WO2020156029A1 - 一种单分子定量检测方法及检测系统 - Google Patents

一种单分子定量检测方法及检测系统 Download PDF

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WO2020156029A1
WO2020156029A1 PCT/CN2020/070283 CN2020070283W WO2020156029A1 WO 2020156029 A1 WO2020156029 A1 WO 2020156029A1 CN 2020070283 W CN2020070283 W CN 2020070283W WO 2020156029 A1 WO2020156029 A1 WO 2020156029A1
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detection
target molecule
nanoparticles
signal
molecule
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PCT/CN2020/070283
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French (fr)
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官志超
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苏州宇测生物科技有限公司
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Priority to CA3128271A priority Critical patent/CA3128271C/en
Priority to US17/042,175 priority patent/US20210025879A1/en
Priority to CN202080000774.8A priority patent/CN111771126B/zh
Priority to JP2021544827A priority patent/JP7402244B2/ja
Priority to AU2020213460A priority patent/AU2020213460B2/en
Priority to EP20747789.4A priority patent/EP3919907A4/en
Publication of WO2020156029A1 publication Critical patent/WO2020156029A1/zh

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    • 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
    • G01N33/54346—Nanoparticles
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • 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
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/552—Glass or silica
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/583—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with non-fluorescent dye label
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y20/00—Nanooptics, e.g. quantum optics or photonic crystals
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites

Definitions

  • This application relates to a quantitative detection method and detection system based on single molecule counting, which can be used for ultra-high-sensitivity quantitative detection of proteins, nucleic acids and small molecules.
  • biomarkers especially those related to major diseases, in the human body is closely related to a variety of biological processes. These biomarkers have low levels in the early stages of disease occurrence, and traditional methods are difficult to achieve accurate and effective detection and analysis. To achieve early and accurate diagnosis of biomarkers (such as DNA, RNA, or enzymes, etc.), ultra-sensitive quantitative detection methods are needed.
  • the detection principle of traditional detection methods is: detection is based on the optical signal of the whole solution, and the detection The obtained light intensity signal is compared with the standard curve to realize quantitative detection.
  • detection equipment has a certain background noise signal, when the target molecule concentration in the sample is low to a certain level, the overall light intensity signal of the solution will be overwhelmed by the background noise signal, resulting in a large deviation in the low value interval, which is difficult to achieve Accurate quantitative analysis. Therefore, these traditional detection methods are difficult to achieve high-sensitivity detection analysis due to the limitation of detection technology principles.
  • Single-molecule detection or digital single-molecule detection adopts a completely different detection strategy from traditional detection methods. That is, the molecule to be tested (protein, small molecule, or nucleic acid molecule) is directly labeled with enzyme molecules, fluorescent dye molecules, or the molecule to be tested is amplified to achieve single-molecule level signal amplification or signal labeling, and then the detection device is used to The single molecule signal after signal amplification is read, and the number of molecules to be tested is directly counted. Finally, the number of molecules to be tested obtained from the detection and the calibration curve are used to achieve single-molecule level quantitative detection.
  • the molecule to be tested protein, small molecule, or nucleic acid molecule
  • Patent Document 1 CN101438146A
  • Patent Document 2 CN102884431A
  • the present application is made in view of the above-mentioned current situation, and aims to provide a novel single-molecule detection method and single-molecule detection system that can realize single-molecule detection through simple single-molecule signal labeling and has low requirements on optical equipment.
  • the inventor of the present application has repeatedly conducted in-depth research, and as a result, discovered a novel single-molecule quantitative detection method and single-molecule detection system based on imaging, high sensitivity and low cost.
  • a single molecule quantitative detection and analysis method including the following steps:
  • the detection antibody is combined with the in-situ signal-enhancing nanoparticles to form a composite material, and then the composite material is added;
  • the in-situ signal-enhancing nanoparticle contains a luminescent material and a nanoparticle carrier, and the particle diameter is 180-480nm;
  • the particle size of the in-situ signal-enhancing nanoparticles is 200-450 nm.
  • the particle size of the in-situ signal-enhancing nanoparticles is 200-350 nm.
  • the particle size of the in-situ signal-enhancing nanoparticles is 220-350 nm, more preferably 220 nm or more and less than 330 nm, most preferably 220 nm or more and less than 300 nm.
  • the target molecules targeted by the technical solution of the present application include proteins, polysaccharides or small molecules with biological activity.
  • the single-molecule quantitative detection method of the present application can be applied to the detection of proteins and nucleic acids, especially the detection of proteins, with ultra-high sensitivity and extremely low cost.
  • the inventor of the present application was surprised to find that in some embodiments, the sensitivity can reach 1 pg/mL; in other embodiments, the sensitivity can reach 100 fg/mL; in some embodiments, the sensitivity It can reach 10fg/mL; the highest sensitivity can reach 1fg/mL level.
  • this application can achieve detection in a short time (only a few minutes of incubation time) on the basis of ensuring high detection sensitivity, thereby shortening the clinical detection time.
  • the single molecule quantitative detection system of the present application based on a specific detection system (especially using specific in-situ signal-enhanced nanoparticles to label the molecule to be tested), ultra-high sensitivity detection is realized in a simple method.
  • the detection sensitivity of the single-molecule quantitative detection system of the present application is far superior to traditional methods, and an improvement of 2-4 orders of magnitude has been achieved.
  • this application breaks the barrier between protein molecule detection and nucleic acid molecule detection. It can detect protein molecules and nucleic acid molecules with one device, especially for proteins with ultra-high sensitivity. checking.
  • a specific detection system such as specific in-situ signal-enhancing nanoparticles
  • the system does not require expensive equipment such as total internal reflection microscopes, and only requires low-cost photosensitive elements such as CCD or CMOS to realize the molecule to be tested.
  • the in-situ imaging greatly reduces the difficulty of single-molecule detection, the cumbersome steps and the requirements for equipment precision, and the single-molecule detection technology can be applied to the field of scientific research and biomedical detection at a low cost.
  • Figure 1 is a schematic diagram of the protein detection method of the application
  • Figure 2 is a schematic diagram of the nucleic acid detection method of this application.
  • Figure 3 is the standard curve obtained in Example 1 (the ordinate, that is, the CPN (copy number) is the number of single molecule signals);
  • Figure 4 is the standard curve obtained in Comparative Example 1;
  • Figure 5 is the standard curve obtained in Comparative Example 2.
  • Figure 6 is the standard curve obtained in Example 2.
  • Figure 7 is the standard curve obtained in Example 11.
  • Figure 8 is a standard curve obtained in Example 14.
  • Figure 9 is a standard curve obtained in Example 15;
  • Figure 10 is the standard curve obtained in Example 16.
  • Figure 11 is the standard curve obtained in Example 17.
  • Figure 12 is a standard curve obtained in Example 18.
  • Figure 13 is the comparison result of clinical samples in Example 18;
  • Figure 14 is the standard curve obtained in Example 19;
  • Figure 15 is the comparison result of clinical samples in Example 19;
  • Figure 16 is a standard curve obtained in Example 20.
  • Fig. 17 is a standard curve obtained in Example 22.
  • the first embodiment of this application is as follows.
  • the single molecule quantitative detection and analysis method includes the following steps:
  • the in-situ signal-enhancing nanoparticles are directly or indirectly combined with the detection antibody to form a composite material in advance, and then the composite material is added to bind to the second site of the target molecule;
  • the in-situ signal-enhancing nanoparticle contains a luminescent material and a nanoparticle carrier, and the particle diameter is 180-480nm;
  • the solid-phase carrier is used for the separation and cleaning of test samples and reagents.
  • the capture antibody is immobilized on the surface of the solid support through physical adsorption or chemical modification, and can bind to a binding site of a target molecule (hereinafter, sometimes referred to as a test molecule) to separate it from the sample.
  • the detection antibody can bind to another binding site of the molecule to be tested.
  • the in-situ signal-enhancing nanoparticle is directly combined with the detection antibody, which means that the detection antibody is directly adsorbed or coupled to the in-situ signal-enhancing nanoparticle through physical adsorption or chemical modification, so as to realize the in-situ signal-enhancing nanoparticle treatment. Measure the functional modification of molecular recognition and labeling.
  • the “in-situ signal-enhancing nanoparticle indirectly binds to the detection antibody” refers to the specific labeling of the in-situ signal-enhancing nanoparticle by binding to the detection antibody (ie, secondary antibody) or biotin-streptavidin system On the detection antibody.
  • the target molecules include proteins, polysaccharides or small molecules with biological activity and complexes of small molecules and proteins.
  • cTnI antigen IL-6 antigen
  • PCT procalcitonin
  • Sema4D signal 4D
  • Nt-proBNP brain natural peptide amino terminal precursor protein
  • tumor markers Vitamin D, vitamin B, folic acid, vitamin D-BSA complex, folic acid-BSA complex, bacteria and viruses.
  • the solid phase carrier is classified according to morphology, and can be magnetic beads, porous plates, centrifuge tubes, chips, micro-scale microspheres, and nano-scale microspheres, etc.; according to material classification, it can be polymers, silica, silicon or their Composites, etc.; according to their functions, they can be magnetic solid-phase carriers and non-magnetic solid-phase carriers.
  • magnetic beads are particularly preferred. In the past, it was believed that the following problems exist when magnetic beads are applied to protein single-molecule quantitative detection: the magnetic beads are suspended in the solution and difficult to quantitatively detect; due to the directionality of the magnetic beads, there is a difference in brightness during imaging.
  • the inventor of the present application has surprisingly discovered that in the case of wanting to perform quantitative single-molecule detection of proteins, compared with the use of glass chips as the carrier, the use of magnetic beads as the carrier can obtain particularly excellent sensitivity and extremely The earth improves the detection efficiency.
  • the surface of the solid-phase carrier is modified with active functional groups that can be covalently coupled with the antibody, such as hydroxyl, carboxyl, amino, succinimidyl ester, sulfonyl (such as tosyl) and their derivative groups.
  • active functional groups such as hydroxyl, carboxyl, amino, succinimidyl ester, sulfonyl (such as tosyl) and their derivative groups.
  • the capture antibodies are classified according to the specific characteristics of the antibodies, and can be one or two of polyclonal antibodies and monoclonal antibodies.
  • the capture antibodies are classified according to sources, and can be one or more of murine antibodies, rabbit antibodies, goat antibodies, and alpaca antibodies. Specifically, Hytest 19C7, Hytest 20C6, Hytest 16A11, Medix 2703, Meridian M86101M, Biospacific A45160, Biospacific G-131-C, etc. can be cited.
  • the detection antibodies are classified according to the specific characteristics of the antibodies, and can be one or two of polyclonal antibodies and monoclonal antibodies.
  • the detection antibody is classified according to the source, and can be one or more of murine antibody, rabbit antibody, goat antibody and alpaca antibody. Specifically, Hytest 16A11, Medix 2704, Meridian M86201M, Biospacific A45502, etc. can be cited.
  • the anti-detection antibody is classified according to the source of the detection antibody, and can be one or more of anti-mouse antibody, anti-rabbit antibody, anti-goat antibody, and anti-alpaca antibody.
  • the anti-detection antibody is classified according to the source, and can be one or more of mouse-derived secondary antibody, rabbit-derived secondary antibody, goat-derived secondary antibody, and alpaca-derived secondary antibody.
  • the in-situ signal-enhancing nanoparticle refers to a material that enhances the fluorescent signal in-situ to a level that can be detected by conventional optical imaging equipment, and it must contain two parts: a luminescent material and a nanoparticle carrier.
  • the nanoparticle carrier plays a very important role. For example, it can bind more luminescent materials to make the luminescence signal stronger; it provides sites for functional modification, which can bind a large number of antibodies to improve the reaction. Activity; It provides the possibility for conventional fluorescence microscope to realize single molecule detection, which cannot be realized without nanoparticle carrier.
  • the nanoparticle carrier is classified according to the material, and can be one or more of silica, polystyrene, polyacrylamide, polymethyl (meth)acrylate, dextran, agarose, and inorganic metal compounds.
  • the nanoparticle carrier is classified according to the structure, and can be one or more of a hollow structure, a core-shell structure, a porous structure, an alloy structure, and a hydrogel structure.
  • the nanoparticle carrier is preferably silica, polyacrylamide, polystyrene, and dextran, and polyacrylamide is particularly preferred.
  • the luminescent material in the in-situ signal-enhancing nanoparticle is also necessary to realize single-molecule detection, and the sensitivity is extremely low when only the nanoparticle carrier is used.
  • the luminescent material may be one or more of fluorescent dye molecules, rare earth elements, rare earth chelates, fluorescent proteins, quantum dots, and upconversion nanoparticles.
  • the luminescent materials are preferably fluoresceins (such as fluorescein isothiocyanate), rhodamines (such as rhodamine green, rhodamine B, etc.), coumarins, quantum dots (such as CdS, CdSe, CdTe, ZnSe) , Rare earth elements (such as Eu, Ce) and their complexes.
  • the luminescent material is adsorbed or wrapped on the surface or inside of the nanoparticle carrier through one or more of covalent modification, chelation, spatial wrapping, hydrophobic interaction and electrostatic adsorption. It should be noted that from the viewpoint of facilitating optical imaging recognition and improving sensitivity, it is preferable that the luminescent material is uniformly wrapped in the inside of the nanoparticle carrier.
  • the in-situ signal-enhancing nanoparticles are preferably fluorescent particles formed by encapsulating fluorescent dye molecules (such as fluorescein) with silica, fluorescent particles formed by encapsulating fluorescent dye molecules (such as fluorescein) with polyacrylamide, and polyphenylene Fluorescent particles made of ethylene coated quantum dots, fluorescent particles made of polystyrene coated rare earth elements or rare earth chelate, fluorescent particles made of dextran coated fluorescent protein, and fluorescent particles made of cross-linked agarose coated quantum dots Particles etc.
  • fluorescent dye molecules such as fluorescein
  • silica fluorescent particles formed by encapsulating fluorescent dye molecules (such as fluorescein) with polyacrylamide
  • polyphenylene Fluorescent particles made of ethylene coated quantum dots fluorescent particles made of polystyrene coated rare earth elements or rare earth chelate
  • fluorescent particles made of dextran coated fluorescent protein and fluorescent particles made of cross-linked agarose coated quantum dots Particles etc.
  • the surface of the in-situ signal-enhancing nanoparticle is modified with active functional groups that can be covalently coupled with antibodies, DNA or RNA, including hydroxyl, carboxyl, amino, sulfhydryl, alkenyl, alkynyl, and succinimide One or more of the ester group and its derivative group.
  • the surface of the in-situ signal-enhancing nanoparticle can be modified with a connecting arm of a certain length;
  • the connecting arm includes a multi-carbon straight chain, a multi-carbon branched chain, a polymer chain, a peptide chain, a protein, and a nucleic acid molecule.
  • the length of the connecting arm is preferably 1-100 nm, more preferably 2-20 nm, and most preferably 5-10 nm.
  • the particle size of the in-situ signal-enhancing nanoparticles needs to be strictly controlled within the range of 180-480nm, such as 190nm, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm , 320nm, 330nm, 340nm, 350nm, 360nm, 370nm, 380nm, 390nm, 400nm, 410nm, 420nm, 430nm, 440nm, 450nm, 460nm and 470nm.
  • 180-480nm such as 190nm, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm , 320nm, 330n
  • the inventor of the present application found that if the particle size of the in-situ signal-enhancing nanoparticles is less than 180nm, such as 150nm, no signal can be detected in conventional optical imaging equipment. If the particle size is greater than 480nm, such as 500nm, the detection sensitivity is very low. , It is difficult to achieve the sensitivity required by the clinic.
  • the particle size of the in-situ signal-enhancing nanoparticles is preferably 200-450nm, more preferably 200-350nm, more preferably 220-350nm, more preferably 220nm or more and less than 330nm, most preferably 220nm or more and less than 300nm . It should be noted that the particle size may be a primary particle size or a secondary particle size.
  • the secondary particle size refers to the particle size formed by the agglomeration of primary particles and secondary particles.
  • the particle size of suitable in-situ signal-enhancing nanoparticles can be obtained by adjusting the mass ratio of fluorescent material to nanoparticle carrier, the type of nanoparticle carrier, the type of fluorescent material, and the type and amount of solvent.
  • the incubation temperature of the sample and the capture antibody is 10-50°C, preferably 20-40°C, particularly preferably 37°C, and the incubation time is 1min-60min, preferably 3min-30min, especially preferred It is 10min ⁇ 30min.
  • the incubation temperature of the detection antibody (or the in situ signal-enhancing nanoparticles with the detection antibody) and the sample is 10-50°C, preferably 20-40°C, particularly preferably 37°C, and incubate The time is 1 minute to 60 minutes, preferably 2 minutes to 30 minutes, and particularly preferably 15 minutes to 25 minutes.
  • the optical imaging equipment mainly includes the following components: excitation light source, objective lens, filter, photosensitive element, data acquisition module, data processing module and dichroic mirror (if it is an upright microscope, there may be no two To the color mirror).
  • the excitation light source is an optical emission device used to excite the reacted sample to an optical signal.
  • the objective lens is used for signal acquisition and amplification of the sample to be tested.
  • the dichroic mirror is used for the reflection of the excitation light path and the collection of the optical signal of the sample.
  • the filter is used for filtering the excitation light waveband and filtering the light signal emitted by the sample.
  • the photosensitive element is used for collecting optical signals of the sample.
  • the data acquisition module is configured to receive the optical signal captured by the photosensitive element and convert it into a digital signal.
  • the data processing module is configured for digital signal conversion and optical image formation and processing.
  • the excitation light source includes one or more of a gas laser, a solid laser, a semiconductor laser, a liquid laser, and a free electron laser.
  • the objective lens is classified according to magnification, including one or more of 1X, 2X, 4X, 5X, 10X, 20X, 40X, 50X, and 100X; the objective lens is classified according to field curvature correction , Including plane objectives or curved objectives.
  • the photosensitive element includes one or both of CCD (Charge Coupled Device) or CMOS (Complementary Metal-Oxide Semiconductor, complementary metal oxide semiconductor).
  • this application adopts a specific detection system, which has low requirements on optical imaging equipment. It is a conventional optical imaging equipment (that is, an optical imaging equipment that does not break the optical diffraction limit), and does not require a total internal reflection fluorescence microscope. , Epi-fluorescence microscope, scanning near-field optical microscope, confocal fluorescence microscope and other expensive imaging equipment that breaks the optical diffraction limit.
  • the concentration of target molecules there are two methods for calculating the concentration of target molecules: single molecule counting mode and fluorescence intensity integration mode.
  • single molecule counting mode the number of bright spots formed by in-situ signal-enhanced nanoparticles in the generated image is directly analyzed and counted, and the number of bright spots is directly or indirectly converted into the target molecule.
  • Concentration information in the sample refers to absolute quantification, that is, conversion into concentration information without calibration curve correction.
  • the so-called “indirect conversion into the concentration information of the target molecule in the sample” refers to the conversion into concentration information by the number of bright spots and the standard curve (or calibration parameter).
  • the area of bright spots formed by in-situ signal-enhancing nanoparticles in the generated image is counted and integrated, and the integration result is divided by a specific parameter, such as averaging each in-situ signal nanometer
  • the average bright spot area formed by the particles or variables related to the bright spot area are converted to obtain the approximate number of in-situ signal-enhancing nanoparticles, and then the value is converted into the target molecule in the sample Concentration information in.
  • the average bright spot area is obtained by counting the bright spot area of a single molecule at a lower concentration and taking the average value.
  • the single molecule counting mode in the low concentration range and the fluorescence intensity integration mode in the high concentration range is important to use the single molecule counting mode in the low concentration range and the fluorescence intensity integration mode in the high concentration range, and then combine the standard curves drawn in these two modes.
  • the above-mentioned dividing line between low concentration and high concentration is generally the concentration when more than one molecule to be tested is bound on the surface of a magnetic bead, or according to the standard curve fitting result, it is preferable that an average of 0.5 is bound on the surface of a magnetic bead.
  • the concentration of one molecule to be tested or the concentration of two molecules to be tested is preferable that an average of 0.5 is bound on the surface of a magnetic bead.
  • the second embodiment of the present application is as follows.
  • a single molecule quantitative detection and analysis method including the following steps:
  • the in-situ signal-enhancing nanoparticle is combined with the detection antibody to form a composite material, and then the composite material is added to the sample to bind the composite material to the second site of the target molecule in the sample;
  • the in-situ signal-enhancing nanoparticle contains a luminescent material and a nanoparticle carrier, and the particle diameter is 180-480nm;
  • the second embodiment only differs in the order of the steps, and other conditions such as in-situ signal enhancement nanoparticles are the same.
  • the third embodiment of the present application is as follows.
  • a single molecule quantitative detection and analysis method including the following steps:
  • a capture probe capable of binding to the target molecule on a solid-phase carrier the capture probe is complementary to the first sequence of the target molecule, and the capture probe is used to capture the target molecule in the sample;
  • a detection probe which can be complementary to the second sequence of the target molecule to form a three-strand hybrid structure of capture probe-target molecule-detection probe, and then add in-situ signal-enhancing nanoparticles;
  • the in situ signal-enhancing nanoparticles can be directly or indirectly bound to the detection probe; or
  • the detection probe is combined with the in-situ signal-enhancing nanoparticles to form a composite material, and then the composite material is added;
  • the in-situ signal-enhancing nanoparticle contains a luminescent material and a nanoparticle carrier, and the particle diameter is 180-480nm;
  • the target molecule includes DNA or RNA.
  • the detection reagent includes a solid phase carrier, a capture probe, a detection probe, and in-situ signal-enhancing nanoparticles.
  • the solid phase carrier is used for the separation and cleaning of the test sample and the reagent.
  • the capture probe is fixed on the surface of the solid support through chemical modification, and can hybridize and combine with a part of the molecule to be tested to separate it from the sample.
  • the detection probe can be hybridized and combined with another part of the molecule to be tested, and the end of the detection probe far from the hybridization position is connected to the in-situ signal-enhancing nanoparticle through a chemical covalent bond.
  • the in-situ signal-enhancing nanoparticles can emit a sufficiently strong optical signal to form an independently distinguishable image signal on the optical imaging device.
  • the solid phase carrier is classified according to morphology, and can be one or more of magnetic beads, porous plates, centrifuge tubes, chips, micro-scale microspheres, and nano-scale microspheres; according to material classification, it can be organic polymers, two One or more of silicon oxide and silicon; according to function classification, it can be one of magnetic solid phase carrier and non-magnetic solid phase carrier.
  • magnetic beads are particularly preferred.
  • the magnetic beads are suspended, making it difficult to quantitatively detect, and there is directionality that makes the brightness difference.
  • the inventors of the present application found that in the case of single-molecule quantitative detection of nucleic acids, compared with the use of glass chips as the carrier, the use of magnetic beads as the carrier can obtain particularly excellent sensitivity and greatly improve the detection effectiveness.
  • the surface of the solid-phase carrier is modified with active functional groups that can be covalently coupled with the probe, including hydroxyl, carboxyl, amino, sulfhydryl, alkenyl, alkynyl, succinimidyl ester groups and their derivatives One or more of the groups.
  • the capture probe can be ribonucleic acid or deoxyribonucleic acid, and its sequence is complementary to a sequence of the molecule to be tested, and can form a double-stranded hybrid.
  • one end of the capture probe is modified with one or more of carboxyl group, amino group, sulfhydryl group and succinimide ester, which can be covalently bound to the surface of the solid support, thereby stably binding to the solid support.
  • the surface of the phase carrier is modified with one or more of carboxyl group, amino group, sulfhydryl group and succinimide ester, which can be covalently bound to the surface of the solid support, thereby stably binding to the solid support.
  • the detection probe can be ribonucleic acid or deoxyribonucleic acid, and its sequence is complementary to another sequence of the molecule to be tested (different from the sequence complementary to the capture probe), and can form a double-stranded hybridization, thereby forming a capture Probe-target molecule-detection probe three-strand hybrid structure.
  • one end of the detection probe is modified with one or more of carboxyl group, amino group, sulfhydryl group and succinimide ester, which can be covalently coupled with the in-situ signal-enhancing nanoparticle, thereby stably binding to the original
  • the bit signal enhances the surface of the nanoparticle.
  • the fourth embodiment of the present application is as follows.
  • a single molecule quantitative detection and analysis method including the following steps:
  • the in-situ signal-enhancing nanoparticle is combined with the detection probe in advance to form a composite material, and then the composite material is complementary to the second sequence of the target molecule in the sample;
  • the in-situ signal-enhancing nanoparticle contains a luminescent material and a nanoparticle carrier, and the particle diameter is 180-480nm;
  • the fourth embodiment only differs in the order of the steps, and other conditions such as in-situ signal-enhancing nanoparticles are the same.
  • the fifth embodiment of the present application is as follows.
  • a single molecule quantitative detection system including:
  • the detection reagent includes (a) a capture antibody that can bind to the first site of a target molecule to capture the target molecule in the sample; (b) a detection antibody that can interact with the second site of the target molecule Site binding and capable of binding with in-situ signal-enhancing nanoparticles; (c) In-situ signal-enhancing nanoparticles, which contain luminescent materials and nanoparticle carriers, and have a particle size of 180-480nm; or
  • the detection reagent includes (a) a capture probe that can bind to the first sequence of the target molecule to capture the target molecule in the sample; (b) a detection probe that can interact with the target molecule The second sequence is combined to form a three-strand hybrid structure of capture probe-target molecule-detection probe; (c) In-situ signal-enhancing nanoparticles, which can be bound to the detection probe and contain luminescent materials and nanoparticle carriers, And the particle size is 180-480nm;
  • Optical imaging equipment which includes an excitation light source and an optical signal acquisition unit.
  • the detection system of the present application includes two parts: detection reagents and optical imaging equipment.
  • the detection reagents include capture antibodies, detection antibodies, and specific in situ signal-enhancing nanoparticles (when used to detect proteins, polysaccharides or small molecules with biological activity), or include capture probes, detection probes and specific in situ Signal enhancement nanoparticles (when used to detect DNA or RNA).
  • the optical imaging device may be a conventional optical imaging device, and it does not require expensive optical imaging devices such as total internal reflection fluorescence microscope, epi-fluorescence microscope, scanning near-field optical microscope, confocal fluorescence microscope, etc., which break the optical diffraction limit.
  • silica fluorescent nanoparticles as an example, the silica fluorescent nanoparticles obtained in each example and comparative example were diluted 1000 times with water, and then 100 ⁇ L was dropped on the surface of the clean silicon wafer, dried, and a small sputtering apparatus was used. 5nm platinum was sputter deposited on the surface, and image analysis was performed using SEM (SU3900 manufactured by Hitachi High-Technologies, Japan) to determine the particle size.
  • the obtained polyacrylamide fluorescent nanoparticles are diluted 1000 times with pure water, and the particle size of the particles is measured using a Malvern particle size analyzer (Zetasizer Nano S90).
  • the combined use of the single molecule counting mode and the fluorescence intensity integration mode can significantly increase the dynamic detection range of the standard curve of the detection marker.
  • the specific implementation method is as follows:
  • the single molecule counting mode is used to draw a standard curve for samples of different concentrations of the molecule to be tested;
  • the concentration of the molecule to be tested exceeds a certain threshold, more than one molecule to be tested may be bound to the surface of a magnetic bead, and single molecule signals are easy to superimpose, leading to deviations in the detection results. Therefore, it is more suitable to use the fluorescence intensity integration mode.
  • Example 1 The magnetic bead method is used for the detection of cTnI antigen molecules in human serum (silica fluorescent nanoparticles: the particle size is 220 nm).
  • the detection result is shown in Figure 3. It can be seen that in this example, the detection range of cTnI is 30fg/mL-10ng/mL. In this interval, the number of single molecule signals (ie CPN) has a good linear relationship with the sample concentration. The lower limit can reach 30fg/mL.
  • Comparative Example 1 The magnetic bead method is used for the detection of cTnI antigen molecules in human serum (silica fluorescent nanoparticles: particle size 150nm).
  • Example 2 The operation was performed in the same manner as in Example 1 except that the surface carboxylated silica fluorescent nanoparticles with a particle size of 150 nm were taken.
  • silica fluorescent nanoparticles with a particle size of 150 nm are used, no fluorescent signal can be detected in the detection device at all, and it can be seen that silica fluorescent nanoparticles of this size cannot be used for single molecule quantitative detection. Since the reagent did not respond to the sample at all, no clinical sample test was performed.
  • Comparative Example 2 The magnetic bead method is used for the detection of cTnI antigen molecules in human serum (silica fluorescent nanoparticles: particle size of 500 nm).
  • Example 2 The operation was performed in the same manner as in Example 1 except that the surface carboxylated silica fluorescent nanoparticles with a particle size of 500 nm were taken.
  • Comparative Example 2 uses particles with a particle size of 500 nm, resulting in a much lower detection sensitivity than Example 1.
  • Example 2 The magnetic bead method is used for the detection of cTnI antigen molecules in human serum (silica fluorescent nanoparticles: the particle size is 350 nm).
  • Example 2 The operation was performed in the same manner as in Example 1 except that the surface carboxylated silica fluorescent nanoparticles with a particle size of 350 nm were taken.
  • the detection result is shown in FIG. 6, and it can be seen that in Example 2, the detection limit of cTnI is 80 fg/mL, and the sensitivity is excellent.
  • Example 1 The operation was performed in the same manner as in Example 1 except that the particle size of the surface carboxylated silica fluorescent nanoparticles used was 160 nm, and the results of the lower detection limit were the same as in Comparative Example 1.
  • Example 1 The operation was performed in the same manner as in Example 1 except that the particle size of the used surface-carboxylated silica fluorescent nanoparticles was 180 nm. The results are shown in Table 1.
  • Example 1 The operation was performed in the same manner as in Example 1 except that the particle size of the used surface carboxylated silica fluorescent nanoparticles was 250 nm. The results are shown in Table 1.
  • Example 1 The operation was performed in the same manner as in Example 1, except that the used surface carboxylated silica fluorescent nanoparticle had a particle size of 295 nm. The results are shown in Table 1.
  • Example 1 The operation was performed in the same manner as in Example 1 except that the used surface carboxylated silica fluorescent nanoparticles had a particle diameter of 300 nm. The results are shown in Table 1.
  • Example 1 The operation was performed in the same manner as in Example 1 except that the particle size of the used surface carboxylated silica fluorescent nanoparticles was 400 nm, and the results are shown in Table 1.
  • Example 1 The operation was performed in the same manner as in Example 1 except that the particle diameter of the used surface carboxylated silica fluorescent nanoparticles was 450 nm. The results are shown in Table 1.
  • Example 1 The operation was performed in the same manner as in Example 1 except that the particle size of the surface carboxylated silica fluorescent nanoparticles used was 480 nm. The results are shown in Table 1.
  • the tosyl-activated glass chip was used to replace the tosyl-activated magnetic beads, except that the operation was performed in the same manner as in Example 1. The results are shown in Table 1.
  • the single-molecule counting mode is used to replace the combined use of the single-molecule counting mode and the fluorescence intensity integration mode in the entire concentration interval, except that the operation is the same as in Example 1.
  • the results are shown in FIG. 7, it can be seen that compared with Example 1, the upper limit of detection is reduced and the detection dynamic range is reduced.
  • the Leica DMi8 fluorescence microscope was used instead of Nikon Eclipse Ti-U, except that the operation was performed in the same manner as in Example 1, and the results are shown in Table 1.
  • the detection antibody modified with biotin is first added to make it contact with the second site of the molecule to be tested. After binding, and then adding streptavidin-modified silica fluorescent nanoparticles, the operation was performed in the same manner as in Example 1, and the results are shown in Table 1.
  • Example 14 Magnetic bead method for the detection of IL-6 antigen molecules (polyacrylamide fluorescent nanoparticles: particle size 220nm)
  • the obtained polyacrylamide fluorescent nanoparticles were diluted 1000 times with pure water, and the particle size was measured using a Malvern particle size analyzer.
  • the particle size of the polyacrylamide fluorescent nanoparticles measured by the Malvern particle size analyzer is 220 nm.
  • the detection result of IL-6 antigen is shown in Figure 8. It can be seen that the diluted sample with a concentration of 0.01pg/mL can be effectively distinguished from the background. After calculation, the detection limit of this example is 0.006pg/mL (ie 6fg/mL ). It can be seen that the sensitivity of the detection method of the present application is extremely excellent.
  • Example 15 Magnetic bead method for detection of cTnI antigen molecules (polyacrylamide fluorescent nanoparticles: particle size 220nm)
  • Example 14 the capture antibody was replaced by Medix 2703 with Hytest 16A11 (that is, the capture antibody for cTnI antigen), and the detection antibody was replaced by Medix 2704 with Hytest 19C7 (that is, the detection antibody for cTnI antigen), and the test molecule was replaced by IL
  • the procedure was the same as in Example 14 except that the -6 antigen was replaced with the cTnI antigen.
  • the detection result of cTnI antigen is shown in Figure 9. It can be seen that the diluted sample with a concentration of 0.01pg/mL can be effectively distinguished from the background. After calculation, the detection limit of this example is 0.008pg/mL (ie 8fg/mL ). It can be seen that the sensitivity of the detection method of the present application is extremely excellent.
  • Example 15 The operation was carried out in the same manner as in Example 15 except that the particle size of the surface carboxylated polyacrylamide fluorescent nanoparticles was 300 nm.
  • the detection result of cTnI antigen is shown in Figure 10. It can be seen that the diluted sample with a concentration of 0.01 pg/mL can be effectively distinguished from the background. After calculation, the detection limit of this example is 0.020 pg/mL (ie 20 fg/mL ). It can be seen that the sensitivity of the detection method of the present application is superior.
  • Example 17 Magnetic bead method for the detection of cTnI antigen molecules (polyacrylamide fluorescent nanoparticles: the secondary particle size is 250nm)
  • the primary and secondary polyacrylamide fluorescent particles they were each diluted 2000 times with pure water, and their particle sizes were measured using a Malvern particle size analyzer.
  • the particle size measurement method is as follows: Take the excess relative to the first-stage particle The secondary particles are allowed to react with the primary particles. The particle size of the secondary particles obtained by the reaction is measured with a Malvern particle size analyzer. As a result, two peaks can be obtained, and the larger peak is used as the particle size of the secondary particles .
  • fetal bovine serum to dilute the concentration of cTnI antigen to 0, 0.01, 0.1, 0.5, 1, 5, 10, 50, and 100 pg/mL.
  • the particle size of the primary and secondary polyacrylamide fluorescent particles is about 80 nm, and the particle size of the secondary polyacrylamide fluorescent particles is about 250 nm.
  • the detection result of the cTnI antigen is shown in Fig. 11. It can be seen that the diluted sample with a concentration of 0.005 pg/mL can be effectively distinguished from the background. After calculation, the detection limit of this embodiment is 0.002 pg/mL (ie, 2fg/mL). It can be seen that the sensitivity of the detection method of the present application is extremely excellent.
  • Example 18 Magnetic bead method for rapid detection of IL-6 antigen molecules (polyacrylamide fluorescent nanoparticles: 220nm in diameter)
  • Example 14 the incubation time in step 3.2 (2) was shortened from 30 min to 3 min, and the incubation time in step 3.2 (3) was shortened from 15 min to 2 min. Other than that, the same operation was performed as in Example 14. .
  • the rapid detection result of IL-6 antigen is shown in Figure 12. It can be seen that even if the detection time is greatly shortened, the diluted sample of 1 pg/mL can be effectively distinguished from the background. After calculation, the detection limit of Example 18 can reach 1 pg/ mL.
  • 20 clinical blood samples were tested in this example. As shown in Figure 13, the abscissa is the reference value measured by the Roche Cobas 6000 automatic electrochemiluminescence equipment, and the ordinate is the detection method obtained by the application. Measurements. Correlation analysis shows that the R 2 value is 0.9887, which proves that the method of this application has a good correlation with the method used by commercially available mature equipment.
  • Example 19 Magnetic bead method for rapid detection of cTnI antigen molecules (polyacrylamide fluorescent nanoparticles: particle size 220nm)
  • Example 15 the incubation time in step 3.2 (2) was shortened from 30 min to 3 min, and the incubation time in step 3.2 (3) was shortened from 15 min to 2 min. Other than that, the same operation was performed as in Example 15 .
  • Example 20 The magnetic bead method is used for the detection of cTnI antigen molecules in human serum (fluorescent microspheres made of polystyrene encapsulated quantum dots (hereinafter referred to as polystyrene fluorescent nanoparticles): particle size is 210 nm).
  • Quantum dots Qdot 605, Thermo
  • surface carboxylated polystyrene microspheres particle size is 210nm, Hangzhou Microbio
  • Example 2 The operation was performed in the same manner as in Example 1, except that the detection antibody-bound polystyrene fluorescent nanoparticles were added in step 3.1 (3).
  • the detection result is shown in Figure 16. It can be seen that in this example, the detection range of cTnI is 50fg/mL ⁇ 100pg/mL. In this interval, the number of single molecule signals has a good linear relationship with the sample concentration, and its detection limit is about 50fg/mL. mL.
  • Embodiment 21 (corresponding to the second embodiment of this application)
  • Example 1 the silica fluorescent nanoparticles bound with the detection antibody are first reacted with the clinical sample, and then the magnetic beads labeled with the capture antibody are added.
  • the operation is the same as in Example 1, except that , The results are shown in Table 1.
  • Example 22 The glass chip method is used for the quantitative detection of DNA molecules in the buffer (silica fluorescent nanoparticles: the particle size is 220 nm).
  • Capture probe sequence NH 2 -TTTTTTTTTTTTTTTGTGTGACATGTTCTAATATAGTCACAT
  • Detection probe sequence TCTGATATAATCTTGTACAGTGTGTTTTTTTTTTTT-NH 2
  • the detection result is shown in FIG. 17. It can be seen that in this example, the detection limit of the DNA template molecule is 0.5 pM, which is close to the detection sensitivity of PCR.
  • Example 23 The magnetic bead method is used for the quantitative detection of DNA molecules in the buffer (silica fluorescent nanoparticles: the particle size is 220 nm).
  • Example 22 except that tosyl-activated M280 magnetic beads were used instead of the low-adsorption glass slide, the same procedure as in Example 22 was performed, and the results are shown in Table 2.
  • the applicant also commented on the in-situ signal-enhancing nanoparticles as fluorescent particles made of polystyrene coated rare earth elements (europium), fluorescent particles made of dextran coated fluorescent protein (GFP), and cross-linked agarose coated quantum
  • fluorescent particles made of dots cadmium sulfide
  • the fluorescent particles made of silica-coated fluorescein were replaced with the above-mentioned three types of fluorescent particles.
  • the sensitivity was also excellent (between 50 fg/mL-5 fg/mL).

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Abstract

一种单分子定量检测方法及检测系统，可用于蛋白质、小分子以及核酸分子的单分子级别的定量检测。该检测方法及检测系统利用具有光学特性的原位信号增强纳米粒子、通过化学修饰以及分子识别技术对待测分子进行标记，使得单分子信号能够被光学成像设备捕获和识别。通过对原位信号增强纳米粒子个数信号进行统计，实现待测分子的超高灵敏度定量检测。

Description

一种单分子定量检测方法及检测系统
相关申请的交叉引用
本申请要求于2019年01月30日提交中国专利局的申请号为CN201910091631.1、名称为“一种单分子定量检测方法及检测系统”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本申请涉及一种基于单分子计数的定量检测方法及检测系统，可用于蛋白质、核酸以及小分子的超高灵敏度定量检测。
背景技术
生物标志物尤其是与重大疾病相关的生物标志物在人体内的含量与多种生物学过程密切相关。这些生物标志物在疾病发生的早期阶段含量较低，传统方法难以实现准确有效的检测分析。要实现生物标志物(例如DNA、RNA或酶等)的早期准确诊断，需要超高灵敏度的定量检测手段辅助。
传统的检测方法(例如用于蛋白质检测的化学发光法、酶联免疫法、荧光免疫法或者用于核酸检测的荧光定量PCR方法)的检测原理为：基于溶液整体的光学信号进行检测，将检测获得的光强度信号与标准曲线进行对比，从而实现定量检测。然而，由于检测设备均存在一定背景噪音信号，当样品中的靶标分子浓度低至一定程度时，溶液整体的光强度信号将被背景噪声信号淹没，导致在低值区间的偏差变大，难以实现准确的定量分析。因此，由于检测技术原理层面上的限制，这些传统检测方法难以实现高灵敏度的检测分析。
单分子检测或者数字化单分子检测采取与传统检测方法完全不同的检测策略。即，通过对待测分子(蛋白质、小分子或核酸分子)直接标记酶分子、荧光染料分子、或者通过使待测分子扩增来实现单分子级别的信号放大或信号标记，然后，使用检测设备对信号放大后的单分子信号进行读取，直接对待测分子的个数进行统计，最终通过检测所得的待测分子的数量、利用标准曲线校正来实现单分子级别的定量检测。
目前已报道的单分子级别的定量检测主要有下述两种策略：
(1)通过单分子标记技术直接在待测分子上标记荧光分子或具有光、电和磁等信号的纳米材料，使用精密度极高的光、电和磁检测设备进行检测，例如可实现单分子荧光响应的全内反射显微镜、近场显微镜和艾里斑聚焦检测设备(即专利文献1中的美国Singulex公司发明的SMC技术所使用的光学设备)等以及可实现单纳米粒子检测的电子显微镜等。这些检测设备由于需要能够识别出几个甚至单个荧光分子信号，因此对设备精密度的要求极高，造成设备成本极为高昂，严重阻碍了这些技术在科研领域和医疗诊断领域中的应用。
(2)通过信号放大的方式，将极微弱的单分子信号放大到容易被设备检测的水平。目前较常见的是数字化PCR和数字化单分子酶催化技术。数字化PCR中，通过聚合酶链式反应直接将待检测的核酸分子以指数级扩增，通过荧光探针实现信号放大；数字化单分子酶催化技术中，在待检测分子上标记具有高催化效率的酶或催化底物，产生具有荧光特性的产物分子，实现信号放大。通常，信号放大的方法对检测设备要求较低，成本相对可控，因而其应用相对于单分子荧光检测较广。但是，无论是数字化PCR或者数字化单分子酶催化技术，其产物信号均较低。为了提高检测的信背比，通常需要将待测分子分散至体积极小(纳升至皮升级别)的液滴中(参见专利文献2)。这使得该类技术操作繁琐，对操作经验的要求高，需要高精密度的液滴生成辅助设备。
此外，尽管目前针对核酸的单分子级别的检测技术处于快速发展期(主要受益于单分子PCR技术的发展)，但本领域中普遍认为与DNA相比，蛋白质的结构比较复杂，难于复制扩增。由于缺乏PCR这类指数级信号放大方法，现有蛋白质检测方法的灵敏度不够高，导致蛋白质的单分子检测技术仍基本处于空白状态，更不用说以常规荧光显微镜即能实现对蛋白质进行单分子检测的技术。
由此可知，现有的单分子检测技术存在检测体系复杂、对检测设备的精密度要求极高、检测试剂的稳定性差、对辅助耗材的精密度要求高、成本难以控制以及无法良好地应用于蛋白质检测等诸多问题。这些问题极大程度地阻碍了单分子检测技术在科研和医疗诊断市场中的应用。
现有技术文献
专利文献1：CN101438146A
专利文献2：CN102884431A
发明内容
[发明要解决的课题]
本申请是鉴于上述现状而做出的，目的在于提供通过简单的单分子信号标记即可实现单分子检测、对光学设备的要求低的新型单分子检测方法和单分子检测系统。
[用于解决课题的手段]
为了解决上述课题，本申请的发明人反复进行了深入研究，结果发现了一种基于成像的、灵敏度高且成本低的新型单分子定量检测方法和单分子检测系统。
本申请的一个技术方案如下。
一种单分子定量检测分析方法，包括如下步骤：
(1)将能够与靶标结合的捕获抗体固定到固相载体上，利用捕获抗体和靶标分子的第一位点结合，从而捕获样品中的靶标分子；
(2)加入检测抗体，所述检测抗体与靶标分子的第二位点结合，然后加入原位信号增强纳米粒子，所述原位信号增强纳米粒子能够与检测抗体结合；或
先将检测抗体与原位信号增强纳米粒子结合而形成复合材料，再将该复合材料加入；
其中，所述原位信号增强纳米粒子含有发光材料和纳米粒子载体，且粒径为180-480nm；
(3)用光学成像设备检测由所述原位信号增强纳米粒子发出的光学信号；
(4)对原位信号增强纳米粒子的个数进行统计，进一步计算得到样品中靶标分子的浓度信息。
优选地，所述原位信号增强纳米粒子的粒径为200-450nm。
优选地，所述原位信号增强纳米粒子的粒径为200-350nm。
进一步优选地，所述原位信号增强纳米粒子的粒径为220-350nm，更优选为220nm以上且小于330nm，最优选为220nm以上且小于300nm。
本申请的该技术方案所针对的靶标分子包括蛋白质、多糖或有生物活性的小分子。
本申请的效果
就本申请的单分子定量检测方法而言，能够以超高的灵敏度和极低的成本应用于蛋白质以及核酸的检测，尤其是蛋白质的检测。在用于蛋白质的检测时，本申请的发明人惊讶地发现，一些实施方式中，灵敏度可以达到1pg/mL；在另一些实施方式中，灵敏度可以达到100fg/mL；在一些实施方式中，灵敏度可以达到10fg/mL；灵敏度最高可以达到1fg/mL级别。此外，本申请能够在保证较高检测灵敏度的基础上以较短的时间(仅用数分钟的孵育时间)实现检测，从而缩短临床上的检测时间。
就本申请的单分子定量检测系统而言，基于特定的检测体系(尤其是利用特定的原位信号增强纳米粒子对待测分子进行标记)，以简单方法实现了超高灵敏度检测。本申请的单分子定量检测系统的检测灵敏度远超传统方法，实现了2-4个数量级的提升。
与现有的单分子检测技术相比，本申请打破了蛋白质分子检测与核酸分子检测之间的屏障，利用一台设备即可检测蛋白质分子和核酸分子，尤其是能够以超高灵敏度用于蛋白质检测中。此外，通过特定的检测体系(如特定的原位信号增强纳米粒子)的使用，所述系统无需全内反射显微镜等昂贵设备，仅需CCD或CMOS这样的低成本感光元件即可实现对待测分子的原位成像，极大地降低了单分子检测的难度、步骤繁琐程度和对设备精密度的要求，能够以低的成本将单分子检测技术应用于科研领域和生物医疗检测领域。
附图说明
图1为本申请的蛋白质检测方法的示意图；
图2为本申请的核酸检测方法的示意图；
图3为实施例1中得到的标准曲线(纵坐标即CPN(copy number)为单分子信号数)；
图4为比较例1中得到的标准曲线；
图5为比较例2中得到的标准曲线；
图6为实施例2中得到的标准曲线；
图7为实施例11中得到的标准曲线；
图8为实施例14中得到的标准曲线；
图9为实施例15中得到的标准曲线；
图10为实施例16中得到的标准曲线；
图11为实施例17中得到的标准曲线；
图12为实施例18中得到的标准曲线；
图13为实施例18中的临床样本比对结果；
图14为实施例19中得到的标准曲线；
图15为实施例19中的临床样本比对结果；
图16为实施例20中得到的标准曲线；
图17为实施例22中得到的标准曲线。
具体实施方式
<第一实施方式>
本申请的第一实施方式如下。
单分子定量检测分析方法，其包括下述步骤：
(1)将能够与靶标分子结合的捕获抗体固定至固相载体上，然后，使所述捕获抗体与样品中的靶标分子的第一位点结合，从而将靶标分子捕获；
(2)加入检测抗体，使其与所述靶标分子的第二位点结合，然后加入原位信号增强纳米粒子，所述原位信号增强纳米粒子直接或间接与检测抗体结合；或者
预先使原位信号增强纳米粒子直接或间接与检测抗体结合而形成复合材料，然后加入该复合材料，与所述靶标分子的第二位点结合；
其中，所述原位信号增强纳米粒子含有发光材料和纳米粒子载体，且粒径为180-480nm；
(3)用光学成像设备检测由所述原位信号增强纳米粒子发出的光学信号；
(4)对原位信号增强纳米粒子的个数进行统计，进一步计算得到样品中靶标分子的浓度信息。
本申请中，所述固相载体用于检测样品与试剂的分离和清洗。所述捕获抗体通过物理吸附或化学修饰的方式固定在固相载体表面，并且能够与靶标分子(以下，有时也称为待测分子)的一个结合位点相结合，使其从样品中分离。
所述检测抗体能够与待测分子的另一个结合位点结合。所述的原位信号增强纳米粒子直接与检测抗体结合，是指检测抗体通过物理吸附或化学修饰的方法，直接吸附或偶联在原位信号增强纳米粒子上，实现原位信号增强纳米粒子对待测分子识别和标记的功能化修饰。所述的“原位信号增强纳米粒子间接与检测抗体结合”是指通过抗检测抗体(即二抗)或生物素-链霉亲和素体系进行结合，将原位信号增强纳米粒子特异性标记在检测抗体上。
所述靶标分子包括蛋白质、多糖或有生物活性的小分子及小分子与蛋白质的复合物。具体而言，可举出cTnI抗原、IL-6抗原、PCT(降钙素原)抗原、Sema4D(信号素4D)抗原、Nt-proBNP(脑自然肽氨基端前体蛋白)抗原、肿瘤标志物、维生素D、维生素B、叶酸、维生素D-BSA复合物、叶酸-BSA复合物、细菌以及病毒等。
所述固相载体按照形态分类，可以为磁珠、多孔板、离心管、芯片、微米尺度微球以及纳米尺度微球等；按照材质分类，可以为聚合物、二氧化硅、硅或它们的复合体等；按照功能分类，可以为磁性固相载体和非磁性固相载体。这些固相载体中，尤其优选磁珠。以往认为，将磁珠应用于蛋白质的单分子定量检测时存在下述问题：磁珠在溶液中处于悬浮状态而难以定量检测；因磁珠存在方向性而导致在成像过程中亮度存在区别。然而，本申请的发明人惊讶地发现，在想要对蛋白质进行单分子定量检测的情况下，与使用玻璃芯片作为载体时相比，使用磁珠作为载体时能够获得尤其优异的灵敏度，并极大地提高了检测效率。
所述固相载体的表面修饰有能够与抗体进行共价偶联的活性官能团，例如羟基、羧基、氨基、琥珀酰亚胺酯基、磺酰基(如甲苯磺酰基)及它们的衍生基团中的一种或多种。
所述捕获抗体按照抗体特异性特性分类，可以为多克隆抗体和单克隆抗体中的一种或两种。所述捕 获抗体按照来源分类，可以为鼠源抗体、兔源抗体、羊源抗体以及羊驼源抗体中的一种或多种。具体而言，可举出Hytest 19C7、Hytest 20C6、Hytest16A11、Medix 2703、Meridian M86101M、Biospacific A45160以及Biospacific G-131-C等。
所述检测抗体按照抗体特异性特性分类，可以为多克隆抗体和单克隆抗体中的一种或两种。所述检测抗体按照来源分类，可以为鼠源抗体、兔源抗体、羊源抗体以及羊驼源抗体中的一种或多种。具体而言，可举出Hytest 16A11、Medix 2704、Meridian M86201M以及Biospacific A45502等。
所述抗检测抗体按照检测抗体来源分类，可以为抗鼠抗体、抗兔抗体、抗羊抗体以及抗羊驼抗体中的一种或多种。所述抗检测抗体按照来源分类，可以为鼠源二抗、兔源二抗、羊源二抗以及羊驼源二抗中的一种或多种。
所述原位信号增强纳米粒子是指在原位(in-situ)将荧光信号增强至能够被常规光学成像设备检测到的水平的材料，其必须含有发光材料和纳米粒子载体这两部分。
所述原位信号增强纳米粒子中，纳米粒子载体发挥非常重要的作用，例如，可以结合更多的发光材料，使得发光信号更强；为官能化修饰提供位点，能够结合大量抗体，提高反应活性；为常规荧光显微镜实现单分子检测提供可能性，如果没有纳米粒子载体则无法实现单分子检测。所述纳米粒子载体按照材料分类，可以为二氧化硅、聚苯乙烯、聚丙烯酰胺、聚(甲基)丙烯酸甲酯、葡聚糖、琼脂糖以及无机金属化合物中的一种或多种。所述纳米粒子载体按照结构分类，可以为中空结构、核壳结构、多孔结构、合金结构以及水凝胶结构中的一种或多种。其中，从使发光材料均匀分布、使发光材料的亮度高的观点考虑，纳米粒子载体优选为二氧化硅、聚丙烯酰胺、聚苯乙烯和葡聚糖，尤其优选聚丙烯酰胺。
所述原位信号增强纳米粒子中的发光材料也是实现单分子检测所必须的，仅有纳米粒子载体时灵敏度极低。所述发光材料可以为荧光染料分子、稀土元素、稀土螯合物、荧光蛋白、量子点以及上转换纳米粒子中的一种或多种。发光材料优选为荧光素类(如异硫氰酸荧光素)、罗丹明类(如罗丹明绿、罗丹明B等)、香豆素类、量子点类(如CdS、CdSe、CdTe、ZnSe)、稀土元素(如Eu、Ce)及其配合物等。所述发光材料通过共价修饰、螯合作用、空间包裹、疏水作用以及静电吸附作用中的一种或多种而吸附或包裹于纳米粒子载体表面或内部。需要说明，从有利于光学成像识别和提高灵敏度的观点考虑，优选的是，发光材料被均匀地包裹于纳米粒子载体的内部。
本申请中，原位信号增强纳米粒子优选为二氧化硅包裹荧光染料分子(如荧光素)而成的荧光粒子、聚丙烯酰胺包裹荧光染料分子(如荧光素)而成的荧光粒子、聚苯乙烯包裹量子点而成的荧光粒子、聚苯乙烯包裹稀土元素或稀土鳌合物而成的荧光粒子、葡聚糖包裹荧光蛋白而成的荧光粒子以及交联琼脂糖包裹量子点而成的荧光粒子等。
需要说明的是，上述发光材料和纳米粒子载体可以进行各种组合。
本申请中，所述原位信号增强纳米粒子的表面修饰有能够与抗体、DNA或RNA共价偶联的活性官能团，包括羟基、羧基、氨基、巯基、烯基、炔基、琥珀酰亚胺酯基团及其衍生基团中的一种或多种。
本申请中，所述原位信号增强纳米粒子的表面可以修饰有一定长度的连接臂；所述连接臂包括多碳直链、多碳支链、聚合物链、肽链、蛋白质以及核酸分子。所述连接臂的长度优选为1-100nm，进一步优选为2-20nm，最优选为5-10nm。
本申请中，原位信号增强纳米粒子的粒径需要严格控制在180-480nm范围内，例如为190nm、200nm、210nm、220nm、230nm、240nm、250nm、260nm、270nm、280nm、290nm、300nm、310nm、320nm、330nm、340nm、350nm、360nm、370nm、380nm、390nm、400nm、410nm、420nm、430nm、440nm、450nm、460nm以及470nm。本申请的发明人发现，若原位信号增强纳米粒子的粒径小于180nm，例如150nm，则在常规光学成像设备中检测不到任何信号，若粒径大于480nm，例如500nm，则检测灵敏度非常低，难以达到临床所要求的灵敏度。其中，所述原位信号增强纳米粒子的粒径优选为200-450nm，进一步优选为200-350nm，更优选为220-350nm，更优选为220nm以上且小于330nm，最优选为220nm以上且小于300nm。需要说明的是，所述粒径可以是一次粒径，也可以是二次粒径。所述二次粒径是指一级颗粒与二级颗粒团聚后形成的粒径。
合适的原位信号增强纳米粒子的粒径可通过对荧光材料与纳米粒子载体的质量比、纳米粒子载体的种类、荧光材料的种类以及溶剂的种类和用量等参数进行调节而得到。
本申请的步骤(1)中，样品与捕获抗体的孵育温度为10～50℃，优选为20～40℃，尤其优选为37℃，孵育时间为1min～60min，优选为3min～30min，尤其优选为10min～30min。本申请的步骤(2)中，检测抗体(或结合有检测抗体的原位信号增强纳米粒子)与样品的孵育温度为10～50℃，优选为20～40℃，尤其优选为37℃，孵育时间为1min～60min，优选为2min～30min，尤其优选为15min～25min。
本申请中，所述光学成像设备主要包括以下部件：激发光源、物镜、滤光片、感光元件、数据采集模块、数据处理模块以及二向色镜(若为正置显微镜，则也可以没有二向色镜)。其中，所述激发光源是用于将反应后的样品激发出光学信号的光学发射装置。所述物镜用于待测样品的信号采集和放大。所述二向色镜用于激发光路的反射和样品光学信号的采集。所述滤光片用于激发光波段的过滤和样品发射光信号的过滤。所述感光元件用于样品光学信号的采集。所述数据采集模块配置为接收感光元件捕获的光学信号，并转换为数字信号。所述数据处理模块配置为数字信号的转换以及光学图像的形成和处理。
在该设备的一些实施方式中，所述激发光源包括气体激光器、固体激光器、半导体激光器、液体激光器以及自由电子激光器中的一种或几种。在该设备的一些实施方式中，所述物镜按照倍率分类，包括1X、2X、4X、5X、10X、20X、40X、50X以及100X中的一种或几种；所述物镜按照场曲校正分类，包括平面物镜或曲面物镜。在该设备的一些实施方式中，所述感光元件包括CCD(Charge Coupled Device，电荷耦合装置)或CMOS(Complementary Metal-Oxide Semiconductor，互补金属氧化物半导体)中的一种或两种。
如前文所述，本申请通过采用特定的检测体系，从而对光学成像设备的要求低，为常规的光学成像设备(即未突破光学衍射极限的光学成像设备)即可，无需全内反射荧光显微镜、落射荧光显微镜、扫描近场光学显微镜、共聚焦荧光显微镜等昂贵的、突破了光学衍射极限的成像设备。
本申请中，靶标分子浓度的计算方式有单分子计数模式和荧光强度积分模式这两种。其中，就所述单分子计数模式而言，直接对生成的图像中的由原位信号增强纳米粒子形成的亮斑个数进行分析和统计，通过亮斑个数直接或间接换算为靶标分子在样品中的浓度信息。所谓“直接换算为靶标分子在样品中的浓度信息”，是指绝对定量，也即，无需标准曲线校正即可换算为浓度信息。所谓“间接换算为靶标分子在样品中的浓度信息”，是指通过亮斑个数和标准曲线(或校正参数)换算为浓度信息。就所述荧光强度积分模式而言，对生成的图像中的由原位信号增强纳米粒子形成的亮斑面积进行统计和积分，通过将积分结果除以特定参数，例如平均每个原位信号纳米粒子所形成的平均亮斑面积或亮斑面积相关变量(例如幂方、开方以及多项式等)，从而换算得到原位信号增强纳米粒子的近似个数，再将该数值换算为靶标分子在样品中的浓度信息。其中，平均亮斑面积是通过在较低浓度下对单个分子的亮斑面积进行统计并取平均值而得到的。从获得更大的检测动态范围的方面考虑，重要的是，在低浓度区间使用单分子计数模式并在高浓度区间使用荧光强度积分模式，然后将在这两种模式下绘制的标准曲线合并，从而绘制完整的标准曲线。需要说明，上述低浓度与高浓度的分界线一般为一个磁珠表面上结合有超过一个的待测分子时的浓度，也可以根据标准曲线拟合结果优选为一个磁珠表面上平均结合有0.5个待测分子时的浓度或2个待测分子时的浓度。
<第二实施方式>
本申请的第二实施方式如下。
一种单分子定量检测分析方法，包括如下步骤：
(1)使检测抗体与样品中的靶标分子的第二位点结合，然后加入原位信号增强纳米粒子，所述原位信号增强纳米粒子能够直接或间接与检测抗体结合；或者
先使原位信号增强纳米粒子与检测抗体结合而形成复合材料，再将该复合材料加入样品中，使该复合材料与样品中的靶标分子的第二位点结合；
其中，所述原位信号增强纳米粒子含有发光材料和纳米粒子载体，且粒径为180-480nm；
(2)将能够与靶标分子结合的捕获抗体固定至固相载体上，然后，使所述捕获抗体与所述靶标分子的第一位点结合，从而将靶标分子捕获；
(3)用光学成像设备检测从所述原位信号增强纳米粒子发出的光学信号；
(4)对原位信号增强纳米粒子的个数进行统计，进一步计算得到样品中靶标分子的浓度信息。
第二实施方式与第一实施方式相比，仅在步骤的实施顺序上存在区别，其他条件如原位信号增强纳 米粒子等均相同。
<第三实施方式>
本申请的第三实施方式如下。
一种单分子定量检测分析方法，包括如下步骤：
(1)将能够与靶标分子结合的捕获探针固定到固相载体上，所述捕获探针与靶标分子的第一序列互补，利用捕获探针捕获样品中的靶标分子；
(2)加入检测探针，所述检测探针能够与靶标分子的第二序列互补，形成捕获探针—靶标分子—检测探针的三链杂交结构，然后加入原位信号增强纳米粒子；所述原位信号增强纳米粒子能够直接或间接与检测探针结合；或
先将检测探针与原位信号增强纳米粒子结合形成而复合材料，再将该复合材料加入；
其中，所述原位信号增强纳米粒子含有发光材料和纳米粒子载体，且粒径为180-480nm；
(3)用光学成像设备检测所述原位信号增强纳米粒子发出的光学信号；
(4)统计原位信号增强纳米粒子的个数，进一步计算得到样品中靶标分子的浓度信息。
本实施方式中，靶标分子包括DNA或RNA。
所述检测试剂包括固相载体、捕获探针、检测探针以及原位信号增强纳米粒子。其中，所述固相载体用于检测样品与试剂的分离和清洗。所述捕获探针通过化学修饰而固定在固相载体表面，可与待测分子的一部分进行杂交结合，使其从样品中分离。所述检测探针可与待测分子的另一部分进行杂交结合，检测探针的远离杂交位置的一端通过化学共价键与原位信号增强纳米粒子相连接。所述原位信号增强纳米粒子能够发出足够强的光学信号，在所述光学成像设备上形成独立可辨别的图像信号。
所述固相载体按照形态分类，可以为磁珠、多孔板、离心管、芯片、微米尺度微球以及纳米尺度微球中的一种或多种；按照材质分类，可以为有机聚合物、二氧化硅以及硅中的一种或多种；按照功能分类，可以为磁性固相载体和非磁性固相载体中的一种。这些固相载体中，尤其优选磁珠。以往认为，将磁珠应用于核酸的定量检测时存在下述问题：磁珠悬浮而难以定量检测，且存在方向性而使亮度存在区别。本申请的发明人发现，在想要对核酸进行单分子定量检测的情况下，与使用玻璃芯片作为载体时相比，使用磁珠作为载体时能够获得尤其优异的灵敏度，并极大地提高了检测效率。
本申请中，所述固相载体的表面修饰有能够与探针共价偶联的活性官能团，包括羟基、羧基、氨基、巯基、烯基、炔基、琥珀酰亚胺酯基团及其衍生基团中的一种或多种。
本申请中，所述捕获探针可以是核糖核酸或脱氧核糖核酸，其序列与待测分子的一段序列彼此互补，能够形成双链杂交。
本申请中，所述捕获探针的一端修饰有羧基、氨基、巯基以及琥珀酰亚胺酯中的一种或几种，能够与固相载体的表面发生共价结合，从而稳定地结合在固相载体的表面。
本申请中，所述检测探针可以是核糖核酸或脱氧核糖核酸，其序列与待测分子的另一段序列(不同于与捕获探针互补的序列)互补，能够形成双链杂交，从而形成捕获探针—靶标分子—检测探针三链杂交结构。
本申请中，所述检测探针的一端修饰有羧基、氨基、巯基以及琥珀酰亚胺酯中的一种或几种，能够与原位信号增强纳米粒子共价偶联，从而稳定结合于原位信号增强纳米粒子的表面。
关于其他条件，例如原位信号增强纳米粒子、光学成像设备以及靶标分子浓度的计算方式等，与上述第一实施方式相同。
<第四实施方式>
本申请的第四实施方式如下。
一种单分子定量检测分析方法，包括如下步骤：
(1)使检测探针与样品中的靶标分子的第二序列互补，然后加入原位信号增强纳米粒子，所述原位信号增强纳米粒子直接或间接与检测探针结合；或者
预先使原位信号增强纳米粒子与检测探针结合而形成复合材料，然后使该复合材料与样品中的靶标分子的第二序列互补；
其中，所述原位信号增强纳米粒子含有发光材料和纳米粒子载体，且粒径为180-480nm；
(2)将能够与靶标分子结合的捕获探针固定至固相载体上，然后，使所述捕获探针与所述靶标分子的第一序列互补，从而将靶标分子捕获；
(3)用光学成像设备检测从所述原位信号增强粒子发出的光学信号；
(4)对原位信号增强粒子的个数进行统计，进一步计算得到样品中靶标分子的浓度信息。
第四实施方式与第三实施方式相比，仅在步骤的实施顺序上存在区别，其他条件如原位信号增强纳米粒子等均相同。
<第五实施方式>
本申请的第五实施方式如下。
一种单分子定量检测系统，包括：
(1)检测试剂，
所述检测试剂包括(a)捕获抗体，所述捕获抗体能够与靶标分子的第一位点结合从而捕获样品中的靶标分子；(b)检测抗体，所述检测抗体能够与靶标分子的第二位点结合，并能够与原位信号增强纳米粒子结合；(c)原位信号增强纳米粒子，其含有发光材料和纳米粒子载体，且粒径为180-480nm；或者
所述检测试剂包括(a)捕获探针，所述捕获探针能够与靶标分子的第一序列结合从而捕获样品中的靶标分子；(b)检测探针，所述检测探针能够与靶标分子的第二序列结合，形成捕获探针—靶标分子—检测探针的三链杂交结构；(c)原位信号增强纳米粒子，其能够结合于检测探针上，含有发光材料和纳米粒子载体，且粒径为180-480nm；
(2)光学成像设备，其包括激发光源及光学信号采集单元。
本申请的检测系统包含检测试剂和光学成像设备这两部分。所述检测试剂包含捕获抗体、检测抗体和特定的原位信号增强纳米粒子(用于检测蛋白质、多糖或有生物活性的小分子时)，或者包含捕获探针、检测探针和特定的原位信号增强纳米粒子(用于检测DNA或RNA时)。所述光学成像设备为常规的光学成像设备即可，无需全内反射荧光显微镜、落射荧光显微镜、扫描近场光学显微镜、共聚焦荧光显微镜等昂贵的、突破了光学衍射极限的光学成像设备。
[实施例]
以下，举出实施例和比较例进一步详细地说明本申请，但本申请并不限定于此。
1、原位信号增强纳米粒子的粒径的测定
以二氧化硅荧光纳米粒子为例，将各实施例和比较例中得到的二氧化硅荧光纳米粒子用水稀释1000倍，然后取100μL滴加在洁净硅片表面，晾干，使用小型溅射仪在其表面溅射沉积5nm的铂，使用SEM(日本日立高新技术公司制SU3900)进行成像分析，求出粒径。
以聚丙烯酰胺荧光纳米粒子为例，用纯水将得到的聚丙烯酰胺荧光纳米粒子稀释1000倍，使用马尔文粒度分析仪(Zetasizer Nano S90)测定粒子的粒径。
2、单分子成像
使用常规荧光显微镜例如尼康Eclipse Ti-U荧光显微镜进行单分子成像，此外，也可采用尼康Eclipse Ti系列的其他荧光显微镜、莱卡DMi8荧光显微镜等。
3、标准曲线绘制方法
本申请中，单分子计数模式与荧光强度积分模式的联合使用能够显著提高检测标志物标准曲线的动态检测范围。具体实施方法如下：
在待测分子的浓度较低时，磁珠数量多于与磁珠结合的待测分子数量，因此使用单分子计数模式对不同浓度的待测分子样本进行标准曲线绘制；
当待测分子的浓度超过一定阈值时，一个磁珠表面可能结合有1个以上的待测分子，单分子信号容易叠加，导致检测结果出现偏差，因此更适合使用荧光强度积分模式。
具体而言，当一张成像图片中的单分子数量不超过设定的阈值时，使用单分子计数模式进行标准曲线的绘制；当一张成像图片中的单分子数量超过设定的阈值时，使用荧光强度积分模式，并将总荧光强度面积除以每个分子的平均荧光强度面积，换算为“近似单分子数量”，从而进行标准曲线绘制。
最后，将利用单分子计数模式得到的标准曲线与利用荧光强度积分模式得到的标准曲线合并，并利 用拟合公式进行曲线拟合，绘制完整的标准曲线。
实施例1：磁珠法用于人血清中cTnI抗原分子的检测(二氧化硅荧光纳米粒子：粒径为220nm)。
1、实验组分
甲苯磺酰基活化M280磁珠(Thermo)、捕获抗体(Hytest 19C7)、检测抗体(Hytest 16A11)、硅烷偶联剂(APTES)、氨水、正硅酸乙酯(TEOS)、异硫氰酸荧光素(FITC)、丁二酸酐、待测血清样本、PBS缓冲液、Buffer C(3mM(NH 4) 2SO 4溶于10mM PBS缓冲液，pH＝7.4)、Buffer D(0.01％NaCl，0.5％BSA溶于10mM PBS，pH＝7.4)、Buffer E(0.0088％NaCl，0.1％BSA溶于10mM PBS，pH＝7.4)、N-羟基琥珀酰亚胺(NHS)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)、微球保存液、样品稀释液以及PBS洗液。
2、制备方法
2.1、二氧化硅荧光纳米粒子的合成和表面羧基化修饰
(1)在平底塑料合成瓶中加入16.5mL无水乙醇、1.2mL氨水、0.9mL超纯水、0.7mL TEOS、0.3μL APTES、0.6mg FITC，并混匀，以400rpm的转速在50℃下搅拌5小时，经20mL无水乙醇清洗(5次)并离心后，获得内部包裹有FITC的二氧化硅微球，使其悬浮于20mL无水乙醇中。
(2)将等摩尔的APTES和丁二酸酐溶于1mL DMF中，常温下搅拌3小时后，添加到(1)中得到的悬浮有二氧化硅微球(其内部均匀分布有FITC)的无水乙醇溶液，加入2mL超纯水和1.2mL氨水，以400rpm的转速在50℃下搅拌5小时，经20mL无水乙醇清洗(3次)并离心后，继续用20mL超纯水清洗(3次)并离心，然后重新悬浮于20mL超纯水中，获得粒径为220nm的经表面羧基化的二氧化硅荧光纳米粒子。
2.2、磁珠与捕获抗体的共价偶联
(1)取166.6μL 30mg/mL的经甲苯磺酰基活化的M280磁珠，使用10mM PBS缓冲液清洗5次，移除缓冲液。
(2)取100μg捕获抗体(Hytest 19C7)，用150μL 10mM PBS缓冲液稀释后加入到(1)的磁珠中，混合均匀，加入100μL Buffer C，于37℃旋转混匀，反应1h。
(3)使用10mM PBS缓冲液清洗5次，加入1mL Buffer D进行封闭反应。于37℃旋转混匀，反应1h。
(4)使用10mM PBS缓冲液清洗5次，使用250μL Buffer E保存，待用。
2.3、检测抗体与二氧化硅荧光纳米粒子的共价偶联
(1)取10μL的二氧化硅荧光纳米粒子，加入40μL PBS缓冲液超声1min。
(2)取0.005g EDC，溶于50μL PBS缓冲液，另取0.0135g NHS溶于150μL PBS缓冲液。
(3)以12000rpm的速度将二氧化硅荧光纳米粒子离心，去除上清液，加入50μL PBS缓冲液重悬，超声1min后加入2.5μL EDC溶液，超声1min后再加入7.5μL NHS溶液，混匀后于37℃旋转混匀反应15min，以12000rpm的转速离心15min，去除上清液，使用50μL PBS缓冲液将纳米粒子重悬。
(4)加入20μg检测抗体(Hytest 16A11)，于37℃旋转混匀反应2h。
(5)加入25μL Buffer D，于37℃封闭反应1h后，以12000rpm离心15min，使用100μL Buffer E重悬保存。
3、实验方法
标准曲线的制作
(1)使用胎牛血清将cTnI抗原的浓度分别稀释为0、0.01、0.1、0.5、1、5、10以及100pg/mL。
(2)将标记有捕获抗体的磁珠稀释为1mg/mL，取50μL，分别加入50μL(1)中得到的各浓度的样品，于37℃孵育60min。使用100μL清洗缓冲液洗三次，洗去残余样品，吸干上清液。
(3)加入10μL结合有检测抗体的二氧化硅荧光纳米粒子，于37℃孵育45min，使用清洗缓冲液洗4次，洗去残余的二氧化硅荧光纳米粒子，去除上清液。
(4)加入5μL检测液将磁珠重悬，转移至检测孔，使用磁铁将磁珠吸引至检测孔的底部，使用荧光显微镜(尼康Eclipse Ti-U)进行单分子成像，联合使用单分子计数模式和荧光强度积分模式来完成后续单分子计数统计和分析。
(5)完成一系列浓度检测，每个浓度点重复6次，根据检测结果绘制标准曲线，计算每个点的CV％值。
4、实验结果
标准曲线的结果
检测结果如图3所示，可知该实施例中，cTnI的检测范围为30fg/mL—10ng/mL，在该区间内单分子信号数(即CPN)与样品浓度呈良好的线性关系，其检测下限可达30fg/mL。
比较例1：磁珠法用于人血清中cTnI抗原分子的检测(二氧化硅荧光纳米粒子：粒径为150nm)。
1、制备方法
1.1、二氧化硅荧光纳米粒子的合成和表面羧基化修饰
(1)在平底塑料合成瓶中加入16.5mL无水乙醇、1.2mL氨水、0.9mL超纯水、0.5mL TEOS、0.3μL APTES、0.6mg FITC混匀，以400rpm转速于50℃搅拌5小时，经20mL无水乙醇清洗(5次)并离心后，获得内部包裹有FITC的二氧化硅微球，使其悬浮于20mL无水乙醇中。
(2)将等摩尔的APTES与丁二酸酐溶于1mL DMF中，常温下搅拌3小时后，添加到(1)中得到的悬浮有二氧化硅微球的无水乙醇溶液，加入2mL超纯水和1.2mL氨水，以400rpm转速于50℃搅拌5小时，经20mL无水乙醇清洗(3次)并离心后，继续用20mL超纯水清洗(3次)并离心，然后重新悬浮于20mL超纯水中，得到粒径为150nm的经表面羧基化的二氧化硅荧光纳米粒子。
1.2、磁珠与捕获抗体的共价偶联
与实施例1的相应步骤同样地进行操作。
1.3、检测抗体与二氧化硅荧光纳米粒子的共价偶联
除了取粒径为150nm的经表面羧基化的二氧化硅荧光纳米粒子外，与实施例1的相应步骤同样地进行操作。
2、实验方法
标准曲线的制作
与实施例1的相应步骤同样地进行操作。
3、实验结果
由图4可知，当使用粒径为150nm的二氧化硅荧光纳米粒子时，在检测设备中完全检测不到任何荧光信号，可见该尺寸的二氧化硅荧光纳米粒子无法用于单分子定量检测。由于试剂对样品完全无响应，因此未进行临床样本测试。
比较例2：磁珠法用于人血清中cTnI抗原分子的检测(二氧化硅荧光纳米粒子：粒径为500nm)。
1、制备方法
1.1、二氧化硅荧光纳米粒子的合成和表面羧基化修饰
(1)在平底塑料合成瓶中加入16.5mL无水乙醇、1.0mL氨水、0.9mL超纯水、1.2mL TEOS、0.3μL APTES、0.6mg FITC混匀，以400rpm转速于50℃搅拌5小时，经20mL无水乙醇清洗(5次)并离心后，获得内部包裹有FITC的二氧化硅微球，使其悬浮于20mL无水乙醇中。
(2)将等摩尔的APTES与丁二酸酐溶于1mL DMF中，常温下搅拌3小时后，添加到(1)中得到的悬浮有二氧化硅微球的无水乙醇溶液，加入2mL超纯水和1.2mL氨水，以400rpm转速于50℃搅拌5小时，经20mL无水乙醇清洗(3次)并离心后，继续用20mL超纯水清洗(3次)并离心，然后重新悬浮于20mL超纯水中，得到粒径为500nm的经表面羧基化的二氧化硅荧光纳米粒子。
1.2、磁珠—捕获抗体的共价偶联
与实施例1的相应步骤同样地进行操作。
1.3、检测抗体与二氧化硅荧光纳米粒子的共价偶联
除了取粒径为500nm的经表面羧基化的二氧化硅荧光纳米粒子外，与实施例1的相应步骤同样地进行操作。
2、实验方法
标准曲线的制作
与实施例1的相应步骤同样地进行操作。
3、实验结果
检测结果如图5所示，可知比较例2中，cTnI的检测下限仅为0.1ng/mL(即100pg/mL)，灵敏度较差。比较例2因使用粒径为500nm的粒子，导致检测灵敏度远远低于实施例1。
实施例2：磁珠法用于人血清中cTnI抗原分子的检测(二氧化硅荧光纳米粒子：粒径为350nm)。
1、制备方法
1.1、二氧化硅荧光纳米粒子的合成和表面羧基化修饰
(1)在平底塑料合成瓶中加入16.5mL无水乙醇、1.0mL氨水、0.9mL超纯水、0.7mL TEOS、0.3μL APTES、0.6mg FITC混匀，以400rpm转速于50℃搅拌5小时，经20mL无水乙醇清洗(5次)并离心后，获得内部包裹有FITC的二氧化硅微球，使其悬浮于20mL无水乙醇中。
(2)将等摩尔的APTES与丁二酸酐溶于1mL DMF中，常温下搅拌3小时后，添加到(1)中得到的悬浮有二氧化硅微球的无水乙醇溶液，加入2mL超纯水和1.2mL氨水，以400rpm转速于50℃搅拌5小时，经20mL无水乙醇清洗(3次)并离心后，继续用20mL超纯水清洗(3次)并离心，然后重新悬浮于20mL超纯水中，得到粒径为350nm的经表面羧基化的二氧化硅荧光纳米粒子。
1.2、磁珠与捕获抗体的共价偶联
与实施例1的相应步骤同样地进行操作。
1.3、检测抗体—二氧化硅荧光纳米粒子的共价偶联
除了取粒径为350nm的经表面羧基化的二氧化硅荧光纳米粒子外，与实施例1的相应步骤同样地进行操作。
2、实验方法
标准曲线的制作
与实施例1的相应步骤同样地进行操作。
3、实验结果
检测结果如图6所示，可知实施例2中，cTnI的检测下限为80fg/mL，灵敏度优异。
比较例3
除了采用的经表面羧基化的二氧化硅荧光纳米粒子的粒径为160nm以外，与实施例1同样地进行操作，其检测下限的结果与比较例1相同。
实施例3
除了采用的经表面羧基化的二氧化硅荧光纳米粒子的粒径为180nm以外，与实施例1同样地进行操作，将其结果示于表1。
实施例4
除了采用的经表面羧基化的二氧化硅荧光纳米粒子的粒径为250nm以外，与实施例1同样地进行操作，将其结果示于表1。
实施例5
除了采用的经表面羧基化的二氧化硅荧光纳米粒子粒径为295nm以外，与实施例1同样地进行操作，将其结果示于表1。
实施例6
除了采用的经表面羧基化的二氧化硅荧光纳米粒子的粒径为300nm以外，与实施例1同样地进行操作，将其结果示于表1。
实施例7
除了采用的经表面羧基化的二氧化硅荧光纳米粒子的粒径为400nm以外，与实施例1同样地进行操作，将其结果示于表1。
实施例8
除了采用的经表面羧基化的二氧化硅荧光纳米粒子的粒径为450nm以外，与实施例1同样地进行操作，将其结果示于表1。
实施例9
除了采用的经表面羧基化的二氧化硅荧光纳米粒子的粒径为480nm以外，与实施例1同样地进行操 作，将其结果示于表1。
实施例10
在固相载体与捕获抗体的共价偶联步骤中，采用甲苯磺酰基活化的玻璃芯片替换甲苯磺酰基活化的磁珠，除此以外，与实施例1同样地进行操作，将其结果示于表1。
实施例11
在对单分子成像的数据进行处理的步骤中，在整个浓度区间内采用单分子计数模式替换单分子计数模式与荧光强度积分模式的联用，除此以外，与实施例1同样地进行操作，结果如图7所示，可知与实施例1相比，检测上限降低，检测动态范围减小。
实施例12
在进行单分子成像的步骤中，采用莱卡DMi8荧光显微镜替换尼康Eclipse Ti-U，除此以外，与实施例1同样地进行操作，将其结果示于表1。
实施例13
在实施例1的步骤2.3和3.1中，不预先形成检测抗体与二氧化硅荧光纳米粒子的复合物，而是先加入修饰有生物素的检测抗体，使其与待测分子的第二位点结合，然后加入修饰有链霉亲和素的二氧化硅荧光纳米粒子，除此以外，与实施例1同样地进行操作，将其结果示于表1。
实施例14：磁珠法用于IL-6抗原分子的检测(聚丙烯酰胺荧光纳米粒子：粒径为220nm)
1、实验组分
甲苯磺酰基活化的M280磁珠(Thermo)、IL-6捕获抗体(Medix 2703)、IL-6检测抗体(Medix 2704)、丙烯酰胺、N,N-亚甲基双丙烯酰胺、丙烯酸、己烷、琥珀辛酯磺酸钠、月桂醇聚氧乙烯醚35(Brij 35)、亚硫酸氢纳、过硫酸铵(引发剂)、四甲基乙二胺、丙烯酰胺—(PEG) 8—荧光素、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)、N-羟基琥珀酰亚胺(NHS)、乙醇、待测血清样本、PBS缓冲液、Buffer C(3mM(NH 4) 2SO 4溶于10mM PBS缓冲液，pH＝7.4)、Buffer D(0.01％NaCl，0.5％BSA溶于10mM PBS，pH＝7.4)、Buffer E(0.0088％NaCl，0.1％BSA溶于10mM PBS，pH＝7.4)、N-羟基琥珀酰亚胺(NHS)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)、荧光粒子保存液以及样品稀释液以及PBS洗液
2、制备方法
2.1、羧基化聚丙烯酰胺荧光纳米粒子的合成以及其与检测抗体的复合物的合成
(1)将1mL含有23％丙烯酰胺、3％N,N-亚甲基双丙烯酰胺、2％丙烯酸以及1％丙烯酰胺—(PEG) 8—荧光素的10mM PBS缓冲液加入至20mL己烷中，然后加入2％Brij-35，室温下以1000rpm的转速搅拌过夜。加入50μL 10％亚硫酸钠溶液、24μL 10％过硫酸铵和12μL四甲基乙二胺。室温下继续搅拌2小时。在室温下，让己烷完全挥发，使用10mM PBS缓冲液使挥发后剩下的聚丙烯酰胺荧光粒子重新悬浮，用100kD超滤管(Millipore)滤去表面活性剂和残余单体。再一次使用100μL 10mM PBS缓冲液使其重悬。
(2)取10μL(1)中制得的聚丙烯酰胺荧光纳米粒子的悬浮液，用10mM PBS缓冲液将其稀释至100μL，加入10μL含有0.5％EDC的水溶液和5μL含有0.5％NHS的水溶液，于37℃活化1小时，使用100kD超滤管去除残余的活化剂，加入100μL 10mM PBS缓冲液使其重悬，然后加入20μg IL-6检测抗体(Medix 2704)，于37℃反应2小时。使用150KD超滤管去除残余的抗体，并使用100μL 10mM PBS缓冲液使其重悬，将超滤和重悬步骤重复一次，将得到的聚丙烯酰胺荧光纳米粒子与检测抗体的复合物于4℃保存备用。
2.2、磁珠与捕获抗体的共价偶联
(1)取166.6μL 30mg/mL的经甲苯磺酰基活化的M280磁珠，使用10mM PBS缓冲液清洗5次，移除缓冲液。
(2)取100μg捕获抗体(Medix 2703)，使用150μL 10mM PBS缓冲液稀释后加入到(1)的磁珠中，混合均匀，加入100μL Buffer C，于37℃旋转混匀，反应1h。
(3)使用10mM PBS缓冲液清洗5次，加入1mL Buffer D进行封闭反应。于37℃旋转混匀，反应1h。
(4)使用10mM PBS缓冲液清洗5次，使用250μL Buffer E保存，待用。
3、实验方法
3.1、聚丙烯酰胺荧光纳米粒子的粒径测定
用纯水将得到的聚丙烯酰胺荧光纳米粒子稀释1000倍，使用马尔文粒度分析仪测定粒子的粒径。
3.2、IL-6抗原检测
(1)使用胎牛血清将IL-6抗原的浓度分别稀释为0、0.01、0.1、0.5、1、5、10、50以及100pg/mL。
(2)将标记有捕获抗体的磁珠稀释为0.1mg/mL，取50μL，然后分别加入50μL的(1)中得到的各浓度的样品，于37℃孵育30min。使用100μL的清洗缓冲液洗三次，洗去残余样品，吸干上清液。
(3)加入10μL结合有检测抗体的聚丙烯酰胺荧光纳米粒子，于37℃孵育15min，使用清洗缓冲液洗4次，洗去残余的聚丙烯酰胺荧光纳米粒子，去除上清液。
(4)加入5μL检测液将磁珠重悬，转移至检测孔，使用磁铁将磁珠吸引至检测孔的底部，使用荧光显微镜(尼康Eclipse Ti-U)进行单分子成像，利用荧光强度积分模式来完成后续单分子计数统计与分析。
(5)完成一系列浓度检测，每个浓度点重复3次，根据检测结果绘制标准曲线，计算每个点的CV％值。
4、实验结果
4.1、聚丙烯酰胺荧光纳米粒子的粒径测定结果
由马尔文粒度分析仪测得的聚丙烯酰胺荧光纳米粒子的粒径为220nm。
4.2、IL-6抗原检测结果
IL-6抗原的检测结果如图8所示，可知浓度为0.01pg/mL的稀释样本能够有效地与背景区分开，经计算，该实施例的检测下限为0.006pg/mL(即6fg/mL)。可见本申请的检测方法的灵敏度极为优异。
实施例15：磁珠法用于cTnI抗原分子的检测(聚丙烯酰胺荧光纳米粒子：粒径为220nm)
在实施例14中，将捕获抗体由Medix 2703替换为Hytest 16A11(即cTnI抗原的捕获抗体)，将检测抗体由Medix 2704替换为Hytest 19C7(即cTnI抗原的检测抗体)，将待测分子由IL-6抗原替换为cTnI抗原，除此以外，与实施例14同样地操作。结果，cTnI抗原的检测结果如图9所示，可知浓度为0.01pg/mL的稀释样本能够有效地与背景区分开，经计算，该实施例的检测下限为0.008pg/mL(即8fg/mL)。可见本申请的检测方法的灵敏度极为优异。
实施例16
除了采用的经表面羧基化的聚丙烯酰胺荧光纳米粒子的粒径为300nm以外，与实施例15同样地进行操作。结果，cTnI抗原的检测结果如图10所示，可知浓度为0.01pg/mL的稀释样本能够有效地与背景区分开，经计算，该实施例的检测下限为0.020pg/mL(即20fg/mL)。可见本申请的检测方法的灵敏度较优异。
实施例17：磁珠法用于cTnI抗原分子的检测(聚丙烯酰胺荧光纳米粒子：二次粒径为250nm)
1、实验组分
甲苯磺酰基活化M280磁珠(Thermo)、捕获抗体(Hytest 16A11)、检测抗体(Hytest 19C7)、链霉亲和素(SA，Roche)、丙烯酰胺以及丙烯酰胺—(PEG) 8—生物素以及丙烯酰胺—(PEG) 8—荧光素。
2、制备方法
2.1、一级聚丙烯酰胺荧光粒子的合成和羧基化修饰
(1)将1mL含有23％丙烯酰胺、3％N,N-亚甲基双丙烯酰胺、2％丙烯酸以及1％丙烯酰胺—(PEG) 8—生物素和1％丙烯酰胺—(PEG) 8—荧光素的10mM PBS缓冲液加入到20mL己烷中，加入4％Brij-35，室温下以1200rpm的转速搅拌过夜。加入50μL 10％亚硫酸钠溶液、24μL 10％过硫酸铵和12μL四甲基乙二胺。室温下继续搅拌2小时。在室温下，让己烷完全挥发，使用10mM PBS缓冲液使挥发后剩下的聚丙烯酰胺荧光粒子重新悬浮，使用100kD超滤管滤去表面活性剂和残余单体。使用100μL 10mM PBS缓冲液重悬。
(2)取15μL(1)中制得的聚丙烯酰胺荧光纳米粒子的悬浮液，用10mM PBS缓冲液将其稀释至100μL，加入10μL含有0.5％EDC的水溶液和5μL含有0.5％NHS的水溶液，于37℃活化1小时，使用100kD超滤管去除残余活化剂，加入100μL 10mM PBS缓冲液重悬，加入20μg检测抗体，于37℃反应 2小时。使用150KD超滤管去除残余抗体，并使用100μL 10mM PBS缓冲液重悬，再将超滤和重悬步骤重复一次，得到的一级聚丙烯酰胺纳米粒子悬浮液于4℃保存，备用。
2.2、二级聚丙烯酰胺荧光粒子的合成和羧基化修饰
(1)将1mL含有23％丙烯酰胺、3％N,N-亚甲基双丙烯酰胺、2％丙烯酸以及1％丙烯酰胺—(PEG) 8—荧光素的10mM PBS缓冲液加入至20mL己烷中，加入4％Brij-35，室温下以1200rpm的转速搅拌过夜。加入50μL 10％亚硫酸钠溶液、24μL 10％过硫酸铵和12μL四甲基乙二胺。室温下继续搅拌2小时。在室温下，让己烷完全挥发，使用10mM PBS缓冲液将挥发后剩下的聚丙烯酰胺荧光粒子重悬，使用100kD超滤管滤去表面活性剂和残余单体。使用100μL 10mM PBS缓冲液重悬。
(2)取15μL(1)中制得的聚丙烯酰胺荧光粒子用10mM PBS缓冲液稀释至100μL，加入10μL含有0.5％EDC的水溶液和5μL含有0.5％NHS的水溶液，于37℃活化1小时，使用100kD超滤管去除残余活化剂，加入100μL 10mM PBS缓冲液重悬，然后加入10μg链霉亲和素，于37℃反应2小时。使用150KD超滤管去除残余的链霉亲和素，并使用100μL 10mM PBS缓冲液重悬，再将超滤和重悬步骤重复一次，得到的二级聚丙烯酰胺荧光粒子悬浮液于4℃保存，备用。
2.3、磁珠与捕获抗体的共价偶联
与实施例1的相应步骤同样地进行操作。
3、实验方法
3.1、聚丙烯酰胺荧光粒子的粒径测定
关于一级和二级聚丙烯酰胺荧光粒子，用纯水将它们各自稀释2000倍，使用马尔文粒度分析仪测定它们的粒径。关于二次聚丙烯酰胺荧光粒子(即以一级粒子为核、以二级粒子为壳的团簇颗粒)的粒径，其粒径的测定方法如下：取相对于一级粒子而言过量的二级粒子，使其与一级粒子反应，针对反应得到的二次粒子，用马尔文粒度分析仪测定其粒径，结果可得到两个峰，将较大的峰作为二次粒子的粒径。
3.2、cTnI抗原检测
(1)使用胎牛血清将cTnI抗原的浓度稀释为0、0.01、0.1、0.5、1、5、10、50以及100pg/mL。
(2)将标记有捕获抗体的磁珠稀释为0.1mg/mL，取50μL，分别加入50μL的(1)中得到的各浓度的样品，然后加入稀释了100倍的一级聚丙烯酰胺荧光粒子10μL，于37℃孵育30min。使用100μL清洗缓冲液洗三次，洗去残余样品，吸干上清液。
(3)加入稀释了100倍的二级聚丙烯酰胺荧光粒子10μL，于37℃孵育15min，使用清洗缓冲液洗4次，洗去残余的聚丙烯酰胺荧光粒子，去除上清液。
(4)加入5μL检测液将磁珠重悬，转移至检测孔，使用磁铁将磁珠吸引至检测孔的底部，使用荧光显微镜(尼康Eclipse Ti-U)进行单分子成像，利用单分子计数模式与荧光强度积分模式的联用来完成后续单分子计数统计和分析。
(5)完成一系列浓度检测，每个浓度点重复3次，根据检测结果绘制标准曲线，计算每个点的CV％值。
4、实验结果
4.1、聚丙烯酰胺荧光粒子的粒径
一级和二级聚丙烯酰胺荧光粒子的粒径均约为80nm，二次聚丙烯酰胺荧光粒子的粒径约为250nm。
4.2、cTnI抗原检测
cTnI抗原的检测结果如图11所示，可知浓度为0.005pg/mL的稀释样本能够有效地与背景区分开，经计算，本实施例的检测下限为0.002pg/mL(即2fg/mL)。可见本申请的检测方法的灵敏度极为优异。
实施例18：磁珠法用于IL-6抗原分子的快速检测(聚丙烯酰胺荧光纳米粒子：粒径为220nm)
在实施例14中，将步骤3.2(2)中的孵育时间由30min缩短为3min，将步骤3.2(3)中的孵育时间由15min缩短为2min，除此以外，与实施例14同样地进行操作。
IL-6抗原的快速检测结果如图12所示，可知即使将检测时间大幅缩短，1pg/mL的稀释样本也能够有效地与背景区分开，经计算，实施例18的检测下限可达到1pg/mL。此外，本实施例中还测试了20个临床血液样本，由图13所示，横坐标为由Roche Cobas 6000全自动电化学发光设备测得的参考值， 纵坐标为由本申请的检测方法得到的测量值。经相关分析可知，R 2值为0.9887，证明本申请的方法与市售的成熟设备所使用的方法具有良好的相关性。
实施例19：磁珠法用于cTnI抗原分子的快速检测(聚丙烯酰胺荧光纳米粒子：粒径为220nm)
在实施例15中，将步骤3.2(2)中的孵育时间由30min缩短为3min，将步骤3.2(3)中的孵育时间由15min缩短为2min，除此以外，与实施例15同样地进行操作。
cTnI抗原的快速检测结果如图14所示，可知即使将检测时间大幅缩短，1pg/mL的稀释样本也能够有效地与背景区分开，经计算，实施例19的检测下限可达到0.5pg/mL。此外，本实施例中还测试了20个临床血液样本，由图15所示，横坐标为由雅培Abbott i1000sr全自动电化学发光设备测得的参考值，纵坐标为由本申请的检测方法得到的测量值。经相关分析可知，R 2值为0.9842，证明本申请的方法与市售的成熟设备所使用的方法具有良好的相关性。
实施例20：磁珠法用于人血清中cTnI抗原分子的检测(聚苯乙烯包裹量子点而成的荧光微球(以下称为聚苯乙烯荧光纳米粒子)：粒径为210nm)。
1、实验组分
量子点(Qdot 605，Thermo)、表面羧基化的聚苯乙烯微球(粒径为210nm，杭州微著生物)
2、制备方法
2.1、聚苯乙烯荧光纳米粒子
(1)称取0.1g表面羧基化的聚苯乙烯微球分散于5mL 0.25％的SDS中，加入Qdot 605和1mL二氯甲烷，使用超声仪于室温超声1小时。
(2)进行旋蒸以除去溶剂，对剩下的液体进行离心，去除上清液。用乙醇将回收的固体清洗三次，接着用超纯水清洗5次，之后用10mL超纯水使其重悬，保存。
2.2、磁珠与捕获抗体的共价偶联
与实施例1的相应步骤同样地进行操作。
2.3、检测抗体与聚苯乙烯荧光纳米粒子的共价偶联
(1)取(2.1)中制得的聚苯乙烯荧光纳米粒子5μL，加入40μL PBS缓冲液超声1min。
(2)取0.01g EDC，溶于50μL PBS缓冲液中，另取0.015g NHS溶于150μL PBS缓冲液中。
(3)以10000rpm的速度进行离心，去除上清液，加入50μL PBS缓冲液重悬，超声1min后加入2.5μL EDC溶液和7.5μL NHS溶液，混匀后于37℃旋转混匀反应15min，以12000rpm的转速离心15min，去除上清液，使用50μL PBS缓冲液将纳米粒子重悬。
(4)加入5μg检测抗体(Hytest 16A11)，于37℃旋转混匀反应2h。
(5)加入25μL Buffer D，于37℃封闭反应1h后，以12000rpm离心15min，使用100μL Buffer E重悬保存。
3、实验方法
标准曲线的制作
除了在步骤3.1(3)中加入结合有检测抗体的聚苯乙烯荧光纳米粒子外，与实施例1同样地进行操作。
4、实验结果
检测结果如图16所示，可知该实施例中，cTnI的检测范围为50fg/mL～100pg/mL，在该区间内单分子信号数与样品浓度成良好的线性关系，其检测下限约50fg/mL。
实施例21(对应本申请的第二实施方式)
在实施例1的步骤3.1和3.2中，先使结合有检测抗体的二氧化硅荧光纳米粒子与临床样本反应，再加入标记有捕获抗体的磁珠，除此以外，与实施例1同样地操作，将其结果示于表1中。
实施例22：玻璃芯片法用于缓冲液中DNA分子的定量检测(二氧化硅荧光纳米粒子：粒径为220nm)。
1、实验组分
低吸附载玻片(Thermo)、捕获探针(上海生工合成，序列见下文)、检测探针(上海生工合成，序列见下文)、DNA模板(上海生工合成，序列见下文)、硅烷偶联剂(APTES)、氨水、正硅酸乙酯(TEOS)、 异硫氰酸荧光素(FITC)、丁二酸酐、双端羧基化聚乙二醇以及PBS缓冲液、
捕获探针序列：NH 2-TTTTTTTTTTTGTGTGACATGTTCTAATATAGTCACAT
检测探针序列：TCTGATATAATCTTGTACAGTGTGTTTTTTTTTT-NH 2
DNA模板序列：
Figure PCTCN2020070283-appb-000001
2、制备方法
2.1、二氧化硅荧光纳米粒子的合成和表面羧基化修饰
与实施例1的相应步骤同样地进行操作。
2.2、载玻片表面活化和捕获探针的修饰
(1)取一低吸附载玻片(Thermo)，在超纯水中超声1小时，在无尘条件下于70℃烘干12小时。使用等离子体清洗机清洗载玻片表面，在载玻片表面产生具有高活性的羟基。
(2)将活化后的载玻片浸渍于浓度为1％的APTES中，于37℃反应2小时，使得载玻片表面氨基化，用超纯水对载玻片的表面清洗5次，于37℃烘干待用。
(3)配置0.5％的双端羧基化聚乙二醇，加入5倍量的EDC和10倍量的NHS，活化10分钟后将(2)中得到的载玻片浸渍于已活化的聚乙二醇溶液中。反应15分钟后，用超纯水对载玻片的表面清洗5次，然后用氮气吹干。
(4)在载玻片的表面上滴加300μL 1μM的捕获探针，室温下反应1小时，使用超纯水清洗5次，除去未反应的捕获探针。
(5)将结合有捕获探针的载玻片浸渍于Buffer D中，于37℃孵育过夜，使用超纯水清洗5次，用氮气吹干，于50℃过夜烘干，待用。
2.3、检测探针与二氧化硅荧光纳米粒子的共价偶联
(1)取10μL二氧化硅荧光纳米粒子，加入40μL PBS缓冲液超声1min。
(2)取0.01g EDC，溶于50μL PBS缓冲液，另取0.025g NHS溶于150μL PBS缓冲液。
(3)以12000rpm的速度将二氧化硅荧光纳米粒子离心，去除上清液，加入50μL PBS缓冲液重悬，超声1min后加入2.5μL EDC溶液，超声1min后再加入7.5μL NHS溶液，混匀后于37℃旋转混匀反应15min，以12000rpm的转速离心15min，去除上清液，使用50μL PBS缓冲液将纳米粒子重悬。
(4)加入10μL 10μM检测探针，于37℃旋转混匀反应2h。
(5)加入25μL Buffer D，于37℃封闭反应1h后，以12000rpm离心15min，使用100μL Buffer E重悬保存。
3、实验方法
标准曲线的制作
(1)使用样品稀释液将DNA模板分子的浓度稀释为0、1、10、50、100以及1000pM。
(2)将50μL样品滴加至载玻片的反应区域内，常温下反应30min。
(3)加入50μL的结合有检测探针的二氧化硅荧光纳米粒子，常温下孵育30min，使用清洗缓冲液洗4次，洗去残余的二氧化硅荧光纳米粒子，去除上清液。
(4)使用荧光显微镜(尼康Eclipse Ti-U)进行单分子成像，利用单分子计数模式与荧光强度积分模式的联用来完成后续单分子计数统计与分析。
(5)完成一系列浓度检测，每个浓度点重复6次，根据检测结果绘制标准曲线，计算每个点的CV％值。
4、实验结果
检测结果如图17所示，可知本实施例中，DNA模板分子的检测下限为0.5pM，与PCR检测灵敏度接近。
实施例23：磁珠法用于缓冲液中DNA分子的定量检测(二氧化硅荧光纳米粒子：粒径为220nm)。
在实施例22中，用甲苯磺酰基活化M280磁珠替换低吸附载玻片，除此以外，与实施例22同样地操作，将其结果示于表2中。
此外，申请人还对原位信号增强纳米粒子分别为聚苯乙烯包裹稀土元素(铕)而成的荧光粒子、葡 聚糖包裹荧光蛋白(GFP)而成的荧光粒子以及交联琼脂糖包裹量子点(硫化镉)而成的荧光粒子的情况进行了实验。具体而言，在实施例1中将二氧化硅包裹荧光素而成的荧光粒子替换为上述三种荧光粒子，结果发现灵敏度也较为优异(在50fg/mL-5fg/mL之间)。
表1
Figure PCTCN2020070283-appb-000002
表2
Figure PCTCN2020070283-appb-000003

Claims (14)

  1. 一种单分子定量检测分析方法，包括如下步骤：
    (1)将能够与靶标分子结合的捕获抗体固定到固相载体上，利用捕获抗体与靶标分子的第一位点结合，从而捕获样品中的靶标分子；
    (2)加入检测抗体，所述检测抗体与靶标分子的第二位点结合，然后加入原位信号增强纳米粒子，所述原位信号增强纳米粒子能够直接或间接与检测抗体结合；或者
    先将检测抗体与原位信号增强纳米粒子结合而形成复合材料，再将该复合材料加入；
    其中，所述原位信号增强纳米粒子含有发光材料和纳米粒子载体，且粒径为180-480nm；
    (3)用光学成像设备检测所述原位信号增强纳米粒子发出的光学信号；
    (4)对原位信号增强纳米粒子的个数进行统计，进一步计算得到样品中靶标分子的浓度信息。
  2. 一种单分子定量检测分析方法，包括如下步骤：
    (1)使检测抗体与样品中的靶标分子的第二位点结合，然后加入原位信号增强纳米粒子，所述原位信号增强纳米粒子能够直接或间接与检测抗体结合；或者
    先使原位信号增强纳米粒子与检测抗体结合而形成复合材料，再将该复合材料加入样品中，使该复合材料与样品中的靶标分子的第二位点结合；
    其中，所述原位信号增强纳米粒子含有发光材料和纳米粒子载体，且粒径为180-480nm；
    (2)将能够与靶标分子结合的捕获抗体固定至固相载体上，然后，使所述捕获抗体与所述靶标分子的第一位点结合，从而将靶标分子捕获；
    (3)用光学成像设备检测从所述原位信号增强纳米粒子发出的光学信号；
    (4)对原位信号增强纳米粒子的个数进行统计，进一步计算得到样品中靶标分子的浓度信息。
  3. 根据权利要求1或2所述的单分子定量检测分析方法，其特征在于，所述原位信号增强纳米粒子的粒径为200-450nm，优选为200-350nm，进一步优选为220-350nm。
  4. 根据权利要求1或2所述的单分子定量检测分析方法，其特征在于，所述靶标分子包括蛋白质、多糖或有生物活性的小分子。
  5. 根据权利要求1或2所述的单分子定量检测分析方法，其特征在于，步骤(4)采用如下方法来计算样品中靶标分子的浓度：
    直接对生成的图像中的由原位信号增强纳米粒子形成的亮斑个数进行分析和统计，通过亮斑个数直接或间接换算为靶标分子在样品中的浓度信息；或
    对生成的图像中的由原位信号增强纳米粒子形成的亮斑面积进行统计和积分，通过将积分结果除以平均每个原位信号增强纳米粒子所形成的平均亮斑面积，从而换算得到原位信号增强纳米粒子的近似个数，再将该数值换算为靶标分子在样品中的浓度信息。
  6. 根据权利要求1～5中任一项所述的单分子定量检测分析方法，其特征在于，所述固相载体为磁珠、多孔板、离心管、芯片、微米尺度微球、纳米尺度微球等，所述原位信号增强纳米粒子中的发光材料为荧光素或量子点，所述原位信号增强纳米粒子中的纳米粒子载体为二氧化硅、聚丙烯酰胺、聚苯乙烯或聚甲基丙烯酸甲酯，在步骤(4)中，将单分子计数模式与荧光强度积分模式联用。
  7. 一种单分子定量检测分析方法，包括如下步骤：
    (1)将能够与靶标分子结合的捕获探针固定到固相载体上，所述捕获探针与靶标分子的第一序列互补，利用捕获探针捕获样品中的靶标分子；
    (2)加入检测探针，所述检测探针能够与靶标分子的第二序列互补，形成捕获探针—靶标分子—检测探针的三链杂交结构，然后加入原位信号增强纳米粒子；所述原位信号增强纳米粒子能够直接或间接与检测探针结合；或
    先将检测探针与原位信号增强纳米粒子结合形成而复合材料，再将该复合材料加入；
    其中，所述原位信号增强纳米粒子含有发光材料和纳米粒子载体，且粒径为180-480nm；
    (3)用光学成像设备检测所述原位信号增强纳米粒子发出的光学信号；
    (4)对原位信号增强纳米粒子的个数进行统计，进一步计算得到样品中靶标分子的浓度信息。
  8. 一种单分子定量检测分析方法，包括如下步骤：
    (1)使检测探针与样品中的靶标分子的第二序列互补，然后加入原位信号增强纳米粒子，所述原位信号增强纳米粒子直接或间接与检测探针结合；或者
    预先使原位信号增强纳米粒子与检测探针结合而形成复合材料，然后使该复合材料与样品中的靶标分子的第二序列互补；
    其中，所述原位信号增强纳米粒子含有发光材料和纳米粒子载体，且粒径为180-480nm；
    (2)将能够与靶标分子结合的捕获探针固定至固相载体上，然后，使所述捕获探针与所述靶标分子的第一序列互补，从而将靶标分子捕获；
    (3)用光学成像设备检测从所述原位信号增强粒子发出的光学信号；
    (4)对原位信号增强粒子的个数进行统计，进一步计算得到样品中靶标分子的浓度信息。
  9. 根据权利要求7或8所述的单分子定量检测分析方法，其特征在于，所述原位信号增强纳米粒子的粒径为200-450nm，优选为200-350nm，进一步优选为220-350nm。
  10. 根据权利要求7或8所述的单分子定量检测分析方法，其特征在于，所述靶标分子包括DNA或RNA。
  11. 根据权利要求7或8所述的单分子定量检测分析方法，其特征在于，步骤(4)采用如下方法计算样品中靶标分子的浓度：
    直接对生成的图像中的由原位信号增强纳米粒子形成的亮斑个数进行分析和统计，通过亮斑个数直接或间接换算为靶标分子在样品中的浓度信息；或者
    对生成的图像中的由原位信号纳米粒子形成的亮斑面积进行统计和积分，将积分结果除以平均每个原位信号纳米粒子所形成的平均亮斑面积，从而换算得到原位信号增强纳米粒子的近似个数，再将该数值换算为靶标分子在样品中的浓度信息。
  12. 一种单分子定量检测系统，包括：
    (1)检测试剂，
    所述检测试剂包括(a)捕获抗体，所述捕获抗体能够与靶标分子的第一位点结合从而捕获样品中的靶标分子；(b)检测抗体，所述检测抗体能够与靶标分子的第二位点结合，并能够与原位信号增强纳米粒子结合；(c)原位信号增强纳米粒子，其含有发光材料和纳米粒子载体，且粒径为180-480nm；或者
    所述检测试剂包括(a)捕获探针，所述捕获探针能够与靶标分子的第一序列结合从而捕获样品中的靶标分子；(b)检测探针，所述检测探针能够与靶标分子的第二序列结合，形成捕获探针—靶标分子—检测探针的三链杂交结构；(c)原位信号增强纳米粒子，其能够结合于检测探针上，含有发光材料和纳米粒子载体，且粒径为180-480nm；
    (2)光学成像设备，其包括激发光源及光学信号采集单元。
  13. 如权利要求12所述的单分子定量检测分析系统，其特征在于，所述原位信号增强纳米粒子的粒径为200-450nm，优选为200-350nm，进一步优选为220-350nm。
  14. 如权利要求12所述的单分子定量检测分析系统，其特征在于，所述光学成像设备还包括数据处理单元，所述数据处理单元具有数据采集模块，其配置为接收感光元件所捕获的光学信号，并转换为数字信号；数据处理模块，配置为数字信号的转换和光学图像的形成与处理。
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