Article pubs.acs.org/IECR
A Novel DNA Biosensor Based on a Pencil Graphite Electrode Modified with Polypyrrole/Functionalized Multiwalled Carbon Nanotubes for Determination of 6‑Mercaptopurine Anticancer Drug Hassan Karimi-Maleh,*,† Fahimeh Tahernejad-Javazmi,† Necip Atar,‡ Mehmet Lütfi Yola,§ Vinod Kumar Gupta,∥,⊥,¶ and Ali A. Ensafi# †
Department of Chemistry, Graduate University of Advanced Technology, Kerman 76311-33131, Iran Department of Chemical Engineering, Pamukkale University, Denizli, Turkey § Department of Metallurgical and Materials Engineering, Sinop University, Sinop, Turkey ∥ Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee-247667, India ⊥ Center for Environment & Water, The Research Institute, King Fahd University of Petroleum and Minerals, Dhahran-31261, Saudi Arabia ¶ Department of Applied Chemistry, University of Johannesburg, Johannesburg, South Africa # Department of Chemistry, Isfahan University of Technology, Isfahan 84156-83111, Iran ‡
ABSTRACT: A novel and sensitive biosensor employing immobilized DNA on a pencil graphite electrode modified with polypyrrole/functionalized multiwalled carbon nanotubes for the determination of 6-mercaptopurine (6-MP) is presented. In the first step, we modified the pencil graphite surface with polypyrrole and functionalized multiwalled carbon nanotubes (MWCNT/ COOH). The developed electrode was characterized by scanning electron microscopy, atomic force microscopy, reflection− absorption infrared spectroscopy, X-ray photoelectron spectroscopy, and electrochemical impedance spectroscopy. In the other step, we used decreases in the oxidation responses of guanine and adenine as a sign of the interaction of 6-MP with salmon sperm double-stranded DNA using differential pulse voltammetry. The signal of guanine oxidation was linear with respect to the 6-MP concentration in the range of 0.2−100 μmol L−1 with a detection limit of 0.08 μmol L−1. The modified electrode was utilized for the determination of 6-MP in real samples.
1. INTRODUCTION The interaction of DNA with electroactive compounds is significant in terms of pharmaceutical, environmental, and drug detection. Recently, several techniques have been studied to investigate the effects of proteins and drugs on gene expression.1 DNA binding compounds are significant materials in the recognition of anticancer and antibacterial drugs.2,3 The investigations of DNA biosensors are currently under intense review because of their great properties such as sensitivity, selectivity, simplicity, rapidity, and low cost.2 The anticancer drug 6-mercaptopurine (6-MP) inhibits the biosynthesis of adenine nucleotides by acting as an antimetabolite. In the human body, 6-MP is converted to the corresponding ribonucleotide, which is a potent inhibitor of the conversion of inosinic acid to adenine. 6-MP as a thiopurine category was utilized to treat leukemia.4 It is not recommended for pregnant women, but several reports indicate that the use of 6-MP by pregnant women shows no increase in fetal abnormalities. In addition, during the first trimester of pregnancy, women using 6-MP have an incidence of abortion. Scientific studies have shown that 6-MP is cancer-causing in humans.5 Several techniques such as spectrophotometry,6 fluorescence, 4 high-performance liquid chromatography (HPLC),7,8 capillary electrophoresis,9 and electrochemical methods10,11 have been reported for detection of 6-MP in biological and pharmaceutical samples. © 2015 American Chemical Society
The modification of electrode surfaces is one of the important developments in recent years.12−15 The process has many benefits such as drug detection and increase in the electron transfer rate at the electrode surface.16−30 In the present study, we developed a differential pulse voltammetry (DPV) method to study the electrochemical behavior of 6-MP with double-stranded DNA (ds-DNA) on a novel pencil graphite electrode (PGE) modified with polypyrrole (PP) and functionalized multiwalled carbon nanotubes (MWCNTs). The developed PP/MWCNTs/PGE biosensor was used to investigate the interaction of 6-MP with ds-DNA. The biosensor in this study was compared with previous reports. According to the results, the biosensor demonstrated a lower limit of detection with good selectivity for the determination of 6-MP. This sensitive and selective electrochemical DNA biosensor suggests new progress for the determination of drugs in the future.
2. EXPERIMENTAL SECTION 2.1. Chemicals. Salmon sperm ds-DNA was bought from Fluka and suspended in Tris-EDTA buffer (pH 7). Pyrrole Received: Revised: Accepted: Published: 3634
November 9, 2014 January 28, 2015 March 24, 2015 March 24, 2015 DOI: 10.1021/ie504438z Ind. Eng. Chem. Res. 2015, 54, 3634−3639
Article
Industrial & Engineering Chemistry Research
water solution (hot water; 35 °C). Some selected values of the above solutions were used for the analysis. Drug-free human urine was obtained from healthy or nonhealthy volunteers (from children with cancer, chronic lymphocytic leukemia). Urine samples were stored in a refrigerator. A 10 mL aliquot of the sample was centrifuged for 10 min at 2000 rpm. The supernatant was diluted five times with Tris-HCl buffer (pH 7.0). The solution was transferred into the voltammetric cell. The standard addition method was utilized for 6-MP detection in real samples.
(Aldrich) was distilled under vacuum. All other reagents, including acetic acid (CH 3 COOH), sodium acetate (NaCH3COO), ethylenediaminetetraacetic acid (EDTA), sodium chloride (NaCl), and sodium hydroxide (NaOH), were of analytical grade from Fluka or Merck. Doubly distilled water was used throughout. Multiwalled carbon nanotubes (>80% MWCNT/COOH basis, d × l = 9.5 nm × 1.5 μm; >8% carboxylic acid functionalized) from Aldrich were used as the substrate for the preparation of the electrodes. 2.2. Apparatus. All of the electrochemical measurements were carried out using an PGSTAT 302N potentiostat/ galvanostat (Autolab, Utrecht, The Netherlands) connected to a three-electrode cell and Metrohm stand. A platinum wire was used as the counter electrode. PP/MWCNTs/PGE and Ag/AgCl/KCl(sat) were utilized as the working and reference electrodes, respectively. Electrochemical impedance spectroscopy (EIS) experiments were performed with IviumStat and IviumStat.XR analyzers. The electrodes were characterized versus the 1.0 mM Fe(CN)63−/4− redox couple. EIS data were measured from 0.1 Hz to 100 kHz at a wave amplitude of 10 mV and an electrode potential of 0.195 V. The infrared spectra were recorded with a Bruker Tensor 27 DTGS detector. X-ray photoelectron spectroscopy (XPS) analysis was performed on a PHI VersaProbe 5000 spectrometer (Physical Electronics, ULVAC-PHI, Inc., Japan/USA) with monochromatized Al Kα radiation (1486.6 eV). The samples for XPS measurements were prepared on clean glass slides by placing one drop of the nanostructure on the slide and then allowing it to air-dry. In order to characterize the surfaces, tapping-mode atomic force microscopy (AFM) was used (Nano Magnetics Instruments, Oxford, UK). The surfaces were installed on a sample holder, and a sample area of 2 μm × 2 μm was shown with a 128 × 128 pixels resolution. The scan rate was 2 μm s−1. Scanning electron microscopy (SEM) was performed on an EVO 50 analytic microscope (Zeiss, Oberkochen, Germany). A digital pH/mV meter (Metrohm model 710) was used for pH measurements. 2.3. Electrode Modification. The composite films were prepared through the following procedure: Functionalized multiwalled carbon nanotubes (MWCNT/COOH) (0.1 g) were dispersed in 0.1 M dodecylbenzenesulfonic acid and sonicated for 1.5 h. Then pyrrole in a certain feeding mass ratio with respect to MWCNT/COOH was dissolved in this emulsion solution under ultrasonic stirring for 30 min at room temperature. Finally, the composite films were synthesized via electropolymerization by cyclic voltammetry at a scan rate of 100 mV/s between +0.50 V and +1.20 V. 2.4. Immobilization of ds-DNA. First, the electrodes were cleaned in ultrapure water two times and then in 50:50 (v/v) isopropyl alcohol/acetonitrile (IPA/MeCN) solution. Then the surface of the PGE was pretreated by applying a potential of +1.20 V for 90 s. After modification of the electrode with polypyrrole and multiwalled carbon nanotubes, the ds-DNA was immobilized on the PP/MWCNTs/PGE by applying a potential of +0.50 V for different selected times from a stirred (300 rpm) solution containing 20.0 μg mL−1 ds-DNA in an acetate buffer (0.5 M, pH 4.8) containing 0.02 mol L−1 NaCl. The electrode was then rinsed with the acetate buffer for 15 s to remove unbound ds-DNA. 2.5. Real Sample Preparation. Five tablets of 6-MP (labeled 50 mg per tablet; Korea United Pharma, Seoul, South Korea) were homogenized. After that, 100 mg of the powder was dissolved with ultrasonication in 100 mL of 1:1 ethanol/
3. RESULTS AND DISCUSSION 3.1. Characterization of the Electrode Surface. As we know, pyrrole can be polymerized electrochemically at a potential of about 0.8 V. The first step in the electrochemical polymerization of pyrrole is the generation of the radical cation. Chain propagation may then proceed by the reaction of two radical cations, pairing the spins and eliminating two protons to produce the neutral dimer. The electropolymerization of polypyrrole was carried out by cyclic voltammetry (CV) in the potential range between 0.5 and 1.2 V at a scan rate of 100 mV/s over 50 cycles (data not shown). The peak current decreased with increasing number of CV cycles, indicating that the film of the polypyrrole was deposited on the MWCNT/ PGE surface. Figure 1 shows the impedance plots for (a) bare PGE, (b) PP/PGE, and (c) PP/MWCNTs/PGE surfaces in 1.0 mM
Figure 1. EIS responses of (a) bare PGE, (b) PP/PGE, and (c) PP/ MWCNTs/PGE electrodes in the presence of 1.0 mM [Fe(CN)6]3−/4− (1:1) in 0.1 M KCl.
[Fe(CN)6]3−/4− (1:1) solution in 0.1 M KCl. The values of the charge transfer resistance (Rct) were calculated for bare PGE, PP/PGE, and PP/MWCNTs/PGE as 75.0, 60.0, and 50.0 Ω, respectively. It is therefore clear that the PP/MWCNTs/PGE can increase the electron transfer compared with bare PGE and PP/PGE. Figure 2 shows the SEM images obtained to investigate the morphologies of the produced surfaces. The smooth surface of the bare PGE is shown in Figure 2A. Figure 2B shows the electrodeposition of polypyrrole onto the PGE. Figure 2C shows the electrode surface after modification with MWCNT layers. On the other hand, Figure 2D shows the surface after a layer of electrodeposition was covered onto the MWCNT/PGE electrode by electropolymerization of polypyrrole. The results show that the MWCNTs and polypyrrole were modified on the PP/MWCNTs/PGE. Figure 3 shows AFM images of the bare PGE and PP/MWCNTs/PGE surfaces. The surface deepness of the PP/MWCNTs/PGE increased. This result indicates that 3635
DOI: 10.1021/ie504438z Ind. Eng. Chem. Res. 2015, 54, 3634−3639
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Industrial & Engineering Chemistry Research
Figure 2. SEM images of (A) bare PGE, (B) PP/PGE, (C) MWCNTs/PGE, and (D) PP/MWCNTs/PGE.
Figure 3. AFM images of (A) bare PGE and (B) PP/MWCNTs/PGE.
polymerization was homogeneously accomplished on the MWCNT/PGE electrode. The infrared spectrum of PP/MWCNTs/PGE (Figure 4) indicates that the peak of OH stretches is around 3200 cm−1. The peaks of the aromatic C−H stretches are around 2950 cm−1, and the peaks of aromatic CC bends are around 1585 cm−1. The peaks around 1350 cm−1 correspond to the symmetric C−O−C stretches. The XPS survey and C1s core-level spectra of PP/ MWCNTs/PGE are shown in Figure 5. The three peaks at around 287, 410, and 520 eV in the survey spectrum correspond to C1s, N1s, and O1s, respectively. As shown in the Figure 5 inset, the peaks at 284.2, 285.8, 286.6, and 287.5 eV correspond to C−C, C−O, CO, and O−CO of the C1s core-level spectrum, respectively. 3.2. Investigation of the Interaction of 6-MP and DNA. As shown in Figure 6, the DPV scans of DNA at the surface of the PP/MWCNTs/PGE electrode nicely exhibit the oxidation
Figure 4. FTIR spectrum of PP/MWCNTs/PGE.
of guanine (ca. +0.92 V) and adenine (ca. +1.26 V) in the absence (1) and presence of different concentrations of 6-MP 3636
DOI: 10.1021/ie504438z Ind. Eng. Chem. Res. 2015, 54, 3634−3639
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Industrial & Engineering Chemistry Research
Figure 5. XPS spectra of PP/MWCNTs/PGE. Figure 7. Dependence of the DPV oxidation signals of guanine and adenine on the ds-DNA concentration. Conditions: ds-DNA immobilization on PP/MWCNTs/PGE at +0.50 V during 200 s.
Figure 6. DPV scans for the interaction of 6-MP with ds-DNAmodified PP/MWCNTs/PGE in solutions containing 0.0, 0.2, 0.95, 2.2, 3.1, and 4.0 μmol L−1 6-MP (labeled as 1−6, respectively). Figure 8. Effect of accumulation time of ds-DNA at the PP/ MWCNTs/PGE surface on the guanine and adenine oxidation signals in solution containing 20.0 mg L−1 ds-DNA.
(2−6). The addition of 6-MP causes a considerable diminution in the guanine and adenine oxidation peak currents (curves 2− 6). The binding of 6-MP to DNA bases inhibits the oxidation of guanine and adenine. To obtain the best conditions for the electrochemical response of the biosensor, we optimized the immobilized dsDNA concentration at the surface of the modified electrode. Figure 7 shows a plot of guanine and adenine oxidation current signals as functions of the ds-DNA concentration after 200 s accumulation at 0.50 V at the surface of the PP/MWCNTs/ PGE in acetate buffer solution. As can be seen, the peak currents of guanine and adenine increased with increasing concentration of ds-DNA up to 20 mg/L, and then the currents became constant. Therefore, 20 mg/L ds-DNA was chosen as the optimum concentration for the determination of and interaction with 6-MP. Also, we optimized the accumulation time of ds-DNA at the PP/MWCNTs/PGE surface. The results showed that the maximum current was obtained with an accumulation time of 200 s, after which the peak currents of guanine and adenine are constant (see Figure 8). It was anticipated that the binding of the 6-MP to ds-DNA would be dependent on the interaction time. The guanine and adenine peak currents decreased as the time increased up to 150 s, after which the guanine and adenine peak currents leveled off and remained constant up to 250 s (Figure 9). Therefore, in all of the subsequent experiments, an interaction time of 150 s was used.
Figure 9. Effect of incubation time of 4.0 μmol L−1 6-MP with dsDNA-modified PP/MWCNTs/PGE on the guanine and adenine oxidation signals.
The differential pulse voltammograms of guanine showed two linear ranges. The plot of the peak current versus 6-MP concentration was linear for 6-MP concentrations of 0.2−8.0 and 8.0−100 μmol L−1 (data not shown). The same results were obtained for adenine signals. The detection limit was 3637
DOI: 10.1021/ie504438z Ind. Eng. Chem. Res. 2015, 54, 3634−3639
Article
Industrial & Engineering Chemistry Research determined as 0.08 μmol L−1 6-MP according to the definition of YLOD = YB + 3σ. The developed DNA biosensor was also compared with other voltammetric methods (Table 1). The developed biosensor demonstrated a lower limit of detection with good selectivity.
there was no important error with 95% of confidence level between the biosensor and HPLC.
4. CONCLUSION The developed PP/MWCNTs/PGE shows good electroactivity due to high surface area. 6-MP binds to the sites of ds-DNA in the interaction of 6-MP with the ds-DNA immobilized on the PP/MWCNTs/PGE. The novel DNA biosensor was used as a highly sensitive electrochemical sensor in 6-MP detection. The developed electrochemical biosensor shows good selectivity, linearity, simplicity, and efficiency in target detection. The linearity range between 6-MP and ds-DNA was obtained as 0.2−100 μmol L−1. After that, the biosensor was applied to 6MP detection in real samples.
Table 1. Comparison of the Efficiencies of Some Electrochemical Methods in the Determination of 6-MP linear dynamic range (μmol L−1)
limit of detection (μmol L−1)
electrode
method
pH
glassy carbon glassy carbon carbon paste carbon paste carbon paste
amperometry
7.0
0.4−100
0.2
31
amperometry
7.0
0.4−100
0.2
32
DPV
9.0
0.09−350
0.06
10
SWVa
4.0
0.5−900
0.1
5
■
DPA
7.0
0.2−100.0
0.08
this work
*Tel.: +98 911 2540112 (mobile). Fax: +98 341 2121018. Email:
[email protected].
a
ref
AUTHOR INFORMATION
Corresponding Author
Notes
Square-wave voltammetry.
The authors declare no competing financial interest.
■
Long-term stability is one of the most important properties for biosensor applications. The stability of the PP/MWCNTs/ PGE was investigated by DPV. After the biosensor was stored in the refrigerator at 4 °C for 15 days, the potentials of the oxidation peaks for guanine and adenine in the DPV scans remained at the same positions, and the peak currents decreased by only about 5.6% of the initial current response (after averaging the signals of adenine and guanine). The relative standard deviation (RSD) for the guanine signals after interaction with 2.0 μmol L−1 6-MP was 5.3% (n = 7). Also, the RSD for the adenine signals after interaction with 2.0 μmol L−1 6-MP was 5.2%. In the selectivity experiment, the effects of several species were investigated in the presence of 5.0 μmol L−1 6-MP. A tolerance limit with an approximate relative error of ±8 was obtained at the maximum concentrations of matrix components, including 1000-fold excesses of glucose, fructose, lactose, sucrose, methanol, and ethanol and 500-fold excesses of urea, glycine, valine, methionine, leucine, alanine, and glycine. Therefore, the developed DNA biosensor has satisfactory selectivity for 6-MP analysis in the presence of other foreign species. HPLC was performed to evaluate the validity of the electrochemical biosensor (Table 2). Table 2 shows the results by biosensor and HPLC. According to the results of paired ttest,
REFERENCES
(1) Erdem, A.; Ozsoz, M. Electrochemical DNA Biosensors Based on DNA−Drug Interactions. Electroanalysis 2002, 14, 965−974. (2) Erdem, A.; Ozsoz, M. Interaction of the Anticancer Drug Epirubicin with DNA. Anal. Chim. Acta 2001, 437, 107−114. (3) Yola, M. L.; Ozaltin, N. Electrochemical studies on the interaction of an antibacterial drug nitrofurantoin with DNA. J. Electroanal. Chem. 2011, 653, 56−60. (4) Wang, L.; Zhang, Z. The study of oxidization fluorescence sensor with molecular imprinting polymer and its application for 6mercaptopurine (6-MP) determination. Talanta 2008, 76, 768−771. (5) Keyvanfard, M.; Khosravi, V.; Karimi-Maleh, H.; Alizad, K.; Rezaei, B. Voltammetric Determination of 6-Mercaptopurine Using a Multiwall Carbon Nanotubes Paste Electrode in the Presence of Isoprenaline as a Mediator. J. Mol. Liq. 2013, 177, 182−189. (6) Besada, A.; Tadros, N. B.; Gawargious, Y. A. Copper(II)− Neocuproine as Colour Reagent for Some Biologically Active Thiols: Spectrophotometric Determination of Cysteine, Penicillamine, Glutathione, and 6-Mercaptopurine. Microchim. Acta 1989, 99, 143−146. (7) Lavi, L. E.; Holcenberg, J. S. A Rapid and Sensitive HighPerformance Liquid Chromatographic Assay for 6-Mercaptopurine Metabolites in Red Blood Cells. Anal. Biochem. 1985, 144, 514−521. (8) Boulieu, R.; Dervieux, T. High-Performance Liquid Chromotographic Determination of Methyl 6-Mercaptopurine Nucleotides (Me6-MPN) in Red Blood Cells: Analysis of Me6-MPN per se or Me6-MPN Derivative? J. Chromatogr., B 1999, 730, 273−276.
Table 2. Determination of 6-MP in real samples (n = 3)a found (μM) sample
added (μM)
expected (μM)
biosensor
HPLC
Fex
Ftab
tex
ttab
tablet
− 5.0 10.0 − 50.0 70.0 −
1.0 5.0 10.0 − 50.0 70.0 −
1.1 ± 0.2 4.8 ± 0.4 10.3 ± 0.3
