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Selective and sensitive furazolidone biosensor based on DNA-modified TiO2-reduced graphene oxide
Applied Surface Science 356 (2015) 301–307
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Selective and sensitive furazolidone biosensor based on DNA-modified TiO2 -reduced graphene oxide Ali A. Ensafi ∗ , Mahboobeh Sohrabi, Mehdi Jafari-Asl, Behzad Rezaei Department of Analytical Chemistry, Faculty of Chemistry, Isfahan University of Technology, Isfahan 84156-83111, Iran
a r t i c l e
i n f o
Article history: Received 2 May 2015 Received in revised form 25 July 2015 Accepted 11 August 2015 Available online 13 August 2015 Keywords: DNA-biosensor Furazolidone TiO2 -reduced graphene oxide modified electrode Differential pulse voltammetry
a b s t r a c t In this study, a new methodology was used to develop a novel and sensitive electrochemical DNA biosensor for determination of furazolidone. This biosensor was fabricated from TiO2 -reduced graphene oxide (rGO) modified-carbon paste electrode (CPE) decorated with ds-DNA. The oxidation signals of guanine and adenine were used as probes to study the interactions, using differential pulse voltammetry. By interaction of furazolidone with the ds-DNA, the oxidation peaks current of guanine and adenine were decreased at the modified-CPE. A linear dependence of the guanine and adenine oxidation signals with furazolidone concentration were observed in the range of 1.0–150.0 pmol L−1 with a detection limit of 0.55 and 0.43 pmol L−1 based on guanine and adenine signals, respectively. The relative standard deviation for five replicate measurements of 10.0 pmol L−1 furazolidone was 4.3%. The influence of potential interfering compounds on the selectivity was studied. This biosensor was successfully applied for determination of trace amounts of furazolidone in real samples. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Furazolidone (FU), chemically known 3-{[(5-nitro-2-furyl) methylene] amino}-1,3-oxazolidin-one, is a class of antibacterial and antiprotozoal [1]. Furazolidone and nitrofuran derivatives have been applied in medicine for the treatment and control of common infections in animals and humans, for more than 30 years [2]. It has been reported that nitro compounds can contribute to a reversible one electron process due to the formation of the nitro radical anion (RNO2 −• ) and an irreversible three electron process corresponding to the formation of the hydroxylamine (RNHOH) in aprotic media, according to the following equations [3,4]. •
RNO2 + 1e RNO− 2 −•
RNO2 + 3e + 4H+ → RNHOH + H2 O
(1) (2)
Free radicals are reactive species that cause DNA damage [5,6]. Furazolidone potentially behaves as a cytotoxic drug [7]. Therefore, trace amounts determination of furazolidone is of great importance. Several methods have been reported to detect furazolidone, including HPLC [8], LC–MS [9], LC–MS/MS [10], ELISA [11], spectrophotometry [12] and fluorimetry [13,14]. However, most of
∗ Corresponding author. E-mail address: Ensafi@cc.iut.ac.ir (A.A. Ensafi). http://dx.doi.org/10.1016/j.apsusc.2015.08.085 0169-4332/© 2015 Elsevier B.V. All rights reserved.
them are time consuming procedures, require expensive instruments, suffer from interfering compounds, and/or do not have suitable detection limit. Electrochemical methods are inexpensive, highly sensitive and have long-term reliability and reproducibility [15]. Few electrochemical methods have been reported for the determination of furazolidone including differential pulse voltammetry with detection limit of 7.6 mg L−1 [16], spectroelectrochemistry with detection limit of 25 g L−1 [17], square-wave voltammetry with detection limit of 5.2 g L−1 [18], and cyclic voltammetry with detection limit of 33 mol L−1 [19]. DNA and DNA–drug interactions have been extensively studied over recent decades [20,21]. The interaction of hazardous compounds and oxidizing substances with DNA can be irreversible, causing instability and subsequently, breaking of the hydrogen bonds, and opening of the double helix [22]. Irreversible damage can be monitored electrochemically either by using changes in the oxidation peaks of guanine and adenine or by appearance of 8-oxo guanine characteristic peak [23]. To study DNA–drug interactions, electrochemical methods can provide rapid and low cost methods for the determination of drugs [24]. The amounts of immobilized DNA (as a probe) directly influence on the sensor’s accuracy, sensitivity and selectivity [25]. The most common materials that have been used as suitable supports are carbon based electrodes such as glassy carbon, carbon fiber, graphite and carbon paste [26]. Graphene is a two-dimensional (2D) sheet of sp2 hybridized carbon atoms tightly packed in a honeycomb lattice. Graphene has
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several unique properties such as excellent electronic conductivity, high specific surface area, high mechanical-flexibility [27], high charge mobility and thermal conductivity [28,29]. Therefore, it has stimulated interest in applications for graphene-based nanocomposites including photovoltaics, phtocatalysis, nanoelectronics, and sensors [29]. Modified reduced graphene oxide is used as electrochemical sensor for detection of inorganic [30,31] and organic compounds [32–34]. Titanium dioxide (TiO2 ) nanoparticles act as an effectual agent in biosensing applications due to its good essential biocompatibility, particular affinity to biomolecules and high reactivity. Owing to its high conductivity and low cost, TiO2 has become an attractive electrode material in different forms such as nanoparticles, nanoneedles and nanotubes. It can be decorated on graphene or other carbon material to fabricate electrochemical DNA-based biosensors [35]. In particular, using grapheme–TiO2 nanocomposites as a bed exploiting has several benefits such as high conductivity to electron transferring plus more active sites for the immobilization of DNA [36,37]. In addition, carbon paste electrode (CPE) was selected for the preparation of DNA biosensor due to a low background current, a wide potential window, distinct advantage of renewability with simple polishing and a simple fabrication procedure [38]. Also presence of nanocomposites such as graphene oxide and TiO2 can promote the active sites and the electron transfer reactions [39]. In this study, first, TiO2 nanoparticle was decorated on reduce graphene oxide (rGO) using hydrothermal method. Then, it was characterized by several techniques such as field emission scanning electron microscopy (FE-SEM), X-ray diffraction (XRD) and FT-IR. Then, TiO2 @rGO modified CPE was prepared and the ds-DNA was immobilized on the surface of the TiO2 @rGO-CPE, to fabricate the DNA biosensor (ds-DNA/TiO2 @rGO-CPE). To determine furazolidone, the oxidation signals of adenine and guanine were used as probes, using differential pulse voltammetry (DPV). The decreases in the electrochemical signals of the oxidation peaks current of guanine and adenine after their interaction with furazolidone were used as analytical signals. As mentioned before, the amounts of immobilized DNA (as a probe) directly influence the sensor’s accuracy, sensitivity and selectivity. Presence of both rGO and TiO2 nanoparticles promote the active sites to dramatically improve the immobilization quality and quantity of DNA, by electrostatic interaction, on the electrode surface. The new biosensor exhibits a better sensitivity for trace analysis than those reported previously. The new DNA-biosensor has high selectivity, sensitivity and fast response with low detection limit for detection for furazolidone in real samples.
2. Experimental 2.1. Chemicals All solutions were prepared using reagent grade chemicals and used as received without further purification. Doubly distilled water was used for preparation of solutions. Furazolidone, titanium isopropoxide (Ti(Oi Pr)4 , 98%), titanium dioxide nanoparticles (TiO2 NPs, 30 nm), and DMF were purchased from Fluka. Reagent grade Tris–HCl, CH3 COOH, CH3 COONa, H3 PO4 , NaCl, NaOH, paraffin and graphite powders (70 m) were purchased from Merck. Salmon sperm ds-DNA was purchased from Sigma–Aldrich (Cat. No.: D1626) with G-C% content of 41.2% and molecular mass of 1.3 × 106 Da. Furazolidone stock solution (1.0 mmol L−1 ) was prepared in phosphate buffer solution (PBS, pH 8.0) in the presence of 10% (v/v) DMF and stored at 4 ◦ C. PBS buffer solution was prepared by appropriate mixing of stock solution of 0.10 mol L−1 H3 PO4 and
0.20 mol L−1 NaOH solutions. The solutions pH were adjusted by addition of sodium hydroxide and/or H3 PO4 solution. Salmon sperm ds-DNA stock solution (100 mg L−1 ) was prepared by dissolving in Tris–HCl buffer (pH 7.0). More dilute solutions of the ds-DNA were prepared with 0.5 mol L−1 acetate buffer (pH 4.8) containing 0.02 mol L−1 NaCl. 2.2. Apparatus Electrochemical analysis, including voltammetric and impedimetric measurements, were performed using an Autolab PGSTAT 12, potentiostat/galvanostat that connected to a three-electrode cell (Metrohm, Model 663 VA stand) with GPES 4.9 software package (Eco Chemie, The Netherlands). The raw data was treated using Savitzky and Golay filter (level 2, from the GPES software) followed by the GPES software for moving average baseline correction with a “peak width” of 0.01. A conventional three-electrode cell was used with the modified working electrode (ds-DNA/TiO2 @rGOCPE), a saturated Ag/AgCl reference electrode and a platinum wire as an auxiliary electrode. A Metrohm 827 pH-meter with a glass electrode was used for pH measurements. UV–vis spectra were recorded with a double beam spectrophotometer (JASCO Model V-750) using 1.0 cm quartz cells. X-ray diffraction experiments were carried out with a Bruker D8 /Advance X-ray diffractometer and with Cu-K␣ radiation. Field emission scanning electron microscopy (FE-SEM) was performed on a Philips XL-30 at an accelerating voltage of 20 kV accomplished on a Philips. Fourier transform IR spectra were recorded using a JASCO FT-IR (680 plus) spectrometer. 2.3. Synthesis of graphene oxide and TiO2 @rGO nanocomposite Graphite oxide (GO) was prepared using modified Staudenmaier method [40]. For this purpose, 1.0 g of graphite powder (with a particle size of 70 m and purity of 99.999%) was added to a mixed solution of 20 mL conc. sulfuric acid, 10 mL conc. nitric acid and 10.0 g potassium chlorate. Then, the mixture was stirred continuously for approximately 100 h. Consequently, the solid was rinsed with 5 wt% HCl aqueous solution and repeatedly washed with deionized water until the pH of the filtrate became neutral. Then, the solid was dried at room temperature. Finally, the graphite oxide was dispersed in water and placed in an ultrasonic bath for 2 h to convert to exfoliated graphene oxide (EGO). Hydrothermal process was used for preparation of TiO2 @rGO. To get a homogenous colloidal suspension of exfoliated graphene oxide, 100 mg EGO was dispersed into 150 mL ethanol under ultrasonication, for 1 h. Then, 5 mL Ti(Oi Pr)4 was slowly added to the EGO suspension and the mixture was ultrasonicated for 1 h. The result mixture was refluxed at 120 ◦ C for 12 h. Finally, the product was separated by filtration and washed with deionized water. The product was dried at room temperature for one night. The TiO2 @rGO nanocomposite was achieved in the form of gray powder [41]. 2.4. Preparation of TiO2 @rGO modified carbon paste electrode (TiO2 @rGO-CPE) Unmodified CPE was prepared by adding 0.30 g paraffin oil to 0.70 g graphite powder and thoroughly hand-mixing in a mortar and pestle to produce a homogenous carbon paste. The result paste was then inserted into the bottom of a polyethylene syringe (geometrical surface area: 0.31 cm2 ) and it was smoothed on a weighing paper. Electrical contact was made by pushing a copper wire down the polyethylene syringe into the back of the mixture. When necessary, a new surface was obtained by pushing an excess of the paste out of the tube and polishing it on a weighing paper.
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TiO2 @rGO-CPE was prepared by mixing 0.30 g paraffin oil with 0.70 g graphite powder and 0.10 g TiO2 @rGO. The result mixture was dispersed in ethanol (for better homogeneity). The mixture was stirred by a magnetic stirrer till the solvent evaporated completely. The result paste then was inserted into the bottom of a polyethylene syringe (surface area, 0.31 cm2 ) and then it was smoothed on a weighing paper. When necessary, a new surface was obtained by pushing an excess of the paste out of the syringe followed by polishing it on a weighing paper. 2.5. Immobilization of ds-DNA on TiO2 @rGO-CPE and electrochemical determination First, the surface of the modified carbon paste electrode (TiO2 @rGO-CPE) was smoothed by polishing with a weighing paper. Then, the surface of the modified CPE was pretreated by applying 1.70 V for 200 s in 0.1 mol L−1 acetate buffer solution (pH 4.8). The TiO2 @rGO-CPE was immersed into a stirred (at constant rate, 200 rpm) solution of ds-DNA (25.0 mg L−1 in 0.5 mol L−1 acetate buffer containing 0.02 mol L−1 NaCl, pH 4.8) and a potential of 0.50 V vs. Ag/AgCl was applied for 240 s. The electrode was gently washed with 0.1 mol L−1 acetate buffer (pH 4.8) to remove unbounded DNA. This DNA-biosensor was designated as ds-DNA/TiO2 @rGO-CPE. For electrochemical detection of furazolidone with the biosensor, first, ds-DNA/TiO2 @rGO-CPE was immersed into 10 mL of 0.1 mol L−1 acetate buffer (pH 4.8). A positive-going differential pulse potential scan (from +0.40 to +1.40) was performed by applying a step potential of 10 mV, pulse amplitude of 70 mV and modulation time of 0.05 s. The oxidation signals of the guanine and adenine were recorded using differential pulse voltammetry (DPV) and tagged as blank signals (Ib ). Then, DNA/TiO2 @rGO-CPE was inserted into 10 mL of phosphate buffer (pH 8.0, at the optimum conditions) containing different concentrations of furazolidone while the solution was stirred at 200 rpm for 240 s at an open circuit system. After accumulation of furazolidone on the biosensor surface, the ds-DNA/TiO2 @rGO-CPE was rinsed with distilled water and placed into a 10 mL of the acetate buffer solution (pH 4.8). The guanine and adenine signals were recorded using DPV to get the sample signals (Is ). 2.6. Preparation of real samples Blood serum was obtained from hospitalized patients and urine samples were collected from the urine of a healthy person, stored at −20 ◦ C until analysis. Different amounts of furazolidone were spiked into the test samples before the treatment. In order to precipitate proteins in the blood serum samples, 5 mL of the sample was treated with 0.1 mL perchloric acid (HClO4 , 20%, v/v). Then, the mixture was vortexed for further 45 s and then centrifuged at 6000 rpm for 10 min. The separated solution was diluted two times with the phosphate buffer solution (pH 8.0) and transferred into the electrochemical cell to be analyzed without further pretreatment. The furazolidone was measured according to the recommended procedure, using standard addition method. 3. Results and discussion 3.1. TiO2 @rGO nanocomposite characterizations To investigate the physical properties of the TiO2 @rGO, different spectroscopic methods such as XRD, FE-SEM and FT-IR were used. FT-IR spectra of TiO2 (a), reduce graphene oxide (b) and TiO2 –rGO (c), in the range of 400–4000 cm−1 , are shown in Fig. 1A. The characteristic peaks of rGO including O H stretching at 3300–3600 cm−1 , aromatic C C at 1600 cm−1 , and C O stretching at 1100 cm−1
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were distinctly observed in the FT-IR spectrum of rGO. The band of the prepared TiO2 at 580 cm−1 is assigned to Ti O Ti stretching, 1630 cm−1 is OH bending and 3250 cm−1 is due to the O H stretching. In the spectrum of TiO2 @rGO (Fig. 1A-c) most of the peaks in the spectra of rGO and TiO2 still exist. In addition, the broad band between 600 and 1000 cm−1 clearly represents the vibration of Ti O stretching in TiO2 -based composite [42]. The absorption band appearing at 1590 cm−1 shows skeletal vibration of graphene sheets [43]. These results support that TiO2 stabilized well at the surface of rGO during the synthesis of TiO2 –rGO with the hydrothermal process. The XRD pattern of TiO2 (a), rGO (b) and TiO2 @rGO (c) are shown in Fig. 1B. The diffraction peak for rGO at 25.14◦ that is indicated to the (0 0 2) plane confirms formation of rGO from GO [44]. The diffraction peaks existing at 2 values of TiO2 nanoparticles at about 25.3◦ , 37.9◦ , 47.8◦ , and 54.4◦ are observed for all of the diffraction patterns, the XRD pattern of the free TiO2 , which obviously match well with the anatase phase (JCPDS 21-1272). In the patterns of the as-prepared TiO2 –rGO the major diffraction lines can be indexed to the anatase phase, suggesting the complete formation of anatase TiO2 during the hydrothermal process. These results confirm that the preparation of TiO2 –rGO with the hydrothermal process is successful. The average particle size of the TiO2 –rGO was estimated according to the Scherer’s equation, D = 0.9/(ˇ2 ·cos ), where D is the average particle size, is the Xray wavelength (0.15418 nm for Cu radiation), ˇ2 is the full width at the half-maximum (FWHM) and is the angle corresponding to the peak maximum. The calculated value of the average particles size was about 63 nm. Fig. 1C illustrates FE-SEM surface morphologies and structures of rGO (a) and TiO2 @rGO (b). The accumulation between TiO2 and rGO in TiO2 @rGO can be pictured from the FE-SEM image, in which TiO2 nanoparticles, some of the nanoparticles have size of less than 50 nm, and are compactly decorated on the graphene substrate. 3.2. Characterization of the modified carbon paste electrode Preliminary modification of the CPE was used to enhance the electrode conductivity and to form an interface with multiple sites for the DNA immobilization. rGO is drastically oxygenated, bearing epoxide and hydroxyl groups on its basal planes and carboxyl groups at the sheet edges [45]. These oxygen containing groups can effectively interact with the hard Ti4+ Lewis acid, which promotes the intercalation and graft of Ti species into GO layers [46,47]. Moreover, positively charged TiO2 –rGO nanocomposite (by applying a potential of 0.50 V vs. Ag/AgCl was applied for 240 s) adsorbed the negatively charged DNA in the solution interface could be absorbed by the DNA at the TiO2 –rGO surface to fabricate ds-DNA/TiO2 @rGO. The surface morphology of the stepwise fabrication of the modified electrode was investigated using FE-SEM (Fig. 2A). Fig. 2A-a shows the FE-SEM image of the unmodified CPE. The graphite layers and empty space between the layers were well seen in this image. It can be seen that the effective surface area of the modified working electrode is dramatically increased with addition of highly conductive TiO2 @rGO (Fig. 2A-b). When DNA is immobilized on the surface of TiO2 @rGO-CPE, the surface morphology is changed again (Fig. 2A-c). Cyclic voltammograms of different electrodes in 5.0 mmol L−1 K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ], containing 0.1 mol L−1 KNO3 , were recorded with a scan rate of 20 mV s−1 (Fig. 2B). The unmodified CPE has a pair of well-defined voltammetric peaks with a cathodic peak potential (Epc ) of −0.145 V and an anodic peak potential (Epa ) of +0.448 V with peak-to-peak separation (Ep ) of 0.593 V. TiO2 @rGO modified carbon paste electrode has a pair of well-defined voltammetric peaks with peak-to-peak separation of 0.396 V. Moreover, the peak-to-peak separation of TiO2 @rGO-CPE
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Fig. 1. (A) FT-IR spectra, (B) XRD pattern of (a) TiO2 , (b) rGO, and (c) TiO2 @rGO and (C) FE-SEM images of (a) rGO and (b) TiO2 @rGO.
Fig. 2. (A) FE-SEM images of (a) unmodified CPE, (b) TiO2 @rGO-CPE, and (c) ds-DNA/TiO2 @rGO-CPE, (B) cyclic voltammograms and (C) impedance spectra (Nyquist plot) of (a) unmodified CPE, (b) TiO2 @rGO-CPE, and (c) ds-DNA/TiO2 @rGO-CPE in 5.0 mmol L−1 [Fe(CN)6 ]3−/4− containing 0.1 mol L−1 KNO3 .
reduced, whereas the peak current of TiO2 @rGO-CPE significantly increased. Therefore, TiO2 @rGO modified carbon paste electrode improves the electron transfer reactions vs. the unmodified CPE. When negatively charged ds-DNA was immobilized at the surface of TiO2 @rGO-CPE, the electron transfer kinetic (peak-to-peak separation) and the redox peak currents of K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] slowly decreased. Fig. 2C shows the Nyquist plots for the unmodified and modified CPE at different conditions. A solution containing 5.0 mmol L−1
K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ], in the presence of 0.10 mol L−1 KNO3 , was used as a probe at a polarization potential of 0.10 V and in the frequency range of 5 mHz to 100 KHz with an amplitude of 10 mV. As shown in Fig. 2C, the charge transfer resistance (Rc.t ) of the electrode decreased dramatically after modification of the CPE with TiO2 @rGO, which is due to the acceleration of the electron transfer. This is due to the promotion of the electron exchange between K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] and the electrode surface. The charge transfer resistance of ds-DNA/TiO2 @rGO-CPE increased in
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Fig. 3. Differential pulse voltammograms of the interaction of furazolidone with the ds-DNA at ds-DNA/TiO2 @rGO-CPE. The oxidation signals of guanine and adenine after interaction with different concentrations of furazolidone as: 1.0, 2.5, 5.0, 7.5, 10.0, 25.0 and 50.0 pmol L−1 (from up to down).
Fig. 4. UV–vis spectra of 0.01 mol L−1 furazolidone before (a) and after (c) interaction with 25.0 mg L−1 ds-DNA, and (b) 25.0 mg L−1 ds-DNA.
the probe solution, indicating the formation of the negatively charged DNA layer on the TiO2 @rGO-CPE surface.
Table 1 Effect of potential interfering substances on the determination of 10 pmol L−1 furazolidone.
3.3. Optimization of the experimental parameters, interaction between furazolidone with ds-DNA/TiO2 @rGO-CPE The interaction between furazolidone and the ds-DNA was inspected by DPV technique. The guanine and adenine oxidation signals in DNA appeared around +0.95 and +1.26 V, respectively. Fig. 3 shows the oxidation signals of guanine and adenine at the ds-DNA/TiO2 @rGO-CPE, after their interactions with furazolidone. Different concentrations of furazolidone were used to interact with the ds-DNA at the surface of ds-DNA/TiO2 @rGO-CPE. It was observed that with increasing in the furazolidone concentration, the oxidation currents of guanine and adenine were decreased. The decrease in the oxidation signals of guanine and adenine, after interaction with furazolidone, could be attributed to the interaction of furazolidone with the ds-DNA at ds-DNA/TiO2 @rGO-CPE surface, resulting the helix destabilization and strand breakage [48]. In order to get the highest sensitivity and the lowest detection limit with the differential pulse voltammetric method, the influence of various parameters affect the response of the biosensor including the modifier percentage, ds-DNA concentration, accumulation time and immobilized potential were optimized. The influence of the modifier (TiO2 @rGO) percentage on the oxidation signals of guanine and adenine at CPE was studied. The results are illustrated in Supplementary materials (Fig. S1A). The results confirm that increasing the percentage of TiO2 @rGO causes an increase in the positive sites, to have more immobilized DNA at the electrode surface. Fig. S1B shows the influence of the ds-DNA concentration, in the fabrication step of the biosensor, on the response of dsDNA/TiO2 @rGO-CPE using the peaks current of the guanine and adenine. The results demonstrated that the guanine and adenine signals increased as DNA concentration rose up to 25.0 mg L−1 , and then leveled off. This is due to the fact that the sites on the electrode surface were filled with the ds-DNA. Therefore, 25.0 mg L−1 of ds-DNA was selected and used to prepare the DNA-modified electrode. To immobilize the ds-DNA at TiO2 @rGO-CPE, different potentials were applied at the electrode between −0.20 and +0.70 V. More details in this study are summarized in Supplementary materials (Fig. S1C). Based on the results, +0.50 V was selected as an optimum immobilization potential. The effect of accumulation time on the immobilization of the ds-DNA at TiO2 @rGO-CPE surface was
Species
Tolerance limit (w/w)
SCN− , sodium citrate, glucose, ascorbic acid NH4 OH, 4-nitrophenel Uric acid, thiourea, dopamine, acetaminophen
500 250 100
also studied (Fig. S1D). The results showed that the oxidation signals of guanine and adenine increased till 240 s, and then leveled off. Therefore, 240 s was considered as an optimum accumulation time. After preparation of the biosensor, the effect of sample solution pH (in the presence of furazolidone) on the guanine and adenine signals at ds-DNA/TiO2 @rGO-CPE was studied too. The results showed that the sensitivity of the biosensor was increased by increasing the solution pH up to 8.0 (Fig. S2A). Moreover, the interaction of furazolidone with the ds-DNA depends on the incubation time. More details in this study are summarized in supplementary materials (Fig. S2B). The results confirmed that by increasing the incubation time till 240 s, the oxidation signals of guanine and adenine were decreased (the sensitivity increased) and then it almost leveled off for higher incubation time. Therefore, 240 s was selected as a suitable incubation time (Fig. S2B). 3.4. UV–visible spectrophotometry study and calculation of the association constant of furazolidone with the ds-DNA UV–visible absorption spectrophotometry was employed to investigate the furazolidone–DNA interaction. The absorption spectra of furazolidone, ds-DNA and ds-DNA ds-DNA-furazolidone (after its interaction with ds-DNA) are shown in Fig. 4. The maximum absorbance of furazolidone is located at 217 nm as illustrated in (Fig. 4a), whereas the maximum absorbance of the ds-DNA is located at 260 nm (Fig. 4b). The absorbance spectra of the mixture of furazolidone and the ds-DNA are shown in (Fig. 4c) with a maximum absorbency of 272 nm. Moreover, the absorbance of ds-DNA decreases after its interaction with furazolidone. The red shift on the ds-DNA spectrum and decreasing in the absorbance intensity demonstrated a strong interaction between furazolidone and the ds-DNA, due to the intercalative binding of furazolidone to the ds-DNA. To calculate of the association constant between furazolidone with the ds-DNA, it is assumed that the DNA and furazolidone only
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Table 2 A systematic comparison on the unmodified CPE, TiO2 @rGO, and ds-DNA/TiO2 @rGO for the electrochemical detection of furazolidone. Electrode
Peak potential (V)
Dynamic range
Detection limit
Sensitivity
Interfering compounds
Unmodified CPE
−0.48
2–30 mol L−1
0.5 mol L−1
−0.015
TiO2 @rGO-CPE
−0.44
0.05–20 mol L−1
0.01 mol L−1
−1.84
DNA/TiO2 @rGO-CPE
Guanine oxidation Adenine oxidation
1–150 pmol L−1
0.55 pmol L−1 0.43 pmol L−1
−0.0544 −0.0704
O2 , ascorbic acid, dopamine, uric acid, urea, acetaminophen O2 , ascorbic acid, dopamine, uric acid, urea, acetaminophen No interfere
0.95 1.25
produce a single complex as DNA.Fum according to the following reaction: DNA + mFu ↔ DNA.Fum
(3)
The UV absorption is most sensitive to the equilibrium constant of DNA.Fum complex [49]. The decreasing value of the UV absorption (in 265 nm) is proportional to the concentration of DNA.Fum complex. KA =
[DNA.Fum ] [DNA][Fu]m
(4)
Totally absorption of solution = εDNA.Fu [DNA.Fum ] + εDNA [DNA]. From Eq. (4), we can obtain: εDNA εDNA A0 = + εDNA + εDNA.Fu (A0 − A) (εDNA + εDNA.Fu )(1/KA [Fu]m )
(5)
If m = 1, by keeping the DNA concentration at 20 mg L−1 and varying the concentration of Fu, a plot of A0 /(A0 − A) as a function of 1/[Fu]m is linear with a slope of DNA /(DNA + DNA.Fu )(1/KA ). The results of KA = 1.04 × 106 M−1 were obtained in the concentration range of the applied furazolidone. 4. Figures of merit DPV was used, with a pulse width of 50.0 ms, pulse amplitude of 50 mV and scan rate of 20 mV s−1 , to check the linear dynamic range and limit of detection of the ds-DNA/TiO2 @rGO-CPE for furazolidone detection. The oxidation peaks current of guanine and adenine at ds-DNA/TiO2 @rGO-CPE were depended on the furazolidone concentration. The results approved that guanine and adenine oxidation currents were decreased with increasing the furazolidone concentration in the range of 1.0–150.0 pmol L−1 , with a correlation equations of I (A) = –0.0544C (pmol L−1 ) + 0.9568 (R2 = 0.9959, n = 5) and I (A) = –0.0704C (pmol L−1 ) + 1.1641 (R2 = 0.9917, n = 5) for guanine and adenine signals, respectively. The limit of detection (3Sb/m) based on guanine and adenine peaks current were obtained as 0.55 and 0.43 pmol L−1 of furazolidone, respectively. Five replicates DPV measurements of furazolidone concentration of 10.0 and 100.0 pmol L−1 showed relative standard deviations of 4.3%, 4.8% and 3.4%, 4.7% for guanine and adenine signals, respectively. Table 1 shows a systematic comparison of the three electrodes containing unmodified-CPE, TiO2 @rGO and ds-DNA/TiO2 @rGO in terms of the detection sensitivity and selectivity of the electrochemical detection of furazolidone. 5. Interference study Before evaluating the performance of the biosensor for analysis of furazolidone in real sample, the effect of potential interfering substances were studied by addition of potential interfering substances into a solution containing 10.0 pmol L−1 furazolidone at the optimum conditions. The potential interfering compounds
Table 3 Recoveries of furazolidone in blood serum and urine samples. Sample
Sample no
Blood serum
1
–
2 3
10.0 75.0
9.8 ± 0.4 75.9 ± 2.0
1 2 3
– 10 75
Detection limit 9.7 ± 0.5 74.8 ± 1.5
Urine
Furazolidone added (pmol L−1 )
Found by biosensor (pmol L−1 ) Detection limit
Recovery (%)
– 98.6 101.2 – 98.6 99.7
were chosen from a group of substances commonly found with furazolidone in pharmaceuticals and biological fluids. The tolerance limit was defined as the maximum concentration of the interfering substance that caused an error less than 5% for the detection of the furazolidone. The oxidation peaks current of guanine and adenine were compared before and after interaction of ds-DNA/TiO2 @rGO-CPE with the sample solutions containing furazolidone in the presence and absence of the potential interfering compounds. The results are given in Table 2, indicating that the biosensor has good selectivity for determination of furazolidone.
6. Real sample analysis In order to evaluate the performance of the biosensor for analysis of furazolidone in real samples, furazolidone in blood serum and urine samples were measured at the optimum conditions using differential pulse voltammetry. The results are given in Table 3, which shows that the new biosensor is able to measure furazolidone in different real samples.
7. Conclusion In this article, a selective DNA electrochemical biosensor was developed by modification of a carbon paste electrode with TiO2 reduced graphene oxide and ds-DNA for the determination of furazolidone in real sample. The result demonstrated that the new nanocomposite acts as active sites to fabricate the DNA-biosensor. The TiO2 –graphene nanocomposites provide good adsorptivity, conductivity and active sites for the immobilization of the ds-DNA. After interaction of furazolidone with the ds-DNA at the electrode surface, a decrease in the adenine and guanine oxidation signals were observed. The advantages of the suggested method are low detection limit, wide linear dynamic range, the sensitivity, the selectivity and its applicability. The ds-DNA/TiO2 @rGO-CPE is not only suitable to examine the interaction of ds-DNA with furazolidone, but also can be used to detect DNA damage caused by furazolidone.
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Selective and sensitive furazolidone biosensor based on DNA-modified TiO2 -reduced graphene oxide Ali A. Ensafi ∗ , Mahboobeh Sohrabi, Mehdi Jafari-Asl, Behzad Rezaei Department of Analytical Chemistry, Faculty of Chemistry, Isfahan University of Technology, Isfahan 84156-83111, Iran
a r t i c l e
i n f o
Article history: Received 2 May 2015 Received in revised form 25 July 2015 Accepted 11 August 2015 Available online 13 August 2015 Keywords: DNA-biosensor Furazolidone TiO2 -reduced graphene oxide modified electrode Differential pulse voltammetry
a b s t r a c t In this study, a new methodology was used to develop a novel and sensitive electrochemical DNA biosensor for determination of furazolidone. This biosensor was fabricated from TiO2 -reduced graphene oxide (rGO) modified-carbon paste electrode (CPE) decorated with ds-DNA. The oxidation signals of guanine and adenine were used as probes to study the interactions, using differential pulse voltammetry. By interaction of furazolidone with the ds-DNA, the oxidation peaks current of guanine and adenine were decreased at the modified-CPE. A linear dependence of the guanine and adenine oxidation signals with furazolidone concentration were observed in the range of 1.0–150.0 pmol L−1 with a detection limit of 0.55 and 0.43 pmol L−1 based on guanine and adenine signals, respectively. The relative standard deviation for five replicate measurements of 10.0 pmol L−1 furazolidone was 4.3%. The influence of potential interfering compounds on the selectivity was studied. This biosensor was successfully applied for determination of trace amounts of furazolidone in real samples. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Furazolidone (FU), chemically known 3-{[(5-nitro-2-furyl) methylene] amino}-1,3-oxazolidin-one, is a class of antibacterial and antiprotozoal [1]. Furazolidone and nitrofuran derivatives have been applied in medicine for the treatment and control of common infections in animals and humans, for more than 30 years [2]. It has been reported that nitro compounds can contribute to a reversible one electron process due to the formation of the nitro radical anion (RNO2 −• ) and an irreversible three electron process corresponding to the formation of the hydroxylamine (RNHOH) in aprotic media, according to the following equations [3,4]. •
RNO2 + 1e RNO− 2 −•
RNO2 + 3e + 4H+ → RNHOH + H2 O
(1) (2)
Free radicals are reactive species that cause DNA damage [5,6]. Furazolidone potentially behaves as a cytotoxic drug [7]. Therefore, trace amounts determination of furazolidone is of great importance. Several methods have been reported to detect furazolidone, including HPLC [8], LC–MS [9], LC–MS/MS [10], ELISA [11], spectrophotometry [12] and fluorimetry [13,14]. However, most of
∗ Corresponding author. E-mail address: Ensafi@cc.iut.ac.ir (A.A. Ensafi). http://dx.doi.org/10.1016/j.apsusc.2015.08.085 0169-4332/© 2015 Elsevier B.V. All rights reserved.
them are time consuming procedures, require expensive instruments, suffer from interfering compounds, and/or do not have suitable detection limit. Electrochemical methods are inexpensive, highly sensitive and have long-term reliability and reproducibility [15]. Few electrochemical methods have been reported for the determination of furazolidone including differential pulse voltammetry with detection limit of 7.6 mg L−1 [16], spectroelectrochemistry with detection limit of 25 g L−1 [17], square-wave voltammetry with detection limit of 5.2 g L−1 [18], and cyclic voltammetry with detection limit of 33 mol L−1 [19]. DNA and DNA–drug interactions have been extensively studied over recent decades [20,21]. The interaction of hazardous compounds and oxidizing substances with DNA can be irreversible, causing instability and subsequently, breaking of the hydrogen bonds, and opening of the double helix [22]. Irreversible damage can be monitored electrochemically either by using changes in the oxidation peaks of guanine and adenine or by appearance of 8-oxo guanine characteristic peak [23]. To study DNA–drug interactions, electrochemical methods can provide rapid and low cost methods for the determination of drugs [24]. The amounts of immobilized DNA (as a probe) directly influence on the sensor’s accuracy, sensitivity and selectivity [25]. The most common materials that have been used as suitable supports are carbon based electrodes such as glassy carbon, carbon fiber, graphite and carbon paste [26]. Graphene is a two-dimensional (2D) sheet of sp2 hybridized carbon atoms tightly packed in a honeycomb lattice. Graphene has
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several unique properties such as excellent electronic conductivity, high specific surface area, high mechanical-flexibility [27], high charge mobility and thermal conductivity [28,29]. Therefore, it has stimulated interest in applications for graphene-based nanocomposites including photovoltaics, phtocatalysis, nanoelectronics, and sensors [29]. Modified reduced graphene oxide is used as electrochemical sensor for detection of inorganic [30,31] and organic compounds [32–34]. Titanium dioxide (TiO2 ) nanoparticles act as an effectual agent in biosensing applications due to its good essential biocompatibility, particular affinity to biomolecules and high reactivity. Owing to its high conductivity and low cost, TiO2 has become an attractive electrode material in different forms such as nanoparticles, nanoneedles and nanotubes. It can be decorated on graphene or other carbon material to fabricate electrochemical DNA-based biosensors [35]. In particular, using grapheme–TiO2 nanocomposites as a bed exploiting has several benefits such as high conductivity to electron transferring plus more active sites for the immobilization of DNA [36,37]. In addition, carbon paste electrode (CPE) was selected for the preparation of DNA biosensor due to a low background current, a wide potential window, distinct advantage of renewability with simple polishing and a simple fabrication procedure [38]. Also presence of nanocomposites such as graphene oxide and TiO2 can promote the active sites and the electron transfer reactions [39]. In this study, first, TiO2 nanoparticle was decorated on reduce graphene oxide (rGO) using hydrothermal method. Then, it was characterized by several techniques such as field emission scanning electron microscopy (FE-SEM), X-ray diffraction (XRD) and FT-IR. Then, TiO2 @rGO modified CPE was prepared and the ds-DNA was immobilized on the surface of the TiO2 @rGO-CPE, to fabricate the DNA biosensor (ds-DNA/TiO2 @rGO-CPE). To determine furazolidone, the oxidation signals of adenine and guanine were used as probes, using differential pulse voltammetry (DPV). The decreases in the electrochemical signals of the oxidation peaks current of guanine and adenine after their interaction with furazolidone were used as analytical signals. As mentioned before, the amounts of immobilized DNA (as a probe) directly influence the sensor’s accuracy, sensitivity and selectivity. Presence of both rGO and TiO2 nanoparticles promote the active sites to dramatically improve the immobilization quality and quantity of DNA, by electrostatic interaction, on the electrode surface. The new biosensor exhibits a better sensitivity for trace analysis than those reported previously. The new DNA-biosensor has high selectivity, sensitivity and fast response with low detection limit for detection for furazolidone in real samples.
2. Experimental 2.1. Chemicals All solutions were prepared using reagent grade chemicals and used as received without further purification. Doubly distilled water was used for preparation of solutions. Furazolidone, titanium isopropoxide (Ti(Oi Pr)4 , 98%), titanium dioxide nanoparticles (TiO2 NPs, 30 nm), and DMF were purchased from Fluka. Reagent grade Tris–HCl, CH3 COOH, CH3 COONa, H3 PO4 , NaCl, NaOH, paraffin and graphite powders (70 m) were purchased from Merck. Salmon sperm ds-DNA was purchased from Sigma–Aldrich (Cat. No.: D1626) with G-C% content of 41.2% and molecular mass of 1.3 × 106 Da. Furazolidone stock solution (1.0 mmol L−1 ) was prepared in phosphate buffer solution (PBS, pH 8.0) in the presence of 10% (v/v) DMF and stored at 4 ◦ C. PBS buffer solution was prepared by appropriate mixing of stock solution of 0.10 mol L−1 H3 PO4 and
0.20 mol L−1 NaOH solutions. The solutions pH were adjusted by addition of sodium hydroxide and/or H3 PO4 solution. Salmon sperm ds-DNA stock solution (100 mg L−1 ) was prepared by dissolving in Tris–HCl buffer (pH 7.0). More dilute solutions of the ds-DNA were prepared with 0.5 mol L−1 acetate buffer (pH 4.8) containing 0.02 mol L−1 NaCl. 2.2. Apparatus Electrochemical analysis, including voltammetric and impedimetric measurements, were performed using an Autolab PGSTAT 12, potentiostat/galvanostat that connected to a three-electrode cell (Metrohm, Model 663 VA stand) with GPES 4.9 software package (Eco Chemie, The Netherlands). The raw data was treated using Savitzky and Golay filter (level 2, from the GPES software) followed by the GPES software for moving average baseline correction with a “peak width” of 0.01. A conventional three-electrode cell was used with the modified working electrode (ds-DNA/TiO2 @rGOCPE), a saturated Ag/AgCl reference electrode and a platinum wire as an auxiliary electrode. A Metrohm 827 pH-meter with a glass electrode was used for pH measurements. UV–vis spectra were recorded with a double beam spectrophotometer (JASCO Model V-750) using 1.0 cm quartz cells. X-ray diffraction experiments were carried out with a Bruker D8 /Advance X-ray diffractometer and with Cu-K␣ radiation. Field emission scanning electron microscopy (FE-SEM) was performed on a Philips XL-30 at an accelerating voltage of 20 kV accomplished on a Philips. Fourier transform IR spectra were recorded using a JASCO FT-IR (680 plus) spectrometer. 2.3. Synthesis of graphene oxide and TiO2 @rGO nanocomposite Graphite oxide (GO) was prepared using modified Staudenmaier method [40]. For this purpose, 1.0 g of graphite powder (with a particle size of 70 m and purity of 99.999%) was added to a mixed solution of 20 mL conc. sulfuric acid, 10 mL conc. nitric acid and 10.0 g potassium chlorate. Then, the mixture was stirred continuously for approximately 100 h. Consequently, the solid was rinsed with 5 wt% HCl aqueous solution and repeatedly washed with deionized water until the pH of the filtrate became neutral. Then, the solid was dried at room temperature. Finally, the graphite oxide was dispersed in water and placed in an ultrasonic bath for 2 h to convert to exfoliated graphene oxide (EGO). Hydrothermal process was used for preparation of TiO2 @rGO. To get a homogenous colloidal suspension of exfoliated graphene oxide, 100 mg EGO was dispersed into 150 mL ethanol under ultrasonication, for 1 h. Then, 5 mL Ti(Oi Pr)4 was slowly added to the EGO suspension and the mixture was ultrasonicated for 1 h. The result mixture was refluxed at 120 ◦ C for 12 h. Finally, the product was separated by filtration and washed with deionized water. The product was dried at room temperature for one night. The TiO2 @rGO nanocomposite was achieved in the form of gray powder [41]. 2.4. Preparation of TiO2 @rGO modified carbon paste electrode (TiO2 @rGO-CPE) Unmodified CPE was prepared by adding 0.30 g paraffin oil to 0.70 g graphite powder and thoroughly hand-mixing in a mortar and pestle to produce a homogenous carbon paste. The result paste was then inserted into the bottom of a polyethylene syringe (geometrical surface area: 0.31 cm2 ) and it was smoothed on a weighing paper. Electrical contact was made by pushing a copper wire down the polyethylene syringe into the back of the mixture. When necessary, a new surface was obtained by pushing an excess of the paste out of the tube and polishing it on a weighing paper.
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TiO2 @rGO-CPE was prepared by mixing 0.30 g paraffin oil with 0.70 g graphite powder and 0.10 g TiO2 @rGO. The result mixture was dispersed in ethanol (for better homogeneity). The mixture was stirred by a magnetic stirrer till the solvent evaporated completely. The result paste then was inserted into the bottom of a polyethylene syringe (surface area, 0.31 cm2 ) and then it was smoothed on a weighing paper. When necessary, a new surface was obtained by pushing an excess of the paste out of the syringe followed by polishing it on a weighing paper. 2.5. Immobilization of ds-DNA on TiO2 @rGO-CPE and electrochemical determination First, the surface of the modified carbon paste electrode (TiO2 @rGO-CPE) was smoothed by polishing with a weighing paper. Then, the surface of the modified CPE was pretreated by applying 1.70 V for 200 s in 0.1 mol L−1 acetate buffer solution (pH 4.8). The TiO2 @rGO-CPE was immersed into a stirred (at constant rate, 200 rpm) solution of ds-DNA (25.0 mg L−1 in 0.5 mol L−1 acetate buffer containing 0.02 mol L−1 NaCl, pH 4.8) and a potential of 0.50 V vs. Ag/AgCl was applied for 240 s. The electrode was gently washed with 0.1 mol L−1 acetate buffer (pH 4.8) to remove unbounded DNA. This DNA-biosensor was designated as ds-DNA/TiO2 @rGO-CPE. For electrochemical detection of furazolidone with the biosensor, first, ds-DNA/TiO2 @rGO-CPE was immersed into 10 mL of 0.1 mol L−1 acetate buffer (pH 4.8). A positive-going differential pulse potential scan (from +0.40 to +1.40) was performed by applying a step potential of 10 mV, pulse amplitude of 70 mV and modulation time of 0.05 s. The oxidation signals of the guanine and adenine were recorded using differential pulse voltammetry (DPV) and tagged as blank signals (Ib ). Then, DNA/TiO2 @rGO-CPE was inserted into 10 mL of phosphate buffer (pH 8.0, at the optimum conditions) containing different concentrations of furazolidone while the solution was stirred at 200 rpm for 240 s at an open circuit system. After accumulation of furazolidone on the biosensor surface, the ds-DNA/TiO2 @rGO-CPE was rinsed with distilled water and placed into a 10 mL of the acetate buffer solution (pH 4.8). The guanine and adenine signals were recorded using DPV to get the sample signals (Is ). 2.6. Preparation of real samples Blood serum was obtained from hospitalized patients and urine samples were collected from the urine of a healthy person, stored at −20 ◦ C until analysis. Different amounts of furazolidone were spiked into the test samples before the treatment. In order to precipitate proteins in the blood serum samples, 5 mL of the sample was treated with 0.1 mL perchloric acid (HClO4 , 20%, v/v). Then, the mixture was vortexed for further 45 s and then centrifuged at 6000 rpm for 10 min. The separated solution was diluted two times with the phosphate buffer solution (pH 8.0) and transferred into the electrochemical cell to be analyzed without further pretreatment. The furazolidone was measured according to the recommended procedure, using standard addition method. 3. Results and discussion 3.1. TiO2 @rGO nanocomposite characterizations To investigate the physical properties of the TiO2 @rGO, different spectroscopic methods such as XRD, FE-SEM and FT-IR were used. FT-IR spectra of TiO2 (a), reduce graphene oxide (b) and TiO2 –rGO (c), in the range of 400–4000 cm−1 , are shown in Fig. 1A. The characteristic peaks of rGO including O H stretching at 3300–3600 cm−1 , aromatic C C at 1600 cm−1 , and C O stretching at 1100 cm−1
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were distinctly observed in the FT-IR spectrum of rGO. The band of the prepared TiO2 at 580 cm−1 is assigned to Ti O Ti stretching, 1630 cm−1 is OH bending and 3250 cm−1 is due to the O H stretching. In the spectrum of TiO2 @rGO (Fig. 1A-c) most of the peaks in the spectra of rGO and TiO2 still exist. In addition, the broad band between 600 and 1000 cm−1 clearly represents the vibration of Ti O stretching in TiO2 -based composite [42]. The absorption band appearing at 1590 cm−1 shows skeletal vibration of graphene sheets [43]. These results support that TiO2 stabilized well at the surface of rGO during the synthesis of TiO2 –rGO with the hydrothermal process. The XRD pattern of TiO2 (a), rGO (b) and TiO2 @rGO (c) are shown in Fig. 1B. The diffraction peak for rGO at 25.14◦ that is indicated to the (0 0 2) plane confirms formation of rGO from GO [44]. The diffraction peaks existing at 2 values of TiO2 nanoparticles at about 25.3◦ , 37.9◦ , 47.8◦ , and 54.4◦ are observed for all of the diffraction patterns, the XRD pattern of the free TiO2 , which obviously match well with the anatase phase (JCPDS 21-1272). In the patterns of the as-prepared TiO2 –rGO the major diffraction lines can be indexed to the anatase phase, suggesting the complete formation of anatase TiO2 during the hydrothermal process. These results confirm that the preparation of TiO2 –rGO with the hydrothermal process is successful. The average particle size of the TiO2 –rGO was estimated according to the Scherer’s equation, D = 0.9/(ˇ2 ·cos ), where D is the average particle size, is the Xray wavelength (0.15418 nm for Cu radiation), ˇ2 is the full width at the half-maximum (FWHM) and is the angle corresponding to the peak maximum. The calculated value of the average particles size was about 63 nm. Fig. 1C illustrates FE-SEM surface morphologies and structures of rGO (a) and TiO2 @rGO (b). The accumulation between TiO2 and rGO in TiO2 @rGO can be pictured from the FE-SEM image, in which TiO2 nanoparticles, some of the nanoparticles have size of less than 50 nm, and are compactly decorated on the graphene substrate. 3.2. Characterization of the modified carbon paste electrode Preliminary modification of the CPE was used to enhance the electrode conductivity and to form an interface with multiple sites for the DNA immobilization. rGO is drastically oxygenated, bearing epoxide and hydroxyl groups on its basal planes and carboxyl groups at the sheet edges [45]. These oxygen containing groups can effectively interact with the hard Ti4+ Lewis acid, which promotes the intercalation and graft of Ti species into GO layers [46,47]. Moreover, positively charged TiO2 –rGO nanocomposite (by applying a potential of 0.50 V vs. Ag/AgCl was applied for 240 s) adsorbed the negatively charged DNA in the solution interface could be absorbed by the DNA at the TiO2 –rGO surface to fabricate ds-DNA/TiO2 @rGO. The surface morphology of the stepwise fabrication of the modified electrode was investigated using FE-SEM (Fig. 2A). Fig. 2A-a shows the FE-SEM image of the unmodified CPE. The graphite layers and empty space between the layers were well seen in this image. It can be seen that the effective surface area of the modified working electrode is dramatically increased with addition of highly conductive TiO2 @rGO (Fig. 2A-b). When DNA is immobilized on the surface of TiO2 @rGO-CPE, the surface morphology is changed again (Fig. 2A-c). Cyclic voltammograms of different electrodes in 5.0 mmol L−1 K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ], containing 0.1 mol L−1 KNO3 , were recorded with a scan rate of 20 mV s−1 (Fig. 2B). The unmodified CPE has a pair of well-defined voltammetric peaks with a cathodic peak potential (Epc ) of −0.145 V and an anodic peak potential (Epa ) of +0.448 V with peak-to-peak separation (Ep ) of 0.593 V. TiO2 @rGO modified carbon paste electrode has a pair of well-defined voltammetric peaks with peak-to-peak separation of 0.396 V. Moreover, the peak-to-peak separation of TiO2 @rGO-CPE
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Fig. 1. (A) FT-IR spectra, (B) XRD pattern of (a) TiO2 , (b) rGO, and (c) TiO2 @rGO and (C) FE-SEM images of (a) rGO and (b) TiO2 @rGO.
Fig. 2. (A) FE-SEM images of (a) unmodified CPE, (b) TiO2 @rGO-CPE, and (c) ds-DNA/TiO2 @rGO-CPE, (B) cyclic voltammograms and (C) impedance spectra (Nyquist plot) of (a) unmodified CPE, (b) TiO2 @rGO-CPE, and (c) ds-DNA/TiO2 @rGO-CPE in 5.0 mmol L−1 [Fe(CN)6 ]3−/4− containing 0.1 mol L−1 KNO3 .
reduced, whereas the peak current of TiO2 @rGO-CPE significantly increased. Therefore, TiO2 @rGO modified carbon paste electrode improves the electron transfer reactions vs. the unmodified CPE. When negatively charged ds-DNA was immobilized at the surface of TiO2 @rGO-CPE, the electron transfer kinetic (peak-to-peak separation) and the redox peak currents of K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] slowly decreased. Fig. 2C shows the Nyquist plots for the unmodified and modified CPE at different conditions. A solution containing 5.0 mmol L−1
K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ], in the presence of 0.10 mol L−1 KNO3 , was used as a probe at a polarization potential of 0.10 V and in the frequency range of 5 mHz to 100 KHz with an amplitude of 10 mV. As shown in Fig. 2C, the charge transfer resistance (Rc.t ) of the electrode decreased dramatically after modification of the CPE with TiO2 @rGO, which is due to the acceleration of the electron transfer. This is due to the promotion of the electron exchange between K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] and the electrode surface. The charge transfer resistance of ds-DNA/TiO2 @rGO-CPE increased in
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Fig. 3. Differential pulse voltammograms of the interaction of furazolidone with the ds-DNA at ds-DNA/TiO2 @rGO-CPE. The oxidation signals of guanine and adenine after interaction with different concentrations of furazolidone as: 1.0, 2.5, 5.0, 7.5, 10.0, 25.0 and 50.0 pmol L−1 (from up to down).
Fig. 4. UV–vis spectra of 0.01 mol L−1 furazolidone before (a) and after (c) interaction with 25.0 mg L−1 ds-DNA, and (b) 25.0 mg L−1 ds-DNA.
the probe solution, indicating the formation of the negatively charged DNA layer on the TiO2 @rGO-CPE surface.
Table 1 Effect of potential interfering substances on the determination of 10 pmol L−1 furazolidone.
3.3. Optimization of the experimental parameters, interaction between furazolidone with ds-DNA/TiO2 @rGO-CPE The interaction between furazolidone and the ds-DNA was inspected by DPV technique. The guanine and adenine oxidation signals in DNA appeared around +0.95 and +1.26 V, respectively. Fig. 3 shows the oxidation signals of guanine and adenine at the ds-DNA/TiO2 @rGO-CPE, after their interactions with furazolidone. Different concentrations of furazolidone were used to interact with the ds-DNA at the surface of ds-DNA/TiO2 @rGO-CPE. It was observed that with increasing in the furazolidone concentration, the oxidation currents of guanine and adenine were decreased. The decrease in the oxidation signals of guanine and adenine, after interaction with furazolidone, could be attributed to the interaction of furazolidone with the ds-DNA at ds-DNA/TiO2 @rGO-CPE surface, resulting the helix destabilization and strand breakage [48]. In order to get the highest sensitivity and the lowest detection limit with the differential pulse voltammetric method, the influence of various parameters affect the response of the biosensor including the modifier percentage, ds-DNA concentration, accumulation time and immobilized potential were optimized. The influence of the modifier (TiO2 @rGO) percentage on the oxidation signals of guanine and adenine at CPE was studied. The results are illustrated in Supplementary materials (Fig. S1A). The results confirm that increasing the percentage of TiO2 @rGO causes an increase in the positive sites, to have more immobilized DNA at the electrode surface. Fig. S1B shows the influence of the ds-DNA concentration, in the fabrication step of the biosensor, on the response of dsDNA/TiO2 @rGO-CPE using the peaks current of the guanine and adenine. The results demonstrated that the guanine and adenine signals increased as DNA concentration rose up to 25.0 mg L−1 , and then leveled off. This is due to the fact that the sites on the electrode surface were filled with the ds-DNA. Therefore, 25.0 mg L−1 of ds-DNA was selected and used to prepare the DNA-modified electrode. To immobilize the ds-DNA at TiO2 @rGO-CPE, different potentials were applied at the electrode between −0.20 and +0.70 V. More details in this study are summarized in Supplementary materials (Fig. S1C). Based on the results, +0.50 V was selected as an optimum immobilization potential. The effect of accumulation time on the immobilization of the ds-DNA at TiO2 @rGO-CPE surface was
Species
Tolerance limit (w/w)
SCN− , sodium citrate, glucose, ascorbic acid NH4 OH, 4-nitrophenel Uric acid, thiourea, dopamine, acetaminophen
500 250 100
also studied (Fig. S1D). The results showed that the oxidation signals of guanine and adenine increased till 240 s, and then leveled off. Therefore, 240 s was considered as an optimum accumulation time. After preparation of the biosensor, the effect of sample solution pH (in the presence of furazolidone) on the guanine and adenine signals at ds-DNA/TiO2 @rGO-CPE was studied too. The results showed that the sensitivity of the biosensor was increased by increasing the solution pH up to 8.0 (Fig. S2A). Moreover, the interaction of furazolidone with the ds-DNA depends on the incubation time. More details in this study are summarized in supplementary materials (Fig. S2B). The results confirmed that by increasing the incubation time till 240 s, the oxidation signals of guanine and adenine were decreased (the sensitivity increased) and then it almost leveled off for higher incubation time. Therefore, 240 s was selected as a suitable incubation time (Fig. S2B). 3.4. UV–visible spectrophotometry study and calculation of the association constant of furazolidone with the ds-DNA UV–visible absorption spectrophotometry was employed to investigate the furazolidone–DNA interaction. The absorption spectra of furazolidone, ds-DNA and ds-DNA ds-DNA-furazolidone (after its interaction with ds-DNA) are shown in Fig. 4. The maximum absorbance of furazolidone is located at 217 nm as illustrated in (Fig. 4a), whereas the maximum absorbance of the ds-DNA is located at 260 nm (Fig. 4b). The absorbance spectra of the mixture of furazolidone and the ds-DNA are shown in (Fig. 4c) with a maximum absorbency of 272 nm. Moreover, the absorbance of ds-DNA decreases after its interaction with furazolidone. The red shift on the ds-DNA spectrum and decreasing in the absorbance intensity demonstrated a strong interaction between furazolidone and the ds-DNA, due to the intercalative binding of furazolidone to the ds-DNA. To calculate of the association constant between furazolidone with the ds-DNA, it is assumed that the DNA and furazolidone only
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Table 2 A systematic comparison on the unmodified CPE, TiO2 @rGO, and ds-DNA/TiO2 @rGO for the electrochemical detection of furazolidone. Electrode
Peak potential (V)
Dynamic range
Detection limit
Sensitivity
Interfering compounds
Unmodified CPE
−0.48
2–30 mol L−1
0.5 mol L−1
−0.015
TiO2 @rGO-CPE
−0.44
0.05–20 mol L−1
0.01 mol L−1
−1.84
DNA/TiO2 @rGO-CPE
Guanine oxidation Adenine oxidation
1–150 pmol L−1
0.55 pmol L−1 0.43 pmol L−1
−0.0544 −0.0704
O2 , ascorbic acid, dopamine, uric acid, urea, acetaminophen O2 , ascorbic acid, dopamine, uric acid, urea, acetaminophen No interfere
0.95 1.25
produce a single complex as DNA.Fum according to the following reaction: DNA + mFu ↔ DNA.Fum
(3)
The UV absorption is most sensitive to the equilibrium constant of DNA.Fum complex [49]. The decreasing value of the UV absorption (in 265 nm) is proportional to the concentration of DNA.Fum complex. KA =
[DNA.Fum ] [DNA][Fu]m
(4)
Totally absorption of solution = εDNA.Fu [DNA.Fum ] + εDNA [DNA]. From Eq. (4), we can obtain: εDNA εDNA A0 = + εDNA + εDNA.Fu (A0 − A) (εDNA + εDNA.Fu )(1/KA [Fu]m )
(5)
If m = 1, by keeping the DNA concentration at 20 mg L−1 and varying the concentration of Fu, a plot of A0 /(A0 − A) as a function of 1/[Fu]m is linear with a slope of DNA /(DNA + DNA.Fu )(1/KA ). The results of KA = 1.04 × 106 M−1 were obtained in the concentration range of the applied furazolidone. 4. Figures of merit DPV was used, with a pulse width of 50.0 ms, pulse amplitude of 50 mV and scan rate of 20 mV s−1 , to check the linear dynamic range and limit of detection of the ds-DNA/TiO2 @rGO-CPE for furazolidone detection. The oxidation peaks current of guanine and adenine at ds-DNA/TiO2 @rGO-CPE were depended on the furazolidone concentration. The results approved that guanine and adenine oxidation currents were decreased with increasing the furazolidone concentration in the range of 1.0–150.0 pmol L−1 , with a correlation equations of I (A) = –0.0544C (pmol L−1 ) + 0.9568 (R2 = 0.9959, n = 5) and I (A) = –0.0704C (pmol L−1 ) + 1.1641 (R2 = 0.9917, n = 5) for guanine and adenine signals, respectively. The limit of detection (3Sb/m) based on guanine and adenine peaks current were obtained as 0.55 and 0.43 pmol L−1 of furazolidone, respectively. Five replicates DPV measurements of furazolidone concentration of 10.0 and 100.0 pmol L−1 showed relative standard deviations of 4.3%, 4.8% and 3.4%, 4.7% for guanine and adenine signals, respectively. Table 1 shows a systematic comparison of the three electrodes containing unmodified-CPE, TiO2 @rGO and ds-DNA/TiO2 @rGO in terms of the detection sensitivity and selectivity of the electrochemical detection of furazolidone. 5. Interference study Before evaluating the performance of the biosensor for analysis of furazolidone in real sample, the effect of potential interfering substances were studied by addition of potential interfering substances into a solution containing 10.0 pmol L−1 furazolidone at the optimum conditions. The potential interfering compounds
Table 3 Recoveries of furazolidone in blood serum and urine samples. Sample
Sample no
Blood serum
1
–
2 3
10.0 75.0
9.8 ± 0.4 75.9 ± 2.0
1 2 3
– 10 75
Detection limit 9.7 ± 0.5 74.8 ± 1.5
Urine
Furazolidone added (pmol L−1 )
Found by biosensor (pmol L−1 ) Detection limit
Recovery (%)
– 98.6 101.2 – 98.6 99.7
were chosen from a group of substances commonly found with furazolidone in pharmaceuticals and biological fluids. The tolerance limit was defined as the maximum concentration of the interfering substance that caused an error less than 5% for the detection of the furazolidone. The oxidation peaks current of guanine and adenine were compared before and after interaction of ds-DNA/TiO2 @rGO-CPE with the sample solutions containing furazolidone in the presence and absence of the potential interfering compounds. The results are given in Table 2, indicating that the biosensor has good selectivity for determination of furazolidone.
6. Real sample analysis In order to evaluate the performance of the biosensor for analysis of furazolidone in real samples, furazolidone in blood serum and urine samples were measured at the optimum conditions using differential pulse voltammetry. The results are given in Table 3, which shows that the new biosensor is able to measure furazolidone in different real samples.
7. Conclusion In this article, a selective DNA electrochemical biosensor was developed by modification of a carbon paste electrode with TiO2 reduced graphene oxide and ds-DNA for the determination of furazolidone in real sample. The result demonstrated that the new nanocomposite acts as active sites to fabricate the DNA-biosensor. The TiO2 –graphene nanocomposites provide good adsorptivity, conductivity and active sites for the immobilization of the ds-DNA. After interaction of furazolidone with the ds-DNA at the electrode surface, a decrease in the adenine and guanine oxidation signals were observed. The advantages of the suggested method are low detection limit, wide linear dynamic range, the sensitivity, the selectivity and its applicability. The ds-DNA/TiO2 @rGO-CPE is not only suitable to examine the interaction of ds-DNA with furazolidone, but also can be used to detect DNA damage caused by furazolidone.
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