基于共振瑞利散射血清蛋白测试技术及POCT仪器的研究
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摘要
血清中蛋白质浓度在临床中有重要诊断意义，在蛋白质分析领域，广泛应用紫外可见分光光度计、荧光分光光度计等精密仪器，这些仪器适合医院化验室使用，但由于它们的复杂、昂贵、体积庞大、耗电量高等因素，而无法满足现场检测POCT (Point of CareTesting)的要求。POCT是临床诊断技术中发展的趋势之一，它使用的仪器操作简便、工作稳定、适于携带。针对这种迫切需要，本文提出了一种基于共振瑞利散射（RRS）适合现场检测的血清蛋白质测试新方法。
论文介绍了RRS国内外的研究现状、基本概念和基本理论；采用荧光分光光度计和紫外－可见分光光度计研究了四羧基酞菁锌－蛋白质和四氨基酞菁铜体系的共振光散射光谱和吸收光谱进行，在pH 3.6的Britton－Robinson（B－R）缓冲溶液中，蛋白质与四羧基酞菁锌发生相互作用，使体系在波长475nm处产生共振散射增强；进一步分析和实验表明：该溶液强吸收波长为420nm附近的蓝紫光波段，在该激励光作用下，其共振波长处会产生RRS，在一定蛋白质浓度范围内溶液的RRS强度与蛋白质的含量成比例，可以利用四羧基酞菁锌为光谱探针的共振散射法来测定血清蛋白，其线性检出范围为10～50mg/mL，检出限为0.001mg/mL；在此基础上，确定了主体以405nm宽禁带半导体激光器为激励光源，以475nm窄带带通滤光片为单色器，以基于蓝光增强光敏二极管的低噪声高增益光电放大器为探测器的血清蛋白质POCT测试装置的总体方案。
论文所做工作还包括：对金属酞菁配合物的合成工艺和它们红外光谱表征的研究，采用4－羧基邻苯二甲酸酐和尿素为原料，钼酸铵为催化剂，固相法合成四羧基酞菁锌和四氨基酞菁铜，并通过红外光谱的表征得到证实；体系的稳定性、酸度对体系的影响、缓冲溶液用量、离子强度、干扰物质等影响检测灵敏度因素的讨论；血清蛋白质POCT测试装置样机的研制，并用它来对血清蛋白质进行测试。
实验结果表明：新开发的血清蛋白质测试装置具有体积小、成本低、功耗小、使用方便等一系列优点，改进后可望在现场检测得到广阔的应用。
Serμm protein concentration in clinical diagnosis has important significance. In theprotein analysis field UV－VIS spectrophotometer, fluorescence spectrophotometer and otherprecision instrμments are applied widely. These instrμments are suitable for hospital lab use,but due to their complex, expensive, bulky and power consμmption factors, they are unable tomeet the requirements of the POCT (Point of Care Testing). POCT is one of the developmenttrends in the clinical diagnostic techniques, whose instrμments require easy operation, steadywork and compact size. In this dissertation, aimed at the urgent requirement a novel POCTmethod of serμm protein was proposed.
The domestic and international research situation, the basic concept and theory of theresonance Rayleigh scattering (RRS) were introduced. Using fluorescence spectrophotometerand UV-VIS spectrophotometer the RRS spectra and the absorption spectra of tetra-carboxyliczinc phthalocyanine-protein and tetra-amino copper phthalocyanine-protein systems werestudied. In the pH 3.6 Britton-Robision (B-R) buffer solution, the interaction betweentetra-carboxylic zinc phathlocyanine and proteins yielded strongly enhanced RRS signals atwavelength 475 nm. Further analysis and experiment showed that the strong absorptionwavelength of the solution is nearby 420 nm blue-violet band and the solution irradiated withexcitation light at the resonance wavelength will produce the RRS. The enhanced intensity ofsolution RRS is proportional to the concentration of proteins within a range of proteinconcentration. Thus tetra-carboxylic zinc phthalocyanine can be used as a probe to determinethe total proteins in hμman serμm samples. Under optimal conditions, the linear range ofhμman serμm albμmin is 10~50mg/mL, and the detection limit is 0.001mg/mL. On this basis,the scheme for POCT serμm proteins measurement device with detecting RRS signal oftetra-carboxylic zinc phthalocyanine-protein solution was designed. The design mainlyadopted a GaN-based blue-violet laser diode of 405nm wavelength as an excitation lightsource, a 475nm narrow bandpass filter as a monochromator, and a low-noise, high gainphotoelectric amplifier with blue enhanced photodiode as a detector.
In this dissertation the author studied the synthetic process of metal phthalocyaninecomplexes and their infrared spectrμm characterization; synthetized tetra-carboxylic zincphthalocyanine and tetra-amino copper phthalocyanine using solid phase synthesis with4-carboxyl phthalic anhydride and urea as raw materials, ammoniμm molybdate as catalystand confirmed them through the characterization of infrared spectrμm; discussed the factorsaffecting the detecting sensitivity including: system stability, acidity on the system, buffersolution volμme, ionic strength and interfering substances; developed the prototype of POCTserμm proteins measurement device; measured the serμm proteins with the prototype.
It is proved that the newly developed serμm proteins measurement device has a lot ofadvantages such as small size, low cost, low power consμmption, easy to use, strongrepetition and reliability, etc. After further improvement the device can be expected to havebroad application prospects in POCT.
论文介绍了RRS国内外的研究现状、基本概念和基本理论；采用荧光分光光度计和紫外－可见分光光度计研究了四羧基酞菁锌－蛋白质和四氨基酞菁铜体系的共振光散射光谱和吸收光谱进行，在pH 3.6的Britton－Robinson（B－R）缓冲溶液中，蛋白质与四羧基酞菁锌发生相互作用，使体系在波长475nm处产生共振散射增强；进一步分析和实验表明：该溶液强吸收波长为420nm附近的蓝紫光波段，在该激励光作用下，其共振波长处会产生RRS，在一定蛋白质浓度范围内溶液的RRS强度与蛋白质的含量成比例，可以利用四羧基酞菁锌为光谱探针的共振散射法来测定血清蛋白，其线性检出范围为10～50mg/mL，检出限为0.001mg/mL；在此基础上，确定了主体以405nm宽禁带半导体激光器为激励光源，以475nm窄带带通滤光片为单色器，以基于蓝光增强光敏二极管的低噪声高增益光电放大器为探测器的血清蛋白质POCT测试装置的总体方案。
论文所做工作还包括：对金属酞菁配合物的合成工艺和它们红外光谱表征的研究，采用4－羧基邻苯二甲酸酐和尿素为原料，钼酸铵为催化剂，固相法合成四羧基酞菁锌和四氨基酞菁铜，并通过红外光谱的表征得到证实；体系的稳定性、酸度对体系的影响、缓冲溶液用量、离子强度、干扰物质等影响检测灵敏度因素的讨论；血清蛋白质POCT测试装置样机的研制，并用它来对血清蛋白质进行测试。
实验结果表明：新开发的血清蛋白质测试装置具有体积小、成本低、功耗小、使用方便等一系列优点，改进后可望在现场检测得到广阔的应用。
Serμm protein concentration in clinical diagnosis has important significance. In theprotein analysis field UV－VIS spectrophotometer, fluorescence spectrophotometer and otherprecision instrμments are applied widely. These instrμments are suitable for hospital lab use,but due to their complex, expensive, bulky and power consμmption factors, they are unable tomeet the requirements of the POCT (Point of Care Testing). POCT is one of the developmenttrends in the clinical diagnostic techniques, whose instrμments require easy operation, steadywork and compact size. In this dissertation, aimed at the urgent requirement a novel POCTmethod of serμm protein was proposed.
The domestic and international research situation, the basic concept and theory of theresonance Rayleigh scattering (RRS) were introduced. Using fluorescence spectrophotometerand UV-VIS spectrophotometer the RRS spectra and the absorption spectra of tetra-carboxyliczinc phthalocyanine-protein and tetra-amino copper phthalocyanine-protein systems werestudied. In the pH 3.6 Britton-Robision (B-R) buffer solution, the interaction betweentetra-carboxylic zinc phathlocyanine and proteins yielded strongly enhanced RRS signals atwavelength 475 nm. Further analysis and experiment showed that the strong absorptionwavelength of the solution is nearby 420 nm blue-violet band and the solution irradiated withexcitation light at the resonance wavelength will produce the RRS. The enhanced intensity ofsolution RRS is proportional to the concentration of proteins within a range of proteinconcentration. Thus tetra-carboxylic zinc phthalocyanine can be used as a probe to determinethe total proteins in hμman serμm samples. Under optimal conditions, the linear range ofhμman serμm albμmin is 10~50mg/mL, and the detection limit is 0.001mg/mL. On this basis,the scheme for POCT serμm proteins measurement device with detecting RRS signal oftetra-carboxylic zinc phthalocyanine-protein solution was designed. The design mainlyadopted a GaN-based blue-violet laser diode of 405nm wavelength as an excitation lightsource, a 475nm narrow bandpass filter as a monochromator, and a low-noise, high gainphotoelectric amplifier with blue enhanced photodiode as a detector.
In this dissertation the author studied the synthetic process of metal phthalocyaninecomplexes and their infrared spectrμm characterization; synthetized tetra-carboxylic zincphthalocyanine and tetra-amino copper phthalocyanine using solid phase synthesis with4-carboxyl phthalic anhydride and urea as raw materials, ammoniμm molybdate as catalystand confirmed them through the characterization of infrared spectrμm; discussed the factorsaffecting the detecting sensitivity including: system stability, acidity on the system, buffersolution volμme, ionic strength and interfering substances; developed the prototype of POCTserμm proteins measurement device; measured the serμm proteins with the prototype.
It is proved that the newly developed serμm proteins measurement device has a lot ofadvantages such as small size, low cost, low power consμmption, easy to use, strongrepetition and reliability, etc. After further improvement the device can be expected to havebroad application prospects in POCT.
引文
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