Phospholipids biotinylated

Urease avidin-urease Urea Phospholipids biotinylated phospholipids SS PPY PP ISFET glass Amperometric pH ISFET 130-132... 

Biotinylated liposomes usually are created by modification of PE components with an amine-reactive biotin derivative, for example NHS-LC-Biotin (Chapter 11, Section 1). The NHS ester reacts with the primary amine of PE residues, forming an amide bond linkage (Figure 22.19). A better choice of biotinylation agent may be to use the NHS-PEG -biotin compounds (Chapter 18), because the hydrophilic PEG spacer provides better accessibility in the aqueous environment than a hydrophobic biotin spacer. Since the modification occurs at the hydrophilic end of the phospholipid molecule, after vesicle formation the biotin component protrudes out from the liposomal surface. In this configuration, the surface-immobilized biotins are able to bind (strept)avidin molecules present in the outer aqueous medium. 

The results summarized above were obtained by using fluorescence based assays employing phospholipid vesicles and fluorescent labeled lipopeptides. Recently, surface plasmon resonance (SPR) was developed as new a technique for the study of membrane association of lipidated peptides. Thus, artificial membranes on the surface of biosensors offered new tools for the study of lipopeptides. In SPR (surface plasmon resonance) systemsI713bl changes of the refractive index (RI) in the proximity of the sensor layer are monitored. In a commercial BIAcore system1341 the resonance signal is proportional to the mass of macromolecules bound to the membrane and allows analysis with a time resolution of seconds. Vesicles of defined size distribution were prepared from mixtures of lipids and biotinylated lipopeptides by extruder technique and fused with a alkane thiol surface of a hydrophobic SPR sensor. 

M. Snejdarkova, M. Rehak and M. Otto, Design of a glucose minisensor based on streptavidin glucose-oxidase complex coupling with self-assembled biotinylated phospholipid membrane on solid support, Anal. Chem., 65 (1993) 665-668. 

FIGURE 6.7. Epifluorescent images of (a) Texas Red labeled streptavidin confined in aqueous compartments above solid supported phospholipid bilayers containing 2 mol% biotinylated lipids. Each box contains a different aqueous phase small molecule as described in the text, (b) Same system as in (a), but fluorescence is excited by total internal reflection revealing that streptavidin is surface bound in boxes 1, 2, and 4, while the surface in box 3 remains protein free. 

Ringsdorf and coworkers used monolayer techniques for the creation of lipid functionalized streptavidin layers.252,253 In their pioneering work, a monolayer of biotinylated phospholipids initially created a two-dimensional docking matrix for the immobilization of streptavidin. In an effort to further expand this system while maintaining control over its structure, bis-biotinylated bifunctional linkers were employed. Addition of these linkers to concanavalin A created a new protein-based bidentate linker. Upon addition to the monolayer of the lipid-functionalized streptavidin a second biotilynated protein layer was created. Upon the further addition of streptavidin, they achieved in a step-by-step construction process well-defined alternating protein triple layers of streptavidin and concanavalin A (Figure 7.37b). A similar approach has more recently been used to crosslink... 

In another report, Michaelis-Menten parameters of ALP have been obtained using a one-shot Lineweaver-Burk (reciprocal) plot. This can be achieved by simultaneously measuring the conversion of 12 independent concentrations of the substrate (4-methylumbelliferyl phosphate) created on-chip. The enzyme was streptavidin-conjugated ALP that was linked to biotinylated phospholipid bilayers coated inside PDMS microchannels. The blue fluorescence of the enzymatic product, 7-hydroxy-4-methyl coumarin, was measured [ 1043]. The surface-bound enzyme was found to have a lower (sixfold) turnover rate than the free enzyme in solutions. After diffusion mixing between two streams (substrate and buffer)... 

Synthesis of Biotinylated Phospholipid. Biotin was covalently attached to dimyristoylphosphatidylethanolamine (DMPE) according to the procedure of Bayer et (8, 9). Our adaptation of their procedure has been described previously (7). 

Dissolution of Biotinylated Phospholipid in Nonionic Surfactant Solution. Solutions of the nonionic surfactant octaethylene-glycol mono-n-dodecylether (C12E8) at a concentration of 10-3 M in standard buffer were used to solubilize DMPE-B to obtain solutions of 5 x lO" M and 1.25 X 10" M of the modified phospholipid. The phospholipid was suspended in 100 ml of the C12E8 solutions and the mixture was vigorously stirred with heating to a uniform temperature of 60-70°C for about 45 minutes whereupon all of the DMPE-B was solubilized. Solutions of underivatized phospholipid in C12E8 were prepared in a similar fashion. 

Cytochrome b5 Electrotransduction Biotinylated phospholipidsipids-streptavidin maleimide-phospholipids Au Amperometric 128... 

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