bio sphere
Using dispersion to detect binding
A
chance observation led Jay Groves and his colleagues at the University of California, Berkeley, and the Lawrence Berkeley National Laboratory to develop a new label-free assay for detecting ligand–receptor binding (Nature 2004, 427, 139–141). “We were studying a colloid as a way of working with [biological] membranes and fortuitously discovered that [the colloid] had phase transitions,” says Groves. “We realized that the phase transition was a very sensitive readout of what was going on at the membrane surface.” The researchers coated silica particles with phospholipid bilayers containing membrane proteins. When these coated particles were added to a water-filled well, they floated above the bottom surface by electrostatic repulsion and formed a colloidal substance. The colloid can exist in various phases, which are differentiated by how tightly the particles are packed together. “We thought it was interesting that [the colloid] exists in multiple phases, but my student [Mike Baksh] was convinced that he could predict, just by looking at the phase, how much of a particular ligand was in solution,” says Groves. “Then, it turned into a little bet.” At Baksh’s insistence, the researchers studied this colloid much more closely. They discovered that by making slight changes to the lipid composition of the bilayers, they could prime the colloid to be near a phase transition. With small perturbations, such as the addition of a protein that binds to a membrane component, phase transitions occurred. Groves and his colleagues soon realized that these interesting changes could form the basis of a new, easy-to-implement assay for ligand–receptor binding on a membrane. Membrane proteins are difficult to study because they often have 368 A
The new assay can be different properties (a) conducted with simple, after they are recommonly available equipmoved from a bioment. A plate reader is logical membrane. the only instrument that “This has been a is required. The assay is serious issue for also more sensitive than the development surface plasmon resonance of drugs against 0s to protein binding events, membrane targets says Groves. He adds that in the past,” says (b) his method is “certainly Groves. sensitive enough to pick At the start of up biologically relevant the assay, the partiinteractions.” cles are condensed, Paul Cremer at Texas but they are almost A&M University comready to transition mends the team’s ingenuinto a less tightly ity and says the assay is a packed phase. When 60 s novel concept. “It’s always a ligand is added extremely exciting when that binds to a mole- When a ligand is added, the colnew techniques can be adcule in the memloid transitions from (a) a convanced, and I think that brane, the colloid densed phase to (b) a dispersed this was an excellent idea,” becomes dispersed phase. (Adapted with permission. says Cremer. He adds, as the silica beads Copyright 2004 Macmillan Magahowever, that its practical move away from zines Ltd.) utility still must be proven. each other. Although Robert Corn at the University of these events are visible by conventional California, Irvine, says the work is an light microscopy, Groves and colleagues excellent first step and will become have developed software to track the widely applicable if the sensitivity of process quantitatively. the assay is increased. Groves agrees The researchers tested the assay by that the sensitivity could be increased, incorporating a fluorescently labeled lipid into the membrane. Although fluo- and his group is developing ways to do this. rescence is not necessary for detection, The researchers also are optimizing the labeled lipid was initially used to enother aspects of the assay. They are still sure that the particles were well coated trying to determine the best lipid comwith membrane material. When an antipositions, and the membrane-coated parbody that binds to the fluorophore was ticles must be stabilized for long-term added to the condensed colloid, the storage. Groves and his team also plan particles dispersed. In separate experito detect the binding of multiple memments, Groves and colleagues incorpobrane-associated molecules with this rated two different proteins into the method. “That is something for which membranes. Phase transitions only octhere are no high-throughput assays, to curred when the appropriate ligand was my knowledge,” says Groves. a added to the colloid; this result demon—Katie Cottingham strates that the assay is specific.
A N A LY T I C A L C H E M I S T R Y / O C T O B E R 1 , 2 0 0 4
© 2004 AMERICAN CHEMICAL SOCIETY
