Craig AspinwallAssociate Professor
Building: OC 322
Education and Appointments
Chemical Analysis of Cellular Transduction Using Sensors and Separations
Analytical Chemistry, Bioanalytical Chemistry, Biological Chemistry, Biophysics, Cell Physiology, Sensors, Instrumentation
Secretion of hormones and neurotransmitters (NTs) is critical for biological function in higher organisms. Defective regulation of hormone and NT levels leads directly to a wide range of human disorders, including diabetes, depression, addiction. Quantification of hormones and NTs provides key benchmarks for diagnosis and prognosis of disease and dysfunction while also enabling elucidation of the molecular basis of disease. While a number of key advances have been made in the arena of cellular analysis, the vast majority of physiologically-relevant analytes cannot be detected with sufficient sensitivity, temporal and/or spatial resolution. The primary focus of our research is to develop innovative analytical approaches that enable detection of key biomolecules and to subsequently apply those methods to develop a better understanding of complex biological signaling pathways and the underlying molecular basis for biological function. To realize these goals, we utilize a multidisciplinary approach that encompasses a wide range of research areas including materials chemistry, polymer chemistry, nanotechnology, biochemistry, cell biology, biotechnology and biophysical chemistry. This research program provides a unique, interdisciplinary training environment whereby students develop a number of fundamental and applied research skills and talents resulting in an integrated educational experience. A subset of our research projects are outlined below.
Porous nanoshells for intracellular sensing and manipulation: We are developing a highly-innovative sensor utilizing novel porous nanoshell architectures with enhanced chemical and physical stability that we will utilize to investigate cellular signal in pancreatic α - and β- cells. Highly-stable nanoshells are prepared via formation of a polymer scaffold within a phospholipid membrane via direct UV-photopolymerization of a) reactive phospholipid monomers or b) an interpenetrating polymer network prepared from reactive hydrophobic monomers partitioned into the bilayer lamella. Size-selective pores are integrated into the membrane via the native phospholipid monomer packing (bisSorbPC) or integration of pore-forming peptides. As seen in Figure 1, the porous phospholipid nanoshell (PPN) functions in a manner analogous to a nanometer sized dialysis membrane and thus can be used to encapsulate a number of selective indicator chemistries, e.g. enzymes, aptamers, FRET-probes, etc. and to exclude large molecular weight species in the external environment. The size-selectivity of the pore facilitates transport of small molecular weight analyte species, irrespective of charge, while excluding large molecular weight compounds e.g. proteins and enzymes that may degrade sensor performance. Thus the PPN serves as a protective shell for encapsulated sensor materials. Our development of the PPN platform centers on key molecular signals in the physiologically important, insulin-secreting pancreatic β-cell and glucagon-secreting pancreatic α-cell. Once developed, we will utilize PPNs to monitor dynamic changes in intracellular signal, including glucose, pyruvate, and inositol hexakisphosphate (IP6) as a function of extracellular modulators, particularly glucose
Biomimetic, polymer lipid stationary phases for advanced chromatographic separations: We are developing a highly innovative series of membrane protein-functionalized, highly stable phospholipid bilayer (PLB) matrices for highly specific separation, detection and identification of membrane protein ligands from complex biomolecular solutions. Incorporation of membrane proteins into stabilized PLB stationary phases provides a novel basis for rapid separation and identification of physiologically and pharmacologically important analytes, while IC functionalized detector cells facilitates functional screening of IC modulators (Fig. 2). The enhanced stability of the PLB provides significant improvements in reproducibility among analyses and eliminates the considerable variability often encountered in cell-based assays. Further, decreased non-specific adsorption of PLB-coated surfaces will facilitate analysis of more complex samples and matrices. We are leveraging our considerable expertise in polymeric phospholipid materials and chemical separations to develop these key enabling technologies that facilitate the analysis of complex physiological and pharmacological molecular libraries and facilitate the next generation of biomolecular separations that can be utilized to investigate new hypotheses in the arena of IC and GPCR regulation.
GPCR-Ion channel functionalized sensors for pharmacological and physiological screening. In collaboration with Prof. Saavedra, we are developing stabilized ion channel (IC) functionalized sensors for label-free detection of hormones and neurotransmitters. ICs serve as regulators between the intracellular and extracellular environments via ion conductance and ligand modulation. Electrophysiological (EP) measurement of ligand-modulated ion flux across an IC-functionalized membrane is a highly sensitive, label-free method for detecting IC agonist and antagonist activity. In collaboration with the Vivaudou group at IBS in Grenoble, France, we are developing sensor platforms utilizing chimeric ion channels that are responsive to a range of NTs and hormones for cellular and clinical investigations (Fig. 3).
|1. Aspinwall, C.A. Huang, L.; Lakey, J.R.T.; Kennedy, R.T. “ Comparison of amperometric methods for detection of exocytosis from single pancreatic beta-cells of different species.” Anal. Chem. 71: 5551-5556, 1999.
2. Ross, E.E.; Mansfield, E.; Huang, Y.; Aspinwall, C.A. “In situ Fabrication of 3-Dimensional Chemical Patterns in Fused Silica Separation Capillaries with Polymerized Phospholipids.” J. Am. Chem. Soc. 127: 16756-7, 2005.
3. Braun, K.L.; Hapurachchi, S.; Fernandez, F.; Aspinwall, C.A. “Fast Hadamard Transform Capillary Electrophoresis for On-line, Time-Resolved Chemical Monitoring.” Anal. Chem. 78: 1628-1635, 2006.
4. Hapuarchchi, S.; Premeau, S.; Aspinwall, C.A. “High Speed Capillary Zone Electrophoresis with Online Photolytic Optical Injection.” Anal. Chem. 78: 3674-3680, 2006.
5. Cheng, Z.; D’Ambruoso, G.D.; Aspinwall, C.A. “Stabilized Porous Phospholipid Nanoshells” Langmuir 22: 9507-9511, 2006.
6. Mansfield, E.; Ross, E.E.; Aspinwall, C.A. “Preparation and Characterization of Cross-linked Phospholipid Bilayer Capillary Coatings for Protein Separations” Anal. Chem. 79: 3135-3141, 2007.
7. Roberts, D.L.; Ma, Y.; Bowles, S.E.; Janczak, C.M.; Pyun, J.; Saavedra, S.S.; Aspinwall, C.A. “Polymer-stabilized phospholipid vesicles with a controllable, pH-dependent disassembly mechanism” Langmuir 25: 1908-1910, 2009.
8. Heitz, B.A.; Xu, J.; Hall, H.K., Jr.; Aspinwall, C.A.Saavedra, S.S. “Enhanced long-term stability for single ion channel recordings using suspended poly(lipid) bilayers.” J. Am. Chem. Soc., 131: 6662-6663, 2009.
9. Heitz, B.A.; Jones I.W.; Hall, H.K. Jr.; Aspinwall, C.A. Saavedra, S.S. “Fractional polymerization of a suspended planar bilayer creates a fluid, highly stable membrane for ion channel recordings.” J. Am. Chem. Soc. 132: 7086-7093, 2010.
10. Heitz, B.A.; Xu, J.; Jones, I.W.; Keogh, J.P.; Comi, T.J.; Hall, H.K.; Aspinwall, C.A.Saavedra, S.S. “Polymerized Planar Suspended Lipid Bilayers for Single Ion Channel Recordings: Comparison of Several Dienoyl Lipids.” Langmuir 27: 1882-1890, 2011.
11. Janczak, C.M. and Aspinwall, C.A. “Composite Nanoparticles: The Best of Two Worlds.” Anal. Bioanal. Chem. 402: 83-88, 2012.
12. Gallagher, E.S.; Comi, T.J.; Braun, K.L.; Aspinwall, C.A. “Online Photolytic Optical Gating of Caged Fluorophores in Capillary Zone Electrophoresis Utilizing an Ultraviolet Light Emitting Diode.” Electrophoresis, In press, 2012.
13. Muhandiramlage, T.P.; Cheng, Z.L.; Roberts, D.L.; Keogh, J.P.; Hall Jr.,H.K.; Aspinwall, C.A. “Determination of pore sizes and relative porosity in porous nanoshell architectures using dextran retention with single monomer resolution and proton permeation.” Anal. Chem., In press, 2012.