Biomedical Engineering Reference
In-Depth Information
response. Carbohydrate presenting lipids also comprise one of the primary
recognition elements of bacterial pathogenesis. Unlike conventional receptor-
ligand interactions, the presentation of carbohydrate ligands in itself has a
significant influence on the recognition event [25, 26]. The high a
nity and
specificity of carbohydrate mediated recognition are achieved through multi-
ple simultaneous interactions between multiple copies of proteins with multi-
ple carbohydrate ligands. Non-cell based methods for studying carbohydrate
recognition have to consider the appropriate presentation of the ligand and
its surface density such that it mimics ligand presentation at the cell surface.
Supported membranes that are laterally fluid enable this biomimetic presen-
tation enabling processes such as ligand clustering. Membrane microarrays are
well suited for studying carbohydrate mediated recognition by combining the
multiplexing ability, miniaturization and convenience afforded by microarray
technology with the biomimetic environment provided by supported mem-
branes.
We have demonstrated the fabrication of lipid microarrays containing gan-
gliosides and described their use for detecting bacterial toxins and for the
screening of potential inhibitors [20]. Gangliosides are a class of carbohydrate
derivatized lipids that comprise approximately 5-10% of the lipid composi-
tion of the plasma membrane of neuronal and glial cells. The interaction of the
cholera and tetanus toxins with the GM1 and GT1b gangliosides, respectively,
are two well-studied examples of ganglioside-toxin interactions.
Microarrays of gangliosides were made by printing sonicated dispersions
of dilaurylphosphatidylcholine (DLPC) containing gangliosides (4 mol%). Fig-
ures 16.5a-f show fluorescence images of these arrays treated with solutions
of toxins. When the array is treated with a solution of fluorescently labelled
cholera toxin (FITC-CTx) (Fig. 16.5b) or the tetanus toxin (FITC-TTx)
(Fig. 16.5c), strong fluorescence is observed from microspots containing the
GM1 and GT1b gangliosides, respectively. Specific inhibition of binding of
FITC-CTx to GM1 microspots is observed when the solution contains excess
unlabelled cholera toxin (compare Figs. 16.5d, e, f). This inhibition is dose
dependent and yields an IC50 value of
20 nM (data not shown).
These studies demonstrate the use of membrane microarrays for the mul-
tiplexed detection of toxins and the screening of potential inhibitors. The
development of membrane microarrays for this application is especially perti-
nent given the recent concerns about biological warfare and the emergence of
bacterial resistance to antibiotics.
∼
16.3 Conclusions
Molecules in the membrane direct events both inside the cell and between
cells, and there is hardly any aspect of cell viability that is not influenced
by recognition events at the cell membrane. It is therefore not surprising
