Covalent Immobilization of Glycosylated Proteins Previously Adsorbed Through Weak Reversible Reactions on a Mixed Epoxi Boronic Acid APBA Self Assembled Monolayer

Starting from electrodes as in Subheading 3.4., boronic acids are incorporated into SAMs to reversibly adsorb glycosylated proteins through their carbohydrate moiety. A mixed monolayer of boronates and epoxy groups makes the adsorption irreversible. Basically, the aminophenylboronic groups promote and accelerate the covalent attachment of glycoproteins through the epoxy groups.

3.7.1. Amidation of the Thioctic Acid With Aminophenyl Boronic Acid and Epoxy Groups

1. Incubate overnight the gold covered with a SAM-TOA-NHS in undiluted DADOO.

2. Remove the unreacted DADOO by washing with ethanol.

3. Dip the gold electrode in undiluted Epi to produce a monolayer of epoxy groups.

4. Dip the electrode in ethanol 5 min to eliminate unreacted Epi. Repeat the wash with ethanol.

5. Incubate Epi-terminated electrodes in 0.16 MAPBA in acetonitrile:water (1:5), pH 8.0, for 8 h. This protocol produces mixed monolayers with phenyl-boronate and epoxy groups.

6. To obtain an estimation of the remaining epoxy groups on this mixed SAM, incubate the electrode overnight in 2 mM P-aminoethyl ferrocene in DMF. Eliminate unreacted P-aminoethyl ferrocene by washing with solvent.

7. Insert the electrode in the electrochemical cell and run a cyclic voltammogram in the range of 150 to 500 mV. The integrated charge of the anodic cyclic voltammetry peak allows the calculation of remaining epoxy groups.

3.7.2. Immobilization ofHorseradish Peroxidase

1. Incubate the modified electrode in 0.2 mg/mL HRP solution in 50 mMbicarbon-ate buffer for 24 h.

2. Wash the electrode for 30 min in the same buffer.

3.7.3. Measurement of the Enzymatic Activity

The enzyme peroxidase catalyzes the reduction of H2O2 to H2O with an electron donor (e.g., thionine). In the assay, a 0.01 mM thionine in 0.05 M phosphate buffer, KCl 0.1 M, pH 7.5, is reduced electrochemically and supplies electrons to the peroxidase enzyme molecule, which in its catalytic cycle donates two electrons to H2O2. Therefore, the course of the enzymatic reaction can be followed by CV analysis of the reduced thionine reaction product, using the same methodology described in the reference.

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