Novel biosensors using intact liposome microarrays.

摘要

We have developed protocols to array individual, intact small unilamellar vesicles (liposomes) onto chemically modified microwell substrates. The substrates have microarrays of 1.2 mm diameter wells in a square pattern fabricated by photolithography and reactive ion etching on a silicon wafer coated with silicon dioxide layer. The background of the wells is modified using polyethylene glycol terminated silane self assembled monolayer using contact printing with a PDMS stamp and the wells are modified to have Neutravidin(TM) in subsequent steps. Liposomes of about 1mm diameter, with 5% Biotin lipid in the lipid bilayer, are exposed to these substrates, which results in the selective attachment of intact individual liposomes into wells by a 'Biotin-Neutravidin' interaction. We envision the use of these arrays as biosensors using membrane proteins or receptors incorporated in the lipid bilayer of the arrayed liposomes.; This thesis also describes the use of this platform to display a cell membrane ganglioside and its use in detecting the presence of a corresponding toxin. The lipid bilayer of the liposomes is modified to have 5% monosialoganglioside GM1, which is used as an antigen and a highly selective receptor for cholera toxin. After arraying these liposomes individually into the chemically modified microwell substrates, these arrays are exposed to fluorescently tagged Cholera Toxin Subunit B to verify selective attachment of cholera toxin to GM1 liposomes. Various control experiments involving exposure of Cholera Toxin Subunit B to chemically modified microwell substrates with no liposomes and with liposomes without GM1 receptors, are performed to show high selectivity of cholera toxin towards GM1 receptor modified liposomes arrayed on microwell substrates. Various steps involved in the protocol are confirmed using Atomic Force Microscopy, Fluorescence Microscopy, Particle Size Analysis, Zeta Potential measurements and Confocal Laser Scanning Microscopy.