PubMed · 14101980
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M D MELAMED, N M GREEN. 1963. AVIDIN. 2. PURIFICATION AND COMPOSITION.. https://doi.org/10.1042/bj0890591
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The design of chemically well-defined, machinable surfaces containing neuroactive molecules offers potential for fundamental neuroscience and clinical neural engineering applications. Here we report the assembly and characterization of silicon platforms containing a tethered form of muscimol. Muscimol, an analog of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), is a potent agonist at postsynaptic GABA(A) and GABA(C) receptors. Surfaces were assembled using covalent avidin conjugation to silanized silicon followed by high-affinity avidin-biotin binding of a biotinylated derivative of muscimol (muscimol-biotin). Contact angle measurements, ellipsometry, and X-ray photoelectron spectroscopy (XPS) were conducted to characterize the wettability, thickness, and chemical composition of progressively deposited surface layers. The data demonstrate successful incorporation of a neurotransmitter analog as part of a layered, silicon-based structure possessing robust and specific biomolecular composition. These findings represent a step toward the design of platforms for applications involving control and modulation of neural signaling.
Luminescent semiconductor quantum dot (QD)-based optical biosensors have the potential to overcome many of the limitations associated with using conventional organic dyes for biodetection. We have previously demonstrated a hybrid QD-protein-based fluorescence resonance energy transfer (FRET) sensor. Although the QD acted as an energy donor and a protein scaffold in the sensor, recognition and specificity were derived from the proteins. Transitioning this hybrid prototype sensor into flow cells and integrated devices will require a surface-immobilization strategy that allows the QD-based sensor to sample the environment and still maintain a distinct protein-covered QD architecture. We demonstrate a self-assembled strategy designed to accomplish this. Using glass slides coated with a monolayer of neutravidin (NA) as the template, QDs with maltose binding protein (MBP) and avidin coordinated to their surface were attached to the glass slides in discrete patterns using an intermediary bridge of biotinylated MBP or antibody linkers. Control of the surface location and concentration of the QD-protein-based structures is demonstrated. The utility of this self-assembly strategy is further demonstrated by assembling a QD-protein structure that allows the QDs to engage in FRET with a dye located on the surface-covering protein.
Two luminescent ruthenium(II) polypyridine complexes containing a biotin moiety [Ru(bpy)(2)(L1)](PF(6))(2) (1) and [Ru(bpy)(2)(L2)](PF(6))(2) (2) (bpy = 2,2'-bipyridine; L1 = 4-(N-((2-biotinamido)ethyl)amido)-4'-methyl-2,2'-bipyridine; L2 = 4-(N-((6-biotinamido)hexyl)amido)-4'-methyl-2,2'-bipyridine) have been synthesized and characterized, and their photophysical and electrochemical properties have been studied. Upon photoexcitation, complexes 1 and 2 display intense and long-lived triplet metal-to-ligand charge-transfer ((3)MLCT) (dpi(Ru) --> pi*(L1 or L2)) emission in fluid solutions at 298 K and in low-temperature glass. We have studied the binding of these ruthenium(II) biotin complexes to avidin by 4'-hydroxyazobenzene-2-carboxylic acid (HABA) assays, luminescence titrations, competitive assays using native biotin, and quenching experiments using methyl viologen. On the basis of the results of these experiments, a homogeneous competitive assay for biotin has been investigated.