Bioelectrochemistry 101 (2015) 52–57
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Bioelectrochemistry journal homepage: www.elsevier.com/locate/bioelechem
Electrochemical determination of biophenol oleuropein using a simple label-free DNA biosensor Maryam Mohamadi a,b,⁎, Ali Mostafavi a, Masoud Torkzadeh-Mahani c,⁎⁎ a b c
Department of Chemistry, Shahid Bahonar University of Kerman, Kerman, Iran Young Research Society, Shahid Bahonar University of Kerman, Kerman, Iran Department of Biotechnology, Institute of Science, High Technology and Environmental Science, Graduate University of Advance Technology, Kerman, Iran
a r t i c l e
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Article history: Received 25 December 2013 Received in revised form 8 July 2014 Accepted 13 July 2014 Available online 18 July 2014 Keywords: Oleuropein DNA biosensor Voltammetry Olive leaf
a b s t r a c t Oleuropein (Ole), naturally occurring phenolic compound found in olive products, is well known for its benefits for human health. In the present work, a simple, sensitive and rapid determination of Ole was achieved using a label-free electrochemical DNA biosensor. The application was related to the molecular interaction between Ole and double-stranded DNA (dsDNA). So, the voltammetric behavior of Ole at the surface of a DNA-immobilized chitosan-modified carbon paste electrode was studied using differential pulse voltammetry (DPV) where the oxidation peak current of Ole was measured as an analytical signal. A considerable increase was observed in the oxidation signal of Ole at the DNA-coated electrode compared with the DNA-free electrode, indicating the pre-concentration of Ole due to the interaction with the surface-confined DNA layer. In order to use the proposed sensor for real samples, different parameters affecting Ole signal such as, immobilization time and potential, accumulation time and pH, and stripping pH were optimized. Under optimized experimental conditions, a linear concentration range of 0.30–12 μmol L−1 with a detection limit of 0.090 μmol L−1 was obtained for Ole determination. The proposed biosensor was successfully applied to the determination of Ole in olive leaf extract and human serum samples. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The term phenolic compound embraces a wide range of plant substances which possess in common an aromatic ring bearing one or more hydroxyl substituents [1]. Phenolic components of olive products, such as olive oil and nutriceuticals containing olive leaf extracts, have been reported to be beneficial to health [2]. The most abundant biophenol and the major bioactive compound in olive leaves is oleuropein (Scheme 1), a natural product of the secoiridoid group [3,4]. Chemically, oleuropein is the ester of elenolic acid and 3,4dihydroxyphenyl ethanol (hydroxytyrosol) [2]. Studies have shown that oleuropein possesses a wide range of pharmacological properties, including antioxidant [5], anti-inflammatory [6], antiatherogenic [7], anti-cancer [8,9], antimicrobial [10], and antiviral [11], and for these reasons, it is commercially available as food supplement in the Mediterranean countries. The quantification of Ole has been reported with reversed-phase HPLC/fluorescence detection [2], HPLC/diode array detection system [12] and liquid chromatography–electrospray ionization/tandem mass ⁎ Corresponding author. Tel./fax: +98 3413222033. ⁎⁎ Corresponding author. Tel.: +98 342 622 6611; fax: +98 342 622 6617. E-mail addresses: [email protected] (M. Mohamadi), [email protected] (M. Torkzadeh-Mahani).
http://dx.doi.org/10.1016/j.bioelechem.2014.07.003 1567-5394/© 2014 Elsevier B.V. All rights reserved.
spectrometry method [13] in plasma samples and with mid-infrared spectroscopy combined with chemometric analyses [3], reversedphase HPLC/UV detection [14] in olive leaves. Electrochemical methods provide rapid, simple and sensitive alternatives in the analysis of bioactive. They are low-cost and usually do not require time consuming sample preparation. Here an electrochemical DNA biosensor is proposed to determine Ole. Biosensors are devices combining a biological component with a detector component. Biosensors contain three parts [15,16]: (1) the sensitive elements (biologically-derived material), (2) the transducer or detector element that transforms the detected signal into a readable, quantified output, and (3) the signal processor that displays the transformed signal in a user-friendly way. Recently, the field of electrochemical biosensors has been speeding up dramatically [17,18]. Due to their high selectivity, rapidness and low cost instrumentation, electrochemical methods have witnessed wide applications not only in fundamental studies, but also in the practice [19]. Electrochemical DNA biosensors contain a nucleic acid recognition layer, which is immobilized on an electrochemical transducer. DNA biosensors have been used for the determination of low-molecular weight compounds with affinity for nucleic acids and for the detection of the hybridization reaction. The first application uses the molecular interaction between surface-linked DNA and the target pollutants or drugs to develop a simple device for rapid screening of toxic or similar compounds. The DNA-trapped compounds can be
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was completely dried under reduced pressure in a rotary evaporator. The resulting extract was then freeze-dried. For electrochemical determination, 0.0010 g of the dry extract was dissolved in distilled water up to a volume of 25 mL. 1 mL of this solution was then transferred to the electrochemical cell containing 0.5 mol L− 1 phosphate buffer (pH 5.0) with a total volume of 10 mL and Ole was determined using the proposed biosensor.
2.4. Preparation of human serum sample
Scheme 1. Chemical structure of oleuropein.
detected either directly [20,21] (if they are electroactive molecules) or via changes in electrochemical DNA signal [22–24]. We have applied an electrochemical DNA biosensor based on the immobilization of double-stranded deoxyribonucleic acid (dsDNA) on the surface of a chitosan-modified carbon paste electrode for the rapid, sensitive and selective determination of oleuropein. Since oleuropein is an electroactive species, the oxidation peak current of the accumulated oleuropein molecules at the electrode surface was measured as analytical signal, using differential pulse voltammetry (DPV). This signal was associated with the oxidation of the catechol group of Ole [25,26]. Direct determination of Ole benefits the selectivity of the electrochemical method whereas measurement of the changes made in the oxidation signal of DNA after binding Ole, possesses no such selectivity. 2. Experimental 2.1. Instrumentation All voltammograms were obtained using an Autolab PGSTAT 101 electrochemical system from Metrohm (Herisau, Switzerland) interfaced with a personal computer for data acquisition and potential control. A three-electrode assembly was employed: a glass cell containing an Ag/AgCl electrode as reference electrode, a platinum wire counter electrode and a carbon paste electrode (CPE) as the working electrode. A Metrohm 827 pH meter supplied with a combination glassreference electrode was used for pH measurements. 2.2. Chemicals and reagents Chitosan (CHIT) was purchased from Acros (Geel, Belgium). Pure graphite powder was obtained from Merck (Darmstadt, Germany). HPLC-grade oleuropein powder was obtained from Sigma (Saint Louis, MO, USA). A 2.0 mmol L−1 stock solution of oleuropein was prepared by dissolving an appropriate amount of the powder in distilled water and stored at 2–8 °C. Deoxyribonucleic acid (DNA) sodium salt was purchased from Acros and its stock solution was prepared by dissolving solid DNA in distilled water and kept frozen. The concentration of DNA was determined according to the absorbance at 260 nm. The molar extinction coefficient (ε260) was found to be 6600 L mol− 1 cm− 1 (per P or nucleotide unit) [27]. 2.3. Preparation of olive leaf extract An ethanolic extract of olive (Oleaeuropaea; variety of Sevillano) leaf was prepared in the Razi Herbal Medicines Research Center (Lorestan, Iran) as described by Esmaeili-Mahani and his coworkers [28]. Briefly, the air-dried olive leaves were ground into fine powder. This powder was extracted twice with 80% ethanol. After the filtration of the collective ethanol extract, the crude extract
Human serum sample was stored at 4 °C until analyzed. The sample was prepared according to the literature [29] with minor modifications. 5 mL of the serum sample was transferred into each of the centrifuge tubes containing different known amounts of Ole and then mixed well with 10 mL of methanol to precipitate the blood proteins. After centrifugation at 3000 rpm for 30 min and separation of the precipitated proteins, the clear supernatant layer was filtered through the 0.45-μm Millipore filter and then diluted to 50 mL with 0.5 mol L−1 phosphate buffer (pH 5.0).
2.5. Electrode preparation A chitosan-modified carbon paste electrode (CHIT/CPE) was prepared by mixing graphite powder and chitosan (90/10 w/w %) and wetting with paraffin oil. The paste was carefully hand-mixed in a mortar and then packed into one end of a glass tube (3.4 mm (i.d.)). The electrical contact was provided via a copper wire connected to the paste in the inner hole of the tube. The electrode surface was gently smoothed by rubbing it on a piece of filter paper until it was shiny. When necessary, a new surface was obtained by pushing a bit of the paste out of the tube. A bare CPE was also prepared by following the procedure explained above without the incorporation of chitosan in the paste matrix.
2.6. DNA immobilization A 10 mL of 0.5 mol L−1 acetate buffer solution (pH 4.8) containing 0.040 mmol L−1 DNA was stirred for 5 min after immersing CHIT/CPE and applying 0.50 V. The electrode was then washed with blank acetate buffer solution for 10 s to remove unbound DNA. Thus a DNA-coated CHIT/CPE was obtained. The DNA was similarly immobilized on the surface of bare CPE to prepare DNA-coated CPE.
2.7. Cyclic voltammetry of K3[Fe(CN)6] The cyclic voltammogram of 5.0 mmol L−1 K3Fe(CN)6 + 5.0 mmol L−1 K4Fe(CN)6 in 0.10 mol L− 1 KCl was recorded from − 200 to 700 mV using a scan rate of 100 mV s−1.
2.8. Electrochemical measurements Before any measurement, the DNA-coated CHIT/CPE was incubated with various concentrations of Ole in 0.5 mol L− 1 phosphate buffer (pH 5.0). The accumulation was carried out at an open circuit system for 10 min. The electrode was then thoroughly rinsed with distilled water and placed in 10 mL of 0.5 mol L− 1 phosphate buffer (pH 6.0). Voltammetric transduction was now performed by DPV under the following conditions: pulse amplitude 0.05 V and scan rate 10 mV s−1. A peak current related to the concentration of Ole at about + 210 mV (corresponding to the Ole oxidation) was taken as analytical signal.
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M. Mohamadi et al. / Bioelectrochemistry 101 (2015) 52–57
I/µA
100.0
b
electrodes enable the detection of the immobilization of the DNA on the surface of CHIT/CPE. The amino groups of chitosan electrostatically interact and form a stable complex with negatively charged phosphate backbone of DNA [30].
80.0 60.0
a
40.0 20.0 0.0 -0.2 0 -20.0
-0.4
3.2. Voltammetric behavior of Ole at the surface of DNA-coated CHIT/CPE
c 0.2
0.4
0.6
0.8
E/V (Ag/AgCl)
-40.0 -60.0 -80.0 -100.0
Fig. 1. Cyclic voltammogram of 5.0 mmol L−1 K3Fe(CN)6 + 5.0 mmol L−1 K4Fe(CN)6 in 0.10 mol L−1 KCl at the surface of (a) bare CPE, (b) CHIT/CPE and (c) DNA-coated CHIT/ CPE using a scan rate of 100 mV s−1. (CHIT: chitosan).
3. Results and discussion 3.1. CV study on the immobilization of DNA on CHIT/CPE 4− redox couple as an Cyclic voltammograms of Fe(CN)3− 6 /Fe(CN)6 indicator for DNA immobilization, were studied at the surface of bare CPE, CHIT/CPE and DNA-coated CHIT/CPE. The results obtained are shown in Fig. 1. It is clear that, the peak currents of both cathodic and anodic waves at CHIT/CPE were almost double the peak currents at bare CPE. Reversibility was also improved. After the immobilization of DNA at the surface of CHIT/CPE, both peak currents were dramatically diminished with an increase of 48 mV in peak-to-peak potential separation. Due to electrostatic repulsion, the negatively charged phosphate backbone of the DNA prevents 4− the redox couple (Fe(CN)3− 6 /Fe(CN)6 ) from reaching the electrode surface, leading to a decrease in the oxidation and reduction signals. Consequently, the differences between the CV profiles for these
10.0
c 8.0
Fig. 2A displays the oxidation signals of Ole after accumulating at the surface of DNA-coated CPE (curve a), CHIT/CPE (curve b) and DNA-coated CHIT/CPE (curve c). As can be seen, the anodic signal was tripled when the surface of CHIT/CPE was coated with DNA. This enhanced sensitivity is a result of a pre-concentration due to the adsorption of Ole at the electrode surface through a strong interaction with the immobilized DNA layer. As mentioned in literature [30], the affinity of chitosan toward the negatively charged backbone of DNA greatly increases the content of DNA coating the electrode surface and subsequently the content of accumulated Ole which in turn, results in an increase of the voltammetric signal when comparing curves a and c. Furthermore, the positive shift observed in the peak potential of Ole at DNA-coated CHIT/CPE relative to CHIT/CPE, may propose an intercalative mode of interaction between Ole and DNA. In other words, the intercalative binding of Ole with the surface-confined DNA layer adsorbs and pre-concentrates this biophenol in the surface of DNA-coated CHIT/CPE resulted in the enhanced sensitivity [31]. It is noteworthy that complementary studies such as spectroscopic ones should be performed for investigating the nature of the interaction (intercalation or not). 3.3. Optimization of experimental variables To obtain maximum response from the proposed biosensor, the working conditions were optimized. 3.3.1. Amount of DNA The amount of DNA immobilized on the electrode surface plays an important role in this approach: the more the immobilized DNA, the more the Ole accumulated at the DNA-coated electrode and, the electrochemical signal increases significantly. So, the effect of the concentration of DNA on the peak current of Ole was investigated. It was observed that the peak current increased rapidly with the increase of DNA concentration up to 0.040 mmol L−1 and then tended to be stable. Accordingly, in subsequent experiments the immobilization of DNA was performed with 0.040 mmol L−1 DNA. 3.3.2. Immobilization potential Since the accumulation of DNA is influenced by the imposed potential to the electrode during the immobilization step, the potential applied to the system for the immobilization of DNA on the CHIT/CPE was optimized. For this purpose, the DNA was immobilized on the
I/µA
6.0
4.0
b I /µA
2.0
a 0.0 0
0.2
0.4
E/V (Ag/AgCl)
8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 2
4
6
8
10
pH Fig. 2. DP voltammograms obtained after accumulation of Ole at (a) DNA-coated CPE, (b) CHIT/CPE and (c) DNA-coated CHIT/CPE. Conditions: accumulation medium 0.50 mol L−1 phosphate buffer pH 5.0, accumulation time 10 min, stripping medium 0.5 mol L−1 phosphate buffer pH 6.0. (CHIT: chitosan, Ole: oleuropein).
Fig. 3. Effect of accumulation pH on the Ipa of 4.0 μmol L−1 Ole at DNA-coated CHIT/CPE surface. Conditions were the same as given in Fig. 8 except accumulation pH. (CHIT: chitosan, Ole: oleuropein).
M. Mohamadi et al. / Bioelectrochemistry 101 (2015) 52–57
A
55
B 14.0
0.4
3
8
E/V (Ag/AgCl)
12.0
I /µA
10.0 8.0 6.0 4.0
0.3 E = -0.053pH + 0.535 R² = 0.998
0.2
0.1
2.0 0
0.0 0
0.1
0.2
0.3
0.4
0.5
0.6
0
2
4
E/V (Ag/AgCl)
6
8
10
pH
Fig. 4. (a) Effect of stripping pH on the Ipa of 5.0 μmol L−1 Ole at DNA-coated CHIT/CPE surface. Conditions were the same as given in Fig. 8 except stripping pH (3.0, 4.0, 5.0, 6.0, 7.0 and 8.0). (b) Anodic peak potential of Ole as a function of pH, extracted from the curves given in (a). (CHIT: chitosan, Ole: oleuropein).
polished electrode using different potentials within a range of 0 to 0.70 V. According to the results obtained, the potential around 0.50 V vs. Ag/Ag Cl was obviously favorable for obtaining the maximum accumulation of Ole resulted in the maximum peak current. Therefore, the potential of 0.50 V was selected as optimal immobilization potential.
3.3.3. Immobilization time The effect of time was investigated on the immobilization of DNA and subsequent accumulation of Ole at the DNA biosensor. The obtained results indicated that maximum DPV signal of Ole was achieved when the optimal potential was applied to the DNA solution stirred for 300 s. Therefore, 300 s is considered the optimal time for immobilization.
3.3.4. Accumulation pH pH can influence the interaction between the Ole and the dsDNA coating the electrode surface, and further accumulation. Accordingly, dependence of the oxidation signal of the accumulated Ole on pH was studied over the range of 3.0–10.0 in 0.5 M phosphate buffer. Fig. 3 illustrates the corresponding results. As one can see, the response current decreases dramatically when pH increases from 5.5 to 10.0. It is stable in the interval 4.5 to 5.5 and for pHs lower than 4.5, the
response again decreases. pH around 5.0 was selected for the remainder of the experiments.
3.3.5. Accumulation time The dependence of the anodic peak current of Ole on the stirring time was investigated. The anodic peak current increased markedly with accumulation time up to 8.0 min. With longer stirring time, the rate of increase diminished. With the sensitivity and the analytical time considered, a time of 10 min was chosen as the optimum accumulation time.
3.3.6. Stripping pH The effect of stripping pH on the biosensor response was investigated in the range of 3.0–8.0. The results are shown in Fig. 4A and reveals that the stripping pH considerably affects the biosensor performance. Typically, at pHs higher than 6.0, DPV signals decline and broaden. Since the maximum signal is observed for stripping at pH of 6.0, this pH was selected as optimum. In addition, a negative shift is observed in the anodic peak potential with increasing pH. Fig. 4B depicts the linear relationship between the peak potential and pH with the linear regression
B
A 30.0
30 25.0 25 20
15.0
I/µA
I/µA
20.0
i 10.0
10
a
5.0
y = 2.190x - 0.188 R² = 0.998
5
0.0
0 0
-5.0
15
0.2
0.4
E/V (Ag/AgCl)
0.6
0.0
5.0
10.0
15.0
C/µmol L-1
Fig. 5. (a) DP voltammograms of Ole obtained at DNA-coated CHIT/CPE at different concentrations (a–i): 0.3, 0.6, 1.0, 1.5, 2.0, 4.0, 6.0, 9.0 and 12 μmol L−1, respectively. Other conditions were the same as given in Fig. 8. (b) Calibration curve for Ole determination extracted from the voltammograms given in Fig. 11A. (CHIT: chitosan, Ole: oleuropein).
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Table 1 Determination of Ole in serum and olive leaves extract using the proposed method. Sample
Added (μM)
Found (μM)a
Recovery (%)
Extract of olive leaves
0 1.0 5.0 0 5.0 10.0
2.7 ± 0.2 3.7 ± 0.2 7.6 ± 0.3 – 4.9 ± 0.2 10.3 ± 0.3
– 100 98
Human serum
a
Acknowledgment This work was supported by grants from the Research Council of Shahid Bahonar University of Kerman (M/342/41).
References 98 103
Mean ± standard deviation (n = 3).
equation of E/V = −0.053 pH + 0.535 and the correlation coefficient (R2) of 0.998. As one can see, the slope is close to the theoretical value of − 0.059 V/pH, indicating that the electrode process is an equal proton-electron transfer [32]. That is, there are two electrons and two protons associated with catechol group, involved in the electrode process of Ole.
3.4. Responsecharacteristicsof DNA-coated CHIT/CPE DP voltammetry was adopted in the experiments and the peak current of the oxidation wave of Ole at 210 mV was used as the detecting signal. Fig. 5A displays the DPV response of the DNAcoated CHIT/CPE for different Ole concentrations. The typical calibration curve for Ole is shown in Fig. 5B. It is clear that the anodic peak currents are linearly correlated with Ole concentrations in the 0.30– 12 μmol L − 1 range. The linear regression equation is I =2.190 C0.188 (R2 = 0.998) where I is the anodic peak current (μA) and C is the concentration of Ole (μmol L − 1 ). Using 3s b/m and 10sb /m (where sb is the standard deviation of blank measurements and m is the slope of the calibration curve), the limit of detection and limit of quantification were calculated to be 0.090 and 0.29 μmol L− 1, respectively. Repeatability of the sensor was estimated with five replicate measurements at 1.5 and 7.0 μmol L− 1 of Ole, and resulted in RSDs of 6.0 and 3.8%.
3.5. Analytical application In order to investigate the possibility of applying the proposed biosensor to the quantification of Ole, extracts of olive leaves and human serum were analyzed using the calibration curve method. In order to verify the accuracy of the approach, the standard addition method was applied for the determination of Ole in spiked samples. As shown in Table 1, the recoveries obtained were acceptable. In addition, total value of Ole was calculated to be 359.4 mg per g of dry extract which is in good agreement with that obtained by HPLC. The results suggest that, it is feasible to apply the proposed electrochemical DNA biosensor to quantitatively determine the concentration of Ole in biological and plant samples.
4. Conclusion A simple, rapid, reliable and sensitive DNA biosensor was proposed for the determination of oleuropein based on the interaction between this biophenol compound and dsDNA. Chitosan, a biocompatible, biodegradable and non-toxic cationic polymer that forms polyelectrolyte complexes with DNA, was used for DNA immobilization on the electrode surface. The oxidation peak current of Ole was measured as an analytical signal for direct determination of the Ole molecules accumulated on the surface of DNA-coated CHIT/CPE. In this way, one can benefit the selectivity of electrochemical methods.
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Bioelectrochemistry journal homepage: www.elsevier.com/locate/bioelechem
Electrochemical determination of biophenol oleuropein using a simple label-free DNA biosensor Maryam Mohamadi a,b,⁎, Ali Mostafavi a, Masoud Torkzadeh-Mahani c,⁎⁎ a b c
Department of Chemistry, Shahid Bahonar University of Kerman, Kerman, Iran Young Research Society, Shahid Bahonar University of Kerman, Kerman, Iran Department of Biotechnology, Institute of Science, High Technology and Environmental Science, Graduate University of Advance Technology, Kerman, Iran
a r t i c l e
i n f o
Article history: Received 25 December 2013 Received in revised form 8 July 2014 Accepted 13 July 2014 Available online 18 July 2014 Keywords: Oleuropein DNA biosensor Voltammetry Olive leaf
a b s t r a c t Oleuropein (Ole), naturally occurring phenolic compound found in olive products, is well known for its benefits for human health. In the present work, a simple, sensitive and rapid determination of Ole was achieved using a label-free electrochemical DNA biosensor. The application was related to the molecular interaction between Ole and double-stranded DNA (dsDNA). So, the voltammetric behavior of Ole at the surface of a DNA-immobilized chitosan-modified carbon paste electrode was studied using differential pulse voltammetry (DPV) where the oxidation peak current of Ole was measured as an analytical signal. A considerable increase was observed in the oxidation signal of Ole at the DNA-coated electrode compared with the DNA-free electrode, indicating the pre-concentration of Ole due to the interaction with the surface-confined DNA layer. In order to use the proposed sensor for real samples, different parameters affecting Ole signal such as, immobilization time and potential, accumulation time and pH, and stripping pH were optimized. Under optimized experimental conditions, a linear concentration range of 0.30–12 μmol L−1 with a detection limit of 0.090 μmol L−1 was obtained for Ole determination. The proposed biosensor was successfully applied to the determination of Ole in olive leaf extract and human serum samples. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The term phenolic compound embraces a wide range of plant substances which possess in common an aromatic ring bearing one or more hydroxyl substituents [1]. Phenolic components of olive products, such as olive oil and nutriceuticals containing olive leaf extracts, have been reported to be beneficial to health [2]. The most abundant biophenol and the major bioactive compound in olive leaves is oleuropein (Scheme 1), a natural product of the secoiridoid group [3,4]. Chemically, oleuropein is the ester of elenolic acid and 3,4dihydroxyphenyl ethanol (hydroxytyrosol) [2]. Studies have shown that oleuropein possesses a wide range of pharmacological properties, including antioxidant [5], anti-inflammatory [6], antiatherogenic [7], anti-cancer [8,9], antimicrobial [10], and antiviral [11], and for these reasons, it is commercially available as food supplement in the Mediterranean countries. The quantification of Ole has been reported with reversed-phase HPLC/fluorescence detection [2], HPLC/diode array detection system [12] and liquid chromatography–electrospray ionization/tandem mass ⁎ Corresponding author. Tel./fax: +98 3413222033. ⁎⁎ Corresponding author. Tel.: +98 342 622 6611; fax: +98 342 622 6617. E-mail addresses: [email protected] (M. Mohamadi), [email protected] (M. Torkzadeh-Mahani).
http://dx.doi.org/10.1016/j.bioelechem.2014.07.003 1567-5394/© 2014 Elsevier B.V. All rights reserved.
spectrometry method [13] in plasma samples and with mid-infrared spectroscopy combined with chemometric analyses [3], reversedphase HPLC/UV detection [14] in olive leaves. Electrochemical methods provide rapid, simple and sensitive alternatives in the analysis of bioactive. They are low-cost and usually do not require time consuming sample preparation. Here an electrochemical DNA biosensor is proposed to determine Ole. Biosensors are devices combining a biological component with a detector component. Biosensors contain three parts [15,16]: (1) the sensitive elements (biologically-derived material), (2) the transducer or detector element that transforms the detected signal into a readable, quantified output, and (3) the signal processor that displays the transformed signal in a user-friendly way. Recently, the field of electrochemical biosensors has been speeding up dramatically [17,18]. Due to their high selectivity, rapidness and low cost instrumentation, electrochemical methods have witnessed wide applications not only in fundamental studies, but also in the practice [19]. Electrochemical DNA biosensors contain a nucleic acid recognition layer, which is immobilized on an electrochemical transducer. DNA biosensors have been used for the determination of low-molecular weight compounds with affinity for nucleic acids and for the detection of the hybridization reaction. The first application uses the molecular interaction between surface-linked DNA and the target pollutants or drugs to develop a simple device for rapid screening of toxic or similar compounds. The DNA-trapped compounds can be
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was completely dried under reduced pressure in a rotary evaporator. The resulting extract was then freeze-dried. For electrochemical determination, 0.0010 g of the dry extract was dissolved in distilled water up to a volume of 25 mL. 1 mL of this solution was then transferred to the electrochemical cell containing 0.5 mol L− 1 phosphate buffer (pH 5.0) with a total volume of 10 mL and Ole was determined using the proposed biosensor.
2.4. Preparation of human serum sample
Scheme 1. Chemical structure of oleuropein.
detected either directly [20,21] (if they are electroactive molecules) or via changes in electrochemical DNA signal [22–24]. We have applied an electrochemical DNA biosensor based on the immobilization of double-stranded deoxyribonucleic acid (dsDNA) on the surface of a chitosan-modified carbon paste electrode for the rapid, sensitive and selective determination of oleuropein. Since oleuropein is an electroactive species, the oxidation peak current of the accumulated oleuropein molecules at the electrode surface was measured as analytical signal, using differential pulse voltammetry (DPV). This signal was associated with the oxidation of the catechol group of Ole [25,26]. Direct determination of Ole benefits the selectivity of the electrochemical method whereas measurement of the changes made in the oxidation signal of DNA after binding Ole, possesses no such selectivity. 2. Experimental 2.1. Instrumentation All voltammograms were obtained using an Autolab PGSTAT 101 electrochemical system from Metrohm (Herisau, Switzerland) interfaced with a personal computer for data acquisition and potential control. A three-electrode assembly was employed: a glass cell containing an Ag/AgCl electrode as reference electrode, a platinum wire counter electrode and a carbon paste electrode (CPE) as the working electrode. A Metrohm 827 pH meter supplied with a combination glassreference electrode was used for pH measurements. 2.2. Chemicals and reagents Chitosan (CHIT) was purchased from Acros (Geel, Belgium). Pure graphite powder was obtained from Merck (Darmstadt, Germany). HPLC-grade oleuropein powder was obtained from Sigma (Saint Louis, MO, USA). A 2.0 mmol L−1 stock solution of oleuropein was prepared by dissolving an appropriate amount of the powder in distilled water and stored at 2–8 °C. Deoxyribonucleic acid (DNA) sodium salt was purchased from Acros and its stock solution was prepared by dissolving solid DNA in distilled water and kept frozen. The concentration of DNA was determined according to the absorbance at 260 nm. The molar extinction coefficient (ε260) was found to be 6600 L mol− 1 cm− 1 (per P or nucleotide unit) [27]. 2.3. Preparation of olive leaf extract An ethanolic extract of olive (Oleaeuropaea; variety of Sevillano) leaf was prepared in the Razi Herbal Medicines Research Center (Lorestan, Iran) as described by Esmaeili-Mahani and his coworkers [28]. Briefly, the air-dried olive leaves were ground into fine powder. This powder was extracted twice with 80% ethanol. After the filtration of the collective ethanol extract, the crude extract
Human serum sample was stored at 4 °C until analyzed. The sample was prepared according to the literature [29] with minor modifications. 5 mL of the serum sample was transferred into each of the centrifuge tubes containing different known amounts of Ole and then mixed well with 10 mL of methanol to precipitate the blood proteins. After centrifugation at 3000 rpm for 30 min and separation of the precipitated proteins, the clear supernatant layer was filtered through the 0.45-μm Millipore filter and then diluted to 50 mL with 0.5 mol L−1 phosphate buffer (pH 5.0).
2.5. Electrode preparation A chitosan-modified carbon paste electrode (CHIT/CPE) was prepared by mixing graphite powder and chitosan (90/10 w/w %) and wetting with paraffin oil. The paste was carefully hand-mixed in a mortar and then packed into one end of a glass tube (3.4 mm (i.d.)). The electrical contact was provided via a copper wire connected to the paste in the inner hole of the tube. The electrode surface was gently smoothed by rubbing it on a piece of filter paper until it was shiny. When necessary, a new surface was obtained by pushing a bit of the paste out of the tube. A bare CPE was also prepared by following the procedure explained above without the incorporation of chitosan in the paste matrix.
2.6. DNA immobilization A 10 mL of 0.5 mol L−1 acetate buffer solution (pH 4.8) containing 0.040 mmol L−1 DNA was stirred for 5 min after immersing CHIT/CPE and applying 0.50 V. The electrode was then washed with blank acetate buffer solution for 10 s to remove unbound DNA. Thus a DNA-coated CHIT/CPE was obtained. The DNA was similarly immobilized on the surface of bare CPE to prepare DNA-coated CPE.
2.7. Cyclic voltammetry of K3[Fe(CN)6] The cyclic voltammogram of 5.0 mmol L−1 K3Fe(CN)6 + 5.0 mmol L−1 K4Fe(CN)6 in 0.10 mol L− 1 KCl was recorded from − 200 to 700 mV using a scan rate of 100 mV s−1.
2.8. Electrochemical measurements Before any measurement, the DNA-coated CHIT/CPE was incubated with various concentrations of Ole in 0.5 mol L− 1 phosphate buffer (pH 5.0). The accumulation was carried out at an open circuit system for 10 min. The electrode was then thoroughly rinsed with distilled water and placed in 10 mL of 0.5 mol L− 1 phosphate buffer (pH 6.0). Voltammetric transduction was now performed by DPV under the following conditions: pulse amplitude 0.05 V and scan rate 10 mV s−1. A peak current related to the concentration of Ole at about + 210 mV (corresponding to the Ole oxidation) was taken as analytical signal.
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M. Mohamadi et al. / Bioelectrochemistry 101 (2015) 52–57
I/µA
100.0
b
electrodes enable the detection of the immobilization of the DNA on the surface of CHIT/CPE. The amino groups of chitosan electrostatically interact and form a stable complex with negatively charged phosphate backbone of DNA [30].
80.0 60.0
a
40.0 20.0 0.0 -0.2 0 -20.0
-0.4
3.2. Voltammetric behavior of Ole at the surface of DNA-coated CHIT/CPE
c 0.2
0.4
0.6
0.8
E/V (Ag/AgCl)
-40.0 -60.0 -80.0 -100.0
Fig. 1. Cyclic voltammogram of 5.0 mmol L−1 K3Fe(CN)6 + 5.0 mmol L−1 K4Fe(CN)6 in 0.10 mol L−1 KCl at the surface of (a) bare CPE, (b) CHIT/CPE and (c) DNA-coated CHIT/ CPE using a scan rate of 100 mV s−1. (CHIT: chitosan).
3. Results and discussion 3.1. CV study on the immobilization of DNA on CHIT/CPE 4− redox couple as an Cyclic voltammograms of Fe(CN)3− 6 /Fe(CN)6 indicator for DNA immobilization, were studied at the surface of bare CPE, CHIT/CPE and DNA-coated CHIT/CPE. The results obtained are shown in Fig. 1. It is clear that, the peak currents of both cathodic and anodic waves at CHIT/CPE were almost double the peak currents at bare CPE. Reversibility was also improved. After the immobilization of DNA at the surface of CHIT/CPE, both peak currents were dramatically diminished with an increase of 48 mV in peak-to-peak potential separation. Due to electrostatic repulsion, the negatively charged phosphate backbone of the DNA prevents 4− the redox couple (Fe(CN)3− 6 /Fe(CN)6 ) from reaching the electrode surface, leading to a decrease in the oxidation and reduction signals. Consequently, the differences between the CV profiles for these
10.0
c 8.0
Fig. 2A displays the oxidation signals of Ole after accumulating at the surface of DNA-coated CPE (curve a), CHIT/CPE (curve b) and DNA-coated CHIT/CPE (curve c). As can be seen, the anodic signal was tripled when the surface of CHIT/CPE was coated with DNA. This enhanced sensitivity is a result of a pre-concentration due to the adsorption of Ole at the electrode surface through a strong interaction with the immobilized DNA layer. As mentioned in literature [30], the affinity of chitosan toward the negatively charged backbone of DNA greatly increases the content of DNA coating the electrode surface and subsequently the content of accumulated Ole which in turn, results in an increase of the voltammetric signal when comparing curves a and c. Furthermore, the positive shift observed in the peak potential of Ole at DNA-coated CHIT/CPE relative to CHIT/CPE, may propose an intercalative mode of interaction between Ole and DNA. In other words, the intercalative binding of Ole with the surface-confined DNA layer adsorbs and pre-concentrates this biophenol in the surface of DNA-coated CHIT/CPE resulted in the enhanced sensitivity [31]. It is noteworthy that complementary studies such as spectroscopic ones should be performed for investigating the nature of the interaction (intercalation or not). 3.3. Optimization of experimental variables To obtain maximum response from the proposed biosensor, the working conditions were optimized. 3.3.1. Amount of DNA The amount of DNA immobilized on the electrode surface plays an important role in this approach: the more the immobilized DNA, the more the Ole accumulated at the DNA-coated electrode and, the electrochemical signal increases significantly. So, the effect of the concentration of DNA on the peak current of Ole was investigated. It was observed that the peak current increased rapidly with the increase of DNA concentration up to 0.040 mmol L−1 and then tended to be stable. Accordingly, in subsequent experiments the immobilization of DNA was performed with 0.040 mmol L−1 DNA. 3.3.2. Immobilization potential Since the accumulation of DNA is influenced by the imposed potential to the electrode during the immobilization step, the potential applied to the system for the immobilization of DNA on the CHIT/CPE was optimized. For this purpose, the DNA was immobilized on the
I/µA
6.0
4.0
b I /µA
2.0
a 0.0 0
0.2
0.4
E/V (Ag/AgCl)
8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 2
4
6
8
10
pH Fig. 2. DP voltammograms obtained after accumulation of Ole at (a) DNA-coated CPE, (b) CHIT/CPE and (c) DNA-coated CHIT/CPE. Conditions: accumulation medium 0.50 mol L−1 phosphate buffer pH 5.0, accumulation time 10 min, stripping medium 0.5 mol L−1 phosphate buffer pH 6.0. (CHIT: chitosan, Ole: oleuropein).
Fig. 3. Effect of accumulation pH on the Ipa of 4.0 μmol L−1 Ole at DNA-coated CHIT/CPE surface. Conditions were the same as given in Fig. 8 except accumulation pH. (CHIT: chitosan, Ole: oleuropein).
M. Mohamadi et al. / Bioelectrochemistry 101 (2015) 52–57
A
55
B 14.0
0.4
3
8
E/V (Ag/AgCl)
12.0
I /µA
10.0 8.0 6.0 4.0
0.3 E = -0.053pH + 0.535 R² = 0.998
0.2
0.1
2.0 0
0.0 0
0.1
0.2
0.3
0.4
0.5
0.6
0
2
4
E/V (Ag/AgCl)
6
8
10
pH
Fig. 4. (a) Effect of stripping pH on the Ipa of 5.0 μmol L−1 Ole at DNA-coated CHIT/CPE surface. Conditions were the same as given in Fig. 8 except stripping pH (3.0, 4.0, 5.0, 6.0, 7.0 and 8.0). (b) Anodic peak potential of Ole as a function of pH, extracted from the curves given in (a). (CHIT: chitosan, Ole: oleuropein).
polished electrode using different potentials within a range of 0 to 0.70 V. According to the results obtained, the potential around 0.50 V vs. Ag/Ag Cl was obviously favorable for obtaining the maximum accumulation of Ole resulted in the maximum peak current. Therefore, the potential of 0.50 V was selected as optimal immobilization potential.
3.3.3. Immobilization time The effect of time was investigated on the immobilization of DNA and subsequent accumulation of Ole at the DNA biosensor. The obtained results indicated that maximum DPV signal of Ole was achieved when the optimal potential was applied to the DNA solution stirred for 300 s. Therefore, 300 s is considered the optimal time for immobilization.
3.3.4. Accumulation pH pH can influence the interaction between the Ole and the dsDNA coating the electrode surface, and further accumulation. Accordingly, dependence of the oxidation signal of the accumulated Ole on pH was studied over the range of 3.0–10.0 in 0.5 M phosphate buffer. Fig. 3 illustrates the corresponding results. As one can see, the response current decreases dramatically when pH increases from 5.5 to 10.0. It is stable in the interval 4.5 to 5.5 and for pHs lower than 4.5, the
response again decreases. pH around 5.0 was selected for the remainder of the experiments.
3.3.5. Accumulation time The dependence of the anodic peak current of Ole on the stirring time was investigated. The anodic peak current increased markedly with accumulation time up to 8.0 min. With longer stirring time, the rate of increase diminished. With the sensitivity and the analytical time considered, a time of 10 min was chosen as the optimum accumulation time.
3.3.6. Stripping pH The effect of stripping pH on the biosensor response was investigated in the range of 3.0–8.0. The results are shown in Fig. 4A and reveals that the stripping pH considerably affects the biosensor performance. Typically, at pHs higher than 6.0, DPV signals decline and broaden. Since the maximum signal is observed for stripping at pH of 6.0, this pH was selected as optimum. In addition, a negative shift is observed in the anodic peak potential with increasing pH. Fig. 4B depicts the linear relationship between the peak potential and pH with the linear regression
B
A 30.0
30 25.0 25 20
15.0
I/µA
I/µA
20.0
i 10.0
10
a
5.0
y = 2.190x - 0.188 R² = 0.998
5
0.0
0 0
-5.0
15
0.2
0.4
E/V (Ag/AgCl)
0.6
0.0
5.0
10.0
15.0
C/µmol L-1
Fig. 5. (a) DP voltammograms of Ole obtained at DNA-coated CHIT/CPE at different concentrations (a–i): 0.3, 0.6, 1.0, 1.5, 2.0, 4.0, 6.0, 9.0 and 12 μmol L−1, respectively. Other conditions were the same as given in Fig. 8. (b) Calibration curve for Ole determination extracted from the voltammograms given in Fig. 11A. (CHIT: chitosan, Ole: oleuropein).
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Table 1 Determination of Ole in serum and olive leaves extract using the proposed method. Sample
Added (μM)
Found (μM)a
Recovery (%)
Extract of olive leaves
0 1.0 5.0 0 5.0 10.0
2.7 ± 0.2 3.7 ± 0.2 7.6 ± 0.3 – 4.9 ± 0.2 10.3 ± 0.3
– 100 98
Human serum
a
Acknowledgment This work was supported by grants from the Research Council of Shahid Bahonar University of Kerman (M/342/41).
References 98 103
Mean ± standard deviation (n = 3).
equation of E/V = −0.053 pH + 0.535 and the correlation coefficient (R2) of 0.998. As one can see, the slope is close to the theoretical value of − 0.059 V/pH, indicating that the electrode process is an equal proton-electron transfer [32]. That is, there are two electrons and two protons associated with catechol group, involved in the electrode process of Ole.
3.4. Responsecharacteristicsof DNA-coated CHIT/CPE DP voltammetry was adopted in the experiments and the peak current of the oxidation wave of Ole at 210 mV was used as the detecting signal. Fig. 5A displays the DPV response of the DNAcoated CHIT/CPE for different Ole concentrations. The typical calibration curve for Ole is shown in Fig. 5B. It is clear that the anodic peak currents are linearly correlated with Ole concentrations in the 0.30– 12 μmol L − 1 range. The linear regression equation is I =2.190 C0.188 (R2 = 0.998) where I is the anodic peak current (μA) and C is the concentration of Ole (μmol L − 1 ). Using 3s b/m and 10sb /m (where sb is the standard deviation of blank measurements and m is the slope of the calibration curve), the limit of detection and limit of quantification were calculated to be 0.090 and 0.29 μmol L− 1, respectively. Repeatability of the sensor was estimated with five replicate measurements at 1.5 and 7.0 μmol L− 1 of Ole, and resulted in RSDs of 6.0 and 3.8%.
3.5. Analytical application In order to investigate the possibility of applying the proposed biosensor to the quantification of Ole, extracts of olive leaves and human serum were analyzed using the calibration curve method. In order to verify the accuracy of the approach, the standard addition method was applied for the determination of Ole in spiked samples. As shown in Table 1, the recoveries obtained were acceptable. In addition, total value of Ole was calculated to be 359.4 mg per g of dry extract which is in good agreement with that obtained by HPLC. The results suggest that, it is feasible to apply the proposed electrochemical DNA biosensor to quantitatively determine the concentration of Ole in biological and plant samples.
4. Conclusion A simple, rapid, reliable and sensitive DNA biosensor was proposed for the determination of oleuropein based on the interaction between this biophenol compound and dsDNA. Chitosan, a biocompatible, biodegradable and non-toxic cationic polymer that forms polyelectrolyte complexes with DNA, was used for DNA immobilization on the electrode surface. The oxidation peak current of Ole was measured as an analytical signal for direct determination of the Ole molecules accumulated on the surface of DNA-coated CHIT/CPE. In this way, one can benefit the selectivity of electrochemical methods.
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