Research
Lipid Microarrays:
There
is an increasing interest in the development of microarray based assays
for high-throughput analysis and detection of biomolecules such as DNA
and proteins. Array-based analyses have proven extremely useful
for DNA detection and sequencing. The general versatility of
microarrays for multi-variable, high-throughput analysis has also been
applied to solve analytical problems in the emerging field of
proteomics. One area of chemical and biological analysis which
could potentially benefit from the use of microarrays is the study of
biological membranes. For example, the ability to form stable
lipid membrane arrays would be useful for studies on membrane
associated proteins, which account for up to two-thirds of known drug
targets. Microfluidics has proven to be a promising method for
producing patterned lipid bilayers; however, the 2D nature of these
devices limits the addressable elements in an array to linear channels
on the surface. We have developed a means to create multicomponent
lipid bilayer arrays using a 3D continuous flow microspotter (CFM)
system developed in the laboratory of Prof. Bruce Gale (Department of
Engineering, University of Utah). The high density production of
lipid arrays has potential applications in many fields such as
biosensing, drug discovery, proteomics and clinical diagnostics.
Label-Free Detection of Proteins and Small-Moleclues:
Analytical methods which can quantify the interaction of proteins and
low molecular weight drug compounds with biological membranes in a
label-free manner are needed to address a growing number of questions
in the areas of pharmaceutics and biotechnology. What is ne
eded is a
noninvasive method to detect drug and protein association to the lipid
membrane without the use of an extrinsic label. The nonlinear technique
of second harmonic generation (SHG) may hold the answer to
this
problem. We have successfully demonstrated that SHG is a viable tool for label-free detection of protein adsorption to biological membranes. Our future goals are to expand the use of SHG and related nonlinear optical methods for the detection not only of proteins but small molecule-drugs with biological membranes. These studies are being coupled with our capabilities to produce multi-component lipid bilayer arrays (as discussed above) to provide an efficient high-throughput, label-free assay for the interrogation of protein and small molecule interactions with membranes. The practical implementation of these technologies will have a significant impact on the pharmacological screening of drug candidates, and the investigation of drug-membrane
interactions.
Lipid Bilayer Structure and Dynamics:
We are also pursuing research aimed at understanding the complex interplay between the movement of lipid species across the cellular membrane and the establishment of lipid compositional asymmetry. A full understanding of the mechanism by which
membrane
asymmetry is achieved and maintained in cellular systems has not been
realized to date; primarily due to the difficulty of
studying membrane biophysical phenomena in a non-destructive or
non-perturbing fashion. It has been suggested that lipid membrane
asymmetry is maintained by unidirectional lipid transporters, in
conjunction with a high energetic barrier to translocation which limits
the rate at which lipids might spontaneously translocate across the
membrane. However, a growing number of publications demonstrate cases
of rapid spontaneous translocation of phospholipids.The connection between lipid compositional asymmetry and flip-flop in planar supported lipid bilayers is being explored using a novel application of sum-frequency vibrational spectroscopy (SFVS) developed by our group to selectively probe the asymmetry in a planar-supported lipid bilayer (PSLB). This new surface analytical method allows for the direct detection of lipid flip-flop without
the
need for a fluorescent or spin-labeled lipid probe, which can alter the
measured translocation rates. The goal of this research is to use this
surface analytical tool to address some of the central issues
concerning the transbilayer movement and establishment of lipid
asymmetry in bilayer systems. Our work is attempting to address the
effect of phospholipid fatty acid alkyl chain length and saturation on
the rate of flip-flop, and the effect of headgroup chemistry and charge
on lipid migration. In addition, the coupling of lipid flip-flop
energetics to the establishment of lipid asymmetry is also being
explored. Theses studies are aimed at providing physical insight into
the mechanism of lipid compositional asymmetry.