【作者】 李伟；

【导师】 许丹科；

【作者基本信息】 南京大学 ， 分析化学， 2015， 博士

【摘要】 发展生物标志物的简便、高灵敏、高选择性的检测方法是实现重大疾病早期诊断和治疗的关键。金纳米粒子具有独特的尺寸效应和良好的生物相容性,且易于制备和生物功能化修饰,因此适合与生物识别元件相偶联构建各种功能化的金纳米生物探针。与传统的检测探针相比,金纳米探针不仅可以增强其对靶标物质的识别和结合能力,而且可以放大信号并提供光学、电化学等多种检测途径,已成为纳米生物传感检测中极为重要的新型分析工具。本论文紧紧围绕适配体-金纳米探针的表面等离子共振、荧光猝灭等重要物理性质以及其优异的化学催化性能的应用开展了一系列深入研究,包括采用共价和非共价两种偶联方式设计和制备适配体-金/银纳米探针、金纳米探针催化还原有色底物(染料或非染料分子)的筛选与评价,金纳米荧光探针的激活策略等。在此基础上,我们将研制的金纳米探针作为新型分子工具用于增强靶标蛋白质的识别和检测,发展了一些新颖高效的蛋白质光学分析检测方法。主要研究成果如下:1.基于金纳米粒子催化甲基橙还原的比色检测新方法传统的比色生物分析主要依赖于天然蛋白酶或人工模拟酶及其底物的使用。针对这一问题,我们研究中使用金纳米粒子为催化剂、以有机偶氮染料分子—甲基橙为有色底物,基于适配体功能化金纳米粒子催化甲基橙还原褪色反应发展了一种新型的无酶化放大比色分析方法。此方法首先利用适配体修饰的磁性粒子从混合物中将待测蛋白质分子捕获、分离和富集至磁性粒子表面,然后加入适配体修饰的金纳米粒子形成三明治复合物;复合物中金纳米粒子可催化甲基橙的还原褪色反应实现目标蛋白质的放大比色检测。该方法简单、快速、高效,成功地实现了凝血酶的高灵敏和高特异性检测,其浓度定量检测范围为45 pM-455 pM。通过肉眼观察,凝血酶的检测限约为320 pM,借助于紫外-可见分析仪器,检测限可进一步降低至30 pM。该方法无需传统的各种酶或银/金增强试剂的使用,为构建无酶化生物检测方法提供了新的途径。2.基于金纳米粒子催化亚甲基蓝和4-硝基苯酚还原的比色检测新方法为了拓宽上述比色方法的应用并进一步解决日前有限的酶-底物反应体系,我们首先分析了基于纳米粒子催化有色底物还原褪色反应构建的比色方法的理论基础和影响检测灵敏度的各种因素,然后使用金纳米粒子为催化剂,选择非偶氮染料分子—亚甲基蓝和非染料分子—-硝基苯酚作为有色反应底物,分别建立了动力学法和终点法两种检测方式来实现放大比色检测。理论分析表明,构建这种分析方法的关键是催化活性粒子不存在时的还原褪色速率应当接近于零或远小于粒子存在时的反应速率;通过提高催化反应的放大因子(kcat△t)和被分析物与受体之间的亲和常数以及选择高消光系数的有色底物等手段均可以提高检测灵敏度。4-硝基苯酚为有色底物时,体系需要酸化终止反应和碱化恢复颜色等步骤,采用终点法来实现凝血酶的检测,其浓度定量检测范围为182 pM-1090pM,检测限为91 PM。亚甲基蓝为有色底物时,体系无需酸化终止和粒子磁分离去除等步骤,采用动力学法来实现凝血酶的检测,其浓度定量检测范围为68 pM-455 pM,检测限为10 PM。该类分析方法的成功开发和多种可供选择的催化活性粒子和底物在一定程度上克服了传统放大比色方法中对有限的酶-底物反应体系的严重依赖。3.聚腺苷酸尾化适配体-金/银纳米探针的研究与应用经典的制备适配体-金/银纳米粒子复合物的方法需要对适配体进行化学修饰,操作过程复杂且成本很高,目前主要依赖于巯基化适配体通过金/银-硫键自组装在粒子表面获得。针对这一问题,我们使用聚腺苷酸(PolyA)尾化适配体替代巯基化适配体,基于PolyA序列与金/银纳米粒子表面强的吸附作用力,分别制备了凝血酶适配体-金纳米粒子复合物(Apt15-PolyA-AuNPs)和血小板衍生生长因子(PDGF-BB)适配体-银纳米粒子复合物(Apt44-PolyA-AgNPs),并将其作为检测探针分别用于凝血酶和PDGF-BB的均相比色检测。基于目标诱导金纳米探针交联聚集的比色检测原理,Apt1 5-PolyA-AuNPs探针对凝血酶表现出较好的响应灵敏度,比以前报道的巯基化Apt-AuNPs探针提高了1个多数量级。采用类似的检测原理,Apt44-PolyA-AgNPs也可应用于PDGF-BB检测,室温下孵育10min时,检测限是2.5 nM。该方法仅需要测定银纳米探针在SPR吸收峰处的吸光度变化即可实现蛋白质的定量检测,明显优于经常使用的双波长吸光度比值法。4.基于聚腺苷酸尾化适配体-金纳米荧光探针的荧光检测新方法为了拓宽聚腺苷酸(PolyA)尾化DNA-金纳米粒子复合物的应用并开发环境友好的策略来实现DNA链从金纳米粒子表面的解离,我们首先证实了PolyA-Au/Ag表面具有较强的集聚亲和力,使得PolyA尾化DNA-AuNPs/AgNPs探针可用于非均相生物分析;然后制备了可激活型PolyA尾化、荧光标记适配体-金纳米荧光探针(F-Apt-PolyA-AuNPs),并使用碘离子和硫代硫酸根作为配体取代试剂实现了 PolyA尾化DNA链从金纳米粒子表面的解离。由于金纳米粒子和荧光基团之间的荧光共振能量转移(FRET)被破坏,原来处于猝灭状态("OFF")的金纳米探针被显著激活("ON"),因此荧光信号获得了极大的恢复。根据这一新的发现,将该可激活型金纳米荧光探针应用于蛋白质检测,发展了一种新颖高效的荧光适配体传感分析方法。该方法能够高特异地检测凝血酶,线性检测范围可达两个数量级(0.174nM-17.4nM),检测限为0.089 nM。值得注意的是,与传统的巯基化合物取代试剂(如2-巯基乙醇和二硫苏糖醇)相比,碘离子具有无毒、无臭味、环保、低成本和容易存储等优点。5.基于金纳米荧光复合探针的荧光检测新方法为了提高金纳米粒子表面荧光分子的负载量并开发通用的、环境友好的策略来激活被金纳米粒子猝灭的荧光分子,我们制备了表面修饰有适配体和小分子异硫氰酸荧光素(FITC)的金纳米荧光复合探针(Apt-AuNP-FITC),尝试使用铁氰化钾和碘离子的混合物作为通用的荧光激活(或恢复)试剂来刻蚀溶解体系中的猝灭剂—金纳米粒子,并将该金纳米探针用于蛋白质的荧光检测。研究表明,该金纳米荧光复合探针表面可负载数千个荧光分子,比传统的荧光标记DNA链的负载量提高了 1-2数量级。所用的Apt-AuNP-FITC探针能与疾病标志物凝血酶高效结合,荧光染料FITC仍处于猝灭状态("OFF"),当向检测体系中加入荧光激活试剂后,金纳米粒子猝灭剂可在几分钟内迅速溶解并成功释放、激活金表面高效富聚的FITC染料("ON"),荧光强度与样品中凝血酶的浓度成比例,从而实现了蛋白质的高灵敏检测。该方法的线性检测范围可达到两个多数量级(21.7 pM-3478 pM),检测限为4.7pM。更重要的是,这种简单、高效、通用的刻蚀溶解激活金纳米探针的策略不仅避免了氰化钾、2-巯基乙醇和二硫苏糖醇等有毒或有臭味试剂的使用,而且该试剂环境友好程度高,配制后可长期保存。更多还原

【Abstract】 The development of highly sensitive and specific detection methods for protein biomarker is crucial for the early diagnosis of diseases and clinical treatment.Nucleic acid aptamers have emeraged as promising alternatives to antibodies for achieving the protein detection.Particularly,the combination of aptamer and nanomaterials permits the design of versatile hybrid sensing materials with great target specificity and selectivity and tunable signal outputs.By coupling the molecular recognition properties of aptamers with the physicochemical properties of AuNPs,aptamer-gold nanoparticle(Apt-AuNPs)conjugates provide a powerful biosensing platform for various protein bioassays.Our research work mainly focuses on the design and construction of gold nanoprobes with different structures,and their applications in protein detection.Taking full advantage of the unique optical properties of AuNPs including surface plasmon resonance and fluorescence quenching,and their excellent catalytic properties,we developed some novel colorimetric and fluorescent methods for protein detection with high sensitivity and specificity.1.Enzyme-free amplified colorimetric bioassays based on gold nanoparticle-catalyzed reductive decolorization of azo dyesTraditional amplified colorimetric bioassays relied heavily on the use of natural proteases and artificial enzymes.To solve this problem,we developed a novel,enzyme-free amplified colorimetric bioassay for protein detection,which took advantage of aptamer-functionalized paramagnetic microparticles(PMPs)for target capture,enrichment and separation,and aptamer-conjugated gold nanoparticle(AuNP)-catalyzed reductive decolorization of azo dye—methyl orange(MO)to generate the colorimetric signals.It was demonstrated that this colorimetric bioassay could enable simple,cost-effective,sensitive and specific thrombin detection,which exhibited a linear detection range of 45 pM to 455 pM with a detection limit of 30 pM.Importantly,this novel method did not require the use of any enhancing solutions and enzyme-substrate systems,and thus provide new avenues for constructing enzyme-free bioassays.2.Enzyme-free amplified colorimetric bioassays based on gold nanoparticle-catalyzed reductive decolorization of methylene blue and 4-nitrophenolTo further broaden the applications of the above colorimetric strategy using reductive decolorization reaction catalyzed by nanoparticle,herein we mainly focus on its fundmental principle and the influence factors on detection sensitivity and selection of colored substrates.A theoretical analysis showed that the detection sensitivity was favored by a high binding affinity between analyte and receptor(Kd),a high amplification factor(kcatAt),and a high extinction coefficient(ε)of the used colored substrate.With 4-nitrophenol(4-NP)as the non-dye colored substrate,colorimetric bioassay of thrombin was achieved by the endpoint method with a linear detection range of 182 pM to1090 pM and a detection limit of 91 pM.In particular,when using methylene blue(MB)as the substrate,a more convenient and efficient kinetic-based colorimetric thrombin bioassay was achieved without the steps of the acidification termination and magnetic removal of particles,which exhibited a linear detection range of 68 pM to 455 pM with a detection limit of 10 pM.Importantly,to some extent,such novel colorimetric methods overcome the heavy dependence on the limited enzyme-substrate systems.3.Fabrication of PolyA-tailed aptamer-gold/silver nanoprobes and their applications in colorimetric bioassaysDue to no requirement of thiol modification,PolyA-tailed DNA-gold nanoparticle conjugates are emerging as promising hybrid sensing materials for fabricating convenient and cost-effective bioassays.Herein,based on the colorimetric principle of target-induced cross-linking assembly,we demonstrated that PolyA-based AuNPs and AgNPs conjugates could act as the detection probe for homogeneous protein bioassays.Experimental results showed that these colorimetric bioassays using PolyA-based nanoparticle probes have better detection sensitivity than those using thiolated counterparts.More interestingly,over the traditional absorbance-ratio method of dual wavelength,it was found that AgNPs-based plasmonic protein sensing merely required the measurement of absorbance at only one wavelength for quantitative protein detecion and thus provided a more convenient analytical strategy.4.Activatable PolyA-tailed aptamer-gold nanoprobe for fluorescent turn-on bioassays using iodide-induced ligand displacementDepending on the strong AuNPs(or AgNPs)-PolyA interaction coming from its multipoint attachment nature and collective binding affinity,we demonstrated that PolyA-tailed DNA-AuNPs(or AgNPs)conjugates could serve as detection probe for heterogeneous protein bioassays.Moreover,we designed a new activatable PolyA-tailed,fluorophore-labeled AuNP probe,which could be turned on by destroying the fluorescence resonance energy transfer(FRET)between AuNPs and fluorophores.Interestingly,it was found that iodide and thiosulfate could act as the ligand displacing reagents to effectively detach PolyA-tailed and fluorophore-labeled DNA strands,from AuNP surface via a divide-and-conquer strategy,leading to the fluorescence recovery of AuNP-quenched fluorophores.Based on the unique fluorescence "OFF-to-ON" properties of activatable PolyA-based AuNP probe,we developed a novel and cost-effective fluorescence turn-on aptasensing platform for highly sensitive and specific thrombin detection.The proposed fluorescent bioassay exhibited a wide dynamic range from 0.174 nM to 17.4 nM with a detection limit of 0.089 nM.More importantly,employment of iodide as the displacing reagent offers many outstanding advantages over those thiolated compounds,including non-toxic,non foul-smelling,environmentally-benign,low cost and ease of storage.5.Oxidative dissolution-enabled fluorescent turn-on bioassays based on activatable gold nanoprobeWe prepared a novel AuNP-based fluorescence-activatable probe by co-assembling the aptamers and small fluorescein isothiocyanate(FITC)molecules onto the AuNP surface,which carried thousands of fluorescent reporter molecules,and meanwhile possessed the target recognition ability.Moreover,it was found that the mixture of iodide and hexacyanoferrate(HCF(Ⅲ))could be qualified as the efficient fluorescence-activated reagent toward the fluorescence recovery of AuNP-quenched FITC molecules by oxidative dissolution of AuNP quencher.Based on this new discovery,we developed a novel and cost-effective fluorescence turn-on aptasensing platform for highly sensitive and specific thrombin detection.The proposed fluorescent bioassay exhibited a wide dynamic range from 21.7 pM to 3478 pM with a detection limit of 4.7 pM.More importantly,this efficient,general gold dissolution-enabled fluorescence-activated strategy not only avoids the use of toxic cyanide-based etching solution and foul-smelling thiolated compounds,but also possesses many outstanding advantages,including environmentally-benign,low cost and ease of storage.更多还原