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3. Results and Discussion
3.1. Electrode fouling and surface regeneration
Although several authors report electrode fouling associated
with phenols determination using tyrosinase sensors
[9,10,15,33,43], this process was never been the focus of any
investigation. For this reason, electrodes fouling studies were
performed in this work. The process was characterized applying
cyclic voltammetry, amperometry, EQCM, and optical microscopy.
Fig. 1A shows the cyclic voltammogram resulting of the reversible
reduction and oxidation of quinone at a bare spectrally pure
graphite electrode with quinone production in situ by tyrosinase
catalyzed oxidation of catechol. The decrease of the cathodic and
anodic peak currents Ipc and Ipa respectively with successive
CV-cycling allows suggesting that an insulating polymer film
which hinders the direct electron transfer and/or the diffusion of
quinone molecules to the electrode surface could be formed. Fig.1B
demonstrates electrode fouling dependence on the number of
potentials scans and the analyte concentration. As expected, the
analyte concentration augmentation favored electrode fouling,
which led to greatest sensor response decrease. Cathodic peak current dropped by 28.66% and 44.21%, and anodic peak current
dropped by 22.02% and 26.12%, respectively, with catechol
concentration increase from 2 mmol L1 to 3 mmol L1 after
25CV potential scans.
The mass of the products of the electroenzymatic reactions,
deposited onto the electrode surface, as well as the rate of
electrode fouling were evaluated by means of an EQCM. The
technique is well suited for the quantification of the electrochemically
induced mass changes at the electrode surfaces, associated
with processes of polymers growth and interfacial phenomena
[44–48]. To accomplish the measurements, catechol 6 mmol L1
was injected into the EQCM cell containing PBS (0.1 mol L1
, pH
6.5), and native tyrosinase. Catechol was enzymatically converted
to quinone, which was reduced at a potential of 0.00V vs. Ag,
AgCl/KClsat, maintained constant. Resonance frequency change of
the quartz crystal over time was simultaneously monitored, as
shown in Fig. 2. The decrease in the resonant frequency, and
respectively the increase in electrode mass confirmed the
formation of an insoluble film, which finally blocks further
quinone reduction, even if the gold electrode of the EQCM served
as a working electrode instead of the previously used spectrally
pure graphite, which could affect the reactions pathway. Mass
change value at the conditions of Fig. 2 was determined by using
the Sauerbrey equation [49]. It was found to be equal to 57 ng. The
rate of electrode fouling was determined by the slope of the linear
part of the obtained curve in coordinates of frequency change vs.
time and was estimated at 0.051 Hz s1
, i.e. 0.07 ng s1 or
0.035 ng s
1 cm2
.
Electrode surface analysis was performed using high resolution
optical microscopy. The obtained optical micrograph images of a
bare graphite electrode before and after a prolonged repetitive
scanning the electrode potential in the range from +0.4 V to -0.2 V
vs. Ag, AgCl/KClsat in phosphate buffer solution (0.1 mol L1
, pH 6.5)
containing catechol 8 mmol L1 and native tyrosinase, catalyzing
catechol enzymatic oxidation to quinone, are presented in Fig. 3.
Visible surface modification, attributed to surface fouling, was
observed.
The adopted strategy to overcome fouling problems consisted
in the application of an appropriate pulse potential waveform. As
the cathodic peak potential for quinone reduction equals
+0.1700.001V vs. Ag, AgCl/KClsat (Fig. 1), the detection potential
was set much more negative, at 0.00 V vs. Ag, AgCl/KClsat to ensure
faster reduction rate. The optimum cleaning potential for in situ
regeneration of the electrode surface was found to be +1.40 V vs.
Ag, AgCl/KClsat. According to data reported in the literature, fouled
electrodes could be reactivated by anodic polarization at high
potentials [50–57]. It is considered that electrode regeneration
could be attributed to the simultaneous action of the produced
hydroxyl radicals and the released oxygen which provoke, As demonstrated in the literature, the prolonged reactivation
time enhances electrode regeneration [57]. Hence, the detection
step potential was applied only for 66.64 ms, but the cleaning
potential step duration was 166.6 ms. These values were selected
as multiples of the electric power line period of 16.66 ms to
minimize the noise.
The RSD, which is used to represent the precision of the
determinations carried out applying the chosen set of pulsed
potential parameters, was found to be 2.97% (n=5, 0.6 mmol L1
catechol). For comparison, the RSD characterizing the precision of
the pulsed amperometric detections performed applying a
cleaning potential of +1.20V/Ag, AgCl (n=5, 0.6 mmol L1 catechol),
and of the amperometric detections without cleaning (n=5,
0.6 mmol L1 catechol) were 4.45% and 6.53%, respectively.
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