Development of scanning probe surface plasmon resonance microscopy

面对当前生物分析领域的挑战，需要开发新的分析方法。量化单个细胞或一小群细胞释放的化学信使与细胞形态的关系，将有助于理解疾病的生物学机制。然而，需要传感器的微型化才能在单个细胞的胞外空间实现蛋白质的量化。表面等离子体共振(SPR)生物传感器具有局部监测蛋白质的潜力，其空间分辨率比细胞大小更好。因此，我们的研究将集中在开发一种对蛋白质表现出强烈SPR响应的微型等离子体传感器。将SPR微型生物传感器集成在原子力显微镜(AFM)悬臂梁的顶端，将能够同时测量细胞外空间中细胞和细胞形态的蛋白质。我们将把“扫描探针SPR显微镜”应用到一个模型系统中。趋化因子是一种信使蛋白质。它们最近被认为是调控癌症生长和转移的信号机制的主要贡献者。然而，目前尚不清楚趋化因子是如何迁移和感染其他细胞的。因此，我们的目标是利用扫描探针SPR显微镜来量化从癌细胞到健康细胞的趋化因子梯度。为了达到我们的目标，我们将专注于通过优化各种实验参数来优化SPR微型生物传感器对趋化因子的响应：悬臂材料和几何形状，支撑SPR微型生物传感器的金属薄膜(Au或Ag)的成分和厚度，以及数据分析方法。传统的SPR传感器也将进行优化，以最大限度地提高细胞培养基样中低浓度趋化因子所产生的反应。这些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了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.