Development of scanning probe surface plasmon resonance microscopy

新的分析方法的发展是需要面对当前的挑战，在生物分析。从单个细胞或一小群细胞释放的化学信使的定量与细胞形态的关系将有助于理解疾病的生物学机制。然而，需要传感器的固定化以实现单个细胞的细胞外空间中的蛋白质定量。表面等离子体共振（SPR）生物传感器表现出的潜力，局部监测蛋白质的空间分辨率优于细胞的大小。因此，我们的研究将集中在开发一个微等离子体传感器表现出强烈的SPR响应的蛋白质。SPR微生物传感器的原子力显微镜（AFM）悬臂的尖端上的集成将使细胞和细胞形态的细胞外空间中的蛋白质的同时测量。我们将应用“扫描探针SPR显微镜”的模型系统。趋化因子是一种信使蛋白。它们最近被确定为调节癌症生长和转移的信号传导机制的主要贡献者。然而，它仍然是未知的趋化因子如何迁移和感染其他细胞。 因此，我们的目标是利用扫描探针SPR显微镜的量化的趋化因子梯度从癌细胞到健康细胞。为了达到我们的目标，我们将专注于优化SPR微生物传感器的响应趋化因子通过优化各种实验参数：悬臂梁材料和几何形状，组成和厚度的金属薄膜（Au或Ag）支持SPR微生物传感器，和数据分析方法。常规SPR传感器也将被优化以最大化由细胞培养基样品中的低浓度趋化因子产生的响应。该传感器将专门用于监测趋化因子CXCL12。这种重要的趋化因子参与乳腺癌转移的生物学机制。

The development of novel analytical methodologies is required to face the current challenges in bioanalysis. The quantification of the chemical messengers released from a single cell or a small group of cells in relation with cellular morphology will help understand the biological mechanism of diseases. However, miniaturisation of sensors is required to achieve protein quantification in the extracellular space of a single cell. Surface Plasmon Resonance (SPR) biosensors exhibit the potential to locally monitor proteins with spatial resolution better than the size of a cell. Hence, our research will focus on developing a micro-plasmonic sensor exhibiting a strong SPR response to proteins. The integration of the SPR micro-biosensor on the tip of an Atomic Force Microscopy (AFM) cantilever will enable the simultaneous measurement of proteins in the extracellular space above cells and cellular morphology. We will apply "scanning probe SPR microscopy" to a model system. A chemokine is a messenger protein. They have been recently identified as a major contributor to the signalling mechanism regulating cancer growth and metastasis. However, it remains unknown how chemokines migrate and infect other cells. Thus, we aim at utilising scanning probe SPR microscopy for the quantification of the chemokine gradient from cancerous to healthy cells. To reach our objectives, we will focus on the optimisation of the response of SPR micro-biosensors to chemokines by optimising various experimental parameters: the cantilever material and geometry, the composition and the thickness of the metal thin film (Au or Ag) supporting the SPR micro-biosensor, and the data analysis method. Conventional SPR sensors will also be optimised to maximise the response generated by low concentration of chemokines in cell culture media samples. The sensors will be specifically applied to monitor the chemokine CXCL12. This chemokine of importance is involved in the biological mechanism of breast cancer metastasis.