[Cu(phen)2]2+ acts as electrochemical indicator and anchor to immobilize probe DNA in electrochemical DNA biosensor

Analytical Biochemistry 492 (2016) 56e62

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[Cu(phen)2]2þ acts as electrochemical indicator and anchor to immobilize probe DNA in electrochemical DNA biosensor Linlin Yang a, 1, Xiaoyu Li a, 1, Xi Li a, *, Songling Yan a, Yinna Ren a, Mengmeng Wang a, Peng Liu a, Yulin Dong a, Chaocan Zhang b, ** a

Department of Chemistry, School of Chemistry, Chemical Engineering, and Life Science, Wuhan University of Technology, Wuhan 430070, People's Republic of China School of Materials Science and Engineering, Wuhan University of Technology, Wuhan 430070, People's Republic of China

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 June 2015 Received in revised form 8 September 2015 Accepted 14 September 2015 Available online 25 September 2015

We demonstrate a novel protocol for sensitive in situ label-free electrochemical detection of DNA hybridization based on copper complex ([Cu(phen)2]2þ, where phen ¼ 1,10-phenanthroline) and graphene (GR) modified glassy carbon electrode. Here, [Cu(phen)2]2þ acted advantageously as both the electrochemical indicator and the anchor for probe DNA immobilization via intercalative interactions between the partial double helix structure of probe DNA and the vertical aromatic groups of phen. GR provided large density of docking site for probe DNA immobilization and increased the electrical conductivity ability of the electrode. The modification procedure was monitored by electrochemical impedance spectroscopy (EIS). Square-wave voltammetry (SWV) was used to explore the hybridization events. Under the optimal conditions, the designed electrochemical DNA biosensor could effectively distinguish different mismatch degrees of complementary DNA from one-base mismatch to noncomplementary, indicating that the biosensor had high selectivity. It also exhibited a reasonable linear relationship. The oxidation peak currents of [Cu(phen)2]2þ were linear with the logarithm of the concentrations of complementary target DNA ranging from 1  1012 to 1  106 M with a detection limit of 1.99  1013 M (signal/noise ¼ 3). Moreover, the stability of the electrochemical DNA biosensor was also studied. © 2015 Elsevier Inc. All rights reserved.

Keywords: Electrochemical DNA biosensor Copper complex Graphene Square-wave voltammetry

Simple, rapid, sensitive, and label-free DNA diagnosis has attracted great attention with the continuous development of biotechnology in a widespread range of applications, including molecular diagnostics, clinical medicines, environmental monitoring, and forensic analysis [1e4]. Consequently, a great deal of strategies such as fluorescence [5], electrochemiluminescence [6], polymerase chain reaction [7], surface plasmon resonance spectroscopy [8], and electrochemistry [9,10] have been developed for the analysis of DNA hybridization. Among them, the electrochemical methods have been widely used due to their rapidness,

Abbreviations used: phen, 1,10-phenanthroline; GR, graphene; GCE, glassy carbon electrode; PBS, phosphate buffer solution; GO, graphene oxide; EIS, electrochemical impedance spectroscopy; SWV, square-wave voltammetry. * Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (X. Li), [email protected] (C. Zhang). 1 These authors contributed equally to this study and share first authorship. http://dx.doi.org/10.1016/j.ab.2015.09.011 0003-2697/© 2015 Elsevier Inc. All rights reserved.

high sensitivity, low cost, and ease of operation [11,12]. The development of highly sensitive and convenient electrochemical DNA sensors with low detection limits is an area of ever-increasing interest for the greater desire for quick and efficient DNA detection. The detection procedure of electrochemical DNA biosensors consists of two main parts [13]: the immobilization of probe DNA onto the electrode surface to form a biorecognition interface and the electronic signal detection through a transducer that could convert the hybridization events into electronic signals. Because the bare electrode is limited in surface area and cannot directly immobilize probe DNA firmly, this may lead to poor stability and detection limit of the biosensor. Therefore, considerable efforts have been made to improve the electrochemical performance of electrodes. Various kinds of nanostructures (e.g., carbon nanotubes [14,15], graphene [16], gold nanoparticles [17,18]), conducting polymers [19,20], and transition metal complexes [21,22] in particular have fascinated researchers in the fabrication of electrochemical DNA biosensors to implement probe DNA attaching and signal amplification.

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Apart from its specific structure and prominent properties, including electrocatalysis in low potential, reversible property of electrode reaction, and exclusive biological function in living systems [23], transition metal complexes have emerged as one of the promising classes of functional materials in ultrasensitive electrical detection of specific DNA oligonucleotides. In general, transition metal complexes can bind DNA backbone through intercalation, noncovalent groove binding, and electrostatic and some other types of interactions [24e27]. The cofactor agents of those complexes, such as Fe(III), Co(II), Cu(II), and Ru(III), can perform changeable redox signals before and after the hybridization reaction occurs, which can be used as the electroactive molecules to monitor the DNA hybridization process [28]. Most of the reported electrochemical DNA biosensors so far employed just the transition metal complexes as indicator; the probe immobilization requires other multiple steps, which might prolong the assay time and impair the analytical performance of the sensors so as to restrict their wide application. Considering these factors, in this study we used a specific DNA oligonucleotide that could form doublestranded chain partly by itself as the probe and copper complex ([Cu(phen)2]2þ, where phen ¼ 1,10-phenanthroline) acted in versatile roles, not only as the electrochemical indicator but also as the anchor for probe DNA immobilization to simplify the chemosynthesis process and fabricating steps. Because of the unique threedimensional configuration of phen, it could achieve the goal of probe DNA immobilization through the intercalative interactions between the partial double helix structure of probe DNA and the vertical aromatic groups of phen. Meanwhile, [Cu(phen)2]2þ performed the electrochemical response and would be inhibited when the probe DNA hybridized with the complementary DNA because the intercalation could block the charge transfer, which could be used as electroactive indicator in electrochemical DNA biosensors. Graphene (GR), a two-dimensional sheet of sp2 bonded carbon atomics, has attracted increasing attention because of its remarkable electronic and mechanical properties such as excellent electrochemical conductivity, extreme high surface area (even larger than single-walled carbon nanotubes), good biocompatibility, rapid heterogeneous electron transfer rate, and high mechanical strength [29e32]. The introduction of GR has been proven effective in accelerating the electron transfer rate of DNA and in enhancing the electrochemical response [33,34]. For instance, a novel electrochemical deoxyribonucleic acid biosensor was fabricated based on an electrochemical reduced graphene oxide modified electrode with a detection limit of 5.45  1013 M [25]. Zhang and Huang [35] described the construction of a sensitive label-free electrochemical DNA biosensor to detect specific sequence of target DNA with a glassy carbon electrode (GCE) modified with GR, gold

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nanoparticles, and polythionine. The aromatic regions of GR can also offer active sites to interact with other aromatic molecules through pep supramolecular interactions [36], confirming the possibility of attaching [Cu(phen)2]2þ firmly on the GR modified electrode. To sum up, we developed a simple label-free electrochemical DNA biosensor that allows the GCE to be modified with GR and [Cu(phen)2]2þ and employs a chain of partial double helix structured oligonucleotide as probe for DNA detection. The [Cu(phen)2]2þ/GR modified electrode could be functionalized with probe DNA through the intercalative interactions. The protocol of our design is illustrated in Fig. 1. After the exploration of experimental conditions such as the amount of GR, the film thickness of [Cu(phen)2]2þ, the immobilization time for probe DNA, and the hybridization time of target DNA, the functionalized biosensor was capable of detecting sequence-specific target DNA as low as 1013 M as well as recognizing different DNA sequences, including complementary, one-base mismatch, three-base mismatch, and noncomplementary. Without multiple modification procedures, it is believed that the developed electrochemical DNA biosensor could find applications in the fields of medicine and biotechnology. Materials and methods Materials All of the probe DNA and target DNA were purchased from Sangon Biotechnology (Shanghai, China) with HPLC purification and diluted with a phosphate buffer solution (PBS, pH 7.4, 0.1 M NaCl) prior to use. The sequences of probe and complementary DNA used in this work were as follows: Probe DNA (P1): 50 -TACTCACCGGGCTGCAGCCGTTCCGCAG-30 Target DNA (T1): 50 -CTGCGGAACCGGTGAGTA-30 One-base mismatch DNA (T2): 50 -CTGCAGAACCGGTGAGTA-30 Three-base mismatch DNA (T3): 50 -CTGCAGAACTGGTGCGTA-30 Noncomplementary DNA(T4): 50 -TGAGATTCGTACGTCCGT-3ʹ The underlined part of the probe DNA can form double-stranded chain by itself that is used as an anchorfor DNA immobilization. One-base mismatch, three-base mismatch, and noncomplementary DNA were used to investigate the selectivity of the DNA biosensor. Graphite with 44 mm diameter was obtained from Nanjing XianFeng Nano (Nanjing, China). First, the graphite was oxidized according to the Hummers method [37] to get the graphene oxide (GO). Then, hydrazine hydrate was added into the obtained GO suspension to get the reductive GR.

Fig.1. Schematic diagram of the electrochemical DNA sensor fabrication.

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Cu(phen)2Cl2$6H2O was prepared according to the literature method [38]. The ethanolic solution (30 ml) including phen monohydrate (3.96 g, 20 mmol) was added to a hot aqueous solution (30 ml) of CuCl2$2H2O (1.7 g, 10 mmol) under stirring. As the temperature decreased, blue crystals appeared. Then, the crystals were isolated and washed with ethanol three times before vacuum drying. After recrystallization, the Cu(phen)2Cl2$6H2O complexes were obtained. Common reagents such as methanol, KCl, Na2HPO4$12H2O, NaH2PO4$2H2O, CuCl2$2H2O, and 1,10-phenanthroline monohydrate were purchased from Sinopharm Chemical Reagent (Shanghai, China) with analytical grade and were used as received. Apparatus All electrochemical experiments were carried out using a model CHI 660D electrochemical workstation (Shanghai Chenhua Instrument, Shanghai, China) with a three-electrode system, which was composed of a bare or modified GCE as the working electrode, a platinum wire as the counter electrode, and a saturated calomel electrode as the reference electrode. Preparation of [Cu(phen)2]2þ and GR modified GCE Prior to the preparation of the modified electrode, the GCE was polished with 0.3 and 0.05 mm alumina paste, respectively, on a polishing cloth and then washed ultrasonically in double distilled water and acetone, respectively, for approximately 1 min. Then, the GCE was electrochemically cleaned in sulfuric acid solution to remove any remaining impurities. GR suspension was prepared by dispersing 10 mg in 100 ml of double distilled water. The modified electrode was prepared by dropping 8 ml of the above suspensions onto the GCE surface and leaving to dry naturally at room temperature to form graphene thin film. The obtained modified GCE is denoted as GR/GCE. The electropolymerization of [Cu(phen)2]2þ was performed by cycling potential scanning from 0.3e1.6 V for a certain cycle with a scan rate of 100 mV s1 in PBS with 0.5 mM [Cu(phen)2]2þ (pH 7.4). The obtained electrode is denoted as [Cu(phen)2]2þ/GR/GCE. Immobilization of probe DNA on [Cu(phen)2]2þ/GR/GCE The immobilization of the probe DNA was conducted by applying 5 ml of 2  106 M probe DNA on the [Cu(phen)2] 2þ/GR/ GCE surface and keeping at 4 for a period of time. Then, the electrode was rinsed with PBS to remove the unimmobilized probe DNA. The obtained electrode is denoted as P1-DNA/[Cu(phen)2]2þ/ GR/GCE.

1.0  103 and 1.0  105 Hz (signal amplitude ¼ 5.0 mV). Squarewave voltammetry (SWV) were carried out from 0.6 to 0.6 V in PBS. The other SWV parameters were as follows: step potential, 0.004 V; frequency, 50 Hz; amplitude, 10 mV. All of the electrochemical measurements were performed at room temperature. Results and discussion Electrochemical characteristics of modified electrodes EIS is a useful method for probing the surface characteristics of different modified electrodes. As shown in Fig. 2, the bare GCE exhibited a small semicircle in the high-frequency part with an almost straight line in low frequency, indicating that the electrochemical step on the bare GCE was a process of diffusion limiting. The diameter of the semicircular portion corresponds to the electron transfer resistance [39,40]. When GR was assembled onto the surface of the electrode (curve b), the diameter of the semicircle significantly decreased, indicating that GR could promote the electron transfer rate. After the electrodeposition of [Cu(phen)2]2þ on the GR/GCE (curve c) surface, the semicircle almost disappeared, suggesting the further increased conductivity ability caused by the introduction of copper complex. All of these experimental data showed that GR and [Cu(phen)2]2þ film had been successfully attached on the electrode surface and could enhance the charge transfer rate of electrode. Electrochemical behaviors of modified electrodes Compared with linear sweep voltammetry and cyclic voltammetry, SWV exhibited a much broader dynamic range and lower detection limit because of its efficient discrimination of capacitance current [41]. The electrochemical behaviors of different modified electrodes were further investigated by SWV. Fig. 3 displays the responses of [Cu(phen)2]2þ oxidation with different modified electrodes in PBS (pH 7.4) at the potential range from 0.6 to 0.6 V. The bare GCE (curve a) and GR/GCE (curve b) displayed a linear line without the participation of copper complex. In addition, the curve of GR/GCE was higher than that of the bare GCE, indicating that the enlarged surface area by GR improved the electron transfer rate. An oxidation peak appeared with the modification of [Cu(phen)2]2þ on the bare GCE (curve c), indicating that it was reasonable for

DNA hybridization on P1-DNA/[Cu(phen)2]2þ/GR/GCE DNA hybridization reaction was carried out by dropping a certain concentration of target T1-DNA sample solution onto the P1-DNA/[Cu(phen)2]2þ/GR/GCE and hybridizing at 4  C for a period of time. Then, the electrode was washed with PBS to remove the unhybridized DNA. This electrode modified with hybridized DNA is denoted as dsDNA/[Cu(phen)2]2þ/GR/GCE. Hybridization with the other mismatched DNA sequences was done using the same process. Electrochemical measurements Electrochemical impedance spectroscopy (EIS) experiments were performed in 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixture containing 0.1 mol L1 KCl solution with a frequency between

Fig.2. Nyquist figures of 5.0 mmol L1 Fe(CN)3e/4e containing 0.1 mol L1 KCl at bare GCE (a), GR/GCE (b), and [Cu(phen)2]2þ/GR/GCE (c).

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backbone, which could hinder the electron transfer. Therefore, the current showed a small decrease after the attaching of the probe DNA on the electrode surface (curve e). After hybridization with the target T1-DNA sequence (curve f), the formation of the double helix structure made more of the complex insert into the doublestranded DNA backbone and covering on the electrode surface, which hindered the electron transfer between copper ions and the surface of the electrode. Therefore, the electron transfer from phen was inhibited due to its intercalation with formed duplex structure, which caused the further decrease of the electrochemical signal.

Optimization of detection conditions

Fig.3. SWV of different modified electrodes in 0.02 mol L1 PBS (pH 7.4): (a) bare GCE; (b) GR/GCE; (c) [Cu(phen)2]2þ/GCE; (d) [Cu(phen)2]2þ/GR/GCE; (e) P1-DNA/ [Cu(phen)2]2þ/GR/GCE; (f) dsDNA/[Cu(phen)2]2þ/GR/GCE.

[Cu(phen)2]2þ to be regarded as the source of electrochemical signals. The highest oxidation peak current appeared after the electrodeposition of [Cu(phen)2]2þ on GR/GCE (curve d). This could be attributed to the excellent electrochemical performance of GR and the coefficient interaction between those two materials. The immobilization of the probe DNA was achieved by the intercalation between the exserted aromatic groups in [Cu(phen)2]2þ and DNA

To improve the electrochemical performance of [Cu(phen)2]2þ/ GR/GCE, the determination conditions, including the amount of GR, the film thickness of [Cu(phen)2]2þ, the immobilization time for probe DNA, and the hybridization time of target DNA, were explored. The amount of GR was controlled by dropping 8 ml of the GR suspension many times on the electrode surface. The relationship of the oxidation peak current and dropping numbers is shown in Fig. 4A. The oxidation peak current decreased gradually with the increase of dropping numbers from 1 to 4. Thus, dropping one time was chosen for the preparation of [Cu(phen)2]2þ/GR/GCE. The effect of the complex film thickness could be easily investigated by changing the number of scan cycles during the electrodeposition of [Cu(phen)2]2þ. In Fig. 4B, the oxidation peak current showed an increase with the number of scan cycles changed from 10 to 20. After 20 cycles, the peak current clearly decreased. This

Fig.4. Dependence of peak current of [Cu(phen)2]2þ in 0.02 mol L1 PBS containing 0.1 mol L1 KCl as supporting electrolyte at different modified GCE on amount of GR (A), film thickness of [Cu(phen)2]2þ (B), immobilization time of probe DNA (C), and hybrid time of target DNA (D).

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Fig.5. SWV response curves of the electrochemical DNA sensor at different target DNA concentrations. (A) Concentration range of target DNA from bottom to top: 106 to 1012 mol L1. (B) Plots of peak currents versus logarithm of target DNA concentration.

might be because the film was too thick to accelerate the electron transfer. So, 20 cycles was chosen in the electrodeposition of [Cu(phen)2]2þ. The immobilization time of probe DNA and the hybridization time of target DNA were also studied. As shown in Fig. 4C, the immobilization time of 30 min was chosen in order to ensure the maximization of the next DNA hybridization. In Fig. 4D, the peak current of [Cu(phen)2]2þ clearly decreased with increasing hybridization times from 10 to 40 min, indicating that the hybridization reaction was accomplished over 40 min. Thus, 40 min for hybridization was chosen in subsequent experiments.

Sensitivity of electrochemical DNA biosensor The sensitivity of the biosensor for the complementary target DNA (T1) was assessed by the SWV signals of P1-DNA/[Cu(phen)2] 2þ /GR/GCE to the T1-DNA in the concentration range of 1  106 to 1  1012 M. It can be seen in Fig. 5 that the peak current of [Cu(phen)2]2þ decreased with the increasing concentrations of T1DNA, indicating that smaller and smaller amounts of DNA helix structure were formed on the electrode surface. The peak current showed a good linear relationship fit to the logarithm of target DNA concentration with the regression equation of Ip (mA) ¼ 206.8e20.67 lg (c/1012 M), R2 ¼ 0.990, where c is the concentration of T1-DNA. The detection limit was obtained as 1.99  1013 M (signal/noise ¼ 3). For comparison, several results of similar modified electrodes of DNA hybridization determination [42e48] are summarized in Table 1. Clearly, our detection limit and linear range compared favorably with most of these biosensors. This could be mostly attributed to the introduction of GR to the

electrochemical DNA biosensor, which realized the destination of making more [Cu(phen)2]2þ and probe DNA immobilized onto the electrode surface and facilitated the electron transfer from [Cu(phen)2]2þ to the electrode without the involvement of complicated chemical procedures and expensive modified materials. Selectivity of electrochemical DNA biosensor To investigate the selectivity of [Cu(phen)2]2þ/GR/GCE, we employed P1-DNA/[Cu(phen)2]2þ/GR/GCE to hybridize with different sequences of complementary DNA from one-base mismatch to noncomplementary. Fig. 6 displays the electrochemical response of [Cu(phen)2]2þ at those electrodes. The developed electrochemical DNA biosensor displayed a decrease in peak current gradually after the hybridization with noncomplementary DNA sequence (curve b), three-base mismatch DNA sequence (curve c), and one-base mismatch DNA sequence (curve d). In addition, the minimum electrochemical signal appeared when hybridized with complementary target DNA, which could be attributed to the full formation of the double chain. All of these data illustrate that the electrochemical DNA biosensor had good selectivity among different sequences of oligonucleotides. Stability of electrochemical DNA biosensor The stability of the as-prepared biosensor was monitored by detecting P1-DNA/[Cu(phen)2]2þ/GR/GCE every day (stored at 4  C in the refrigerator). No obvious changes of electrical signals (relative standard deviation ¼ 5.14%) could be observed within 1 week,

Table 1 Comparison of similar electrochemical DNA biosensors. Modified electrode

Linear range (M)

Determination limit (M)

Determination method

Reference

PTCA/GR Carbon nanofiber/chitosan layer/GCE ssDNA-probe-wrappedeSWCNTs/GCE Au NRseGO/GCE CTSeCo3O4eGR/CILE PDDA/PDCeSWNTs/GCE AuNPs/PTCA/GR/GCE [Cu(phen)2]2þ/GR/GCE

1012e106 5  1010e4  108 1011e5  109 1014e109 1012e106 1011e106 1013e106 1012e106

5.0  1013 8.8  1011 1012 3.5  1015 4.3  1013 2.6  1012 3.4  1014 1.99  1013

EIS DPV SWV DPV DPV DPV EIS SWV

[42] [43] [44] [45] [46] [47] [48] This work

Note. PTCA, 3,4,9,10-perylene tetracarboxylic acid; GR, graphene; AuNPs, gold nanoparticles; GO, graphene oxide; PDDA, poly(diallyldimethyl ammonium chloride); PDC, 2,6pyridinedicarboxylic acid; CTS, chitosan; CILE, carbon ionic liquid electrode; EIS, electrochemical impedance spectroscopy; DPV, differential pulse voltammetry; SWV, squarewave voltammetry.

L. Yang et al. / Analytical Biochemistry 492 (2016) 56e62

Fig.6. SWV of [Cu(phen)2]2þ at P1-DNA/[Cu(phen)2]2þ/GR/GCE and after hybridization with 1  106 mol L1 (a), noncomplementary oligonucleotides (b), three-base mismatch oligonucleotides (c), one-base mismatch oligonucleotides (d), and complementary target DNA oligonucleotides (e).

demonstrating that the electrochemical DNA biosensor presents good stability. Conclusion A simple approach was introduced to fabricate an electrochemical DNA biosensor based on [Cu(phen)2]2þ and GR modified GCE. [Cu(phen)2]2þ acted advantageously both as the electrochemical indicator and as the anchor for probe DNA immobilization because of its reasonable electroactivity and the intercalative interaction with DNA backbone. In the meantime, GR was used to increase the surface area of electrode to support a high loading of [Cu(phen)2]2þ and the probe DNA and to accelerate the electron transfer rate considering its properties of good electrical conductivity and vast surface-to-bulk ratio. After the optimization of working conditions, the electrochemical DNA biosensor showed a correlation with T1-DNA in the concentration range from 1  106 to 1  1012 M, and the detection limit was obtained as 1.99  1013 M. The electrochemical DNA biosensor also proved to have good discrimination ability to effectively identify one-base mismatch, three-base mismatch, and noncomplementary oligonucleotides in hybridization events. Therefore, this work would be attractive with a new design concept for the genetic target analysis with high sensitivity and selectivity. Acknowledgments This research was supported by the National Natural Science Foundation of China (51273155) and Fundamental Research Funds for the Central Universities (2014-Ia-030). References [1] G. Yang, J.H. Chen, G.N. Chen, A label-free electrochemical biosensor for detection of HIV related gene based on interaction between DNA and protein, Sens. Actuat. B 184 (2013) 113e117. [2] S. Radhakrishnan, C. Sumathi, A. Umar, S.J. Kim, J. Wilson, Polypyrroleepoly(3,4-ethylenedioxythiophene)eAg (PPyePEDOTeAg) nanocomposite films for label-free electrochemical DNA sensing, Biosens. Bioelectron. 47 (2013) 133e140. [3] N. Manjunath, G. Yi, Y. Dang, Newer gene editing technologies toward HIV gene therapy, Viruses 5 (2013) 2748e2766.

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