Microfluidic immunoassay for bacterial toxins with supported phospholipid bilayer membranes on poly(dimethylsiloxane)

Abstract

We report a heterogeneous immunoassay for cholera toxin (CT) using supported bilayer membranes (SBMs) in a poly(dimethylsiloxane) (PDMS) microfluidic device. Phosphatidylcholine membranes assembled on plasma-oxidized PDMS by vesicle fusion bring about favorable surface properties, such as improved wettability and protein resistance. Contact angle measurements show that the lipid membranes can preserve hydrophilic surfaces for hours, whereas untreated substrates rapidly undergo hydrophobic recovery. Fluorescence recovery after photobleaching performed in situ reveals that the membranes have relatively high lateral mobility. Experimental data-fitting to theoretical models yields diffusion coefficients of 1.8 +/- 0.7 microm(2)/s on PDMS and 3.4 +/- 0.8 microm(2)/s on glass. Fluorescence studies utilizing tagged proteins show that SBMs reduce nonspecific adsorption of avidin and BSA on PDMS by 2-3 orders of magnitude, as compared to that on plasma oxidized surfaces. SBMs and their protein-resistant properties are not significantly affected by long flow times, indicating good membrane stability. These studies increase our understanding of the relationship between molecular level interactions and membrane properties, allowing for development of a rapid heterogeneous immunoassay for CT in PDMS microchips with cell surface receptor molecules. Using optimized sample injection and buffer washing conditions, microfluidic immunoassay of CT is complete within 25 min, and a dynamic range over 3 orders of magnitude with a detection limit of 8 fmol of toxin is achieved.