【作者】 陈婧；

【导师】 林新华；

【作者基本信息】 福建医科大学 ， 药学， 2010， 硕士

【摘要】 急性早幼粒细胞白血病是急性髓细胞白血病(AML)FAB分型中的M3亚型,大约占成人AML的10%～15%。95%以上的APL患者具有PML/RARα融合基因。PML/RARα融合基因是急性早幼粒细胞白血病特异的分子学标志,检测PML-RARα融合基因不仅能够协助急性早幼粒细胞白血病诊断,而且对该融合基因定量对微小残留病(MRD)检测也有重要意义。由于传统形态学及常规PCR技术不能对其定量,所以本文设计了一种新的方法检测急性早幼粒细胞白血病PML/RARα融合基因。将电化学DNA生物传感器技术和多通道生物传感芯片技术结合构建出多通道电化学传感芯片对急性早幼粒细胞白血病PML/RARα融合基因进行检测。电化学DNA生物传感器是近几年迅速发展起来的一种全新的生物传感器,具有灵敏度高、响应快、微型化、价格低廉等优点。多通道生物传感芯片也是最近几年在生命科学研究领域崭露头角的一项新技术。多通道生物传感芯片是由16个通道构成,每一通道都是一个传感器的探头,包括一个工作电极,参比电极及辅助电极,因此每个通道都能构成一个独立的传感器。将二者结合能够实现一次操作多次检测,具有快速、高效及并行处理的能力,具有广泛的应用前景。脱氧核糖核酸(DNA,为英文Deoxyribonucleic acid的缩写),又称去氧核糖核酸,是染色体的主要化学成分,同时也是组成基因的材料。DNA是由许多脱氧核苷酸残基按一定顺序彼此用3’,5’-磷酸二酯键相连构成的长链。大部分DNA以双螺旋结构存在,为双链长分子。高温下,双链DNA变性成为两条单链能够重新杂交形成双链,从而会阻碍探针与目标DNA的杂交。核糖核酸(缩写为RNA,即Ribonucleic Acid),由核糖核苷酸经磷酯键缩合而成长链状分子。与DNA不同,RNA一般为单链长分子,不形成双螺旋结构。因此,不存在变性与复性,而能够与探针充分杂交。因此,本文以急性早幼粒细胞白血病PML/RARαmRNA为检测目标,希望能够提高灵敏度。本文设计了两种结构的电化学探针(茎环结构探针和“三明治”结构探针),并分别构建了两种不同的多通道电化学传感芯片,用于急性早幼粒细胞白血病PML/RARαmRNA的检测,同时考察了传感芯片的特异性、稳定性等性能。首先,根据急性早幼粒细胞白血病融合基因的碱基序列设计出“三明治”结构DNA探针。将电化学技术和酶联免疫分析技术相结合构建出的“三明治”结构的多通道电化学传感芯片可以识别和定量检测溶液中人工合成的PML/RARα融合基因片断。结果表明,在最佳条件下,测定mRNA的线性范围为0.1 pM- 200 pM,检测限为1.2×10-14 M。研究还发现该传感器能够很好地识别互补链与不匹配链,具有较好的特异性。可以实现对急性早幼粒细胞白血病中PML/RARα融合基因mRNA定性定量的测定。另外,根据急性早幼粒细胞白血病融合基因的碱基序列设计出茎环结构DNA探针并且采用电化学酶联免疫分析技术构建出发夹结构探针修饰的多通道电化学传感芯片对人工合成的PML/RARα融合基因片断进行检测。结果表明:在优化杂交条件下,该传感芯片测定mRNA的浓度线性范围为0.5 pM- 300 pM,检测限为5.0×10-14 M。能很好的识别杂交溶液中的完全互补序列与单碱基突变序列,甚至能够很好识别种类的单碱基不匹配序列(A碱基不匹配、G碱基不匹配、C碱基不匹配),可以实现对急性早幼粒细胞白血病中PML/RARα融合基因定性定量的测定。更多还原

【Abstract】 Acute promyelocytic leukemia (APL) accounts for approximately 10% to15% of all cases of acute myeloid leukemia (AML). 95% of APL patients have PML/RARαfusion gene which is APL specific signal of the molecular biology. The detection of PML/RARαfusion gene not only assists the diagnosis of APL, but the quantitation of the fusion gene also has the important significance to the detection of minimal residual disease (MRD). Because the traditional morphology and routine PCR technology can’t quantitate, we designed a novel method to detect APL PML/RARαmRNA in this study. Based on electrochemical DNA biosensor and multi-channel biosensor chip, the multi-channel electrochemical chip was developed for detection of PML/RARαmRNA in APL. Electrochemical DNA biosensor is a novel biosensor which developed rapidly in recent years, with the advantages of fast detection, simplity, sensitivity, low price, and so on. The multi-channel biosensor chip is also a new technology which is coming to the forefront of life science in the recent years. There are 16 wells on the multi-channel biosensor chip, and every tunnel is a detecting head, including a working electrode, reference electrode and auxiliary electrode. Therefore, each tunnel could be a independent sensor. Combining the functions of both, the multichannel electrochemical chip can implement that once operation and multiple detection, which is of fast, high efficient and simultaneous treatment, which is of wide prospect.Deoxyribonucleic acid, or DNA, is a nucleic acid molecule that contains the genetic instructions used in the development and functioning of all known living organisms. Many deoxynucleotide residues which are linked together with 3’, 5’-phosphodiester bond in turn make up the long strands, named DNA . Bulk of DNA, as a long-chain molecule, were discovered to be a double helix. Under high temperature, the DNA duplex is melting into the single-strand DNA(ssDNA) which can rehybridize into double strands(dsDNA) thus to interfere the target DNA hybridization between and DNA probe. Ribonucleic Acid, or RNA, is a long chain molecule which forms by the condensation of ribonucleotide through phosphate ester linkage. RNA is general a long-chain molecule not a double helix, which is different with DNA. Accordingly, RNA could hybridize fully with DNA probe withou denaturation and renaturation. Therefore, we take for APL PML/RARαmRNA as the testing targets.Two novel DNA electrochemical probes (hairpin probe and Sandwich-type probe) were respectively designed and developed the multichannel electrochemical chips for detection of PML/RARαmRNA in acute promyelocytic leukemia (APL) in this article. The stability and the specificity of these multichannel electrochemical chips were also been investigated.Therein, Sandwich-type DNA electrochemical probe was designed according to APL PML/RARαmRNA fragment in this study. And then, based on the electrochemical enzyme immunoassay, the relationship between amperometric current and the concentration of synthetic target mRNA complementary strand is linear in the range of 0.1 pM to 200 pM with the detection limit of 1.2×10-14 M. At the same time, the method that demonstrates its simple and good specificity to distinguish the target sequences, single-base mismatched sequences, PML sequences, RARαsequences and non-complementary sequences. It is possible to qualitatively and quantitatively detect PML/RARαmRNA in APL.Besides, hairpin DNA electrochemical probe (HP) was designed according to APL PML/RARαmRNA fragment. And then, based on the electrochemical enzyme immunoassay, the hairpin probe modified multichannel electrochemica chip was developed for detection of PML/RARαfusion gene in APL in this study. The results indicate that, under the optimal conditions, the relationship between amperometric current and the concentration of synthetic target mRNA complementary strand is linear in the range of 0.5 pM to 300 pM with the detection limit of 5.0×10-14 M. Otherwise, the biosensor shows a good specificity to distinguish the complementary sequences and the different mismatched sequences, even the different kinds of the single-base mutation sequences (A-base mismatch, G-base mismatch and C-base mismatch). The obvious difference of results after hybridization demonstrated this multichannel electrochemical chip could detect PML/RARαmRNA qualitatively and quantitatively.更多还原