【作者】 胡顺；

【导师】 骆海明；

【作者基本信息】 华中科技大学 ， 生物医学工程， 2024， 博士

【摘要】 阿尔茨海默病（Alzheimer’s disease,AD）是一种疾病进程不可逆的神经退行性疾病,其早期诊断可以改善AD治疗效果从而延缓病情的发展。血浆中AD核心标志物（β-淀粉样蛋白和磷酸化tau）可以反映脑内与Aβ沉积和tau蛋白异常相关的病理变化。然而,由于AD疾病的复杂性和异质性,AD临床前期患者的血液中AD标志物缺乏敏感性,导致其病程评估的准确度降低。因此,针对AD临床前期患者病程评估不准确的问题,本论文设计了同时检测血浆中多种AD生物标志物的超灵敏检测方法,研究了其检测灵敏度和特异度,并通过对临床样本的测试评价其区分AD不同病程阶段的检测效果。本文主要内容如下:（1）利用淀粉样蛋白β（β-amyloid,Aβ）对金属基纳米酶酶活性的调节作用,设计一种二氧化锰纳米酶传感器阵列,实现了六种Aβ肽(Aβ_1-40、Aβ_1-42、Aβ_1-42原纤维、Aβ_1-42纤维、AβE22G和AβArc原纤维)的同步检测。本纳米酶传感器阵列能在一小时内对六种Aβ肽进行同步检测,其动态检测范围为0.01～500 n M。通过双盲实验验证本传感器阵列的实用性,30个未标记类别的Aβ肽均被准确分类。此外,本传感器阵列也可以用于血清检测环境中六种Aβ肽的准确识别。（2）基于免疫聚合酶链式反应（Polymerase chain reaction,PCR）的信号放大技术,开发一种免疫磁性外泌体PCR的检测方法,实现了临床血浆外泌体上Aβ_1-42、Aβ_1-40、p-tau¹⁸¹和p-tau^396,404的超灵敏检测。通过降低检测的背景信号、优化反应体系中抗体-DNA的稀释比例、免疫结合时间、洗涤次数,本检测方法可以对AD标志物进行高灵敏度地定量检测,其定量检测的最低检测浓度为10 fg/m L,其检测灵敏度比传统酶联免疫吸附测定（Enzyme-Linked Immunosorbnent Assay,ELISA）检测提高了1000倍。在血浆外泌体（CD63⁺外泌体）检测中Aβ_1-42可以有效区分AD患者和健康者,其受试者工作特性曲线下面积（Area under the curve,AUC）为0.993,其检测的真阳性率为95.0%,分类的灵敏度为95.0%、特异度为95.0%。（3）将生物传感器阵列的多维检测与免疫磁性外泌体PCR的超灵敏检测优势相结合,建立一种多重检测方法——免疫磁性外泌体PCR传感器阵列,实现了血浆外泌体上多种AD生物标志物(Aβ_1-42、Aβ_1-40、p-tau¹⁸¹、p-tau²¹⁷、p-tau^396,404和GFAP)的同步检测。传感单元与传统传感器阵列的设计不同,本方法利用抗体-DNA偶联物设计的引物探针作为传感单元,引入引物探针对模板DNA的特异性,可检测浓度下限低至1.0 fg/m L。使用本方法测量临床血浆外泌体样本（n=150）中的六种标志物水平,通过机器学习算法辅助数据分析,对所检测的临床样本进行AD不同疾病阶段的区分,其分类准确度高达98.6%。本方法能有效区分临床队列中健康与临床前期主观认知下降（Subjective cognitive decline,SCD）阶段（AUC=0.99）,其分类的灵敏度为89.5%、特异度为100%、精确度为100%、准确度为94.3%。更多还原

【Abstract】 Alzheimer’s disease（AD）is a neurodegenerative disorder with an irreversible disease process.Early diagnosis of AD improves the effectiveness of AD treatment and thus delays the progression of the disease.Plasma AD core markers（β-amyloid and phosphorylated tau）can reflect pathological changes in the brain associated with Aβdeposition and abnormal tau protein.However,due to the complexity and heterogeneity of AD disease,AD markers in the blood of patients with preclinical AD lack sensitivity,resulting in reduced accuracy in assessing the course of the disease.Therefore,aiming at the problem of inaccurate assessment of the patients’disease course with preclinical AD,in this thesis,we designed an ultra-sensitive detection method for simultaneously detecting multiple biomarkers of AD in plasma,studied its detection sensitivity and specificity,and evaluated its detection effect of distinguishing different stages of AD disease course by detecting clinical samples.The main contents of this paper are as follows:（1）Taking advantage of the regulatory effect of amyloidβ（Aβ）on the enzymatic activity of metal-based nanoenzymes,a manganese dioxide nanoenzyme sensor array was designed to achieve simultaneous detection of six Aβpeptides(Aβ_1-40,Aβ_1-42,Aβ_1-42protofibrils,Aβ_1-42 fibrils,AβE22G,and AβArc protofibrils).The nanoenzyme sensor array could accurately detect six Aβspecies in parallel within one hour,with a detection dynamic range of 0.01 to 500 n M.The practicability of the sensor array was verified by double-blind experiments,and 30 unlabeled samples were classified accurately.In addition,the sensor array can also be used for accurate identification of six Aβpeptides in serum assay environment.（2）Based on the signal amplification technique of immuno-polymerase chain reaction（PCR）,we developed an immunomagnetic exosomal PCR assay to achieve ultrasensitive detection of Aβ_1-42,Aβ_1-40,p-tau¹⁸¹,and p-tau^396,404 in plasma exosomes.By reducing the background signal in the detection and optimizing the dilution ratio of antibody–DNA,immunobinding time,and washing steps in the reaction system,this detection method can perform high-sensitivity quantitative detection of AD markers with a minimum detectable concentration of 10 fg/m L.And its detection sensitivity is 1000 times higher than that of traditional Enzyme-Linked Immunosorbnent Assay（ELISA）detection.In plasma exosome（CD63⁺exosome）assay,Aβ_1-42 can effectively differentiate between AD patients and healthy individuals,with an area under the receiver operating characteristic curve（AUC）of0.993,a true positive rate of 95.0%,and a classification sensitivity of 95.0%and a specificity of 95.0%.（3）Combining the multi-dimensional detection of biosensor array with the ultra-sensitive detection advantage of immunomagnetic exosomal PCR,a multiplex detection,immunomagnetic exosomal PCR sensor array（imEPA）was established to enable the simultaneous detection of multiple AD biomarkers(Aβ_1-42,Aβ_1-40,p-tau¹⁸¹,p-tau²¹⁷,p-tau^396,404 and GFAP)on plasma exosomes.Unlike the design of traditional sensor arrays,imEPA was designed with a primer probe designed using an antibody-DNA conjugation as the sensing unit,which introduces the specificity of the primer probe for template DNA,resulting in a detection limit of imEPA as low as 1.0 fg/m L.The method was used to measure the levels of six markers in clinical plasma exosomes samples（n=150）,and machine learning algorithms assisted with data analysis to differentiate between different disease stages of AD in the clinical samples tested,with a classification accuracy of 98.6%.This method can effectively distinguish healthy and preclinical subjective cognitive decline（SCD）stages in the clinical cohort（AUC=0.99）,corresponding to 17 true positive samples.The sensitivity,specificity,precision and accuracy of the classification were 89.5%,100%,100% and 94.3%respectively.更多还原