagulant to prevent blood clotting. , And then centrifuge at 3000-6000 rpm to isolate the serum as a specimen;
  • Mass spectrometry detection The target molecular solution is qualitatively and quantitatively obtained by mass spectrometry.
  • Example 1 Simultaneous detection of multiple proteins and genes to reduce the window period method
  • Acid elution Add 0.5ml of elution buffer (0.1M glycine, 0.5M sodium chloride, pH3.0, 0.05% Tween20), shake for 1min, collect the eluate, and immediately add 250 ⁇ L 1M TrisHCL (pH8.0) The elution was repeated three times and the eluate was collected separately.
  • elution buffer 0.1M glycine, 0.5M sodium chloride, pH3.0, 0.05% Tween20
  • Mass spectrometric detection qualitative and quantitative detection of multiple proteins (P24 antigen, P24 antibody, CD4 and virus envelope antibody) using a mass spectrometer.
  • serum was added to nucleic acid extraction magnetic beads, lysed by lysate, magnetic beads washed and eluted, and then reverse transcription-PCR amplified.
  • the detection method can simultaneously detect and analyze RNA, p120 and p24 antigens, CD4, membrane protein antibodies and molecules of the antigen, and can comprehensively understand the patient's condition and give the patient a reasonable treatment plan.
  • Non-gastric cancer serum as the control
  • aptamers were obtained by screening the exponentially enriched ligand evolution system.
  • the same aptamers of three tumors were selected as tumor marker aptamers. .
  • Acid elution Add 0.5ml of elution buffer (0.1M glycine, 0.5M sodium chloride, pH3.0, 0.05% Tween20), shake for 1min, collect the eluate, and immediately add 250 ⁇ L 1M TrisHCL (pH8.0) ( Neutralize the standard peptide containing the target molecule for detection, repeat the elution three times, and collect the eluate as a mixture of multiple tumor markers.
  • elution buffer 0.1M glycine, 0.5M sodium chloride, pH3.0, 0.05% Tween20
  • 250 ⁇ L 1M TrisHCL pH8.0
  • Mass spectrometry detection qualitative and quantitative detection of multiple target molecules using mass spectrometers and standard peptide molecules in the detection solution.
  • Marker target molecules extracted from three tumor aptamers were identified as common marker molecules by mass spectrometry as tumor markers, confirming the occurrence of tumors; special marker molecules possessed by three tumors, Marker for tumor classification.
  • Gastric cancer serum (non-gastric cancer serum as a control) was used as a composite target molecule, and an exponentially enriched ligand evolution system was used to screen aptamers to obtain gastric cancer marker aptamers.
  • Acid elution Add 0.5ml of elution buffer (0.1M glycine, 0.5M sodium chloride, pH3.0, 0.05% Tween20), shake for 1min, collect the eluate, and immediately add 250 ⁇ L 1M TrisHCL (pH8.0) ( Neutralize the standard peptide containing the target molecule for detection, repeat the elution three times, and collect the eluate as a mixture of multiple tumor markers.
  • elution buffer 0.1M glycine, 0.5M sodium chloride, pH3.0, 0.05% Tween20
  • 250 ⁇ L 1M TrisHCL pH8.0
  • Mass spectrometry detection qualitative and quantitative detection of multiple target molecules using mass spectrometers and standard peptide molecules in the detection solution.
  • the invention provides a method and a kit for simultaneous detection of magnetic beads-aptamers-multi-target molecules, belonging to the field of molecular biology. It includes the screening of specific nucleic acid aptamers, the quantitative and qualitative detection of target molecule groups by mass spectrometry, and the data processing and analysis report of the data platform to achieve the detection of related multi-target molecule groups. A clearer response to the development of the body, or the overall development of a function.
  • the advantage of the invention is that specific target nucleic acid is used to catch related target molecule groups, and qualitative and quantitative analysis through mass spectrometry can relatively completely reflect the function change of the body or tissue; the large amount of data greatly improves the accuracy of omics analysis. Therefore, the invention is of great significance for proteomics and genomics research and clinical molecular detection.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Genetics & Genomics (AREA)
  • General Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Biophysics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Medical Informatics (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Databases & Information Systems (AREA)
  • Artificial Intelligence (AREA)
  • Epidemiology (AREA)
  • Bioethics (AREA)
  • Software Systems (AREA)
  • Public Health (AREA)
  • Data Mining & Analysis (AREA)
  • Evolutionary Computation (AREA)

Abstract

本发明属于分子生物学领域，基于靶分子配体和核酸配基的分子识别，结合磁分离纯化，通过质谱的分子定性定量分析，实现多分子组检测和多功能组信息的统一，能检测出机体或组织的分子组和功能组的相关性。本发明利用磁分离简单完成靶分子组的分离；利用适配体的特异性和高亲和性可有效获得靶分子组；质谱有效的进行多分子的定性定量，实现二次分子检测，提高检测的准确性；多分子的统一定性定量可较真实的反应分子间的相互关系，同时也揭示机体功能的相互关系，利于蛋白质组学和基因组学研究与临床分子检测。

Description

磁珠-核酸适配体-多靶分子同时检测的方法及试剂盒 技术领域
[根据细则91更正 30.08.2018]　
本发明涉及磁珠-核酸适配体-多靶分子同时检测的方法及试剂盒，尤其是涉及一种通过多分子样品的标志物核酸适配体组捕捉样品中的标志物分子利用激光解析飞行质谱对检测标志物进行定性定量，再利用数据平台的分析处理得到样品综合体系的检测分析结果，具体的说是涉及对样品整体特征分子检测和综合体系分析。
背景技术
早期蛋白质检测是以抗体同特定待检物蛋白质的低解离常数和一定的特异性为基础，抗体捕捉方法是简易方便的筛选方法。抗体捕捉法中，抗原包被于固相支持物，然后用抗体去结合抗原，洗板去掉未结合的抗体，最后用和结合抗体特异识别的标记分子去检测结合抗体，许多抗体捕捉法是利用间接法来检测抗体。例如，检测抗体是鼠抗体，检测分子可能是带检测标记的兔抗鼠抗体。传统的检测标记包括放射性同位素，染料和作用于底物产生可检测分子如色原的酶。
抗原捕捉检测法是检测样品中有无抗原。首先抗体先结合到支持物上，然后抗原加入和抗体反应形成复合物，最后检测复合物。也可以抗原抗体先反应形成复合物后，再结合到固相支持物上，然后检测复合物。
ELISA是广为人知的免疫检测法，1971年一问世，就启动了诊断方法的革命，传统的ELISA技术似三明治夹心法，即两抗体共同结合到某一抗原。捕捉抗体同样品中的抗原结合，再加入同抗原结合的偶连酶的检测抗体反应形成捕捉抗体—抗原—检测抗体“三明治”复合物，最后测偶连酶活性显示检测结果。
抗体检测法具有较大的应用价值，但是他的检测范围受捕捉抗体和抗原反应的Kd值限 制，在实践中，检测底线大约是Kd值的1％，当待分析物浓度降低到这个可能检测限时，捕捉到待分析物抗体百分率不足以产生相对于信噪比的可检测信号。因此，用荧光或化学发光检测系的抗体检测法的检测下限约1pg/ml(10-4M对平均分子量50，000道尔顿的蛋白质)。
[根据细则91更正 30.08.2018]　
核酸与蛋白质在细胞内相互作用是普遍现象。核酸能折叠形成二级结构和三级结构，这对其与蛋白质相互结合作用非常重要。通过使核苷酸序列多样化而使体外检测核酸蛋白质相互作用方法成熟。SELEX技术被用来分离所选靶标的核苷酸配体，这些配体被称为配基或适配体，意即核酸可以形成一定结构并适配入靶分子的口袋，SELEX技术就是利用该原理筛选靶分子配基的方法。
[根据细则91更正 30.08.2018]　
Gold等在1995年(Gold L，et al.Annu Rev Biochem,64:763--797)应用SELEX筛选出的系统性红斑狼疮特异抗体的RNA和ssDNA配基，不仅对系统性红斑狼疮进行诊断，而且进行病情监测和疗效检验。Gold等又在1999年(Gold L，et al.Diagn Dec；4(4):381-8)在配基微阵分子诊断应用研究中,对配基微阵分辨率进行研究，均显出核酸配基检测有巨大的应用前景。但是目前的配基检测都是采用直接对配基PCR扩增放大检测的方法。这种方法操作复杂，需要将配体和配基分离，且灵敏度低(由于配体和配基分离后，配基纯度及残留配体和配基的再结合阻断DNA复制)和准确性差。
SELEX技术开始后，许多靶标的核苷酸配基已被筛选出，尤其是已知能和核酸结合的许多蛋白质可作为SELEX技术的较合适靶标，如T4DNA聚合酶、噬菌体R17被膜蛋白，大肠杆菌rho因子，大肠杆菌核糖体蛋白S1，苯丙氨酸-tRNA合成酶，识别RNA的自身免疫抗体，E2F转录因子，不同的HIV相关蛋白。
SELEX技术可筛选出许多不同蛋白配基的事实引发了配基应用的拓展，可作为单抗和多抗产品的替代品应用于诊断和治疗。配基代替抗体应用于诊断已显示其价值。DNA聚合酶的配基已被用于热启动PCR来诊断低拷贝的复制子，提高PCR敏感性和保真性。配基也被用 于促进实验方法。中性弹性蛋白的酶配体荧光标记后用于流式细胞仪检测弹性酶浓度，中性弹性蛋白酶配基尚用于鼠肺炎症诊断模型体内诊断。
在酶联寡核苷酸方法(ELDNA)中，一个或多个抗体被对抗原有高亲和力高特异性的配基取代。这样的配基可通过SELEX技术体外筛选获得，见美国专利WO96/40991和WD97/38134描述了酶联寡核苷酸方法，其中用核苷酸配基代替夹心法中的检测抗体或捕捉抗体。通常，夹心法用传统的酶联检测抗体检测捕捉分子-抗原复合物，然而用报告酶分子标记寡核苷酸，这需要化学合成步骤和额外的劳动。上述用抗体试剂的方法本身也存在着困难。
[根据细则91更正 30.08.2018]　
上两个专利中也提到捕捉分子-靶分子-检测分子复合物中核酸配基PCR扩增检测系统；利用常用报告分子如酶、生物素等的PCR引物来扩增扩增子，这样做是提高了配体的数量，但是也带来了不利：需进一步把扩增的配基和不纯的核苷酸引物二聚体分离。传统的凝胶分离需大量人工劳作。DNA和RNA配基都存在着这样的问题。用标记的引物不能解决不纯问题和引物二聚体扩增问题，因此，无法定量。
[根据细则91更正 30.08.2018]　
尽管上述进步显著，但诊断方法仍需提高灵敏度，减少人工操作，改进动态监测以便快速分析样品中靶标的存在与否及对其定量。
[根据细则91更正 30.08.2018]　
MALDI-TOF MS是近年来发展起来的一种软电离新型有机质谱。已成为检测和鉴定多肽、蛋白质、多糖、核苷酸、糖蛋白、高聚物以及多种合成聚合物的强有力工具，原理是:当用一定强度的激光照射样品与基质形成的共结晶薄膜，基质从激光中吸收能量，基质-样品之间发生电荷转移使得样品分子电离，电离的样品在电场作用下加速飞过飞行管道，根据到达检测器的飞行时间不同而被检测，即测定离子的质量电荷之比与离子的飞行时间成正比来检测离子。MALDI-TOF-MS的中心技术就是依据样品的质荷比(m/z)的不同来进行检测，并测得样品分子的分子量。
根据MALDI-TOF MS的原理，MALDI-TOF MS具有灵敏度高、准确度高、分辨率高、图谱简明、质量范围广及速度快等特点，在操作上制样简便、可微量化、大规模、并行化和 高度自动化处理待检生物样品，而且在测定生物大分子和合成高聚物应用方面有特殊的优越性。近年来已成为检测和鉴定多肽、蛋白质、多糖、核苷酸、糖蛋白、高聚物以及多种合成聚合物的强有力工具。如应用MALDI-TOF MS测定蛋白质酶解的肽质量指纹图谱(PMF)、源后裂解(PSD)碎片离子图谱、并结合质谱网络数据库检索，可获得多肽、蛋白质的序列。应用MALDI-TOF MS对基因组单核苷酸多态性(SNPs)进行分析检测，可区分和鉴别相对分子质量达7,000左右(含20多个碱基)、仅存在1个碱基差别的不同DNA。特别值得指出的是，MALDI-TOF MS已成为生命科学领域蛋白质组研究中必不可缺的重要关键技术之一。
随着科学技术的快速发展，尤其是大数据处理技术进步，只要有足够的数据信息量，就能促进类似人体分子功能机制的深入了解和应用。
发明内容
血清肿瘤标志物的检测可作为一种非介入性的诊断方法，在肿瘤诊断中发挥着重要作用，因此，建立一种简单、快捷、灵敏、便于动态检测的血清肿瘤标志物组的诊断方法，是肿瘤早期诊断和防治的关键。本发明的目的在于提出涉及可以利用核酸配基组钓到检测液中的标志物组，并分离得到标志物组，再通过MALDI-TOF MS和生物信息学等，对标志物组进行定性、定量和组学分析的相关方案，可用于血清和体液等复合样品检测。
本发明的一种方案通过以下技术方案实现：一种磁珠-适配体-多靶分子同时检测的方法，用于多标志物同时检测分析，该方法包括下述步骤：
(1)孵育：磁珠-适配体和标本一起孵育，形成磁珠-适配体-靶分子复合物；
(2)酸洗脱：加入洗脱缓冲液,晃动，收集洗脱液，立即加入内含有检测靶分子的标准肽段的TrisHCL中和；
(3)质谱定性定量检测指定的靶分子；
(4)通过数据平台分析系统进行分析。
优选的，步骤(1)所述标本包括血清、尿液、体液，以及细胞和组织悬液中的至少一种。
优选的，步骤(1)中的适配体是针对所述标本利用指数富集的配基系统进化技术筛选获得的特异性核酸适配体组。
优选的，步骤(2)中酸洗脱分离适配体和靶分子的洗脱效率为98％以上。
优选的，步骤(2)中所述标准肽段是指为准确对指定蛋白肽段进行定性定量，在质谱样本中带进的参照标准肽段。
优选的，步骤(3)所述靶分子是经过实验筛选获得的具有特异性标志的靶分子。
优选的，步骤(4)根据检测到的指定蛋白的量效关系而推测功能机制，作出分析报告。
优选的，步骤(1)所述标本通过以下方法制备：
按静脉穿刺法采取所需血量，立即卸下针头，将血液注入盛有抗凝剂的试管内，立即轻轻摇动，使血液和抗凝剂混匀，以防血液凝固，然后在3000-6000转/min下离心，分离血清即得。
[根据细则91更正 30.08.2018]　
优选的，在步骤(1)中，所述磁珠-适配体-靶分子复合物通过以下方法形成：将100μL所述标本与50μL所述磁珠-适配体一起37℃孵育30分钟，经3χSSC洗涤一次，20倍体积的0.4mM Binding Buffer洗涤3次，磁分离即得。
优选的，在步骤(2)中，包括以下步骤：在步骤(1)所述磁珠-适配体-靶分子复合物中加入0.5ml洗脱缓冲液,晃动1min，收集洗脱液，立即加入250μL、pH为8.0、1M的TrisHCL中和，重复洗脱三次，混合三次洗脱中和液，即得质谱靶分子检测液。
本发明另一种方案通过以下技术方案实现：一种磁珠-适配体-多靶分子同时检测的试剂盒，包括：
试剂1：5-10mL、pH值为7.4的0.01-0.1M磷酸盐缓冲液，内溶50％粒径5-50nm的链霉亲和素琼脂磁珠微粒；
[根据细则91更正 30.08.2018]　
试剂2：5-10mL、pH值为7.4的0.4mM 1×Binding Buffer缓冲液，内溶有0.003-0.3ug/L生物素化的特异性核酸适配体；
试剂3：洗脱缓冲液。
优选的，所述洗脱缓冲液包括甘氨酸、氯化钠和Tween20。
本发明再一种方案通过以下技术方案实现：一种磁珠-核酸适配体-质谱多分子检测分析系统，包括：
至少一第一试剂，该第一试剂包括表面具有特异性核酸适配体组的磁珠分离载体；
至少一第二试剂，该第二试剂包括分子洗脱液；
至少一仪器检测平台，该平台包括质谱仪；
至少一个数据平台，该平台包括数据分析仪器和相应软件；
其中，所述特异性核酸适配体组是针对复合物样品特异性分子筛选出的特异性核酸适配体组，所述磁珠分离载体由所述特异性核酸适配体组包被到磁珠上形成并具特异性结合标志物能力；
所述仪器检测平台用于对结合后的磁珠洗涤中和液，经激光解析飞行质谱检测样品中的特定分子的含量；
所述数据分析软件用于对质谱检测到的特定靶分子量效关系进行分析，以确定分子相互关系及功能相关性。
优选的，所述特异性核酸适配体组由指数级富集的配基进化系统筛选获得。
优选的，所述质谱仪用于对所述特异性核酸适配体组结合的多靶分子进行定性定量检测。
优选的，所述数据分析软件是针对某一疾病检测物中发病分子功能机制，特定标志物的性质和含量，通过数据分析软件进行分析判断得出发病的几率和发病机制的参考结果。
优选的，该检测分析系统用于对某一疾病和生物功能根据特征分子定性定量检测的量 效关系通过自动分析体系给出的分析报告体系。
上述多标志物同时检测并给出结果分析的方法具有以下有益效果：
以磁珠为载体通过磁珠上的特异性核酸适配体与靶分子(非核酸分子)结合，再通过质谱进行定性定量分析，及数据归纳分析得出组学分析结果，可对多种非核酸配体的信号进行检测和/或生物信息学分析，具有快速、灵敏度高、特异性强、完整的多配体组微阵检测、归纳分析和人为操作简单，可全程机械化完成等特点。
利用磁珠(琼脂磁珠等)作为载体，该载体具有附着靶分子，对检测分子进行磁性分离，可机械加样，分离，洗脱，孵育等，再经质谱定性定量得到相关分子的特征谱系，利用数据平台的处理分析给出相应的结果。
琼脂磁珠具有成本低，生物相容性好，能保持结合分子的优异空间结果等特性，其结构是由琼脂包裹顺磁磁铁(四氧化铁)周围，经脱脂奶粉封闭，外加活性基团，如羧基、环氧基和氨基等活性基团，具有很好的结合生物分子的能力。
本发明的特异性核酸适配体是根据需要利用指数级富集的配基进化系统(SELEX)技术，针对不同的样品筛选获得的特异性核酸适配体分子，该核酸适配体可结合样品中的特异性分子，该分子是所在样品的生物体异常功能所形成，所以通过对该分子的定性和定量，可间接判断生物所产生的异常功能(即发病机制)。
[根据细则91更正 30.08.2018]　
利用洗脱液洗涤，该洗涤液为强离子酸性洗脱液(0.1M甘氨酸，0.5M氯化钠，pH3.0，0.05％Tween20)晃动1min，利用磁结合，收集洗脱液，立即加入250μL 1M TrisHCL(pH8.0)中和，重复三次混合在一起提供做质谱。在酸性中和液中加入有特定蛋白肽段的标准样品，为特定蛋白定性定量用。
利用质谱仪和标准肽段对特定蛋白进行定性定量。利用特定的数据分析平台进行数据处理分析，给出参考结果。数据平台是根据SELEX筛选获得的特异性适配体，用适配体提取获得的特异性分子，经研究获得疾病等发病机制功能，再挑选功能中特定标志蛋白组， 通过标志蛋白组中蛋白性质和数量，可判断发病和/或生理机制。数据平台就是根据质谱测得的各分子的性质和数量及发病和/或生理机制的关系，建立的分析平台。
所用检测仪器可机械化完成全部检测过程，包括：自动加样、孵育、洗涤、质谱仪进行相对定性定量检测，数据平台进行数据处理、发报告等。加样、加试剂、洗涤、质谱检测及多分子数据处理，结果分析等，可全机械化完成，机械化程度高；通过样品多分子检测，可储存大量信息，可较全面反映机体整体情况，基本功能机制。
本发明的特异性核酸适配体是根据需要利用指数级富集的配基进化系统(SELEX)技术，针对不同的样品筛选获得的特异性核酸适配体分子；该核酸适配体可结合样品中的特异性分子，该分子是所在样品的生物体异常功能所形成，所以通过对分子的定性和定量，可间接判断生物所产生的异常功能(如发病机制)；质谱检测是非常好的多分子检测技术，可以成体系的检测适配体钓取的样品特定靶分子，并极好的保持各分子间的量效关系，对全面了解生物机体功能具有非常大的帮助。
本发明灵敏度高、特异性强，是利用配体配基的特异结合，使检测的特异性增强；质谱检测采集数据量大，具备准确的信息采集和处理系统。由于本发明仪器的信息采集和处理系统是通过质谱检测完成，故有准确的数据报告。
检测时使用的器皿单一，操作过程简单，数据采集及处理可在全封闭的情况下完成，有效保证环境的安全洁净。由于在反应时只需将配体和配基室温孵育45分钟就能完成配体和配基的结合反应，因此操作简便，便于一般实验室或临床检验科室的普及应用；利用本发明组装的检测试剂盒或构建的生物芯片可以广泛应用于基础研究与临床检测，可带来一定的经济效益与社会效益。
综上所述，本发明的磁珠-核酸适配体-质谱多分子联合检测方法同时检测蛋白(等非核酸靶分子)和基因的试剂盒是利用核酸信标配基分子(该分子已申请《新型配体检测方法》CN1521272专利)的配基与蛋白配体结合，形成配体-核酸信标配基分子复合物，使靶 分子配体信息转换成核酸信标信息，使靶分子的信息与核酸基因信息统一，再利用实时定量-PCR对靶分子和核酸基因进行同时检测。本发明的试剂盒通过采用特异性适配体组，磁分离技术进行靶分子的提取分离，不同点是采用质谱进行多靶分子信息的采集，针对复合样本进行多分子信息的采集，实现多靶分子的定性定量，可更好的为了解生物体功能提供完整的数据信息。该试剂盒不仅操作方法简单，成本低，而且检测技术具有快速、灵敏度高、特异性强、分子信息量大和可机械化完成等特点。对病原体，基因和蛋白质等组学的研究具有重要意义。
附图说明
下面结合附图和具体实施例对本发明作进一步详细说明。
图1为本发明实施例1的示意图；
[根据细则91更正 30.08.2018]　
图2为本发明实施例2的示意图。
[根据细则91更正 30.08.2018]　
图3为本发明实施例3的示意图。
具体实施方式
作为一种示例，本实施例多标志物同时检测的方法包括以下步骤：
(1)将检测标本脱脂，利用降温至4℃20分钟3000转离心，弃去上层油脂后剩余部分。
[根据细则91更正 30.08.2018]　
(2)将处理后的样品加入到0.4mM的Binding Buffer具有特异性核酸适配体组磁珠的缓冲液中结合；
(3)将结合后的磁珠用PH7.4的3χSSC缓冲液洗涤一遍，再用10倍体积的BB洗涤三次；
(4)将磁珠用碱缓冲液洗脱结合的特异性标志物，加入1/2体积酸性缓冲液中和，形成靶分子提取液；
(以上(2)-(4)步骤可由磁珠洗涤提取仪等一次性完成)。
[根据细则91更正 30.08.2018]　
(5)将靶分子提取液经激光解析飞行质谱等检测样品中的特定蛋白分子的含量；
(6)将检测的特定蛋白分子含量，经数据平台分析推理，确定疾病的发病机理和推荐治疗方案。
作为试剂盒的一种示例，其包括以下试剂：
试剂1：5-10mL、pH值为7.4的0.01-0.1M磷酸盐缓冲液，内溶50％粒径5-50nm的链霉亲和素琼脂磁珠微粒；
[根据细则91更正 30.08.2018]　
试剂2：5-10mL、pH值为7.4的0.4mM 1×Binding Buffer缓冲液，内溶有0.003-0.3ug/L生物素化的特异性核酸适配体；
试剂3：洗脱缓冲液。
[根据细则91更正 30.08.2018]　
具体地，琼脂磁珠通过羧基(或环氧基)连接链霉亲和素，再连接生物素化的适配体形成的靶分子结合载体(含0.01％叠氮钠的防腐剂)；
[根据细则91更正 30.08.2018]　
0.4mM pH值为7.4的Banding Buffer是适配体和靶分子结合的缓冲液(含0.01％叠氮钠的防腐剂)；(400000ml 1×BB的配制：138mmol/L NaCl，2.7mmol/L KCl，8.1mmol/L Na2HPO4，1.1mmol/L KH2PO4，1mmol/L MgCl2，用HCl调节溶液的PH值至7.4，加水定容至1L，高压蒸汽灭菌20min，室温保存)。
PH7.4的3χSSC缓冲液洗涤；
碱性洗脱液加入两倍体积的洗脱缓冲液(0.1M甘氨酸，0.5M氯化钠，pH3.0，0.05％Tween20),晃动1min，收集洗脱液；
[根据细则91更正 30.08.2018]　
酸性中和液(含标准肽段试剂)加入1/2倍体积的酸性中和液(250μL 1M TrisHCL(pH8.0))中和。
作为另一种示例，本实施例的磁珠-核酸适配体-质谱多分子检测方法，用包被特异性适配体的磁珠分离提取靶分子组，通过质谱定性定量及数据平台分析，给出检测结果，该方法包括下述步骤：
1.标本制备：按静脉穿刺法采取所需血量，立即卸下针头，将血液注入盛有抗凝剂的试管内，立即轻轻摇动，使血液和抗凝剂混匀，以防血液凝固，然后在3000-6000转/min下离心，分离血清为标本；
[根据细则91更正 30.08.2018]　
2.磁珠-适配体-靶分子复合物的形成：将100μL标本与50μL磁珠-适配体一起37℃孵育30分钟，经3χSSC洗涤一次，20倍体积的0.4mM Binding Buffer洗涤3次，磁分离获得磁珠-适配体-靶分子复合物；
[根据细则91更正 30.08.2018]　
3.靶分子样品的制备：在磁分离的磁珠-适配体-靶分子复合物中加入0.5ml洗脱缓冲液(0.1M甘氨酸，0.5M氯化钠，pH3.0，0.05％Tween20),晃动1min，收集洗脱液，立即加入250μL 1M TrisHCL(pH8.0)中和(含可供定性定量的标准肽段分子)，重复洗脱三次，混合三次洗脱中和液，即为质谱靶分子检测液。
4.质谱检测：通过质谱对靶分子液进行分子定性定量获得靶分子信息。
[根据细则91更正 30.08.2018]　
5.数据平台数据分析和获得分析报告。
下面结合具体实施方式对本发明进行进一步的描述：
实施例1：多蛋白和基因同时检测降低窗口期方法
[根据细则91更正 30.08.2018]　
参考图1：通过对蛋白的定性定量及基因的定性定量分析，得出艾滋病感染病情，如:窗口期、静默期和爆发期等。
(血清)样本制备
[根据细则91更正 30.08.2018]　
首先备齐用物，贴好标签，核对无误后按静脉穿刺法采取所需血量，立即卸下针头，将血液注入盛有抗凝剂的试管内，立即轻轻摇动，使血液和抗凝剂混匀，以防血液凝固。离心3000-6000转/min分离血清为样本，4℃存放。
P24抗原,P24抗体,CD4和病毒包膜抗体适配体的制备
分别以P24抗原,P24抗体,CD4和病毒包膜抗体为靶分子，利用指数级富集的配基进化系统筛选获得的特异性核酸适配体；
磁珠-适配体-多蛋白(P24抗原,P24抗体,CD4和病毒包膜抗体等)夹心复合物的形成
[根据细则91更正 30.08.2018]　
首先取链霉亲和素磁珠,加入生物素化的适配体(包含P24抗原,P24抗体,CD4和病毒包膜抗体等的适配体),孵育30分钟,用含0.05％Tween20的PBS洗涤3次，3分钟/次，即形成磁珠-适配体复合物；取10-100μL的上述琼脂磁珠复合物10-100μL加入血清100-1000μL标本，孵育30分钟,结合形成磁珠-适配体-多蛋白复合物。再利用电磁极吸附磁珠，分离除去未结合的血清，吸出血清后，用洗涤液0.01-0.1M PBS，含Tween200.01-0.1M 400μL，洗涤3-12次，3-6min/次后。
质谱检测液的制备
[根据细则91更正 30.08.2018]　
酸洗脱：加入0.5ml洗脱缓冲液(0.1M甘氨酸，0.5M氯化钠，pH3.0，0.05％Tween20),晃动1min，收集洗脱液，立即加入250μL 1M TrisHCL(pH8.0)中和，重复洗脱三次，分别收集洗脱液。
[根据细则91更正 30.08.2018]　
质谱检测；用质谱仪进行多蛋白定性定量检测(P24抗原,P24抗体,CD4和病毒包膜抗体)。
基因检测，将血清加入到核酸提取磁珠，经裂解液裂解，磁珠洗涤和洗脱，进行反转录-PCR扩增。
数据采集，处理和分析。
打印检测报告。
该检测方法可对RNA、p120和p24抗原、CD4，膜蛋白抗体和抗原的分子同时检测分析，能够对病人病情有全面的了解，给病人一个合理的治疗方案。
实施例二：多肿瘤标志物分子检测方法
[根据细则91更正 30.08.2018]　
参考图2：通过肽段的量效关系，确定肿瘤的发生，和癌症类型：胃癌、肺癌和肝癌等。
(血清)样本制备
[根据细则91更正 30.08.2018]　
首先备齐用物，贴好标签，核对无误后按静脉穿刺法采取所需血量，立即卸下针头，将血液注入盛有抗凝剂的试管内，立即轻轻摇动，使血液和抗凝剂混匀，以防血液凝固。离心3000转/min分离血清为样本，4℃存放。
肿瘤标志物适配体的制备
分别以胃癌、肝癌和肺癌血清为复合靶分子(非胃癌血清为对照)，利用指数级富集的配基进化系统筛选获得适配体，取三种肿瘤相同适配体为肿瘤标志适配体。
磁珠-肿瘤标志物适配体复合物的制备
[根据细则91更正 30.08.2018]　
取链霉亲和素磁珠,加入生物素化的肿瘤标志物适配体,孵育30分钟,用含0.05％Tween20的PBS洗涤3次，3分钟/次，即形成磁珠-适配体复合物；
磁珠-适配体-多肿瘤标志物夹心复合物的制备
[根据细则91更正 30.08.2018]　
取10-100μL的上述琼脂磁珠-肿瘤标志物适配体复合物10-100μL加入血清100-1000μL标本，孵育30分钟,结合形成磁珠-适配体-多蛋白复合物。再利用电磁极吸附磁珠，分离除去未结合的血清，吸出血清后，用洗涤液0.01-0.1M PBS，含Tween20 0.01-0.1M 400μL，洗涤3-12次，3-6min/次后。
多肿瘤标志物的制备
[根据细则91更正 30.08.2018]　
酸洗脱：加入0.5ml洗脱缓冲液(0.1M甘氨酸，0.5M氯化钠，pH3.0，0.05％Tween20),晃动1min，收集洗脱液，立即加入250μL 1M TrisHCL(pH8.0)(含有检测靶分子的标准肽段)中和，重复洗脱三次，收集洗脱液为多肿瘤标志物混合液。
质谱检测；用质谱仪和检测液中的标准肽段分子进行多靶分子的定性定量检测。
数据采集，处理和分析：以三种肿瘤适配体提取的标志靶分子，经质谱确认为共同的标志物分子为肿瘤标志物，确认有肿瘤的发生；三种肿瘤具有的特殊标志物分子，为肿瘤分类标志。
打印检测报告。
实施例三：胃癌发病机制多标志物分子检测方法
[根据细则91更正 30.08.2018]　
参考图2：通过肽段的量效关系，确定肿胃癌的发病机制。
(血清)样本制备
[根据细则91更正 30.08.2018]　
首先备齐用物，贴好标签，核对无误后按静脉穿刺法采取所需血量，立即卸下针头，将血液注入盛有抗凝剂的试管内，立即轻轻摇动，使血液和抗凝剂混匀，以防血液凝固。离心3000转/min分离血清为样本，4℃存放。
胃癌发病机制多标志物分子适配体的制备
以胃癌血清(非胃癌血清为对照)为复合靶分子，利用指数级富集的配基进化系统筛选获得适配体，为胃癌标志物适配体。
磁珠-胃癌标志物适配体复合物的制备
[根据细则91更正 30.08.2018]　
取链霉亲和素磁珠,加入生物素化的胃癌标志物适配体,孵育30分钟,用含0.05％Tween20的PBS洗涤3次，3分钟/次，即形成磁珠-适配体复合物；
磁珠-适配体-胃癌标志物夹心复合物的制备
[根据细则91更正 30.08.2018]　
取10-100μL的上述琼脂磁珠-胃癌标志物适配体复合物10-100μL加入血清100-1000μL标本，孵育30分钟,结合形成磁珠-适配体-胃癌标志物复合物。再利用电磁极吸附磁珠，分离除去未结合的血清，吸出血清后，用洗涤液0.01-0.1M PBS，含Tween20 0.01-0.1M 400μL，洗涤3-12次，3-6min/次后。
多胃癌标志物的制备
[根据细则91更正 30.08.2018]　
酸洗脱：加入0.5ml洗脱缓冲液(0.1M甘氨酸，0.5M氯化钠，pH3.0，0.05％Tween20),晃动1min，收集洗脱液，立即加入250μL 1M TrisHCL(pH8.0)(含有检测靶分子的标准肽段)中和，重复洗脱三次，收集洗脱液为多肿瘤标志物混合液。
质谱检测；用质谱仪和检测液中的标准肽段分子进行多靶分子的定性定量检测。
[根据细则91更正 30.08.2018]　
数据采集，处理和分析，是以胃瘤适配体提取其发病机制中的标志靶分子为参考，经质谱确认分析，归纳得出胃癌的发病的机制。
打印检测报告。
工业应用性
[根据细则91更正 30.08.2018]　
本发明提出了磁珠-适配体-多靶分子同时检测方法及试剂盒，属于分子生物学领域。它包括特异性核酸适配体的筛选，质谱对靶分子组的定量定性检测，以及通过数据平台的数据处理和分析报告，实现相关多靶分子组的检测，通过多靶分子的数据分析，可较清楚的反应机体，或某一功能的整体发生发展的情况。本发明的优点是利用特异性核酸钓取相关的靶分子组，通过质谱定性定量分析，可相对完整反应机体或组织的功能变化；数据量大，极大地提高了组学分析的准确性。因此，该项发明对蛋白质组学和基因组学研究与临床分子检测具有重要意义
应当理解的是，对本领域普通技术人员来说，可以根据上述说明加以改进或变换，而所有这些改进和变换都应属于本发明所附权利要求的保护范围。
上面对本发明专利进行了示例性的描述，显然本发明专利的实现并不受上述方式的限制，只要采用了本发明专利的方法构思和技术方案进行的各种改进，或未经改进将本发明专利的构思和技术方案直接应用于其它场合的，均在本发明的保护范围内。

Claims (16)

  1. 一种磁珠-适配体-多靶分子同时检测的方法，其特征在于，包括下述步骤：
    (1)孵育：磁珠-适配体和标本一起孵育，形成磁珠-适配体-靶分子复合物；
    (2)酸洗脱：加入洗脱缓冲液,晃动，收集洗脱液，立即加入内含有检测靶分子的标准肽段的TrisHCL中和；
    (3)质谱定性定量检测指定的靶分子；
    (4)通过数据平台分析系统进行分析。
  2. 根据权利要求1所述的方法，其特征在于，步骤(1)所述标本包括血清、尿液、体液，以及细胞和组织悬液中的至少一种。
  3. 根据权利要求1所述的方法，其特征在于，步骤(1)中的适配体是针对所述标本利用指数富集的配基系统进化技术筛选获得的特异性核酸适配体组。
  4. 根据权利要求1所述的方法，其特征在于，步骤(2)中酸洗脱分离适配体和靶分子的洗脱效率为98％以上。
  5. 根据权利要求1所述的方法，其特征在于，步骤(2)中所述标准肽段是指为准确对指定蛋白肽段进行定性定量，在质谱样本中带进的参照标准肽段。
  6. 根据权利要求1所述的方法，其特征在于，步骤(3)中所述靶分子是经过实验筛选获得的具有特异性标志的靶分子。
  7. 根据权利要求1所述的方法，其特征在于，步骤(4)根据检测到的指定蛋白的量效关系而推测功能机制，作出分析报告。
  8. 根据权利要求1所述的方法，其特征在于，步骤(1)所述标本通过以下方法制备：
    按静脉穿刺法采取所需血量，立即卸下针头，将血液注入盛有抗凝剂的试管内，立即轻轻摇动，使血液和抗凝剂混匀，以防血液凝固，然后在3000-6000转/min下离心，分离血清即得。
  9. [根据细则91更正 30.08.2018]
    根据权利要求1所述的方法，其特征在于，在步骤(1)中，所述磁珠-适配体-靶分 子复合物通过以下方法形成：
    将100μL所述标本与50μL所述磁珠-适配体一起37℃孵育30分钟，经3χSSC洗涤一次，20倍体积的0.4mM Binding Buffer洗涤3次，磁分离即得。
  10. 根据权利要求1所述的方法，其特征在于，在步骤(2)中，包括以下步骤：
    在步骤(1)所述磁珠-适配体-靶分子复合物中加入0.5ml洗脱缓冲液,晃动1min，收集洗脱液，立即加入250μL、pH为8.0、1M的TrisHCL中和，重复洗脱三次，混合三次洗脱中和液，即得质谱靶分子检测液。
  11. [根据细则91更正 30.08.2018]
    一种磁珠-适配体-多靶分子同时检测的试剂盒，其特征在于，包括：
    试剂1：5-10mL、pH值为7.4的0.01-0.1M磷酸盐缓冲液，内溶50％粒径5-50nm的链霉亲和素琼脂磁珠微粒；
    试剂2：5-10mL、pH值为7.4的0.4mM 1×Binding Buffer缓冲液，内溶有0.003-0.3ug/L生物素化的特异性核酸适配体；
    试剂3：洗脱缓冲液。
  12. 根据权利要求11所述的试剂盒，其特征在于，所述洗脱缓冲液包括甘氨酸、氯化钠和Tween20。
  13. 一种磁珠-核酸适配体-质谱多分子检测分析系统，其特征在于，包括：
    至少一第一试剂，该第一试剂包括表面具有特异性核酸适配体组的磁珠分离载体；
    至少一第二试剂，该第二试剂包括分子洗脱液；
    至少一仪器检测平台，该平台包括质谱仪；
    至少一个数据平台，该平台包括数据分析仪器和相应软件；
    其中，所述特异性核酸适配体组是针对复合物样品特异性分子筛选出的特异性核酸适配体组，所述磁珠分离载体由所述特异性核酸适配体组包被到磁珠上形成并具特异性结合标志物能力；
    所述仪器检测平台用于对结合后的磁珠洗涤中和液，经激光解析飞行质谱检测样品中的特定分子的含量；
    所述数据分析软件用于对质谱检测到的特定靶分子量效关系进行分析，以确定分子相互关系及功能相关性。
  14. 根据权利要求13所述的磁珠-核酸适配体-质谱多分子检测分析系统，其特征在于，所述特异性核酸适配体组由指数级富集的配基进化系统筛选获得。
  15. 根据权利要求13所述的磁珠-核酸适配体-质谱多分子检测分析系统，其特征在于，所述质谱仪用于对所述特异性核酸适配体组结合的多靶分子进行定性定量检测。
  16. 根据权利要求13所述的磁珠-核酸适配体-质谱多分子检测分析系统，其特征在于，该检测分析系统用于对某一疾病和生物功能根据特征分子定性定量检测的量效关系通过自动分析体系给出的分析报告体系。
PCT/CN2018/090472 2018-06-08 2018-06-08 磁珠-核酸适配体-多靶分子同时检测的方法及试剂盒 WO2019232797A1 (zh)

Priority Applications (6)

Application Number Priority Date Filing Date Title
DE112018007705.5T DE112018007705T5 (de) 2018-06-08 2018-06-08 Magnetisches Perlen-Nukleinsäure Aptamer-Multitargetmolekül-Simultan-Nachweisverfahren und Kit
AU2018426414A AU2018426414A1 (en) 2018-06-08 2018-06-08 Method and kit for simultaneous detection of multi target molecules using magnetic bead-aptamer conjugate
KR1020207034485A KR20210013569A (ko) 2018-06-08 2018-06-08 자기 비드-핵산 앱타머-다중 표적 분자를 동시에 검출하는 방법 및 키트
PCT/CN2018/090472 WO2019232797A1 (zh) 2018-06-08 2018-06-08 磁珠-核酸适配体-多靶分子同时检测的方法及试剂盒
CN201880003651.2A CN110168376A (zh) 2018-06-08 2018-06-08 磁珠-核酸适配体-多靶分子同时检测的方法及试剂盒
US16/658,092 US11293918B2 (en) 2018-06-08 2019-10-20 Method and kit for simultaneous detection of multi target molecules using magnetic bead-aptamer conjugate

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/090472 WO2019232797A1 (zh) 2018-06-08 2018-06-08 磁珠-核酸适配体-多靶分子同时检测的方法及试剂盒

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US16/658,092 Continuation US11293918B2 (en) 2018-06-08 2019-10-20 Method and kit for simultaneous detection of multi target molecules using magnetic bead-aptamer conjugate

Publications (1)

Publication Number Publication Date
WO2019232797A1 true WO2019232797A1 (zh) 2019-12-12

Family

ID=67645399

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/090472 WO2019232797A1 (zh) 2018-06-08 2018-06-08 磁珠-核酸适配体-多靶分子同时检测的方法及试剂盒

Country Status (6)

Country Link
US (1) US11293918B2 (zh)
KR (1) KR20210013569A (zh)
CN (1) CN110168376A (zh)
AU (1) AU2018426414A1 (zh)
DE (1) DE112018007705T5 (zh)
WO (1) WO2019232797A1 (zh)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112964868A (zh) * 2021-02-05 2021-06-15 华中农业大学 一种基于磁分离同时检测多种目标物的生化分析方法
CN114942323A (zh) * 2022-04-08 2022-08-26 宁波海壹生物科技有限公司 一种血管内皮生长因子(vegf)检测试剂盒及其制备和使用方法
CN115718134A (zh) * 2021-08-24 2023-02-28 四川大学 一种基于镧系纳米探针同时检测多种乳腺癌生物标志物的分析方法
CN115786326A (zh) * 2022-11-04 2023-03-14 重庆医科大学 一种病毒核酸提取试剂盒及利用该试剂盒提取完整病毒颗粒核酸的方法
CN117169519A (zh) * 2023-10-26 2023-12-05 艾康生物技术(杭州)有限公司 用于检测样本中tt3和/或tt4的解离剂和试剂盒

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113624724A (zh) * 2020-05-07 2021-11-09 廖世奇 一种适配体分子信标对靶分子的多元检测分析方法
WO2022252745A1 (zh) * 2021-06-02 2022-12-08 重庆医科大学 一种免疫分子病毒颗粒检测试剂盒
CN113249449B (zh) * 2021-06-28 2021-10-12 中国农业大学 一种基于延伸发光引物的实时定量PCR测定Kd值的方法
CN114324557B (zh) * 2021-12-03 2024-05-10 融智生物科技（青岛）有限公司 一种基于MALDI-TOF MS的ζ-珠蛋白的检测方法
CN114441749B (zh) * 2022-01-28 2022-09-30 安源基因科技(上海)有限公司 一种新型多元适体分子在磁珠免疫化学发光afp中的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110124015A1 (en) * 2006-07-18 2011-05-26 Weihong Tan Aptamer-based methods for identifying cellular biomarkers
CN104450713A (zh) * 2014-04-11 2015-03-25 中国人民解放军军事医学科学院基础医学研究所 一种特异识别异质性核糖核蛋白A2/B1(hnRNPA2/B1)的寡核苷酸适配体C6-8的序列和应用
CN105018590A (zh) * 2015-01-30 2015-11-04 廖世奇 蛋白配体和基因同时检测试剂盒及应用
CN106353499A (zh) * 2016-09-14 2017-01-25 燕山大学 肺癌血清标志物适配体和基因同时检测试剂盒及应用

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101130814A (zh) * 2007-08-30 2008-02-27 西北师范大学 核酸配基组芯片及其制备方法
JP6195840B2 (ja) 2012-10-22 2017-09-13 ユニバーサル・バイオ・リサーチ株式会社 複数種の目的物質を同時に検出又は定量するための分析方法及び分析キット
CN103134852A (zh) * 2013-02-04 2013-06-05 复旦大学 一种核酸适配体靶上富集和激光酶解检测蛋白质的方法
WO2016018934A1 (en) * 2014-07-29 2016-02-04 Companion Dx Reference Lab, Llc (Texas) Biomarkers and morphology based aptamer selection of same
CN105548127B (zh) 2016-02-03 2018-06-05 环境保护部南京环境科学研究所 能同时大范围原位测量泥水界面溶氧的拼接式检测设备及其检测方法
CN106226523A (zh) * 2016-07-04 2016-12-14 福建广生堂药业股份有限公司 一种免疫标志物的检测方法、试剂和检测试剂盒

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110124015A1 (en) * 2006-07-18 2011-05-26 Weihong Tan Aptamer-based methods for identifying cellular biomarkers
CN104450713A (zh) * 2014-04-11 2015-03-25 中国人民解放军军事医学科学院基础医学研究所 一种特异识别异质性核糖核蛋白A2/B1(hnRNPA2/B1)的寡核苷酸适配体C6-8的序列和应用
CN105018590A (zh) * 2015-01-30 2015-11-04 廖世奇 蛋白配体和基因同时检测试剂盒及应用
CN106353499A (zh) * 2016-09-14 2017-01-25 燕山大学 肺癌血清标志物适配体和基因同时检测试剂盒及应用

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112964868A (zh) * 2021-02-05 2021-06-15 华中农业大学 一种基于磁分离同时检测多种目标物的生化分析方法
CN115718134A (zh) * 2021-08-24 2023-02-28 四川大学 一种基于镧系纳米探针同时检测多种乳腺癌生物标志物的分析方法
CN114942323A (zh) * 2022-04-08 2022-08-26 宁波海壹生物科技有限公司 一种血管内皮生长因子(vegf)检测试剂盒及其制备和使用方法
CN115786326A (zh) * 2022-11-04 2023-03-14 重庆医科大学 一种病毒核酸提取试剂盒及利用该试剂盒提取完整病毒颗粒核酸的方法
CN115786326B (zh) * 2022-11-04 2023-10-03 重庆医科大学 一种病毒核酸提取试剂盒及利用该试剂盒提取完整病毒颗粒核酸的方法
CN117169519A (zh) * 2023-10-26 2023-12-05 艾康生物技术(杭州)有限公司 用于检测样本中tt3和/或tt4的解离剂和试剂盒
CN117169519B (zh) * 2023-10-26 2024-01-30 艾康生物技术(杭州)有限公司 用于检测样本中tt3和/或tt4的解离剂和试剂盒

Also Published As

Publication number Publication date
US11293918B2 (en) 2022-04-05
KR20210013569A (ko) 2021-02-04
US20200116711A1 (en) 2020-04-16
CN110168376A (zh) 2019-08-23
AU2018426414A1 (en) 2020-12-10
DE112018007705T5 (de) 2021-03-25

Similar Documents

Publication Publication Date Title
WO2019232797A1 (zh) 磁珠-核酸适配体-多靶分子同时检测的方法及试剂盒
EP2290100B1 (en) Kits for displacement Sandwich Immuno-PCR
WO2017181339A1 (zh) 蛋白配体和基因同时检测方法及试剂盒
US9983203B2 (en) Method for protein analysis
Minakshi et al. Single-Cell proteomics: technology and applications
CN111777696B (zh) 一种特异性可逆富集新生蛋白质的方法
Hung et al. Microfluidic platforms for discovery and detection of molecular biomarkers
CN112415195A (zh) 检测新型冠状病毒双靶点的试剂盒及其应用
US20200149117A1 (en) Kit and method for detecting target-tumor serum aptamer complex
CN105510591B (zh) 一种利用抗体修饰免疫pcr反应的检测试剂盒及检测方法
WO2008043256A1 (fr) Kit d'essai de détection de groupes de biomarqueurs à variantes ou modifiés avec des groupes d'anticorps et par spectrométrie de masse et procédé associé
WO2015023068A1 (ko) 당단백질의 탈당화 검출을 통한 암 마커 스크리닝 방법 및 간세포암 마커
CN111235243A (zh) 一种肿瘤特异性外泌体/胞外囊泡的定量方法
WO2022201000A1 (en) Biological sample preparation and analysis
CN113528611A (zh) 一种蛋白检测试剂盒及检测方法
WO2017078787A1 (en) Protein quantitation in multicellular tissue specimens
US20240230633A1 (en) Enzyme-conjugated magnetic beads suspended in internal standard buffer
US20230143949A1 (en) Microbeads and uses thereof
Beckman et al. Single-cell proteomics in blood samples
WO2023059731A2 (en) Single molecule assays for ultrasensitive detection of analytes
CN116148365A (zh) 一种SARS-CoV-2 S1蛋白的检测方法
CN116804196A (zh) 一种用于检测hcmv病毒的核酸适配体、试剂盒及应用
CN117025613A (zh) Eb病毒的核酸适配体检测试剂盒
EP3008469A2 (en) Methods for predicting rheumatoid arthritis treatment response
CN113999890A (zh) 基于核酸适配体探针的vegf识别方法及检测vegf的试剂盒

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18921710

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2018426414

Country of ref document: AU

Date of ref document: 20180608

Kind code of ref document: A

122 Ep: pct application non-entry in european phase

Ref document number: 18921710

Country of ref document: EP

Kind code of ref document: A1