WO2021180129A1 - 基于荧光标记流式单分子计数的蛋白质含量计量基准方法 - Google Patents

基于荧光标记流式单分子计数的蛋白质含量计量基准方法 Download PDF

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WO2021180129A1
WO2021180129A1 PCT/CN2021/080008 CN2021080008W WO2021180129A1 WO 2021180129 A1 WO2021180129 A1 WO 2021180129A1 CN 2021080008 W CN2021080008 W CN 2021080008W WO 2021180129 A1 WO2021180129 A1 WO 2021180129A1
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protein
molecule
fluorescent
solution
labeled
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French (fr)
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武利庆
杨彬
高运华
王迪
刘亚辉
金有训
王晶
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中国计量科学研究院
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1434—Optical arrangements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/38—Diluting, dispersing or mixing samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1486—Counting the particles

Definitions

  • the invention relates to the field of biochemical detection, in particular to a protein content measurement standard method based on fluorescent labeling flow-type single-molecule counting.
  • Metrology is an activity that achieves unity of units and accurate and reliable measurement values. Researching and establishing high-accuracy measurement methods is one of the important contents of metrology research.
  • the benchmark measurement method is a measurement method with the highest metrological quality. Its operation can be fully described and understood. The final uncertainty can be expressed in SI units, and the measurement result does not depend on the measurement standard being measured.
  • the benchmark method is the basis for forming the source of the value.
  • Protein content is the basic attribute of protein. It describes the number of defined "protein molecules" and is the basic value of protein measurement. It accounts for more than 80% of various protein testing items. Therefore, in order to ensure the accuracy and comparability of protein content detection results, it is necessary to study and establish a high-accuracy protein content measurement method to achieve accurate transmission of protein quality values, so as to achieve accurate and comparable detection results, realize the mutual communication and mutual recognition of detection results, and guarantee The purpose of fair trade and protecting the health of the people.
  • Protein content measurement methods can be divided into conventional measurement methods and high-accuracy measurement (potential) reference measurement methods according to measurement accuracy.
  • Common measurement (potential) reference methods include isotope dilution mass spectrometry, mass balance method, and quantitative nuclear magnetic method. According to the measurement principle, it can be divided into titration, spectroscopy, chromatography, mass spectroscopy, electrophoresis, spectroscopy, comprehensive methods, etc.
  • Kjeldahl nitrogen determination and trace Kjeldahl nitrogen determination are titration methods; biuret Method, Folin-phenol method (Lowry method), Coomassie brilliant blue method, etc. belong to colorimetric spectroscopy methods.
  • the upper level standard is required during the measurement process, it can be divided into two types: absolute measurement method and relative measurement method of protein content.
  • the absolute measurement method of protein content does not require the same standard substance as a standard during the measurement process, while the relative measurement method requires the corresponding standard substance to draw a standard curve during the measurement process, or quantify by bracket method or single-point method.
  • Mass balance method, quantitative nuclear magnetic method, and Kjeldahl method are absolute measurement methods, while most protein content measurement methods such as isotope dilution mass spectrometry, liquid chromatography, colorimetry, etc. are relative measurement methods.
  • IDMS is a method of chemical analysis using stable isotopes. When determining protein content, this method adds a certain amount of isotope-labeled compounds to the sample. These isotope-labeled compounds can be isotope-labeled elements, amino acids, peptides or proteins . After the isotopes are uniformly mixed with the sample, the hydrolysis or enzymatic hydrolysis is performed, and then the ratio of the non-labeled substance and the labeled substance after the reaction is detected by mass spectrometry, so that the protein can be accurately quantified.
  • Mass Balance Method is an absolute measurement method for high-purity solid protein content, and its measurement results have a small uncertainty. It takes the content of the main component as 1, and then uses various techniques to measure and deduct the inorganic components, organic impurities, volatile components, and moisture contained in it one by one, so as to perform absolute quantification of the substance. This method is widely used in the purity determination of organic high-purity small molecules. In the accurate quantification of proteins, it is only used for the determination of peptides or small proteins. In general, due to the complex composition of the protein, this method is used in the accurate determination of protein content. The application of is still very limited.
  • Quantitative nuclear magnetic resonance technology is proposed in recent years on the basis of nuclear magnetic resonance technology, adding quantitative known markers to the sample, and then generating the signal according to the molecular weight of the test substance, the selected quantitative integral signal The number of protons can be quantitatively studied by substituting it into the calculation formula.
  • qNMR has the characteristics of fast analysis speed and simple pretreatment. Affected by the overlap of spectral peaks, quantitative nuclear magnetic technology can only be used for accurate quantification of small peptides or proteins.
  • the purpose of the invention of this application overcomes the shortcomings of the existing measurement methods, and provides a technique for directly measuring the protein content in the solution based on the fluorescent-labeled flow-type single-molecule counting technology. There is no need to rely on any standard substance in the measurement process, and the measurement result can be It is directly traceable to the SI unit, which conforms to the definition of the measurement benchmark method.
  • step (1) Use the diluent of step (1) to dilute the purified fluorescently labeled protein molecules to a concentration level of 100-1000 molecules/ ⁇ L, and the dilution factor is D 2 to obtain a fluorescently labeled protein diluted by D 2 times Solution
  • M The molar mass of the protein to be tested, g/mol
  • the protein is a protein with a purity greater than 99%, using SDS-PAGE, gel exclusion High performance liquid chromatography, reversed phase high performance liquid chromatography, ion exchange high performance liquid chromatography, chip electrophoresis, capillary electrophoresis or two-dimensional electrophoresis to detect the purity of the protein to be tested.
  • the diluent solution contains: 0.1%-10% of the volume of the diluent solution and 0.1% of the volume of the diluent solution ⁇ 30% organic solvent, surfactant is Tween 20 or HEPES; organic solvent is acetonitrile, methanol or isopropanol; buffer salt is phosphate, acetate or borate; adjust dilution with phosphoric acid, acetic acid or boric acid
  • the pH value of the liquid solution is such that the isoelectric point of the diluent solution is the same as the isoelectric point of the protein to be tested, and the crude concentration is measured by ultraviolet absorption, Coomassie brilliant blue, Bradford or high performance liquid chromatography.
  • the diluent solution contains 10 mM to 100 mM phosphate; or contains 0.1_M to 1.0_M acetate; or contains 0.1_M ⁇ 1.0M borate.
  • the wavelength of the fluorescent dye is consistent with the wavelength of the laser light source of the single-molecule analyzer and the sensitive wavelength of the detector, and the fluorescent dye includes: Alexa Fluor 647 , APC-Cy7, Bodipy 650/665-X, Cy5.1 8, Cy5 TM , Indodicarbocyanine (DiD), SYTO 62, SYTO 63, Thiadicarbocyanine (DiSC3), TO-PRO-3, TOTO-3 or Mltralite; fluorescent dye
  • the binding method to the protein molecule is covalent coupling labeling or non-covalent coupling labeling.
  • the protein molecule to be tested is labeled with a fluorescent dye far exceeding the stoichiometric ratio to ensure that each protein to be tested is labeled with a fluorescent dye.
  • the measured protein molecules are all labeled with fluorescent dyes, and at the same time, the fluorescent dyes and fluorescently labeled proteins remain stable within 7-30 days.
  • step (3) the separation of fluorescent-labeled protein and excess fluorescent dye is performed by gel exclusion filtration, molecular sieve, and reversed-phase high-efficiency liquid It is carried out by means of phase chromatography or ion exchange chromatography.
  • gel exclusion filtration or molecular sieve is used for separation, the molecular weight cut-off of gel exclusion filtration or molecular sieve is less than 1/10 of the molecular weight of the protein to be measured.
  • the fluorescently labeled protein is collected and stored in a dark environment, and the container for collecting and storing the fluorescently labeled protein is an opaque container.
  • the opaque container is a container that is shielded with a light-shielding material
  • the light-shielding material is aluminum foil or tin foil.
  • step (4) the purified fluorescent-labeled protein is diluted with a diluent, and then the diluted fluorescent-labeled protein is sent to In the single molecule analyzer of step (5), the diluted fluorescently labeled protein solution is directly flow-counted to obtain the single molecule counting result w.
  • the diluted fluorescently labeled protein solution continues to be diluted until the single molecule count result w is in the range of 1000 ⁇ 10000/min, at this time the protein concentration is 100 ⁇ 1000/ ⁇ L, record the dilution factor as D 2 , and get the diluted D 2 times the fluorescently labeled protein solution.
  • step (5) The method for absolute measurement of protein content based on fluorescent-labeled flow single-molecule counting of the present invention, wherein: in step (5), the following method is used to determine the mass flow rate:
  • step (6) when the fluorescent-labeled protein molecules are uniformly distributed in the pipeline, the detection probability is composed of laser spots and capillaries The ratio of the volume of the detection area to the volume of the geometric area occupied by the liquid in the pipeline.
  • the detection probability is each in the detection area composed of the laser spot and the pipeline.
  • Figure 1 is a schematic diagram of the experimental process
  • Figure 2 shows the relative ratio of phenylalanine and valine at different hydrolysis times
  • Figure 3 shows the uncertainty component of the bovine serum albumin solid standard material and the uncertainty of the synthesis standard.
  • the labeled fluorescent protein molecules are detected separately, and the results obtained are compared.
  • the Alexa647 commercial labeling kit is selected as the fluorescent dye.
  • the wavelength of the fluorescent dye is the same as that of the single-molecule analyzer (that is, the laser light source of the Erenna platform is a 650nm laser).
  • the wavelength of the light source is the same as the sensitive wavelength of the detector.
  • the Alexa647 commercial labeling kit is added to the protein solution prepared above, and the fluorescent dye is covalently coupled to the GBW09815 bovine serum albumin molecule to be tested.
  • the fluorescent dye exceeding the stoichiometric ratio is used to label the bovine serum albumin molecules to be tested, to ensure that each GBW09815 bovine serum albumin molecule to be tested is labeled with a fluorescent dye to form a fluorescently labeled GBW09815 bovine serum albumin molecule, and a labeled solution is obtained.
  • Use an opaque container such as: use a light-proof material for aluminum foil or tin foil to collect and store fluorescent dyes and fluorescently labeled GBW09815 bovine serum albumin molecules.
  • fluorescently labeled GBW09815 bovine serum albumin molecules are placed In the refrigerator at -80°C, when the storage time is less than one week, the fluorescently labeled GBW09815 bovine serum albumin molecule is placed in the refrigerator at 4°C or below;
  • the molecular weight cut-off of the gel exclusion filtration is less than 1/10 of the molecular weight of the tested GBW09815 bovine serum albumin to remove excess untested fluorescently labeled GBW09815 bovine serum Fluorescent dyes bound to albumin molecules to obtain purified fluorescently labeled GBW09815 bovine serum albumin molecules;
  • step (1) Use the diluent of step (1) to dilute the purified fluorescently labeled GBW09815 bovine serum albumin molecule, and then send the diluted fluorescently labeled GBW09815 bovine serum albumin molecule to step (5) for single-molecule analysis
  • the diluted fluorescent-labeled GBW09815 bovine serum albumin solution is directly flow-counted, and the single-molecule counting result w is obtained. If the single-molecule counting result w is not within the range of 1000 ⁇ 10000/min, the diluted fluorescent label Continue to dilute the GBW09815 bovine serum albumin solution until the single molecule count result w is in the range of 1000 ⁇ 10000/min.
  • the Erenna platform was used to repeat the determination of the above protein solution 6 times, and the fluorescence count results were 2595, 2734, 2683, 2739, 2724, 2678, 2878, respectively.
  • the average value of the 6 repeated analysis results was 2718.71, and the relative standard deviation was 3.2%.
  • the detection probability p is calculated by the ratio of the volume of the occupied geometric area, that is, the laser detection spot diameter and the capillary diameter are calculated.
  • the capillary is a square with a side length of 100mm, and the laser detection spot size is 5mm.
  • M The molar mass of the protein to be tested, g/mol
  • N A 6.02214076 ⁇ 10 23
  • the molar mass of protein determined by MALDI-TOF M 67229.5g/mol
  • D 1*1068331 ⁇ 10 3
  • f 12.684
  • p 0.01309 into the above formula for calculation, single molecule
  • the result of the counting is 1.953 mg/g, that is, the concentration of the GBW09815 bovine serum albumin solution to be tested is obtained, and the deviation from the IDMS result is -5.2%.
  • the uncertainty component introduced by the counting result w is mainly counting repeatability, which is evaluated by the type A uncertainty evaluation method:
  • the uncertain component introduced by the molar mass is mainly introduced by the molecular weight measurement, and the component repeatedly determined by the molecular weight is evaluated by the type A uncertainty evaluation method:
  • the uncertainty component introduced by the mass axis calibration is calculated based on the value of the reference material certificate:
  • the uncertainty introduced by the dilution factor D is mainly introduced by the balance. According to the calculation of the dilution factor:
  • the uncertainty introduced by the mass flow rate f is calculated according to the following formula, which is mainly introduced by balance weighing, ignoring the uncertainty introduced by time.
  • the uncertainty introduced by the detection probability factor p needs to be calculated based on the geometric shape, but the geometric shape has not been directly measured in this study, but the manufacturer's parameter is quoted, so the uncertainty is estimated to be 3%.
  • the AB company 5500 mass spectrometer was used for isotope dilution mass spectrometry, and the liquid phase conditions used were as follows:
  • the mobile phase gradient is as follows:
  • the mass spectrum signal adopts the multi-reaction monitoring mode.
  • proline, valine, and phenylalanine the following ion pairs are detected respectively:
  • m sample weigh the mass of the sample
  • R sample the ratio of the peak area of the amino acid to the isotope-labeled amino acid in the sample
  • I 1 The mass ratio of the amino acid and the isotope-labeled amino acid in the low-standard solution
  • I 2 The mass ratio of amino acid and isotope-labeled amino acid in high standard solution
  • R 2 The peak area ratio of the amino acid and the isotope-labeled amino acid in the high-standard solution
  • R 1 The ratio of the peak area of the amino acid to the isotope-labeled amino acid in the low-standard solution
  • c Phe the concentration of phenylalanine in the hydrolysate determined by isotope dilution mass spectrometry
  • MW BSA the relative molecular mass of fluorescently labeled bovine serum albumin
  • MW Phe the relative molecular mass of phenylalanine.
  • the mass concentration of fluorescently labeled bovine serum albumin is 2.059 mg/g.
  • the main consideration is the uncertainty component introduced by weighing, hydrolysis efficiency, method repeatability, and amino acid standard substances in the isotope dilution mass spectrometry process.
  • the main components introduced in weighing include: the uncertainty m_Phe and m_Val introduced by the weighing of phenylalanine and valine standard substances, and the uncertainty introduced by the weighing of aqueous solutions when the phenylalanine and valine standard substances are dissolved m_Phe_Water , M_Val_Water; the uncertainty m_Stock_Phe, m_Stock_Val introduced by weighing the standard substance solution of phenylalanine and valine when preparing the standard; the uncertainty introduced by the weighing solution when preparing the standard m_stock_AA_Water; weighing phenylalanine when preparing the marker mixture
  • the uncertainty introduced by each balance weighing is calculated according to the minimum division and rectangular distribution.
  • the uncertainty of the purity of the amino acid standard substance is quoted from the certificate, and the relative uncertainty of the Val and Phe measurement introduced by the hydrolysis efficiency is based on the final measurement result. It is estimated that, in addition to the uncertainty component introduced by the repeatability of the method, for the calculation of the bovine serum albumin content through the determination of Phe and Val content, the synthetic standard uncertainty is calculated according to the following formula:
  • the uncertainty of the bovine serum albumin solid determination method adopts the A-type evaluation method, and the standard deviation of the 6 analysis results is calculated:
  • the method proposed in the present invention can achieve single-molecule level detection, and its sensitivity is based on traditional isotope dilution mass spectrometry.
  • the quasi-method can't reach it.
  • the method for absolute measurement of protein content based on fluorescent-labeled flow-type single-molecule counting of the present invention can be widely used in the field of biochemical detection.

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Abstract

基于荧光标记流式单分子计数蛋白质含量绝对测量方法包括：将待测蛋白质纯品用稀释液配制成或分步稀释至0.1mg/g-1mg/g，稀释倍数记录D 1；对稀释后蛋白质分子进行荧光标记，使荧光染料与每个蛋白质分子结合；将溶液中标记好的荧光标记蛋白质与过量荧光染料进行分离；用稀释液对纯化好的荧光标记蛋白质继续稀释至100-1000分子/μL浓度水平，稀释倍数为D 2，测量溶液质量流速f，用单分子分析仪对荧光标记蛋白质溶液直接进行流式计数，得到单分子计数结果w；根据由单分子分析仪照射出来激光斑点和毛细管几何尺寸算出检测概率p；根据单分子计数结果w、质量流速f、稀释倍数D和检测概率p，计算蛋白质溶液质量浓度。

Description

基于荧光标记流式单分子计数的蛋白质含量计量基准方法 技术领域
本发明涉及生物化学检测领域，特别是涉及一种基于荧光标记流式单分子计数的蛋白质含量计量基准方法。
背景技术
计量是实现单位统一、量值准确可靠的活动，研究建立高准确度的测量方法是计量学研究的重要内容之一。基准测量方法是一种具有最高计量学品质的测量方法，其操作可以被完全地描述和理解，最终不确定度可以用SI单位表述，测量结果不依赖被测量的测量标准。基准方法是形成量值源头的依据。
随着生命科学和生物技术的发展，生命科学已经经历了从“描述生物学”向“实验生物学”再向“创造生物学”发展的过程，当今的生物学已经成为精准定量的科学。没有对生命体各类生命现象的精确测量，便难以对生命过程进行全方位的调控与干预。蛋白质作为一类重要的生物大分子，是生命活动的主要承担者，在体内各个生命活动如营养代谢、酶催化、激素、免疫、遗传、变异等过程中均离不开蛋白质功能的发挥。鉴于蛋白质生物体内的重要作用，它已经成为众多领域的检测靶标。例如在体外诊断中，常见的体外诊断项目中有一半以上检测对象为蛋白质；在食品安全中，160多种食品含有可以导致过敏反应的食品过敏原，其中大于90％的过敏原都是蛋白质，根据国内食品标签标识管理规定，相应的蛋白质过敏原均应被检测并被标识；在生物医药领域，目前已经批准150多个蛋白质药物上市，400多种蛋白质药物处于临床研究阶段，3000多种处于临床前研究阶段。不论是体外诊断，还是食品安全与用药安全，这些都关乎大众健康与国计民生，相关领域蛋白质检验结果的准确可比则是保证大众健康与安全的基石！
通过建立量值溯源传递体系来保证检测结果的准确一致已经成为共识，是计量界的通行做法并在传统的物理计量与化学计量领域得到广泛和成功的应用。量值溯源传递体系的建立一是要有量值的源头，二是要有量值传递方法，尤其是要有具有较高测量准确度的“基标准”方法，才能保证国家基准复现的量值能够准确的传递到下一级的标准物质或工作计量器具上。高准确度量值传递方法的缺乏将导致即使国家基准能够复现出量值，也无法准确的传递下去，从而使 得保证检测结果的准确可比成为空谈。因此，高准确度的计量方法研究在计量及量值溯源传递中具有举足轻重的作用。
蛋白质含量是蛋白质的基本属性，它描述了被定义的“蛋白质分子”数量的多少，是蛋白质测量的基本量值，在各类蛋白质检测项目中占到了80％以上。因此，要保证蛋白质含量检测结果的准确可比，必须研究建立高准确度的蛋白质含量计量方法，才能实现蛋白质量值的准确传递，从而达到检测结果准确可比、实现检测结果的互通与互认、保证贸易公平、保护***健康的目的。
蛋白质含量测量方法按照测量准确度可以分为常规测量方法和高准确度的计量(潜)基准测量方法，常用的计量(潜)基准方法包括同位素稀释质谱法、质量平衡法和定量核磁法。按照测量原理可以分为滴定法、光谱法、色谱法、质谱法、电泳法、波谱法、综合法等，例如，常用的凯氏定氮、微量凯氏定氮等属于滴定法；双缩脲法、Folin-酚法(Lowry法)、考马斯亮蓝法等属于比色光谱法。按照测量过程中是否需要上一级标准，可以分为蛋白质含量绝对测量方法和相对测量方法两类。蛋白质含量绝对测量方法在测量过程中不需要同样的标准物质作为标准，而相对测量方法在测量过程中需要相应的标准物质绘制标准曲线，或者通过括弧法或单点法进行定量。质量平衡法、定量核磁法、凯氏定氮法属于绝对测量方法，而同位素稀释质谱法、液相色谱法、比色法等大多数蛋白质含量测量方法都属于相对测量方法。
IDMS是应用稳定同位素进行化学分析的一种方法，在测定蛋白质含量时，该方法将一定量的同位素标记化合物添加到样品中，这些同位素标记化合物可以是同位素标记的元素、氨基酸、肽段或蛋白质。待同位素与样品混合均匀后进行水解或酶解的操作，再通过质谱技术检测反应后非标记物和标记物的比例，由此对蛋白质进行准确定量。由于该方法所采用的内标物与待测物具有几乎相同的物理化学性质，在分离和分析过程中始终在一起，因此能够消除掉前处理过程和分析过程中的***误差。通过对非标记物和标记物比例的精确测量及所加入稀释剂的准确称量，有效地保证了高精度和高准确度。一旦稀释剂与样品反应平衡，同位素比值恒定，在保证测量操作正确的情况下检测结果很难受到影响，具有很高的稳定性。而高灵敏度的质谱可以提高IDMS的检测水平，进行微量、痕量以及超痕量的分析。
质量平衡法(Mass Balance Method)是一种高纯固体蛋白质含量绝对测量方法，其测量结果具有很小的不确定度。它将主成分的含量作为1，然后采用各种技术对其中含有的无机成分、有机杂质、挥发性成分、水分等逐一进行测定并扣除，从而对物质进行绝对定量。该方法在有机高纯小分子的纯度测定中应用广泛，在蛋白质准确定量中，仅用于肽段或小蛋白质的测定， 总的来说，由于蛋白质组成复杂，该方法在蛋白质含量准确测定中的应用还十分有限。
定量核磁共振技术(qNMR)是近些年提出的在核磁共振技术的基础上，向样品中加入定量已知的标记物，后根据被测物的分子量、选定的定量积分信号以及产生该信号的质子数，代入计算公式边可以定量研究。此外，作为新型的定量技术，qNMR有分析速度快、预处理简单等特点。受到谱峰重叠的干扰，定量核磁技术也只能用于小的肽段或蛋白质的准确定量。
上述这些方法在进行蛋白质定量时，同样需要使用一种化学品的标准物质来做标准，其量值不是直接溯源到SI单位的；同时，上述测定方法蛋白质含量的测定结果是基于其一级序列的蛋白质浓度，不能反映出蛋白质的活性；另外，同位素稀释质谱测定过程中需要将蛋白质分解成小分子如氨基酸或者肽段以后进行测定，在水解或者酶解过程中，可能引入一定的不确定度。
因此，建立一种能够不依赖任何标准品直接对目标蛋白质进行定量的基准方法是十分必要和迫切的。
发明内容
本申请的发明目的克服现有测量方法的缺陷，而提供一种基于荧光标记流式单分子计数技术对溶液中蛋白质含量进行直接测定的技术，在测定过程中不必依赖任何标准品，测定结果可以直接溯源到SI单位，符合计量基准方法的定义。
为了完成本申请的发明目的，本申请采用以下技术方案：
(1)用含有表面活性剂、有机溶剂和缓冲盐的水溶液作为稀释液，将蛋白质固体配制成0.1mg/g-2mg/g的待测蛋白质溶液，记录稀释倍数D 1＝1；或用上述稀释液来稀释经过粗测的浓度的待测蛋白质溶液至0.1mg/g-2mg/g，记录稀释倍数D 1，得浓度为0.1mg/g-2mg/g的待测蛋白质溶液；
(2)将荧光染料加入到上述浓度为0.1mg/g-2mg/g的待测蛋白质溶液中，对溶液中的蛋白质分子进行标记，使荧光染料与每个蛋白质分子充分结合，形成荧光标记蛋白质分子，得到标记好的溶液；
(3)将标记好的溶液中的荧光标记蛋白质分子与过量荧光染料进行分离，收集被截留的荧光标记蛋白质分子组分，除去过量的未结合的染料，得到纯化好的荧光标记蛋白质分子；
(4)用步骤(1)的稀释液对纯化好的荧光标记蛋白质分子进行稀释，稀释到100-1000分子/μL的浓度水平，稀释倍数为D 2，得到稀释了D 2倍的荧光标记蛋白质溶液；
(5)采用单分子分析仪对稀释D 2倍的荧光标记蛋白质溶液直接进行流式计数,得到单分 子计数结果w，单位为min -1，为了测定单分子分析仪的质量流速，用纯水在指定时间内流过单分子分析仪的质量和上述指定的时间之比，得到单分子分析仪的质量流速f，单位为mg/min；
(6)根据由单分子分析仪照射出来的激光斑点和毛细管组成的检测区域与液体占据的几何区域的体积之比，计算出检测概率p；
(7)根据单分子计数结果w、质量流速f、稀释倍数D和检测概率p，计算蛋白质溶液的质量浓度：
Figure PCTCN2021080008-appb-000001
其中：
c——待测蛋白质的质量浓度，mg/g；
M——待测蛋白质的摩尔质量，g/mol；
D——进行单分子计数分析时的稀释倍数，D＝D 1×D 2,无量纲；
N A——阿伏伽德罗常数，mol -1；
f——质量流速，mg/min；
w——荧光分子计数，min -1；
p——检测概率，无量纲。
本发明的基于荧光标记流式单分子计数的蛋白质含量绝对测量方法，其中：在所述步骤(1)中，所述的蛋白质为纯度大于99％的蛋白质，用SDS-PAGE、凝胶排阻高效液相色谱、反相高效液相色谱、离子交换高效液相色谱、芯片电泳、毛细管电泳或双向电泳来检测待测蛋白质的纯度。
本发明的基于荧光标记流式单分子计数的蛋白质含量绝对测量方法，其中：所述的稀释液溶液包含：占稀释液溶液体积0.1％～10％的表面活性剂和占稀释液溶液体积0.1％～30％的有机溶剂，表面活性剂为吐温20或HEPES；有机溶剂为乙腈、甲醇或异丙醇；缓冲盐为磷酸盐、醋酸盐或硼酸盐；用磷酸、乙酸或硼酸调整稀释液溶液的pH值，使稀释液溶液的等电点与待测蛋白质的等电点相同，所述粗测的浓度为用紫外吸收、考马斯亮蓝、Bradford或高效液相色谱方法进行的。
本发明的基于荧光标记流式单分子计数的蛋白质含量绝对测量方法，其中：所述的稀释液溶液包含10mM～100mM的磷酸盐；或者包含0.1_M～1.0_M的醋酸盐；或者包含0.1_M～1.0M的硼酸盐。
本发明的基于荧光标记流式单分子计数的蛋白质含量绝对测量方法，其中：所述荧光染料的波长与单分子分析仪的激光光源波长和检测器的敏感波长一致，荧光染料包括：Alexa Fluor 647、APC-Cy7、Bodipy 650/665-X、Cy5.1 8、Cy5 TM，Indodicarbocyanine(DiD)、SYTO 62、SYTO 63、Thiadicarbocyanine(DiSC3)、TO-PRO-3、TOTO-3或Μltralite；荧光染料与蛋白质分子的结合方式为共价偶联标记或非共价偶联标记，在待测蛋白质进行荧光标记时，用远远超过化学计量比的荧光染料对待测蛋白质分子进行标记，确保每个待测蛋白质分子均被荧光染料标记，同时，荧光染料及荧光标记后的蛋白质在7-30天内保持稳定。
本发明的基于荧光标记流式单分子计数的蛋白质含量绝对测量方法，其中：在步骤(3)中，荧光标记蛋白质与过量荧光染料进行分离是采用凝胶排阻过滤、分子筛、反相高效液相色谱或离子交换色谱的方式进行的，当使用凝胶排阻过滤或分子筛进行分离时，凝胶排阻过滤或分子筛的截留分子量小于待测蛋白质分子量的1/10，在荧光标记蛋白质和荧光染料分离时，为了保持荧光标记蛋白质的稳定和荧光量子产率，在避光的环境下对荧光标记蛋白质进行收集和保存，收集和保存荧光标记蛋白质的容器为不透明的容器。
本发明的基于荧光标记流式单分子计数的蛋白质含量绝对测量方法，其中：所述不透明的容器为用避光材料遮蔽的容器，避光材料为铝箔纸或锡箔纸，超过一周保存时间的荧光标记蛋白质放置在-80℃的冰箱中，一周以内保存时间的荧光标记蛋白质放置在4℃或4℃以下的冰箱中。
本发明的基于荧光标记流式单分子计数的蛋白质含量绝对测量方法，其中：在步骤(4)中，用稀释液对纯化好的荧光标记蛋白质进行稀释，然后将稀释后的荧光标记蛋白质，送入步骤(5)的单分子分析仪中，对稀释后的荧光标记蛋白质溶液直接进行流式计数,得到单分子计数结果w，如果单分子计数结果w不在1000～10000/min的范围内，对稀释后的荧光标记蛋白质溶液继续进行稀释，直到单分子计数结果w在1000～10000/min的范围内，此时蛋白质的浓度为100～1000/μL，记录稀释倍数为D 2，得到稀释了D 2倍的荧光标记蛋白质溶液。
本发明的基于荧光标记流式单分子计数的蛋白质含量绝对测量方法，其中：在步骤(5)中，用以下方法来测定质量流速：
取纯水在室温下平衡30min～1h，然后在样品板内的n个进样孔中加入足量纯水，保证在每次进样过程中不会将进样孔中的纯水完全吸干，在开始测定前，用精密天平称量样品板和所加入的纯水质量，记为m 1，然后，对上述n个独立的纯水样本进行进样计数并记录计数时间t min，等待单分子分析仪将n个样本测量完成后，从单分子分析仪中取出样品板，再次称量样 品板和剩余液体的质量，记为m 2，根据公式(2)计算单位采样时间内的质量流速：
Figure PCTCN2021080008-appb-000002
本发明的基于荧光标记流式单分子计数的蛋白质含量绝对测量方法，其中：在步骤(6)中，当荧光标记蛋白质分子在管路内呈现均匀分布时，检测概率就是由激光斑点和毛细管组成的检测区域的体积与管路内液体所占据的几何区域的体积之比，当荧光标记蛋白质分子在管路内呈现不均匀分布时，检测概率为由激光斑点和管路组成的检测区域内各点分子密度(ρ 1)对空间坐标的三重积分与管路内液体占据的体积内各点分子密度(ρ)对空间坐标的三重积分之比：
Figure PCTCN2021080008-appb-000003
下面结合具体实施例对本发明的蛋白质含量计量基准技术作进一步说明。
附图说明
图1为实验流程示意图；
图2为不同水解时间下苯丙氨酸和缬氨酸的相对比例；
图3为牛血清白蛋白质固体标准物质不确定度分量与合成标准不确定度。
具体实施方式
如图1所示，用本发明的基于荧光标记流式单分子计数的蛋白质含量绝对测量方法与同位素稀释质谱的测量方法，对标记了荧光蛋白分子分别进行检测，所得到的结果进行比较。
实施例
本发明的基于荧光标记流式单分子计数的蛋白质含量绝对测量方法包括以下步骤：
(1)用凝胶排阻高效液相色谱检测待测GBW09815牛血清白蛋白质的纯度，确保GBW09815牛血清白蛋白质的纯度大于99％，称量GBW09815牛血清白蛋白质固体1.769mg标准物质，用1g左右的稀释液将待测GBW09815牛血清白蛋白质配制成至0.1mg/g-2mg/g的待测蛋白溶液，上述稀释液含有1％的HEPES的表面活性剂、0.1％的乙腈有机溶剂和50mmol/l磷酸盐的缓冲水溶液，并用磷酸将上述稀释液溶液的pH值调整至6.8，使稀释液溶液的等电点与待测蛋白质的等电点相同，记录稀释倍数D 1＝1，得到浓度为0.1mg/g-2mg/g的待测蛋白 质溶液；
(2)为了保证蛋白质分子的标记效率能够接近100％，选用Alexa647商品化标记试剂盒作为荧光染料，该荧光染料的波长与单分子分析仪(即是Erenna平台的激光光源为650nm激光器)的激光光源波长和检测器的敏感波长一致，将Alexa647商品化标记试剂盒加入到上述配制的蛋白质溶液中，荧光染料以共价偶联标记的方式与待测GBW09815牛血清白蛋白分子结合，用远远超过化学计量比的荧光染料对待测牛血清白蛋白分子进行标记，确保每个待测GBW09815牛血清白蛋白分子均被荧光染料标记，形成荧光标记GBW09815牛血清白蛋白分子，得到标记好的溶液，用不透明的容器如：用避光材料为铝箔纸或锡箔纸遮蔽的容器收集和保存荧光染料和荧光标记GBW09815牛血清白蛋白分子，在保存时间超过一周时，荧光标记GBW09815牛血清白蛋白分子放置在-80℃的冰箱中，在保存时间在一周以内时，荧光标记GBW09815牛血清白蛋白分子放置在4℃或4℃以下的冰箱中；
(3)将标记好的溶液通过凝胶排阻过滤，凝胶排阻过滤的截留分子量小于待测GBW09815牛血清白蛋白质分子量的1/10，以除去过量的未被待测荧光标记GBW09815牛血清白蛋白分子结合的荧光染料，得到纯化好的荧光标记GBW09815牛血清白蛋白分子；
(4)用步骤(1)的稀释液对纯化好的荧光标记GBW09815牛血清白蛋白分子进行稀释，然后将稀释后的荧光标记GBW09815牛血清白蛋白分子，送入步骤(5)的单分子分析仪中，对稀释后的荧光标记GBW09815牛血清白蛋白溶液直接进行流式计数,得到单分子计数结果w，如果单分子计数结果w不在1000～10000/min的范围内，对稀释后的荧光标记GBW09815牛血清白蛋白溶液继续进行稀释，直到单分子计数结果w在1000～10000/min的范围内，此时GBW09815牛血清白蛋白的浓度为100～1000/μL，记录稀释倍数为D 2，得到稀释了D 2＝1068331×10 3倍的荧光标记GBW09815牛血清白蛋白溶液；
(5)采用单分子分析仪(即是Erenna平台的激光光源为650nm激光器)对稀释D 2倍的荧光标记GBW09815牛血清白蛋白溶液直接进行流式计数,得到单分子计数结果w，单位为min -1，
用Erenna平台对上述蛋白溶液重复测定6次，荧光计数结果分别为2595、2734、2683、2739、2724、2678、2878，6次重复分析结果的平均值为2718.71，相对标准偏差为3.2％，
为了测定单分子分析仪的质量流速，用以下方法来测定质量流速：
首先，取纯水在室温下平衡30min，在384孔板中连续24孔中加入50μL上述纯水，称量整板质量m 1；接着运行单分子测定程序，设置采样时间为60s，由于上述纯水中不含有荧光标记的蛋白质分子，因此程序会一直计数直到60s的终止时间；运行完成后再次称量整板质量 m 2；根据下面的公式(2)计算出准确的质量流速，其中t取值为1分钟，n为24，
Figure PCTCN2021080008-appb-000004
重复4次分析，结果如下：
Figure PCTCN2021080008-appb-000005
Figure PCTCN2021080008-appb-000006
Figure PCTCN2021080008-appb-000007
Figure PCTCN2021080008-appb-000008
取4次流速分析的平均值：(11.7375+12.625+13.300+13.075)/4＝12.684(mg/min)
(6)、计算检测概率p:在本次测量中，荧光标记GBW09815牛血清白蛋白分子在管路内呈均匀分布，因此，可以由激光斑点和毛细管组成的检测区域的体积与管路内液体所占据的几何区域的体积之比来计算检测概率p，即通过激光检测斑点直径和毛细管直径进行计算，根据厂家提供数据，毛细管为边长为100mm的正方形，激光检测斑点大小为5mm，考虑聚焦的影响，
Figure PCTCN2021080008-appb-000009
(7)根据上述单分子计数结果w、质量流速f、稀释倍数D和检测概率p，计算蛋白质溶液的质量浓度：
Figure PCTCN2021080008-appb-000010
其中：
c——待测蛋白质的质量浓度，mg/g；
M——待测蛋白质的摩尔质量，g/mol；
D——进行单分子计数分析时的稀释倍数，D＝D 1×D 2,无量纲；
N A——阿伏伽德罗常数，mol -1；
f——质量流速，mg/min；
w——荧光分子计数，min -1；
p——检测概率，无量纲。
将N A＝6.02214076×10 23，根据MALDI-TOF测定的蛋白质的摩尔质量M＝67229.5g/mol，D＝1*1068331×10 3，f＝12.684，p＝0.01309入上式进行计算，单分子计数的结果为1.953mg/g，即得到待测GBW09815牛血清白蛋白溶液的浓度，与IDMS结果偏差为-5.2％。
不确定度评定
根据下面的计算公式对单分子计数的不确定度进行评定：
Figure PCTCN2021080008-appb-000011
由计数结果w引入的不确定度分量主要为计数重复性，采用A类不确定度评定方法进行评定：
Figure PCTCN2021080008-appb-000012
由摩尔质量引入的不确定的分量主要由分子量测量引入，由分子量重复测定的分量采用A类不确定度评定方法进行评定：
Figure PCTCN2021080008-appb-000013
由质量轴校准引入的不确定度分量根据标准物质证书值进行计算：
Figure PCTCN2021080008-appb-000014
因此，由摩尔质量引入的不确定度分量：
Figure PCTCN2021080008-appb-000015
稀释因子D引入的不确定度主要由天平引入，根据稀释因子的计算：
Figure PCTCN2021080008-appb-000016
Figure PCTCN2021080008-appb-000017
根据SI单位定义，阿伏伽德罗常数N A的不确定度为0，即：
Figure PCTCN2021080008-appb-000018
质量流速f引入的不确定度根据下面公式进行计算，主要由天平称量引入，忽略由时间引入的不确定度。
Figure PCTCN2021080008-appb-000019
Figure PCTCN2021080008-appb-000020
由检测概率因子p引入的不确定度需要根据几何形状进行计算，但是几何形状本研究中尚未进行直接测定，而是引用的厂家参数，因此估计其不确定度为3％。
因此，最终测量结果的不确定度由以上不确定度分量合成：
Figure PCTCN2021080008-appb-000021
取包含因子k＝2，则扩展不缺定度为：
U r＝ku r,c＝6.6％
为突出本发明实施例的突出效果，进行同位素稀释质谱法的对比试验：
对比例1
荧光标记牛血清白蛋白质含量的同位素稀释质谱定量
用纯水稀释上述步骤(3)荧光标记牛血清白蛋白质溶液至0.1_mg/mL，取20μL稀释的荧光标记牛血清白蛋白质溶液，按照水解后理论产生的氨基酸的量，加入同浓度的标记氨基酸混合溶液，然后浓缩离心至完全干燥，再加入6mol/L的盐酸，通氮气除氧后密封，在(110.0±0.5)℃的烘箱中进行水解。水解完成后用氮气吹干，接着用0.1mol/L的盐酸复溶，溶液经过0.45μm滤膜过滤后上机测定。
采用AB公司5500质谱仪进行同位素稀释质谱测定，所用液相条件如下：
进样量：3μL
流动相A：0.1％TFA的水溶液
流动相B：MeCN
色谱柱：Phenomenex KINETEX C18色谱柱(150mm×2mm)
流动相梯度如下：
表1_HPLC-IDMS流动相及梯度
Figure PCTCN2021080008-appb-000022
质谱信号采用多反应监测模式，对于脯氨酸、缬氨酸、苯丙氨酸的检测，分别检测以下离子对：
表2 氨基酸及离子对
Figure PCTCN2021080008-appb-000023
1、水解时间的优化
在进行同位素稀释质谱测定荧光标记牛血清白蛋白质之间，首先对水解条件进行了优化。取荧光标记牛血清白蛋白质样品，分别水解不同的时间后上机测定缬氨酸和苯丙氨酸的相对比例，结果如图2所示：
从图2可以看出，在经过48小时的水解之后苯丙氨酸和缬氨酸的含量都到达了一个平台，水解时间的进一步延长会略微导致其相对比例的下降，因此水解时间定在48小时。
2、同位素稀释质谱测定
取6个牛血清白蛋白质样品按照前述溶液配制、水解及同位素稀释质谱测定步骤进行测定。同位素稀释质谱的测定结果按照下面的公式进行计算：
Figure PCTCN2021080008-appb-000024
上式中：
P：氨基酸标准物质的纯度；
P H：牛血清白蛋白质水解效率；
m 样：称量样品的质量；
R 样：样品中氨基酸和同位素标记氨基酸的峰面积比；
I 1：低标溶液中氨基酸和同位素标记氨基酸的质量比；
I 2：高标溶液中氨基酸和同位素标记氨基酸的质量比；
R 2：高标溶液中氨基酸和同位素标记氨基酸的峰面积比；
R 1：低标溶液中氨基酸和同位素标记氨基酸的峰面积比；
M：样品质量。
然后根据同位素稀释质谱测定的氨基酸溶液的浓度，代入下式中计算牛血清白蛋白质的质量分数：
Figure PCTCN2021080008-appb-000025
式中：
c Phe：同位素稀释质谱法测定的水解液中苯丙氨酸的浓度；
MW BSA：荧光标记牛血清白蛋白质的相对分子质量；
M Total：溶液总质量；
MW Phe：苯丙氨酸的相对分子质量。
采用苯丙氨酸和缬氨酸的含量计算荧光标记牛血清白蛋白质的质量分数，总共选取6个荧光标记牛血清白蛋白质样品进行测定，测定结果如表6所示：
表3_荧光标记牛血清白蛋白质量浓度测定结果(mg/g)
1 2 3 4 5 6 平均 CV％
2.026 1.999 2.101 2.085 2.059 2.086 2.059 1.9
因此，荧光标记牛血清白蛋白质的质量浓度为2.059mg/g。
3、定量结果的不确定度评定
采用同位素稀释质谱方法进行定值时，主要考虑在同位素稀释质谱测定过程中由称量、水 解效率、方法重复性、氨基酸标准物质等引入的不确定度分量。
称量引入的主要分量包括：苯丙氨酸、缬氨酸标准物质称量引入的不确定度m_Phe、m_Val，溶解苯丙氨酸和缬氨酸标准物质时水溶液称量引入的不确定度m_Phe_Water、m_Val_Water；配制标准时称量苯丙氨酸和缬氨酸标准物质溶液引入的不确定度m_Stock_Phe、m_Stock_Val；配制标准时称量溶液引入的不确定度m_stock_AA_Water；配制标记物混合液时称量苯丙氨酸和缬氨酸标记物溶液引入的不确定度m_stock_LPhe、m_stock_LVal；配制标记物时称量水溶液引入的不确定度m_stock_LAA_Water；配制同位素稀释高低标准时称量氨基酸标准物质混合溶液引入的不确定度m_AA_solution1、m_AA_solution2，配制同位素稀释高低标准时称量氨基酸标记物混合溶液映入的不确定度m_LAA_solution1、m_LAA_solution2，称量荧光标记牛血清白蛋白质溶液引入的不确定度m_sample；在荧光标记牛血清白蛋白质水解液中称量加入标记物混合溶液时引入的不确定度m_sample_LAA_solution；苯丙氨酸和缬氨酸标准物质纯度引入的不确定度Purity_Phe、Purity_Val；同位素稀释质谱测量苯丙氨酸和缬氨酸浓度时由于水解效率引入的不确定度Hydro_Eff_Phe、Hydro_Eff_Val；以及方法重复性引入的不确定度。上述这些不确定度分量可以用图3表示：
根据同位素稀释质谱法的计算公式
Figure PCTCN2021080008-appb-000026
以及样品溶液的配制过程，根据下式计算灵敏系数：
Figure PCTCN2021080008-appb-000027
每次天平称量引入的不确定度按照最小分度和矩形分布计算，氨基酸标准物质纯度的不确定度引自证书，由水解效率引入的Val和Phe测定的相对不确定度均按照最终测定结果的1％估计，除方法重复性引入的不确定度分量外，对于通过Phe和Val含量测定计算牛血清白蛋白质含量，根据下面的公式计算合成标准不确定度：
Figure PCTCN2021080008-appb-000028
得：
u r,Val＝1.1％；u r,Phe＝1.02％
Figure PCTCN2021080008-appb-000029
牛血清白蛋白质固体定值方法的不确定度采用A类评价方法，以6次分析结果的标准方差进行计算：
Figure PCTCN2021080008-appb-000030
Figure PCTCN2021080008-appb-000031
取包含因子k＝2(95％置信水平)，则扩展不确定度可以表示为：
U r＝k×u r，c＝2×1.1％＝2.2％
以上对比例和实施例进行试验结果对比，得出以下结论：
(1)采用本发明提出的基于荧光标记流式单分子计数的方法，对荧光标记蛋白质定量的结果与传统同位素稀释质谱计量基准方法测定的结果在不确定度范围内等效一致；
(2)同位素稀释质谱实时过程中，同样需要氨基酸标准物质作为测量标准，不是直接对样品中的蛋白质进行定量，而发明专利在测定过程中不依赖任何标准品即可对样品中的蛋白质浓度进行测定；
(3)本发明提出的方法可实现单分子水平的检测，其灵敏度是传统同位素稀释质谱计量基
准方法所达不到的。
工业实用性
本发明的基于荧光标记流式单分子计数蛋白质含量绝对测量方法可以在生物化学检测领域中被广泛使用。
以上描述是对本发明的解释，不是对发明的限定，本发明所限定的范围参见权利要求，在不违背本发明的精神的情况下，本发明可以作任何形式的修改。

Claims (10)

  1. 一种基于荧光标记流式单分子计数的蛋白质含量绝对测量方法，其特征在于：包括以下步骤：
    (1)用含有表面活性剂、有机溶剂和缓冲盐的水溶液作为稀释液，将蛋白质固体配制成0.1mg/g-2mg/g的待测蛋白质溶液，记录稀释倍数D 1＝1；或用上述稀释液来稀释经过粗测的浓度的待测蛋白质溶液至0.1mg/g-2mg/g，记录稀释倍数D 1，得浓度为0.1mg/g-2mg/g的待测蛋白质溶液；
    (2)将荧光染料加入到上述浓度为0.1mg/g-2mg/g的待测蛋白质溶液中，对溶液中的蛋白质分子进行标记，使荧光染料与每个蛋白质分子充分结合，形成荧光标记蛋白质分子，得到标记好的溶液；
    (3)将标记好的溶液中的荧光标记蛋白质分子与过量荧光染料进行分离，收集被截留的荧光标记蛋白质分子组分，除去过量的未结合的染料，得到纯化好的荧光标记蛋白质分子；
    (4)用步骤(1)的稀释液对纯化好的荧光标记蛋白质分子进行稀释，稀释到100-1000分子/μL的浓度水平，稀释倍数为D 2，得到稀释了D 2倍的荧光标记蛋白质溶液；
    (5)采用单分子分析仪对稀释D 2倍的荧光标记蛋白质溶液直接进行流式计数,得到单分子计数结果w，单位为min -1，为了测定单分子分析仪的质量流速，用纯水在指定时间内流过单分子分析仪的质量和上述指定的时间之比，得到单分子分析仪的质量流速f，单位为mg/min；
    (6)根据由单分子分析仪照射出来的激光斑点和毛细管组成的检测区域与液体占据的几何区域的体积之比，计算出检测概率p；
    (7)根据单分子计数结果w、质量流速f、稀释倍数D和检测概率p，计算待测蛋白质溶液的质量浓度：
    Figure PCTCN2021080008-appb-100001
    其中：
    c——待测蛋白质的质量浓度，mg/g；
    M——待测蛋白质的摩尔质量，g/mol；
    D——进行单分子计数分析时的稀释倍数，D＝D 1×D 2,无量纲；
    N A——阿伏伽德罗常数，mol -1；
    f——质量流速，mg/min；
    w——荧光分子计数，个数/min；
    p——检测概率，无量纲。
  2. 根据权利要求1所述的基于荧光标记流式单分子计数的蛋白质含量绝对测量方法，其特征在于：在所述步骤(1)中，所述的蛋白质为纯度大于99％的蛋白质，用SDS-PAGE、凝胶排阻高效液相色谱、反相高效液相色谱、离子交换高效液相色谱、芯片电泳、毛细管电泳或双向电泳来检测待测蛋白质的纯度，所述粗测的浓度为用紫外吸收、考马斯亮蓝、Bradford或高效液相色谱方法进行的。
  3. 根据权利要求2所述的基于荧光标记流式单分子计数的蛋白质含量绝对测量方法，其特征在于：所述的稀释液溶液包含：占稀释液溶液体积0.1％～10％的表面活性剂和占稀释液溶液体积0.1％～30％的有机溶剂，表面活性剂为吐温20或HEPES；有机溶剂为乙腈、甲醇或异丙醇；缓冲盐为磷酸盐、醋酸盐或硼酸盐；用磷酸、乙酸或硼酸调整稀释液溶液的pH值，使稀释液溶液的等电点与待测蛋白质的等电点相同。
  4. 根据权利要求2所述的基于荧光标记流式单分子计数的蛋白质含量绝对测量方法，其特征在于：所述的稀释液溶液包含10mM～100mM的磷酸盐；或者包含0.1M～1.0M的醋酸盐；或者包含0.1M～1.0M的硼酸盐。
  5. 根据权利要求4所述基于荧光标记流式单分子计数的蛋白质含量绝对测量方法，其特征在于：所述荧光染料的波长与单分子分析仪的激光光源波长和检测器的敏感波长一致，荧光染料包括：Alexa Fluor 647、APC-Cy7、Bodipy 650/665-X、Cy5.1 8、Cy5 TM，Indodicarbocyanine(DiD)、SYTO 62、SYTO 63、Thiadicarbocyanine(DiSC3)、TO-PRO-3、TOTO-3或Ultralite；荧光染料与蛋白质分子的结合方式为共价偶联标记或非共价偶联标记，在待测蛋白质进行荧光标记时，用远远超过化学计量比的荧光染料对待测蛋白质分子进行标记，确保每个待测蛋白质分子均被荧光染料标记，同时，荧光染料及荧光标记后的蛋白质在7-30天内保持稳定。
  6. 根据权利要求5所述基于荧光标记流式单分子计数的蛋白质含量绝对测量方法，其特征在于：在步骤(3)中，荧光标记蛋白质与过量荧光染料进行分离是采用凝胶排阻过滤、分子筛、反相高效液相色谱或离子交换色谱的方式进行的，当使用凝胶排阻过滤或分子筛进行分离时，凝胶排阻过滤或分子筛的截留分子量小于待测蛋白质分子量的1/10，在荧光标记蛋白质和荧光染料分离时，为了保持荧光标记蛋白质的稳定和荧光量子产率，在避光的环境下对荧光 标记蛋白质进行收集和保存，收集和保存荧光标记蛋白质的容器为不透明的容器。
  7. 根据权利要求6所述基于荧光标记流式单分子计数的蛋白质含量绝对测量方法，其特征在于：所述不透明的容器为用避光材料遮蔽的容器，避光材料为铝箔纸或锡箔纸，超过一周保存时间的荧光标记蛋白质放置在-80℃的冰箱中，一周以内保存时间的荧光标记蛋白质放置在4℃或4℃以下的冰箱中。
  8. 根据权利要求7所述的基于荧光标记流式单分子计数的蛋白质含量绝对测量方法，其特征在于：在步骤(4)中，用稀释液对纯化好的荧光标记蛋白质进行稀释，然后将稀释后的荧光标记蛋白质，送入步骤(5)的单分子分析仪中，对稀释后的荧光标记蛋白质溶液直接进行流式计数,得到单分子计数结果w，如果单分子计数结果w不在1000～10000/min的范围内，对稀释后的荧光标记蛋白质溶液继续进行稀释，直到单分子计数结果w在1000～10000/min的范围内，此时蛋白质的浓度为100～1000/μL，记录稀释倍数为D 2，得到稀释了D 2倍的荧光标记蛋白质溶液。
  9. 根据权利要求8所述的基于荧光标记流式单分子计数的蛋白质含量绝对测量方法，其特征在于：在步骤(5)中，用以下方法来测定质量流速：
    取纯水在室温下平衡30min～1h，然后在样品板内的n个进样孔中加入足量纯水，保证在每次进样过程中不会将进样孔中的纯水完全吸干，在开始测定前，用精密天平称量样品板和所加入的纯水质量，记为m 1，然后，对上述n个独立的纯水样本进行进样计数并记录计数时间t min，等待单分子分析仪将n个样本测量完成后，从单分子分析仪中取出样品板，再次称量样品板和剩余液体的质量，记为m 2，根据公式(2)计算单位采样时间内的质量流速：
    Figure PCTCN2021080008-appb-100002
    它的单位为g/min。
  10. 根据权利要求9所述的基于荧光标记流式单分子计数的蛋白质含量绝对测量方法，其特征在于：在步骤(6)中，当荧光标记蛋白质分子在管路内呈现均匀分布时，检测概率就是由激光斑点和毛细管组成的检测区域的体积与管路内液体所占据的几何区域的体积之比，当荧光标记蛋白质分子在管路内呈现不均匀分布时，检测概率为由激光斑点和管路组成的检测区域内各点分子密度ρ 1对空间坐标的三重积分与管路内液体占据的体积内各点分子密度ρ对空间坐标的三重积分之比：
    Figure PCTCN2021080008-appb-100003
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