基于有机合成和核酸化学的生物医学检测体系研究
摘要
DNA检测在现代生物医学中正在发挥越来越重要的作用。高选择性、高灵敏度的DNA检测体系在分子诊断、环境监测、疾病控制等方面都有应用。现有检测技术往往应用生物、有机、纳米技术,将DNA序列信息转化为各种可读的信号。近年来本领域的研究尽管取得了巨大成就,但具有高选择性和高灵敏度,简易便携,容易操作的DNA检测方法仍然有待开发。
本论文报道了一种基于水蒸气冷凝的DNA检测方法。这种检测方法依赖于在目标DNA存在的情况下,通过核酸扩增,表面亲水程度发生改变,导致表面凝结出现水滴后表面形态发生变化,从而影响光线的漫反射／散射,形成肉眼可见的信号。实验证实这种方法灵敏度高,可以检测1微升溶液中600个目标DNA分子；选择性好,能够区分6种单一碱基错配的目标DNA；且能够进行多重检测。这种方法通过点样方法制备DNA芯片,不需要标记分子,适用于集成化,模块化检测技术。本检测技术理论上可以拓展应于其他生物分子的检测。
论文报道了一种通过生物酶将合成的人工修饰核苷酸引入DNA序列的方法。利用DNA聚合酶对脱氧脲苷三磷酸的5位修饰具有相对的容忍性,我们设计了一种含有2-溴异丁酰基团的脲嘧啶脱氧核苷类似物。以市售的(+)-5-碘-2'-脱氧脲苷为起始原料,通过5步合成反应成功得到目标产物,产物通过1H-NMR,13C-NMR,31P-NMR,元素分析,电喷雾质谱和高分辨质谱表征。进一步通过三种特殊设计的引物延伸实验证明该化合物能够被四种DNA聚合酶作为底物识别利用,从而将人工合成的核苷酸引入DNA序列中。设计的实验避免采用常规放射自显影的方法。
论文还报道了一些DNA检测相关技术。将上述合成的分子以滚换扩增的方式引入至DNA序列中后,通过原子转移自由基聚合(ATRP)的方式进行DNA检测；报道了在单一温度下一步合成掺杂型或合金型量子点的方法；还报道了一种在单一温度下进行的连接酶链反应(LCR),将其与滚换扩增的DNA检测方法相结合,可以检测出1微升溶液中的600个目标DNA分子。
Sequence-specific DNA detection is now becoming the key diagnostic tool in modern biomedical tests. Highly sensitive and selective biological detection systems have been developed for molecular diagnostics, environmental monitoring, and disease prevention, etc. These systems use biological, organic and nanomaterials, which translates sequence information facilely into a variety of read-out formats. Despite great progress, however, to our knowledge, sentivity of a single molecular detection level yet field portable, easy to operate DNA detection methods still awaits to be discovered.
We have developed a surface condensation of water strategy for the on-chip detection of DNA. This strategy relies on a target-driven alteration of surface wettability, and consequently, a transition of morphological state of and light propagation mode in the surface-condensed water, for the signaling of target. High sensitivity, high selectivity, and multiplexed analysis capability have been achieved in a label-free, direct spot array fabrication/visualization/imaging format, thus offering significant assay advantages over conventional diagnostic systems. Given recent advances in the tailored control of morphological state of surface-condensed water, the underlying principle described herein is likely extendable to the detection of other types of biological targets.
We have developed a method for the synthesis of artificial nucleotide and proved its incorporation into DNA strand by various DNA polymerases. Utilizing the enzyme's relatively tolerance for modification at the5-position of deoxyuridine triphosphate. We designed a uridine analogue containing2-Bromoisobutyryl functionalities. Starting from commercially available (+)-5-Iodo-2'-deoxyuridine, through a five step synthetic procedure, the target compound was successfully synthesized and charactered by1H-NMR,13C-NMR,31P-NMR, Elemental analysis, ESI-MS and HRMS. Further the recognization of the compound by four DNA polymerases was verified by three specially designed primer extension reactions. These designed reactions avoided the use of common autoradiography techniques.
We have also developed other DNA detection related techniques. The utilization of the above synthesized compound was used in rolling circle amplification-ATRP related DNA detection systems. We also reported a method for one temperature synthesis of doped and alloyed Zn-Cd-Se nanocrystals. We have developed a one temperature ligase chain reaction method and by coupling with rolling circle amplification, the method was able to detect DNA at very low concentrations (600DNA molecules in1milliliter).
本论文报道了一种基于水蒸气冷凝的DNA检测方法。这种检测方法依赖于在目标DNA存在的情况下,通过核酸扩增,表面亲水程度发生改变,导致表面凝结出现水滴后表面形态发生变化,从而影响光线的漫反射／散射,形成肉眼可见的信号。实验证实这种方法灵敏度高,可以检测1微升溶液中600个目标DNA分子；选择性好,能够区分6种单一碱基错配的目标DNA；且能够进行多重检测。这种方法通过点样方法制备DNA芯片,不需要标记分子,适用于集成化,模块化检测技术。本检测技术理论上可以拓展应于其他生物分子的检测。
论文报道了一种通过生物酶将合成的人工修饰核苷酸引入DNA序列的方法。利用DNA聚合酶对脱氧脲苷三磷酸的5位修饰具有相对的容忍性,我们设计了一种含有2-溴异丁酰基团的脲嘧啶脱氧核苷类似物。以市售的(+)-5-碘-2'-脱氧脲苷为起始原料,通过5步合成反应成功得到目标产物,产物通过1H-NMR,13C-NMR,31P-NMR,元素分析,电喷雾质谱和高分辨质谱表征。进一步通过三种特殊设计的引物延伸实验证明该化合物能够被四种DNA聚合酶作为底物识别利用,从而将人工合成的核苷酸引入DNA序列中。设计的实验避免采用常规放射自显影的方法。
论文还报道了一些DNA检测相关技术。将上述合成的分子以滚换扩增的方式引入至DNA序列中后,通过原子转移自由基聚合(ATRP)的方式进行DNA检测；报道了在单一温度下一步合成掺杂型或合金型量子点的方法；还报道了一种在单一温度下进行的连接酶链反应(LCR),将其与滚换扩增的DNA检测方法相结合,可以检测出1微升溶液中的600个目标DNA分子。
Sequence-specific DNA detection is now becoming the key diagnostic tool in modern biomedical tests. Highly sensitive and selective biological detection systems have been developed for molecular diagnostics, environmental monitoring, and disease prevention, etc. These systems use biological, organic and nanomaterials, which translates sequence information facilely into a variety of read-out formats. Despite great progress, however, to our knowledge, sentivity of a single molecular detection level yet field portable, easy to operate DNA detection methods still awaits to be discovered.
We have developed a surface condensation of water strategy for the on-chip detection of DNA. This strategy relies on a target-driven alteration of surface wettability, and consequently, a transition of morphological state of and light propagation mode in the surface-condensed water, for the signaling of target. High sensitivity, high selectivity, and multiplexed analysis capability have been achieved in a label-free, direct spot array fabrication/visualization/imaging format, thus offering significant assay advantages over conventional diagnostic systems. Given recent advances in the tailored control of morphological state of surface-condensed water, the underlying principle described herein is likely extendable to the detection of other types of biological targets.
We have developed a method for the synthesis of artificial nucleotide and proved its incorporation into DNA strand by various DNA polymerases. Utilizing the enzyme's relatively tolerance for modification at the5-position of deoxyuridine triphosphate. We designed a uridine analogue containing2-Bromoisobutyryl functionalities. Starting from commercially available (+)-5-Iodo-2'-deoxyuridine, through a five step synthetic procedure, the target compound was successfully synthesized and charactered by1H-NMR,13C-NMR,31P-NMR, Elemental analysis, ESI-MS and HRMS. Further the recognization of the compound by four DNA polymerases was verified by three specially designed primer extension reactions. These designed reactions avoided the use of common autoradiography techniques.
We have also developed other DNA detection related techniques. The utilization of the above synthesized compound was used in rolling circle amplification-ATRP related DNA detection systems. We also reported a method for one temperature synthesis of doped and alloyed Zn-Cd-Se nanocrystals. We have developed a one temperature ligase chain reaction method and by coupling with rolling circle amplification, the method was able to detect DNA at very low concentrations (600DNA molecules in1milliliter).
引文
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