Université de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France.
Biosens Bioelectron. 2013 May 15;43:148-54. doi: 10.1016/j.bios.2012.12.023. Epub 2012 Dec 20.
Surface Plasmon Resonance imaging (SPRi) is a label free technique typically used to follow biomolecular interactions in real time. SPRi offers the possibility to simultaneously investigate numerous interactions and is dedicated to high throughput analysis. However, precise determination of binding constants between partners is not highly reliable. We report here a dendrimer functionalization of gold surface that significantly improves selectivity of the detection of protein-DNA interactions. We showed that amino-gold surface functionalization with phosphorus dendrimers of fourth generation (G4) allowed complete coverage of the gold surface and the increase of the surface roughness. We optimized the conditions for DNA probe deposition to allow accurate detection of a well-known protein-DNA interaction involved in bacterial chromosome segregation. Using this G4-functionalized surface, the specificity of the SPRi response was significantly improved allowing discrimination between protein and DNA interactions of different strengths. Kinetic constants similar to those obtained with other techniques currently used in molecular biology were only obtained with the G4 dendrimer functionalized surface. This study demonstrated the benefit of using dendrimeric surfaces for sensitive high throughput SPRi analysis.
表面等离子体共振成像（SPRi）是一种无标记技术，通常用于实时跟踪生物分子相互作用。SPRi 可同时研究多种相互作用，专门用于高通量分析。然而，对伙伴之间结合常数的精确确定并不十分可靠。我们在这里报告了金表面的树枝状大分子功能化，这显著提高了蛋白质-DNA 相互作用检测的选择性。我们表明，第四代（G4）磷树枝状大分子的氨基金表面功能化允许金表面的完全覆盖和表面粗糙度的增加。我们优化了 DNA 探针沉积的条件，以允许准确检测涉及细菌染色体分离的众所周知的蛋白质-DNA 相互作用。使用这种 G4 功能化表面，SPRi 响应的特异性得到了显著提高，允许区分不同强度的蛋白质和 DNA 相互作用。只有使用 G4 树枝状大分子功能化表面才能获得与目前在分子生物学中使用的其他技术相似的动力学常数。这项研究证明了使用树枝状大分子表面进行敏感的高通量 SPRi 分析的好处。
