Fabrication of Phospholipid Bilayer-Coated Microchannels for On-Chip Immunoassays

Abstract

Herein we describe a new class of microfluidic immunoassays based upon solid supported lipid bilayers. Two-dimensionally fluid bilayer material, which can accommodate multivalent binding between surface-bound ligands and aqueous receptors, was coated on the surface of poly(dimethylsiloxane) microchannels. The bilayers contained dinitrophenyl (DNP)-conjugated lipids for binding with bivalent anti-DNP antibodies. Twelve independent data points of surface coverage versus bulk protein concentration could be made simultaneously by forming a linear array of channels and flowing fluorescently labeled antibodies into them. This enabled an entire binding curve to be obtained in a single experiment. The measured apparent binding constant for the DNP/anti-DNP system was 1.8 microM. The methodology for performing heterogeneous assays developed here not only produces rapid results but also requires much less protein than traditional procedures and eliminates some standard sources of experimental error.

Multiplexing Ligand−Receptor Binding Measurements by Chemically Patterning Microfluidic Channels

“…6,39,40 Briefly, the appropriate lipid mixtures were combined in organic solvent. Chloroform was used to dissolve most lipids; however, methanol was used as the solvent for GM1 as this glycolipid is not soluble in pure chloroform.…”

“…This created an evanescent wave above the interface, which exponentially decayed to its 1/e value over ~70 nm. 6,43 This allowed the proteins bound to the SLBs to be studied with high specificity. All images were collected using Metamorph software (Universal Imaging Corp.) and transferred to Microsoft Excel and Sigma Plot for further processing.…”

“…5 In previous work it has been demonstrated that microfluidic devices can be designed for measuring binding affinities in multivalent systems. [5][6][7][8][9][10][11][12][13][14] In our previous setup, ligands were incorporated into supported lipid bilayers (SLBs) coated on the walls and floors of polydimethylsiloxane/glass microchannels. Linear arrays of channels were then monitored by total internal reflection fluorescence microscopy (TIRFM) 15 to obtain equilibrium dissociation constants in one-shot assays.…”

Fabrication and Characterization of Spatially Defined, Multiple Component, Chemically Functionalized Domains in Enclosed Silica Channels Using Cross-Linked Phospholipid Membranes

“…In particular, self-assembly of SPBs on surfaces increases their versatility and has been exploited in situations where planar lipid deposition techniques, for example, Langmuir-Blodgett (LB) and Langmuir-Schaefer (LS), can not be used. 27 Incorporation of chemically functionalized lipids provides a basis for immobilization of proteins or other biologically active compounds in SPBs through both covalent and noncovalent interactions. 26 Moreover, SPBs support the function and integrity of many membrane proteins, allowing investigation of ligand-receptor interactions.…”

Surface modification in microchip electrophoresis

“…The main focus is on the improvement of electrophoretic separations by the reduction of analyte-wall interactions and the manipulation of electroosmosis. Approaches for surface derivatization of microfluidic chips in order to introduce additional functionality such as immunoassays [33][34][35][36], stationary phases for electrochromatography [37][38][39][40] and immobilized reagents [41] and enzymes [42,43] will not be discussed in detail.…”