(203d) A Lipobead Microarray Made by Microfluidic Entrapment
This presentation will describe the design of a bead-based, microfluidic array platform for the display of cell membrane sequestered receptors, to be used for the high-throughput, multiplexed screening of these receptors with extracellular target molecules. The design will be of multiple use, but our particular focus is to utilize the platform as a sensor for the detection and identification of bacterial exotoxins (pathogens) by displaying in the platform the cell membrane glycolipids which are selectively bound by the toxins as their first step in cell invagination.
Our array platform is a microfluidic cell fabricated by soft lithography out of polydimethyl siloxane (PDMS) with the surfaces of the fluidic pathways functionalized with polyethylene glycol (PEG) to prevent nonspecific adsorption of biomolecules. The cell consists of a single rectangular channel in which an obstacle course of capture elements (traps) have been arrayed to capture microbeads. The channel is approximately 60 microns in height and 1- 3 mm in width. The microbeads are 40 microns in diameter glass particles. Each trap consists of two concave elements to form a wine cup-shaped enclosure. Each element is separated from the other by a 25 microns gap in width, so that the flow can pass through the wine cup-shaped trap and a certain proportion of total streamlines can carry the lipobeads into the trap. Their capture is undertaken by streaming the microbeads through the cell, allowing the particles to be entrapped by the capture elements. In principle, the cell can be partially filled with one set of entrapped microbeads, spatially indexed by optical inspection, and then filled sequentially and indexed with other sets of beads to form a complete array of entrapped microbeads.
The microbeads are the hosts for the cell membrane receptors. In order to accommodate the strict requirement that the receptors need to be sequestered in a lipid bilayer to retain their selective and high affinity binding, lipid bilayers of DMPC are first formed around the particles to form lipobeads. This is undertaken by forming vesicles of DMPC by hydration of dried films of the phospholipid, and then fusing the vesicles to the silica surface. The receptors are sequestered in the bilayers by constituting the film with the receptors, or, in the case of delicate proteins, inserting them afterwards into the formed bilayer.
A functioning microfluidic array is constituted by trapping different sets of lipobeads, with each set displaying a different receptor. After entrapment, analyte solutions with labeled targets are flowed through the cell, and microbeads binding the labeled target can be identified using wide field fluorescence microscopy. The receptor on the microbead surface is identified by the array registrycompiled during the filling of the traps. The small size of the microbeads and the array allows for a miniaturization which is at least an order of magnitude smaller in size than can be accomplished with conventional microarrays set-up by using robotic fluidic dispensing to form spotted arrays.
In this presentation, we demonstrate the utility of this platform for multiplexed screening using biotin-streptavidin as an example of a selective biomolecular interaction. Biotin conjugated lipids ? the receptor - are sequestered into a DMPC lipobead bilayer, and displayed in the array by fluidic entrapment. A second set of lipobeads, not containing the biotin ?receptor? is also displayed by sequential entrapment. An analyte containing fluorescently labeled Neutravidin is then flowed through the cell. The Neutravidin selectively binds only to the microbeads containing biotin displayed on their surface as evidenced by the localized fluorescence on only the biotin lipobeads.