三螺旋电致化学发光生物传感器的研制及应用
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摘要
三螺旋DNA是在双螺旋DNA的基础上形成的,三螺旋结构在研发新的生物分子工具以及治疗试剂等方面具有巨大的潜力,可以对特定序列的双链DNA予以识别,以及对基因调节,包括干扰基因转录、复制和重组。三螺旋DNA的研究有助于深入理解细胞过程,揭示某些基因疾病的形成机理,为建立基因疾病新疗法提供全新的思路。
本论文以适体探针作为蛋白质与小分子的特异识别分子,结合具有灵敏的信号转换功能的电致化学发光技术,与三螺旋DNA杂交技术相结合,通过对DNA序列的设计来选择性的识别和检测凝血酶、腺苷的浓度,设计简单灵活,为蛋白质和小分子的研究提供了新的手段和思路。本论文主要内容包括以下三个方面:
1研究了一种新颖的基于二茂铁猝灭联吡啶钌发光的三螺旋电致化学发光(ECL)生物传感器,首先将标记联吡啶钌的DNA自组装到金电极上,然后与特定序列的DNA、标记二茂铁羧酸的DNA,在绑定剂存在的条件下杂交,形成三螺旋DNA结构。然后对合成的发光标记物的浓度进行了标定,以及对探针溶液进行了表征,和对绑定剂进行了选择,初步形成了一种可行的三螺旋DNA生物传感器,为接下来的凝血酶和腺苷的检测提供了一种可行的方法。
2在前期工作的基础了,我们改进了实验方法,研究了一种以适体为基础的ECL-三螺旋DNA生物传感器,使之用于凝血酶浓度的检测。在凝血酶存在下,DNA-3中凝血酶的适体部分与凝血酶结合,标有二茂铁的DNA-3被解离下来,二茂铁基团远离联吡啶钌基团,从而使联吡啶钌发光强度增强,以发光强度的变化值为信号实现了对凝血酶浓度的高灵敏度的检测。凝血酶浓度的线性范围是是4.0×10~(-15) ~ 3.0×10~(-14)mol L~(-1),检测限可达1.0 f mol L~(-1)。同时,设计了双螺旋检测凝血酶的对比试验,实验结果显示,我们的三螺旋生物传感器比其灵敏度提高了100倍。本方法对提高蛋白质检测的特异性与灵敏度提供了一条有效的途径。
3在以上两种DNA传感器的研究基础上,我们探索了第三种传感器,将标记联吡啶钌的腺苷适体链固定到金电极表面,选择三螺旋特征链和标记二茂铁基团的DNA链与之互补,形成三螺旋结构,在腺苷的作用下,联吡啶钌标记的探针与腺苷结合,形成腺苷-适体的发夹式复合结构,从而联吡啶钌更接近于电极表面而远离二茂铁,使发光强度大大增强。以发光强度的变化值为信号对腺苷的浓度进行检测,在最佳条件下,腺苷浓度的线性范围是1.0×10~(-9) ~ 1.0×10~(-8) mol L~(-1),检测限可达2.7×10~(-10) mol L~(-1)。并进行了双螺旋对比试验,三螺旋生物传感器比双螺旋的灵敏度提高了大约10倍。同时我们用胞苷和尿苷对其选择性进行了研究,发现我们研制的传感器具有较好的选择性。本方法为电化学生物传感器检测小分子的方法提供了一种新的思路。
Triplex DNA is formed on the basis of the duplex DNA. The structure of triple-helix is with great potential in the development of new biological molecule tools and treatment reagents. It can identify specific sequences of duplex DNA as well as on gene regulation, including interference with transcription, replication and reorganization. The research of triplex DNA will help further understanding cellular processes, reveal the formation mechanism of genetic diseases, and provide a new method to new treatments for the establishment of these diseases.
The main contents of this paper were to combine sensitive signal conversion function of ECL technology, triple helix DNA hybridization technique with the aptamer probes for protein and molecule specific recognition, and prepared a new ECL biosensor, which was designed by DNA sequence to identify and selectively detect the concentration of adenosine or thrombin. This method was simple and flexible. It provided new tools and ideas for protein and small molecule research. This thesis included the following three parts:
1 A novel triplex electrochemiluminescence biosensor based on the ferrocene quenching of tris(2,2'-bipyridyl)ruthenium luminescence was developed. First the DNA tagged with tris(2,2'-bipyridyl)ruthenium was self-assembled onto the surface of gold electrodes. And then it hybrided with a specific sequence of DNA and ferrocene (Fc) labeled DNA, in the presence of binding agent to form the structure of triplex DNA. The concentration of the synthesized of luminous markers was calibrated, and the probe solution was characterized, and the binding agent was choosed. Finally a viable triplex DNA biosensor was constructed for the next detection of thrombin and adenosine.
2 On the basis of preliminary work, a novel ECL-triplex aptamer-based biosensing method for the determination of thrombin was developed based on a structure-switching ECL-quenching mechanism, so that for the detection of thrombin concentrations. In the presence of thrombin, the aptamer sequence prefered to form the aptamer-thrombin complex and the switch of the binding partners occured in conjunction with the generation of a strong ECL signal owing to the dissociation of FcDNA. The luminous intensity changed with the concentration of thrombin can be tested, the linear range was 4.0×10~(-15) to 3.0×10~(-14)mol L~(-1). A detection limit of 1.0 f mol L~(-1) of thrombin was achieved with high sensitivity. At the same time, a duplex ECL contrast test was designed, the results showed that the sensitivity of our triplex biosensor increased more than 100-fold. The method provided an effective way to improve the specificity and sensitivity for protein detection.
3 On the base of the above two sensors, a novel ECL-triplex aptamer-based biosensing method for the determination of adenosine was developed. In the presence of adenosine, the tris(2,2'-bipyridyl)ruthenium labeled DNA-1 probe combined with adenosine to form adenosine - aptamer hairpin complex structure, which is closer to the surface of electrode and away from the ferrocene to increase the luminous intensity. The ECL intensity was increased with the increase of the concentration of adenosine in the range of 1.0×10~(-9) to 1.0×10~(-8) mol L~(-1). The detection limit is 2.7×10~(-10)mol L~(-1). Meanwhile, we made a duplex contrast test, the sensitive of triplex biosensor increased about 10-fold. We used the cytidine and uridine for its selectivity, and we found that our triplex biosensor had good selectivity. The method provided a new thought for the detection of small molecules with electrochemical biosensor.
本论文以适体探针作为蛋白质与小分子的特异识别分子,结合具有灵敏的信号转换功能的电致化学发光技术,与三螺旋DNA杂交技术相结合,通过对DNA序列的设计来选择性的识别和检测凝血酶、腺苷的浓度,设计简单灵活,为蛋白质和小分子的研究提供了新的手段和思路。本论文主要内容包括以下三个方面:
1研究了一种新颖的基于二茂铁猝灭联吡啶钌发光的三螺旋电致化学发光(ECL)生物传感器,首先将标记联吡啶钌的DNA自组装到金电极上,然后与特定序列的DNA、标记二茂铁羧酸的DNA,在绑定剂存在的条件下杂交,形成三螺旋DNA结构。然后对合成的发光标记物的浓度进行了标定,以及对探针溶液进行了表征,和对绑定剂进行了选择,初步形成了一种可行的三螺旋DNA生物传感器,为接下来的凝血酶和腺苷的检测提供了一种可行的方法。
2在前期工作的基础了,我们改进了实验方法,研究了一种以适体为基础的ECL-三螺旋DNA生物传感器,使之用于凝血酶浓度的检测。在凝血酶存在下,DNA-3中凝血酶的适体部分与凝血酶结合,标有二茂铁的DNA-3被解离下来,二茂铁基团远离联吡啶钌基团,从而使联吡啶钌发光强度增强,以发光强度的变化值为信号实现了对凝血酶浓度的高灵敏度的检测。凝血酶浓度的线性范围是是4.0×10~(-15) ~ 3.0×10~(-14)mol L~(-1),检测限可达1.0 f mol L~(-1)。同时,设计了双螺旋检测凝血酶的对比试验,实验结果显示,我们的三螺旋生物传感器比其灵敏度提高了100倍。本方法对提高蛋白质检测的特异性与灵敏度提供了一条有效的途径。
3在以上两种DNA传感器的研究基础上,我们探索了第三种传感器,将标记联吡啶钌的腺苷适体链固定到金电极表面,选择三螺旋特征链和标记二茂铁基团的DNA链与之互补,形成三螺旋结构,在腺苷的作用下,联吡啶钌标记的探针与腺苷结合,形成腺苷-适体的发夹式复合结构,从而联吡啶钌更接近于电极表面而远离二茂铁,使发光强度大大增强。以发光强度的变化值为信号对腺苷的浓度进行检测,在最佳条件下,腺苷浓度的线性范围是1.0×10~(-9) ~ 1.0×10~(-8) mol L~(-1),检测限可达2.7×10~(-10) mol L~(-1)。并进行了双螺旋对比试验,三螺旋生物传感器比双螺旋的灵敏度提高了大约10倍。同时我们用胞苷和尿苷对其选择性进行了研究,发现我们研制的传感器具有较好的选择性。本方法为电化学生物传感器检测小分子的方法提供了一种新的思路。
Triplex DNA is formed on the basis of the duplex DNA. The structure of triple-helix is with great potential in the development of new biological molecule tools and treatment reagents. It can identify specific sequences of duplex DNA as well as on gene regulation, including interference with transcription, replication and reorganization. The research of triplex DNA will help further understanding cellular processes, reveal the formation mechanism of genetic diseases, and provide a new method to new treatments for the establishment of these diseases.
The main contents of this paper were to combine sensitive signal conversion function of ECL technology, triple helix DNA hybridization technique with the aptamer probes for protein and molecule specific recognition, and prepared a new ECL biosensor, which was designed by DNA sequence to identify and selectively detect the concentration of adenosine or thrombin. This method was simple and flexible. It provided new tools and ideas for protein and small molecule research. This thesis included the following three parts:
1 A novel triplex electrochemiluminescence biosensor based on the ferrocene quenching of tris(2,2'-bipyridyl)ruthenium luminescence was developed. First the DNA tagged with tris(2,2'-bipyridyl)ruthenium was self-assembled onto the surface of gold electrodes. And then it hybrided with a specific sequence of DNA and ferrocene (Fc) labeled DNA, in the presence of binding agent to form the structure of triplex DNA. The concentration of the synthesized of luminous markers was calibrated, and the probe solution was characterized, and the binding agent was choosed. Finally a viable triplex DNA biosensor was constructed for the next detection of thrombin and adenosine.
2 On the basis of preliminary work, a novel ECL-triplex aptamer-based biosensing method for the determination of thrombin was developed based on a structure-switching ECL-quenching mechanism, so that for the detection of thrombin concentrations. In the presence of thrombin, the aptamer sequence prefered to form the aptamer-thrombin complex and the switch of the binding partners occured in conjunction with the generation of a strong ECL signal owing to the dissociation of FcDNA. The luminous intensity changed with the concentration of thrombin can be tested, the linear range was 4.0×10~(-15) to 3.0×10~(-14)mol L~(-1). A detection limit of 1.0 f mol L~(-1) of thrombin was achieved with high sensitivity. At the same time, a duplex ECL contrast test was designed, the results showed that the sensitivity of our triplex biosensor increased more than 100-fold. The method provided an effective way to improve the specificity and sensitivity for protein detection.
3 On the base of the above two sensors, a novel ECL-triplex aptamer-based biosensing method for the determination of adenosine was developed. In the presence of adenosine, the tris(2,2'-bipyridyl)ruthenium labeled DNA-1 probe combined with adenosine to form adenosine - aptamer hairpin complex structure, which is closer to the surface of electrode and away from the ferrocene to increase the luminous intensity. The ECL intensity was increased with the increase of the concentration of adenosine in the range of 1.0×10~(-9) to 1.0×10~(-8) mol L~(-1). The detection limit is 2.7×10~(-10)mol L~(-1). Meanwhile, we made a duplex contrast test, the sensitive of triplex biosensor increased about 10-fold. We used the cytidine and uridine for its selectivity, and we found that our triplex biosensor had good selectivity. The method provided a new thought for the detection of small molecules with electrochemical biosensor.
引文
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