Laboratoire Biogéosciences, UMR CNRS-EPHE 6282, University of Burgundy, Dijon, France; Synchrotron SOLEIL, Beamline ANATOMIX, Saint-Aubin, Gif-sur-Yvette, France.
Laboratoire Interdisciplinaire Carnot de Bourgogne (ICB), UMR CNRS 6303, University of Burgundy, Dijon, France.
Acta Biomater. 2023 Sep 15;168:198-209. doi: 10.1016/j.actbio.2023.07.028. Epub 2023 Jul 23.
Spatial localizing of skeletal proteins in biogenic minerals remains a challenge in biomineralization research. To address this goal, we developed a novel in situ mapping technique based on molecular recognition measurements via atomic force microscopy (AFM), which requires three steps: (1) the development and purification of a polyclonal antibody elicited against the target protein, (2) its covalent coupling to a silicon nitride AFM tip ('functionalization'), and (3) scanning of an appropriately prepared biomineral surface. We applied this approach to a soluble shell protein - accripin11 - recently identified as a major component of the calcitic prisms of the fan mussel Pinna nobilis [1]. Multiple tests reveal that accripin11 is evenly distributed at the surface of the prisms and also present in the organic sheaths surrounding the calcitic prisms, indicating that this protein is both intra- and inter-crystalline. We observed that the adhesion force in transverse sections is about twice higher than in longitudinal sections, suggesting that accripin11 may exhibit preferred orientation in the biomineral. To our knowledge, this is the first time that a protein is localized by molecular recognition atomic force microscopy with antibody-functionalized tips in a biogenic mineral. The 'pros' and 'cons' of this methodology are discussed in comparison with more 'classical' approaches like immunogold. This technique, which leaves the surface to analyze clean, might prove useful for clinical tests on non-pathological (bone, teeth) or pathological (kidney stone) biomineralizations. Studies using implants with protein-doped calcium phosphate coating can also benefit from this technology. STATEMENT OF SIGNIFICANCE: Our paper deals with an unconventional technical approach for localizing proteins that are occluded in biominerals. This technique relies on the use of molecular recognition atomic force microscopy with antibody-functionalized tips. Although such approach has been employed in other system, this is the very first time that it is developed for biominerals. In comparison to more classical approaches (such as immunogold), AFM microscopy with antibody-functionalized tips allows higher magnification and keeps the scanned surface clean for other biophysical characterizations. Our method has a general scope as it can be applied in human health, for non-pathological (bone, teeth) and pathological (kidney stone) biomineralizations as well as for bone implants coated with protein-doped calcium phosphate.
生物矿化研究中，骨蛋白在生物矿中的空间定位仍然是一个挑战。为了实现这一目标，我们开发了一种基于原子力显微镜（AFM）分子识别测量的新型原位映射技术，该技术需要三个步骤：（1）针对目标蛋白制备多克隆抗体，（2）将其共价偶联到氮化硅 AFM 探针上（“功能化”），以及（3）扫描适当制备的生物矿化表面。我们将该方法应用于一种可溶性壳蛋白 - accripin11 - 最近被确定为扇贻贝 Pinna nobilis 方解石棱柱的主要成分[1]。多项测试表明，accripin11 均匀分布在棱柱的表面上，也存在于围绕方解石棱柱的有机鞘中，表明该蛋白既存在于晶体内部，也存在于晶体之间。我们观察到横向截面的粘附力大约是纵向截面的两倍，表明 accripin11 可能在生物矿化中表现出优先取向。据我们所知，这是第一次通过抗体功能化尖端的分子识别原子力显微镜在生物矿中对蛋白质进行定位。与更“经典”的方法（如免疫金）相比，我们讨论了这种方法的“优缺点”。与更“经典”的方法（如免疫金）相比，这种方法的优点是留下干净的表面进行分析。该技术可能对非病理性（骨骼、牙齿）或病理性（肾结石）生物矿化的临床测试有用。使用带有蛋白质掺杂的磷酸钙涂层的植入物进行的研究也可以受益于该技术。 意义声明：本文介绍了一种用于定位被生物矿化物掩盖的蛋白质的非常规技术方法。该技术依赖于使用抗体功能化尖端的分子识别原子力显微镜。尽管这种方法已在其他系统中得到应用，但这是第一次将其应用于生物矿化物。与更经典的方法（如免疫金）相比，抗体功能化尖端的 AFM 显微镜允许更高的放大倍数，并保持扫描表面清洁，以便进行其他生物物理特性分析。我们的方法具有广泛的适用性，可应用于人类健康领域，用于非病理性（骨骼、牙齿）和病理性（肾结石）生物矿化以及涂有蛋白质掺杂的磷酸钙的骨植入物。
