RESUMEN
We report a robust and practical method for the preparation of water-soluble luminescent quantum dots (QDs) selectively coupled through an amine or thiol linkage to peptide ligands targeted to G-protein coupling receptors (GPCRs) and demonstrate their utility in whole-cell and single-molecule imaging. We utilized a low molecular weight ( approximately 1200 Da) diblock copolymer with acrylic acids as hydrophilic segments and amido-octyl side chains as hydrophobic segments for facile encapsulation of QDs (QD 595 and QD 514) in aqueous solutions. As proof of principle, these QDs were targeted to the human melanocortin receptor (hMCR) by chemoselectively coupling the polymer-coated QDs to either a hexapeptide analog of alpha-melanocyte stimulating hormone or to the highly potent MT-II ligand containing a unique amine. To label QDs with ligands lacking orthogonal amines, the diblock copolymers were readily modified with water-soluble trioxa-tridecanediamine to incorporate freely available amine functionalities. The amine-functionalized QDs underwent facile reaction with the bifunctional linker NHS-maleimide, allowing for covalent coupling to GPCR-targeted ligands modified with unique cysteines. We demonstrate the utility of these maleimide-functionalized QDs by covalent conjugation to a highly potent Deltorphin-II analog that allowed for selective cell-surface and single-molecule imaging of the human delta-opioid receptor (hDOR).
Asunto(s)
Fragmentos de Péptidos/química , Puntos Cuánticos , Receptores Acoplados a Proteínas G/química , Células Cultivadas/metabolismo , Fluorescencia , Humanos , Técnicas para Inmunoenzimas , Riñón/metabolismo , Membrana Dobles de Lípidos/química , Metalotioneína/química , Metalotioneína/metabolismo , Oligopéptidos/química , Oligopéptidos/metabolismo , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/metabolismo , Polímeros/química , Receptor de Melanocortina Tipo 4/química , Receptor de Melanocortina Tipo 4/metabolismo , Receptores Opioides delta/química , Receptores Opioides delta/metabolismoRESUMEN
Productions of /s/ and /z/ by ten adult speakers were investigated using the electropalatograph (EPG). The participants, ten speech researchers who spoke English as their first language, recorded productions of /s/ and /z/ in nonsense and real words. The maximum contact frame was used as the point of reference to compare tongue/palate contact for each production. Each speaker had alveolar contact, lateral bracing and most had a midline groove for both /s/ and /z/; however, the array of contacted electrodes was unique for each speaker. The groove widths and lengths ranged from 0-3 electrodes. There was significantly greater alveolar tongue/palate contact for /z/ compared to /s/ in word-initial position, but not in word-final position for the following measures: alveolar palatal contact, medial groove width, medial groove length. However, when measures of total palate contact and centre of gravity were considered, there was a complex interaction between the phonemes /s/ and /z/, coarticulation with the vowel, word position, and word context (real and nonsense words).