荧光酮类试剂—钼（VI）光谱探针测定蛋白质的研究
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摘要
本文详细介绍了蛋白质定量分析的一些主要方法及其在生物化学、药物、食品、临床医学方面的应用概况，这些方法包括最早的浊度法、凯氏定氮法，和目前研究最多、应用最广、操作较为简便的分光光度法、荧光光度法，还有新进展起来的共振瑞利散射法、高效液相色谱法、毛细管电泳分析法。实验中选取以苯基荧光酮、水杨基荧光酮为母体、具有不同取代基的苯基荧光酮(PF)、邻硝基苯基荧光酮(o-NPF)、间硝基苯基荧光酮(m-NPF)、邻氯苯基荧光酮(o-CPF)、间氯苯基荧光酮(m-CPF)、对氯苯基荧光酮(p-CPF)、2′、4′-二氯苯基荧光酮(2′、4′-DCPF)、二溴羟基苯基荧光酮(DBH-PF)、水杨基荧光酮(SAF)、5′-硝基水杨基荧光酮(5′-NSF)、3′、5′二溴水杨基荧光酮(3′、5′-DBSF)等十一种三羟基荧光酮类试剂。三羟基荧光酮类试剂-钼(VI)配合物作为定量测定蛋白质的光谱探针，用分光光度法系统地研究了其与蛋白质分子的反应条件，如酸度、介质及用量、显色剂用量、反应时间、离子强度、干扰离子、最大结合数、摩尔吸光系数ε等，建立了一系列定量测定蛋白质的新方法；并首次将微乳液作为介质用于蛋白质测定中，起到了增溶增敏的作用，提高了体系的灵敏度。
试验结果表明，三羟基荧光酮类试剂-钼(VI)配合物作为定量测定蛋白质的光谱探针具有很高的灵敏度。现有文献记载金属离子—染料结合光度法中灵敏度最高的体系是水杨基荧光酮-钼(VI)-BSA体系，其摩尔吸光系数为2.86×10~6Lmol~(-1)cm~(-1)。本论文中新建立的十一种体系中5′-NSF-Mo(VI)-BSA体系、o-NPF-Mo(VI)-BSA体系、DBH-PF-Mo(VI)-BSA体系摩尔吸光系数分别达到：8.71×10~6Lmol(-1)cm(-1)、9.06×10~6Lmol~(-1)cm~(-1)、15.30×10~6Lmol~(-1)cm~(-1)，其摩尔吸光系数分别是水杨基荧光酮-钼(VI)-BSA体系的3.0、3.2、5.4倍。
试验还表明，微乳液比胶束对体系有更好的增溶增敏作用。微乳液体系比胶束体系有较高的摩尔吸光系数，从而验证了微乳液比胶束对体系有更好的增敏作用。
在实验基础上，选取与BSA反应灵敏度较高的5′-NSF-Mo(VI)、o-NPF-Mo(VI)、DBH-PF-Mo(VI)作为光谱探针，应用于人尿样、人血样、奶粉中蛋白质的分析测定上，取得了令人满意的结果。
本论文的系统性、科学性和新颖性：
1．系统性：
本论文系统地研究了具有不同取代基的三羟基荧光酮类试剂-钼(VI)配合物光谱探针与蛋白质分子的反应条件(酸度、介质及用量、显色剂用量、反应时间、离子强度、干扰离子、最大结合数、摩尔吸光系数ε)及取代基、取代基位置对体系灵敏度的影响，为建立高灵敏度定量测定蛋白质的方法奠定了基础。
2．科学性：
摘要
荧光酮类试剂一金属离子配合物光谱探针光度法测定蛋白质灵敏度高且抗干扰能力
强，成为微量蛋白质检测的一种灵敏方法。本论文通过大量的科学实验建立了更灵敏的方
法，灵敏度比文献记载的方法高5.4倍，应用于人尿样、人血样、奶粉中蛋白质的分析
测定上，取得了令人满意的结果。
3.新颖性:
首次将微乳液作为介质用于蛋白质测定中，起到了增溶增敏的作用，提高了体系
的灵敏度;论文中所用的大部分荧光酮类试剂都是首次用于蛋白质的测定，建立了一
系列定量测定蛋白质的新方法。
In this paper, some major quantitative analysis methods of protein and their applications in the fields of biochemistry, medicine, food and clinical medicine are presented in details. These methods mainly include Turbidity Method and Kjeldahl, which are applied in the earlier period; Spetrophotometric Method and Flurospetrophotometric Method, which are presently studied and applied widely, and can be easy operated; and Esonance Rayleigh Scattering Method, High Performance Liquid Chromatographic Method, and Capilary Electrophoresis Method, which are newly developed. In our experiments, eleven tri-hydroxyl fluorone reagents with different substituents are selected on the basis of Phenyl Fluorone(PF) and salicylfluorone(SAF), such as Phenyfluorone(PF), o-Nitrophenylfluorone(o-NPF), Salicylfluorone and etc. The complex of Tri-hydroxyl Fluorone and Mo(VI) is used as a spectral probe for protein determination in quantity. The Spetrophotometric Analytical Methods is applied for the systematic study of the reaction
conditions (such as acidity, media and dosage, dosage of developer, reaction time, ionic strength, interfering ion, and etc.) for Tri-hydroxyl Fluorone-Mo(VI) and protein, and a series of new methods with higher sensitivity for the determination of protein are established. Microemulsion, as the media, is firstly introduced to the determination of protein, which has increased the sensitivity of system with its sensitization and solubilization.
The experimental results indicate that the complex of Tri-hydroxyl Fluorone and Mo(VI), as a spectral probe of protein determination, has very high sensitivity. At present, the relevant reference shows that SAF-Mo(VI)-BSA System has the highest sensitivity in the method of Metallic Ion-dye Spetrophotometry, with the molar absorptivity of 2.86 X 106Lmol-1cm-1. Among the eleven systems studied in this paper, 5'-NSF-Mo(VI)-BSA , o-NPF-Mo(VI)-BSA, DBH-PF-Mo(VI)-BSA have the highest sensitivity, with the molar absorptivities of 8.71 X 106mol-1cm-1, 9.06X106Lmol-1cm-1 and 1.53 X 107Lmol-1cm-1 respectively, which are 3.0, 3.2 and 5.4 times higher than the SAF-Mo(VI)-BSA method.
The experiments also indicate that the microemulsion media has more solubilization and sensitization than micelle media. That the microemulsion media has higher molar absorptivity than that of micelle media proves that it has higher sensitization than micelle media.
Based on the experiments, the 5'-NSF-Mo(VI), o-NPF-Mo(VI), DBH-PF-Mo(VI), as a
spectral probe, have been successfully applied to the determination of protein in human urine, human blood sample and milk powder.
This paper features in systematic, scientific, and creative methods.
1. Systematic feature:
This paper systematically studies the reaction conditions (such as acidity, media and dosage, dosage of developer, reaction time, ionic strength, interfering ion, and etc.) for Tri-hydroxyl Fluorone-Mo(VI) and protein and the effects of substituents on the sensitivity of system, and provides a foundation for quantitative determination of protein with the higher sensitivity.
2. Scientific feature:
The Spetrophotometric Method with Fluorone Reagent-Metallic Ion as spectral probe for the determination of protein becomes a sensitive way in the micro-determination of protein with higher sensitivity and stronger anti-interference. This paper establishes a more sensitive method based on the large amounts of experiments which has indicated 5.4 times higher sensitivity than that of the reference and been successfully applied to the determination of protein in human urine, human blood sample and milk powder.
3. Creative feature:
Microemulsion, as the media, is firstly introduced to the determination of protein, which has increased the sensitivity of system with its sensitization and solubilization. The most Fluorone reagents described in this paper are all firstly introduced for the determination of protein and formed a series of new methods.
试验结果表明，三羟基荧光酮类试剂-钼(VI)配合物作为定量测定蛋白质的光谱探针具有很高的灵敏度。现有文献记载金属离子—染料结合光度法中灵敏度最高的体系是水杨基荧光酮-钼(VI)-BSA体系，其摩尔吸光系数为2.86×10~6Lmol~(-1)cm~(-1)。本论文中新建立的十一种体系中5′-NSF-Mo(VI)-BSA体系、o-NPF-Mo(VI)-BSA体系、DBH-PF-Mo(VI)-BSA体系摩尔吸光系数分别达到：8.71×10~6Lmol(-1)cm(-1)、9.06×10~6Lmol~(-1)cm~(-1)、15.30×10~6Lmol~(-1)cm~(-1)，其摩尔吸光系数分别是水杨基荧光酮-钼(VI)-BSA体系的3.0、3.2、5.4倍。
试验还表明，微乳液比胶束对体系有更好的增溶增敏作用。微乳液体系比胶束体系有较高的摩尔吸光系数，从而验证了微乳液比胶束对体系有更好的增敏作用。
在实验基础上，选取与BSA反应灵敏度较高的5′-NSF-Mo(VI)、o-NPF-Mo(VI)、DBH-PF-Mo(VI)作为光谱探针，应用于人尿样、人血样、奶粉中蛋白质的分析测定上，取得了令人满意的结果。
本论文的系统性、科学性和新颖性：
1．系统性：
本论文系统地研究了具有不同取代基的三羟基荧光酮类试剂-钼(VI)配合物光谱探针与蛋白质分子的反应条件(酸度、介质及用量、显色剂用量、反应时间、离子强度、干扰离子、最大结合数、摩尔吸光系数ε)及取代基、取代基位置对体系灵敏度的影响，为建立高灵敏度定量测定蛋白质的方法奠定了基础。
2．科学性：
摘要
荧光酮类试剂一金属离子配合物光谱探针光度法测定蛋白质灵敏度高且抗干扰能力
强，成为微量蛋白质检测的一种灵敏方法。本论文通过大量的科学实验建立了更灵敏的方
法，灵敏度比文献记载的方法高5.4倍，应用于人尿样、人血样、奶粉中蛋白质的分析
测定上，取得了令人满意的结果。
3.新颖性:
首次将微乳液作为介质用于蛋白质测定中，起到了增溶增敏的作用，提高了体系
的灵敏度;论文中所用的大部分荧光酮类试剂都是首次用于蛋白质的测定，建立了一
系列定量测定蛋白质的新方法。
In this paper, some major quantitative analysis methods of protein and their applications in the fields of biochemistry, medicine, food and clinical medicine are presented in details. These methods mainly include Turbidity Method and Kjeldahl, which are applied in the earlier period; Spetrophotometric Method and Flurospetrophotometric Method, which are presently studied and applied widely, and can be easy operated; and Esonance Rayleigh Scattering Method, High Performance Liquid Chromatographic Method, and Capilary Electrophoresis Method, which are newly developed. In our experiments, eleven tri-hydroxyl fluorone reagents with different substituents are selected on the basis of Phenyl Fluorone(PF) and salicylfluorone(SAF), such as Phenyfluorone(PF), o-Nitrophenylfluorone(o-NPF), Salicylfluorone and etc. The complex of Tri-hydroxyl Fluorone and Mo(VI) is used as a spectral probe for protein determination in quantity. The Spetrophotometric Analytical Methods is applied for the systematic study of the reaction
conditions (such as acidity, media and dosage, dosage of developer, reaction time, ionic strength, interfering ion, and etc.) for Tri-hydroxyl Fluorone-Mo(VI) and protein, and a series of new methods with higher sensitivity for the determination of protein are established. Microemulsion, as the media, is firstly introduced to the determination of protein, which has increased the sensitivity of system with its sensitization and solubilization.
The experimental results indicate that the complex of Tri-hydroxyl Fluorone and Mo(VI), as a spectral probe of protein determination, has very high sensitivity. At present, the relevant reference shows that SAF-Mo(VI)-BSA System has the highest sensitivity in the method of Metallic Ion-dye Spetrophotometry, with the molar absorptivity of 2.86 X 106Lmol-1cm-1. Among the eleven systems studied in this paper, 5'-NSF-Mo(VI)-BSA , o-NPF-Mo(VI)-BSA, DBH-PF-Mo(VI)-BSA have the highest sensitivity, with the molar absorptivities of 8.71 X 106mol-1cm-1, 9.06X106Lmol-1cm-1 and 1.53 X 107Lmol-1cm-1 respectively, which are 3.0, 3.2 and 5.4 times higher than the SAF-Mo(VI)-BSA method.
The experiments also indicate that the microemulsion media has more solubilization and sensitization than micelle media. That the microemulsion media has higher molar absorptivity than that of micelle media proves that it has higher sensitization than micelle media.
Based on the experiments, the 5'-NSF-Mo(VI), o-NPF-Mo(VI), DBH-PF-Mo(VI), as a
spectral probe, have been successfully applied to the determination of protein in human urine, human blood sample and milk powder.
This paper features in systematic, scientific, and creative methods.
1. Systematic feature:
This paper systematically studies the reaction conditions (such as acidity, media and dosage, dosage of developer, reaction time, ionic strength, interfering ion, and etc.) for Tri-hydroxyl Fluorone-Mo(VI) and protein and the effects of substituents on the sensitivity of system, and provides a foundation for quantitative determination of protein with the higher sensitivity.
2. Scientific feature:
The Spetrophotometric Method with Fluorone Reagent-Metallic Ion as spectral probe for the determination of protein becomes a sensitive way in the micro-determination of protein with higher sensitivity and stronger anti-interference. This paper establishes a more sensitive method based on the large amounts of experiments which has indicated 5.4 times higher sensitivity than that of the reference and been successfully applied to the determination of protein in human urine, human blood sample and milk powder.
3. Creative feature:
Microemulsion, as the media, is firstly introduced to the determination of protein, which has increased the sensitivity of system with its sensitization and solubilization. The most Fluorone reagents described in this paper are all firstly introduced for the determination of protein and formed a series of new methods.
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