Carboxylate Particles

Wash particles (e.g., 100 mg of 1 pm carboxylated latex beads) into coupling buffer (i.e., 50 mM MES, pH 6.0 or 50 mM sodium phosphate, pH 7.2 buffers with pH values from pH 4.5 -7.5 may be used with success however, as the pH increases the reaction rate will decrease). Suspend the particles in 5 ml coupling buffer. The addition of a dilute detergent solution may be done to increase particle stability (e.g., final concentration of 0.01 percent sodium dodecyl sulfate (SDS)). Avoid the addition of any components containing carboxylates or amines (such as acetate, glycine, Tris, imidazole, etc.). Also, avoid the presence of thiols (e.g., dithiothreitol (DTT), 2-mercaptoethanol, etc.), as these will react with EDC and effectively inactivate it. 

Dissolve the amine-containing ligand to be coupled in 5 ml coupling buffer at a concentration sufficient to provide a 1- to 10-fold molar excess of ligand over the maximal calculated carboxylate group concentration for the amount and type of beads used. For particle manufacturers reporting a carboxylate concentration in meq/g, this is equivalent to pmol/mg. 

Combine the ligand solution with the particle suspension and mix thoroughly. 

Add 100 mg EDC and mix to dissolve. To facilitate faster dissolution, EDC may be dissolved immediately before use as a concentrated stock solution in reaction buffer and then an aliquot of this solution added to the particle suspension to obtain the correct final concentration. 

React at room temperature 2-4 hours with mixing. 

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Perhaps a better design for a bis-hydrazide compound to modify carboxylate particles would include a short PEG spacer arm between the two hydrazide groups. This type of linker would result in a hydrophilic surface due to the presence of the PEG spacers, while providing the terminal hydrazide functionality necessary for coupling to carbonyl compounds. Unfortunately, this type of compound is not currently available, so the aliphatic bis-hydrazides are the only choice. 

Figure 14.18 Carboxylate-particles or aldehyde-particles can be modified with the carbohydrazide in excess to create a hydrazide-particle that can be used to couple with aldehyde-containing molecules.

Wash the carboxylated particles at least 3 times with DMF by centrifugation. Resuspend in water and wash 3 times with water to remove DMF. Store the particles in water or a suitable buffer at neutral or slightly acidic pH. 

Figure 2. Effect of equilibration time on carboxylic particle expansion at pH = 12.5 as determined by photon correlation spectroscopy. The confidence interval on d is at the 90% probability level. The initial particle size is given by d0 (acrylic latex, 2% AA(ll), d0 = 1120 A).

Carbon-supported (Activated carbon SXplus supplied by NORIT, Sbet = 750 m2.g-l, particle size 0.2-0.1mm) bimetallic and monometallic catalysts were prepared by deposition from a suspension of carboxylate particles in n-heptane chosen as inert organic solvent. Precursors used for the incorporation of the metals were either, palladium(II) acetate (ACROS) and bismuth(lll) oxoacetate, Bi0(02CCH3) (synthesized as described elsewhere [8]), or diammine(pyrazine-2,3-dicarboxylato-N,0)palladium(II) [12] and tris(monohydrogenopyrazine-2,3-dicarboxylato)bismuth(III) (noted Bi(2,3-pzdcH>3) [13]. 

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