植物血凝素与金色葡萄球菌肠毒素刺激人外周血单个核细胞的比较研究

比较了植物血凝素(PHA)与金色葡萄球菌肠毒素(SEA)对外周血单个核细胞(PBMCs)的刺激效果。取健康人外周静脉血,分离得到PBMCs,分别加入PHA及SEA进行刺激并孵育12 h,利用原子力显微镜和倒置荧光显微镜观察细胞形态变化,利用量子点联合共聚焦显微镜观察活化后细胞膜表面CD3、CD69的分布情况,利用流式细胞术检测淋巴细胞活化早期细胞分化抗原CD69分子表达的变化情况。结果显示,丝裂原PHA刺激的淋巴细胞大多成群聚集,而超抗原SEA刺激的淋巴细胞大多呈分散状,且二者形态有明显差异;两组活化后淋巴细胞的体积均大于静息组,且活化过程中发生极化作用,迁移淋巴细胞,形成膜突起;丝裂原和超抗原刺激淋巴细胞12 h后,使淋巴细胞表达CD69抗原分子,但在量表达上存在差异性(PHA:39.5%±8.7%;SEA:8.3%±1.8%),抗原分子CD3和CD69在膜表面呈不均匀分布,且SEA活化后的T淋巴细胞表面受体CD3和CD69分子在空间上形成了微结构域;外周单个核细胞中其它非T细胞在丝裂原或者超抗原的刺激下也能表达活化抗原分子CD69。     

基于原子力显微镜的人外周血CD8+T细胞形貌观察与力谱分析

体外分离人外周血CD8+T细胞,用植物凝结素(PHA)刺激活化,利用原子力显微镜(AFM)观察CD8+T细胞的形貌,并结合针尖修饰技术对力谱进行分析,确定了CD8抗原分子的分布.结果表明,与静息的CD8+T细胞相比,活化后的CD8+T细胞直径和高度增大,细胞表面变得更粗糙;CD8抗原-抗体的相互作用力大约是非特异性黏附力的5倍,活化后的CD8+T细胞上特异性黏附力(即CD8抗原分子位置)分布不均匀,其表面的CD8抗原分子分布以纳米团簇分布为主,CD8分子聚集更为明显,CD8+T细胞上CD8抗原-抗体相互作用力不随着活化而发生明显变化,说明CD8抗原-抗体之间具有高选择性.原子力显微镜为特异性T细胞的抗原识别和活化研究提供了一种新手段,能够使T细胞抗原识别和活化的机制得到更好地阐明.     

化学力显微镜对ω-巯基正十一胺自组装单分子膜的力滴定研究

采用化学力显微术（ＣＦＭ）研究了一种长链氨基自组装单分子膜的力滴定性质,考察了表面基团性质不同的力探针对力滴定的影响,滴定了金包覆的Ｓｉ３Ｎ经－ＯＨ和－ＮＨ２基功能化的金样品之间的粘滞力,得到了作为淀粉 ＰＨ值函数的粘滞力曲线（力滴定曲线）,结果表明,－ＮＨ２和－ＯＨ基功能化的表面ｐＫ１／２分别为８．４和９．０。     

原子力显微镜研究聚丙烯酸的单链力学性质聚丙烯酸的单分子力谱

自从1986年发明原子力显微镜(AFM)以来,AFM已经发展成为应用最为广泛的扫描探针显微镜[1],它给材料科学家、化学家和生物学家提供了一个极为便利的研究手段.目前,原子力显微镜的空间分辨率已经达到原子尺度,同时又具有非常高的力的敏感性,可以探测10 pN的力,这就为研究单分子的性质提供了可能性[2,3].     

非支撑磷脂双层膜制备及温度对其力学性质的影响

结合聚苯乙烯球刻蚀和微机电系统技术加工氮化硅纳米多孔膜, 并在其上用囊泡法制备非支撑磷脂双层膜, 通过温控原子力显微术(AFM)的成像模式和力曲线模式对非支撑磷脂双层膜的形貌和力学性质进行研究. 实验结果表明, 该方法制备的非支撑磷脂双层膜具有流动性, 能进行自我修复, 该特点有利于提供足够的非支撑磷脂双层膜区域用于其性质研究; 非支撑磷脂双层膜的膜破力和粘滞力均随着温度的升高而减小, 即膜的机械稳定性随着温度的升高而降低. 非支撑磷脂双层膜膜破力小于支撑磷脂双层膜的膜破力, 并且非支撑磷脂双层膜粘滞力随温度的变化趋势与支撑磷脂双层膜的变化趋势相反.     

原子力显微镜在聚烯烃研究中的应用

聚烯烃材料因其卓越的性能和较低的价格广泛应用于工农业、医疗卫生、军事、日常生活等领域。为了拓展聚烯烃材料的应用范围,表面功能化聚烯烃、聚烯烃与其他材料的共混物以及聚烯烃/无机纳米复合材料等得到了发展,并成为该领域的研究热点。原子力显微镜(AFM)是利用一个极为尖锐的针尖与样品间的相互作用力进行材料表面的形貌、物理和化学信息探测的一种技术,它在上述聚烯烃研究中发挥着重要作用。表面粗糙度是聚烯烃材料表面功能化研究中的重要参数之一,AFM技术则能够给出准确的表面粗糙度信息。由于AFM技术能够直观地观察各组分的混合状态及相分离情况,因此AFM技术成为聚烯烃与其他材料共混研究的重要手段之一。此外,AFM是一种非常有效的微纳米结构形貌的表征方法,在聚烯烃的结晶研究方面也得到了应用。本文简单介绍了AFM表面形貌观测的工作原理和工作模式,主要阐述AFM在聚烯烃材料研究中的三个主要应用:材料表面粗糙度、共混相分离以及结晶研究。     

利用原子力显微镜探测人正常与慢性淋巴白血病外周血B淋巴细胞

采用Ficoll密度梯度离心法得到人外周血单个核细胞(PBMC),并结合磁珠分选的方法进一步纯化得到正常B淋巴细胞,探索了正常和肿瘤B淋巴细胞之间的差异。通过应用具有高分辨率的原子力显微镜(AFM)对正常人和慢性淋巴白血病人外周血B淋巴细胞进行成像,并对这两种B淋巴细胞的高度、直径、体积及膜表面的颗粒平均高度、平均粗糙度和颗粒分布进行测量,对比观察两组细胞膜表面宏观和纳米结构的变化。结果表明,慢性淋巴白血病B淋巴细胞比正常的B淋巴细胞高大,细胞膜表面颗粒更大且细胞膜粗糙。此外,对这两组淋巴细胞进行了机械性质方面的测量和统计,结果发现慢性淋巴白血病B淋巴细胞粘附力(524.1±160.0)pN比正常B淋巴细胞粘附力(1091±260)pN约小1倍,且癌变的B淋巴细胞硬度明显比正常的小。当正常细胞癌变时,细胞的形貌、超微结构及骨架会发生一定的改变。实验证明应用AFM可在形态学和机械性质上明显区别正常和慢性淋巴白血病B淋巴细胞,为临床诊断慢性淋巴白血病提供新的技术手段。     

原子力显微镜对中性粒细胞与K562细胞超微结构及机械性能的探测

采用原子力显微镜在纳米尺度下对正常中性粒细胞与白血病细胞株K562细胞的表面形貌及细胞的硬度、粘附力进行定性定量分析.结果表明,相比正常中性粒细胞的平均粗糙度(Ra=5.31±1.52 nm),K562细胞的超微结构更为复杂,细胞表面平均粗糙度显著升高(Ra=26.54±8.01 nm).此外,细胞的生物机械特性也有显著差别:中性粒细胞的硬度为9.5±1.3 kPa,AFM针尖与中性粒细胞的非特异性粘附力为135±23.4 pN;K562细胞的硬度为3.0±0.8 kPa,AFM针尖与K562细胞的非特异性粘附力为95±15.6 pN.AFM在单细胞水平上的探测表明,中性粒细胞和K562细胞的超微结构和机械特性均有明显差异.通过对细胞表面超微结构和力学特性的探测可以诊断慢性粒细胞白血病,原子力显微镜有望成为临床肿瘤诊断的工具.     

表面温敏聚合物刷的原子力显微镜和石英晶体微天平研究

通过表面引发的原子转移自由基聚合在硅片表面制备了聚（N-异丙基丙烯酰胺）（PNIPAAm）聚合物刷。用原子力显微镜（AFM）分别研究了PNIPAAm的接枝动力学、温度和溶剂性质对厚度的影响以及PNIPAAm链与原子力针尖间的粘附力。结果表明,PNIPAAm链在硅片表面的生长具有很好的可控性。常温下厚度为33nm的PNIPAAm膜在水溶液中的增加到82.4nm;而在甲醇/水（v/v,1:1）溶液中,PNIPAAm分子链处于坍塌收缩状态,厚度降低为45nm;在55℃下干燥所得厚度则仅为22nm。力-距离测量结果表明,在溶液中,PNIPAAm链与原子力针尖之间的粘附力远小于在干态下的粘附力。用石英晶体微天平（QCM-D）对PNIPAAm的可逆相转变进行了研究,结果表明PNIPAAm分子链随温度变化的构象转变是发生在30-34℃之间的连续过程。     

AFM单分子力谱在真实材料及生物体系中的应用——挑战与机遇并存

基于原子力显微镜技术（AFM）的单分子力谱是研究分子间分子内相互作用的有效手段．为了简化样品体系及数据的解析,真实的生物或材料体系通常被简化,其中的目标分子被提取并桥连于AFM的针尖与固体基片之间进行研究,这是认识真实体系的有效途径．随着技术的不断进步（包括样品固定方法的改进）,使得直接研究真实生物及材料体系中的各种弱相互作用成为可能,此种条件下获得的信息对相关生命过程的调控及高性能材料的设计更具指导意义．本文概述了近几年基于AFM力谱技术在活体细胞以及高分子材料领域的研究进展,分析了存在的主要问题,并对相关领域的未来进行了展望．     

Studies of photosensitive alignment layer based on ladder‐like polysilsequioxane liquid‐crystal devices using atomic force microscopy/force curve

Atomic force microscopy (AFM)/force curve measurements were used to study the photochemical process of UV‐treated (0, 10, 20, 30 and 60 min) organic thin films that were prepared from azobenzene and cinnamate side‐chain co‐grafted ladder‐like polysilsequioxanes (LPS). The morphological data of the thin films describe the changing process on the surface of the thin film. The statistical results of the adhesion force of the thin films further demonstrate the intermolecular characteristics of the thin films. A photosensitive thin film after UV exposure for 20 min would be a better material with a preferred orientation that can be used to make liquid‐crystal devices. Copyright © 2003 John Wiley & Sons, Ltd.     

运用功能化修饰的碳纳米管针尖测量配体-受体的作用力

电弧法自制碳纳米管原子力显微镜针尖,对其末端进行功能化修饰,然后测量配体-受体之间的作用力。运用没有功能化修饰的碳纳米管针尖与修饰有亲和素分子的基底进行接触测量时,没有粘滞力出现;而运用末端修饰生物素分子的碳纳米管针尖测量时,有粘滞力产生。功能化的碳纳米管针尖直接测得的粘滞力均大约200pN,此值符合一对配体生物素和受体亲和素之间的作用力。这一结果很难用传统的针尖获得,功能化修饰的碳纳米管针尖能够克服传统针尖在力测量中的局限,在生物学和化学领域有着广泛的应用前景。     

Adhesion forces between hybrid colloidal particles and concanavalin A

Hybrid particles of poly(methyl methacrylate) and carboxymethylcellulose, PMMA/CMC, were attached to atomic force microscopy cantilevers and probed against concanavalin A (ConA) films formed either on Si wafers or on CMC substrate. Regardless of the substrate, the approach curves showed different inclinations, indicating that the probe first touches a soft surface and then a hard substrate. The distance corresponding to the soft layer was estimated as 20 +/- 10 nm and was attributed to the CMC layers attached to the hybrid particles surfaces. Probing PMMA/CMC particles against ConA adsorbed onto Si wafers yielded retract curves with a sawlike pattern. The average range of adhesion forces (maximum pull-off distance) and mean adhesion force were estimated as 100 +/- 40 nm and -11 +/- 7 nN, respectively, evidencing multiple adhesions between CMC sugar residues and ConA. However, upon probing against ConA adsorbed onto CMC substrates, the mean pull-off distance and mean adhesion force were reduced to 37 +/- 18 nm and -3 +/- 1 nN, respectively, indicating that the ConA molecules immobilized onto CMC films are less available to interact with the hybrid particle than the ConA molecules adsorbed onto Si wafers. Another set of experiments, where PMMA/CMC particle probed against ConA-covered Si wafers in the presence of mannose, showed that the addition of mannose led to a considerable decrease in the mean adhesion force from -11 +/- 7 to -3 +/- 1 nN. Two hypotheses have been considered to explain the effect caused by mannose addition. The first suggested the desorption of ConA from the substrate so that the hybrid particle would probe bare Si wafer (weak adhesion). The second proposed the adsorption of mannose onto the ConA layer so that mannose layer would probe against another mannose layer, leading to low adhesion forces. In situ ellipsometry and capillary electrophoresis have been applied to check the hypotheses.     

The importance of force in microbial cell adhesion

Microbes have evolved sophisticated strategies to colonize biotic and abiotic surfaces. Forces play a central role in microbial cell adhesion processes, yet until recently these were not accessible to study at the molecular scale. Unlike traditional assays, atomic force microscopy (AFM) is capable to study forces in single cell surface molecules and appendages, in their biologically relevant conformation and environment. Recent AFM investigations have demonstrated that bacterial pili exhibit a variety of mechanical responses upon contact with surfaces and that cell surface adhesion proteins behave as force-sensitive switches, two phenomena that play critical roles in cell adhesion and biofilm formation. AFM has also enabled to assess the efficiency of sugars, peptides, and antibodies in blocking cell adhesion, opening up new avenues for the development of antiadhesion therapies against pathogens.     

An atomic force microscopic investigation of electro-sensitive polymer surface

An electro-sensitive poly(2-acrylamide-2-methylpropane sulfonic acid) (PAMPS) film was fabricated by surface-initiated atom transfer radical polymerization (ATRP) method on silicon substrate. Atomic force microscopy (AFM) experiments in contact mode show that friction force and the adhesion force between the AFM tip and the film may change regularly with the alteration of the applied negative bias voltage between them, indicating that the microscopic wettability of the film can be adjusted by external electric field. On the other hand, the AFM experiments in tapping mode reveal that the film may take corresponding phase change under the electric field. These effects were considered to result from the conformational overturn of the sulfonic groups and the adjacent alkyl chains in the electric field.     

Adsorption of ions onto polymer surfaces and its influence on zeta potential and adhesion phenomena

 The adhesion behavior that governs many technologically and biologically relevant polymer properties can be investigated by zeta potential measurements with varied electrolyte concentration or pH. In a previous work [1] it was found that the difference of the adsorption free energies of Cl^- and K⁺ ions correlates with the adhesion force caused by van der Waals interactions, and that the decrease of adhesion strength by adsorption layers can be elucidated by zeta potential measurements. In order to confirm these interrelations, zeta potential measurements were combined with atomic force microscopy (AFM) measurements. Force–distance curves between poly(ether ether ketone) and fluorpolymers, respectively, and the Si₃N₄ tip of the AFM device in different electrolyte solutions were measured and analysed. The adsorption free energy of anions calculated from the Stern model correlates with their ability to prevent the adhesion between the polymer surface and the Si₃N₄ tip of the AFM device. These results demonstrate the influence of adsorption phenomena on the adhesion behavior of solids. The results obtained by AFM confirm the thesis that the electrical double layer of solid polymers in electrolyte solutions is governed by ion adsorption probably due to van der Waals interactions and that therefore van der Waals forces can be detected by zeta potential measurements. Received: 18 November 1997 Accepted: 19 January 1998     

利用AFM和SNOM对淋巴细胞膜表面超微结构及其光学性质的初步研究

利用原子力显微镜(Atomic Force Microscopy，AFM)对淋巴细胞表面形貌进行了形态学的初步研究，观察到了其膜表面其他显微技术所不能发现的超微结构．同时也运用扫描近场光学显微镜(Scanning Near field Optical Microscopy，SNOM)对淋巴细胞进行成像，观察了其对光的透射、吸收等光学性质，并对两种成像方法进行了比较．研究发现：淋巴细胞膜表面凹凸不平，分布着大量直径几十到几百纳米不等的小颗粒；淋巴细胞中央部位有自发荧光现象．结果表明，AFM和SNOM可作为进一步探讨淋巴细胞的结构与功能关系的有力工具．     

Characterizing NF and RO membrane surface heterogeneity using chemical force microscopy

Chemical force microscopy (CFM) was used to characterize the chemical heterogeneity of two commercially available nanofiltration and reverse osmosis membranes. CFM probes were modified with three different terminal functionalities: methyl (CH₃), carboxyl (COOH), and hydroxyl (OH). Chemically distinct information about the membrane surfaces was deduced based on differences in adhesion between the CFM probes and the membrane surfaces using both traditional atomic force microscopy (AFM) force measurements and spatially resolved friction images. Contact angle titration and streaming potential measurements provided general information about surface chemistry and potential, which largely complemented the CFM analyses, but could not match the accuracy of CFM on the atomic level. Using CFM it was found that both membranes were characterized as chemically heterogeneous. Specifically, membrane chemical heterogeneity became more significant as the scan size approached colloidal or micron-sized dimensions. In many instances, the chemically unique regions, contributing to the overall chemical heterogeneity of the membrane surface, were substantially different in chemistry (e.g., hydrophobicity) from that determined for the surface at large from contact angel and streaming potential analyses. Topographical and corresponding CFM images supports previous adhesion studies finding a correlation between surface roughness and the magnitude of adhesion measured with AFM. However, chemical specificity was also significant and in turn measurable with CFM. The implication of these findings for future membrane development is discussed.     

Simple test system for single molecule recognition force microscopy

We have established an easy-to-use test system for detecting receptor-ligand interactions on the single molecule level using atomic force microscopy (AFM). For this, avidin-biotin, probably the best characterized receptor-ligand pair, was chosen. AFM sensors were prepared containing tethered biotin molecules at sufficiently low surface concentrations appropriate for single molecule studies. A biotin tether, consisting of a 6 nm poly(ethylene glycol) (PEG) chain and a functional succinimide group at the other end, was newly synthesized and covalently coupled to amine-functionalized AFM tips. In particular, PEG₈₀₀ diamine was glutarylated, the mono-adduct NH₂-PEG-COOH was isolated by ion exchange chromatography and reacted with biotin succinimidylester to give biotin-PEG-COOH which was then activated as N-hydroxysuccinimide (NHS) ester to give the biotin-PEG-NHS conjugate which was coupled to the aminofunctionalized AFM tip. The motional freedom provided by PEG allows for free rotation of the biotin molecule on the AFM sensor and for specific binding to avidin which had been adsorbed to mica surfaces via electrostatic interactions. Specific avidin-biotin recognition events were discriminated from nonspecific tip-mica adhesion by their typical unbinding force (∼40 pN at 1.4 nN/s loading rate), unbinding length (<13 nm), the characteristic nonlinear force-distance relation of the PEG linker, and by specific block with excess of free d-biotin. The convenience of the test system allowed to evaluate, and compare, different methods and conditions of tip aminofunctionalization with respect to specific binding and nonspecific adhesion. It is concluded that this system is well suited as calibration or start-up kit for single molecule recognition force microscopy.