WO2015018177A1 - 一种基于白蛋白假酯酶水解反应的特异性荧光探针及应用 - Google Patents

一种基于白蛋白假酯酶水解反应的特异性荧光探针及应用 Download PDF

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WO2015018177A1
WO2015018177A1 PCT/CN2014/000334 CN2014000334W WO2015018177A1 WO 2015018177 A1 WO2015018177 A1 WO 2015018177A1 CN 2014000334 W CN2014000334 W CN 2014000334W WO 2015018177 A1 WO2015018177 A1 WO 2015018177A1
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albumin
fluorescent probe
human serum
specific fluorescent
serum albumin
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French (fr)
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杨凌
崔京南
葛广波
刘兆明
冯磊
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中国科学院大连化学物理研究所
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Priority to JP2016526408A priority Critical patent/JP6281727B2/ja
Priority to US14/787,868 priority patent/US9340821B2/en
Priority to EP14834394.0A priority patent/EP3031801A4/en
Publication of WO2015018177A1 publication Critical patent/WO2015018177A1/zh

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    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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    • G01N2333/76—Assays involving albumins other than in routine use for blocking surfaces or for anchoring haptens during immunisation
    • G01N2333/765—Serum albumin, e.g. HSA

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  • the invention belongs to the technical field of medicine, and particularly relates to a specific fluorescent probe based on hydrolysis reaction of albumin pseudoesterase and application thereof.
  • Human serum albumin is the most abundant protein in plasma, accounting for 50-60% of the total protein in plasma [S oc/ze Pharmacol 2005 Nov 25;70(11): 1673- 84.]. Human serum albumin can transport fatty acids, bile pigments, amino acids, steroid hormones, metal ions and many therapeutic molecules in body fluids while maintaining the normal osmotic pressure of the blood. The normal value of albumin in human serum ranges from 34-54 g/L [Mw.m 7.gov. Retrieved 2010-05-12.].
  • liver disease especially cirrhosis
  • nephrotic syndrome tumors, protein-losing enteropathy, burns, and malnutrition
  • serum albumin levels can lead to a decrease in serum albumin levels
  • high-protein diets and chronic dehydration can cause Albumin levels are elevated. Therefore, quantitative determination of albumin content in human blood has been clinically used for disease-related diagnosis. If human albumin is much lower than the normal range, it is necessary to inject human serum albumin.
  • a large number of clinical practices have confirmed that monitoring serum albumin levels has important diagnostic reference value for early diagnosis of liver disease or kidney disease patients and for the recovery of the disease.
  • microalbuminuria in urine is generally used as a marker for kidney disease (such as diabetes, hypertension, glomerulonephritis after streptococcal infection), endothelial dysfunction, heart disease, and venous thromboembolism. And diagnostic indicators. Table 1 below gives the normal range of diagnostic values for human microalbuminuria. Table 1 Diagnostic value of microalbuminuria
  • methods for detecting and quantifying albumin include: electrophoresis, immunoassay, and dye binding methods.
  • electrophoresis time-consuming, poor specificity, could easily lead to overestimation of albumin concentration [Clinical Chemistry: Theory, Analysis, Correlation, Mosby, 5 th edition, 2009.] o and although specific preferred immunization
  • the cost is higher, so there are currently fewer clinical applications.
  • the dye-binding color method is relatively widely used, but the method is based on the binding of small molecules of dyes to partial regions of albumin or the modification of individual amino acids.
  • the body part of the tissue protein can also act similarly to small dye molecules, so its specificity Sex cannot be guaranteed.
  • Bromocresol green and bromocresol purple are two simple, inexpensive and relatively specific dye molecules that are clinically used to quantify albumin.
  • albumin quantification method is necessary.
  • albumin also has a catalytic function, which can catalyze the hydrolysis of compounds with ester bond structure.
  • iBiochem Pharmacol 2010; 79 (5): 784-91 & J Biol Chem 2008; 283 (33): 22582-90) after the reaction, its own partially free amino acid forms a covalent bond with the carboxylic acid group in the ester molecular structure, and simultaneously releases a molecular hydrolysis product containing a free phenolic hydroxyl group.
  • Ge Guangbo et al. also found that some small molecules can be catalyzed by albumin-specific esterase hydrolysis.
  • the GLP-1 receptor agonist Boc5 can only be catalyzed by albumin in humans, while other human esterases are not involved. Hydrolysis of compounds J. Pharm. Sci. 2013; 48: 360 - 9). Recent studies at home and abroad suggest that we can design and develop specific fluorescent probe substrate molecules for albumin esterase activity for the assessment and quantitative analysis of albumin activity.
  • the present invention provides a N-n-butyl-4-hydroxy-1,8-naphthalimide parent C-4 hydroxy substituted dibenzoyl ester derivative as a human serum albumin fluorescent probe substrate
  • the application which is hydrolyzed by the pseudoesterase activity of albumin, produces a hydrolyzed product having a different fluorescence emission spectrum than the prototype.
  • the hydrolysis reaction has the characteristics of high selectivity and easy detection of metabolites.
  • the object of the present invention is to provide a specific fluorescent probe molecule and application based on the hydrolysis reaction of albumin pseudoesterase; using the probe molecule to detect albumin content in human serum, plasma or urine, and detecting the sample in the sample
  • the fluorescence intensity value is used to calculate the albumin concentration; the fluorescence emission wavelength of the prototype and the hydrolyzate is significantly different, and the product is more easily detected.
  • the invention provides a specific fluorescent probe based on the hydrolysis reaction of albumin pseudoesterase, wherein the ester bond of the specific fluorescent probe can be specifically hydrolyzed into a molecule by human serum albumin.
  • N2014/000334 fluorescence emission spectrum significantly different fluorescent products, the content and function of albumin are determined by the content of the product in the system;
  • the specific fluorescent probe is N-n-butyl-4-hydroxy-1,8-naphthyl a dibenzoyl ester derivative in which a hydroxyl group at the C-4 position of the imine precursor is substituted, and its structural formula is as defined
  • R & lt! Is -H, -CH 3, -OCH 3, -OC 2 H 5 any one of, an alkyl group is 28, any of a halogenated alkyl group or a derivative thereof.
  • the invention also provides the application of the specific fluorescent probe for quantitatively determining the human serum albumin content in different samples, and the compound of the above formula (1) is used as an albumin-specific hydrolyzed metabolic substrate, and the hydrolysis reaction is carried out by quantification.
  • the amount of albumin removed per unit time and the amount of hydrolysate produced were measured to determine the content of albumin in different samples (including recombinantly expressed albumin mono-enzyme, human tissue preparation solution, various tissue cells, etc.);
  • the method is -
  • reaction time is 5 - 120 minutes, to ensure that the corresponding hydrolysate of the above substrate reaches the limit of quantitation;
  • the specific fluorescent probe provided by the invention is used for quantitatively determining the content of human serum albumin in different samples, the probe substrate and the hydrolyzate thereof have fluorescence properties, and the fluorescence detector is used for rapid and sensitive detection of the substrate and the product; Fluorescence detection conditions are: excitation wavelength 300 - 500 nm, emission wavelength 410 - 600 nm.
  • the recombinant human serum albumin incubation system was used to investigate N-n-butyl-4-hydroxy-1,8- by specific inhibition experiments, recombinant single-enzyme metabolic reactions, and evidence of enzyme reaction kinetics.
  • the naphthyl halide precursor C-4 hydroxy substituted dibenzoyl ester derivative can be specifically metabolized by albumin (as shown in Figure 4) to form a hydrolysate of C-4 ester bond cleavage.
  • N-n-butyl-4-hydroxy-1,8-naphthalimide precursor C-4 hydroxy substituted dibenzoyl ester derivative can be highly specifically metabolized by albumin Into a metabolic product a hydrolysis product of the C-4 ester bond cleavage;
  • N-n-butyl-4-hydroxy-1,8-naphthalimide precursor C-4 hydroxy substituted dibenzoyl ester derivatives and their hydrolysis products can be chemically synthesized Obtained, the synthesis process is simple and easy;
  • Figure 1 Structural formula of a N-n-butyl-4-hydroxy-1,8-naphthalimide C-4 hydroxy-substituted dibenzoyl ester derivative; wherein A is a fluorescent probe molecule VIII, B Fluorescent probe molecules B, C are fluorescent probe molecules (3, D is fluorescent probe molecule D;
  • reaction solution is subjected to removal of the solvent under reduced pressure, and the residual solid is subjected to silica gel chromatography. Purification, using ethyl acetate-n-hexane (1:3, v/v) as eluent: 241 mg of white solid B;
  • HSA human serum albumin

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Abstract

一种基于白蛋白假酯酶水解反应的特异性荧光探针分子及应用，是基于人血清白蛋白（human serum albumin，HSA）特有的假酯酶活性，即在生理pH值条件下HSA能催化具有酯键结构的荧光探针分子（λ ex=342nm，λ em=416nm）发生类水解反应，其自身的氨基酸与探针分子结构中的羧酸基团形成共价结合，同时释放出含有游离酚羟基的水解产物，该产物是荧光发射谱显著不同于底物分子的荧光母体分子（λ ex=452nm，λ em=564nm），根据检测到荧光母体分子的浓度可以检测体系中对应的HSA的含量。该探针分子可用于测定生物样品中白蛋白的绝对含量。该探针分子的优点在于准确度高，检测灵敏，受环境干扰小，可与各种内源性物质、外源性药物及表面活性剂等兼容。

Description

说 明 书 一种基于白蛋白假酯酶水解反应的特异性荧光探针及应用 技术领域
本发明属于医药技术领域，具体涉及一种基于白蛋白假酯酶水解 反应的特异性荧光探针及应用。
背景技术
人血清白蛋白（human serum albumin, HSA)是血浆中含量最丰 富的蛋白质，约占到血浆中蛋白总量的 50 - 60%【S oc/ze Pharmacol 2005 Nov 25;70(11)： 1673-84.】。 在体液中人血清白蛋白可以运输脂肪 酸、 胆色素、 氨基酸、 类固醇激素、 金属离子和许多治疗分子等， 同 时能维持血液正常的渗透压。 人血清中白蛋白的正常值范围为 34-54 g/L【Mw.m 7.gov. Retrieved 2010-05-12.】。但多种疾病， 比如肝病（尤 其是肝硬化)、 肾病综合症、 肿瘤、 蛋白丢失性肠病、 烧伤以及营养 不良等会导致血清白蛋白的水平下降，而高蛋白饮食以及慢性脱水则 会造成白蛋白水平升高。因此人血中白蛋白含量的定量测定已被临床 用于疾病相关诊断，如果人体白蛋白的含量较正常范围值低很多时就 需要给患者注射人血清白蛋白。大量临床实践已证实，监测血清白蛋 白水平对于肝病或肾病患者的早期诊断以及疾病的愈后都有十分重 要的诊断参考价值。更为重要的是，尿液中出现微量白蛋白一般可作 为肾脏疾病 （像是糖尿病、 高血压、 链球菌感染后的肾小球肾炎）、 血管内皮功能障碍、 心脏疾病以及静脉血栓栓塞的标志物及诊断指 标。 下表 1给出了人体微量蛋白尿诊断值的正常范围。 表 1 微量蛋白尿诊断值
Figure imgf000004_0001
目前， 白蛋白的检测和定量方法包括： 电泳法、免疫法和染料结 合法等方法。其中电泳法耗时耗力， 特异性较差， 易造成白蛋白浓度 的高估 [Clinical Chemistry: Theory, Analysis, Correlation, Mosby, 5th edition, 2009. ]o 而免疫法虽然其特异性较好， 但成本较高， 因此 目前临床应用较少。染料结合显色法应用相对较为广泛，但该方法是 基于染料小分子与白蛋白部分区域的结合作用或个别氨基酸的修饰 作用，机体部分组织蛋白也可与染料小分子发生类似作用， 因而其特 异性无法保障。溴甲酚绿和溴甲酚紫作为简单、便宜且特异性相对较 好的两种染料分子，在临床上被较多的用来定量白蛋白。但由于其灵 敏度较低【 Clinical Chemistry: Theory, Analysis, Correlation, Mosby, 5th edition, 2009.】、 无法保证定量的准确性， 常出现高估 iJ Clin Pathol 2003, 56, 780.】或低估现象 IClin Chim Acta. 1986, 155， 83.】，且不可避免受到生物样品中的非特异性干扰【C¾2 Chem" 2009: 55， 583.】。 因而， 开发一种简单易操作、 特异性高、 准确可靠的白蛋 白定量方法十分必要。 近年来， 国内外学者发现白蛋白还具有催化功能，其可催化具有 酯键结构的化合物发生类水解反应 iBiochem Pharmacol 2010; 79 (5):784-91 & J Biol Chem 2008; 283 (33):22582-90) , 反应后其自身部 分游离氨基酸与酯类分子结构中的羧酸基团形成共价键，同时释放出 含有游离酚羟基的一分子水解产物。葛广波等人也发现部分小分子可 被白蛋白特异性催化发生类酯酶水解反应， 如 GLP-1 受体激动剂 Boc5 在人体内仅可被白蛋白催化， 而人体其它酯酶并不参与该化合 物的水解 J. Pharm. Sci. 2013; 48: 360 - 9)。以上国内外新近研究提 示我们可针对白蛋白的类酯酶活性设计并开发其特异性的荧光探针 底物分子， 用于白蛋白的活性评估及定量分析。
本发明提供了一种 N-正丁基 -4-羟基 -1，8-萘酰亚胺母体 C-4位羟 基取代的联苯甲酰酯类衍生物作为人血清白蛋白荧光探针底物的应 用，其经白蛋白的假酯酶活性水解后可生成荧光发射波谱不同于原型 的水解产物。 该水解反应具有选择性高、 代谢产物易检测等特点。 发明内容
本发明的目的在于提供一种基于白蛋白假酯酶水解反应的特异 性荧光探针分子及应用； 利用该探针分子， 检测人血清、血浆或尿液 中白蛋白含量，通过检测样本中的荧光强度值来计算其中的白蛋白浓 度；其原型和水解产物的荧光发射波长具有明显差异，且产物更易检 测。
本发明提供了一种基于白蛋白假酯酶水解反应的特异性荧光探 针，该特异性荧光探针的酯键可被人血清白蛋白特异性水解为与分子 N2014/000334 荧光发射波谱显著不同的荧光产物，借助体系中产物的含量来确定白 蛋白的含量及功能； 该特异性荧光探针为 N-正丁基 -4-羟基 -1,8-萘酰 亚胺母体 C-4位羟基被取代的联苯甲酰酯类衍生物，其结构通式如式
( 1 )所示， 其中 R!为 -H、 -CH3、 -OCH3、 -OC2H5中的任意一种， 为 2 8烷基， 卤代烷基或其衍生物中的任意一种。
Figure imgf000006_0001
式 （1 )
本发明还提供了所述特异性荧光探针在定量测定不同样本中人 血清白蛋白含量的应用， 采用上述式 （1 )化合物作为白蛋白特异性 的水解代谢底物，进行水解反应，通过定量检测单位时间内底物的消 除量以及水解产物的生成量来测定不同样本（包括重组表达的白蛋白 单酶、 人组织制备液、 各类组织细胞等生物体系） 中白蛋白的含量； 具体测定方法为-
——体系中以 N-正丁基 -4-羟基 -1,8-萘酰亚胺母体 C-4位羟基取 代的联苯甲酰酯类衍生物作为特异性探针底物； 底物浓度选择 1/10 - 10 Km; 单点测定时底物浓度优选 ( m为米氏动力学常数) ——在磷酸缓冲液等常用缓冲液中， 反应温度为 20 - 60Ό之间 (优选反应温度为 37°C ); 孵育体系 pH值介于 5.5 - 10.5之间（优选 pH值为 7.4);
——反应时间为 5 - 120分钟， 在确保以上底物相应的水解产物 达到定量限；
——测定单位时间内底物减少量或水解产物生成量作为白蛋白 活性的评价指标。
本发明提供的特异性荧光探针在定量测定不同样本中人血清白 蛋白含量的应用，探针底物及其水解产物均具有荧光属性，采用荧光 检测器实现底物及产物的快速灵敏检测； 荧光检测条件为：激发波长 300 - 500 nm, 发射波长 410 - 600 nm。
本发明提供的所述特异性荧光探针底物的应用，该特异性探针底 物用于人血清、 血浆或尿液中白蛋白的定量测定。
采用重组表达的人血清白蛋白孵育体系进行考察，通过特异性抑 制实验， 重组单酶代谢反应， 以及酶反应动力学几方面的证据， 证明 N-正丁基 -4-羟基 -1,8-萘酰亚胺母体 C-4位羟基取代的联苯甲酰酯类 衍生物可特异性的经白蛋白水解代谢（如图 4所示）， 生成 C-4位酯 键断裂的水解产物。
选用本发明所述白蛋白的特异性探针检测白蛋白具有以下突出 优势：
(1)高特异性： N-正丁基 -4-羟基 -1,8-萘酰亚胺母体 C-4位羟基取 代的联苯甲酰酯类衍生物可被白蛋白高特异性地代谢成一个代谢产 物， 即 C-4位酯键断裂的水解产物；
(2) 廉价易得: N-正丁基 -4-羟基 -1,8-萘酰亚胺母体 C-4位羟基取 代的联苯甲酰酯类衍生物及其水解产物均可经化学合成获得，合成工 艺简单易行；
(3)高灵敏度：具有 N-正丁基 -4-羟基 -1,8-萘酰亚胺母体结构的化 合物均具有良好的荧光发射光谱特性（410 - 600 nm)，该底物及其水 解代谢产物具有不同的荧光发射光谱特征， 能较好的进行区分检测， 同时可经比率法通过绘制标准曲线进行定量测定。
附图说明
图 1. N-正丁基 -4-羟基 -1,8-萘酰亚胺 C-4羟基被取代的联苯甲酰 酯类衍生物的结构式； 其中， A为荧光探针分子八， B荧光探针分子 B, C为荧光探针分子(3， D为荧光探针分子 D;
图 2. N-正丁基 -4-羟基 -1，8-萘酰亚胺母体 C-4位羟基取代的联苯 甲酰酯的1 H-NMR谱图；
图 3. N-正丁基 -4-羟基 -1，8-萘酰亚胺母体 C-4位羟基取代的联苯 甲酰酯的 13C-NMR谱图；
图 4. N-正丁基 -4-羟基 -1，8-萘酰亚胺母体 C-4位羟基取代的联苯 甲酰酯类衍生物的人重组单酶筛选试验结果；
图 5. N-正丁基 -4-羟基 -1,8-萘酰亚胺母体 C-4位羟基取代的联苯 甲酰酯类衍生物及其脱联苯甲酰代谢产物的荧光发射光谱图（分别在 342 nm或 452 nm进行激发） ；
图 6.脱联苯甲酰水解代谢产物的生成量随白蛋白浓度变化的线 性拟合。
具体实施方式
下面的实施例将对本发明予以进一步的说明，但并不因此而限制 本发明。
实施例 1. N-正丁基 -4-羟基 -1，8-萘酰亚胺母体 C-4位羟基取代的联苯 甲酰酯 （荧光探针分子 A) 的化学合成
( 1 )向 10 mL含有 0.5 mmol的 N-正丁基 -4-羟基 -1,8-萘酰亚胺和 0.625 mmol三乙胺的四氢呋喃溶液中，缓慢滴加 0.6 mM的 4-联苯基 甲酰氯 （溶于 10 mL二氯甲垸中）， 控制温度在 0 °C ;
(2) 冰浴下搅拌 l h后， 反应体系放置在室温过夜反应；
(3 )反应液经过减压除去溶剂，残留的固体采用硅胶色谱法进行 纯化， 采用乙酸乙酯-正己烷（l: 3，v/v)作为洗脱液， 得 113 mg白色 固体 A;
(4)对所得白色固体 A化合物进行结构确证， 其核磁氢谱和碳 谱见图 2和图 3。
实施例 2. N-正丁基 -4-羟基1，8-萘酰亚胺母体 C-4位羟基取代的 4-对 甲基联苯甲酰酯 （荧光探针分子 B) 的化学合成
( 1 )向 10 mL含有 0.5 mmol的 N-正丁基 -4-羟基 -1,8-萘酰亚胺和 0.625 mmol三乙胺的四氢呋喃溶液中，缓慢滴加 0.6 mM的 4-对甲基 联苯基甲酰氯（溶于 10 mL二氯甲烷中）， 控制温度在 0 °C ;
(2)冰浴下搅拌 1 h后， 反应体系放置在室温过夜反应；
(3 )反应液经过减压除去溶剂，残留的固体采用硅胶色谱法进行 纯化， 采用乙酸乙酯-正己垸（1: 3, v/v) 作为洗脱液， 得: 241 mg白 色固体 B;
实施例 3. N-正丁酸 -4-羟基 -1，8-萘酰亚胺母体 C-4位羟基取代的 4-对 甲基联苯甲酰酯（荧光探针分子 C) 的化学合成
( 1 )向 10 mL含有 0.5 mmol的 N-正丁酸 -4-羟基 -1，8-萘酰亚胺和 0.625 mmol三乙胺的四氢呋喃溶液中，缓慢滴加 0.6 mM的 4-对甲基 联苯基甲酰氯（溶于 10 mL二氯甲垸中）， 控制温度在 0 °C ;
(2)冰浴下搅拌 l h后， 反应体系放置在室温过夜反应；
(3 )反应液经过减压除去溶剂，残留的固体采用硅胶色谱法进行 纯化， 采用乙酸乙酯-正己垸（1: 3， v/v)作为洗脱液， 得 95 mg白色 固体 C;
实施例 4. N-正丁基 -4-羟基 -1，8-萘酰亚胺母体 C-4位羟基取代的 4-甲 氧基联苯甲酰酯（荧光探针分子 D) 的化学合成
( 1 )向 10 mL含有 0.5 mmol的 N-正丁基 -4-羟基 -1，8-萘酰亚胺和 0.625 mmol三乙胺的四氢呋喃溶液中，缓慢滴加 0.6 mM的 4-甲氧基 联苯基甲酰氯 （溶于 10 mL二氯甲烷中）， 控制温度在 0 °C ;
(2)冰浴下搅拌 l h后， 反应体系放置在室温过夜反应；
(3 )反应液经过减压除去溶剂，残留的固体采用硅胶色谱法进行 纯化， 采用乙酸乙酯-正己烷（1: 3, v/v)作为洗脱液， 得 143 mg白 色固体 D;
实施例 5.荧光探针分子 A水解代谢的选择性
( 1 )含有 CESlb、 CESlc、 CES2 (5 g/mL)、 乙酰胆碱酯酶（0.1 U/L)、 丁酰胆碱酯酶 （20 U/L)、 血浆 （1%)、 人血清白蛋白 （0.5 mg/mL),小牛血清白蛋白（0.5 mg/mL)的 pH = 6.0的磷酸缓冲液（10 mM) 196 L， 于 37 °C条件下震荡预孵 10分钟；
(2)加入 4 浓度为 0.5 mM的荧光探针分子 A起始反应， 以 使待测样品中的荧光探针分子的终浓度为 10 μΜ; 37 Ό震荡孵育；
(3 ) 30分钟后， 加入 200 冰乙腈， 剧烈震荡后， 终止反应；
(4)分别检测探针分子 A aex= 342 nm, ,m= 416 nm)和水解 产物 Al aex= 452 nm, 2^= 564 ^)在采集波长处的荧光强度值， 计算 A1与 A的荧光强度比值 （见图 4和图 5)。
实施例 6. 白蛋白定量标准曲线的绘制
( 1 )采用 5 mg/mL的人血清白蛋白 （HSA) 的标准溶液， 采用 磷酸缓冲液稀释成不同浓度的标准曲线工作液（0, 10, 20, 50， 100, 200, 300， 400， 500， 600, 700， 800, 900， 1000 mg/L) (见表 2)， 37 °C下预孵 育 10 min;
(2)向每个所述溶液样本（980 L)中加入 20 μL浓度为 0.5 mM 的荧光探针分子 B， 使其终浓度为 10 μΜ; 37°C震荡孵育 30 min， 加 入 1 mL的冰乙腈剧烈震荡 15秒钟终止反应；
Figure imgf000011_0001
180 μL
160 μL
140
120 μL
100 μL
80 μL
60 μΐ 8 40 940 200 mg/L
9 20 960 μL lOOmg/L
10 从 0号管中吸取 100 880 μL 50 mg/L
11 从 0号管中吸取 40 L 940 μ 20 mg/L
12 从 0号管中吸取 20 L 960 μΐ, 10 mg/L
13 ΟμΙ 980 μL Omg/L
(3)分别检测探针分子 Β ( c= 342■， Xem = 416 run)和水解 产物 Bl
Figure imgf000012_0001
lem=564nm)在采集波长处的荧光强度值， 将 Bl与 B的荧光强度比值对 HSA浓度进行线性拟合作图， 建立白 蛋白的定量标准曲线； 曲线方程为 Y = 0.005578xX + 0.04533， 相关 系数平方为 0.9992 (见图 6)。
实施例 7.人血浆样本中白蛋白的定量分析
(1) 吸取 1 (约 1小滴） 人血浆样本， 采用 pH = 7.4的磷酸 缓冲液（10mM) 稀释 200倍， 于 45 条件下震荡预孵 10分钟；
(2)加入加入 4 μ∑浓度为 0.5 mM的荧光探针分子 B起始反应， 以使待测样品中的荧光探针分子的终浓度为 10 μΜ; 45 °C震荡孵育；
(3) 30分钟后， 加入 200 冰乙腈， 剧烈震荡后， 终止反应；
(4)分别检测探针分子 B (Aex= 342 nm, 2em= 416 nm)和水解 产物 Bl
Figure imgf000012_0002
lem=564nm)在采集波长处的荧光强度值， 计算 B1与 B的荧光强度比值；将荧光比值代入实施例 2中的标准曲 线中， 求得血浆中的白蛋白浓度为 46.2 mg/mL。 实施例 8.人尿液样本中白蛋白的定量分析
( 1 )吸取 490 人尿液样本，采用 pH = 7.4的磷酸缓冲液 490 μL 进行稀释， 37 °C下预孵育 lOmin;
(2) 加入 20 的荧光探针分子 C (0.5 mM), 以使待测样品中 的荧光探针分子的终浓度为 10 μΜ·, 37°C震荡孵育 30min，加入 1 mL 的冰乙腈剧烈震荡 15秒钟终止反应；
(3)分别检测探针分子 C Olex= 342 nm， 2em= 416 nm)和水解 产物 CI (lex=452nm, lem=564nm)在采集波长处的荧光强度值， 计算 C1与 C的荧光强度比值；
(4)在标准曲线中找到对应的白蛋白浓度值，计算出待测样品中 的白蛋白浓度为 48mg/L。
实施例 9.重组表达体系中人白蛋白的定量分析
( 1 )称取 10 mg重组表达的人白蛋白样品，用 pH = 7.4的磷酸缓 冲液对其溶解， 37 °( 下配置成 lmg/mL 白蛋白溶液；
(2)加入 20 的荧光探针分子 D (0.5mM)， 以使待测样品中 的荧光探针分子的终浓度为 10 μΜ; 37 ！震荡孵育 30min后， 加入 1 mL的冰乙腈剧烈震荡 15秒钟终止反应；
(3) 分别检测探针分子 D ex= 342 nm, 2em= 416 nm) 和水解 产物 Dl Qex=452nm, Aem=564nm)在采集波长处的荧光强度值， 计算 D1与 D的荧光强度比值；
(4)基于白蛋白标准曲线计算出该样品（1 mg/rnL重组表达白蛋 白溶液） 中白蛋白的含量为 14.9 μΜ。

Claims

权 利 要 求 书
1、 一种基于白蛋白假酯酶水解反应的特异性荧光探针， 其特征 在于：该特异性荧光探针的酯键可被人血清白蛋白特异性水解为与分 子荧光发射波谱显著不同的荧光产物，借助体系中产物的含量来确定 白蛋白的含量及功能；
该特异性荧光探针为 N-正丁基 -4-羟基 -1,8-萘酰亚胺母体 C-4位 羟基被取代的联苯甲酰酯类衍生物， 其结构通式如式（1 ) 所示， 其 中 为-11、 -CH3、 -OCH3. -OC2 中的任意一种， 为 2-( 8烷基， 卤代垸基或其衍生物中的任意一种。
Figure imgf000014_0001
式 （1 )
2、 一种权利要求 1所述特异性荧光探针在定量测定不同样本中 人血清白蛋白含量的应用， 其特征在于： 采用上述式 （1 )化合物作 为白蛋白特异性的水解代谢底物，进行水解反应，通过定量检测单位 时间内底物的消除量以及水解产物的生成量来测定不同样本中白蛋 白的含
3、 按照权利要求 2所述特异性荧光探针在定量测定不同样本中 人血清白蛋白含量的应用，其特征在于： 所述样本为重组表达的白蛋 白单酶、 人组织制备液或各类组织细胞。
4、 按照权利要求 2所述特异性荧光探针在定量测定不同样本中 人血清白蛋白含量的应用， 其特征在于： 所述水解反应条件为： 体系 为磷酸缓冲液； 探针底物的浓度介于 1/10 - 10 m之间； 孵育体系 pH 值介于 5.5 - 10.5之间； 反应温度介于 20 - 60 Ό之间。
5、 按照权利要求 2所述特异性荧光探针在定量测定不同样本中 人血清白蛋白含量的应用，其特征在于：探针底物及其水解产物均具 有荧光属性，采用荧光检测器实现底物及产物的快速灵敏检测； 荧光 检测条件为： 激发波长 300 - 500 nm， 发射波长 410 - 600 nm。
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