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。从单个细胞或一小群细胞中释放的与细胞形态相关的化学信使的定量将有助于了解疾病的生物学机制。然而，在单个细胞的细胞外空间实现蛋白质定量需要传感器的小型化。表面等离子体共振（SPR）生物传感器显示出局部监测蛋白质的潜力，其空间分辨率优于细胞的大小。因此，我们的研究将集中于开发一种对蛋白质具有强SPR响应的微等离子体传感器。SPR微型生物传感器集成在原子力显微镜（AFM）悬臂顶端，可以同时测量细胞上方细胞外空间的蛋白质和细胞形态。我们将“扫描探针SPR显微镜”应用于模型系统。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机制的主要贡献者。然而，趋化因子如何迁移和感染其他细胞仍不清楚。因此，我们的目标是利用扫描探针SPR显微镜定量癌细胞到健康细胞的趋化因子梯度。为了达到我们的目标，我们将通过优化各种实验参数来优化SPR微生物传感器对趋化因子的响应：悬臂材料和几何形状，支持SPR微生物传感器的金属薄膜（Au或Ag）的组成和厚度，以及数据分析方法。传统的SPR传感器也将被优化，以最大限度地提高细胞培养基样品中低浓度趋化因子产生的反应。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.