Preparation of Capacitive Electrodes

In order to develop an assay for measuring real-time enzyme activity by monitoring the degradation of substrates, two different immobilization strategies were used for the preparation of capacitive electrodes.

The first strategy was based on the use of substrate (IgG) covalently immobilized to the electrode surface and then to monitor the degradation. The second strategy involved a reversible immobilization method such that after the assay, the electrode could be regenerated and reused.

Preparation of the capacitive electrodes by using these strategies and activity of the enzyme on these electrodes are schematically shown in Scheme 1.

Preparation of the capacitive electrodes by using different strategies and activity of the enzyme on these electrodes, a IgG-immobilized electrodes and b APBA-immobilized electrodes

For the immobilization of IgG, in the first step, the electrodes were subsequently cleaned with ethanol, de-ionized water, acetone, de-ionized water, and acidic Piranha solution [3:1, H₂SO₄ (95%):H₂O₂ (30%), v/v] respectively for 10 min in each step, in an ultrasonic cleaner. Then, plasma cleaning (Mod. PDC-3XG, Harrick, NY, USA) was applied to the electrodes for 20 min. Afterwards, electro-polymerization of tyramine was performed by using a cyclic voltammetry (CV) in ethanolic solution of 10 mM tyramine with a set potential range of 0–1.5 V (vs Ag/AgCl) and a scan rate of 50 mV s⁻¹ for 15 scans as described before [27]. By this way, free primary amino groups were introduced on the surface of the electrode. Then, the electrodes were rinsed with distilled water and dried with nitrogen gas. In the next step, for the activation of amino groups on the surface, the electrodes were immersed in 5.0% (v/v) glutaraldehyde solution in 10 mM phosphate buffer (pH 7.4) for 1 h at room temperature. Then, electrodes were rinsed with distilled water and dried with nitrogen gas.

In the last step, electrodes were immersed overnight at 4 °C in IgG solution (0.1 mg mL⁻¹) prepared in 10 mM phosphate buffer (pH 7.4). Finally, to cover pinholes in the insulating layer of the gold surface, they were kept in 10 mM of 1-dodecanethiol in ethanol for 20 min. 1-Dodecanethiol treatment does not affect the immobilized protein on the surface and only interacts with the pinholes on the surface. Therefore, it has been used for this purpose in so many previous reports where capacitive sensors are used for detection [28–31].

In the first step, gold electrodes were cleaned by using the same strategy described above in the “Preparation of IgG-Immobilized Capacitive Electrodes” section. In the next step, electro-polymerization of tyramine was performed by cyclic voltammetry (CV) in ethanolic solution of 10 mM tyramine with a set potential range of 0–1.5 V (vs. Ag/AgCl) and a scan rate of 50 mV s⁻¹ for 15 scans. Then, sodium carboxymethyl cellulose was dissolved in 0.05 M morpholinoethanesulfonic acid (MES) buffer (pH 6.0) at 1.0% (w/v). Poly-tyramine-coated electrodes were immersed in this solution for 1 h at room temperature. By this way, carboxyl groups were introduced on the surface of the electrode. Same method has been used successfully for the 3-APBA modification of capacitive gold electrode surface for saccharide modification in our previous study [32].

In the next step, for the activation of carboxyl groups, electrodes were immersed in 1 mL of 0.05 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1.0 mL of 0.03 M N-hydroxysuccinimide sodium salt in MES buffer (pH 6.0) for 2 h. N-Hydroxysuccinimide-activated carboxylic groups were then allowed to bind with the primary amino groups of 3-aminophenylboronic acid (40 mM) in phosphate buffer (10 mM, pH 7.0) overnight, at room temperature [32]. Finally, to cover bare parts of the gold surface, they were kept in 10 mM of 1-dodecanethiol in ethanol for 20 min.