Supported Model Membranes for Biosensing Applications - Optical Oxytocin Binding Assay

Citation:

Kubowicz, A. K. ; Kustanovich, K. ; Gitlin-Domagalska, A. ; Yantchev, V. ; Hurevich, M. ; Yitzchaik, S. ; Jesorka, A. ; Gozen, I. Supported Model Membranes for Biosensing Applications - Optical Oxytocin Binding Assay. Biophys. J. 2020, 118, 232-233.

Abstract:

 

Solid-supported phospholipid bilayers are versatile model structures for mimicking the biological cell membrane, and are increasingly utilized as functional interface components of biosensors and other bio-micro- and nanofluidic devices. In the context of biosensor development, membrane-embedded peptides have gained importance as bio-recognition elements, targeting diverse analytes including proteins, nucleic acids, bacteria, metal ions, enzymes and antibodies. For example, the neuropeptide oxytocin has a key role during labor and lactation as well as in the development of social behavior. Divalent cations such as Zn2+ and Cu2+ vitally affect the activity of oxytocin upon binding. Deviations in the quantity and whereby binding of such ions to oxytocin are associated with diseases; e.g., multiple sclerosis, Alzheimer, and autism spectrum disorder (ASD). We developed an immunofluorescence assay to verify and quantify lipid bilayer membrane-integration of oxytocin-cholesterol conjugate, which was designed and synthesized as membrane-associated recognition element for a surface acoustic resonance (SAR) sensor. In our study, a microfluidic open-volume superfusion device, the Biopen, was used to deposit small unilamellar vesicles, prepared from 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) and oxytocin-conjugated cholesterol, onto a glass surface, where they transformed into extended patches of planar surface-supported bilayer. Thereafter, oxytocin endogenous carrier protein neurophysin-1 (primary antibody) and a fluorescently tagged secondary antibody, were sequentially delivered to the membrane. An antibody binding dependence on oxytocin-concentration was determined by means of fluorescence microscopy, and an optimal concentration for sensor applications was established. The fluorescence assay can be directly transferred to the SAR sensor, where a supported bilayer is established as sensing layer in order to quantify interactions between oxytocin and molecules of interest in a quantitative manner with high sensitivity, fundamentally supporting the development of new diagnostic and therapeutic options for the early detection of neurological and neurodegenerative conditions.

 

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