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。磷脂构成了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，它们形成的单层膜具有非常相似的结构和性质，恰好是生物膜磷脂双层的一半。这是因为液体磷脂层与光滑的液体汞表面直接相容。该体系的最大优点是，由于磷脂层被负载在导电表面上，因此可以用非常紧密的电化学方法来研究它的结构。通过这种方式，可以感觉到与改变单层结构的生物活性化合物的相互作用。缺点是汞电极脆弱，有毒，不适用于现场使用，尽管该系统对生物膜活性物种很敏感。另一个缺点是，汞表面只能以极高的难度成像。本项目采用了上述经过验证的传感系统，并对其进行了以下改进。在硅片上制作了单个和阵列铂(铂)微电极(S)。在每个微电极上电沉积微量的汞，在每个汞/铂电极上沉积一层磷脂单分子膜。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。该项目将利用具有沉积的磷脂单分子层的微电极阵列系统可用于成像的事实。利兹大学的AFM研究已经被用于成像云母支撑的磷脂双层膜中温度引起的相变，显示了成核和生长过程。因此，原子力显微镜系统非常适合于成像单个芯片微电极上磷脂单分子膜的电势诱导相变。这样做很重要，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机制基本相同，具有巨大的物理意义，通过成像可以更好地了解它们。我们还将尝试成像该层与不同电位值的膜活性多肽的相互作用。AFM系统将被开发成成像吸附在单层上的抗菌肽的构象和聚集状态，特别是作为电位变化的函数。当生物膜活性化合物与汞表面的磷脂层相互作用时，它们改变了磷脂层的流动性和组织。这反过来又影响了电势诱导相变的特性。通过快速循环伏安法(RCV)可以非常有效地监测这一点。对分析的干扰将进行表征。该项目将开发模式识别技术来表征单个活性化合物的电化学反应。该项目将在硅片上安装一个传感器，该传感器有可能检测天然水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。已证实的清洁汞/铂电极和更新传感磷脂层的可行性将有助于将该设备整合到具有全自动化和可编程操作的流动系统中。

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.