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

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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.