In this multi-disciplinary project, we will develop a unique electroactive integrated optical channel waveguide (EA-channel IOW) that is functionalized with ligand-gated, ion channel proteins (ICs) reconstituted into a stabilized planar supported lipid bilayer (PSLB). Our long-term objective is to create arrays of these devices on a single platform to enable high throughput, multiplexed screening of ligands that modulate 1C function, including those correlated with human disease states, and new pharmaceutical candidates to treat these diseases. Successful development of this highly integrated, bioassay platform will complement and potentially supplant existing, cumbersome methods for screening libraries of 1C modulators, providing a powerful new tool for drug discovery, clinical diagnostics, and physiological investigations of structure-activity relationships in structurally-related ICs.
The Specific Aims are designed to systematically develop this technology and demonstrate its utility in these important areas, and thus its relevance to human health. 1. New types of polymerizable lipids and poly(lipid) membranes will be created that preserve the activity of incorporated ATP-sensitive K+ (KATP) ion channels and are sufficiently stabilized to enable long-term use and storage. 2. New types of transducer layers will be created from porous sol-gels (PSGs) with embedded conducting polymer (CP) """"""""wires"""""""" and appended crown ether-dye conjugates. These CP-PSG layers will serve as supports for poly(PSLBs) with incorporated KATP channels. Ligand binding to IC-poly(PSLBs) will produce changes in K+ flux across the membrane, which will be detected via K+ binding to the crown, producing both electrochemical and fluorescence responses. 3. Novel EA-channel lOWs will be designed to efficiently excite dyes in the overlying CP-PSG layer, and to efficiently capture their emission into waveguided modes. This approach will enable the backcoupled fluorescence to be detected in the waveguide plane and, when interfaced with an in-plane light source (organic light emitting diode) and photodetector (organic photovoltaic), will lead to creation of chip-like, waveguide arrays for multiplexed, optical/electrochemical surface-binding assays. 4. IC-poly(PSLB)/CP-PSG multilayers and EA-IOWs will be integrated into single channel sensors and then four-channel arrays, and coupled to microfluidic analyte delivery systems. Multiplexed, 4-channel screening of different 1C ligands and 1C structural compositions will demonstrate the utility of this technology for high throughput screening and establish the feasibility of evolving to higher density arrays. ? ? ? ?
|Principalli, Maria A; Lemel, Laura; Rongier, Anaëlle et al. (2017) Functional mapping of the N-terminal arginine cluster and C-terminal acidic residues of Kir6.2 channel fused to a G protein-coupled receptor. Biochim Biophys Acta 1859:2144-2153|
|Orosz, Kristina S; Jones, Ian W; Keogh, John P et al. (2016) Photopolymerization of Dienoyl Lipids Creates Planar Supported Poly(lipid) Membranes with Retained Fluidity. Langmuir 32:1577-84|
|Liang, Boying; Ju, Yue; Joubert, James R et al. (2015) Label-free detection and identification of protein ligands captured by receptors in a polymerized planar lipid bilayer using MALDI-TOF MS. Anal Bioanal Chem 407:2777-89|
|Bright, Leonard K; Baker, Christopher A; Bränström, Robert et al. (2015) Methacrylate Polymer Scaffolding Enhances the Stability of Suspended Lipid Bilayers for Ion Channel Recordings and Biosensor Development. ACS Biomater Sci Eng 1:955-963|
|Martel, Joseph M; Smith, Kyle C; Dlamini, Mcolisi et al. (2015) Continuous Flow Microfluidic Bioparticle Concentrator. Sci Rep 5:11300|
|Baker, Christopher A; Aspinwall, Craig A (2015) Emerging trends in precision fabrication of microapertures to support suspended lipid membranes for sensors, sequencing, and beyond. Anal Bioanal Chem 407:647-52|
|Zhou, Yi; Bright, Leonard K; Shi, Wenqing et al. (2014) Ion channel probes for scanning ion conductance microscopy. Langmuir 30:15351-5|
|Berglund, Erik; Berglund, David; Akcakaya, Pinar et al. (2013) Evidence for Ca(2+)-regulated ATP release in gastrointestinal stromal tumors. Exp Cell Res 319:1229-38|
|Baker, Christopher A; Bright, Leonard K; Aspinwall, Craig A (2013) Photolithographic fabrication of microapertures with well-defined, three-dimensional geometries for suspended lipid membrane studies. Anal Chem 85:9078-86|
|Adem, Seid M; Mansfield, Elisabeth; Keogh, John P et al. (2013) Practical considerations for preparing polymerized phospholipid bilayer capillary coatings for protein separations. Anal Chim Acta 772:93-8|
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