【作者】 王琦；

【导师】 马飞； 张春阳；

【作者基本信息】 山东师范大学 ， 分析化学， 2022， 硕士

【摘要】 基于生物探针的检测方法以其特异性好、精度高、响应快等特点在人类疾病研究中发挥着重要作用。其中荧光探针因具有良好的水溶性、热稳定性、易合成和修饰等优点受到越来越多的关注,基于荧光探针的传感器由于其高灵敏度和良好的选择性,已被开发用于医疗诊断、生化分析、食品安全和环境监测等领域。生化分析领域中,生物标志物的检测是了解和治疗各种疾病的有力工具,在流行病学调查分析、疾病筛查、临床诊断和预后等方面都具有重要价值。因此,我们以DNA甲基转移酶（DNA methyltransferase,MTase）和β位淀粉样前体蛋白裂解酶1（BACE-1,β-分泌酶1）为模型,开发了两种基于酶切响应荧光探针的检测方法。主要研究内容如下:（1）发展了一种基于CRISPR/Cas12a切割双标记核酸荧光探针的信号放大方法,用于检测DNA甲基转移酶。我们设计了一个双标记核酸荧光探针,该探针可被双链DNA（ds DNA）和g RNA触发的CRISPR/Cas12a非特异性消化。当甲基转移酶存在时,ds DNA底物被甲基化,不能被Bss HII核酸内切酶切割。随后,ds DNA底物与g RNA结合激活Cas12a,裂解荧光探针,恢复Cy5荧光,该荧光信号可通过单分子成像准确定量。双标记荧光探针5 nt的长度保证了其高猝灭效率和低背景信号;CRISPR/Cas12a效应器不仅可以将生物识别转换为可检测的荧光信号,还可以轻松实现输出信号的放大。该方法具有很好的灵敏度(检测限为4.61×10^-4 U/m L)和特异性,可进一步应用于抑制剂筛选、酶动力学分析和人血清中DNA MTase活性测定,在临床诊断、药物开发和药物评价等方面具有广阔的应用前景。（2）开发了一种基于金纳米颗粒（Au NP）的荧光探针,用于BACE-1的体外检测和体内成像。我们设计了两种荧光探针,包括Cy5修饰的肽探针和黑洞猝灭剂2（BHQ2）标记的DNA探针。肽探针和DNA探针通过稳定的Au-S自组装在Au NP表面,获得超猝灭Au NP纳米探针,当靶标缺失时背景荧光接近于零,靶标BACE-1存在时催化肽探针裂解并释放大量荧光基团,产生明显的荧光信号,该荧光信号通过单分子计数进行量化。该方法无需任何抗体和分离步骤,即可一步检测BACE-1活性,具有良好的特异性和灵敏度（检测限为26.48 p M）,还可用于BACE-1抑制剂的筛选和动力学参数分析。特别是该纳米探针具有良好的稳定性,可以很容易地转移到活细胞中,并实时成像细胞中BACE-1活性,为BACE-1相关的阿尔茨海默病基础和临床研究提供了有价值的工具。更多还原

【Abstract】 The bioprobe-based methods have the advantages such as good specificity,excellent accuracy and rapid response,and play an important role in human disease research.Due to their good water solubility,thermal stability,and ease of synthesis and modification,fluorescent probes have attracted more and more attention recently.Fluorescent probe-based sensors have been developed for use in a range of fields,including medical diagnostics,biochemical analysis,food safety,and environmental monitoring because of their high sensitivity and good selectivity.In the field of biochemical analysis,the detection of biomarkers is a powerful tool for understanding and treating various diseases,and is of great value for epidemiological investigation and analysis,disease screening,clinical diagnosis and prognosis.Therefore,we developed two biosensors based on enzyme cleavage responsive fluorescent probes using DNA methyltransferase（MTase）andβ-site amyloid precursor protein-cleaving enzyme 1（BACE-1）as models.Main research contents are as follows:（1）We developed a signal amplification method to detect DNA methylase based on probe CRISPR/Cas12a-mediated cleavage of double-labeled nucleic acid fluorescent.We designed a double-labeled nucleic acid fluorescent probe that can be non-specifically digested by double-stranded DNA（ds DNA）activated CRISPR/Cas12a effector.In the presence of DNA methyltransferase,the ds DNA substrate is methylated and it cannot be digested by Bss HII endonuclease.Subsequently,the ds DNA substrate can bind with g RNA to activate the Cas12a,resulting in the cleavage of fluorescent probes and the recovery of Cy5 fluorescence which can be accurately quantified by single-molecule imaging.The length of the double-labeled fluorescent probe is 5 nt,which ensures its high quenching efficiency and low background signal.The CRISPR/Cas12a effector can convert biometric recognition event into a detectable fluorescent signal,enabling the easy amplification of output signal.This assay exhibits good specificity and high sensitivity with a detection limit of 4.61×10^-4 U/m L,and it can be further applied for inhibitor screening,enzyme kinetic analysis,and the measurement of DNA MTase activity in human serum,holding great potential in clinical diagnosis,drug development,and drug evaluation.（2）We developed a fluorescent probes-modified gold nanoparticle（Au NP）nanosensors for in vitro detection and in vivo imaging of BACE-1.We designed two fluorescent probes,including a Cy5-modified peptide probe and a dark quencher 2（BHQ2）-labeled DNA probe.Through the self-assembly of both fluorophore-labeled peptide probes and quencher-labeled assistant DNA probes on the surface of a single Au NP,a superquenched Au NP nanoprobe is obtained with a near-zero background fluorescence.The presence of target BACE-1 induces a distinct fluorescence signal as a result of the BACE-1-catalyzed cleavage of peptide probe and the subsequent release of abundant fluorophore moieties from the Au NP nanoprobe.The fluorescence signal can be directly quantified by single-molecule counting.This nanosensor involves only a single nanoprobe for the one-step homogeneous detection of the BACE-1 activity without the requirements of any antibodies and separation steps,and it possesses good selectivity and high sensitivity with a low detection limit of 26.48 p M.Moreover,it can be employed to screen BACE-1 inhibitors and analyze kinetic parameters.Especially,this nanoprobe possesses good stability and can be easily transferred into live cells for the real-time imaging of cellular BACE-1 activity,providing a new platform for BACE-1 associated research and early diagnosis of Alzheimer′s disease.更多还原