Anal. Chem. 2009, 81, 1146–1153
Regenerable Tethered Bilayer Lipid Membrane Arrays for Multiplexed Label-Free Analysis of Lipid-Protein Interactions on Poly(dimethylsiloxane) Microchips Using SPR Imaging Joseph D. Taylor, Matthew J. Linman, Thomas Wilkop, and Quan Cheng* Department of Chemistry, University of California, Riverside, California 92521 We report a microfabrication approach to generate welldefined, addressable, and regenerable lipid membrane arrays in poly(dimethylsiloxane) (PDMS) microchips for label-free analysis of lipid-protein interactions with surface plasmon resonance imaging (SPRi). The multiplexed detection is demonstrated with a tethered bilayer membrane array built in parallel microchannels. These channels allow multiple measurements to be carried out simultaneously, showing low deviations for element-toelement variation in quantifiable signal. Lipid-conjugated receptors were utilized as model systems for protein binding analysis, and the feasibility of regenerating the tethering sublayer after binding was investigated. The results show that the lipid membrane can be removed effectively by nonionic surfactant Triton X-100. The small variance in SPR signal for the buildup process, i.e., 3 µm2/s) after PEG-induced fusion.28
Regeneration of Tethered Membranes and Detection. Many SPRi analyses focus on the ability to specifically build an effective interface for sensing measurement. Regeneration of the interface, especially post-detection, to provide multiple, reproducible analyses on a single substrate is still challenging. The ability to reuse conventional gold substrates is often hindered by the high cost and effectiveness associated with substrate cleaning, time (i.e., overnight SAM incubation, chemical derivatization), and labor involved. Paths have been sought after to maximize the reusability of the substrate without damaging or denaturing immobilized ligands required for proper binding.34 The supported membranes have some clear advantages over protein-based interfaces because of the transient stability of the membranes, and the key for regeneration is to choose mild conditions that permit complete dissociation of the bound complex while keeping the major structure intact. We recently examined the stability of a number of supported membrane structures on nanoglassy gold surfaces.35 Nonionic surfactant Triton X-100 was found to be effective to quantitatively detach the tBLM from the avidin sublayer. The tBLM could be removed using relatively low concentrations (0.5-1% v/v) of Triton. It appears that the submembraneous reservoir increases the surface area for solvent access, thus contributing to decreased stability against surfactants. The additional loss of favorable interactions (i.e., hydration, electrostatic, steric, and/or long-range van der Waals forces) between the polar head groups of the lipid and a suitable hydrophilic surface may also be a causative factor. This condition was further applied here for membrane array removal and regeneration. Figure 3 is a sensorgram constructed with SPR images for an array element inside a PDMS microchannel. Three consecutive
(32) Lei, S. B.; Tero, R.; Misawa, N.; Yamamura, S.; Wan, L. J.; Urisu, T. Chem. Phys. Lett. 2006, 429, 244–249. (33) Haque, M. E.; McIntosh, T. J.; Lentz, B. R. Biochemistry 2001, 40, 4340– 4348.
(34) Linman, M. J.; Taylor, J. D.; Yu, H.; Chen, X.; Cheng, Q. Anal. Chem. 2008, 80, 4007–4013. (35) Taylor, J. D.; Han, J. H.; Phillips, K. S.; Wang, X.; Feng, P.; Cheng, Q. Langmuir 2008, 24, 8127–8133.
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Figure 2. (a) SPR image of the array taken under s-polarized light conditions; (b) SPR image taken under p-polarized light conditions; (c) normalized “difference” image that eliminates the effect of artifacts visible on the surface; (d) 3D-rendered surface plot of the array to show the depth profile of the microfluidic flow channels. The scale bar is 600 µm.
Figure 3. SPR sensorgram constructed from images that shows the complexation of a single array element within a microfluidic channel and demonstrates the membrane removal, regeneration, and detection for a total of three cycles. 1150
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cycles of vesicle attachment, membrane fusion, protein binding, and membrane removal were monitored. The GM1-containing bilayer membranes were used with 175 µg/mL cholera toxin as the binding protein. The initial baseline represents the deposited biot-BSA protein. Following the deposition, 1% BSA was injected and incubated for surface passivation prior to avidin injection. The injection of BSA displayed an increase in the reflectivity, but once flow resumed, the signal returned to the biot-BSA background, showing no net increase in reflectivity. This demonstrates both adequate surface coverage of biot-BSA and its ability to block potential non-specific adsorption. Avidin was subsequently coinjected with BSA and incubated for a short period of time. When the flow was resumed, the signal decreased slightly from the high concentration of BSA in the microchannel. Once the baseline signal stabilized, small unilamellar vesicles were introduced and captured on the avidin sublayer, followed by triggered fusion with PEG. The large spikes in the sensorgram are due to high concentration of the PEG solution, which induces a sudden change of the solution refractive index. CT was then introduced to the membranes using the co-injection method with BSA, which explains why the signal decreased when flow resumed. Neverthe-
Figure 4. SPR images taken prior to and after CT binding (top) and the difference image (bottom right). On the low left are the plot profiles of the channels for the corresponding concentrations.
less, the magnitude of CT binding signal for all three cycles is highly reproducible (3.7% RSD). A 1% solution of Triton X-100 was then used to disrupt and remove the membrane, resulting in a net change in reflectivity back to the avidin baseline. Clearly each cycle returns to essentially the same baseline (
