共振光散射测定辣根过氧化物酶及功能化碳纳米管在生化分析中的应用
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摘要
酶是由细胞产生的具有催化功能的蛋白质,酶分析在生命科学研究中具有重要的意义,已深刻影响到生物、化学等领域。但是传统的酶检测方法,如放射性标记、酶标记法等,往往操作繁杂,这在一定程度上限制了酶分析的研究。随着生命科学的发展,如何对酶进行快速、简便、高效检测,已经成为研究的热点问题。辣根过氧化物酶是应用最为广泛的一种酶制剂。碳纳米管是90年代发现的一种新型材料,其独特的物理和化学性能,已成为众多领域的研究热点。本文分为两部分,就这两种应用广泛的物质作了研究,为发展其在环境微生物免疫分析中得到更广泛的应用。
第一部分,基于苯酚与H2O2的氧化反应共振光散射技术测定辣根过氧化物酶。
本文建立了一种高灵敏度的测定辣根过氧化物酶的共振光散射分析方法。HRP催化苯酚和H2O2反应生成酚类共轭聚合物,导致共振光散射信号在200～700 nm之间有很大的增强,并在313 nm处达到最大值。基于此现象,建立了一种灵敏的检测HRP的方法。测定HRP的标准曲线的线性范围是0.05～5.0×10-7g/mL,检出限是1.1×10-10 g/mL。
第二部分,碳纳米管的化学修饰及DNA分子功能化。
本文利用简单的方法使羧基化修饰的碳纳米管通过共价作用与单链DNA进行结合,利用具有较强的荧光共轭聚合物PFP与DNA的静电结合作用,实现对碳纳米管DNA分子功能化的验证,建立了一种简便的碳纳米管功能化的方法,为碳纳米管作为功能化荧光探针在免疫分析中的应用打下了基础,发展了碳纳米管在环境微生物的免疫分析方面的应用。
Enzyme is a protein which has catalytic activity and the enzyme analysis has great influence in the field of life science research. But the traditional methods to detect enzyme, such as the radioactive label and the enzyme label, generally have complex operations that has limited the advanced research in enzyme in some degree. With the development of life science research, how to establish rapid, simple and highly effective methods to detect enzyme has been becoming a hot topic. HRP (Horseradish Peroxidase) is one of the enzymes which are most widely used. CNT (Carbon Nanotube) is becoming a hot topic for its unique physical and chemical properties after the 90th. Based on above, this paper is divided into two parts to research the HRP and CNT and prospect on the application in the field of the environmental microbiological immunoassay.
Part one:Determination of HRP based on the oxidation reaction of phenol with H2O2 by using resonance light scattering technique.
A new assay of HRP is proposed by using of resonance light scattering (RLS). The oxidation reaction of phenol with hydrogen peroxide (H2O2) catalyzed by HRP results in a great enhancement of RLS intensity in the wavelength range from 200 nm to 700 nm characterized by the RLS peak around 313 nm. The enhanced intensity of RLS at 313 nm is proportional to the concentration of HRP in the range of 0.05～5.0×10-7 g/mL and the detection limit is 1.1×10-10 g/mL. Therefore, a simple and sensitive method for HRP determination is established.
Part two:Chemical modified CNT and DNA molecular functional CNT.
We developed applications of CNT in the detection of DNA by using high sensitive fluorescence assay. The amount of signal was proportional to the concentration of target oligonucleotide. At the same time, we lay a good foundation for the application of PFP (cationic conjugated polymer) in process of hybridization reaction. If we design other suitable probes and complementary sequences, this assay could be used for other DNA hybridization and SNP analysis.
第一部分,基于苯酚与H2O2的氧化反应共振光散射技术测定辣根过氧化物酶。
本文建立了一种高灵敏度的测定辣根过氧化物酶的共振光散射分析方法。HRP催化苯酚和H2O2反应生成酚类共轭聚合物,导致共振光散射信号在200～700 nm之间有很大的增强,并在313 nm处达到最大值。基于此现象,建立了一种灵敏的检测HRP的方法。测定HRP的标准曲线的线性范围是0.05～5.0×10-7g/mL,检出限是1.1×10-10 g/mL。
第二部分,碳纳米管的化学修饰及DNA分子功能化。
本文利用简单的方法使羧基化修饰的碳纳米管通过共价作用与单链DNA进行结合,利用具有较强的荧光共轭聚合物PFP与DNA的静电结合作用,实现对碳纳米管DNA分子功能化的验证,建立了一种简便的碳纳米管功能化的方法,为碳纳米管作为功能化荧光探针在免疫分析中的应用打下了基础,发展了碳纳米管在环境微生物的免疫分析方面的应用。
Enzyme is a protein which has catalytic activity and the enzyme analysis has great influence in the field of life science research. But the traditional methods to detect enzyme, such as the radioactive label and the enzyme label, generally have complex operations that has limited the advanced research in enzyme in some degree. With the development of life science research, how to establish rapid, simple and highly effective methods to detect enzyme has been becoming a hot topic. HRP (Horseradish Peroxidase) is one of the enzymes which are most widely used. CNT (Carbon Nanotube) is becoming a hot topic for its unique physical and chemical properties after the 90th. Based on above, this paper is divided into two parts to research the HRP and CNT and prospect on the application in the field of the environmental microbiological immunoassay.
Part one:Determination of HRP based on the oxidation reaction of phenol with H2O2 by using resonance light scattering technique.
A new assay of HRP is proposed by using of resonance light scattering (RLS). The oxidation reaction of phenol with hydrogen peroxide (H2O2) catalyzed by HRP results in a great enhancement of RLS intensity in the wavelength range from 200 nm to 700 nm characterized by the RLS peak around 313 nm. The enhanced intensity of RLS at 313 nm is proportional to the concentration of HRP in the range of 0.05～5.0×10-7 g/mL and the detection limit is 1.1×10-10 g/mL. Therefore, a simple and sensitive method for HRP determination is established.
Part two:Chemical modified CNT and DNA molecular functional CNT.
We developed applications of CNT in the detection of DNA by using high sensitive fluorescence assay. The amount of signal was proportional to the concentration of target oligonucleotide. At the same time, we lay a good foundation for the application of PFP (cationic conjugated polymer) in process of hybridization reaction. If we design other suitable probes and complementary sequences, this assay could be used for other DNA hybridization and SNP analysis.
引文
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