【作者】 陈伟；

【导师】 刘正春；

【作者基本信息】 中南大学 ， 微生物学， 2023， 博士

【摘要】 目的金黄色葡萄球菌（Staphylococcus aureus,SA）感染率递增和耐药菌的广泛传播对全球公共卫生构成了严重威胁,因此及时、快速和准确的检测SA并进行有效的治疗对食品安全、环境监测、临床诊疗和预防耐药菌的传播具有重要的意义。本研究围绕目前SA检测与治疗困境,构建几种基于特异性生物识别元件适配体以及检测信号放大策略的高灵敏SA生物传感器,并且构建一种基于细菌外膜囊泡（Extracellular vesicle,EV）的仿生纳米载药体系用于递送肽核酸（Peptide nucleic acid,PNA）,初步探索PNA对SA的反义治疗效果。方法（1）构建基于新型简易放大策略的荧光适配体SA传感器。生物素修饰的适配体通过碱基配对与4条羧基荧光素（carboxyfluorescein,FAM）修饰的部分互补DNA（Partially complementary DNA,pc DNAs）结合形成杂合序列（生物素-适配体&FAM-pc DNAs）,并通过生物素与链霉亲和素固定在磁性纳米粒子（Magnetic nanoparticles,MNP_S）上形成生物素-适配体&FAM-pc DNAs@链霉亲和素-MNP_S检测探针。当适配体特异性识别并结合SA时,检测探针中的4条FAM-pc DNAs信标探针被替换并释放到上清液中,通过测量被替换的信标探针FAM-pc DNAs的荧光强度（λexc/em=492/520 nm）,则可以量化SA的浓度;（2）构建基于氧化还原生物电容的电化学适配体SA传感器。首先在玻碳电极（Glassy carbon electrode,GCE）表面进行壳聚糖（Chitosan,Chi）阴极电沉积,然后在Chi膜上进行邻苯二酚（Catechol,Cat）阳极接枝,结合检测体系中可扩散的氧化还原双介质1,1’-二茂铁二甲醇和氯化六氨合钌（Ⅲ）(1,1’-ferrocene dimethanol-Hexaammine-ruthenium（Ⅲ）chloride,Fc-Ru³⁺),从而构建Cat-Chi-Fc-Ru³⁺氧化还原生物电容体系。醛基修饰的适配体通过与Chi上的氨基进行席夫碱反应从而被固定在电极表面。当适配体特异性识别并结合SA时,SA会限制双介质中离子的迁移,阻止电极电荷转移,CV检测并记录SA所导致的电流峰值信号的降低;（3）构建基于多重放大策略的高灵敏电化学SA传感器。首先生物素标记的适配体1与链霉亲和素修饰的MNP_S结合形成链霉亲和素-MNP_S@生物素-适配体1捕获探针。生物素-适配体2与4段巯基修饰的互补序列（pc DNAs-SH）结合形成杂交体序列pc DNAs-SH&生物素-适配体2,然后将此杂交体序列通过生物素-链霉亲和素结合在MNP_S颗粒上形成pc DNAs-SH&生物素-适配体2@链霉亲和素-MNP_S检测探针。捕获探针捕获样本中的SA,且捕获的SA能将检测探针中的pc DNAs-SH置换下来,进而采用基于Cat-Chi-Fc-Ru³⁺氧化还原电容信号放大策略的电化学传感器对pc DNAs-SH进行检测,电容电量变化（Q₀-Q）/Q₀作为分析指标;（4）PNA的仿生纳米递送及其对SA的反义治疗初探。通过溶胶-凝胶法制备介孔二氧化硅纳米颗粒（Mesoporous Silicon Nanoparticles,MSN）,采用基于亲和树脂层析方法试剂盒提取SA分泌的EV,通过简单的超声处理（50 W,3min）使EV涂层于MSN上,并对MSN@EV的稳定性,生物相容性（溶血实验）以及内化作用进行评估。MSN通过简单的物理吸附方式封装PNA药物,计算包封率与载药量,并初步探索所构建PNA@MSN@EV仿生纳米载药体系的体外释放效果和体外抗菌效果。结果（1）本研究所构建的基于单个SA置换4条信标探针的新型简易放大策略的荧光适配体SA传感器具有良好的特异性、较高的灵敏度（1.23 cfu/m L）和宽线性范围（1～10⁸ cfu/m L）。回收实验进一步验证了所提出的荧光适配体传感器可以检测血液样本中的SA;（2）本研究所构建的基于Cat-Chi-Fc-Ru³⁺氧化还原生物电容信号放大策略的电化学适配体SA传感器表现出良好的性能,检测速度快（25 min）、线性范围宽（10～10⁸ cfu/m L）、灵敏度高（2cfu/m L）,并且还具有良好的再生性、稳定性和特异性。电化学适配体传感器还可以直接定量检测全血样本中的SA;（3）本研究所构建的结合MNP_S的富集分离系统,单个SA置换4条信标探针和Cat-Chi-Fc-Ru³⁺氧化还原生物电容信号放大策略的电化学SA传感器具有单细胞检测能力（<1 cfu/m L）,较强的特异性,且其稳定性好,可重复使用。回收实验进一步验证所构建的电化学传感器可以用于血液样本的检测;（4）采用溶胶-凝胶法所制备的MSN,粒径均匀（100 nm）,且孔径分明（六角形）,用于负载PNA具有较好的包封率（62.90%）和载药量（7.74%）。采用亲和树脂层析法所提取的EV浓度约为1.74 mg/m L。通过简单的超声处理（50 W,3 min）,能使EV很好的涂层于MSN,且其稳定性较好,血液中相容性好。体外抗菌试验初步显示,PNA@MSN@EV对SA表现出增强的抗菌活性,在等效PNA浓度（8μM）下显现较强的杀菌活性,明显高于游离的PNA的杀菌活性。结论（1）通过新型简易放大策略能显著提高技术成熟的荧光适配体传感器的灵敏度,以便其能作为病原体早期检测的有力工具,并且为临床探索病原菌诊断方法提供新的思路。（2）基于Cat-Chi-Fc-Ru³⁺氧化还原生物电容信号放大策略的电化学适配体传感器检测性能好,检测速度快,可实现及时反应和实时监测,展示其在POCT系统中以及整合在便携式、可植入的和/或可穿戴的设备中的潜在应用。（3）基于多重放大策略的高灵敏电化学传感器可以实现单细胞水平检测,稳定性好,可重复使用,能去除样本复杂环境对其干扰,使其能广泛适用于食品安全、环境监测以及临床诊断。（4）EV涂层MSN的仿生纳米载药体系,提供一种提高PNA治疗细菌感染功效的新策略,该技术有望扩展到传统上效果较差的抗生素或易产生抗性的抗生素的递送。图66幅,表19个,参考文献289篇更多还原

【Abstract】 Objective:The increasing prevalence of S.aureus infections and the widespread of drug-resistant bacteria pose a serious threat to global public health.Therefore,timely,rapid and accurate detection of S.aureus and effective treatment are important for food safety,environmental monitoring,clinical treatment and prevention of the spread of drug-resistant bacteria.This study focuses on the current dilemma of S.aureus detection and treatment by constructing several highly sensitive SA biosensors based on specific biorecognition element aptamers and detection signal amplification strategies,and constructing a biomimetic nanodelivery based on bacterial outer membrane vesicles for the delivery of PNA and to explore the antisense therapeutic effect of PNA on S.aureus.Methods:（1）Construction of a fluorescent aptasensor for S.aureus based on a novel simple amplification strategy.The biotin-modified aptamer binder to four FAM-modified pc DNAs via base-pairing to form heterodimeric sequence（biotin-aptamer&FAM-pc DNAs）and was immobilized on streptavidin modified MNP_S by strong affinity interactions between biotin and streptavidin to form biotin-aptamers&FAM-pc DNAs@streptavidin-MNP_S detection probes.When the aptamer specifically recognized and binder to S.aureus,the four FAM-pc DNAs beacon probes in the biotin-aptamers&FAM-pc DNAs@streptavidin-MNP_S detection probes were replaced and released into the supernatant,and the concentration of S.aureus can be quantified by measuring the fluorescence intensity of the replaced beacon probes FAM-pc DNAs（λexc/em=492/520 nm）;（2）Construction of an electrochemical aptasensor for S.aureus based on redox biocapacitance.Chi cathodic electrodeposition was first performed on the surface of GCE,followed by Cat anodic grafting on the Chi film.The redox capacitor amplification system was constructed using Cat-Chi film and Fc-Ru³⁺double mediators.The aldehyde-modified aptamer was immobilized on the electrode surface by the Schiff base reaction with the amino group of Chi.When the aptamer specifically recognized and binder S.aureus,the S.aureus limited the migration of ions in the double dielectric and blocked charge transfer from the electrode.CV detects and records the reduction in peak current signal caused by S.aureus;（3）Construction of a highly sensitive electrochemical sensor for S.aureus based on multiple amplification strategy.Biotin-aptamer 1 binder to streptavidin-modified MNP_S to form the streptavidin-MNP_S@biotin-aptamer 1 capture probe.Biotin-aptamer 2 binder to four pc DNAs-SH to form the hybrid sequence pc DNAs-SH&biotin-aptamer 2,which was then bound to the MNP_S to form pc DNAs-SH&biotin-aptamer 2@streptavidin-MNP_S detection probes through the strong affinity interactions between biotin and streptavidin.The S.aureus which was captured by the streptavidin-MNP_S@biotin-aptamer 1 capture probe in the sample could displaces pc DNAs-SH in the pc DNAs-SH&biotin-adaptor 2@streptavidin-MNP_Sdetection probe.Then we could detect pc DNAs-SH using an electrochemical sensor based on the Cat-Chi-Fc-Ru³⁺redox capacitor amplification system,with capacitance change（Q₀-Q）/Q₀ as the analytical index;（4）Biomimetic bionanodelivery of PNA and its antisense treatment of S.aureus.MSN were first prepared by sol-gel method.The EV secreted by S.aureus was extracted using an affinity resin-based chromatography kit and coated on the MSN by simple sonication（50 W,3 min）.The stability,the biocompatibility（hemolysis assay）and internalization of the MSN@EV were evaluated.The PNA drug was encapsulated by simple physical adsorption of MSN,and the encapsulation rate and drug loading capacity were calculated.The in vitro release and in vitro antimicrobial effects of the constructed PNA@MSN@EV biomimetic nanodelivery system was initially explored.Results:（1）The proposed fluorometric aptasensor based on a novel simple amplification strategy of replacing four beacon probes with one S.aureus has good specificity,ultra-high sensitivity（1.23 cfu/m L）and wide linear range（1～10⁸ cfu/m L）.The recovery test further demonstrated that the proposed fluorometric aptasensor can detect S.aureus in spiked blood samples;（2）The electrochemical aptasensor based on Cat-Chi-Fc-Ru³⁺redox capacitance signal amplification strategy constructed in this study showed good performance with fast detection（25 min）,wide linear range（10～10⁸ cfu/m L）,high sensitivity（2 cfu/m L）,and also good regeneration,stability and specificity.The electrochemical aptasensor can also directly quantify S.aureus in whole blood samples;（3）The proposed electrochemical sensor based on MNP_S enrichment and separation system,an S.aureus replacement four beacon probes and Cat-Chi-Fc-Ru³⁺redox capacitor amplification system showed single cell detection capability（<1 cfu/m L）,strong specificity and it is stable and reusable.The recovery experiments further demonstrated that the constructed electrochemical sensor could be used for the detection of blood samples;（4）The MSN prepared by the sol-gel method has a uniform particle size（100 nm）and a clear pore size（hexagonal）,which could be used to load PNA with good encapsulation efficiency（62.90%）and drug loading（7.74%）.The concentration of EVs extracted by affinity resin chromatography was about 1.74 mg/m L.All characterization showed that EV could be well coated on MSN through simple ultrasonic treatment（50 W,3 min）,and its stability and blood compatibility was good.In vitro antibacterial test showed that PNA@MSN@EV exhibited enhanced antibacterial activity against S.aureus and revealed strong bactericidal activity at equivalent PNA concentration（8μM）,which was significantly higher than that of free PNA.Conclusion:（1）The novel and simple amplification strategy can significantly improve the sensitivity of existing fluorescent aptasensor so that it can be used as a powerful tool for early detection of pathogens and provide new ideas for clinical exploration of pathogenic bacteria diagnostic methods.（2）The Cat-Chi-Fc-Ru³⁺redox capacitor-amplification based electrochemical aptasensor with good detection performance and fast detection speed for timely response and real-time monitoring,demonstrating its potential application in POCT systems and integration in portable,implantable and/or wearable devices.（3）The highly sensitive electrochemical sensor based on multiple amplification strategy enables single cell level monitoring and is stable and reusable,removing interference from the complex environment of the sample,making it widely applicable to food safety,environmental monitoring and clinical diagnosis.（4）The EV-coated MSN biomimetic nanodelivery system provides a novel strategy to enhance PNA for the treatment of bacterial infections,and the technology is expected to the delivery of antibiotics that are traditionally less effective or susceptible to resistance.更多还原