Ebner Andreas, Wildling Linda, Kamruzzahan A S M, Rankl Christian, Wruss Jürgen, Hahn Christoph D, Hölzl Martin, Zhu Rong, Kienberger Ferry, Blaas Dieter, Hinterdorfer Peter, Gruber Hermann J
Institute of Biophysics, J. Kepler University, Altenberger Strasse 69, A-4040 Linz, Austria.
Bioconjug Chem. 2007 Jul-Aug;18(4):1176-84. doi: 10.1021/bc070030s. Epub 2007 May 22.
Functionalization of atomic force microscope (AFM) tips with bioligands converts them into monomolecular biosensors which can detect complementary receptor molecules on the sample surface. Flexible PEG tethers are preferred because the bioligand can freely reorient and locally palpate the sample surface while the AFM tip is moved along. In a well-established coupling scheme [Hinterdorfer et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 3477-3481], a heterobifunctional PEG linker is used to tether thiol-containing bioligands to amino-functionalized AFM tips. Since antibodies contain no free thiol residues, prederivatization with N-succinimidyl 3-(acetylthio)propionate (SATP) is needed which causes a relatively high demand for antibody. The present study offers a convenient alternative with minimal protein consumption (e.g., 5 microg of protein in 50 microL of buffer) and no prederivatization, using a new heterobifunctional cross-linker that has two different amino-reactive functions. One end is an activated carboxyl (N-hydroxysuccinimide ester) which is much faster to react with the amino groups of the tips than the benzaldehyde function on its other end. The reactivity of the latter is sufficient, however, to covalently bind lysine residues of proteins via Schiff base formation. The method has been critically examined, using biotinylated IgG as bioligand on the tip and mica-bound avidin as complementary receptor. These experiments were well reproduced on amino-functionalized silicon nitride chips where the number of specifically bound IgG molecules (approximately 2000 per microm2) was estimated from the amount of specifically bound ExtrAvidin-peroxidase conjugate. For a bioscientific application, human rhinovirus particles were tethered to the tip, very-low-density lipoprotein receptor fragments were tethered to mica, and the specific interaction was studied by force microscopy.
用生物配体对原子力显微镜（AFM）探针进行功能化处理，可将其转化为单分子生物传感器，能够检测样品表面的互补受体分子。柔性聚乙二醇（PEG）连接链是首选，因为在AFM探针沿样品表面移动时，生物配体可以自由重新定向并局部探测样品表面。在一种成熟的偶联方案中[欣特多费尔等人（1996年），《美国国家科学院院刊》93卷，3477 - 3481页]，使用异双功能PEG连接子将含硫醇的生物配体连接到氨基功能化的AFM探针上。由于抗体不含游离硫醇残基，因此需要用N - 琥珀酰亚胺基3 -（乙酰硫基）丙酸酯（SATP）进行预衍生化处理，这导致对抗体的需求量相对较高。本研究提供了一种便捷的替代方法，蛋白质消耗极少（例如，50微升缓冲液中含5微克蛋白质）且无需预衍生化，使用一种具有两种不同氨基反应性功能的新型异双功能交联剂。一端是活化羧基（N - 羟基琥珀酰亚胺酯），它与探针氨基的反应速度比另一端的苯甲醛功能快得多。然而，后者的反应活性足以通过席夫碱形成共价结合蛋白质的赖氨酸残基。该方法已通过严格检验，使用生物素化的IgG作为探针上的生物配体，云母结合的抗生物素蛋白作为互补受体。这些实验在氨基功能化的氮化硅芯片上得到了很好的重现，其中通过特异性结合的抗生物素蛋白 - 过氧化物酶共轭物的量估算出特异性结合的IgG分子数量（每平方微米约2000个）。对于生物科学应用，将人鼻病毒颗粒连接到探针上，将极低密度脂蛋白受体片段连接到云母上，并通过力显微镜研究特异性相互作用。
