Functionalized beads

Figure 9. Immobilization of antibodies, a) directly on functional beads, or b) via crosslinkers (e.g. biotin-streptavidin chemistry). Arraying onto the slide in c) print buffer or d) entrapped in hydrogel.

In another approach, reactive monodisperse porous poly(chloromethylstyrene-co-styrene-co-divinylbenzene) beads have been employed for the preparation of chiral HPLC packings. Thus, reactive chloromethyl groups were derivatized to yield amino functionalized beads onto which both rt-basic and rt-acidic type chiral. selectors, (/ )- -(l-naphthyl)ethylamine and (/ )-A -(3.5-dinitrobenzoyl)phenylglycine, respectively, were attached. The resulting chiral particles were chromatographically tested for the enantioseparation of model SAs. Despite the presence of strongly competitive it-TT-binding sites of the styrenic support these chirally modified beads afforded baseline separations for 2,2,2-trifluoro-l-(9-anthryl) ethanol and Af-(3.5-dinitro-benzoyl) leucine enantiomers, respectively [369. ... 

Most commercial systems like Mustang from Pall and Sartobind from Sartorius make use of functionalized microporous membranes. The fibrils reinforced membranes are (pleated) layered around a porous core. The feed is forced to permeate through the membranes in radial direction. This approach results in high area to volume ratio. The 3M and Mosaic Systems approach is different. Instead of functionalization of a porous support they make use of already functionalized beads, which are embedded in a porous support. In this approach, the beads are responsible for the capacity and selectivity where the porous matrix controls the hydrodynamics. The 3M modules consist of stacked flat sheet or pleated membranes, while Mosaic Systems makes use of porous fibers in which the active particles are embedded (Figure 3.23). 

To observe the dynamics of small structures such as the growth of single actin filaments in solution or on functionalized beads, a specialized TIRF microscope is needed. Critical for TIRF imaging are furthermore a sensitive CCD Camera, high-quality objectives with a high numeric aperture (NA > 1.4), immersion oil (e.g., Leica with a refractive index of 1.518) as well as suitable lasers and filter sets. These days, complete systems for TIRF microscopy can be purchased from manufacturers such as Olympus, Nikon or Leica. 

The intention of the device presented by Lilliehorn et al. was to utilize the miniaturized transducers in combination with the laminar flow conditions in microfluidic systems to create a dynamic arraying device holding several individually controlled transducers as envisioned in Figure 44.21. Here, an array of 16 transducers are loaded with different functionalized beads in a first step. The beads are acoustically trapped in the positions given by the transducers. In a second step, samples are supplied through the orthogonal channels and the response is, for example, read by fluorescence. The transducers are then deactivated and the beads are flushed out of the device and the whole procedure is repeated again for a new set of beads and samples. 

Tentagel, described above, is in wide use today in solid phase syntheses. Polyethylene glycol has also been attached to various other polymers to form support resins. For instance, Frechet and coworkers [37] used cross-linked methacrylate esters of ethylene glycol oligomers in a suspension polymerization to synthesize hydroxyl group functionalized beads. These beads swell well in a variety of polar solvents. Another example is that of Grubbs attaching a ruthenium metathesis catalyst to polyethylene [38] ... 

In analogy with carboxylate-functionalized beads, di-pyridylamide-based resins were synthesized. In contrast to di-pyridylamine, the corresponding amide revealed a high selectivity for both mercury(II) and palladium(II) [10]. This allowed the selective enrichment of these two transition-metal ions from aqueous solutions over a concentration range covering several orders of magnitude [11]. 

Besides the functionalization of the biosensor surface, when micro- or nanoparticles are used (eg, magnetic particles), they also need to be functionalized. For instance, functional beads prepared by emulsion polymerization or dispersion polymerization offer a large specific surface for chemical binding. These small particles can be advantageously used as a mobile substrate for bioassays, or even for in vivo applications, as they can be easily recovered from dispersion (Gijs, 2004). 

After affinity purification of proteins using the functionalized beads, bound proteins need to be digested for further LC/MS analysis. Two different strategies are presented and their advantages are briefly discussed. 

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