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. 2022 Oct 20;23(20):12569.
doi: 10.3390/ijms232012569.

Studies on the Detection of Oleuropein from Extra Virgin Olive Oils Using Enzymatic Biosensors

Alexandra Virginia Bounegru  1 , Constantin Apetrei  1
Affiliations

Studies on the Detection of Oleuropein from Extra Virgin Olive Oils Using Enzymatic Biosensors

Alexandra Virginia Bounegru et al. Int J Mol Sci. .

Abstract

Oleuropein (OLEU) is an important indicator of the quality and authenticity of extra virgin olive oils (EVOO). Electrochemical sensors and biosensors for the detection of oleuropein can be used to test the adulteration of extra virgin olive oils. The present study aimed at the qualitative and quantitative determination of oleuropein in commercial EVOO samples by applying electrochemical techniques, cyclic voltammetry (CV) and square wave voltammetry (SWV). The sensing devices used were two newly constructed enzyme biosensors, supported on single-layer carbon-nanotube-modified carbon screen-printed electrode (SPE/SWCNT) on whose surface tyrosinase (SPE/SWCNT/Tyr) and laccase (SPE/SWCNT/Lac) were immobilized, respectively. The active surfaces of the two biosensors were analyzed and characterized by different methods, cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and Fourier transform infrared spectroscopy (FTIR) and the results confirmed the efficient immobilization of the enzymes. SPE/SWCNT/Tyr was characterized by a low detection limit (LOD = 9.53 × 10-8 M) and a very good sensitivity (0.0718 μA·μM-1·cm-2) over a wide linearity range from 0.49 to 11.22 μM. The process occurring at the biosensor surface corresponds to kinetics (h = 0.90), and tyrosinase showed a high affinity towards OLEU. The tyrosinase-based biosensor was shown to have superior sensitive properties to the laccase-based one. Quantitative determination of OLEU in EVOOs was performed using SPE/SWCNT/Tyr and the results confirmed the presence of the compound in close amounts in the EVOOs analysed, proving that they have very good sensory properties.

Keywords: laccase; oleuropein; olive oil; screen-printed electrode; single-walled carbon nanotube; tyrosinase.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Scheme 1
Scheme 1
Bitterness of oleuropein and products of its chemical transformations. Addapted from [55,56].
Figure 1
Figure 1
Cyclic voltammograms recorded in 10−1 M PBS solution, by the SPE/SWCNT/Lac at pH 5.0 (a) and SPE/SWCNT/Tyr at pH 7.0 (b). Scan rate 0.1 V·s−1.
Figure 2
Figure 2
Nyquist plots of EIS for SPE/SWCNT (black line), SPE/SWCNT/Tyr (red line), SPE-SWCNT-Lac (green line) in 10−1 M KCl and 10−3 M [Fe(CN)6]3−/4− for a frequency range of 0.01 Hz to 10 kHz, amplitude 10 mV. Inset: equivalent circuit applied to fit the impedance spectra.
Figure 3
Figure 3
FTIR for SPE/SWCNT (green line), SPE/SWCNT/Tyr (blue line) and SPE/SWCNT/Lac (red line).
Scheme 2
Scheme 2
Reversible oxidation mechanism of oleuropein.
Figure 4
Figure 4
(a) Cyclic voltammograms of SPE/SWCNT (green line), SPE/SWCNT/Tyr (black line) and SPE/SWCNT-Lac (red line) in PBS solution 10−1 M (pH 7.0) containing 10−4 M OLEU. Scan rate: 0.1 V·s−1. (b) Square wave voltammograms of SPE/SWCNT/Tyr (black line) and SPE/SWCNT-Lac (red line) in PBS solution 10−1 M (pH 7.0) containing 10−4 M OLEU (anodic scan-up and cathodic scan-down).
Figure 5
Figure 5
Cyclic voltammograms of SPE/SWCNT/Lac (a) and SPE/SWCNT/Tyr (b) in 10−4 M OLEU–10−1 M PBS solution. The cyclic voltammograms with different colours correspond to different scan rates.
Figure 6
Figure 6
Linear dependence between cathodic current and scanning rate for SPE/SWCNT/Lac (a) and SPE/SWCNT/Tyr (b).
Figure 7
Figure 7
Square wave voltammograms recorded for SPE/SWCNT/Lac (a) and SPE/SWCNT/Tyr (b) on the concentration range 0.01–28.62 μM OLEU. Different colors correspond to different concentration of the analysed solutions.
Figure 8
Figure 8
Linear fit in the range 0.49–11.22 μM for SPE/SWCNT/Lac (a) and SPE/SWCNT/Tyr (b).
Figure 9
Figure 9
Cathodic current intensity obtained by immersing the same SPE/SWCNT/Tyr biosensor in a solution of 10−4 M OLEU, pH 7.0 over 20 consecutive days. The results are acceptable, considering that enzyme activity can easily degrade.
Figure 10
Figure 10
Square wave voltammograms of SPE-SWCNT-Ty immersed in solutions obtained from the 6 EVOOs selected for analysis: (a) Solaris, (b) Monini mini, (c) Costa D’oro Italiano, (d) Mueloliva, (e) Top Seller and (f) Regina Extra.
Figure 11
Figure 11
Preparation process of the tyrosinase and laccase biosensors based on a carbon-based screen-printed electrode.
See this image and copyright information in PMC

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