Stroh Cordula M, Ebner Andreas, Geretschläger Manfred, Freudenthaler Günter, Kienberger Ferry, Kamruzzahan A S M, Smith-Gill Sandra J, Gruber Hermann J, Hinterdorfer Peter
Institute for Biophysics, Atomic Physics and Surface Science, Johannes Kepler University of Linz, A-4040 Linz, Austria.
Biophys J. 2004 Sep;87(3):1981-90. doi: 10.1529/biophysj.104.043331.
We present a method for simultaneously recording topography images and localizing specific binding sites with nm positional accuracy by combining dynamic force microscopy with single molecule recognition force spectroscopy. For this we used lysozyme adsorbed to mica, the functionality of which was characterized by enzyme immunoassays. The topography and recognition images were acquired using tips that were magnetically oscillated during scanning and contained antibodies directed against lysozyme. For cantilevers with low Q-factor (approximately 1 in liquid) driven at frequencies below resonance, the surface contact only affected the downward deflections (minima) of the oscillations, whereas binding of the antibody on the tip to lysozyme on the surface only affected the upwards deflections (maxima) of the oscillations. The recognition signals were therefore well separated from the topographic signals, both in space (Delta z approximately 5 nm) and time (approximately 0.1 ms). Topography and recognition images were simultaneously recorded using a specially designed electronic circuit with which the maxima (U(up)) and the minima (U(down)) of each sinusoidal cantilever deflection period were depicted. U(down) was used for driving the feedback loop to record the height (topography) image, and U(up) provided the data for the recognition image.
我们提出了一种通过将动态力显微镜与单分子识别力谱相结合，以纳米级位置精度同时记录形貌图像和定位特定结合位点的方法。为此，我们使用了吸附在云母上的溶菌酶，其功能通过酶免疫测定进行表征。形貌和识别图像是使用在扫描过程中进行磁振荡且含有抗溶菌酶抗体的探针采集的。对于在低于共振频率下驱动的低品质因数悬臂梁（在液体中约为1），表面接触仅影响振荡的向下偏转（最小值），而探针上的抗体与表面上的溶菌酶结合仅影响振荡的向上偏转（最大值）。因此，识别信号在空间（Δz约为5 nm）和时间（约0.1 ms）上都与形貌信号很好地分离。使用专门设计的电子电路同时记录形貌和识别图像，通过该电路描绘每个正弦悬臂梁偏转周期的最大值（U(up)）和最小值（U(down)）。U(down)用于驱动反馈回路以记录高度（形貌）图像，而U(up)提供识别图像的数据。
