药物相关基因单核苷酸多态性的电化学分析方法研究

发布时间：2018-05-12 21:41

本文选题：单核苷酸多态性 + 生物传感器　；参考：《重庆医科大学》2017年硕士论文

【摘要】：单核苷酸多态性(single nucleotide polymorphisms,SNP)是被公认的导致基因多样性与复杂性的重要原因。β1肾上腺素能受体基因(ADRB1)发生SNP直接影响高血压患者服用美托洛尔的降压效应。而维生素K环氧化物还原酶复合物亚基1(VKORC1)基因发生SNP与华法林的使用剂量有密切关系。因此,建立全新的SNP分子检测方法可以更好地掌握针对某种药物反应性的个体间差异,从而实现个体化治疗,降低医疗风险和医疗费用。本文通过合成全新的纳米材料,用于修饰电化学DNA传感器,实现了对两种比较常见的临床应用药物相关的SNP的检测,以期精准指导个体化临床用药。研究工作分为以下两部分:第一章应用氧化石墨烯/二氧化铈/铂纳米粒子作为纳米信标对β1肾上腺素能受体基因的电化学检测本实验构建了一个基于氧化石墨烯/二氧化铈/铂纳米粒子(GO/CeO2/PtNPs)作为纳米信标,金纳米粒子/链霉亲和素(Au NPs/SA)作为电极修饰纳米材料形成的DNA传感器,用于血清中ADRB1突变特异性检测。首先以GO为载体,固载了大量的CeO2。接着,在其表面还原了Pt NPs,合成了GO/CeO2/PtNPs获得良好的催化性能。另外,在玻碳电极(GCE)表面沉积一层AuNPs之后修饰SA来连接生物素标记的探针。通过各种条件优化,制备的传感器灵敏度较高,特异性较好,线性范围较宽,重现性较好。线性范围为分为两段:1 fM到100 fM和100 fM到10 nM,最低检测限为0.33 fM。此外,加样回收率在99.4-101.9%之间,说明该传感器可以应用于血清中ADRB1基因突变的检测。第二章应用富勒烯-聚酰胺-胺型树枝状高分子-空心铂作为纳米信标对维生素K环氧化物还原酶复合物亚基1的电化学检测本实验构建了一个基于富勒烯-聚酰胺-胺型树枝状高分子-空心铂(C60NPs-PAMAM-PtPNPs)作为纳米信标,还原性氧化石墨烯四乙烯五胺/金纳米颗粒/链霉亲和素(rGO-TEPA/AuNPs/SA)作为电极修饰纳米材料形成的DNA传感器,用于血清中VKORC1基因突变的特异性检测。C60NPs不仅具有氧化还原活性可以作为信号物质,而且还能作为生物分子的载体来固载PtPNPs。因此,我们合成了C60NPs-PAMAM-PtPNPs。在GCE表面修饰rGO-TEPA后再沉积一层AuNPs,接着修饰SA来连接生物素标记的四面体DNA用于捕获目标DNA。通过条件优化,传感器的灵敏度较高,特异性较好,线性范围较宽,重现性较好。线性范围为1 pM到10 nM,最低检测限为33 fM。此外,加样回收率在96.7-103.94%之间,说明该传感器可以应用于血清中VKORC1基因突变的检测。
[Abstract]:Single nucleotide polymorphisms (SNPs) are recognized as an important cause of gene diversity and complexity. 尾 1 adrenoceptor gene (ADRB1) has a direct impact on the antihypertensive effect of metoprolol on hypertension patients with SNP. The SNP of vitamin K epoxide reductase complex subunit 1 (VKORC1) was closely related to the dosage of warfarin. Therefore, the establishment of a new SNP molecular detection method can better grasp the individual differences in response to a certain drug, so as to achieve individualized treatment, reduce medical risks and medical costs. In this paper, a novel nano-material was synthesized to modify the electrochemical DNA sensor, and the detection of two common clinical drug related SNP was realized, in order to guide the individualized clinical medicine accurately. The research work is divided into two parts as follows: in Chapter 1, a novel method for the electrochemical detection of 尾 _ 1-adrenergic receptor gene by using graphene oxide / cerium oxide / platinum nanoparticles as a nano-beacon has been developed. A novel method based on graphite oxide has been developed for the electrochemical detection of 尾 _ 1-adrenergic receptor gene. The nanocrystalline CEO _ 2 / PtNPs / cerium dioxide / platinum nanoparticles are used as nanoscale beacons. Gold nanoparticles / streptavidin / au NPs / SAs were used as DNA sensors modified with electrodes to detect ADRB1 mutation specificity in serum. At first, a large amount of CEO _ 2 was immobilized on go carrier. Then, Pt NPs were reduced on the surface and GO/CeO2/PtNPs was synthesized to obtain good catalytic performance. In addition, a layer of AuNPs was deposited on the surface of the glassy carbon electrode (GCE) and modified with SA to bind the biotin labeled probe. By optimizing various conditions, the sensor has high sensitivity, good specificity, wide linear range and good reproducibility. The linear range is divided into two segments: 1: 1 FM to 100 FM and 100 FM to 10 nm. The minimum detection limit is 0.33 fM. In addition, the recovery rate was 99.4-101.9%, which indicated that the sensor could be used to detect the mutation of ADRB1 gene in serum. Chapter 2 electrochemical detection of vitamin K epoxide reductase complex subunit 1 using fullerene-polyamide-amine dendrimer and hollow platinum as nanometer beacon Amino-amine type dendritic macromolecule, C60NPs-PAMAM-PtPNPs), used as nanoscale beacons, Reductive graphene tetraethylenetetramethylamine / gold nanoparticles / streptavidin / rGO-TEPA / AuNPs / SA) were used as DNA sensors for the modification of nanomaterials. The specific detection of VKORC1 gene mutation in serum. C60 NPs not only have redox activity but also can be used as carriers of biomolecules to immobilize PtPNPs. Therefore, we synthesized C60NPs-PAMAM-PtPNPs. A layer of AuNPs was deposited after rGO-TEPA was modified on the surface of GCE, and then SA was modified to bind biotin labeled tetrahedron DNA to capture target DNAs. The sensor has high sensitivity, good specificity, wide linear range and good reproducibility. The linear range is from 1 pm to 10 nm, and the minimum detection limit is 33 FM. In addition, the recovery rate was 96.7-103.94%, which indicated that the sensor could be used to detect the mutation of VKORC1 gene in serum.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96;TP212.3

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