基于生物、有机、纳米材料的DNA检测体系研究
摘要
DNA检测体系策略的发展在分子诊断领域起到非常重要的作用愈加广泛的实际需求推动了更加稳定、灵敏性好、选择性强的检测剂、检测体系的不断发展。因此,化学家们在新型检测体系的设计、新型检测材料的制造中,都发挥了极其重要的作用。本文在前人的基础上做了以下几方面的内容：
第一部分
我们设计并发明了一种以细菌作为报告探针的DNA检测体系。这种“体内”信号检测手段包括两种探针：一是与目标DNA部分互补的的磁粒子捕获探针,二是与目标DNA其他序列部分互补的细菌(E.coli)信号探针。在目标DNA存在时,磁粒子捕获探针、目标DNA、细菌信号探针杂交,并形成了“三明治”的结构。通过磁分离的方式,可将杂交得到的E.coli信号探针特异性的分离出来。E.coli在37℃下培养生长,加入定量的异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导增强型绿色荧光蛋白(EGFP)的大量表达,从而将目标DNA的序列信息转化为绿色荧光读出的信号。
第二部分
每种高效的荧光核酸染料都可能因其能稳定与核酸结合,从而改变荧光特性才得以成为可用的荧光染料,从理论上都是不安全的。我们实验室自行设计合成了一种在Zn2+介导下可直接对DNA电泳体系染色的荧光染料,其作用快,检测限低,已得到验证。因此,我们在前人所做工作的基础上,将对我们实验室做出的以Zn2+介导的DNA荧光染料体系进行综合的生物安全性评价。实验结果表明我们自行设计、合成的分子对生物体有一定的致移码突变的作用。
第三部分
带有羧酸、羟基、氨基等多基团的分子修饰在金纳米粒子表面并引发可逆组装已经得到广泛报导。但基于这种体系所产生的实际应用却很少。因此,我们设计并发明一种以多官能团小分子与DNA双修饰的金纳米粒子作为DNA检测探针的体系。因小分子在不同pH、盐浓度条件下产生比较弱的氢键、离子键、疏水键等不稳定的弱相互作用,从而诱导并加速双修饰的金纳米产生可逆聚集,加速DNA检测体系的进程。
Sequence-specific DNA detection has extensive applications in molecular diagnostics, environmental monitoring, and antibioterrorism. Highly sensitive and selective biological systems have been developed by using biological, organic and nanomaterials. These methods exhibit extraordinary accuracy and specificity by virtue of the involvement of highly efficient reactions, even in complex biological media, which translates sequence information facilely into a variety of read-out formats.
Part Ⅰ:
A DNA diagnostic system has been developed by using bacteria as the reporter probe. DNA hybridization-driven magnetic isolation of E. coli, followed by exponential bacterial growth and fluorescent protein expression, allows the translation of target binding event into a vastly amplified signal and a highly sensitive/selective detection platform. The system involves the use of two types of probes: DNA-functionalized magnetic microparticles,(MMPs) and DNA-modified bacteria (harbored with a plasmid encoding enhanced green fluorescence protein, or EGFP). The sandwich structure formed by these two probes and target DNA could be magnetically separated and excessive EGFP could be expressed in the proliferated bacterial population for a facile fluorescent readout.
Part Ⅱ:
We have developed an ultrafast fluorescent staining system for the identification of DNA in gel electrophores with molecular Z1we synthesized in hand. Each efficient fluorescent nucleic acid dye may be mutagenic, because their stable combination with nucleic acids. So we evaluate Z1's genetic toxcity with Ames test. The results show that it can bring out slight frameshift mutation down to the concentration of6μg/mL.
Part Ⅲ:
Modification of small molecules with carboxyl and amino functional groups on AuNP will lead to the reversible assembly of nanoparticles under the change of pH, salt concentration. These systems have been widely reported. But the potential applications in research have been little reported.
In this study, we make use of the weak interactions in nature to promote and accelerate the strong interaction, and the formation of a spontaneous assembly system. We functionalized AuNPs with both DNA and the molecular carboxyl and amino functional groups we synthesized to cause the pH, salt-induced assembly of AuNP, thereby speeding up the DNA hybridization systems. With lower pH and increased salt concentration, the DNA hybridization process can be limited to4min from more than an hour.
第一部分
我们设计并发明了一种以细菌作为报告探针的DNA检测体系。这种“体内”信号检测手段包括两种探针：一是与目标DNA部分互补的的磁粒子捕获探针,二是与目标DNA其他序列部分互补的细菌(E.coli)信号探针。在目标DNA存在时,磁粒子捕获探针、目标DNA、细菌信号探针杂交,并形成了“三明治”的结构。通过磁分离的方式,可将杂交得到的E.coli信号探针特异性的分离出来。E.coli在37℃下培养生长,加入定量的异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导增强型绿色荧光蛋白(EGFP)的大量表达,从而将目标DNA的序列信息转化为绿色荧光读出的信号。
第二部分
每种高效的荧光核酸染料都可能因其能稳定与核酸结合,从而改变荧光特性才得以成为可用的荧光染料,从理论上都是不安全的。我们实验室自行设计合成了一种在Zn2+介导下可直接对DNA电泳体系染色的荧光染料,其作用快,检测限低,已得到验证。因此,我们在前人所做工作的基础上,将对我们实验室做出的以Zn2+介导的DNA荧光染料体系进行综合的生物安全性评价。实验结果表明我们自行设计、合成的分子对生物体有一定的致移码突变的作用。
第三部分
带有羧酸、羟基、氨基等多基团的分子修饰在金纳米粒子表面并引发可逆组装已经得到广泛报导。但基于这种体系所产生的实际应用却很少。因此,我们设计并发明一种以多官能团小分子与DNA双修饰的金纳米粒子作为DNA检测探针的体系。因小分子在不同pH、盐浓度条件下产生比较弱的氢键、离子键、疏水键等不稳定的弱相互作用,从而诱导并加速双修饰的金纳米产生可逆聚集,加速DNA检测体系的进程。
Sequence-specific DNA detection has extensive applications in molecular diagnostics, environmental monitoring, and antibioterrorism. Highly sensitive and selective biological systems have been developed by using biological, organic and nanomaterials. These methods exhibit extraordinary accuracy and specificity by virtue of the involvement of highly efficient reactions, even in complex biological media, which translates sequence information facilely into a variety of read-out formats.
Part Ⅰ:
A DNA diagnostic system has been developed by using bacteria as the reporter probe. DNA hybridization-driven magnetic isolation of E. coli, followed by exponential bacterial growth and fluorescent protein expression, allows the translation of target binding event into a vastly amplified signal and a highly sensitive/selective detection platform. The system involves the use of two types of probes: DNA-functionalized magnetic microparticles,(MMPs) and DNA-modified bacteria (harbored with a plasmid encoding enhanced green fluorescence protein, or EGFP). The sandwich structure formed by these two probes and target DNA could be magnetically separated and excessive EGFP could be expressed in the proliferated bacterial population for a facile fluorescent readout.
Part Ⅱ:
We have developed an ultrafast fluorescent staining system for the identification of DNA in gel electrophores with molecular Z1we synthesized in hand. Each efficient fluorescent nucleic acid dye may be mutagenic, because their stable combination with nucleic acids. So we evaluate Z1's genetic toxcity with Ames test. The results show that it can bring out slight frameshift mutation down to the concentration of6μg/mL.
Part Ⅲ:
Modification of small molecules with carboxyl and amino functional groups on AuNP will lead to the reversible assembly of nanoparticles under the change of pH, salt concentration. These systems have been widely reported. But the potential applications in research have been little reported.
In this study, we make use of the weak interactions in nature to promote and accelerate the strong interaction, and the formation of a spontaneous assembly system. We functionalized AuNPs with both DNA and the molecular carboxyl and amino functional groups we synthesized to cause the pH, salt-induced assembly of AuNP, thereby speeding up the DNA hybridization systems. With lower pH and increased salt concentration, the DNA hybridization process can be limited to4min from more than an hour.
引文
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