The development of a membrane-based biosensor for ICMT: Membrane synthesis, properties & methods of detection

David Holland, Purdue University

Abstract

The membrane design, synthesis, properties, and methods of detection for a biosensor to detect the activity of an integral membrane protein, isoprenylcysteine carboxylmethyltransferase, are described. The membrane component consists of a bolalipid membrane which is utilized to increase the stability of the membrane. The lateral fluidity of membranes, as measured by pulsed-field gradient NMR, composed of a specific bolalipid, C20BAS, is found to be similar to that of monopolar lipid membranes. Based on 2H NMR spectroscopy, the bolalipid and monopolar lipid membranes have dramatically different order profiles. Further, membranes composed of C20BAS and a monopolar lipid, POPC are found to phase separate based on theory, 2H NMR spectroscopy, and atomic force microscopy. This mixing behavior is believed to be due to hydrophobic mismatch of the two domains. Two types of assays to detect the activity of ICMT are described. A compound is synthesized which conjugates self-quenching fluorescein molecules together through a disulfide bond. This compound is also anchored to the membrane through lipid chains for in order to localize the fluorophores close to the solid support to increase the sensitivity of TIRF measurements. This molecular beacon is sensitive to homocysteine, the product of a coupled enzymatic process with ICMT. A second assay to detect the direct enzymatic product of ICMT, S-adenosylhomocystine, is described. This assay utilizes an anti-SAH antibody—SAH—anti-SAH aptamer complex to tether magnetic beads to a microfabricated gold surface for detection with interferometry with a detection limit of 64 pM. Additionally, this sandwich interaction can be utilized to conjugate fluorescent streptavidin to the magnetic bead for detection with flow cytometry.

Degree

Ph.D.

Advisors

Thompson, Purdue University.

Subject Area

Organic chemistry

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