Studies on the Interaction Mechanism of 1,10-Phenanthroline Cobalt(II) Complex with DNA and Preparation of Electrochemical DNA Biosensor

Fluorescence spectroscopy and ultraviolet (UV) spectroscopy techniques coupled with cyclic voltammetry (CV) were used to study the interaction between salmon sperm DNA and 1,10-Phenanthroline cobalt(II) complex, [Co(phen)2(Cl)(H2O)]Cl·H2O, where phen = 1,10-phenanthroline. The interaction between [Co(phen)2(Cl)(H2O)]+ and double-strand DNA (dsDNA) was identified to be intercalative mode. An electrochemical DNA biosensor was developed by covalent immobilization of probe single-strand DNA (ssDNA) related to human immunodeficiency virus (HIV) on the activated glassy carbon electrode (GCE). With [Co(phen)2(Cl)(H2O)]+ being the novel electrochemical hybridization indicator, the selectivity of ssDNA-modified electrode was investigated and selective detection of complementary ssDNA was achieved using differential pulse voltammetry (DPV).


Introduction
Nowadays, considerable attention has been paid on electrochemical DNA biosensor in the diagnosis of genetic diseases and the detection of pathogenic biological species due to the highly sensitive, rapid yet accurate, simple and inexpensive detection technique it offers [1][2][3][4][5]. Electrochemical DNA biosensor is generally based on an electrode with oligonucleotide immobilization. The sequencespecific hybridization of nucleic acids could be detected directly [6][7][8][9] or by DNA intercalator [10][11][12][13], and thus different forms of electrochemical DNA biosensors have been developed now. Among them, the later fashion, using an intercalator as redox label or indicator for the hybridization events, attracts great interest.
Owing to the potential of interacting with the nitrogenous bases of DNA, metal complexes, especial transition metal complexes, have received considerable attention in recent years as remarkable and unique DNA-binding compounds. Researches conducted also showed that transition metal complexes have important biological activities. It was reported that the metal coordination compounds of 1,10phenanthroline (phen) and its derivatives could inhibit tumor growth by interacting with DNA. In order to obtain more insight into the design of highly sensitive reactive probes, diagnostic reagents and new drugs, detailed understanding of the interaction between such complexes and DNA is potentially useful. Yang [14] reported that [Ni(phen) 2 dppz] 2+ and [Co(phen) 2 dppz] 3+ could bind strongly to the DNA and make DNA split in the light, which made instructive suggestions in the designing of new anticancer drugs. Although interaction between cobalt(III) complexes and DNA have been reported in many researches [15][16][17], interaction between 1,10-Phenanthroline cobalt(II) complex and DNA is seldom studied. To our best knowledge, research on the interaction between [Co(phen) 2 (Cl)(H 2 O)]Cl·H 2 O and DNA has not been reported.
In the present paper, fluorescence spectroscopy and ultraviolet (UV) spectroscopy techniques combined with cyclic voltammetry (CV) were used to study the interaction between [Co(phen) 2 (Cl)(H 2 O)]Cl·H 2 O and model DNA, salmon sperm DNA. Results showed that [Co(phen) 2 (Cl)(H 2 O)] + could bind to double-strand DNA (dsDNA) through intercalative binding mode. After probe single-strand DNA (ssDNA) related to human immunodeficiency virus (HIV) was covalently immobilized on the activated glassy carbon electrode (GCD), an electrochemical DNA biosensor using [Co(phen) 2 (Cl)(H 2 O)] + as the electrochemical hybridization indicator was developed and it demonstrated potential in selective detection of the complementary ssDNA. The work might bring further insight on the interaction mechanism between transition metal complexes and DNA and be helpful for further research for designing novel anti-tumor drugs and diagnosis disease.

Instrumentation
The electrochemical measurement was carried out with Model CHI 832B Voltammetric Analyzer (ChenHua Instruments, China). A three-electrode system was employed with Pt wire as the auxiliary electrode, Ag/AgCl/KCl (sat) as reference electrode, and GCE or modified GCE as working electrode. Spectroscopic experiments were conducted on Hitachi F-4500 fluorospectrometer (Hitachi, Japan) and cary 50 UV/Vis spectrometer (Varian, Australian).

Electrochemical study on the interaction between [Co(phen) 2 (Cl)(H 2 O)] + and DNA
The changes on characteristics of cyclic voltammograms (CVs) and the differential pulse voltammograms (DPVs) of the [Co(phen) 2 (Cl)(H 2 O)] + solutions in the absence and presence of salmon sperm DNA were investigated. For cyclic voltammetric scanning, the potential scanning ranged from -0.4 V to 0.6 V and the scanning rate was 0.10 V s -1 . The sample interval was set as 0.001 V and the quiet time was 2 s. For DPV measurment, the initial potential was 0.8 V. The high potential was 0.8 V and the low potential was -0.4 V. The pulse scope was set as 0.004 V and the pulse extent was 0.05 s. The pulse cycle was 0.2 s. The sample interval and the quiet time were 0.001 V and 2 s respectively.

Preparation of the electrochemical DNA sensor
The developed electrochemical DNA biosensor was based on the covalent immobilization of human immunodeficiency virus (HIV) probe single-strand DNA (S 1 ) on the activated electrode. Prior to experiment, the GCE was firstly polished using 1.0 µm, 0.3 µm and 0.05 µm α-Al 2 O 3 suspension respectively and then extensively rinsed in DDW with ultrasonic. Afterwards, the electrode was oxidized at +0.50 V for 1 min in 5.00 × 10 -2 mol L -1 PBS at pH 7.4 followed by thoroughly rinsed with DDW. As previously reported [19], four-step procedure was used for the preparation of the DNA biosensor. Firstly, the electrode was activated by dropping 20 µL 5.00 × 10 -2 mol L -1 PBS (pH 7.4) containing 5.0 × 10 -3 mol L -1 EDC and 8.0 × 10 -3 mol L -1 NHS on the electrode surface. After the solution was air-dried, the activated electrode was rinsed with DDW for several times. Secondly, ssDNA (S 1 ) was immobilized on the electrode surface by dropping ssDNA solution on the activated GCE. After dried under an infrared lamp, the electrode was rinsed with DDW to eliminate the DNA adsorbed. Thirdly, the S 1 -immobilized GCE was immersed in 20 mmol L -1 Tris-HCl buffer (pH 7.0) containing complementary S 2 or uncomplementary S 3 respectively to obtain S 1 -S 2 hybridized GCE and S 1 -S 3 hybridized GCE respectively. Such DNA interaction was allowed at 40 o C for 1 h with stirring. Afterwards, the obtained electrodes were thoroughly rinsed with the same Tris-HCl buffer and DDW successively to eliminate the adsorbed S 2 or S 3 and then dried at room temperature. Fourthly, [Co(phen) 2 (Cl)(H 2 O)] + was accumulated onto the surface by immersing the resulted electrode in the Tris-HCl buffer solution containing [Co(phen) 2 (Cl)(H 2 O)] + at room temperature for 5 min with stirring. After rinsed with the same Tris-HCl buffer and DDW successively, the DPV measurements were performed.

Fluorescence spectroscopic studies of the interaction between [Co(phen) 2 (Cl)(H 2 O)] + and DNA
Fluorescence spectroscopy was used to investigate the interaction between [Co(phen) 2 (Cl)(H 2 O)] + and salmon sperm DNA. Figure 1  To investigate the binding mode between [Co(phen) 2 (Cl)(H 2 O)] + and dsDNA, the fluorescence spectrum of EB-DNA system in the presence of [Co(phen) 2 (Cl)(H 2 O)] + was studied. As shown in Figure 2, EB itself emitted weak fluorescence emission (curve 1). After it intercalated into the double helix of DNA molecule, a significant increase in fluorescence intensity was observed (curve 2). However, distinct quenching of fluorescence occurred when [Co(phen) 2 (Cl)(H 2 O)] + was added (curve [3][4][5]. Moreover, the more the [Co(phen) 2 (Cl)(H 2 O)] + added, the lower the fluorescence emitted. Such fluorescence quenching might be due to the competition between [Co(phen) 2 (Cl)(H 2 O)] + and EB for the binding sites of dsDNA, which made the fluorescence intensity of EB-DNA system weaken [20]. Accordingly, the binding mode of [Co(phen) 2 (Cl)(H 2 O)] + to dsDNA might be similar with EB. So, the intercalative binding between [Co(phen) 2 (Cl)(H 2 O)] + and DNA was supposed.

UV spectroscopic studies of the interaction between [Co(phen) 2 (Cl)(H 2 O)] + and DNA
The hypochromic effect and the bathochromic effect were the identifying marks of the intercalation, as reported by Long et al [21]. To confirm our hypothesis, UV spectroscopy was used to further study the interaction between [Co(phen) 2

Electrochemical study on the interaction between [Co(phen) 2 (Cl)(H 2 O)] + and DNA
To explore the application of [Co(phen) 2 (Cl)(H 2 O)] + in electrochemical DNA biosensors, electrochemical study on [Co(phen) 2 (Cl)(H 2 O)] + and its interaction with DNA were performed. CVs of [Co(phen) 2 (Cl)(H 2 O)] + in the absence and presence of salmon sperm DNA were shown in Figure 4. We could see that there were a couple of quasi-reversible redox voltammetric peaks for [Co(phen) 2 (Cl)(H 2 O)] + (curve 1), indicating the electrochemical activity of [Co(phen) 2 (Cl)(H 2 O)] + . The cathodic peak potential (E pc ) and the anodic peak potential (E pa ) were 0.010 V and 0.077 V respectively. Curves 2-3 were the CVs of [Co(phen) 2 (Cl)(H 2 O)] + in the presence of different concentration of DNA. When the DNA concentration in the solution increased, the peak currents, both I pc and I pa , decreased slightly and small shift to positive potentials for both E pc and E pa occurred. The phenomena mentioned above were further studied by DPV and results were shown in Figure 5. Curve 1 was the differential pulse voltammogram of the [Co(phen) 2 (Cl)(H 2 O)] + solution, while curve 2-3 were the voltagramms when different amounts of DNA were added to the solution. As can be seen, the peak currents also decreased with increasing of DNA. Therefore, intercalative binding mode of [Co(phen) 2 (Cl)(H 2 O)] + to DNA drawn from electrochemical measurement was consistent with that obtained through spectroscopic studies [22].

The selectivity of the DNA electrochemical biosensor
Differential pulse voltammetry was used to study the selectivity of the prepared electrochemical DNA biosensor. Figure 7 showed the representative differential pulse voltammograms obtained in PBS (pH 6.0) with the S 1 -immobilied GCE (curve 1), S 1 -S 2 hybridized GCE (curve 2) and S 1 -S 3 hybridized GCE (curve 3) being the working electrode respectively. S 1 -S 2 hybridized GCE represented that S 1 -immobilied GCE hybridized with the complimentary DNA segment S 2 and S 1 -S 3 hybridized GCE referred to the electrode obtained after S 1 -immobilied GCE hybridized with uncomplimentary DNA segment S 3 . Before DPV measurement, [Co(phen) 2 (Cl)(H 2 O)] + accumulation step for the three electrodes were all performed. No voltammetric response for the S 1 -immobilied GCE was found in curve 1. However, the reduction peak of the S 1 -S 2 hybridized GCE appeared at about -0.144 V, as shown in curve 2, which was the same potential as the bare GCE in the [Co(phen) 2 (Cl)(H 2 O)] + solution. The peak current was 1.378 × 10 -7 A. The phenomenon indicated the intercalation of the indicator, [Co(phen) 2 (Cl)(H 2 O)] + , into the base pairs of the dsDNA resulted from S 1 -S 2 hybridization. In the case of S 1 -S 3 hybridized GCE, no dsDNA was formed because S 3 was uncomplementary to S 1 and no indicator could intercalate. As expected, S 1 -S 3 hybridized GCE displayed no voltammetric response, as shown in curve 3, which indicated the favorable selectivity of the ssDNA modified GCE. In summary, the interaction between [Co(phen) 2 (Cl)(H 2 O)] + and the probing DNA, salmon sperm DNA, was studied by fluorescence spectroscopy, ultraviolet spectroscopy and voltammetry.
[Co(phen) 2 (Cl)(H 2 O)] + can bind to dsDNA by intercalative binding mode. By using [Co(phen) 2 (Cl)(H 2 O)] + as the electrochemical hybridization indicator, the electrochemical DNA biosensor was prepared base on the covalent immobilization of ssDNA and it showed high selectivity in detection of complementary ssDNA.