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Immunosensing Arrays Based on Air-Stable Tethered Lipid Membrane Mimics and Ligand-Gated Ion Channels

Luis G. Casc„o-Pereira, Mark Gebert, William Throndset, Matt Flynn, Meredith Fujdala, Vicky Huynh, Jim Kellis, and Todd Becker. Genencor International, 925 Page Mill Road, Palo Alto, CA 94304

Lipid bilayers on solid substrates have shown great promise as cell membrane mimics. Potential applications include drug discovery, bioelectronics, and biosensors. To date, the fragile nature of biomimetic lipid bilayers have precluded their incorporation into practical devices, such as biosensors, due to their lack of stability. In this paper, we report on tethered lipid bilayers (t-BLM) used in ion-channel switch sensors [1,2] that were made stable against exposure to an air interface when dried for long-term storage. Upon rehydration the t-BLM regains nearly all of its functionality. We will discuss the role of drying process parameters, formulations and provide some mechanistic insights. Our t-BLM is made of phytanyl lipids and is linked to a gold substrate via disulphide bonds. Moreover, the t-BLM possesses an ionic reservoir made of hydrophilic ester linkers suitable for the incorporation of membrane proteins, especially ion-channels. Ion-channels are linked to antibody fragments and are "gated off" upon analyte binding. We characterize our lipid bilayers at different processing stages by electrochemical impedance spectroscopy (EIS), atomic force microscopy (AFM), fluorescence microscopy, fluorescence recovery after photobleaching (FRAP), the ability to incorporate membrane proteins, in our case the ion channel gramicidin-A, and assay dose response. Bilayers stored wet lose physical integrity and functionality within a few weeks. EIS measurements indicate loss of electrical seal due to increased leakage conductance. Tapping AFM reveals lipid bilayer rearrangement leading to desorption and the appearance of bilayer-free defect patches. In contrast, rehydrated bilayers remain insulating even after prolonged dry storage. Further incorporation of gramicidin allows dimer formation and rise in conductance levels. FRAP studies show that fluidity (percent recovery) and mobility (in-plane diffusion constant) of rehydrated bilayers remain comparable to the fresh, never dried, state. The assay dose response exhibits good linearity and sensitivity upon rehydration. This approach enables long-term storage in the dry state, while taking advantage of the self-assembly nature of lipids in their fluid, wet state for device manufacture.

[1] B.A. Cornell, V.L.B. Braach-Maksvytis, L.G. King, P.D.J. Osman, B. Raguse, L. Wieczorekw, R.J. Pace. "A Biosensor That Uses Ion-channel Switches". Nature 387, 580 (1997).

[2] S. Lee, L.G. Cascao-Pereira, R.F.Sala, S.P.Holmes, K.J. Ryan, and T. Becker. "Ion-channel Swtich Array: a Biosensor for Detecting Multiple Pathogens". Industrial Biotechnology 1, 26 (2005).