适配体Au、CdTe纳米粒子功能化修饰及凝血酶电化学传感研究
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摘要
核酸适配体(Aptamers)是人工合成的单链寡核苷酸片段—DNA或RNA,是从含大量自由序列的随机寡核苷酸文库中通过体外筛选分离出来的,它能以高的亲和力及特异性结合各种靶分子(蛋白质、小分子、金属离子、DNA,甚至整个细胞)。由于核酸适体合成简易、稳定性好且易于保存,而且对配体具有强的亲和力和高的结合特异性,在生化分析领域,已被视为一种富有应用前景的探针,可与抗体相媲美,甚至比传统的抗体更具有优势,受到越来越多的重视。凝血酶核酸适体是最早被筛选出来的,它有两条不同的碱基序列,能与凝血酶不同位点发生特异性结合,形成G-四重体结构。本论文以凝血酶适体为模型,发展了一系列凝血酶检测的新方法。主要内容如下：
(1)建立结合Fe3O4/Au磁分离和CdTe纳米粒子核酸适体探针的电化学凝血酶检测新方法。水相法合成了Fe3O4/Au磁性复合微粒以及L-半胱氨酸修饰的CdTe纳米粒子,以前者作为DNA的固定和分离材料,修饰了凝血酶适体(15-mer)的CdTe纳米粒子作为检测探针。首先将巯基修饰的适体互补链通过金-硫键固定在Fe3O4/Au磁性复合微粒上,再让CdTe纳米粒子修饰的适体链与固定探针杂交即完成了凝血酶传感器的制备。凝血酶的存在使CdTe纳米粒子修饰的适体探针从磁性复合微粒上脱落,利用外加磁场分离,通过差分脉冲伏安法检测上清液中Cd2+的量即可达到定量检测凝血酶的目的。该方法简便,灵敏度高,选择性好,成本低,无有害物质,在蛋白质检测中有很大的应用前景。
(2)以亚甲基蓝作为检测探针结合ZrO2/AuNPs构建凝血酶电化学生物传感器。利用亚甲基蓝与DNA分子的特殊性作用,让它作为检测探针,结合ZrO2/AuNPs膜修饰电极对检测信号的放大作用,构建一种简单灵敏的电化学生物传感器,用于凝血酶的检测。将巯基化的适体互补链固定到修饰电极表面作为固定探针,然后让凝血酶适体(29-mer)与其杂交,再让亚甲基蓝插入到双链DNA中或与鸟嘌呤特异性结合,制成凝血酶适体传感器。当电极浸入到凝血酶溶液中,适体即与凝血酶特异性结合成G-四重体结构,从而使亚甲基蓝从电极表面脱落,再检测电极表面亚甲基蓝峰电流的下降值(其降低幅度与凝血酶浓度成一定的比例关系),达到检测凝血酶的目的。本传感器有较宽的线性范围和很低的检测限,并且选择性高,再生能力强,可用于高灵敏检测蛋白质。
(3)应用电化学阻抗法和杂交反应的增敏作用建立高灵敏检测凝血酶的方法。利用凝血酶与它的两条适体形成一种“三明治”夹心结构,以及DNA对电极表面电荷传递能力的影响,采用交流阻抗法,发展一种新的凝血酶的放大检测方法,实现对目标蛋白质的高灵敏检测,有效提高检测的响应性能。
Aptamers are synthetic oligonucleotides DNA or RNA, which are isolated from random-sequence nucleic acid bibraries by "in vitro selection". They can bind to molecular targets with high affinity and selectivity, such as proteins, small molecules, metal ion, DNA, even the whole cells. Owing to aptamer's relative ease of isolation and modification, good stability, the easier storage, the high binding affinity and simplicity of in vitro selection, it have attracted more and more attentions in the area of biochemical analysis. Thrombin aptamer has two kinds of nucleic acid sequence, which can bind to thrombin at different sites with binding affinity to form G-quartet structure. In this paper, thrombin used as a model protein to develop new thrombin detection methods using aptamers. The main work is summarized as follows:
(1) We established a new method for electrochemical detection of thrombin combining Fe3O4/Au magnetic separation and CdTe-aptamer probe. Fe3O4/Au magnetic nanoparticles and L-cysteine modified CdTe nanoparticles were synthesized. Fe3O4/Au magnetic nanoparticles were used as DNA immobilization and separation material, while aptamer modified CdTe nanoparticles used as detection probe. Thiolated modified capture DNA which complementary with thrombin aptamer was anchored onto the surface of Au coated magnetic beads via sulfur-gold affinity. CdTe nanoparticles modified aptamer probe hybridized with capture DNA sequence modified on magnetic beads by hybridization reaction, and then the thrombin aptsensor was obtained. When thrombin added to the detection solution, CdTe nanoparticles modified aptamer was forced to dissociate from the magnetic beads. The thrombin was detected through current peak of Cd2+ in supernate of differential pulse anodic stripping voltammetry (DPV) after separation with an extermal magnetic field. This method is sensitive, no harmful reagents, requiring only a small sample volume and has a great potential for proteins detection.
(2) A new thrombin sensor was development by using methylene blue (MB) as electrochemical probe and ZrO2/AuNPs as signal amplification and magnetic separation material. MB is used as the external electroactive indicator owing to its special interaction with DNA. In this paper, A thiolated capture DNA is immobilized on the surface of modified electrode and then hybridized with thrombin aptamer (29-mer). MB intercalates into the double-strands of nucleic acids and binds specifically to guanine bases, after which the aptasensors were obtained. In the presence of thrombin, thrombin interacted with its aptamer to form G-quadruplex, while MB desorbed from electrode surface, resulting in a decrease in current. The decrement of current is proportional to the amount of thrombin. This aptasensor has wide concentration range, low detection limit, high selectivity and good reproducibility and this aptasensor was able to sensitively detect thrombin.
(3) A highly sensitive detection method was developed by using hybridization amplification and electrochemical impedance technique. Since one thrombin molecule has two active sites for its two kinds of aptamer, the electrode-aptamerⅠ/thrombin/aptamerⅡ-AuNPs sandwich structure was fabricated as the sensing platform. Here, we reported a simple and highly sensitive label-free impedimetric aptasensor for thrombin detection based on the hybridized signal amplification. This strategy could not only improve the detection sensitivity but also provide a simple model for the signal amplification of the impedimetric sensors. Meanwhile, this method exhibits good selectivity.
(1)建立结合Fe3O4/Au磁分离和CdTe纳米粒子核酸适体探针的电化学凝血酶检测新方法。水相法合成了Fe3O4/Au磁性复合微粒以及L-半胱氨酸修饰的CdTe纳米粒子,以前者作为DNA的固定和分离材料,修饰了凝血酶适体(15-mer)的CdTe纳米粒子作为检测探针。首先将巯基修饰的适体互补链通过金-硫键固定在Fe3O4/Au磁性复合微粒上,再让CdTe纳米粒子修饰的适体链与固定探针杂交即完成了凝血酶传感器的制备。凝血酶的存在使CdTe纳米粒子修饰的适体探针从磁性复合微粒上脱落,利用外加磁场分离,通过差分脉冲伏安法检测上清液中Cd2+的量即可达到定量检测凝血酶的目的。该方法简便,灵敏度高,选择性好,成本低,无有害物质,在蛋白质检测中有很大的应用前景。
(2)以亚甲基蓝作为检测探针结合ZrO2/AuNPs构建凝血酶电化学生物传感器。利用亚甲基蓝与DNA分子的特殊性作用,让它作为检测探针,结合ZrO2/AuNPs膜修饰电极对检测信号的放大作用,构建一种简单灵敏的电化学生物传感器,用于凝血酶的检测。将巯基化的适体互补链固定到修饰电极表面作为固定探针,然后让凝血酶适体(29-mer)与其杂交,再让亚甲基蓝插入到双链DNA中或与鸟嘌呤特异性结合,制成凝血酶适体传感器。当电极浸入到凝血酶溶液中,适体即与凝血酶特异性结合成G-四重体结构,从而使亚甲基蓝从电极表面脱落,再检测电极表面亚甲基蓝峰电流的下降值(其降低幅度与凝血酶浓度成一定的比例关系),达到检测凝血酶的目的。本传感器有较宽的线性范围和很低的检测限,并且选择性高,再生能力强,可用于高灵敏检测蛋白质。
(3)应用电化学阻抗法和杂交反应的增敏作用建立高灵敏检测凝血酶的方法。利用凝血酶与它的两条适体形成一种“三明治”夹心结构,以及DNA对电极表面电荷传递能力的影响,采用交流阻抗法,发展一种新的凝血酶的放大检测方法,实现对目标蛋白质的高灵敏检测,有效提高检测的响应性能。
Aptamers are synthetic oligonucleotides DNA or RNA, which are isolated from random-sequence nucleic acid bibraries by "in vitro selection". They can bind to molecular targets with high affinity and selectivity, such as proteins, small molecules, metal ion, DNA, even the whole cells. Owing to aptamer's relative ease of isolation and modification, good stability, the easier storage, the high binding affinity and simplicity of in vitro selection, it have attracted more and more attentions in the area of biochemical analysis. Thrombin aptamer has two kinds of nucleic acid sequence, which can bind to thrombin at different sites with binding affinity to form G-quartet structure. In this paper, thrombin used as a model protein to develop new thrombin detection methods using aptamers. The main work is summarized as follows:
(1) We established a new method for electrochemical detection of thrombin combining Fe3O4/Au magnetic separation and CdTe-aptamer probe. Fe3O4/Au magnetic nanoparticles and L-cysteine modified CdTe nanoparticles were synthesized. Fe3O4/Au magnetic nanoparticles were used as DNA immobilization and separation material, while aptamer modified CdTe nanoparticles used as detection probe. Thiolated modified capture DNA which complementary with thrombin aptamer was anchored onto the surface of Au coated magnetic beads via sulfur-gold affinity. CdTe nanoparticles modified aptamer probe hybridized with capture DNA sequence modified on magnetic beads by hybridization reaction, and then the thrombin aptsensor was obtained. When thrombin added to the detection solution, CdTe nanoparticles modified aptamer was forced to dissociate from the magnetic beads. The thrombin was detected through current peak of Cd2+ in supernate of differential pulse anodic stripping voltammetry (DPV) after separation with an extermal magnetic field. This method is sensitive, no harmful reagents, requiring only a small sample volume and has a great potential for proteins detection.
(2) A new thrombin sensor was development by using methylene blue (MB) as electrochemical probe and ZrO2/AuNPs as signal amplification and magnetic separation material. MB is used as the external electroactive indicator owing to its special interaction with DNA. In this paper, A thiolated capture DNA is immobilized on the surface of modified electrode and then hybridized with thrombin aptamer (29-mer). MB intercalates into the double-strands of nucleic acids and binds specifically to guanine bases, after which the aptasensors were obtained. In the presence of thrombin, thrombin interacted with its aptamer to form G-quadruplex, while MB desorbed from electrode surface, resulting in a decrease in current. The decrement of current is proportional to the amount of thrombin. This aptasensor has wide concentration range, low detection limit, high selectivity and good reproducibility and this aptasensor was able to sensitively detect thrombin.
(3) A highly sensitive detection method was developed by using hybridization amplification and electrochemical impedance technique. Since one thrombin molecule has two active sites for its two kinds of aptamer, the electrode-aptamerⅠ/thrombin/aptamerⅡ-AuNPs sandwich structure was fabricated as the sensing platform. Here, we reported a simple and highly sensitive label-free impedimetric aptasensor for thrombin detection based on the hybridized signal amplification. This strategy could not only improve the detection sensitivity but also provide a simple model for the signal amplification of the impedimetric sensors. Meanwhile, this method exhibits good selectivity.
引文
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