【作者】 冷翠翠；

【导师】 张宜恒；

【作者基本信息】 青岛科技大学 ， 应用化学， 2010， 硕士

【摘要】 本研究构建了掺杂导电油墨制作的丝网印刷电极,并将其用于对特定DNA片段和肿瘤细胞进行识别和测定,为许多疾病的临床早期诊断提供基础性研究。本文着重进行了三种类型的DNA传感器的研制和性质研究:1.丝网印刷电极的构建。合成了形貌、尺寸和电导率适当的聚苯胺纳米管,然后将室温离子液体与所合成的聚苯胺纳米管共同掺杂入导电油墨中,用于制造三电极丝网印刷DNA传感器中的工作电极,再用壳聚糖膜加以覆盖修饰电极。优化了掺杂和修饰条件,在最佳条件下所得的电极具有较低的表面阻抗和较高的电化学灵敏度。2.基于这个传感器建立了一种基于酶放大的DNA夹心检测的方法。将avidin修饰的辣根过氧化物酶(avidin-HRP)固定到杂交好的DNA上,通过HRP催化底物邻氨基酚和双氧水反应产生信号。在实验选定的最佳条件下,得到了目标DNA的线性范围为8×10-14-2×10-13 M,检测限为8.37×10-15 M。另外,三碱基错配序列和完全非互补序列的杂交情况同样被检测。其中,三碱基错配序列显示有微弱的电化学信号而完全非互补序列则无信号检出。这说明我们设计的DNA传感器具有良好的选择性。3.在以上工作的基础上,我们改进了实验方法,研究了另一种灵敏度更高的基于六氨基钌和金纳米粒子放大信号的电化学方法法检测DNA特定序列的生物传感器。首先,将巯基修饰的捕获DNA固定在纳米金修饰的丝网印刷电极表面,之后分别与目标DNA和标记金纳米粒子的3’位巯基修饰的检测探针、探针杂交,形成了“夹心”式的DNA杂交复合物。通过六氨基钌特异性吸附到纳米金结合的单链DNA中,六氨基钌发生氧化还原反应产生信号通过计时电量法检测信号进一步增强了检测的灵敏度。结合以上两种因素,该DNA传感器可检测到10-17的低浓度的目标DNA,并在三碱基错配DNA的测定上显示出良好的选择性。同时,对实验的最佳检测条件进行了选择。在最佳条件下进行检测,得到了目标DNA的8×10-17 M的检测限,检测的线性范围为1.0×10-16 M到1.0×10-14 M。4.我们又深入研究制作了一种可以检测肿瘤细胞的传感器。这种传感器是以Ramos肿瘤细胞的适体识别以及DNA序列的循环复制替换为基础构建的。这一检测方案分两步进行:首先,将适体序列DNA固定在磁珠上后与“信使序列”杂交,Ramos细胞与这一杂交复合体结合导致信使序列DNA释放出来;将发卡序列固定在电极上,释放出的信使序列与其“环”部分杂交,颈环部分被打开,再与金纳米颗粒结合的引物DNA杂交形成杂交复合体,并在DNA复制酶、脱氧三磷酸核苷酸混合物(dNTP)和镁离子存在下引发以引物为起点的DNA聚合反应,反应过程中形成一条与发卡探针互补的新链,同时信使序列DNA被替换而释放到溶液中,找到另一个发卡探针开始新的循环复制。引物序列上的纳米金颗粒同时附有分子条码序列,其上吸附有六氨基钌,通过计时电量法检测六氨基钌的氧化还原信号,进一步增强了检测的灵敏度。结合以上两种因素,该DNA传感器可检测到10-17的低浓度的目标DNA,也可以检测1 ml溶液中含有100个细胞,并在三碱基错配DNA的测定上显示出良好的选择性。同时,对含有其他干扰细胞时该传感器也可以显示出良好的选择性。该研究还对实验的最佳检测条件进行了选择。更多还原

【Abstract】 The main idea of the reseach was to modified the commercial graphite ink (Electrodag 423SS) for manufacture of high sensitivity, high selectivity, single-used and stable screen-printed DNA sensor, which also determinated DNA sequence and tumor cells. It provided a basic theoretical reseach for clinic diagnoses of many diseases at early stage. In this paper, we prepared three types of DNA biosensors which could be summarized as follows:1. Preparation of modified screen-printed electrode (SPE). Polyaniline nanotubes (PANINTs) with appropriate shape, size and conductivity were synthesized, and were doped into the commercial graphite ink together with room-temperature ionic liquid (RTIL) or the printing of the SPE, and chitosan was covered on the electrode surface. A homogeneous, stable and highly conductive electrode surface was thus obtained. The optimal amounts of PANINT, RTIL and chitosan were investigated. 2. The DNA assay based on the SPE described above was established. The target DNA sequences were specifically recognized by the capture probes immobilized on the SPE surface and the reporter probes labeled with HRP through avidin-biotin binding. o-Phenylenediamine was catalyzed by HRP to produce 2,3-diaminophenazine, which generate electrochemical response. The target DNA could be quantitatively detected in the range from 8×10-13-2×10-14 M and a detection limit of 8.37×10-15 M was found for this sensor. The signal for the complementary DNA was much larger and apparently distinguishable from the control signal or the mismatched sequence signal. A good selectivity was thus obtained on this sensor.3. A novel and sensitive DNA sensor for the sequence specific DNA detection based on the signal amplification with gold nanoparticles and hexaamineruthenium(III) chloride (RuHex) was developed on the basis of our preceding work. A DNA detection protocol was established based on this sensor in sandwich hybridization mode. Au nanoparticles were bound to the hybridization complexes through reporter probes, and RuHex was attached to the bar-code DNA on nanoparticles. This sensor was found to be able to distinguish the complementary target DNA at low concentration and the three-base mismatched DNA at higher concentration. The target DNA could be quantitatively detected in the range from 1.0×10-16 M to 1.0×10-14 M, and a detection limit of 8.0×10-17 M was found.4. A new approach for the detecting of tumor cells, Ramos was established based on aptamer-based cell recognition and the isothermal circular DNA strand-displacement polymerization. This assay was carried out in two steps. A hybridization complex of the aptamer and its partially comlementary sequence (“messenger sequence”) were immobilized on the magnetic beads. The recognition of Ramos cells on this complex resulted in the release of the messenger, which triggered a strand-replacement DNA replication for itself, resulting in the multiplication of its concentration. The messenger hybridized with hairpin probes on the electrodes to open their stem parts, to which a gold nanoparticle with primer sequences and bio-bar-code sequences was hybridized, and an isothermal DNA polymerization was initiated, which replaced the intermediary target sequence and released it into the solution. The released messenger sequence found another hairpin probe to begin another strand-replacement polymerization. The signal was generated through the chronocoulometric interrogation of RuHex that was attached on the bio-bar-code sequences. A detection limit of 1.2×10-17 M was found for the DNA sequence detection, and 100 cells for cell detection. This sensor was found to be able to distinguish the complementary target DNA at low concentration and the three-base mismatched DNA at higher concentration. Selectivity in the cell detection was thus obtained on this sensor. The optimal experimental conditions were also explored.更多还原