【作者】 王静；

【导师】 衣馨瑶；

【作者基本信息】 中南大学 ， 分析化学， 2024， 硕士

【摘要】 阿尔茨海默症(Alzheimer’s disease,AD)是一种常见的神经退行性疾病,其症状为记忆力丧失和其他认知能力受损,严重程度足以干扰患者正常生活,最终导致患者死亡。目前全世界有超过3500万名AD患者,随着人口老年化速度的加快,被诊断患有AD的人数仍在不断增加,这将严重威胁全人类的健康。AD起病隐匿、进展缓慢且不确定,在患病的早期并不会出现记忆力受损和认知障碍等症状,因此仅凭临床表型难以识别其病理过程。生物标志物提供了人体内相关病理生理学的客观指标,在AD的早期诊断和治疗中发挥着重要的作用。目前检测AD的可靠生物标志物主要是Aβ,Tau蛋白和apoE,但是Aβ和Tau蛋白在生物样品中浓度极低且存在多种亚型,因此,Aβ和Tau蛋白的检测仍然具有挑战性。apoE等位基因的多态性是AD的主要遗传决定因素,其中,apoEε4是AD的最强遗传风险因素,同时携带两个apoEε4等位基因的携带者患AD的风险高达90%。因此,对apoEε4基因的检测在AD的预防、早期诊断和疾病治疗中发挥重要作用。本论文拟开发两种用于apoEε4基因检测的荧光传感器,主要研究内容如下:(1)基于DNA四面体纳米结构(DNA tetrahedron nanostructure,DTN)的荧光传感方法用于apoEε4等位基因密码子112位(Tc1)和密码子158位(Tc2)附近片段的双信号检测。以噻唑橙(Thiazole Orange,TO)为供体,DTN两个顶点的荧光团(Cy3和Cy5)为受体,在DTN中启动荧光共振能量转移(F(?)rster resonance energy transfer,FRET)过程。当靶标(Tc1和Tc2)存在时,靶标会与DNA四面体的延伸部分杂交形成双链结构,GCGC碱基对被限制性核酸内切酶Hha I特异性切割,荧光团(Cy3和Cy5)被切割下来游离在溶液中,Cy3在570 nm和Cy5在670 nm的荧光信号大幅下降并伴随着TO在533 nm处荧光信号的恢复。该方法具有较宽的线性范围(2 n M-100n M),检测限为0.82 n M(Tc1)和0.77 n M(Tc2),且对与靶标单碱基错配的序列表现出良好的选择性。所提出的方法具有检测时间短、易于操作、高通量等优点。(2)构建了基于成簇的规则间隔短回文重复序列(Clustered regularly interspaced short palindromic repeats,CRISPR)-Cas12a系统的荧光传感器用于对apoEε4基因的检测。将捕获探针(FAM-DP1-biotin和Cy5-DP2-biotin)固定在修饰了链霉亲和素的磁珠表面。当存在目标物时,目标物会与捕获探针杂交形成双链结构,激活的CRISPR-Cas12a无法对形成的DNA双链结构进行切割,磁分离后上清液中测得的荧光信号很低。通过比较荧光信号的变化实现了对于apoEε4基因的灵敏检测。所提出的方法具有较宽的线性范围(50 p M-25 n M),检测限为22.4 p M(Tc1)和32.5 p M(Tc2),对apoE基因的另外两种表型有较好的区分能力。该传感方法操作简单、灵敏度高,可扩展至其他疾病生物标志物的检测。图37幅,表10个,参考文献129篇。更多还原

【Abstract】 As a neurodegenerative disorder,Alzheimer’s disease(AD)is characterized by cognitive dysfunction and behavioral impairment.The symptoms of AD are memory loss and other cognitive impairments that are severe enough to interfere with daily life and eventually lead to death.There are currently more than 35 million AD patients worldwide,and as the population ages faster,the number of people diagnosed with AD is still increasing,which will seriously threaten the health of all humankind.The onset of AD is insidious,slow,and uncertain,and there are no symptoms such as memory impairment and cognitive impairment in the early stage of the disease,so it is difficult to identify its pathological process based on clinical phenotype alone.Biomarkers provide objective indicators of related pathophysiology in the human body,and play an important role in the early diagnosis and treatment of AD.At present,the reliable biomarkers for the detection of AD are mainly Aβ,Tau and apoE,but the concentration of Aβand Tau in biological samples is extremely low and there are multiple subtypes,so the detection of Aβand Tau proteins is still challenging.Polymorphism of apoE alleles is the main genetic determinant of AD,among which apoEε4 is the strongest genetic risk factor for AD,and carriers with two apoEε4 alleles have a 90%risk of developing AD.Therefore,the detection of apoEε4 gene plays an important role in the prevention,early diagnosis,and disease treatment of AD.In this thesis,two fluorescence sensors for apoEε4 gene detection are developed.The main research contents are as follows:(1)A dual-signal fluorescence sensing method is designed based on DNA tetrahedron nanostructure(DTN)for detection of fragments associated with apoEε4 allele near codon 112(Tc1)and codon 158(Tc2).The F(?)rster resonance energy transfer(FRET)process in the DTN is initiated with thiazole orange(TO)served as the donor and the fluorophores(Cy3 and Cy5)at the two vertices of DTN served as the acceptors.In the presence of targets(Tc1 and Tc2),the target hybridizes with an extension of the DNA tetrahedron to form a dual-stranded structure,the GCGC base pairs appended to the DTN are specifically cleaved by Hha I restriction enzyme,the fluorophores(Cy3 and Cy5)are cleaved free in solution,and the fluorescence signal of Cy3 at 570 nm and Cy5 at 670nm decreased significantly,accompanied by the recovery of the fluorescence signal of TO at 533 nm.The method has a wide linear range(2 n M-100 n M),the limit of detection is estimated to be 0.82 n M(Tc1)and0.77 n M(Tc2),and shows good selectivity for single-base mismatched sequences with the targets.The proposed method thus possesses the advantages of short detection time,easy operation,and high-throughput capability.(2)The fluorescence sensor based on clustered regularly interspaced short palindromic repeats(CRISPR)-Cas12a system is constructed for the detection of apoEε4 gene.The capture probes(FAM-DP1-biotin and Cy5-DP2-biotin)modified with biotin and fluorophor are fixed on the surface of streptavidin coated magnetic beads.In the presence of targets,the hybridization of targets and capture probes are facilitated,the activated CRISPR-Cas12a cannot cleave double-stranded DNA,and the fluorescence signal of the supernatant after magnetic separation is very low.The sensitive detection of apoEε4 gene fragments are realized by comparing the changes of fluorescence signals.The proposed method has a wide linear range(50 p M-25 n M),the limit of detection is estimated to be 22.4 p M(Tc1)and 32.5 p M(Tc2),and has a good ability to distinguish the other two phenotypes of apoE gene.The sensing approach possessed the advantages of simple operation,sensitive,and can be extended to the detection for other disease biomarkers.更多还原