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FIG. 10 Schematic representation of the covalent immobilization of a peptide onto a reactive surface. Reactive groups on the peptide and the solid phase are usually ligated via cross-linking reagents.

TABLE 1 Types of Functional Groups and Their Corresponding Ligands Used for Covalent Linkage

Functional group on particle

Ligand group in peptide

Comments

Aldehyde (CHO) Amino (NH2)

Chloro-activated Amino (NH2) (ClCH2)

Thiol (SH)

Thiol (SH)

Amino (NH2) Hydroxyl (OH)

Thiol (SH)

Maleimide, iodoacetyl

Carboxyl (COOH) Amino (NH2)

Amino (NH2) Carboxyl (COOH) Amino (NH2)

Nucleophilic addition No activation step required May require deprotection of the aldehyde group

Nucleophilic substitution No activation step required Nucleophilic substitution No activation step required Requires pH above the pKb of the amino group Reduction

No activation step required Susceptible to oxidation and displacement by other SH groups No activation step required Requires prior modification of peptide Nucleophilic substitution Requires activation step Requires activation step with bifunctional reagent Nucleophilic substitution Requires activation step, often with use of organic solvent

Source: Adapted from Ref. 61.

Some of these latexes with added functional groups require activation steps prior to conjugation with peptide, e.g., those with carboxyl or hydroxyl groups; others must be stored with their functional groups protected until the moment of use, e.g., those with aldehyde groups. The variety of functional groups available allows peptides to be attached covalently not only by their primary amino groups but also by thiol, carboxyl, and hydroxyl groups.

When immobilizing a peptide onto a reactive surface, it is possible to add a nonpeptide molecule to act as a "spacer," i.e., as an extension of the peptide toward the aqueous phase, so preventing the peptide from sinking to the particle surface, and allowing it to gain flexibility and improve its recognition (Fig. 11). Adding a nonpeptide spacer has advantages over adding amino acid residues during peptide synthesis for the same purpose. Hydrophilic spacers, like poly-

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