【作者】 薛亚东；

【导师】 鞠熀先；

【作者基本信息】 南京大学 ， 分析化学， 2010， 博士

【摘要】 细胞是生命体的基本单位,对细胞结构和功能的深入研究是人类征服疾病的关键。细胞膜上的聚糖分子通过与蛋白质和脂类缀合形成糖缀合物而影响蛋白和脂类的功能,介导和调控细胞黏附行为。更重要的是,糖缀合物结构和功能可以主导恶性肿瘤的许多生长特性及侵袭转移能力。因此,原位特异性检测细胞表面活性聚糖,对于揭示糖基化在细胞生理活动中的作用,开发癌症治疗的新方法具有重要的意义。传统研究方法一般是以大量细胞为研究对象,用平均结果反映单个细胞的化学信息。然而,由于生物组织中各种化学成分分布及细胞本身的个体差异性,大量细胞分析常常得出不准确甚至错误的结论。因此直接分析单个细胞的化学成分和生理状态十分重要。本论文结合纳米技术、电化学技术、和化学生物学实现了对细胞表面聚糖的原位电化学检测,研究了药物对细胞表面聚糖表达的影响；并进一步结合扫描电化学显微镜技术和微米／纳米加工技术在单细胞水平上对细胞表面聚糖和凋亡行为进行了图像学监测。本论文主要包括以下四个部分：1.凝集素功能化碳纳米管与细胞表面聚糖动态表达的无标记监测本工作以聚二烯丙基二甲基氯化铵作为连接剂,将凝集素非共价地组装在羧基化的多壁碳纳米管(MWCNTs)表面,构建了一种凝集素功能化的MWCNTs (lectin-MWCNTs)。用扫描电子显微镜、原子力显微镜和接触角分析仪表征了所得新型纳米材料的均一性和生物相容性。该材料同时具有凝集素对细胞表面糖基的特异性识别能力和MWCNTs独特的电子和机械性能。通过在玻碳电极表面构建一层lectin-MWCNTs界面,我们首先发展了一种无标记的电化学细胞传感方法,它对K562细胞检测的线性响应范围为1.0×104至1.0×107cells mL-1。这种细胞传感方法还能用来监测药物处理后,K562细胞表面聚糖表达的动态变化。通过构建lectin-MWCNTs阵列,进一步发展了一种简便的高通量活细胞表面聚糖动态表达分析的光学方法,具有令人满意的速度和低成本。凝集素对碳纳米管的非共价功能化有望用于细胞表面糖基相关的细胞生物学研究。2.电化学凝集素探针的构建与细胞均相传感和表面糖基原位检测通过碳二酰亚胺偶合反应,共价构建了二茂铁-伴刀豆球蛋白A (Fc-ConA)电化学凝集素探针。该探针同时具有凝集素对糖基的特异性识别能力和二茂铁基团良好的电化学性能。利用得到的Fc-ConA探针设计了一种简便的均相细胞传感策略,用以检测白血病K562细胞。在细胞浓度1×104至1×107 cells mL-1范围内线性相关性良好,并且在3σ时计算其检测限为3×103 cells mL-1。由于ConA对细胞表面甘露糖基的特异性识别作用,该凝集素探针还能用以方便估算细胞表面的甘露糖基数目,并测得平均每个K562细胞表面的甘露糖基数约为3×1010个。该策略可进一步用于监测药物作用下细胞表面糖基数目的变化,且得到的细胞表面糖基变化跟传统的流式细胞仪检测结果相吻合。这一策略为细胞的均相灵敏传感和监测细胞对药物应答时表面糖基的表达水平提供了一个有力的平台。3.以氧气为内源性指示剂实时监测细胞纳米级高度变化与活性本工作以氧气作为内源性指示剂,发展了一种SECM实时监测单细胞纳米级高度变化的方法,并间接实现了细胞活性的评估。首先,通过将穿有铂丝的石英毛细管放入硼硅玻璃管中拉制,得到纳米铂盘电极,该电极具有均匀的锥形尖端,最小半径可达5 nm。以铂纳米电极作为SECM探针,在恒高度模式下可以清晰地对磷酸盐生理缓冲液中单个BGC细胞周围的氧气浓度变化进行电化学成像。考虑到引起该浓度变化的原因包括多种因素,本论文设计了一种" step-approaching "策略将总电流变化中归属于反馈电流的部分分离出来,并将此方法应用于研究单个细胞长期的高度变化。SECM测量结果显示BGC细胞暴露于25℃磷酸盐生理缓冲液中2h内的高度变化为55-365 nm(n=8)。经AnnexinⅤ-FITC凋亡试剂盒染色证实,细胞凋亡是引起细胞高度变化的主要原因。因此,所提出的SECM方法可以间接对细胞的活性进行实时监测,以紫杉醇作为模型药物的药敏试验成功验证了本方法监测细胞活性的实用性。本文提供了一种在近生理环境无外源性指示剂下监测活细胞纳米级高度变化的策略,并可用于细胞活性的评估。4.微图案化的单个贴壁细胞表面聚糖的原位电化学成像以人胃癌BGC-823细胞为模型,发展了一种基于SECM的对单个贴壁细胞表面四种聚糖进行原位成像的方法。首先,通过将这些贴壁细胞在聚二甲基硅氧烷(PDMS)薄膜的微孔里图案化,精确地控制局部表面的相互作用,然后用辣根过氧化物酶(HRP)标记的凝集素对细胞表面相应的糖基进行特异性的识别。SECM探针是直径2微米的铂盘电极,通过激光拉制的方法制备而成,具有良好的电化学性能。基于HRP催化H202氧化甲醇基二茂铁(FMA)生成FMA+这一酶催化反应,通过测量SECM针尖上FMA+的还原电流实现了细胞表面糖基的检测。因此,SECM方法无需将贴壁细胞从培养皿底部消化脱落,便可在单细胞水平上对细胞表面的聚糖进行成像。在优化条件下,SECM得到的四种糖基的表达水平显示出统计学上的差异,并且与流式细胞仪的结果相一致。该方法为单细胞水平原位研究活细胞表面糖基化水平提供了一个有力的平台,并有望用于细胞表面糖基表达相关的生物学研究。更多还原

【Abstract】 Cells are the elements of all living organisms, thus deep research into cellular structure and function is the key for the conquest of diseases. The glycan molecules on the membrane surface can dictate proteins and lipid functions through conjugation, mediate and control cellular adhesion behavior and signal transduction. Many of the growth characteristics of malignant tumor cells are manifested by structural and functional changes in the glycans. Thus, specific detection of cell surface glycans is important for unraveling the role of glycosylation change in cellular adhesion and migration, formation and metathesis of malignant tumors, and developing new methods for the treatment of cancer. Traditional research methods only offer the information of the average expression level of a cell population, thus, possibly enshroud the heterogeneity of single cells, which might be indicative of disease in the early stage, leading to the wrong result. The analysis of cellular composition and physiological behavior at the single-cell level is mostly desired for the accurate early warning of disease. In this dissertation, by integrating nanotechnology, surface science and chemical biology, in situ electrochemical detection of cell glycans has been realized. The effect of drugs on the cell surface glycan expression has been investigated. Furthermore, in situ electrochemical imaging method for monitoring cell membrane glycan and cell viability at the single-cell level has also been proposed by combining scanning electrochemical microscopy with the micromachining technique. This dissertation includes the following four parts:1. Noncovalent functionalization of carbon nanotubes with lectin for label-free dynamic monitoring of cell-surface glycan expressionA kind of concanavalin A functionalized multi-walled carbon nanotubes (ConA-MWCNT) was constructed by noncovalent assembly of ConA on carboxylated MWCNT with poly(diallyldimethylammonium) as a linker. The novel nanomaterial was characterized with scanning electron microscopy and atomic force microscopy. It incorporated both the specific recognition ability of lectin for cell-surface mannosyl groups and the unique electronic and mechanical properties of MWCNT. An electrochemical label-free method for cytosensing was proposed by constructing a ConA-MWCNT interface on a glassy carbon electrode, which showed a linear response to K562 cells ranging from 1×104 to 1×107 cells mL-1. The ConA-MWCNT interface could be further used for monitoring of dynamic variation of glycan expression on K562 cells in response to drug. A facile and high-throughput optical method for the analysis of dynamic glycan expression on living cells was also developed by constructing an array of ConA-MWCNTs spots on a glass slide. This method showed acceptable rapidity and low cost. The noncovalent functionalization of MWCNTs with lectins could be potentially applied in cell biological study based on cell-surface glycan expression.2. A simple electrochemical lectin-probe for in situ homogeneous cytosensing and facile evaluation of cell surface glycanThis work constructed a novel electrochemical lectin-probe, ferrocene-concanavalin A (Fc-ConA), for in situ monitoring of cell surface glycan by incorporating the specific recognition ability of lectin to glycan and favorable electrochemical property of ferrocenyl group. The covalent conjugation of ConA with ferrocenyl group was achieved by a carbodiimide coupling reaction and proved with UV-vis absorption spectroscopy and infrared spectroscopy. Cyclic voltammetric behavior of Fc-ConA at glassy carbon electrode demonstrated a reversible diffusion-controlled process. A facile homogeneous cytosensing strategy was then developed by using Fc-ConA probe for detection of K562 cells. The suspending cells specifically captured Fc-ConA via membrane mannosyl groups and decreased the concentration of free Fc-ConA, producing a response correlative with cell number and the content of cell surface glycan. A wide linear response to cells ranging from 1×104 to 1×107 cells mL-1 with a calculated detection limit of 3000 cells mL-1 was obtained. The lectin-probe could be conveniently used to in situ evaluate cell surface glycan. The average number of mannose moieties on single living K562 cell was detected to be 3.0×1010, while this value increased by 81% on drug-treated cells. These results agreed with those from flow cytometric detection. This strategy presented a promising platform for homogeneous sensitive cytosensing and facile monitoring of carbohydrate expression on living cells in response to drugs.3. Real-time monitoring of cell viability by its nanoscale height change with oxygen as endogenous indicatorThis work developed a novel method for real-time monitoring of nanoscale height change of single cell by scanning electrochemical microscopy (SECM) with oxygen as an endogenous indicator. The Pt nanodisk electrode was firstly prepared as a SECM probe by assembling Pt wire-inserted quartz capillary in a glass tube, which produced a taper tip with controllable radius down to 5 nm. The early apoptosis of adherent BGC-823 human gastric carcinoma (BGC) cell could sensitively be monitored by the decrease of oxygen reduction current during the constant-height scan around single BGC cell, which was characterized with AnnexinⅤ-FITC apoptosis detection kit coupled with a confocal fluorescent microscope. A step-approaching method was proposed to separate the negative feedback current from the total reduction current above single living cell for testing the morphological effect of cell. The height change of BGC cells exposed to 25℃PBS in 2 h was estimated to be in the range of 55 to 365 nm (n=8). The practicability of the designed strategy was demonstrated by treating the model cells with paclitaxel and real-time SECM monitoring. This work provided a powerful protocol to monitor the nanoscale height change of living cells in physiological environment for real-time cell viability evaluation.4. In situ electrochemical imaging of membrane glycan expression on micropatterned adherent single cellsA scanning electrochemical microscopic (SECM) method for in situ imaging of four types of membrane glycan motifs on single adherent cells was proposed using BGC-823 human gastric carcinoma (BGC) cells as the model. These adherent cells were firstly micropatterned in the microwell of poly(dimethylsiloxane) membrane for precisely controlling the localized surface interaction, and the membrane glycans were then specifically recognized with corresponding lectins labeled with horseradish peroxidase (HRP). Based on the enzymatic oxidization of ferrocenylmethanol (FMA) by H2O2 to yield FMA+, the glycan expression level was detected by the reduction current of FMA+at the SECM tip. The cell-surface glycans could thus be in situ imaged by SECM at a single-cell level without peeling the cells from culture dish. Under the optimized conditions, four types of membrane glycan motifs showed statistically distinguishable expression levels. The SECM results for different glycan motifs on adherent single cells were consistent with those estimated by flow cytometric assay. This work provides a reliable approach for in situ evaluation of the characteristic glycopattern of single living cells, and can be applied in cell biologic study based on cell surface carbohydrate expression.更多还原