Towards an effective and accessible chip supported biosensor based on modification of monolayer organisation and fluidity

生物膜是一种高度组织化的结构。许多生物活性化合物与生物膜相互作用，并以非常有选择性的方式改变其结构和组织。磷脂形成生物膜的基本骨架结构。当磷脂层被吸附在汞滴电极（HMDE）上时，它们形成具有与生物膜的磷脂双层的一半非常相似的结构和性质的单层。其原因是，流体磷脂层与光滑的液态汞表面直接相容。该系统的最大优点是，吸附的磷脂层的结构可以非常密切地询问电化学，因为它是支持在导电表面上。以这种方式，可以感测与改变单层结构的生物活性化合物的相互作用。缺点是汞电极易碎、有毒，尽管系统对生物膜活性物质敏感，但不适用于现场使用。另一个缺点是，汞表面成像极为困难。本项目采用上述经过验证的传感系统，并以以下方式对其进行修改。在硅晶片上制造单个铂（Pt）微电极和铂（Pt）微电极阵列。在每个微电极上，微量的汞被电沉积，在每个汞/铂电极上，磷脂单层被沉积。通过电极的边缘效应，保证各磷脂层的稳定性。我们将使用硅晶片阵列进行受控磷脂沉积实验，这在HMDE上是不可能的。我们还将尝试其他磷脂沉积方法。该项目将利用沉积磷脂单层的微电极阵列系统可用于成像的事实。原子力显微镜研究在利兹已经被用来成像温度引起的云母支持磷脂双层显示成核和生长过程中的相变。因此，AFM系统非常适合于对基于微电极的单个芯片上的磷脂单层的电势诱导相变进行成像。这样做是很重要的，因为这些相变的发生对磷脂层与生物膜活性物质的相互作用非常敏感。此外，相变的机制与细胞电穿孔中发生的相变基本相同，具有巨大的物理意义，通过它们的成像可以更好地了解它们。我们还将尝试在不同的电位值下成像该层与膜活性肽的相互作用。原子力显微镜系统将被开发为图像的构象和吸附的抗微生物肽的聚集状态的单分子层，特别是作为一个功能的电位变化。当生物膜活性化合物与汞上的磷脂层相互作用时，它们会改变磷脂层的流动性和组织。这反过来又影响了潜在的诱导相变的特性。这可以通过快速循环伏安法（RCV）非常有效地进行电化学监测。将对分析的干扰进行表征。该项目将开发模式识别技术，以模拟对单个活性化合物的电化学响应。该项目将在硅片上提供一种传感器，该传感器有可能检测天然沃茨中低水平的生物膜活性有机化合物，并评估药物化合物的生物膜活性。清洁Hg/Pt电极和更新感测磷脂层的经证实的可行性将促进将该装置并入具有全自动化和可编程操作的流通系统中。

The biological membrane is a highly organised structure. Many biologically active compounds interact with the biological membrane and modify its structure and organisation in a very selective manner. Phospholipids form the basic backbone structure of biological membranes. When phospholipid layers are adsorbed on a mercury drop electrode (HMDE) they form monolayers which have a very similar structure and properties to exactly half the phospholipid bilayer of a biological membrane. The reason for this is that the fluid phospholipid layer is directly compatible with the smooth liquid mercury surface. The great advantage of this system is that the structure of the adsorbed phospholipid layer can be very closely interrogated electrochemically since it is supported on a conducting surface. In this way interactions with biologically active compounds which modify the monolayer's structure can be sensed. The disadvantage is that Hg electrodes are fragile, toxic and have no applicability for field use in spite of the sensitivity of the system to biological membrane active species. Another disadvantage is that the Hg surface can only be imaged with extreme difficulty. This project takes the above proven sensing system and modifies it in the following way. A single and an array of platinum (Pt) microelectrode(s) are fabricated on a silicon wafer. On each microelectrode a minute amount of Hg is electrodeposited and on each Hg/Pt electrode a phospholipid monolayer is deposited. The stability of each phospholipid layer will be ensured through the edge effect of the electrode. We will use the silicon wafer array to carry out controlled phospholipid deposition experiments which are not possible on the HMDE. We shall also try out other methods of phospholipid deposition. The project will exploit the fact that the microelectrode array system with deposited phospholipid monolayers is accessible for imaging. AFM studies at Leeds have already been used to image temperature induced phase changes in mica supported phospholipid bilayers showing nucleation and growth processes. The AFM system is eminently suitable therefore to image the potential induced phase changes of the phospholipid monolayers on the individual chip based microelectrodes. It is important to do this because the occurrence of these phase transitions is very sensitive to the interaction of the phospholipid layer with biomembrane active species.In addition the mechanism of the phase changes which are fundamentally the same as those occurring in the electroporation of cells are of immense physical interest and a greater understanding of them can be gained through their imaging. We shall also attempt to image the interaction of the layer with membrane active peptides at different potential values. The AFM system will be developed to image the conformation and state of aggregation of adsorbed anti-microbial peptides on the monolayer in particular as a function of potential change. When biomembrane active compounds interact with phospholipid layers on Hg they alter the fluidity and organisation of the layers. This in turn affects the characteristics of the potential induced phase transitions. This can be very effectively monitored electrochemically by rapid cyclic voltammetry (RCV). Interferences to the analysis will be characterised. Pattern recognition techniques will be developed to characterise the electrochemical response to individual active compounds.The project will deliver a sensor on a silicon wafer which has the potential to detect low levels of biomembrane active organic compounds in natural waters and to assess the biomembrane activity of pharmaceutical compounds. The proven feasibility of cleaning the Hg/Pt electrode and renewing the sensing phospholipid layer will facilitate the incorporation of the device into a flow through system with a full automation and programmable operation.