Coupling carboxylate particles

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.

Figure 5 Schematic of the attachment of coupling agent to a filler particle. X = Cl, OCHj, carboxyl M = Si, Ti, Zr R = long-chain alkyls or oligomers that contain grafted polar groups, e.g., amides and carboxyl.

Carbodiimide coupling to carboxylate-containing QDs usually involves the use of EDC in a single-step or two-step process to form an amide bond. If a one-step reaction is done, the QD is activated with EDC in the presence of an amine-containing molecule, such as a protein. Many protocols use this method, but it can result in protein polymerization in addition to coupling, because proteins contain both carboxylates and amines. A two-step protocol results in better control of the reaction (Figure 9.61). In the first step, EDC is used in the presence of sulfo-NHS to activate the carboxylates on the particles to intermediate sulfo-NHS esters. After a quick separation step to remove excess reactants, the activated QDs are added to the protein solution to be coupled. This then results in amide bond formation without polymerization of the protein in solution. See Chapter 3, Section 1 and Chapter 14, Section 1 for additional information on this process. 

Figure 9.61 QDs containing carboxylate groups can be coupled to amine-containing proteins or other molecules using the EDC/sulfo-NHS reaction to form amide bond linkages. The intermediate sulfo-NHS ester is negatively charged and will help maintain particle stability due to like charge repulsion between particles.

Polymeric particles can be constructed from a number of different monomers or copolymer combinations. Some of the more common ones include polystyrene (traditional latex particles), poly(styrene/divinylbenzene) copolymers, poly(styrene/acrylate) copolymers, polymethylmethacrylate (PMMA), poly(hydroxyethyl methacrylate) (pHEMA), poly(vinyltoluene), poly(styrene/butadiene) copolymers, and poly(styrene/vinyltoluene) copolymers. In addition, by mixing into the polymerization reaction combinations of functional monomers, one can create reactive or functional groups on the particle surface for subsequent coupling to affinity ligands. One example of this is a poly(styrene/acrylate) copolymer particle, which creates carboxylate groups within the polymer structure, the number of which is dependent on the ratio of monomers used in the polymerization process. 

Figure 14.5 A method of making particles biocompatible includes the use of PEG-based spacers. A lawn of mPEG molecules in interspersed with some longer PEG chains that terminate in carboxylate groups for coupling amine-containing molecules. The result is an extremely hydrophilic surface with low nonspecific binding.

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. 

These Ru(H)bpy32+ fluorescent silica nanoparticles were used to detect single bacterial cells using antibodies conjugated to the surface after functionalization with trimethoxysilyl-propyldiethylenetriamine followed by succinylation to create carboxylates. Specific antibody molecules against E. coli 0157 then were coupled to this modified fluorescent particle using the carbodiimide method with EDC and NHS (Zhao et al., 2004). 

Surface functionalization of silica particles or fluorescent silica particles typically is done using functional alkyl silanes. The process may be used to add a reactive group to the surface of the particles for spontaneous coupling to biomolecules or it may be used to add the appropriate nucleophilic group to the surface, such as an amine or a carboxylate. Silane modification chemistry is discussed in more detail in Chapter 13. 

Carboxylated silica particles may be coupled with amine-containing ligands, such as proteins, using a carbodiimide reaction with EDC. A similar protocol to that previously described for coupling to carboxylate polymer particles may be used. The following protocol is based on the method of Zhao et al. (2004), which was used for immobilizing monoclonal antibodies to E. coli 0157. 

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