The authors thank the DFG (Hu 997/2-2) for financial support. FS acknowledges the Hanns-Seidel-Stiftung (HSS). SKr was supported by the Elitenetzwerk Bayern in the framework of the doctorate program Material Science of Complex Interfaces. SKi thanks the SFB 863 for financial support.

注:参见图2的处理流程，其包括制备，数据采集和数据分析的步骤的概述。 1.试剂设置注:所有化学品必须小心处理，因而白大褂，手套和保护眼睛应该被使用。所有操作都必须在实验室罩中进行。尤其特殊的手套时，应佩戴情况下，氯仿使用。 <ol><li>使用具有低水含量的化学物质，如无水氯仿迅速和存储干燥，但不超过一个星期。同时存储的化学品在-20℃和在氮气或氩气，因为APTES（（3-氨基丙基）triethoxysilan）（ 见表1）和PEG（聚乙二醇）具有吸湿性，PEG是受氧化在空气中。 </li><li>避免了库存到大气中的氧气和水分的频繁接触，制备小等分，理想手套箱系统用氮气情调内重。 </li></ol> 2.设备安装注:为避免交叉污染，使用新鲜的，干净的船只每一步。 <ol><li>清洁玻璃器皿和在30分钟在超声波浴中，在60℃的洗涤剂溶液镊子。 </li><li>冲洗，并从步骤2.1两次用超纯水充分声处理设备。 </li><li>热设备从RCA溶液步骤2.1（超纯水，过氧化氢和氨（5:1:1）），以75℃的烘箱中45分钟，随后用超纯水冲洗。 </li><li>最后，干燥的玻璃器皿和镊子在干燥氮气流下或者在烘箱（100℃，3小时）。 </li></ol> 3.提示功能化注意:使用镊子，容器等由不锈钢，聚四氟乙烯，玻璃或它是在有机溶液中，如果适用的化学稳定的任何其它材料制成。除非另有规定，做好各项步骤为RT.所需的温育溶液的量取决于悬臂的芯片的数量。确保该悬臂浸渍在各时间的各解决方案。 注:使用遮光罩，以避免吸入有机蒸气。 <ol><li>形成于悬臂表面（&#39;激活&#39;）（约0.5小时）OH-组: <ol><li>用镊子把新鲜的悬臂式芯片（材质:单，弹簧常数:10-100 PN /纳米）在一个干净的玻片上，并把它们在等离子室（100 W）。 </li><li>抽空腔室（〜0.1毫巴）。 </li><li>洪水室氧气，并再次撤离。 </li><li>激活等离子体处理（功率:20％，持续时间:15分钟，处理压力:0.25毫巴）。 </li></ol></li><li>氨基硅烷化悬臂（约1小时）: <ol><li>制备2.5毫升APTES溶液（ 见表1）中的玻璃培养皿-在等离子体处理执行此理想。 </li><li>立即出现等离子处理后Y，浸每个悬臂1秒丙酮，并将它们随即在APTES解决方案。 </li><li>孵育15分钟，在RT。 </li><li>在10毫升丙酮，一次在10毫升氯仿中仔细冲洗悬臂芯片的两倍。 </li><li>任选最终步骤:从在干净的载玻片上一步中放置悬臂芯片和烘烤它们30分钟，在70℃。注意，也有表面活化， 例如，UV处理12的替代策略。 </li></ol></li><li> PEG化（约2小时）: 注:执行过程中的氨基硅烷化步骤3.3.1-3.3.4。 NHS和马来酰亚胺基团受到水解在含水环境和PEG本身受氧化在空气中。因此定时（特别是在步骤之间）是一个关键的参数。请参阅13获取更多信息。 <ol><li>制备的氯仿溶液（ 见表1）。 </li><li>为避免结露温暖PEG粉末达RT打开等分，体重适量之前。 </li><li>对于聚合物或脂质与氨基的偶联，分别解决的NHS-PEG-NHS（6 kDa的）和甲基-PEG-NHS（5 kDa的），在氯仿溶液通过涡旋直到它们被完全解决。请参阅表1的浓度。 <ol><li>另外。用于与硫醇基团的聚合物的偶联分别解决MAL-NHS-PEG和甲基PEG-NHS的氯仿溶液中。 </li></ol></li><li>以调节NHS-或马来酰亚胺和甲基封端的PEG分子（:500通常为1）之间有一定的数量比需要混合的解决方案。需要注意的是理想的比例要被迭代的一系列制备-实验周期来确定。 </li><li>孵育悬臂芯片在氯仿饱和气氛中的PEG溶液1小时，以防止蒸发出氯仿。 </li></ol></li><li>探针分子共轭（&gt; 1小时）: 注意:在下面的3.4.1-3.4.3，三个例子为不同的探针分子的AFM针尖的共价偶联了描述。对于每一个分子的协议必须被调整。进一步注意到，与实施例1和3的NHS-化学中使用，而实施例2中的巯基官能化的聚合物PI- 克 -PS经由马来酰亚胺化学偶联于PEG。有关详情请参阅第14。 <ol><li>对聚（氨基酸）聚-D-酪氨酸（40-100 kDa）的<ol><li>溶解在1M的NaOH将聚酪氨酸缀合探针分子。调至浓度为1毫克/毫升。 </li><li>使用旋转脱盐立即柱（7 kDa的MWCO）的功能化之前交换的NaOH溶液硼酸钠缓冲液（pH 8.1） </li><li>用5毫升的氯仿中，然后用5毫升乙醇和最后用5毫升硼酸盐缓冲液的第一清洗悬臂。 </li><li>孵育悬臂芯片1小时的聚酪氨酸硼酸盐缓冲溶液，然后用清水冲洗硼酸盐缓冲剂和超纯水。储存在超纯水中，直到测量。 </li></ol></li><li>对于线性骨干（聚异戊二烯，119 kDa的），具有嫁接侧链（聚苯乙烯，88 kDa的）14聚合物<ol><li>溶解PI- 克 -PS无水氯仿。调节偶联溶液至4毫克/毫升的浓度。 </li><li>冲洗用5毫升氯仿悬臂孵育至少1小时，在缀合溶液。 </li><li>在5毫升氯仿中再次冲洗并在氯仿存储在氯仿 - 饱和的环境（防止氯仿蒸发），直到测量。 </li></ol></li><li>对于脂质教皇<ol><li>在干燥的氯仿溶解教皇。浓度调整至20mM。 </li><li>冲洗用5ml乙醇和5ml超纯水的悬臂1小时，在脂质溶液的温育前的悬臂。 </li><li>温育后，冲洗悬臂用5毫升氯仿，加入5ml ethano的L和5毫升的温，超纯水（按顺序），以除去未结合的探针分子，并摆脱三氯甲烷残留物。立即使用功能化的提示。可替代地，直到需要将它们存储在氯仿中。 </li></ol></li></ol></li></ol><img alt="图1" src="/files/ftp_upload/52456/52456fig1.jpg" /> 图1（A）的示意图表示使用NHS化学的例子的尖端官能化方法。采用通过氨基连接探针分子给小费（B）化学键。 <a href="https://www.jove.com/files/ftp_upload/52456/52456fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 4.表面处理<ol><li>自组装单层（SAM）的 注意:作为表面的第一个例子，一个自组装单层被选择。请参考文献15 </s达&gt;详细信息。 <ol><li>干净的载玻片用洗涤剂溶液，然后两次在超纯水中在超声浴中对每个30分钟。然后，作为附加的清洗步骤，将它们放置在RCA溶液（参见步骤2.3），在75℃下进行15分钟。 </li><li>外套与10纳米的铬镍，并在真空涂布机100纳米金的幻灯片。然后前直接进行下一步再存放在冰箱和清洁，在RCA溶液。 </li><li>孵育前一步骤的幻灯片在12小时为2mM 1-十二/乙醇溶液。该硫醇基团结合到金表面和疏水单层自组装。用氮气流冲洗，用乙醇和超纯水中，然后干燥的幻灯片。确认制得的表面的静态接触角测量的疏水性在用CCD照相机10一个测角器。 </li></ol></li><li>氢终止金刚石 注意:作为该第二个例子的表面上，一氢终止金刚石被选择。如先前16所述进行表面处理。 </li><li>支持的脂质双层 注意:在最后的例子中，表面支持的脂质双层（SLB）用作表面。为了获得这样的表面上，大的单层囊泡（的LUV）选自磷脂P​​OPC的溶液（0.1毫克/毫升）通过挤出法形成。然后将50μl的LUV溶液被放在一个新鲜剥离云母片（A = 1平方厘米），并孵育30分钟。最后该溶液漂洗用20ml超纯水。对于SLBs的编制的详细说明，请参阅17,18。 </li></ol> 5.数据采集注:对于实验中，使用AFM，它提供的液体测量能力。数据采集​​和数据分析方法都适用，无论使用AFM模式。此外，在一些实验中，有利的是要控制。温度的可能性液晶单元内即通过将激光束偏转方法19检测悬臂偏转。弹簧常数分别与热噪声的方法20。 <ol><li>将官能化的悬臂成的AFM悬臂支架，适用于测量流体。 </li><li>放表面（制备见上文）被采样到AFM和用液体覆盖。两者，悬臂和表面现在应浸渍在与悬臂尖端指向表面的液体。注意，在许多情况下，一滴液体就足够了。在流体中的任何情况下，蒸发应该避免， 例如，通过使用封闭的流体单元。 </li><li>如果适用的话，调整控制器上所需的温度。 </li><li>让系统平衡至少一个半小时。 </li><li>为了排除任何表面阻尼效应记录与所述悬臂的悬臂的热噪声频谱远离表面。需要注意的是，通常10以上的光谱必须累积，以获得足够的信噪比。 </li><li>接近表面。需要注意的是，为了节省末梢几何结构和小费官能的方法处理，必须仔细地进行。这可以通过以下方式实现，例如，在接近断续接触模式。 </li><li>确定纳米/伏测量逆光杠杆灵敏度（InvOLS）。通过推动悬臂刀片针对硬表面做到这一点。 <ol><li>通过测量压电行驶距离与在光电二极管电压的变化的斜率确定InvOLS。拟合线的力 - 距离曲线，其中所述悬臂刀片是在与表面接触的部分。在涉及柔软的表面像脂质双层实验，开展对未涵盖与脂质双层（&#39;缺陷点&#39;）的区域这一步。 </li></ol></li><li>通过安装一个谐振子的热噪声谱确定弹簧常数（PN /纳米）。使用自动化的软件功能弹簧常数测定;否则，查阅文献20。 </li><li>开始实验。 <ol><li>记录无数力 - 距离曲线在每个实验条件。通常情况下，用于该实验的参数以下值:5千赫，1微米/秒的尖端速度的采样率，回缩距离为1微米。 注:有时，根据所研究的实验的动力学，可能有必要等待一定量的时间，在与表面接触的前端（&#39;停留时间&#39;）。 注:本实验的具体参数可从以上给出的数值基本上偏离。 </li><li>在长期的测量，通过重新定位所述光电二极管零上的光电二极管上的激光位置不时。 </li><li>可选:每个力曲线后，重新定位AFM尖到另一个位置坐标。 </li><li>在实验结束时，确定灵敏度和弹簧CONSTA再次新台币检查系统的一致性和稳定性。 注:前和实验后的弹簧常数和灵敏度比较也是一种手段，以确保该系统的性能和尖端的特性保持不变在实验的过程中。如果弹簧常数之差不是太大（&lt;= 15％），它们可以被平均化，由于弹簧常数确定的固有的不确定性也是15％左右。对于较大的差别，最好是先找到并与进一步的实验，然后再继续消除病因的改变表观弹簧常数。 </li></ol></li></ol> 6.数据准备注:在其通常进行独立实验的具体类型的转换单位牛顿纳米以及以校正数据中描述了本节的一般数据准备步骤。实验的具体数据分析是BRiefly下面进一步描述，在各自的有代表性的例子一节。 <ol><li>通常情况下，进行所有数据准备和分析由家用写算法， 例如自动步骤，基于所述软件IGOR Pro 6的。 </li><li>原始偏转信号（DEFL）[V]的转换成力通过与InvOLS [纳米/ V]和弹簧常数[NN / nm]的相乘。 </li><li>抵消在这样一种方式，在从表面（空载悬臂）在足够的距离的悬臂上的力变为0 NN的力。 </li><li>计算实际尖端位置z *，这需要在悬臂的弯曲计（测量时相对于表面也称为分离）: <img alt="式（1）" fo:content-width="2in" src="/files/ftp_upload/52456/52456eq1.jpg" /></li><li>偏移Z *在于Z * = 0对应于表面的z轴位置的方式。 注意:在这里，F是作 ​​用在前端，因此弯曲的力S中的悬臂（在步骤6.2确定的。）中，z是测量的z传感器位置（由仪器直接给出）和k表示弹簧常数（在步骤5.8确定）。 </li></ol><img alt="图2" src="/files/ftp_upload/52456/52456fig2.jpg" /> 展示样品制备，数据采集和数据分析图2.工艺流程图。 <a href="https://www.jove.com/files/ftp_upload/52456/52456fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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S., Hugel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Nanomechanical+Properties+of+Lipid+Membranes+are+Significantly+Influenced+by+the+Presence+of+Ethanol.">The Nanomechanical Properties of Lipid Membranes are Significantly Influenced by the Presence of Ethanol.</a> Biophysical Journal. 104 (5), 1049-1055 (2013).</li><li>Putman, C. A. J., De Grooth, B. G., Hulst, N. F., Greve, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+detailed+analysis+of+the+optical+beam+deflection+technique+for+use+in+atomic+force+microscopy.">A detailed analysis of the optical beam deflection technique for use in atomic force microscopy.</a> Journal of Applied Physics. 72 (1), 6-12 (1992).</li><li>Hutter, J. L., Bechhoefer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calibration+of+atomic-force+microscope+tips.">Calibration of atomic-force microscope tips.</a> Review of Scientific Instruments. 64 (7), 1868 (1993).</li><li>Krysiak, S., Liese, S., Netz, R. R., Hugel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+desorption+kinetics+from+single+molecule+force+spectroscopy+studies.">Peptide desorption kinetics from single molecule force spectroscopy studies.</a> Journal of the American Chemical Society. 136 (2), 688-697 (2014).</li><li>Li, I. T. S., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interfacial+free+energy+governs+single+polystyrene+chain+collapse+in+water+and+aqueous+solutions.">Interfacial free energy governs single polystyrene chain collapse in water and aqueous solutions.</a> Journal of the American Chemical Society. 132 (18), 6530-6540 (2010).</li><li>Dudko, O. K., Hummer, G., Szabo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory,+analysis,+and+interpretation+of+single-molecule+force+spectroscopy+experiments.">Theory, analysis, and interpretation of single-molecule force spectroscopy experiments.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (41), 15755-15760 (2008).</li><li>Stetter, F. W. S., Cwiklik, L., Jungwirth, P., Hugel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Lipid+Extraction:+The+Anchoring+Strength+of+Cholesterol+in+Liquid-Ordered+and+Liquid-Disordered+Phases.">Single Lipid Extraction: The Anchoring Strength of Cholesterol in Liquid-Ordered and Liquid-Disordered Phases.</a> Biophysical Journal. 107 (5), 1167-1175 (2014).</li><li>Horinek, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+adsorption+on+a+hydrophobic+surface+results+from+an+interplay+of+solvation,+surface,+and+intrapeptide+forces.">Peptide adsorption on a hydrophobic surface results from an interplay of solvation, surface, and intrapeptide forces.</a> Proc Natl Acad Sci U S A. 105 (8), 2842-2847 (2008).</li><li>Ebner, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionalization+of+probe+tips+and+supports+for+single-molecule+recognition+force+microscopy.">Functionalization of probe tips and supports for single-molecule recognition force microscopy.</a> Topics in current chemistry. 285 (April), 29-76 (2008).</li><li>Geisler, M., Balzer, B. N., Hugel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer+Adhesion+at+the+Solid-Liquid+Interface+Probed+by+a+Single-Molecule+Force+Sensor.">Polymer Adhesion at the Solid-Liquid Interface Probed by a Single-Molecule Force Sensor.</a> Small. 5 (24), 2864-2869 (2009).</li><li>Morfill, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity-matured+recombinant+antibody+fragments+analyzed+by+single-molecule+force+spectroscopy.">Affinity-matured recombinant antibody fragments analyzed by single-molecule force spectroscopy.</a> Biophysical journal. 93 (10), 3583-3590 (2007).</li></ol>

The authors declare that they have no competing financial interests.

在过去的几十年中，单分子实验提供了前所未有的见解的分子机制，竟然是在生命科学及以后的宝贵途径。以实现从SMFS实验，理想地1，并在同一分子用在实验的全过程良好的和有意义的统计数据。与此相反，以与分子的合奏实验，SMFS实验能够检测罕见事件和隐藏的分子状态。的单分子实验的另一个优点是，它们可以通过分子动力学模拟24,25手段进行建模。 上述悬臂?...

在过去的30年中，原子力显微镜（AFM）已被证明是一个有价值的成像技术来研究生物学1,2和3的合成材料和表面，因为它提供了分子的空间分辨率在三个维度，可以在不同的溶剂进行操作环境。此外，AFM单分子力谱（SMFS）使测量力范围从PN为μN政权，并给予前所未有的洞察到例如蛋白质折叠4,5，高分子物理6 - 8，和单分子表面相互作用9 -学习背后的单个分子，而不是分子的集合体12 .The理由是为了避免影响平均，往往掩盖了罕见的事件或隐藏的分子状态。此外，众多的分子参数，如轮廓长度，库恩长度，粘合自由能等可以获得。此详述在下面的实施例。在一个典型的AFM-SMFS实验，探针分子通过接头分子偶联到一个非常尖锐的尖端。尖端本身位于一可弯曲的悬臂的端部。如果针尖被带入与该表面接触的探针分子将与该表面相互作用。通过在尖端，力，因此自由能缩回观察悬臂的偏转，以从表面分离的分子可以被确定。以获得有意义的统计数据，大量的所谓的力 - 距离曲线已被收购。此外，能有真正的单分子实验（ 即 ，使用一个和在整个实验期间相同的探针分子）探针分子应当共价偶联到所述针尖。这里，一个实验协议悬臂官能经由共价键的单个分子呈现。单分子既可以通过氨基或硫基耦L组的针尖。缀合方法可以在广泛的各种溶剂（有机相和水）占所用的聚合物的溶剂化性质来进行。 在第一部分中，一个通用的方案来共价通过接头分子与AFM针尖描述附加一个单一的分子（"探针分子"）。为此目的，有机NHS-或马来酰亚胺化学过程中使用13。随着协议3例如分子，数据采集和数据分析的方法描述和提供了用于进一步阅读参考。的例子的分子是:（线性）聚合物酪氨酸，接枝聚合物PI- 克 -PS和脂质POPE。这包括协议的微小变化，例如共价连接半胱氨酸。另外，一个段是专用不同的表面的制备诸如金刚石表面，CH 3 -self组装单层和脂质双层。这些接口都省恩是很好的参考和实例。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Materials&lt;/strong&gt;</td></tr><tr><td>Hellmanex III alkaline liquid concentrate (detergent solution)</td><td>Hellma</td><td></td><td></td></tr><tr><td>RCA (ultrapure water, hydrogen peroxide (35%), ammonia (32%); 5:1:1(v/v/v))</td><td>Sigma</td><td></td><td></td></tr><tr><td>Vectabond reagent / APTES (3-Aminopropyl)triethoxysilane</td><td>Vectorlabs</td><td></td><td></td></tr><tr><td>Dry acetone (&amp;lt; 50 ppm H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma</td><td></td><td></td></tr><tr><td>Dry chloroform (&gt; 99.9%)</td><td>Sigma</td><td></td><td></td></tr><tr><td>Triethylamine</td><td>Sigma</td><td></td><td></td></tr><tr><td>Ultrapure water</td><td>Biochrom, Germany</td><td></td><td></td></tr><tr><td>Di-sodium tetraborate (&gt;&amp;nbsp;99.5%)</td><td>Biochrom, Germany</td><td></td><td></td></tr><tr><td>Boric Acid</td><td>Biochrom, Germany</td><td></td><td></td></tr><tr><td>Monofunctional &amp;alpha;-methoxy-&amp;omega;-NHS PEG, 5 kDa, &amp;ldquo;methyl-PEG-NHS&amp;rdquo;</td><td>Rapp, Germany</td><td></td><td></td></tr><tr><td>Heterobifunctional &amp;alpha;,&amp;omega;-bis-NHS PEG, 6&amp;nbsp;kDa, &amp;ldquo;NHS-PEG-NHS&amp;rdquo;</td><td>Rapp, Germany</td><td></td><td></td></tr><tr><td>Heterobifunctional &amp;alpha;-maleimidohexanoic- &amp;omega;-NHS PEG, 5 kDa, &amp;ldquo;Mal-PEG-NHS&amp;rdquo;</td><td>Rapp, Germany</td><td></td><td></td></tr><tr><td>Probe molecule (polymer, lipid, &lt;em&gt;etc.&lt;/em&gt;)</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>Sufficient amount of glass crystallising dishes with spout (10&amp;nbsp;ml), glass Petri dishes (500 &amp;micro;l) and glass lids</td><td>VWR International GmbH, Germany</td><td></td><td>&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Laboratory oven model UF30</td><td>Memmert, Germany</td><td></td><td></td></tr><tr><td>Temperature controlled sonicator</td><td>VWR International GmbH, Germany</td><td></td><td></td></tr><tr><td>Plasma system &quot;Femto&quot;, 100 W</td><td>Diener, Germany</td><td></td><td></td></tr><tr><td>One separate glass syringe for each organic solvent</td><td>VWR International GmbH, Germany</td><td></td><td></td></tr><tr><td>Vortex mixer</td><td>VWR International GmbH, Germany</td><td></td><td></td></tr><tr><td>Microcentrifuge tubes (0.5 ml or 1.5 ml)</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Pipettes: 10-100 &amp;micro;l, 50-200 &amp;micro;l and 100-1,000&amp;nbsp;&amp;micro;l</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>AFM with temperature controlled fluid cell (&lt;em&gt;e.g.&lt;/em&gt; MFP-3D with BioHeater)</td><td>Asylulm Research, Santa Barbara</td><td></td><td></td></tr><tr><td>Soft SiN cantilevers cantilever, typically made from silicon nitride (SiN) (spring constant less than 100 pN/nm, &lt;em&gt;e.g.&lt;/em&gt; MLCT)</td><td>Bruker AXS, Santa Barbara</td><td></td><td></td></tr></tbody></table>

investigating single molecule adhesion by atomic force spectroscopy

原子力谱是研究分子在表面和界面的​​理想工具。一个试验性协议，以夫妻种类繁多的单分子的共价键上的原子力显微镜针尖呈现。同时针尖被钝化，以防止尖端和基板，这是一个先决条件，研究附着在针尖单个分子之间的非特异性相互作用。分析，以确定的粘合力，粘合长度，并在固体表面上和生物界面这些分子不久提出并提供用于进一步阅读外部引用的自由能。实施例的分子是聚（氨基酸）聚酪氨酸，接枝聚合物PI- 克 -PS和磷脂POPE（1-棕榈酰-2-油酰- SN -glycero -3-磷酸）。这些分子是从不同的表面，如CH 3 -SAMs，氢封端的金刚石解吸并支持的各种溶剂的条件下的脂质双层。最后，该力分光单分子实验的优点进行了讨论，包括装置，以决定是否确实是一个单一分子已经研究了实验。

在下文中，其结果为上述实施例的分子，即该聚合物的聚（氨基酸）聚酪氨酸，接枝聚合物PI- 克 -PS和磷脂POPE，现介绍。首先对各实施例，实验的具体细节的数据采集和数据准备设置。然后，示例性结果实验，其中这些分子是从不同的表面上解吸（CH 3 -SAMs，氢封端的金刚石和脂质双层）被示出。的粘附力，粘合长度和自由能的测定进行了介绍。 实施例1:...

Watch this Scientific Journal Video about Investigating Single Molecule Adhesion by Atomic Force Spectroscopy at JoVE.com

Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

A protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. Procedures and examples to determine the adhesion force and free energy of these molecules on solid supports and bio-interfaces are provided.

调查单分子粘附的原子力谱

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single ...

investigating-single-molecule-adhesion-by-atomic-force-spectroscopy

Research

JoVE Journal

Engineering

10.3K 次观看.  Technische Universität München. A protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. Procedures and examples to determine the adhesion force and free energy of these molecules on solid supports and bio-interfaces are provided.

视频: Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate using the Atomic Force Microscope (AFM), can provide key insights into physical interactions present within colloidal systems. Colloidal systems are widely existent in several applications including, pharmaceuticals, foods, paints, paper, soil and minerals, detergents, printing and much more.1-3 Furthermore, colloids can exist in many states such as emulsions, foams and suspensions. Using colloidal probe nanoscopy one can obtain key information on the adhesive properties, binding energies and even gain insight into the physical stability and coagulation kinetics of the colloids present within. Additionally, colloidal probe nanoscopy can be used with biological cells to aid in drug discovery and formulation development. In this paper we describe a method for conducting colloidal probe nanoscopy, discuss key factors that are important to consider during the measurement, and show that both quantitative and qualitative data that can be obtained from such measurements.

Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems.

粒子粒子相互作用采用胶体探针纳米显微定量和定性检查

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate ...

科研

化学

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

粘附频率含量为原位跨交界的分子相互作用，在细胞与细胞界面的动力学分析

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

生物工程

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. Atomic force microscopy (AFM) can address this problem by enabling stretching and relaxation of single protein molecules, which gives direct evidence of specific unfolding and refolding characteristics. AFM-based single-molecule force-spectroscopy (AFM-SMFS) provides a means to consistently measure high-energy conformations in proteins that are not possible in traditional bulk (biochemical) measurements. Although numerous papers were published to show principles of AFM-SMFS, it is not easy to conduct SMFS experiments due to a lack of an exhaustively complete protocol. In this study, we briefly illustrate the principles of AFM and extensively detail the protocols, procedures, and data analysis as a guideline to achieve good results from SMFS experiments. We demonstrate representative SMFS results of single protein mechanical unfolding measurements and we provide troubleshooting strategies for some commonly encountered problems.

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

原子力显微镜对单蛋白质分子的力谱

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

生物化学

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

单分子力谱中蛋白质共价固定化的研究

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase.

洞察氨基酸和多肽与无机材料的使用相互作用的单分子力谱

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

调查纤维素酶与基于AFM的单分子力谱受体 - 配体系统

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

原子力显微镜成像和支持的脂质双层力谱

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Covalent Attachment of Single Molecules for AFM-based Force Spectroscopy

Atomic force microscopy (AFM)-based single molecule force spectroscopy is an ideal tool for investigating the interactions between a single polymer and surfaces. For a true single molecule experiment, covalent attachment of the probe molecule is essential because only then can hundreds of force-extension traces with one and the same single molecule be obtained. Many traces are in turn necessary to prove that a single molecule alone is probed. Additionally, passivation is crucial for preventing unwanted interactions between the single probe molecule and the AFM cantilever tip as well as between the AFM cantilever tip and the underlying surface. The functionalization protocol presented here is reliable and can easily be applied to a variety of polymers. Characteristic single molecule events (i.e., stretches and plateaus) are detected in the force-extension traces. From these events, physical parameters such as stretching force, desorption force and desorption length can be obtained. This is particularly important for the precise investigation of stimuli-responsive systems at the single molecule level. As exemplary systems poly(ethylene glycol) (PEG), poly(N-isopropylacrylamide) (PNiPAM) and polystyrene (PS) are stretched and desorbed from SiOx (for PEG and PNiPAM) and from hydrophobic self-assembled monolayer surfaces (for PS) in aqueous environment.

Covalent attachment of probe molecules to atomic force microscopy (AFM) cantilever tips is an essential technique for the investigation of their physical properties. This allows us to determine the stretching force, desorption force and length of polymers via AFM-based single molecule force spectroscopy with high reproducibility.

基于AFM的力光谱单分子共价附价

Atomic force microscopy (AFM)-based single molecule force spectroscopy is an ideal tool for investigating the interactions between a single polymer and ...

Atomic Force Microscopy Cantilever-Based Nanoindentation: Mechanical Property Measurements at the Nanoscale in Air and Fluid

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by the probe tip and its contact area with the sample can be quantified, it is possible to determine the nanoscale mechanical properties (e.g., elastic or Young&#39;s modulus) of the surface being probed. A detailed procedure for performing quantitative AFM cantilever-based nanoindentation experiments is provided here, with representative examples of how the technique can be applied to determine the elastic moduli of a wide variety of sample types, ranging from kPa to GPa. These include live mesenchymal stem cells (MSCs) and nuclei in physiological buffer, resin-embedded dehydrated loblolly pine cross-sections, and Bakken shales of varying composition.
Additionally, AFM cantilever-based nanoindentation is used to probe the rupture strength (i.e., breakthrough force) of phospholipid bilayers. Important practical considerations such as method choice and development, probe selection and calibration, region of interest identification, sample heterogeneity, feature size and aspect ratio, tip wear, surface roughness, and data analysis and measurement statistics are discussed to aid proper implementation of the technique. Finally, co-localization of AFM-derived nanomechanical maps with electron microscopy techniques that provide additional information regarding elemental composition is demonstrated.

Quantifying the contact area and force applied by an atomic force microscope (AFM) probe tip to a sample surface enables nanoscale mechanical property determination. Best practices to implement AFM cantilever-based nanoindentation in air or fluid on soft and hard samples to measure elastic modulus or other nanomechanical properties are discussed.

原子力显微镜悬臂式纳米压痕:空气和流体中纳米级机械性能测量

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by ...

工程学

Application of Atomic Force Microscopy to Detect Early Osteoarthritis

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.

We present a method to investigate early osteoarthritic changes at the cellular level in articular cartilage by using atomic force microscopy (AFM).

原子力显微镜在早期骨关节炎检测中的应用

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in ...

调查单分子粘附的原子力谱

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