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Horseradish peroxidase (HRP) is immobilized in three easy steps on SiO(2) surfaces with the help of a polycationic second generation dendronized polymer (denpol) and the biotin-avidin system. This stepwise immobilization process is monitored and quantitatively analyzed with the transmission interferometric adsorption sensor. Partially biotinylated denpol is first adsorbed onto SiO(2) , followed by addition of avidin and then of biotinylated HRP. Denpols in their molecular structure combine properties of polymers as well as dendrimers which are found to be of clear advantage for this type of non-covalent enzyme immobilization. With respect to the reproducibility of the adsorption process and with respect to the stability of the adsorbed polymer layer, the denpol is superior to α-poly-D-lysine which is used as a reference polymer. Furthermore, HRP immobilized with the denpol on commercial glass slides remains considerably more active upon storage as compared to HRP immobilized with the help of α-poly-D-lysine with a similar number of repeating units. The ease of the denpol-mediated HRP immobilization and the high stability of the immobilized enzyme are promising for bioanalytical applications.

Four UV/Vis spectrophotometric methods for the quantification of a-polylysine (a-PL) are described and discussed. The methods are based on different chemical reactivities of a-PL allowing the indirect determination of a-PL concentrations in aqueous solutions down to 1-2 mg mL À1 . The four methods are the trypan blue (TB) assay, the 2,4,6-trinitrobenzene sulfonate (TNBS) assay, the orthophthalaldehyde (OPA) assay and the bicinchoninic acid (BCA) assay. The TB assay is based on the polycationic character of a-PL in acidic aqueous solutions, in which the non-covalent binding with the oligoanionic dye TB leads to a precipitation of the dye and a concomitant decrease in the intensity of the blue color of the solution. Both the TNBS and the OPA assay utilize the nucleophilicity of the amino groups in alkaline solutions resulting in trinitrophenylated amino groups and isoindole derivatives, respectively. Finally, in the BCA assay the reductive properties of the peptide bonds are used to reduce copper(II) to copper(I) which eventually forms a stable, purple 1 : 2 complex with bicinchoninic acid. For each method, mechanistic aspects are discussed and detailed experimental protocols are provided. With respect to the level of minimum quantification the TB and the OPA assays are best. Scheme 1 Structural formulae of L-lysine (L-Lys), D-lysine (D-Lys), a-poly(L-Lys) (a-PLL), a-poly(D-Lys) (a-PDL), 3-poly(L-Lys) (3-PLL), and 3-poly(D-Lys) (3-PDL).

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