Borchers Kirsten, Weber Achim, Brunner Herwig, Tovar Günter E M
Laboratory for Biomimetic Surfaces, Fraunhofer-Institute for Interfacial Engineering and Biotechnology & Institute for Interfacial Engineering, University Stuttgart, Nobelstr. 12, 70569 Stuttgart, Germany.
Anal Bioanal Chem. 2005 Nov;383(5):738-46. doi: 10.1007/s00216-005-3396-9. Epub 2005 Aug 11.
Nanostructured core-shell particles with tailor-made affinity surfaces were used to generate microstructured affinity surfaces by microspotting the particles to form densely packed amorphous nanoparticle layers. These layers provided a large reactive surface for the specific binding of protein ligands from aqueous solution. Biofunctional core-shell particles were synthesized for this purpose that consisted of a silica core with a diameter of 100 nm and an organic shell a few nm thick. The nanoparticle core was prepared by sol-gel chemistry and the shell formed in suspension by organosilane chemistry. The shell provided amino groups or carbonyl groups at its outer surface for subsequent covalent immobilization of streptavidin, rabbit IgG antibodies or goat IgG antibodies. AlexaFluor 647-conjugated and biotinylated cytochrome C and CyDye-labeled anti-rabbit IgG and anti-goat IgG were probed as model analytes. The core-shell nanoparticles were spotted using a pin-ring micro-arrayer onto microscope glass slides that were coated with a polycation monolayer by dip-coating prior to nanoparticle deposition. Amorphous particle layers of well-defined thicknesses in the range of 100 nm to 2 microm were obtained by printing aqueous particle suspensions containing 5-500 mg/mL (0.5-50 wt%) of silica particles. The specific affinity of the plotted nanoparticulate capture surface was demonstrated by binding Cy3-labeled donkey anti-rabbit IgG and Cy5-labeled mouse anti-goat IgG to immobilized rabbit IgG and goat IgG particles. The signal intensity per spot increased for any given analyte concentration when the amount of particles per spot was augmented. This was attributed to the increasing integration of receptor molecules per surface footprint, which shifted the binding equilibrium towards the formation of the receptor-ligand complex. Additionally, the locally-increased supply of receptor molecules at the nanoparticulate microchip surface resulted in a wide dynamic range of 4 fM-20 nM (covering six orders of magnitude).
具有定制亲和表面的纳米结构核壳颗粒通过微点样这些颗粒以形成紧密堆积的无定形纳米颗粒层来生成微结构亲和表面。这些层为从水溶液中特异性结合蛋白质配体提供了大的反应表面。为此合成了生物功能核壳颗粒，其由直径为100nm的二氧化硅核和几纳米厚的有机壳组成。纳米颗粒核通过溶胶-凝胶化学制备，壳通过有机硅烷化学在悬浮液中形成。壳在其外表面提供氨基或羰基，用于随后将链霉亲和素、兔IgG抗体或山羊IgG抗体共价固定。将AlexaFluor 647共轭和生物素化的细胞色素C以及CyDye标记的抗兔IgG和抗山羊IgG作为模型分析物进行检测。使用针环微阵列仪将核壳纳米颗粒点样到显微镜载玻片上，该载玻片在纳米颗粒沉积之前通过浸涂涂覆有聚阳离子单层。通过印刷含有5-500mg/mL（0.5-50wt%）二氧化硅颗粒的水性颗粒悬浮液，获得了厚度在100nm至2μm范围内的明确定义的无定形颗粒层。通过将Cy3标记的驴抗兔IgG和Cy5标记的小鼠抗山羊IgG与固定的兔IgG和山羊IgG颗粒结合，证明了所绘制的纳米颗粒捕获表面的特异性亲和力。当每个点的颗粒量增加时，对于任何给定的分析物浓度，每个点的信号强度都会增加。这归因于每个表面足迹中受体分子的整合增加，这使结合平衡向受体-配体复合物的形成方向移动。此外，纳米颗粒微芯片表面受体分子的局部供应增加导致了4fM-20nM的宽动态范围（涵盖六个数量级）。
