Catalytic activity of glucose oxidase after dielectrophoretic immobilization on nanoelectrodes.

Dielectrophoresis (DEP) is an AC electrokinetic effect that is proven to be effective for the immobilization of not only cells, but also of macromolecules, for example, antibodies and enzyme molecules. In our previous work, we have already demonstrated the high catalytic activity of immobilized horseradish peroxidase after DEP. To evaluate the suitability of the immobilization method for sensing or research in general, we want to test it for other enzymes, too. In this study, glucose oxidase (GOX) from Aspergillus niger was immobilized on TiN nanoelectrode arrays by DEP. Fluorescence microscopy showed the intrinsic fluorescence of the immobilized enzymes flavin cofactor on the electrodes. The catalytic activity of immobilized GOX was detectable, but a fraction of less than 1.3% of the maximum activity that was expected for a full monolayer of immobilized enzymes on all electrodes was stable for multiple measurement cycles. Therefore, the effect of DEP immobilization on the catalytic activity strongly depends on the used enzyme.

Activity of AC electrokinetically immobilized horseradish peroxidase.

Dielectrophoresis (DEP) is an AC electrokinetic effect mainly used to manipulate cells. Smaller particles, like virions, antibodies, enzymes, and even dye molecules can be immobilized by DEP as well. In principle, it was shown that enzymes are active after immobilization by DEP, but no quantification of the retained activity was reported so far. In this study, the activity of the enzyme horseradish peroxidase (HRP) is quantified after immobilization by DEP. For this, HRP is immobilized on regular arrays of titanium nitride ring electrodes of 500 nm diameter and 20 nm widths. The activity of HRP on the electrode chip is measured with a limit of detection of 60 fg HRP by observing the enzymatic turnover of Amplex Red and H2 O2 to fluorescent resorufin by fluorescence microscopy. The initial activity of the permanently immobilized HRP equals up to 45% of the activity that can be expected for an ideal monolayer of HRP molecules on all electrodes of the array. Localization of the immobilizate on the electrodes is accomplished by staining with the fluorescent product of the enzyme reaction. The high residual activity of enzymes after AC field induced immobilization shows the method's suitability for biosensing and research applications.