【作者】 张华；

【导师】 王青； 王柯敏；

【作者基本信息】 湖南大学 ， 分析化学， 2017， 硕士

【摘要】 生物标志物的检测已成为研究热点之一,然而,大多生物标志物以非常低的浓度存在,例如循环microRNAs(miRNAs)在血液中仅以femtomolar甚至更低水平存在,因而有必要构建高灵敏度的生物传感器。鉴于单一的放大方法难以满足低浓度生物标志检测的要求,多重放大技术的发展已成为趋势。本论文选择miRNA-221和肌红蛋白作为模式目标物,将催化发夹组装、超级三明治技术以及酶催化放大技术等相结合构建了高灵敏的生物传感器,并在此基础上,简化操作步骤以及检测设备,构建了便携式的生物传感器,具体如下:1、基于催化发夹组装(CHA)和超级三明治技术相结合的新型电化学传感器用于miRNA-221的检测研究。一方面,CHA可以实现目标miRNA-221循环利用,实现信号放大;另一方面,通过在电极表面形成的超级三明治结构可以负载多个辣根过氧化物酶(HRP)修饰的信号探针,进而实现电化学信号的放大。在等温双重放大技术作用下,可检测到低至0.6 pM(S/N=3)miRNA-221。此外,该传感器具有高选择性,能够将miRNA-221从同源miRNA中区分开来。更重要的是,该传感器能够用于细胞内提取的总RN A中miRN A-221的检测,其结果与qRT-PCR方法得到的结果基本一致,这意味着该电化学传感器有望应用于临床分析领域。并且该电化学传感器避免了纳米颗粒的引入,克服了纳米颗粒的局限性。2、便携式比色核酸适配体传感器用于肌红蛋白的检测研究。该传感器一方面基于超级三明治技术和HRP-DNA催化放大技术实现了信号的增强,另一方面,该比色核酸适配体传感器的整个过程可以直接在Ep管内完成,只需要简单的几步,操作简便。并且利用便携式光纤传感器作为检测器,不需要大型仪器设备和专业技术人员。该传感器可检测低至3 nM(S/N=3)的肌红蛋白,能够满足临床检测要求。由于光纤传感器的光源为调制光,可使该方法免受外界光的影响。该工作有望发展成为一种新型即时检测方法。更多还原

【Abstract】 The detection of biomarkers has become one of the hotspots.However,most of biomarkers existed at very low concentrations.For example,circulating microRNAs(miRNAs)are present at femtomolar or even lower levels in blood.Thus,it is necessary to construct high sensitive biosensors.Given that single amplification strategy cannot meet the requirements for detecting low concentrations of biomarkers,the development of multiple amplification technology has become a trend.In this thesis,miRNA-221 and myoglobin were chosen as model target,high sensitive biosensors for the detection of biomarker were proposed based on the combination of catalyzed hairpin assembly,supersandwich amplification and enzyme catalytic amplification strategies.In addition,a portable biosensor was prepared to simplify the operation steps and equipment.The details were as follows:1.A novel isothermal electrochemical biosensor was proposed for the sensitive detection of miRNA-221 based on the ingenious combination of the target-catalyzed hairpin assembly(CHA)and supersandwich amplification strategies.On the one hand,CHA can achieve the cyclicality of the target miRNA,which led to signal amplification;on the other hand,the signal probe was modified with horseradish peroxidase(HRP)to form a supersandwich multiplex HRP-DNA label on the electrode,which could achieve an amplified electrochemical signal.Using the isothermal dual signal amplification strategies,as low as 0.6 pM(S/N=3)miRNA-221 could be detected.In addition,this biosensor showed high selectivity and could discriminate miRNA-221 from the homologous miRNAs.Note that human miRNA from cancer cells could also be detected and the results were in excellent agreement with those obtained using qRT-PCR.That it is to say,the proposed biosensor has great potential for broad applications in the field of clinical analysis.Given that the biosensor avoided the introduction of nanoparticles,the limitation of using the nanoparticles was overcome.2.A simple and portable colorimetric aptasensor was proposed for sensitive myoglobin detection.On the one hand,the colorimetric aptasensor achieved the signal enhancement by using supersandwich amplification and HRP-DNA catalytic amplification strategies.On the other hand,the entire process of the colorimetric aptasensor can be done directly in the Ep tube,requiring a few simple steps and simple operation.Moreover,with a portble fiber optic sensor as a detector,it did not need large equipment and professional technical personnel.More importantly,the colorimetric aptasensor could detect as low as 3 nM(S/N=3)myoglobin,which was lower than clinical cutoff for myoglobin in healthy patients.Since the light source was modulated light,fiber optic sensor was not affected by external light.This work was expected to develop into a novel point of care testing.更多还原