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NaA zeolite modified carbon paste electrode as a DNA biosensor
Sensors and Actuators B 181 (2013) 319–325
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Preparation of Ag/NaA zeolite modified carbon paste electrode as a DNA biosensor Seyed Naser Azizi a,∗ , Sara Ranjbar a , Jahan Bakhsh Raoof b , Ezat Hamidi-Asl b a b
Analytical Division, Faculty of Chemistry, University of Mazandaran, Babolsar, Iran Eletroanalytical Chemistry Research Laboratory, Department of Analytical Chemistry, Faculty of Chemistry, University of Mazandaran, Babolsar, Iran
a r t i c l e
i n f o
Article history: Received 2 November 2012 Received in revised form 3 February 2013 Accepted 6 February 2013 Available online 14 February 2013 Keywords: Methylene blue DNA hybridization Ag/NaA zeolite Modified carbon paste electrode DNA biosensor
a b s t r a c t In this research, we reported a method for synthesis of NaA zeolite. NaA zeolite has been successfully prepared within 30 min by microwave heating without using any conventional hydrothermal treatment and due to the exchange process the silver-loaded NaA microsized zeolites was produced. The synthesized zeolite was characterized using X-ray diffraction, scanning electronic microscopy and FT-IR techniques. The modified carbon paste electrode was prepared by incorporation of Ag(I) zeolite in the carbon paste matrix. Differential pulse voltammetry (DPV) was employed for development of electrochemical DNA hybridization biosensors based on Ag/NaA zeolite modified carbon paste electrode (Ag/ZMCPE) and methylene blue (MB) as electroactive label was used. The sensors rely on immobilization of a 15-mer single stranded oligonucleotide probe for detection of target DNA, as a model. The hybridization event was evaluated by DPV. Moreover, Ag/ZMCPE showed better advantages than NaA zeolite modified carbon paste electrode (ZMCPE). Under optimized experimental conditions, limit of detection was calculated 4.00 × 10−12 M. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Zeolites are an important group of crystalline aluminosilicates currently available for various fields of applications. These minerals are widely used as sorbents, ion exchangers, catalysts and catalyst supports [1,2]. The catalytic nature and activity, and other properties of zeolites can be greatly improved by cation exchange [3]. Zeolite-modified electrodes (ZMEs) form a subcategory of the so-called “chemically modified electrodes” (CMEs), which were largely studied and promoted by Murray and coworkers [4,5]. ZME can be exploited as electrochemical sensors in relation with zeolite’s properties, e.g., ion exchange capacity, molecular selectivity, and catalyst-assisted reactivity. Moreover, metal ion-doped zeolites allow exploitation of ion-exchange capacity of zeolite for the development of electrochemical sensors for the sensing non-electroactive inorganic or organic species [6]. In recent years, there has been considerable interest in developing electrochemical DNA biosensors for the rapid and inexpensive diagnosis of genetic diseases, forensic analysis and other applications [7,8]. DNA biosensors consist of a biological recognition
∗ Corresponding author at: Analytical Division, Faculty of Chemistry, University of Mazandaran, P.O. Box 47416-95447, Babolsar, Iran. Tel.: +98 1125342350; fax: +98 1125342350. E-mail address: [email protected] (S.N. Azizi). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.02.026
layer, usually single stranded DNA and a transducer converting the recognition event into a measurable signal. Optical, piezoelectric or electrochemical instruments are often used in DNA biosensors as transducers [9–11]. Electrochemical methods, in particular, provide sensitive, cost effective and rapid way of analysis [12,13]. The detection is accomplished by immobilization of single stranded DNA onto electrode surface and hybridization of a target DNA sequence present in the sample. The method is very efficient and specific, because DNA sensor can detect an analyte even in the presence of a mixture of many different nucleic acid fragments [14]. Methylene blue (MB) has been widely used as an electroactive label to monitor the DNA hybridization reaction [15–20] because less MB can bind to double strand DNA (dsDNA) compared to single strand DNA (ssDNA) [18]. The single mismatches in oligonucleotides were investigated by Kelly et al. [21] based on charge transport from the intercalated MB through selfassembled oligonucleotide. Erdem et al. studied the interaction between MB and ssDNA and dsDNA immobilized on gold electrode [22,23] and on carbon paste electrode [16–18]. The electrochemical detection of hybridization based on peptide nucleic acid probes with MB on carbon paste and modified gold electrode were also reported by Erdem group [24,25]. This article reports method for synthesizing NaA zeolite, studies of the electrochemical behavior of Ag(I) incorporated in zeolite-modified carbon paste electrode and the application of the modified electrode for electrochemical DNA biosensor.
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2. Experimental methods 2.1. Regents and material The NaA zeolite was synthesized in our laboratory. Sodium aluminate was from Fison. Graphite powder, sodium metasilicate pentahydrate, silver nitrate were purchased from Fluka. Sodium hydroxide used in this work was also purchased from Merck. All reagents were of analytical grade and used as received without further purification. In this work a 15-mer oligonucleotide as the probe and its complementary as the target nucleotide were used. Some oligonucleotides were utilized as non-complementary oligonucleotides. All oligonucleotides were synthesized by MW G-BIOTECH Company (Germany). The sequences of these oligonucleotides are as below: Probe DNA
5 -TGG GGA TGC AGA ACT-3
Complementary DNA
5 -AGT TCT GCA TCC CCA-3
Non-complementary DNAs NC1:
5 -GTT ACT GTT GTA GAT ACT AC-3
NC2
5 -GTA GTA TCT ACC ACA GTA AC-3
Stock solution of the oligonucleotides (100 M) were prepared with TE buffer solution (10 mM Tris–HCl, 1 mM EDTA, pH 8.00) and kept frozen. More diluted solutions of the oligonucleotides were prepared using 0.50 M acetate buffer solution (pH 4.80) containing 20 mM NaCl. Other chemicals were of analytical reagent grade. Distilled, deionized and sterilized water was used in all solution preparation. Each measurement consisted of immobilization of probe and detection of target DNA (immobilization/detection cycle) carried out on a fresh ZMCPE and Ag/ZMCPE surface. All the experiments were performed at room temperature in an electrochemical cell. 2.2. Instrumentation The X-ray diffractograms of zeolitic sample was measured using an X-ray diffractometer (XRD, GBC MMA Instrument, = 1.5418 A˚ and 28 mA). FT-IR spectrum was recorded at room temperature using FT-IR spectrometer (Vector 22-Bruker), in the range of 450–2000 cm−1 . Scanning electron microscopy (SEM) was done on selected samples to determine the crystallite size and morphology using a JEOL JXA-840 SEM. Electrochemical experiments were performed using AUTOLAB PGSTAT 30 electrochemical analysis system and GPES 4.9 software package (Eco Chemie, The Netherlands). The utilized three electrode system was composed of a ZMCPE and Ag/ZMCPE (surface area of 0.015 cm2 ) as the working electrode, a saturated calomel electrode (SCE) as the reference electrode for DPV and a platinum wire as the auxiliary electrode. 3. Procedure 3.1. Synthesis and characterization of NaA zeolite Sodium aluminate and sodium metasilicate pentahydrate were used as Al and Si sources, respectively, the sodium source were NaOH pellets. A sodium hydroxide solution was prepared which split into two portions prior to any other experiments. In the first step, a suitable amounts of sodium aluminate (NaAlO2 , 61 wt% of Al2 O3 , Merck) was added and stirred until clear. To the second half of the sodium hydroxide solution, suitable amounts of sodium metasilicate pentahydrate (SiO2 28%, Na2 O 29%, H2 O 43%) was added. The dissolution of the sodium metasilicate was very slow at room temperature; therefore, heating assistance was applied to the sample at 70 ◦ C for 1 h which is then filtered. The two
clear solutions were then recombined and the resulting white gel was shaken until homogeneous. The synthesis of zeolite NaA was attempted under only microwave heating [26]. A suitable composition of gels was employed for microwave heating at 30 and 60 min. The resulting white powder products were filtered and rinsed with distilled water before drying at 60 ◦ C for overnight. The XRD pattern is shown in Fig. 1A. Fig. 1A shows that the crystallization of NaA zeolite was completed in 30 min microwave irradiation and with increasing microwave irradiation time to 60 min; it displayed a phase transformation of zeolite NaA into sodalite. All the characteristic reference IR bands of the zeolite NaA with 30 min microwave irradiation are observed for the synthesized NaA (Fig. 1B). The reference IR wave numbers were given as 1002, 672, 557 and 464 cm−1 for NaA zeolite [27,28]. The broad band at about 3446 cm−1 and band at about 1652 cm−1 are attributed to zeolitic water, whereas the band at about 1002 cm−1 is due to asymmetrical vibrations related between tetrahedral. SEM micrograph of the synthesized zeolite sample with 30 min microwave irradiation is illustrated in Fig. 1C, which the formation of cubical crystallites with the smaller size of zeolite crystals. This is most likely due to the accelerated rate of heating and gel dissolution, which means the gel, is consumed quickly, forming more nuclei from which crystals grow. As limited reagents are left after the nuclei formation period, they produce many smaller crystals. The fact that the gel is heated for a shorter time also means that the time for crystal formation is not enough. Results showed that this method is a faster, simpler, higher purity and very energy efficient method. 3.2. Preparation of the working electrode For preparing the ionic silver–zeolite sample containing 33 wt% silver (Ag–NaA-33), 2 g of NaA zeolite was washed by distilled water and then stirred for 6 h with 160 mL AgNO3 (0.05 M) at 35 ◦ C. To have a completely ion-exchanged silver–zeolite, the samples were again mixed with fresh AgNO3 solution at the same conditions. Finally, the Ag/NaA zeolite particles were filtered and then dried at 50 ◦ C in an oven. All the steps were carried out in the absence of direct light. SEM micrograph of Ag/NaA zeolite particles is showed in Fig. 1D. The silver content of ion-exchanged samples was determined by EDS. The sodium elements were detected in minor amount by EDS in Ag+ /NaA samples, indicating the complete ion exchange of NaA zeolite (Fig. 1E). The ZMCPE (zeolite modified carbon paste electrode) and Ag/ZMCPE (Ag/NaA zeolite modified carbon paste electrode) was prepared by mixing NaA-zeolite and Ag/NaA-zeolite and high viscosity paraffin (density = 0.88 g cm−3 ) from Fluka in a ratio of 5:95%, 15:85%, 30:70% (w/w) of NaA zeolite and Ag/NaA zeolite to graphite powder in a mortar. Fig. 2 shows SEM and EDS of ZMCPE and Ag/ZMCPE in a ratio of 30:70% (w/w) NaA zeolite and Ag/NaA zeolite to graphite powder. As seen in this figure, zeolite particles were dispersed into the carbon paste very well. A portion of the resulting paste was then inserted in the bottom of a glass tube. The electrical connection was implemented by a copper wire lead fitted into the glass tube. The surface of the resulting paste electrodes were smoothed on a weighing paper and rinsed carefully with distilled water. 3.3. Electrochemical activation of the Ag/ZMCPE and ZMCPE The polished electrode was pretreated at optimized potential of 1.80 V vs. SCE for 5 min for electrochemical activation of electrode
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Fig. 1. (A) XRD pattern of the sample prepared by (a) 30 min, (b) 60 min microwave irradiation. (B) FT-IR spectrum of NaA zeolite. (C) SEM image of synthesized NaA zeolite by 30 min microwave irradiation. (D) SEM image of synthesized Ag/NaA zeolite. (E) The EDS spectra of ion-exchanged silver–zeolite.
surface. Pretreatment was carried out in 0.50 M acetate buffer solution (pH 4.80) containing 20 mM of NaCl without stirring. 3.4. Immobilization of probe on the Ag/ZMCPE and ZMCPE For immobilization of probe on Ag/ZMCPE and ZMCPE, following activation, the working electrode was immersed in 0.50 M acetate buffer solution (pH 4.8) containing 1 M probe and 20 mM of NaCl. After that, 0.5 V potential vs. SCE was applied to the electrode for 5 min into the stirred solution (with 200 rpm) at room temperature. Then, the electrode was rinsed with sterilized and deionized water.
3.6. MB accumulation on the Ag/ZMCPE and ZMCPE MB was accumulated on the probe-modified Ag/ZMCPE and ZMCPE by immersing the modified electrode into the 20 mM Tris–HCl buffer (pH 7.00) containing 20 mM MB and 20 mM NaCl for 5 min with 200 rpm stirring without applying any potential to the electrode. Once MB was accumulated, the electrode was rinsed with 20 mM Tris–HCl buffer (pH 7.00) for 5 s. The same protocol was applied for the accumulation of MB on the bare-electrode and probe-modified electrodes following hybridization with complementary or non-complementary oligonucleotides.
3.7. Voltammetric measurements 3.5. Hybridization Hybridization reaction was conducted by immersing the probe captured electrode into a stirred hybridization solution (0.5 M acetate buffer pH 4.8) containing 3 M of target oligonucleotide and 20 mM of NaCl, for 5 min, while the electrode potential was held at 0.50 V vs. SCE. The electrode was washed with sterilized and deionized water to remove the non-hybridized DNA. For hybridization of probe with non-complementary sequences, the same strategy was carried out.
Electrochemical investigation was carried out using DPV in 20 mM of Tris–HCl buffer (pH 7.00) solution and scanning the electrode potential between 0.00 and −0.50 V vs. SCE at pulse amplitude of 50 mV. The raw data were treated using the Savitzky and Golay filter (level 2) of GPES software, followed by the GPES software moving average baseline correction using a “peak width” of 0.01. Repetitive measurements were carried out following renewing the electrode surface by cutting and polishing of the electrode.
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4.1.2. Effect of activation time In order to optimize activation time of the working electrode, different activation time periods were used (N = 3). The accumulation of probe on the Ag/ZMCPE was performed according to the procedure described in Section 3. Fig. 3B displays the DPV response of probe modified Ag/ZMCPE versus activation time. As seen in this figure, the probe signal was increased with increasing the activation time. But after 5 min, the working electrode surface will be saturated of DNA. Therefore, stirring of the electrode in the DNA solution more than this period cusses desorption of accumulated probe from electrode surface. This behavior is reported in pervious published paper in the field of electrochemical DNA biosensors [10,30]. Thus, 5 min is adequate and optimized time for activation of the Ag/ZMCPE surface in order to accumulate almost maximum amount of DNA probe. 4.1.3. Effect of immobilization potential Considering that adsorption and immobilization of the probe on the electrode surface is very important for DNA biosensors function, and on the other hand, accumulation of DNA probes is influenced by the imposed potential to the electrode during the accumulation, the influence of imposed potential on the electrochemical behavior of probe was investigated using DPV. For this, we studied the imposed potentials ranging between −1.0 and 1.0 V vs. SCE (N = 3) (Fig. 3C). As shown in the figure, the potential around 0.50 V vs. SCE was obviously favorable for obtaining the maximum peak current for oxidation of guanine. Therefore, potential of 0.50 V vs. SCE was selected as optimum immobilization potential.
Fig. 2. EDS and SEM of (A) ZMCPE and (B) Ag/ZMCPE.
4.1.4. Effect of immobilization time The effect of immobilization time of the probe on the activated Ag/ZMCPE was studied using DPV. Fig. 3D shows DPV signal of probe versus the immobilization time (N = 3). As can be seen, the amount of adsorbed DNA rises with increasing adsorption time and starts to saturate at approximately 300 s.
4. Result and discussion 4.2. Interaction of MB with working electrode surface 4.1. Influence of electrochemical pretreatment of Ag/ZMCPE 4.1.1. Effect of activation potential Electrochemical pretreatment is usually required for activation of the working electrode surface [29]. Electrochemical pretreatment is commonly conducted either at negative or positive potentials [30]. In this study, potentiostatic method was used for activation of the electrode surface. In order to find an optimum activation potential, the polished Ag/ZMCPE was activated at different potentials within a wide voltage range (i.e., from −1.0 to 2.0 V vs. SCE, N = 3) and accumulation of probe DNA was conducted as described in Section 3.4. Fig. 3A shows the DPV response of probe immobilized on the activated Ag/ZMCPE as a probe at imposed potential ranging between −1.0 and 2.0 V vs. SCE. As seen in this figure, activity of the electrode improved when potentials exceeded from 0.5 V vs. SCE until reached to its maximum value at 1.80 V vs. SCE and then decreased at more positive potentials. When potential is ranged from −1.0 V to +1.8 V, the positive charges of electrode surface is increased. The more positive charge, the more electrostatic attraction. Therefore, the higher amount accumulation of DNA probe takes place. In potential more than +1.8 V not only the oxidation of solvent is started, but also the oxidation of DNA itself will be started on the electrode. Therefore, potential of +1.8 V vs. SCE is selected as optimum activation potential. This behavior is as same as pervious published paper in the field of electrochemical DNA biosensors [10,30].
Fig. 4 shows the DPV response of accumulated MB at bare activated 5% ZMCPE and Ag/ZMCPE. As seen in Fig. 4, there are significant differences between the signals of MB accumulated on the ZMCPE and Ag/ZMCPE. The higher signals obtained in the presence of silver could be due to an electrocatalytic effect of Ag+ on the MB reduction process. The catalytic effect of silver micro and nanoparticles on MB reduction has been studied by Yuan et al. [31]. Mehrgardi and Enayati Ahangar report the use of Ag nanoparticles as redox reporter for the amplified electrochemical detection of single base mismatches [32]. In order to further investigate about the interaction of MB with DNA on the Ag/ZMCPE and ZMCPE surface, six electrodes were made with different ratio of Ag/NaA zeolite and NaA zeolite. Differential pulse voltammetry was selected as simple and sensitive electrochemical technique. Fig. 5A shows the differential pulse voltammograms of accumulated MB on the probe modified ZMCPE and Ag/ZMCPE with different ratio of zeolite/graphite powder (W/W). Curve (a), (b) and (c) is related to 5%, 15% and 30% of ZMCPE, respectively. Also, curve (d), (e) and (f) is related to Ag/ZMCPE with 5%, 15% and 30% of zeolite/graphite powder, respectively. As seen, Ag/ZMCPE with 30% (w/w) of zeolite to graphite powder shows the highest reduction signal of MB. The best ratio for preparation of electrode is 30:70% (w/w) of Ag/NaA-zeolite to graphite powder. This observation clearly confirmed that MB has strong affinity for ssDNA and a considerable amount of MB accumulates on the probe modified Ag/ZMCPE,
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Fig. 3. The variations of DPV signal of the immobilized probe DNA on the activated Ag/ZMCPE vs. activation potentials (A) and activation times (B). The variations of DPV response of immobilized probe DNA on the activated Ag/ZMCPE vs. immobilization potential (C) and immobilization time (D). The concentration of probe DNA in solution was 1 M, other conditions for Ag/ZMCPE activation, probe DNA immobilization and DPV measurements as described in Section 3.
so that interaction of MB with ssDNA could be electrochemically detected and used for investigation of hybridization event. 4.3. Electrochemical detection of hybridization The height of differential pulse voltammetry of accumulated MB on the Ag/ZMCPE was chosen to investigate hybridization event between DNA probe and its complementary target. Fig. 5B shows the DPV voltammograms for accumulated MB at probe modified electrode before hybridization (curve a) and after hybridization in 15 ng/L solutions of complementary (curve b), and non-complementary oligonucleotides (curves c and d), respectively. The highest MB reduction signal was observed with the probe-modified Ag/ZMCPE because MB has a strong affinity for free guanine bases in ssDNA and hence the greatest amount of MB
accumulated on the Ag/ZMCPE surface. After hybridization of probe with complementary oligonucleotide target, a significant decrease in the MB signal was observed (curve b). This may be attributed to less MB accumulation on the double stranded DNA caused by the inaccessibility of MB to the guanine bases [16–18] or may be due to a steric inhibition of the reducible groups of MB packed between the bulky double helix of the DNA hybrids [15]. It is concluded that the decrease in the MB signal after hybridization represents the extent of the hybridization event at the electrode surface. Having observed this (remarkable difference between the reduction signals of MB obtained with dsDNA and ssDNA conditions) the probe-modified Ag/ZMCPE was used for the hybridization investigation between probe and noncomplementary oligonucleotides. As seen in Fig. 5B (curve c and d), the interaction between these non-complementary oligonucleotides and immobilized probe did not lead to MB signal decrease, due to negligible hybridization, and the signal was nearly equal to that of the probe signal. This result indicates that only a complementary sequence could form a double-stranded DNA on the probe-modified Ag/ZMCPE and causes a significant decrease in the accumulation of MB. 4.4. Detection limit of probe immobilization
Fig. 4. Differential pulse voltammograms of accumulated MB at bare activated (a) 5% ZMCPE and (b) 5% Ag/ZMCPE.
In order to determinate the detection limit (LOD), different concentrations of probe (0.002–60 nM) was immobilized on the Ag/ZMCPE, then the reduction signals of accumulated MB on the Ag/ZMCPE was investigated by DPV technique (N = 3). As shown in Fig. 6A and B, the MB reduction peak is obviously increased with increasing the amount of the probe. Fig. 6A illustrates the DPV signals obtained after immobilization of increasing levels of DNA (0.002–60 nM). Well defined peaks were obtained over a flat background. The variation of the voltammetric response vs. probe concentration is shown as Fig. 6B. The calibration graph is shown
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Fig. 5. (A) Differential pulse voltammograms of accumulated MB on the probe modified working electrode: (a) 5%, (b) 15%, (c) 30% (w/w) ZMCPE; (d) 5%, (e) 15%, (f) 30% (w/w) Ag/ZMCPE (w/w: zeolite/graphite powder). (B) Differential pulse voltammograms of accumulated MB on the probe-modified Ag/ZMCPE (a); after hybridization with complementary target sequence (b); non-complementary sequences (c) and (d).
in inset of Fig. 6B. There is a linear dependence between DPV signal and probe concentration between 0.002 and 0.008 nM with correlation coefficient of 0.99. The detection limit was calculated 4.00 × 10−12 M upon 3Sb /m (m, the slope of the standard curve
and Sb , standard deviation chart). These calculated detection limits prove that efficiency of biosensor improve in presence of zeolite modified with silver ion in the carbon paste. 5. Conclusions The results suggest a DNA hybridization biosensor based on using of Ag/NaA zeolite as a modifier and MB as an electroactive marker. Ag/NaA zeolite modified carbon paste electrode possess advantages of ease of preparation and easy renewable of surface. This study demonstrated that Ag/NaA zeolite modified carbon paste electrode had some advantages over NaA zeolite modified carbon paste electrode. Finally the selectivity of the biosensor was investigated using complementary and some non-complementary sequences. Our results demonstrated that the DNA biosensor had a good selectivity and sensitivity. Also this electrode has a suitable detection limit of probe immobilization toward other DNA hybridization sensors. This is the first report for using of zeolite in modified carbon paste electrode and using in DNA biosensor. On the other hand this study can be promising approaches to DNA biosensors. Todays, innovative and novel methods in DNA hybridization sensor play an important role in medical, forensic, agricultural, and environmental sciences. Therefore, Technological platforms that provide sensors of high sensitivity, selectivity and stability are in high demand. References
Fig. 6. (A) Differential pulse voltammograms of accumulated MB on the probe Ag/ZMCPE that was modified with different probe concentration (a) 0.002, (b) 0.004, (c) 0.006, (d) 0.008, (e) 0.1, (f) 0.2, (g) 0.4, (h) 0.6, (i) 10, (j) 20, (k) 40, (l) 60 nM; (B) differential pulse voltammograms responses vs. probe concentration; inset: related calibration plot at probe concentration range 0.002–0.008 nM.
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Biographies
Seyed Naser Azizi is a full professor of analytical chemistry, Mazandaran University, Babolsar, Iran. PhD from Durham University (England) in analytical chemistry, 1996–2001. His research interests are NMR studies of silicates, aluminosilicates, synthesis of zeolites and characterizations methods of zeolites, preparation of adsorbents and carbon active and nano particles.
Sara Ranjbar obtained her MSc in analytical chemistry in August 2011 from Mazandaran University, Babolsar, Iran; BSc in pure chemistry, Yazd University, Yazd, Iran, 2006. During her MSc degree she has worked in interdisciplinary project between spectroscopy, material chemistry and electrochemistry. In her project she has developed a new DNA biosensor with Ag zeolite modified carbon paste electrode. Also, she has experience about synthesis of nanoparticle and different variety of zeolite.
Jahan Bakhsh Raoof is a full professor of analytical chemistry, Mazandaran University, Babolsar, Iran. BS from Tabriz University, 1988; MS from Tabriz University in analytical chemistry, 1990; PhD from Tabriz University in analytical chemistry, 1996. His research interests cover electrochemical sensors, biosensors, electrochemical behavior of nanoparticles and fuel cell.
Ezat Hamidi-Asl obtained her PhD in analytical chemistry in March 2012 from Mazandaran University, Babolsar, Iran; MSc from Damghan University, Damghan, Iran, 2006; BSc in chemistry, Ferdowsi University, Mashhad, Iran, 2003. At the moment, she is postdoc researcher at the fellowship of Iranian nanotechnology initiative council in the field of synthesis of nano materials and using in DNA sensors. She has published several papers in field of bioelectrochemistry. Her research interest covers nanobiotechnology, spectroscopy, electrochemistry, DNA and RNA sensors and aptamers.
Contents lists available at SciVerse ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Preparation of Ag/NaA zeolite modified carbon paste electrode as a DNA biosensor Seyed Naser Azizi a,∗ , Sara Ranjbar a , Jahan Bakhsh Raoof b , Ezat Hamidi-Asl b a b
Analytical Division, Faculty of Chemistry, University of Mazandaran, Babolsar, Iran Eletroanalytical Chemistry Research Laboratory, Department of Analytical Chemistry, Faculty of Chemistry, University of Mazandaran, Babolsar, Iran
a r t i c l e
i n f o
Article history: Received 2 November 2012 Received in revised form 3 February 2013 Accepted 6 February 2013 Available online 14 February 2013 Keywords: Methylene blue DNA hybridization Ag/NaA zeolite Modified carbon paste electrode DNA biosensor
a b s t r a c t In this research, we reported a method for synthesis of NaA zeolite. NaA zeolite has been successfully prepared within 30 min by microwave heating without using any conventional hydrothermal treatment and due to the exchange process the silver-loaded NaA microsized zeolites was produced. The synthesized zeolite was characterized using X-ray diffraction, scanning electronic microscopy and FT-IR techniques. The modified carbon paste electrode was prepared by incorporation of Ag(I) zeolite in the carbon paste matrix. Differential pulse voltammetry (DPV) was employed for development of electrochemical DNA hybridization biosensors based on Ag/NaA zeolite modified carbon paste electrode (Ag/ZMCPE) and methylene blue (MB) as electroactive label was used. The sensors rely on immobilization of a 15-mer single stranded oligonucleotide probe for detection of target DNA, as a model. The hybridization event was evaluated by DPV. Moreover, Ag/ZMCPE showed better advantages than NaA zeolite modified carbon paste electrode (ZMCPE). Under optimized experimental conditions, limit of detection was calculated 4.00 × 10−12 M. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Zeolites are an important group of crystalline aluminosilicates currently available for various fields of applications. These minerals are widely used as sorbents, ion exchangers, catalysts and catalyst supports [1,2]. The catalytic nature and activity, and other properties of zeolites can be greatly improved by cation exchange [3]. Zeolite-modified electrodes (ZMEs) form a subcategory of the so-called “chemically modified electrodes” (CMEs), which were largely studied and promoted by Murray and coworkers [4,5]. ZME can be exploited as electrochemical sensors in relation with zeolite’s properties, e.g., ion exchange capacity, molecular selectivity, and catalyst-assisted reactivity. Moreover, metal ion-doped zeolites allow exploitation of ion-exchange capacity of zeolite for the development of electrochemical sensors for the sensing non-electroactive inorganic or organic species [6]. In recent years, there has been considerable interest in developing electrochemical DNA biosensors for the rapid and inexpensive diagnosis of genetic diseases, forensic analysis and other applications [7,8]. DNA biosensors consist of a biological recognition
∗ Corresponding author at: Analytical Division, Faculty of Chemistry, University of Mazandaran, P.O. Box 47416-95447, Babolsar, Iran. Tel.: +98 1125342350; fax: +98 1125342350. E-mail address: [email protected] (S.N. Azizi). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.02.026
layer, usually single stranded DNA and a transducer converting the recognition event into a measurable signal. Optical, piezoelectric or electrochemical instruments are often used in DNA biosensors as transducers [9–11]. Electrochemical methods, in particular, provide sensitive, cost effective and rapid way of analysis [12,13]. The detection is accomplished by immobilization of single stranded DNA onto electrode surface and hybridization of a target DNA sequence present in the sample. The method is very efficient and specific, because DNA sensor can detect an analyte even in the presence of a mixture of many different nucleic acid fragments [14]. Methylene blue (MB) has been widely used as an electroactive label to monitor the DNA hybridization reaction [15–20] because less MB can bind to double strand DNA (dsDNA) compared to single strand DNA (ssDNA) [18]. The single mismatches in oligonucleotides were investigated by Kelly et al. [21] based on charge transport from the intercalated MB through selfassembled oligonucleotide. Erdem et al. studied the interaction between MB and ssDNA and dsDNA immobilized on gold electrode [22,23] and on carbon paste electrode [16–18]. The electrochemical detection of hybridization based on peptide nucleic acid probes with MB on carbon paste and modified gold electrode were also reported by Erdem group [24,25]. This article reports method for synthesizing NaA zeolite, studies of the electrochemical behavior of Ag(I) incorporated in zeolite-modified carbon paste electrode and the application of the modified electrode for electrochemical DNA biosensor.
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2. Experimental methods 2.1. Regents and material The NaA zeolite was synthesized in our laboratory. Sodium aluminate was from Fison. Graphite powder, sodium metasilicate pentahydrate, silver nitrate were purchased from Fluka. Sodium hydroxide used in this work was also purchased from Merck. All reagents were of analytical grade and used as received without further purification. In this work a 15-mer oligonucleotide as the probe and its complementary as the target nucleotide were used. Some oligonucleotides were utilized as non-complementary oligonucleotides. All oligonucleotides were synthesized by MW G-BIOTECH Company (Germany). The sequences of these oligonucleotides are as below: Probe DNA
5 -TGG GGA TGC AGA ACT-3
Complementary DNA
5 -AGT TCT GCA TCC CCA-3
Non-complementary DNAs NC1:
5 -GTT ACT GTT GTA GAT ACT AC-3
NC2
5 -GTA GTA TCT ACC ACA GTA AC-3
Stock solution of the oligonucleotides (100 M) were prepared with TE buffer solution (10 mM Tris–HCl, 1 mM EDTA, pH 8.00) and kept frozen. More diluted solutions of the oligonucleotides were prepared using 0.50 M acetate buffer solution (pH 4.80) containing 20 mM NaCl. Other chemicals were of analytical reagent grade. Distilled, deionized and sterilized water was used in all solution preparation. Each measurement consisted of immobilization of probe and detection of target DNA (immobilization/detection cycle) carried out on a fresh ZMCPE and Ag/ZMCPE surface. All the experiments were performed at room temperature in an electrochemical cell. 2.2. Instrumentation The X-ray diffractograms of zeolitic sample was measured using an X-ray diffractometer (XRD, GBC MMA Instrument, = 1.5418 A˚ and 28 mA). FT-IR spectrum was recorded at room temperature using FT-IR spectrometer (Vector 22-Bruker), in the range of 450–2000 cm−1 . Scanning electron microscopy (SEM) was done on selected samples to determine the crystallite size and morphology using a JEOL JXA-840 SEM. Electrochemical experiments were performed using AUTOLAB PGSTAT 30 electrochemical analysis system and GPES 4.9 software package (Eco Chemie, The Netherlands). The utilized three electrode system was composed of a ZMCPE and Ag/ZMCPE (surface area of 0.015 cm2 ) as the working electrode, a saturated calomel electrode (SCE) as the reference electrode for DPV and a platinum wire as the auxiliary electrode. 3. Procedure 3.1. Synthesis and characterization of NaA zeolite Sodium aluminate and sodium metasilicate pentahydrate were used as Al and Si sources, respectively, the sodium source were NaOH pellets. A sodium hydroxide solution was prepared which split into two portions prior to any other experiments. In the first step, a suitable amounts of sodium aluminate (NaAlO2 , 61 wt% of Al2 O3 , Merck) was added and stirred until clear. To the second half of the sodium hydroxide solution, suitable amounts of sodium metasilicate pentahydrate (SiO2 28%, Na2 O 29%, H2 O 43%) was added. The dissolution of the sodium metasilicate was very slow at room temperature; therefore, heating assistance was applied to the sample at 70 ◦ C for 1 h which is then filtered. The two
clear solutions were then recombined and the resulting white gel was shaken until homogeneous. The synthesis of zeolite NaA was attempted under only microwave heating [26]. A suitable composition of gels was employed for microwave heating at 30 and 60 min. The resulting white powder products were filtered and rinsed with distilled water before drying at 60 ◦ C for overnight. The XRD pattern is shown in Fig. 1A. Fig. 1A shows that the crystallization of NaA zeolite was completed in 30 min microwave irradiation and with increasing microwave irradiation time to 60 min; it displayed a phase transformation of zeolite NaA into sodalite. All the characteristic reference IR bands of the zeolite NaA with 30 min microwave irradiation are observed for the synthesized NaA (Fig. 1B). The reference IR wave numbers were given as 1002, 672, 557 and 464 cm−1 for NaA zeolite [27,28]. The broad band at about 3446 cm−1 and band at about 1652 cm−1 are attributed to zeolitic water, whereas the band at about 1002 cm−1 is due to asymmetrical vibrations related between tetrahedral. SEM micrograph of the synthesized zeolite sample with 30 min microwave irradiation is illustrated in Fig. 1C, which the formation of cubical crystallites with the smaller size of zeolite crystals. This is most likely due to the accelerated rate of heating and gel dissolution, which means the gel, is consumed quickly, forming more nuclei from which crystals grow. As limited reagents are left after the nuclei formation period, they produce many smaller crystals. The fact that the gel is heated for a shorter time also means that the time for crystal formation is not enough. Results showed that this method is a faster, simpler, higher purity and very energy efficient method. 3.2. Preparation of the working electrode For preparing the ionic silver–zeolite sample containing 33 wt% silver (Ag–NaA-33), 2 g of NaA zeolite was washed by distilled water and then stirred for 6 h with 160 mL AgNO3 (0.05 M) at 35 ◦ C. To have a completely ion-exchanged silver–zeolite, the samples were again mixed with fresh AgNO3 solution at the same conditions. Finally, the Ag/NaA zeolite particles were filtered and then dried at 50 ◦ C in an oven. All the steps were carried out in the absence of direct light. SEM micrograph of Ag/NaA zeolite particles is showed in Fig. 1D. The silver content of ion-exchanged samples was determined by EDS. The sodium elements were detected in minor amount by EDS in Ag+ /NaA samples, indicating the complete ion exchange of NaA zeolite (Fig. 1E). The ZMCPE (zeolite modified carbon paste electrode) and Ag/ZMCPE (Ag/NaA zeolite modified carbon paste electrode) was prepared by mixing NaA-zeolite and Ag/NaA-zeolite and high viscosity paraffin (density = 0.88 g cm−3 ) from Fluka in a ratio of 5:95%, 15:85%, 30:70% (w/w) of NaA zeolite and Ag/NaA zeolite to graphite powder in a mortar. Fig. 2 shows SEM and EDS of ZMCPE and Ag/ZMCPE in a ratio of 30:70% (w/w) NaA zeolite and Ag/NaA zeolite to graphite powder. As seen in this figure, zeolite particles were dispersed into the carbon paste very well. A portion of the resulting paste was then inserted in the bottom of a glass tube. The electrical connection was implemented by a copper wire lead fitted into the glass tube. The surface of the resulting paste electrodes were smoothed on a weighing paper and rinsed carefully with distilled water. 3.3. Electrochemical activation of the Ag/ZMCPE and ZMCPE The polished electrode was pretreated at optimized potential of 1.80 V vs. SCE for 5 min for electrochemical activation of electrode
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Fig. 1. (A) XRD pattern of the sample prepared by (a) 30 min, (b) 60 min microwave irradiation. (B) FT-IR spectrum of NaA zeolite. (C) SEM image of synthesized NaA zeolite by 30 min microwave irradiation. (D) SEM image of synthesized Ag/NaA zeolite. (E) The EDS spectra of ion-exchanged silver–zeolite.
surface. Pretreatment was carried out in 0.50 M acetate buffer solution (pH 4.80) containing 20 mM of NaCl without stirring. 3.4. Immobilization of probe on the Ag/ZMCPE and ZMCPE For immobilization of probe on Ag/ZMCPE and ZMCPE, following activation, the working electrode was immersed in 0.50 M acetate buffer solution (pH 4.8) containing 1 M probe and 20 mM of NaCl. After that, 0.5 V potential vs. SCE was applied to the electrode for 5 min into the stirred solution (with 200 rpm) at room temperature. Then, the electrode was rinsed with sterilized and deionized water.
3.6. MB accumulation on the Ag/ZMCPE and ZMCPE MB was accumulated on the probe-modified Ag/ZMCPE and ZMCPE by immersing the modified electrode into the 20 mM Tris–HCl buffer (pH 7.00) containing 20 mM MB and 20 mM NaCl for 5 min with 200 rpm stirring without applying any potential to the electrode. Once MB was accumulated, the electrode was rinsed with 20 mM Tris–HCl buffer (pH 7.00) for 5 s. The same protocol was applied for the accumulation of MB on the bare-electrode and probe-modified electrodes following hybridization with complementary or non-complementary oligonucleotides.
3.7. Voltammetric measurements 3.5. Hybridization Hybridization reaction was conducted by immersing the probe captured electrode into a stirred hybridization solution (0.5 M acetate buffer pH 4.8) containing 3 M of target oligonucleotide and 20 mM of NaCl, for 5 min, while the electrode potential was held at 0.50 V vs. SCE. The electrode was washed with sterilized and deionized water to remove the non-hybridized DNA. For hybridization of probe with non-complementary sequences, the same strategy was carried out.
Electrochemical investigation was carried out using DPV in 20 mM of Tris–HCl buffer (pH 7.00) solution and scanning the electrode potential between 0.00 and −0.50 V vs. SCE at pulse amplitude of 50 mV. The raw data were treated using the Savitzky and Golay filter (level 2) of GPES software, followed by the GPES software moving average baseline correction using a “peak width” of 0.01. Repetitive measurements were carried out following renewing the electrode surface by cutting and polishing of the electrode.
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4.1.2. Effect of activation time In order to optimize activation time of the working electrode, different activation time periods were used (N = 3). The accumulation of probe on the Ag/ZMCPE was performed according to the procedure described in Section 3. Fig. 3B displays the DPV response of probe modified Ag/ZMCPE versus activation time. As seen in this figure, the probe signal was increased with increasing the activation time. But after 5 min, the working electrode surface will be saturated of DNA. Therefore, stirring of the electrode in the DNA solution more than this period cusses desorption of accumulated probe from electrode surface. This behavior is reported in pervious published paper in the field of electrochemical DNA biosensors [10,30]. Thus, 5 min is adequate and optimized time for activation of the Ag/ZMCPE surface in order to accumulate almost maximum amount of DNA probe. 4.1.3. Effect of immobilization potential Considering that adsorption and immobilization of the probe on the electrode surface is very important for DNA biosensors function, and on the other hand, accumulation of DNA probes is influenced by the imposed potential to the electrode during the accumulation, the influence of imposed potential on the electrochemical behavior of probe was investigated using DPV. For this, we studied the imposed potentials ranging between −1.0 and 1.0 V vs. SCE (N = 3) (Fig. 3C). As shown in the figure, the potential around 0.50 V vs. SCE was obviously favorable for obtaining the maximum peak current for oxidation of guanine. Therefore, potential of 0.50 V vs. SCE was selected as optimum immobilization potential.
Fig. 2. EDS and SEM of (A) ZMCPE and (B) Ag/ZMCPE.
4.1.4. Effect of immobilization time The effect of immobilization time of the probe on the activated Ag/ZMCPE was studied using DPV. Fig. 3D shows DPV signal of probe versus the immobilization time (N = 3). As can be seen, the amount of adsorbed DNA rises with increasing adsorption time and starts to saturate at approximately 300 s.
4. Result and discussion 4.2. Interaction of MB with working electrode surface 4.1. Influence of electrochemical pretreatment of Ag/ZMCPE 4.1.1. Effect of activation potential Electrochemical pretreatment is usually required for activation of the working electrode surface [29]. Electrochemical pretreatment is commonly conducted either at negative or positive potentials [30]. In this study, potentiostatic method was used for activation of the electrode surface. In order to find an optimum activation potential, the polished Ag/ZMCPE was activated at different potentials within a wide voltage range (i.e., from −1.0 to 2.0 V vs. SCE, N = 3) and accumulation of probe DNA was conducted as described in Section 3.4. Fig. 3A shows the DPV response of probe immobilized on the activated Ag/ZMCPE as a probe at imposed potential ranging between −1.0 and 2.0 V vs. SCE. As seen in this figure, activity of the electrode improved when potentials exceeded from 0.5 V vs. SCE until reached to its maximum value at 1.80 V vs. SCE and then decreased at more positive potentials. When potential is ranged from −1.0 V to +1.8 V, the positive charges of electrode surface is increased. The more positive charge, the more electrostatic attraction. Therefore, the higher amount accumulation of DNA probe takes place. In potential more than +1.8 V not only the oxidation of solvent is started, but also the oxidation of DNA itself will be started on the electrode. Therefore, potential of +1.8 V vs. SCE is selected as optimum activation potential. This behavior is as same as pervious published paper in the field of electrochemical DNA biosensors [10,30].
Fig. 4 shows the DPV response of accumulated MB at bare activated 5% ZMCPE and Ag/ZMCPE. As seen in Fig. 4, there are significant differences between the signals of MB accumulated on the ZMCPE and Ag/ZMCPE. The higher signals obtained in the presence of silver could be due to an electrocatalytic effect of Ag+ on the MB reduction process. The catalytic effect of silver micro and nanoparticles on MB reduction has been studied by Yuan et al. [31]. Mehrgardi and Enayati Ahangar report the use of Ag nanoparticles as redox reporter for the amplified electrochemical detection of single base mismatches [32]. In order to further investigate about the interaction of MB with DNA on the Ag/ZMCPE and ZMCPE surface, six electrodes were made with different ratio of Ag/NaA zeolite and NaA zeolite. Differential pulse voltammetry was selected as simple and sensitive electrochemical technique. Fig. 5A shows the differential pulse voltammograms of accumulated MB on the probe modified ZMCPE and Ag/ZMCPE with different ratio of zeolite/graphite powder (W/W). Curve (a), (b) and (c) is related to 5%, 15% and 30% of ZMCPE, respectively. Also, curve (d), (e) and (f) is related to Ag/ZMCPE with 5%, 15% and 30% of zeolite/graphite powder, respectively. As seen, Ag/ZMCPE with 30% (w/w) of zeolite to graphite powder shows the highest reduction signal of MB. The best ratio for preparation of electrode is 30:70% (w/w) of Ag/NaA-zeolite to graphite powder. This observation clearly confirmed that MB has strong affinity for ssDNA and a considerable amount of MB accumulates on the probe modified Ag/ZMCPE,
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Fig. 3. The variations of DPV signal of the immobilized probe DNA on the activated Ag/ZMCPE vs. activation potentials (A) and activation times (B). The variations of DPV response of immobilized probe DNA on the activated Ag/ZMCPE vs. immobilization potential (C) and immobilization time (D). The concentration of probe DNA in solution was 1 M, other conditions for Ag/ZMCPE activation, probe DNA immobilization and DPV measurements as described in Section 3.
so that interaction of MB with ssDNA could be electrochemically detected and used for investigation of hybridization event. 4.3. Electrochemical detection of hybridization The height of differential pulse voltammetry of accumulated MB on the Ag/ZMCPE was chosen to investigate hybridization event between DNA probe and its complementary target. Fig. 5B shows the DPV voltammograms for accumulated MB at probe modified electrode before hybridization (curve a) and after hybridization in 15 ng/L solutions of complementary (curve b), and non-complementary oligonucleotides (curves c and d), respectively. The highest MB reduction signal was observed with the probe-modified Ag/ZMCPE because MB has a strong affinity for free guanine bases in ssDNA and hence the greatest amount of MB
accumulated on the Ag/ZMCPE surface. After hybridization of probe with complementary oligonucleotide target, a significant decrease in the MB signal was observed (curve b). This may be attributed to less MB accumulation on the double stranded DNA caused by the inaccessibility of MB to the guanine bases [16–18] or may be due to a steric inhibition of the reducible groups of MB packed between the bulky double helix of the DNA hybrids [15]. It is concluded that the decrease in the MB signal after hybridization represents the extent of the hybridization event at the electrode surface. Having observed this (remarkable difference between the reduction signals of MB obtained with dsDNA and ssDNA conditions) the probe-modified Ag/ZMCPE was used for the hybridization investigation between probe and noncomplementary oligonucleotides. As seen in Fig. 5B (curve c and d), the interaction between these non-complementary oligonucleotides and immobilized probe did not lead to MB signal decrease, due to negligible hybridization, and the signal was nearly equal to that of the probe signal. This result indicates that only a complementary sequence could form a double-stranded DNA on the probe-modified Ag/ZMCPE and causes a significant decrease in the accumulation of MB. 4.4. Detection limit of probe immobilization
Fig. 4. Differential pulse voltammograms of accumulated MB at bare activated (a) 5% ZMCPE and (b) 5% Ag/ZMCPE.
In order to determinate the detection limit (LOD), different concentrations of probe (0.002–60 nM) was immobilized on the Ag/ZMCPE, then the reduction signals of accumulated MB on the Ag/ZMCPE was investigated by DPV technique (N = 3). As shown in Fig. 6A and B, the MB reduction peak is obviously increased with increasing the amount of the probe. Fig. 6A illustrates the DPV signals obtained after immobilization of increasing levels of DNA (0.002–60 nM). Well defined peaks were obtained over a flat background. The variation of the voltammetric response vs. probe concentration is shown as Fig. 6B. The calibration graph is shown
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Fig. 5. (A) Differential pulse voltammograms of accumulated MB on the probe modified working electrode: (a) 5%, (b) 15%, (c) 30% (w/w) ZMCPE; (d) 5%, (e) 15%, (f) 30% (w/w) Ag/ZMCPE (w/w: zeolite/graphite powder). (B) Differential pulse voltammograms of accumulated MB on the probe-modified Ag/ZMCPE (a); after hybridization with complementary target sequence (b); non-complementary sequences (c) and (d).
in inset of Fig. 6B. There is a linear dependence between DPV signal and probe concentration between 0.002 and 0.008 nM with correlation coefficient of 0.99. The detection limit was calculated 4.00 × 10−12 M upon 3Sb /m (m, the slope of the standard curve
and Sb , standard deviation chart). These calculated detection limits prove that efficiency of biosensor improve in presence of zeolite modified with silver ion in the carbon paste. 5. Conclusions The results suggest a DNA hybridization biosensor based on using of Ag/NaA zeolite as a modifier and MB as an electroactive marker. Ag/NaA zeolite modified carbon paste electrode possess advantages of ease of preparation and easy renewable of surface. This study demonstrated that Ag/NaA zeolite modified carbon paste electrode had some advantages over NaA zeolite modified carbon paste electrode. Finally the selectivity of the biosensor was investigated using complementary and some non-complementary sequences. Our results demonstrated that the DNA biosensor had a good selectivity and sensitivity. Also this electrode has a suitable detection limit of probe immobilization toward other DNA hybridization sensors. This is the first report for using of zeolite in modified carbon paste electrode and using in DNA biosensor. On the other hand this study can be promising approaches to DNA biosensors. Todays, innovative and novel methods in DNA hybridization sensor play an important role in medical, forensic, agricultural, and environmental sciences. Therefore, Technological platforms that provide sensors of high sensitivity, selectivity and stability are in high demand. References
Fig. 6. (A) Differential pulse voltammograms of accumulated MB on the probe Ag/ZMCPE that was modified with different probe concentration (a) 0.002, (b) 0.004, (c) 0.006, (d) 0.008, (e) 0.1, (f) 0.2, (g) 0.4, (h) 0.6, (i) 10, (j) 20, (k) 40, (l) 60 nM; (B) differential pulse voltammograms responses vs. probe concentration; inset: related calibration plot at probe concentration range 0.002–0.008 nM.
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Biographies
Seyed Naser Azizi is a full professor of analytical chemistry, Mazandaran University, Babolsar, Iran. PhD from Durham University (England) in analytical chemistry, 1996–2001. His research interests are NMR studies of silicates, aluminosilicates, synthesis of zeolites and characterizations methods of zeolites, preparation of adsorbents and carbon active and nano particles.
Sara Ranjbar obtained her MSc in analytical chemistry in August 2011 from Mazandaran University, Babolsar, Iran; BSc in pure chemistry, Yazd University, Yazd, Iran, 2006. During her MSc degree she has worked in interdisciplinary project between spectroscopy, material chemistry and electrochemistry. In her project she has developed a new DNA biosensor with Ag zeolite modified carbon paste electrode. Also, she has experience about synthesis of nanoparticle and different variety of zeolite.
Jahan Bakhsh Raoof is a full professor of analytical chemistry, Mazandaran University, Babolsar, Iran. BS from Tabriz University, 1988; MS from Tabriz University in analytical chemistry, 1990; PhD from Tabriz University in analytical chemistry, 1996. His research interests cover electrochemical sensors, biosensors, electrochemical behavior of nanoparticles and fuel cell.
Ezat Hamidi-Asl obtained her PhD in analytical chemistry in March 2012 from Mazandaran University, Babolsar, Iran; MSc from Damghan University, Damghan, Iran, 2006; BSc in chemistry, Ferdowsi University, Mashhad, Iran, 2003. At the moment, she is postdoc researcher at the fellowship of Iranian nanotechnology initiative council in the field of synthesis of nano materials and using in DNA sensors. She has published several papers in field of bioelectrochemistry. Her research interest covers nanobiotechnology, spectroscopy, electrochemistry, DNA and RNA sensors and aptamers.