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single-layer MoS2 nanosheets nanocomposite modified carbon paste electrode
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Highly sensitive biosensor for detection of DNA nucleobases: Enhanced electrochemical sensing based on polyaniline/single-layer MoS2 nanosheets nanocomposite modified carbon paste electrode Meisam Sadeghi , Mohsen Jahanshahi , Hamedreza Javadian PII: DOI: Reference:
S0026-265X(19)30439-4 https://doi.org/10.1016/j.microc.2019.104315 MICROC 104315
To appear in:
Microchemical Journal
Received date: Revised date: Accepted date:
21 February 2019 9 August 2019 6 October 2019
Please cite this article as: Meisam Sadeghi , Mohsen Jahanshahi , Hamedreza Javadian , Highly sensitive biosensor for detection of DNA nucleobases: Enhanced electrochemical sensing based on polyaniline/single-layer MoS2 nanosheets nanocomposite modified carbon paste electrode, Microchemical Journal (2019), doi: https://doi.org/10.1016/j.microc.2019.104315
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HIGHLIGHTS A simple analytical method for the determination of DNA nucleobases. Enhanced sensitivity for electrochemical detection using PANI-MoS2/CPE. The effects of supporting electrolyte, concentration, and pH were investigated.
Low limit of detection of the electrochemical biosensor with favorable R2.
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Highly sensitive biosensor for detection of DNA nucleobases: Enhanced electrochemical sensing based on polyaniline/single-layer MoS2 nanosheets nanocomposite modified carbon paste electrode Meisam Sadeghia, Mohsen Jahanshahia, Hamedreza Javadianb,* a
Nanotechnology Research Institute, Faculty of Chemical Engineering, Babol Noushirvani University of Technology, Babol, Iran b
Universitat Politécnica de Catalunya, Department of Chemical Engineering, ETSEIB, Diagonal 647, 08028 Barcelona, Spain *
Corresponding author.
Email addresses: [email protected]; [email protected] Abstract Robust detection of DNA as a molecule that encodes genetic instructions is getting significant attention in recent years due to its promising applications in diseases diagnosis and treatment, forensic analysis, food safety evaluation, environmental monitoring, and so on. In this research, a simple, rapid and multiplex electrochemical method based on single-layer molybdenum disulfide (MoS2) nanosheets sensing platform and differential pulse voltammetry (DPV) method for detection of DNA nucleobases (DNANBs) was set up without any pretreatment or separation process. Hence, the carbon paste electrode modified with MoS2 (MoS2/CPE) was selected, and the effects of different parameters such as supporting electrolyte composition and pH were optimized to obtain the best peak potential separation and high sensitivity. Then, the individual, respective and simultaneous determinations of DNANBs were performed by controlling the certain experimental conditions. The results exhibited the excellent sensitivity, low limit of detection, and high selectivity of the electrochemical
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biosensor. Moreover, the peak current values were linearly dependent on the concentration of DNANBs. For comparison, common methods for determination of DNANBs were analyzed based on the absorption of ultraviolet light and Raman spectroscopy. Finally, the proposed electrode was modified with polyaniline (PANI-MoS2/CPE) as a new biosensor for the determination of DNANBs, exhibiting good reproducibility and stability. Keywords: Electrochemical method, Carbon paste electrode, DNA nucleobases detection, Raman and Uv-vis spectroscopy, MoS2 nanosheets, Polyaniline/MoS2 nanocomposites.
1. Introduction Human chromosome, a linear biopolymer consisting nucleotide units (DNA and RNA), is considered as an important biological macromolecule that plays a crucial role in the formation of genetic instructions and guides the development of biological function by the replication and transcription of genetic information [1-4]. Guanine (G), adenine (A), thymine (T) and cytosine (C) are the important components of DNA. It is assumed that the mole percentages of G and A are equal to those of C and T, respectively [5]. The abnormal changes of these nucleobases in organisms results in the deficiency and mutation of immunity system, leading to various diseases such as epilepsy, cancer and HIV infection, etc [6,7]. Hence, the development of a simple and accurate analytical method to determine the concentration of G, A, T, and C is so much important for bio-analytical chemistry and life sciences. The common instrumental methods used for nucleobases detection are separation methods, such as electrophoresis, mass spectrometry, chromatography, etc [8-14]. The most alternative method for the mentioned methods to detect nucleobases is electrochemical detection, because nucleobases could be oxidized and/or reduced on polarized and renewable electrode surface [15]. In this method, polarographic signals coming from A and G are used in 3
DNA biosensors for process evaluation. It is mainly due to lower oxidation potentials of A and G than C and T [15]. It is an appropriate way to overcome the disadvantages of other methods such as complicated sample preparation processes, high cost and long runs [7]. To analyze the changes in G, A, T, and C, a series of comprehensive studies on their components are important. By individual analysis of each base components, the intact base combination in DNA can be measured [16-19]. Furthermore, in the structure explanation of nucleic acids and their nucleotide building blocks such as A, G, C, and T, Raman spectroscopy and UV are as the most important tools [20]. Intensive efforts have been performed to apply different electrode materials for detection of nucleobases. The varied electronic structure of transition metal dichalcogenides (TMDs) possess, ranging from semi-conducting (MoS2, WS2) to semi-metals (VS2, TiS2) and superconductors (TaSe2, NbSe2) makes them ideal for applying in electrochemical biosensors. MoS2 is a type of transition metal sulfides constructed by stacking covalently S–Mo-S bond through weak Van der Waals interactions. As a 2D layer structure analogous to graphene, MoS2 has recently become a popular material regarding its outstanding properties, such as being a transistor [21,22], supercapacitor [23], electro-analyzer [24-26], and thermal conductor [27]. The optical and electronic properties of MoS2 change as the number of layers decreases, mainly due to the electronic band transformation from indirect gap to direct gap [28]. In addition, researchers have indicated that aromatic compounds could physisorb the basal plane of MoS2. According to the above-mentioned properties of MoS2, a nucleic acid sequence detection method with the single-layer MoS2 nanosheets as sensing platform can be proposed [29,30]. Generally, an ideal electrochemical sensor should possess a wide potential range and high sensitivity and electroactivities. It is possible to improve the sensitivity of MoS2-based 4
electrodes. PANI-based composite materials have been widely applied in the field of electrochemical sensor utilization due to tunable properties, easy synthesis, environmental stability, inexpensive monomer, variable oxidation state, effective mass/charge transfer, broad range conductivity, nanoarchitecture and good electroactivity [31]. It has been proved by researchers that the electrochemical performance of electrode materials can be greatly increased by adding PANI [32–36]. Therefore, to enhance the electrochemical performance of the single-layer MoS2 nanosheets, their combination with PANI to form the PANI-MoS2/CPE sensor can be carried out. In this present work, the feasibility of the MoS2/CPE to develop a biosensor by a simple preparation procedure was investigated. The fabricated electrode was used in different buffer solutions by DPV method. To our knowledge, the application of MoS2/CPE biosensor for the multiplex detection of G, A, T, and C by electrochemical method has not been reported yet. Afterwards, the sensitivity of the MoS2/CPE was improved. The PANI-MoS2/CPE was prepared to modify the biosensor. The effect of various mass ratios of PANI in the reaction system, ultrasonication time, and the response signals of analytes for the determination of purine bases (G and A) were investigated. 2. Materials and methods 2.1. Reagents and instruments The bulk molybdenum disulfide (MoS2, 99.0%), and G, A, T and C bases were purchased from Merck. The stock solutions were prepared by dissolving them into 0.1 M NaOH solution and kept in a refrigerator at 4 °C. The working solutions were freshly prepared by the appropriate dilution of the stock solutions. NaAc, HAc, NaH2PO4, Na2HPO4, H3PO4, and NaOH were purchased from Beijing Chemical Reagent Company (Beijing, China). 0.1 M acetate buffer solutions (ABS) at various pH values were prepared by mixing 0.1 M NaAc and 5
0.1 M HAc. 0.1 M phosphate buffer solutions (PBS) at various pH values were prepared by mixing 0.1 M NaH2PO4, 0.1 M Na2HPO4 and 0.1 M H3PO4. Tris-HCl buffer solutions (TBS) of various pH were obtained by mixing 0.1 M Tris and 0.1 M HCl. The ultrasonic homogenizer (Advanced Equipment Engineering Company, IRAN) was used for ultrasonic exfoliation, and the obtained nanocomposites were analyzed. DPV was employed to record the electrochemical oxidation of G, A, T and C. The electrochemical measurement was performed using an Auto lab Potentiostat-Galvanostat (Metrohm Herisau, Switzerland), and the NOVA 1.8 software (Metrohm Herisau, Switzerland) was employed for the data evaluation. Three conventional electrode cells including CPE as a working electrode, Ag/Agcl as a reference electrode, and platinum wire as a counter electrode were used in this work. All experiments were carried out at room temperature (27±1 ◦C). Double distilled water and Ultrapure water were used for preparing the solutions and the fabrication of the electrodes, respectively. 2.2. Fabrication of the modified electrodes The CPE was fabricated using a compact mixture of a carbon powder, namely highly pure graphite with particle size typically in micrometers without using any special equipment. The term carbon paste electrode means a setup of carbon paste fixed in a suitable body, which may be of quite different construction. The preparation of the PANI-MoS2/CPE electrodes was according to the method reported by Zhang et al. [37]. Different mass ratios of PANI and MoS2 were mixed and dispersed in ultrapure water. The mixtures were ultrasonicated for a given time until the homogeneous nanocomposites of PANI-MoS2 were formed. Then, 20 μL of each mixture containing the nanocomposites were dripped on the surface of the CPEs and finally,
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the prepared electrodes were dried in the air. In a similar way, MoS2/CPE and PANI/CPE were fabricated without the existence of the PANI and MoS2, respectively. 2.3. Electrochemical, Raman and UV-visible analyses Differential pulse voltammetry (DPV) parameters were set as follows: The pulse amplitude, pulse width, and pulse period were set as 0.05 V, 0.06 s, and 0.2 s, respectively. The reported result for each electrode was the mean value of the three parallel measurements. The results were
analyzed
by
Raman
spectrometer
(Jasco
NRS-5100,
USA),
and
UV-vis
spectrophotometer (DR6000, Hach, USA). 3. Results and discussion This section was divided into two parts, both aiming towards the molecular identification of DNANBs by means of the electrochemical method, Raman spectroscopy, and UV-vis spectrophotometer. In the first part, the electrochemical results of DNANBs analyzed by Raman spectroscopy and UV-vis spectrophotometer were compared. In the second part, the investigation was focused on enhancing the electrochemical sensing of DNANBs by the ultrasonic exfoliation method. 3.1. Sensing behavior of the MoS2/CPE towards DNANBs supported by Raman and UV-vis spectra 3.1.1. Influence of pH The influence of pH value on the oxidation behaviors of 12 μM G, 12 μM A, 120 μM T and 120 μM C was studied by recording DPV of MoS2/CPE in 0.1 M ABS in range of 3–12 (Fig. 1). Both the oxidation peak potentials and peak currents were related to solution pH value. As can be seen in Fig. 1, the oxidation peak potentials of G, A, T, and C shifted negatively by increasing pH value. The plots of Ep versus pH values show the straight lines at different pH values. The change observed in the slope of the lines may be due to the deprotonation of 7
DNANBs. It can be seen that the peak current of G decreases by increasing pH value, while the peak current of A increases by changing pH value from 3 to 4 and then, decreases at higher pH values. The peak currents of T and C increase in the range from 3 to 7 and 6, respectively, reaching the maximum values at pH = 7 and 6, respectively, and then, decline. Although the modified electrode showed an excellent electrocatalytic activity towards the oxidation of DNANBs at wide range of pH, pH=7.4 (physiological value) was selected as optimum pH value in order to obtain the best results for simultaneous determination of DNANBs after considering the situation of each base at different pH values. 3.1.2. Influence of supporting electrolytes In order to investigate the electrochemical behaviors of 12 μM G, 12 μM A, 120 μM T and 120 μM C, the DPV of MoS2/CPE electrode was carried out in the different supporting electrolytes of 0.1 M of ABS, PBS and Tris-HCl (pH=7). As can be seen in Fig. 2, in contrast with the observed oxidation peaks of G and A, no obvious oxidation peaks of T and C are observed by using PBS and Tris-HCl. When ABS is used as supporting electrolyte, the oxidation peaks appeared towards G, A, T and C are more obvious than those in PBS and Tris-HCl, indicating that the solution of ABS is more appropriate for the electron transfer in the oxidation reaction process of DNANBs. The appeared oxidation peaks could somehow be applied for the determination of G, A, T, and C. 3.1.3. Individual determination of DNANBs The DPVs of the modified electrode in a buffer solution containing different concentrations of DNANBs were recorded. The DPVs and calibration plots of DNANBs in Fig. 3 demonstrate that DNANBs can be determined purely (without any supporting substrate) in the concentration range of 0.1–12 µM for G and A, and 1.2–136 µM for T and C under the investigated conditions. The values of limit of detection (LOD), linear dynamic range (LDR), 8
coefficient of determination (R2) and sensitivity of the modified electrode towards DNANBs determinations in Table 1 indicate its good sensitivity, applicability over a wide concentration range, and favorable LOD values. 3.1.4. Simultaneous determination of DNANBs Fig. 4 shows the results of DPVs and calibration curves for simultaneous determination of DNANBs in the quaternary solutions with various concentrations by diluting 12 μM G, 12 μM A, 120 μM T and 120 μM C. The relationship between the oxidation peak currents of DNANBs and their concentrations is approximately linear in the range of 0.1–10 µM for G and A, and 1.2–100 µM for T and C. When G, A and T are determined individually, the reason for the differences in the parts of the linear regression of the calibration curves could be attributed to the change in the control factor of the electrode surface reaction. At lower concentrations of G and A, the surface reaction of the electrode is a diffusion controlled process, and at their higher concentrations, their electro-oxidation on the electrode surface change to an adsorption controlled process. In the case of C, inverse results are obtained. The obtained LODs, LDRs, coefficient of determination (R2) and sensitivity for DNANBs are listed in Table 1. 3.1.5. Determination of DNANBs at different concentrations The electrochemical response of DNANBs in the buffer solution was performed at different concentrations of each base under the optimum conditions by bare CPE, while the concentration of other DNANBs was constant. As shown in Fig. 5, by increasing the concentration of each base, the voltammetric response increases without any change in the response of other bases (synergistic effect), indicating that the electrochemical signals of G, A, T, and C are independent of each other. As it can be seen, when the concentration of G changes from 1 to 12 μM in the presence of 12 μM A, 120 μM T and 120 μM C, four individual oxidation peaks towards G, A, T, and C at different potentials are observed. Similar 9
experiments were performed for A, T and C. The results show that the proposed method can be used to individually determine each base in the presence of other bases without any interference. 3.1.6. Raman spectra of DNANBs, the MoS2 and DNANBs adsorbed onto the MoS2 By using Raman spectroscopy (a sensitive technique for the study of biological materials with different electronic band structures depending on the number of layers), valuable information about the chemical composition, the secondary structure that presents in the macromolecules and the chemical structure of surrounding subunits are obtained. Due to the major disadvantage of this technique related to the small scattering cross-section of biological molecules, solutions with high concentrations are used. Using picosecond excitation at 532 nm, it was possible to obtain Raman spectra of DNANBs (Chemical structures in Fig. 6). As mentioned above, many investigations have been done for the interpretation of Raman spectra of DNANBs. The spectra of DNANBs are shown in Fig. 7 [38]. Fig. 8 shows the Raman spectrum of the MoS2. The main Raman bands in the off-resonance spectra correspond to the zone-center first-order E12g mode at ~383 cm-1 and A1g mode at ~ 408 cm-1 [39]. The Raman spectra of DNANBs adsorbed onto the MoS2 are shown in Fig. 9. The spectrum of A-MoS2 is presented in Fig. 9a. The signal of the symmetric ring breathing mode (722 cm−1) is very strong in the normal Raman spectrum. Furthermore, the Raman spectrum indicates a strong contribution of several bands associated with NH2 and N9−H deformation modes, specifically the NH2 rocking band at 1248 cm−1, in-plane NH2 scissoring vibrations at 1334 cm−1, and N9−H bending modes at 1483 cm−1 [40]. The spectrum of G-MoS2 is shown in Fig. 9b. The Raman spectrum of G is different from the other nucleobases in the range of 1200-1600 cm−1, such as the obvious signal of the NH2 scissoring at around 1550 cm−1 and the 10
absence of the in-plane NH bending mode at 1387 cm−1 and 1231 cm-1 [40]. The most important differences are found in the ranges below 800 cm−1. The ring breathing mode at 645 cm−1 is very strong. Fig. 9c shows the Raman spectrum of C-MoS2 obtained from the most obvious signals of the C−N stretching mode at 1275 cm−1 and the ring breathing mode at 786 cm−1 [40], confirming the adsorption of C onto the MoS2. Fig. 9d presents the Raman spectrum of T-MoS2 in which the signal observed at 1371 cm−1 is related to the bending vibrations of CH3 and C6-H, and the signal at 1674 cm−1 is assigned to the stretching bands of C2=O and C4=O [40]. The results of Raman spectra confirm the adsorption of DNANBs onto the MoS2. 3.1.7. UV-vis spectra The UV absorption spectra of DNANBs were recorded in the range of 180–300 nm. The photostability of nucleobases (pure and adsorbed onto the single-layer MoS2 was investigated by UV irradiation experiments. The UV spectrum of MoS2 is shown in Fig. 10. Two characteristic absorption peaks of MoS2 are clearly observed in the range of 250–450 nm, corresponding to the direct excitonic transitions of the MoS2 originated from the energy split of valence-band and spin-orbital coupling [41]. In Fig. 11, the UV spectra of pure bases are compared with bases adsorbed onto the MoS2. According to Fig. 11a, the UV spectrum of A shows a strong and broad band with maximum absorption at around 260 nm. The spectrum of A-MoS2 is also observed in the range of 250–256 nm. As can be seen in Fig. 11b, after adsorption of T onto MoS2, a red-shifted about 5–10 nm is occurred. Also, the peak intensities of T and T-MoS2 are lower than those of A and A-MoS2. The UV absorption spectrum of C is strongly dependent on pH value. Hence, at pH=7.4, the UV absorption spectrum of C is indistinct and does not show obvious absorption bands, and any changes in the bands in the range of approximately 240-300 are more than other ranges (Fig. 11c). The UV absorption
11
spectrum of G (Fig. 11d) is different from other nucleobases due to the existence of two distinct electronic states, resulting in the formation of two bands with the maximum transition wavelengths of about 250 and 275 nm. The obvious decrease in the band after adsorption of G may be due to its chemical adsorption onto the MoS2. The results of UV-vis spectra confirm the results obtained by Raman spectra. 3.2. Sensing behavior of the PANI-MoS2/CPE prepared by ultrasonic exfoliation method The CPE and MoS2/CPE electrodes appear no obvious redox peaks due to their weak conductivity. Therefore, to intensify the sensitivity of the MoS2 nanosheets for the determination of DNANBs, the immobilization of PANI on the electrode surface was performed through the homogeneous dispersion of PANI on the surface of MoS2 nanosheets by the ultrasonic exfoliation method to prevent any aggregation in the composite. The synergistic effect between MoS2 and PANI electrocatalytic activity demonstrated that the electrochemical properties of MoS2 could be improved by the utilization of PANI particles. Due to the better sensitivity results of the MoS2/CPE electrode towards A and G, the electrochemical behaviors of 0.1 mmol L−1 A and G in acetate buffer solution at pH=7 were investigated by the PANI-MoS2/CPE. The electrochemical responses of PANI-MoS2/CPE in Figs. 12a and 12b for A and G, respectively, reveal the best peak current compared with CPE, PANI/CPE, and MoS2/CPE. This could be allocated to the strong π−π* interactions and electrostatic adsorption that cause better electron exchange between the free bases and the electrode surface. 3.2.1. Effect of mass ratio of MoS2:PANI The electrochemical behaviors of A and G by the PANI-MoS2 modified electrodes were investigated in acetate (pH=7), and the DPVs of PANI at the modified MoS2/CPE before and after PANI immobilization with different mass ratios are shown in Fig. 13 (1:1, 1:2, 1:3, and 12
1:4). The oxidation peak current value (Ip) was used to evaluate the optimum mass ratio. As can be seen in Fig. 13, the mass ratio of 1:3 shows the best electrocatalytic activity for A and G due to the largest peak currents. 3.2.2. Effect of ultrasonication time The effect of ultrasonication time of the PANI/MoS2 nanocomposite on the electrocatalytic behaviors was evaluated. As shown in Fig. 14, Ip increases by increasing the ultrasonication time of PANI-MoS2 nanocomposite up to 5 h and then, decreases. The results show that the appropriate sonication time can prevent the MoS2 nanosheets from aggregation and consequently, better results of Ip can be obtained. The obtained PANI/MoS2 nanocomposite with unique structure and abundant negative sites can improve the catalytic activity of A and G. 3.2.3. Effect of DNANBs concentration The electrochemical behaviors of A and G at different concentrations (0.05−100 μmol L−1) were investigated under the optimized conditions. As shown in Fig. 15, when A and G are determined individually, the signals increase at higher concentrations. Two linear regions were obtained in the calibration curves. The obtained linear regression equations of A and G are as follows: IpA = 0.133 C + 3.741 (R2 = 0.991)
(1)
IpG = 0.09 C + 2.725 (R2 = 0.993)
(2)
The detection limits are 3.0 × 10−9 mol L−1 and 5.0 × 10−9 mol L−1 for A and G, respectively. When the concentrations of A and G are increased simultaneously, the corresponding peak currents increase. Furthermore, the two oxidation peaks in Fig. 16 at +1.014 V and +0.718 V approximately exhibit two linear segments with the linear regression equations as following:
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IpA = 0.110 C + 3.542 (R2 = 0.990)
(3)
IpG = 0.065 C + 2.777 (R2 = 0.982)
(4)
The detection limits are 4.5 × 10−9 mol L−1 and 6.3 × 10−9 mol L−1 for A and G, respectively. Therefore, this method can sensitively detect A and G in a large concentration range. The results showed that based on the MoS2 nanosheets, the biosensor could detect A and G. Compared with blank MoS2 nanosheets, their significantly improved electrochemical activity could be due to higher active sites by introducing PANI, indicating that the PANI-MoS2/CPE shows an acceptable potential to be used as an excellent sensing platform for highly sensitive determination of DNANBs. 3.2.4. Stability and reproducibility of the modified electrode Ten modified electrodes were used to investigate their reproducibility by detecting 0.1 mmol L1
A and G. The stability of the electrodes was also evaluated by keeping them at room
temperature for 15 days. The results revealed that the values of relative standard deviation (RSD) for A and G oxidation peak currents are 4.06% and 3.76%, respectively, confirming the long-term stability and good reproducibility of the modified electrode. Acknowledgements The authors are thankful for the financial support received from Babol Noushirvani University of Technology. 4. Conclusion In this research, a simple, selective and sensitive electrochemical sensing platform for the determination of G, A, T and C nucleobases based on the MoS2 nanosheets was set up and developed without any separation or pretreatment process. Four separated oxidation peaks towards DNANBs were obtained that exhibited a rational linear range and acceptable detection limit. Moreover, the oxidation behavior of DNANBs characterized by Raman spectroscopy and 14
UV-vis spectrophotometer showed satisfactory results and proved the applicability of this method for providing a new platform for genetic information research and clinical diagnosis. Furthermore, it was demonstrated that the proposed sensing platform could be modified with PANI. Hence, PANI/MoS2 was prepared via the ultrasonication of the different mass ratios of PANI and MoS2, and the oxidation of DNANBs was investigated. The abundant negative charge and special structure of the prepared nanocomposite made it possible to adsorb the positively charged molecules. The modified electrode showed excellent catalytic activity, high sensitivity, and LOD towards DNANBs at a wide range of pH and ultrasonication time. Due to the special and unique chemical and physical properties of the prepared electrode, it can be applied as an excellent sensing platform for the detection of DNANBs. References [1] Y. Cho, S.K. Min, J. Yun, W.Y. Kim, A. Tkatchenko, K.S. Kim, Noncovalent interactions of DNA bases with naphthalene and graphene, J. Chem. Theory Comput. 9 (2013) 2090–2096. [2] S. Ketabi, S.M. Hashemianzadeh, M. Moghimi, W. asks, Study of DNA base-Li doped SiC nanotubes in aqueous solutions: a computer simulation study, J. Mol. Model. (2013), 19 (4) 1605–1615. [3] N. Goswami, A. Giri, S.K. Pal, MoS2 nanocrystals confined in a DNA matrix exhibiting energy transfer, Langmuir. 29 (2013) 11471–11478. [4] Shin-ichi Tanaka, M. Taniguchi, T. Kawai, Selective adsorption of DNA onto SiO2 surface in SiO2/SiH pattern, Jpn. J. Appl. Phys. 43 (2004) 7346–7349. [5] A.B. Farimani, K. Min, N.R. Aluru, DNA base detection using a single-layer MoS2, ACS Nano, 8 (2014) 7914–7922. [6] P.N. Bartlett, Bioelectrochemistry: Fundamentals, Experimental, Techniques, and Applications. John Wiley & Sons, Ltd. (2008). 15
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Fig. 1. Influence of pH on the (a) peak potentials (Ep) and (b) peak currents (Ip) of DNANBs.
20
Fig. 2. DPVs of 12 μM G, 12 μM A, 120 μM T and 120 μM C in 0.1 M electrolyte at pH=7.
Fig. 3. DPVs and calibration curves for the individual determination of 0.1-12 μM G, 0.1-12 μM A, 1.2-136 μM T, and 1.2-136 μM C.
21
Fig. 4. DPVs and calibration curves for the simultaneous determination of 0.1-10 μM G, 0.110 μM A, 1.2-72 μM T, and 1.2-72 μM C.
22
Fig. 5. DPVs at various concentrations of (a) G in the presence of 12 μM A, 120 μM T, and 120 μM C, (b) A in the presence of 12 μM G, 120 μM T and 120 μM C, (c) T in the presence of 12 μM G, 12 μM A and 120 μM C, and (d) C in the presence of 12 μM G, 12 μM A and 120 μM T.
Fig. 6. Structures and atom labeling of DNANBs.
23
Fig. 7. Raman spectra of DNANBs.
24
Fig. 8. Raman spectrum of the MoS2.
25
Fig. 9. Raman spectra of (a) A, (b) G, (c) C, and (d) T adsorbed onto the MoS2 in acetate buffer (pH=7) (Excitation=532 nm, photon flux density=1.4×1027 photons cm-2s-1; acquisition time=1 s, scale bars=300 cps and DNANBs concentration=5×10-5 M.
26
Fig. 10. UV-vis absorption spectrum of the MoS2.
Fig. 11. UV-vis absorption spectra of DNANBs adsorbed onto the MoS2 in acetate buffer (pH 7). The concentrations of DNANBs are constant.
27
Fig. 12. DPVs of 1.0×10−4 mol L-1 (a) A and (b) G recorded by different electrodes.
Fig. 13. DPVs of 1.0×10−4 mol L−1 (a) A and (b) G recorded at different mass ratios of MoS2:PANI.
28
Fig. 14. DPVs of 1.0×10-4 mol L-1 (a) A and (b) G recorded at different ultrasonication time of PANI-MoS2 nanocomposite.
Fig. 15. DPVs and calibration curves of (a) A and (b) G in ABS with various concentrations in the range of 0.05-100 μmol L−1 by PANI-MoS2 (1:3, 5 h)/CPE.
29
Fig. 16. DPVs and calibration curves for the simultaneous determination of A and G in ABS by the PANI-MoS2 (1:3, 5h)/CPE with the concentration in the range of 0.05-100 μmol L−1.
30
Table 1. Linear regression analysis and limit of detection by DPV for the quantification of individual and quaternary mixture of DNA bases.
Process
DNA bases
LDR
LOD
Sensitivity
mol L-1)µ(
mol L-1)µ(
A µmol L-1)µ(
(R2)
Guanine
0.9911
0.1-12
0.015
0.3877
Adenine
0.9924
0.1-12
0.015
0.3289
Cytosine
0.9968
1.2-136
0.140
0.0175
Thymine
0.9926
1.2-136
0.140
0.0499
Guanine
0.9961
0.1-10
0.015
0.5412
Adenine
0.9902
0.1-10
0.015
0.3712
Cytosine
0.9925
1.2-72
0.140
0.0444
Thymine
0.9895
1.2-72
0.140
0.0210
Individual
Quaternary
31
Highly sensitive biosensor for detection of DNA nucleobases: Enhanced electrochemical sensing based on polyaniline/single-layer MoS2 nanosheets nanocomposite modified carbon paste electrode Meisam Sadeghi , Mohsen Jahanshahi , Hamedreza Javadian PII: DOI: Reference:
S0026-265X(19)30439-4 https://doi.org/10.1016/j.microc.2019.104315 MICROC 104315
To appear in:
Microchemical Journal
Received date: Revised date: Accepted date:
21 February 2019 9 August 2019 6 October 2019
Please cite this article as: Meisam Sadeghi , Mohsen Jahanshahi , Hamedreza Javadian , Highly sensitive biosensor for detection of DNA nucleobases: Enhanced electrochemical sensing based on polyaniline/single-layer MoS2 nanosheets nanocomposite modified carbon paste electrode, Microchemical Journal (2019), doi: https://doi.org/10.1016/j.microc.2019.104315
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HIGHLIGHTS A simple analytical method for the determination of DNA nucleobases. Enhanced sensitivity for electrochemical detection using PANI-MoS2/CPE. The effects of supporting electrolyte, concentration, and pH were investigated.
Low limit of detection of the electrochemical biosensor with favorable R2.
1
Highly sensitive biosensor for detection of DNA nucleobases: Enhanced electrochemical sensing based on polyaniline/single-layer MoS2 nanosheets nanocomposite modified carbon paste electrode Meisam Sadeghia, Mohsen Jahanshahia, Hamedreza Javadianb,* a
Nanotechnology Research Institute, Faculty of Chemical Engineering, Babol Noushirvani University of Technology, Babol, Iran b
Universitat Politécnica de Catalunya, Department of Chemical Engineering, ETSEIB, Diagonal 647, 08028 Barcelona, Spain *
Corresponding author.
Email addresses: [email protected]; [email protected] Abstract Robust detection of DNA as a molecule that encodes genetic instructions is getting significant attention in recent years due to its promising applications in diseases diagnosis and treatment, forensic analysis, food safety evaluation, environmental monitoring, and so on. In this research, a simple, rapid and multiplex electrochemical method based on single-layer molybdenum disulfide (MoS2) nanosheets sensing platform and differential pulse voltammetry (DPV) method for detection of DNA nucleobases (DNANBs) was set up without any pretreatment or separation process. Hence, the carbon paste electrode modified with MoS2 (MoS2/CPE) was selected, and the effects of different parameters such as supporting electrolyte composition and pH were optimized to obtain the best peak potential separation and high sensitivity. Then, the individual, respective and simultaneous determinations of DNANBs were performed by controlling the certain experimental conditions. The results exhibited the excellent sensitivity, low limit of detection, and high selectivity of the electrochemical
2
biosensor. Moreover, the peak current values were linearly dependent on the concentration of DNANBs. For comparison, common methods for determination of DNANBs were analyzed based on the absorption of ultraviolet light and Raman spectroscopy. Finally, the proposed electrode was modified with polyaniline (PANI-MoS2/CPE) as a new biosensor for the determination of DNANBs, exhibiting good reproducibility and stability. Keywords: Electrochemical method, Carbon paste electrode, DNA nucleobases detection, Raman and Uv-vis spectroscopy, MoS2 nanosheets, Polyaniline/MoS2 nanocomposites.
1. Introduction Human chromosome, a linear biopolymer consisting nucleotide units (DNA and RNA), is considered as an important biological macromolecule that plays a crucial role in the formation of genetic instructions and guides the development of biological function by the replication and transcription of genetic information [1-4]. Guanine (G), adenine (A), thymine (T) and cytosine (C) are the important components of DNA. It is assumed that the mole percentages of G and A are equal to those of C and T, respectively [5]. The abnormal changes of these nucleobases in organisms results in the deficiency and mutation of immunity system, leading to various diseases such as epilepsy, cancer and HIV infection, etc [6,7]. Hence, the development of a simple and accurate analytical method to determine the concentration of G, A, T, and C is so much important for bio-analytical chemistry and life sciences. The common instrumental methods used for nucleobases detection are separation methods, such as electrophoresis, mass spectrometry, chromatography, etc [8-14]. The most alternative method for the mentioned methods to detect nucleobases is electrochemical detection, because nucleobases could be oxidized and/or reduced on polarized and renewable electrode surface [15]. In this method, polarographic signals coming from A and G are used in 3
DNA biosensors for process evaluation. It is mainly due to lower oxidation potentials of A and G than C and T [15]. It is an appropriate way to overcome the disadvantages of other methods such as complicated sample preparation processes, high cost and long runs [7]. To analyze the changes in G, A, T, and C, a series of comprehensive studies on their components are important. By individual analysis of each base components, the intact base combination in DNA can be measured [16-19]. Furthermore, in the structure explanation of nucleic acids and their nucleotide building blocks such as A, G, C, and T, Raman spectroscopy and UV are as the most important tools [20]. Intensive efforts have been performed to apply different electrode materials for detection of nucleobases. The varied electronic structure of transition metal dichalcogenides (TMDs) possess, ranging from semi-conducting (MoS2, WS2) to semi-metals (VS2, TiS2) and superconductors (TaSe2, NbSe2) makes them ideal for applying in electrochemical biosensors. MoS2 is a type of transition metal sulfides constructed by stacking covalently S–Mo-S bond through weak Van der Waals interactions. As a 2D layer structure analogous to graphene, MoS2 has recently become a popular material regarding its outstanding properties, such as being a transistor [21,22], supercapacitor [23], electro-analyzer [24-26], and thermal conductor [27]. The optical and electronic properties of MoS2 change as the number of layers decreases, mainly due to the electronic band transformation from indirect gap to direct gap [28]. In addition, researchers have indicated that aromatic compounds could physisorb the basal plane of MoS2. According to the above-mentioned properties of MoS2, a nucleic acid sequence detection method with the single-layer MoS2 nanosheets as sensing platform can be proposed [29,30]. Generally, an ideal electrochemical sensor should possess a wide potential range and high sensitivity and electroactivities. It is possible to improve the sensitivity of MoS2-based 4
electrodes. PANI-based composite materials have been widely applied in the field of electrochemical sensor utilization due to tunable properties, easy synthesis, environmental stability, inexpensive monomer, variable oxidation state, effective mass/charge transfer, broad range conductivity, nanoarchitecture and good electroactivity [31]. It has been proved by researchers that the electrochemical performance of electrode materials can be greatly increased by adding PANI [32–36]. Therefore, to enhance the electrochemical performance of the single-layer MoS2 nanosheets, their combination with PANI to form the PANI-MoS2/CPE sensor can be carried out. In this present work, the feasibility of the MoS2/CPE to develop a biosensor by a simple preparation procedure was investigated. The fabricated electrode was used in different buffer solutions by DPV method. To our knowledge, the application of MoS2/CPE biosensor for the multiplex detection of G, A, T, and C by electrochemical method has not been reported yet. Afterwards, the sensitivity of the MoS2/CPE was improved. The PANI-MoS2/CPE was prepared to modify the biosensor. The effect of various mass ratios of PANI in the reaction system, ultrasonication time, and the response signals of analytes for the determination of purine bases (G and A) were investigated. 2. Materials and methods 2.1. Reagents and instruments The bulk molybdenum disulfide (MoS2, 99.0%), and G, A, T and C bases were purchased from Merck. The stock solutions were prepared by dissolving them into 0.1 M NaOH solution and kept in a refrigerator at 4 °C. The working solutions were freshly prepared by the appropriate dilution of the stock solutions. NaAc, HAc, NaH2PO4, Na2HPO4, H3PO4, and NaOH were purchased from Beijing Chemical Reagent Company (Beijing, China). 0.1 M acetate buffer solutions (ABS) at various pH values were prepared by mixing 0.1 M NaAc and 5
0.1 M HAc. 0.1 M phosphate buffer solutions (PBS) at various pH values were prepared by mixing 0.1 M NaH2PO4, 0.1 M Na2HPO4 and 0.1 M H3PO4. Tris-HCl buffer solutions (TBS) of various pH were obtained by mixing 0.1 M Tris and 0.1 M HCl. The ultrasonic homogenizer (Advanced Equipment Engineering Company, IRAN) was used for ultrasonic exfoliation, and the obtained nanocomposites were analyzed. DPV was employed to record the electrochemical oxidation of G, A, T and C. The electrochemical measurement was performed using an Auto lab Potentiostat-Galvanostat (Metrohm Herisau, Switzerland), and the NOVA 1.8 software (Metrohm Herisau, Switzerland) was employed for the data evaluation. Three conventional electrode cells including CPE as a working electrode, Ag/Agcl as a reference electrode, and platinum wire as a counter electrode were used in this work. All experiments were carried out at room temperature (27±1 ◦C). Double distilled water and Ultrapure water were used for preparing the solutions and the fabrication of the electrodes, respectively. 2.2. Fabrication of the modified electrodes The CPE was fabricated using a compact mixture of a carbon powder, namely highly pure graphite with particle size typically in micrometers without using any special equipment. The term carbon paste electrode means a setup of carbon paste fixed in a suitable body, which may be of quite different construction. The preparation of the PANI-MoS2/CPE electrodes was according to the method reported by Zhang et al. [37]. Different mass ratios of PANI and MoS2 were mixed and dispersed in ultrapure water. The mixtures were ultrasonicated for a given time until the homogeneous nanocomposites of PANI-MoS2 were formed. Then, 20 μL of each mixture containing the nanocomposites were dripped on the surface of the CPEs and finally,
6
the prepared electrodes were dried in the air. In a similar way, MoS2/CPE and PANI/CPE were fabricated without the existence of the PANI and MoS2, respectively. 2.3. Electrochemical, Raman and UV-visible analyses Differential pulse voltammetry (DPV) parameters were set as follows: The pulse amplitude, pulse width, and pulse period were set as 0.05 V, 0.06 s, and 0.2 s, respectively. The reported result for each electrode was the mean value of the three parallel measurements. The results were
analyzed
by
Raman
spectrometer
(Jasco
NRS-5100,
USA),
and
UV-vis
spectrophotometer (DR6000, Hach, USA). 3. Results and discussion This section was divided into two parts, both aiming towards the molecular identification of DNANBs by means of the electrochemical method, Raman spectroscopy, and UV-vis spectrophotometer. In the first part, the electrochemical results of DNANBs analyzed by Raman spectroscopy and UV-vis spectrophotometer were compared. In the second part, the investigation was focused on enhancing the electrochemical sensing of DNANBs by the ultrasonic exfoliation method. 3.1. Sensing behavior of the MoS2/CPE towards DNANBs supported by Raman and UV-vis spectra 3.1.1. Influence of pH The influence of pH value on the oxidation behaviors of 12 μM G, 12 μM A, 120 μM T and 120 μM C was studied by recording DPV of MoS2/CPE in 0.1 M ABS in range of 3–12 (Fig. 1). Both the oxidation peak potentials and peak currents were related to solution pH value. As can be seen in Fig. 1, the oxidation peak potentials of G, A, T, and C shifted negatively by increasing pH value. The plots of Ep versus pH values show the straight lines at different pH values. The change observed in the slope of the lines may be due to the deprotonation of 7
DNANBs. It can be seen that the peak current of G decreases by increasing pH value, while the peak current of A increases by changing pH value from 3 to 4 and then, decreases at higher pH values. The peak currents of T and C increase in the range from 3 to 7 and 6, respectively, reaching the maximum values at pH = 7 and 6, respectively, and then, decline. Although the modified electrode showed an excellent electrocatalytic activity towards the oxidation of DNANBs at wide range of pH, pH=7.4 (physiological value) was selected as optimum pH value in order to obtain the best results for simultaneous determination of DNANBs after considering the situation of each base at different pH values. 3.1.2. Influence of supporting electrolytes In order to investigate the electrochemical behaviors of 12 μM G, 12 μM A, 120 μM T and 120 μM C, the DPV of MoS2/CPE electrode was carried out in the different supporting electrolytes of 0.1 M of ABS, PBS and Tris-HCl (pH=7). As can be seen in Fig. 2, in contrast with the observed oxidation peaks of G and A, no obvious oxidation peaks of T and C are observed by using PBS and Tris-HCl. When ABS is used as supporting electrolyte, the oxidation peaks appeared towards G, A, T and C are more obvious than those in PBS and Tris-HCl, indicating that the solution of ABS is more appropriate for the electron transfer in the oxidation reaction process of DNANBs. The appeared oxidation peaks could somehow be applied for the determination of G, A, T, and C. 3.1.3. Individual determination of DNANBs The DPVs of the modified electrode in a buffer solution containing different concentrations of DNANBs were recorded. The DPVs and calibration plots of DNANBs in Fig. 3 demonstrate that DNANBs can be determined purely (without any supporting substrate) in the concentration range of 0.1–12 µM for G and A, and 1.2–136 µM for T and C under the investigated conditions. The values of limit of detection (LOD), linear dynamic range (LDR), 8
coefficient of determination (R2) and sensitivity of the modified electrode towards DNANBs determinations in Table 1 indicate its good sensitivity, applicability over a wide concentration range, and favorable LOD values. 3.1.4. Simultaneous determination of DNANBs Fig. 4 shows the results of DPVs and calibration curves for simultaneous determination of DNANBs in the quaternary solutions with various concentrations by diluting 12 μM G, 12 μM A, 120 μM T and 120 μM C. The relationship between the oxidation peak currents of DNANBs and their concentrations is approximately linear in the range of 0.1–10 µM for G and A, and 1.2–100 µM for T and C. When G, A and T are determined individually, the reason for the differences in the parts of the linear regression of the calibration curves could be attributed to the change in the control factor of the electrode surface reaction. At lower concentrations of G and A, the surface reaction of the electrode is a diffusion controlled process, and at their higher concentrations, their electro-oxidation on the electrode surface change to an adsorption controlled process. In the case of C, inverse results are obtained. The obtained LODs, LDRs, coefficient of determination (R2) and sensitivity for DNANBs are listed in Table 1. 3.1.5. Determination of DNANBs at different concentrations The electrochemical response of DNANBs in the buffer solution was performed at different concentrations of each base under the optimum conditions by bare CPE, while the concentration of other DNANBs was constant. As shown in Fig. 5, by increasing the concentration of each base, the voltammetric response increases without any change in the response of other bases (synergistic effect), indicating that the electrochemical signals of G, A, T, and C are independent of each other. As it can be seen, when the concentration of G changes from 1 to 12 μM in the presence of 12 μM A, 120 μM T and 120 μM C, four individual oxidation peaks towards G, A, T, and C at different potentials are observed. Similar 9
experiments were performed for A, T and C. The results show that the proposed method can be used to individually determine each base in the presence of other bases without any interference. 3.1.6. Raman spectra of DNANBs, the MoS2 and DNANBs adsorbed onto the MoS2 By using Raman spectroscopy (a sensitive technique for the study of biological materials with different electronic band structures depending on the number of layers), valuable information about the chemical composition, the secondary structure that presents in the macromolecules and the chemical structure of surrounding subunits are obtained. Due to the major disadvantage of this technique related to the small scattering cross-section of biological molecules, solutions with high concentrations are used. Using picosecond excitation at 532 nm, it was possible to obtain Raman spectra of DNANBs (Chemical structures in Fig. 6). As mentioned above, many investigations have been done for the interpretation of Raman spectra of DNANBs. The spectra of DNANBs are shown in Fig. 7 [38]. Fig. 8 shows the Raman spectrum of the MoS2. The main Raman bands in the off-resonance spectra correspond to the zone-center first-order E12g mode at ~383 cm-1 and A1g mode at ~ 408 cm-1 [39]. The Raman spectra of DNANBs adsorbed onto the MoS2 are shown in Fig. 9. The spectrum of A-MoS2 is presented in Fig. 9a. The signal of the symmetric ring breathing mode (722 cm−1) is very strong in the normal Raman spectrum. Furthermore, the Raman spectrum indicates a strong contribution of several bands associated with NH2 and N9−H deformation modes, specifically the NH2 rocking band at 1248 cm−1, in-plane NH2 scissoring vibrations at 1334 cm−1, and N9−H bending modes at 1483 cm−1 [40]. The spectrum of G-MoS2 is shown in Fig. 9b. The Raman spectrum of G is different from the other nucleobases in the range of 1200-1600 cm−1, such as the obvious signal of the NH2 scissoring at around 1550 cm−1 and the 10
absence of the in-plane NH bending mode at 1387 cm−1 and 1231 cm-1 [40]. The most important differences are found in the ranges below 800 cm−1. The ring breathing mode at 645 cm−1 is very strong. Fig. 9c shows the Raman spectrum of C-MoS2 obtained from the most obvious signals of the C−N stretching mode at 1275 cm−1 and the ring breathing mode at 786 cm−1 [40], confirming the adsorption of C onto the MoS2. Fig. 9d presents the Raman spectrum of T-MoS2 in which the signal observed at 1371 cm−1 is related to the bending vibrations of CH3 and C6-H, and the signal at 1674 cm−1 is assigned to the stretching bands of C2=O and C4=O [40]. The results of Raman spectra confirm the adsorption of DNANBs onto the MoS2. 3.1.7. UV-vis spectra The UV absorption spectra of DNANBs were recorded in the range of 180–300 nm. The photostability of nucleobases (pure and adsorbed onto the single-layer MoS2 was investigated by UV irradiation experiments. The UV spectrum of MoS2 is shown in Fig. 10. Two characteristic absorption peaks of MoS2 are clearly observed in the range of 250–450 nm, corresponding to the direct excitonic transitions of the MoS2 originated from the energy split of valence-band and spin-orbital coupling [41]. In Fig. 11, the UV spectra of pure bases are compared with bases adsorbed onto the MoS2. According to Fig. 11a, the UV spectrum of A shows a strong and broad band with maximum absorption at around 260 nm. The spectrum of A-MoS2 is also observed in the range of 250–256 nm. As can be seen in Fig. 11b, after adsorption of T onto MoS2, a red-shifted about 5–10 nm is occurred. Also, the peak intensities of T and T-MoS2 are lower than those of A and A-MoS2. The UV absorption spectrum of C is strongly dependent on pH value. Hence, at pH=7.4, the UV absorption spectrum of C is indistinct and does not show obvious absorption bands, and any changes in the bands in the range of approximately 240-300 are more than other ranges (Fig. 11c). The UV absorption
11
spectrum of G (Fig. 11d) is different from other nucleobases due to the existence of two distinct electronic states, resulting in the formation of two bands with the maximum transition wavelengths of about 250 and 275 nm. The obvious decrease in the band after adsorption of G may be due to its chemical adsorption onto the MoS2. The results of UV-vis spectra confirm the results obtained by Raman spectra. 3.2. Sensing behavior of the PANI-MoS2/CPE prepared by ultrasonic exfoliation method The CPE and MoS2/CPE electrodes appear no obvious redox peaks due to their weak conductivity. Therefore, to intensify the sensitivity of the MoS2 nanosheets for the determination of DNANBs, the immobilization of PANI on the electrode surface was performed through the homogeneous dispersion of PANI on the surface of MoS2 nanosheets by the ultrasonic exfoliation method to prevent any aggregation in the composite. The synergistic effect between MoS2 and PANI electrocatalytic activity demonstrated that the electrochemical properties of MoS2 could be improved by the utilization of PANI particles. Due to the better sensitivity results of the MoS2/CPE electrode towards A and G, the electrochemical behaviors of 0.1 mmol L−1 A and G in acetate buffer solution at pH=7 were investigated by the PANI-MoS2/CPE. The electrochemical responses of PANI-MoS2/CPE in Figs. 12a and 12b for A and G, respectively, reveal the best peak current compared with CPE, PANI/CPE, and MoS2/CPE. This could be allocated to the strong π−π* interactions and electrostatic adsorption that cause better electron exchange between the free bases and the electrode surface. 3.2.1. Effect of mass ratio of MoS2:PANI The electrochemical behaviors of A and G by the PANI-MoS2 modified electrodes were investigated in acetate (pH=7), and the DPVs of PANI at the modified MoS2/CPE before and after PANI immobilization with different mass ratios are shown in Fig. 13 (1:1, 1:2, 1:3, and 12
1:4). The oxidation peak current value (Ip) was used to evaluate the optimum mass ratio. As can be seen in Fig. 13, the mass ratio of 1:3 shows the best electrocatalytic activity for A and G due to the largest peak currents. 3.2.2. Effect of ultrasonication time The effect of ultrasonication time of the PANI/MoS2 nanocomposite on the electrocatalytic behaviors was evaluated. As shown in Fig. 14, Ip increases by increasing the ultrasonication time of PANI-MoS2 nanocomposite up to 5 h and then, decreases. The results show that the appropriate sonication time can prevent the MoS2 nanosheets from aggregation and consequently, better results of Ip can be obtained. The obtained PANI/MoS2 nanocomposite with unique structure and abundant negative sites can improve the catalytic activity of A and G. 3.2.3. Effect of DNANBs concentration The electrochemical behaviors of A and G at different concentrations (0.05−100 μmol L−1) were investigated under the optimized conditions. As shown in Fig. 15, when A and G are determined individually, the signals increase at higher concentrations. Two linear regions were obtained in the calibration curves. The obtained linear regression equations of A and G are as follows: IpA = 0.133 C + 3.741 (R2 = 0.991)
(1)
IpG = 0.09 C + 2.725 (R2 = 0.993)
(2)
The detection limits are 3.0 × 10−9 mol L−1 and 5.0 × 10−9 mol L−1 for A and G, respectively. When the concentrations of A and G are increased simultaneously, the corresponding peak currents increase. Furthermore, the two oxidation peaks in Fig. 16 at +1.014 V and +0.718 V approximately exhibit two linear segments with the linear regression equations as following:
13
IpA = 0.110 C + 3.542 (R2 = 0.990)
(3)
IpG = 0.065 C + 2.777 (R2 = 0.982)
(4)
The detection limits are 4.5 × 10−9 mol L−1 and 6.3 × 10−9 mol L−1 for A and G, respectively. Therefore, this method can sensitively detect A and G in a large concentration range. The results showed that based on the MoS2 nanosheets, the biosensor could detect A and G. Compared with blank MoS2 nanosheets, their significantly improved electrochemical activity could be due to higher active sites by introducing PANI, indicating that the PANI-MoS2/CPE shows an acceptable potential to be used as an excellent sensing platform for highly sensitive determination of DNANBs. 3.2.4. Stability and reproducibility of the modified electrode Ten modified electrodes were used to investigate their reproducibility by detecting 0.1 mmol L1
A and G. The stability of the electrodes was also evaluated by keeping them at room
temperature for 15 days. The results revealed that the values of relative standard deviation (RSD) for A and G oxidation peak currents are 4.06% and 3.76%, respectively, confirming the long-term stability and good reproducibility of the modified electrode. Acknowledgements The authors are thankful for the financial support received from Babol Noushirvani University of Technology. 4. Conclusion In this research, a simple, selective and sensitive electrochemical sensing platform for the determination of G, A, T and C nucleobases based on the MoS2 nanosheets was set up and developed without any separation or pretreatment process. Four separated oxidation peaks towards DNANBs were obtained that exhibited a rational linear range and acceptable detection limit. Moreover, the oxidation behavior of DNANBs characterized by Raman spectroscopy and 14
UV-vis spectrophotometer showed satisfactory results and proved the applicability of this method for providing a new platform for genetic information research and clinical diagnosis. Furthermore, it was demonstrated that the proposed sensing platform could be modified with PANI. Hence, PANI/MoS2 was prepared via the ultrasonication of the different mass ratios of PANI and MoS2, and the oxidation of DNANBs was investigated. The abundant negative charge and special structure of the prepared nanocomposite made it possible to adsorb the positively charged molecules. The modified electrode showed excellent catalytic activity, high sensitivity, and LOD towards DNANBs at a wide range of pH and ultrasonication time. Due to the special and unique chemical and physical properties of the prepared electrode, it can be applied as an excellent sensing platform for the detection of DNANBs. References [1] Y. Cho, S.K. Min, J. Yun, W.Y. Kim, A. Tkatchenko, K.S. Kim, Noncovalent interactions of DNA bases with naphthalene and graphene, J. Chem. Theory Comput. 9 (2013) 2090–2096. [2] S. Ketabi, S.M. Hashemianzadeh, M. Moghimi, W. asks, Study of DNA base-Li doped SiC nanotubes in aqueous solutions: a computer simulation study, J. Mol. Model. (2013), 19 (4) 1605–1615. [3] N. Goswami, A. Giri, S.K. Pal, MoS2 nanocrystals confined in a DNA matrix exhibiting energy transfer, Langmuir. 29 (2013) 11471–11478. [4] Shin-ichi Tanaka, M. Taniguchi, T. Kawai, Selective adsorption of DNA onto SiO2 surface in SiO2/SiH pattern, Jpn. J. Appl. Phys. 43 (2004) 7346–7349. [5] A.B. Farimani, K. Min, N.R. Aluru, DNA base detection using a single-layer MoS2, ACS Nano, 8 (2014) 7914–7922. [6] P.N. Bartlett, Bioelectrochemistry: Fundamentals, Experimental, Techniques, and Applications. John Wiley & Sons, Ltd. (2008). 15
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Fig. 1. Influence of pH on the (a) peak potentials (Ep) and (b) peak currents (Ip) of DNANBs.
20
Fig. 2. DPVs of 12 μM G, 12 μM A, 120 μM T and 120 μM C in 0.1 M electrolyte at pH=7.
Fig. 3. DPVs and calibration curves for the individual determination of 0.1-12 μM G, 0.1-12 μM A, 1.2-136 μM T, and 1.2-136 μM C.
21
Fig. 4. DPVs and calibration curves for the simultaneous determination of 0.1-10 μM G, 0.110 μM A, 1.2-72 μM T, and 1.2-72 μM C.
22
Fig. 5. DPVs at various concentrations of (a) G in the presence of 12 μM A, 120 μM T, and 120 μM C, (b) A in the presence of 12 μM G, 120 μM T and 120 μM C, (c) T in the presence of 12 μM G, 12 μM A and 120 μM C, and (d) C in the presence of 12 μM G, 12 μM A and 120 μM T.
Fig. 6. Structures and atom labeling of DNANBs.
23
Fig. 7. Raman spectra of DNANBs.
24
Fig. 8. Raman spectrum of the MoS2.
25
Fig. 9. Raman spectra of (a) A, (b) G, (c) C, and (d) T adsorbed onto the MoS2 in acetate buffer (pH=7) (Excitation=532 nm, photon flux density=1.4×1027 photons cm-2s-1; acquisition time=1 s, scale bars=300 cps and DNANBs concentration=5×10-5 M.
26
Fig. 10. UV-vis absorption spectrum of the MoS2.
Fig. 11. UV-vis absorption spectra of DNANBs adsorbed onto the MoS2 in acetate buffer (pH 7). The concentrations of DNANBs are constant.
27
Fig. 12. DPVs of 1.0×10−4 mol L-1 (a) A and (b) G recorded by different electrodes.
Fig. 13. DPVs of 1.0×10−4 mol L−1 (a) A and (b) G recorded at different mass ratios of MoS2:PANI.
28
Fig. 14. DPVs of 1.0×10-4 mol L-1 (a) A and (b) G recorded at different ultrasonication time of PANI-MoS2 nanocomposite.
Fig. 15. DPVs and calibration curves of (a) A and (b) G in ABS with various concentrations in the range of 0.05-100 μmol L−1 by PANI-MoS2 (1:3, 5 h)/CPE.
29
Fig. 16. DPVs and calibration curves for the simultaneous determination of A and G in ABS by the PANI-MoS2 (1:3, 5h)/CPE with the concentration in the range of 0.05-100 μmol L−1.
30
Table 1. Linear regression analysis and limit of detection by DPV for the quantification of individual and quaternary mixture of DNA bases.
Process
DNA bases
LDR
LOD
Sensitivity
mol L-1)µ(
mol L-1)µ(
A µmol L-1)µ(
(R2)
Guanine
0.9911
0.1-12
0.015
0.3877
Adenine
0.9924
0.1-12
0.015
0.3289
Cytosine
0.9968
1.2-136
0.140
0.0175
Thymine
0.9926
1.2-136
0.140
0.0499
Guanine
0.9961
0.1-10
0.015
0.5412
Adenine
0.9902
0.1-10
0.015
0.3712
Cytosine
0.9925
1.2-72
0.140
0.0444
Thymine
0.9895
1.2-72
0.140
0.0210
Individual
Quaternary
31