Electrochemical Detection and Control of Interactions between DNA

Division of Environmental Material Science, Graduate School of Environmental Science, Hokkaido University, Sapporo, ... Publication Date (Web): May 23...
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Environ. Sci. Technol. 2006, 40, 4240-4244

Electrochemical Detection and Control of Interactions between DNA and Electroactive Intercalator Using a DNA-Alginate Complex Film Modified Electrode YUKO MATSUMOTO, NORIFUMI TERUI, AND SHUNITZ TANAKA* Division of Environmental Material Science, Graduate School of Environmental Science, Hokkaido University, Sapporo, Hokkaido, 060-0810, Japan

The surface of a glassy carbon electrode was modified with a DNA-alginate complex film in which water-soluble DNA was encapsulated into a calcium-alginate gel. The resulting modified electrode (DAFE) was used to detect ethidium bromide (EtBr), after its accumulation on the electrode. The intercalative behavior of EtBr for dsDNA in the film was investigated by measuring the electrode response based on the intercalation of EtBr separated from nonspecific interactions (electrostatic interaction). The accumulation of EtBr in the dsDAF was enhanced by applying a negative potential below -200 mV at the dsDAFE. When a positive potential above +700 mV was applied to the dsDAFE for a constant time with stirring in a Tris buffer solution, the amount accumulated decreased. These results indicate that it is possible to electrochemically control the accumulation and release of EtBr when a dsDAFE is used. In addition, the accumulation and detection of EtBr in spiked river water samples and daunomycin, an antitumor agent, is described.

Introduction DNA is an extremely important material in living organisms, and investigations of DNA are rapidly proceeding as the center of the fields of genetic engineering and molecular biology. DNA is an ultrahigh molecular weight material and is present in large quantities in the natural world. It is also considered to be a functional material that can specifically or selectively interact with various chemical substances. It is known that the binding of certain compounds with DNA control cellular functions such as DNA replication and gene expression. The structure of DNA can be perturbed by interaction with some types of drugs, carcinogens, mutagens, and dyes, and such intercalations can have toxic or therapeutic effects. The binding of compounds with DNA can be classified into three major types of binding modes: intercalation, groove binding, and covalent cross-linking (1). Among these types of interactions, it is well-known that the structure of planar substances containing several aromatic rings can be often intercalated into the stacking of base pairs in DNA (2). Included among such harmful chemicals are many planar organic molecules. Consequently, DNA would * Corresponding author phone and fax: e-mail: [email protected]. 4240

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be expected to function as a removal agent for such harmful chemicals. DNA is abundantly present in the milt of salmon or scallops, and most of these molecules are wasted. Therefore, utilizing DNA such as the milt of a salmon, which is discarded as an industrial waste, could be used to protect our environment against pollution from organic substances. However, DNA is water-soluble and has a low mechanical strength. Hence, the utilization of DNA as a removal agent has been limited. Thus, the means for converting watersoluble DNA into an insoluble form would be of interest. Up to this time, DNA-alginate complexes (3), DNA-lipid complexes (4), DNA immobilized by ultraviolet of irradiation (5), and so on have been developed, and their use as functional materials has been examined. The DNA-alginate complex can be included in this class, as the nature and structure of DNA is negligibly affected by immobilization as an alginate complex, that is, the intercalation of carcinogens as EtBr and environmental hormones as benzo[a]pyrene into DNA encapsulated in alginate gel was observed in a similar manner as free DNA by spectrum analyses for the complexes (6). Alginic acid, which makes up the skeleton of the complex, is a nontoxic polysaccharide, consisting of mannuronic acid and guluronic acid units, and shows gelling properties in the presence of divalent cations (7). For example, calciumalginate gel particles produced by the bridge between alginate and Ca ions can be mainly prepared by two methods (i.e., dripping and emulsification (8)). It is also reported that the pore size of the net made with the bridge between alginate and Ca ions of this particle (9) determines the encapsulation or release of macromolecules such as proteins (10, 11) and that small molecules have high a diffusion coefficient in calcium-alginate gel particles (12). Tanaka et al. reported that the release of proteins larger than albumin from calcium-alginate particle gels was hindered by the alginate net (11). Therefore, DNA, an ultra-polymer material, can also be successfully immobilized in calcium-alginate gel particles (6). However, little information is currently available regarding calcium-alginate gel particles including DNA, and no study using electrochemical analysis has yet been reported. In this study, a glassy carbon electrode was modified with a DNA-alginate complex film. The interaction (especially, intercalation) between a planar organic molecule and DNA in the film was electrochemically confirmed by observing cyclic voltammograms (CV) of the electroactive compound that had been intercalated into the film. In this experiment, ethidium bromide (EtBr) was employed as an electroactive intercalator and a model substance to examine the intercalative behavior of DNA in the DAFE. EtBr is a planar aromatic dye that readily intercalates between base pairs of DNA and is frequently used to detect DNA (13). Thus, an electrochemical investigation of the interaction between EtBr and DNA-alginate film on the electrode was carried out. In addition, an attempt was made to electrochemically control the accumulation and release of EtBr in the film. The increase in the rate of accumulation of EtBr into DNA was observed by applying a negative potential. The possibility of film regeneration was also investigated by applying a positive potential to release EtBr from the film instead of adding an organic solvent or highly basic polycations such as a protamine. The DAFE was also applied to the accumulation and detection of EtBr spiked to river water samples and to daunomycin, an antitimor agent.

Experimental Procedures Apparatus. All voltammetric measurements and the application of a constant potential were carried out using a 10.1021/es060084x CCC: $33.50

 2006 American Chemical Society Published on Web 05/23/2006

CV-50W Voltammetric Analyzer (Bioanalytical Systems Inc. (BAS)). A three-electrode system was used in all electrochemical experiments. A glassy carbon electrode with a diameter of 3 mm (Model No. 11-2012, BAS) was used as the working electrode. Prior to use, the electrode was immersed in 0.1 M nitric acid for 5 min and polished sequentially with a 0.3 and 0.05 µm alumina slurry. After cleaning ultrasonically for 1 min, a potential of +1200 mV was applied to the electrode in a stirred, aqueous 0.5 M NaOH solution for 10 min to eliminate the irreversible adsorption of EtBr to the electrode (14). A platinum wire was used as a counter electrode, and a Ag/AgCl electrode (Model No. 11-2020, BAS) was used as a reference electrode. The measurement of pH was performed using a pH meter (M-13, HORIBA, Ltd., Kyoto, Japan). Reagents. Double-stranded (ds) DNA derived from salmon milt (P-1051, MW 5 000 000-) was purchased from Yuki Fine Chemical Co., Ltd. (Tokyo, Japan). EtBr with a purity of 95% was obtained from Acros Organics (A016477601). Sodium alginate (300-400cP), calcium chloride, dihydrate, potassium hexacyanoferrate (II) trihydrate, hexaammineruthenium (III) chloride, and tris(hydroxylmethyl)-aminomethane were purchased from Wako Pure Chemical Co. (Osaka, Japan). Daunomycin hydrochloride was obtained from Sigma Chemical Co. (St. Louis, MO). Tris buffer solution at pH 7.4 for washing or making up the sample was prepared with 0.1 M Tris and 0.1 M HCl. The sample solution was deoxygenated by deaeration with highquality nitrogen gas. All chemicals were prepared from analytical grade reagents, and all solutions were prepared using deionized water purified with a Millipore Milli-Q system. Preparation of DNA-Alginate Complex Film Modified Electrode (DAFE). The DNA-alginate complex film modified electrode (DAFE) as a working electrode was prepared as follows: first, a 5 mg/mL alginate solution including 0.5 mg/ mL DNA was prepared. Then, 4 µL of the DNA-alginate mixed solution was dropped on the surface of a pretreated glassy carbon electrode followed by drying in an oven at 30 °C. The electrode was covered with a piece of the dialysis membrane (MWCO: 10 000, Spectrum Labs. Inc.) by a rubber ring. When the membrane/DNA-alginate mixture/electrode was immersed in a 1% calcium chloride solution for more than 2 h, a DNA-alginate complex gel film was formed between the membrane and the electrode. Finally, by immersing the modified electrode in deionized water to remove excess calcium chloride from the resulting film, a DAFE was successfully prepared. In this paper, dsDAFE is defined as an electrode modified with a film including double-stranded (ds) DNA, while ssDAFE indicates the one modified with film including single-stranded (ss) DNA. ssDNA was prepared by the repetitive boiling of a solution of DNA and alginate for 10 min and then rapidly cooling the solution to 4 °C. Electrochemical Measurement of EtBr in DNA-Alginate Complex Film (DAF). The experimental procedure for the electrochemical measurement of EtBr taken into the DAF is illustrated in Figure 1. The DAFE was first immersed in a Tris buffer solution containing EtBr with stirring for a constant time (step 1). The DAFE was next washed by soaking in a Tris buffer solution not including EtBr with stirring for 10 min (step 2). CV measurements of EtBr strongly attached to the DAF were then carried out in another Tris buffer solution (step 3). For the estimation of the total interaction, that is, both strong (intercalative) and weak (electrostatic and so on) interactions between the DAF and the EtBr, the CV measurements were carried out in a Tris buffer solution containing EtBr without the washing process after step 1. The effect of electrochemical accumulation was studied by measuring the current response of EtBr obtained in step 3 via step 2 after applying a negative potential in the adsorption process

FIGURE 1. Experimental procedure for the electrochemical measurement of EtBr in DAF.

FIGURE 2. Cyclic voltammograms of (A) 0.1 mM Ru(NH3)63+ and (B) 0.1 mM Fe(CN)64- in Tris buffer solution at DAFE (a), AFE (b), and GCE (c). The scan rate is 100 mV s-1. of step 1. The determination of the electrochemical release of EtBr from DAF (especially dsDNA) was performed by immersing the DAFE in a Tris buffer solution and applying a positive potential for a constant time with stirring after step 2, and the effect was confirmed in step 3.

Results and Discussion Electrochemical Characterization of Modified Electrode. The electrochemical characterization of the modified electrode involved the use of Ru(NH3)63+ and Fe(CN)64- as representative cation and anion markers for the electrode reaction. The electrode response was compared with a bare glassy carbon electrode (GCE), an alginate film modified electrode (AFE), and a DNA-alginate complex film modified electrode (DAFE). The incorporation of Ru(NH3)63+ into AFE and DAFE was demonstrated on the basis of measurements of cyclic voltammograms as shown in Figure 2A. These CVs were measured after immersing AFE or DAFE in 0.1 mM Ru(NH3)63+ in 0.1 M Tris buffer solution for 30 min. The peak currents of Ru(NH3)63+ at both AFE and DAFE were clearly enhanced as compared with that at a bare GCE, and the incorporating ability of DAFE for Ru(NH3)63+ was slightly higher than that for AFE. The increase in the peak current can be attributed to the anionic phosphate backbone of the DNA. On the other hand, when CVs were measured after immersing AFE and DAFE in 0.1 mM Fe(CN)64- in 0.1 M Tris VOL. 40, NO. 13, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Cyclic voltammograms of 0.1 mM EtBr in Tris buffer solution without (A) and with (B) a washing process. (a) ssDAFE, (b) dsDAFE, (c) AFE, and (d) GCE. The scan rate was 100 mV s-1. buffer solution for 30 min, the peak current for Fe(CN)64- at both the AFE and the DAFE was smaller than that at GCE, as shown in Figure 2B. The decrease in peak current can be attributed to the electrostatic repulsion between negative charges on the surface of the modified electrode and Fe(CN)64-. It was concluded that the DAFE has negative charges arising from both the phosphate groups of DNA and the carboxyl groups of alginic acid, and therefore, the DAFE can attract cationic substances, but anionic substances will not interact with the electrode. Interaction of EtBr with DAFE or AFE. The interaction of planar aromatic compounds with DNA in the film was electrochemically investigated using electroactive EtBr. Figure 3A shows the CVs for EtBr measured at four different electrodes (ssDAFE, dsDAFE, AFE, and GCE). The measurement was carried out after immersing these electrodes in 0.1 mM EtBr in a Tris buffer solution with stirring for 30 min. The CV measurements for EtBr at ssDAFE, dsDAFE, and AFE gave 3.4, 2.5, and 1.5 times larger responses, respectively, than that of GCE. One of the reasons for the increase in these peak currents against GCE is an accumulative effect based on an electrostatic interaction between the anionic portions of AF and phosphate groups of DNA and cationic Et+. In addition, ssDAF and dsDAF have a specific affinity that Et+ can interact face-to-face with the relatively electron-rich nucleobases since the phenanthridinium ring of Et+ is electron-poor. Moreover, Et+ can be intercalated into the double helix of dsDNA. Using methylene blue (MB) as an intercalator, Kara et al. reported that the decrease in the accessibility of guanine bases, for binding to MB, in the double helix of dsDNA led to a decrease in the voltammetric reduction signals of MB (15). Thus, the decrease in the voltammetric response of EtBr observed for the dsDAFE as compared with that for the ssDAFE in this study would be analogous to the effect reported by Kara et al. Figure 3B shows CVs measured using the four types of electrodes after washing them by immersion in 0.1 mM EtBr in a Tris buffer solution with stirring for 30 min. The voltammetric response of EtBr at ssDAFE, AFE, and GCE was reduced abruptly after this washing process. However, the voltammetric response of EtBr at the dsDAFE was still 4242

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FIGURE 4. (A) Cyclic voltammogram of 10 µM EtBr in Tris buffer solution at the dsDAFE (scan rate ) 10, 50, 100, 200, 300, and 500 mV s-1). (B) The plots of the oxidation peak current of EtBr at dsDAFE with the square root of the potential sweep rate and with the potential sweep rate. maintained after the washing process. Since EtBr interacts weakly with ssDAFE and AFE, it would be removed from the film during the washing process. EtBr, which strongly interacts with dsDNA by intercalation, is not removed from the film (dsDAF) by this brief washing. The cyclic voltammograms of 10 µM EtBr at dsDAFE are shown in Figure 4A. The peak currents of EtBr measured after the washing process were enhanced with an increase in the potential sweep rate. The peak currents attributed to the oxidation of EtBr are linearly dependent on the potential sweep rate but not the square root of the potential sweep rate in the range of 10-500 mV/s (Figure 4B). This indicates that the electrode process of EtBr at the dsDAFE is regulated by the EtBr retained at the electrode surface by intercalation with the dsDAFE but not the result of diffusion from the solution (16). This confirms that EtBr strongly interacts with the dsDAF and responds electrochemically well at the dsDAFE. Taking these results into account, it can be concluded that the electrode response of EtBr obtained in this study is based on a strong interaction, mainly intercalation, of EtBr into the base pairs of the dsDNA. Accumulation Time and Applied Potential for the Accumulation of EtBr. The intercalative behavior of EtBr at dsDAFE was investigated from measurements of the peak current of EtBr as a function of accumulation time and applied potential. Figure 5 shows the relationship between the peak current measured using 100, 10, and 5 µM EtBr at dsDAFE and the accumulation time. When the measurements were carried out using 100 and 10 µM EtBr, the times required to reach the saturated peak current were about 15 and 120 min, respectively. The lower the concentration of EtBr, the more time required for the saturated response. This is because a long period of time is needed to fill all binding sites in DNA with EtBr at low concentrations. The relationship between the accumulation time of EtBr and the applied accumulation potentials was also investigated

FIGURE 5. Comparison between natural potential and applied potential at -200 mV on the oxidation peak current of EtBr as a function of accumulation time. White squares, circles, and triangles indicate 100, 10, and 5 µM EtBr at a natural potential, respectively. Black circles and triangles indicate 10 and 5 µM EtBr at -200 mV, respectively.

FIGURE 7. (A) Cyclic voltammograms of 0.1 mM EtBr in Tris buffer at the dsDAFE after intercalative accumulation (solid line) and after applying a potential of +1000 mV (bold line). The scan rate was 100 mV s-1. (B) The response of oxidation peak current in Tris buffer for five repetitive experiments for accumulation and release of EtBr.

FIGURE 6. Histogram of the peak current obtained by cyclic voltammetry measured in Tris buffer at dsDAFE after applying a positive potential (500, 600, 700, 800, 900, and 1000 mV) in Tris buffer solution with stirring for 10 min via steps 1 and 2. by measuring the peak currents of EtBr accumulated at the dsDAFE via steps 2 and 3 after applying a negative potential in the accumulation process of step 1. An increase in the rate of accumulation of EtBr was observed by applying a negative potential of -200 mV as shown by the black circles and triangles in Figure 5. The same result was obtained by applying a potential at -400 mV, although the data are not superposed on the graph. These results indicate that applying a negative potential increases the rate of accumulation of EtBr at dsDAFE. Electrochemical Release of EtBr from dsDAF. If the EtBr that accumulates in DAF can be removed, the DAF could be regenerated and recycled as a removal agent. Hence, the desorption of EtBr from DAF is an important issue. In general, once EtBr intercalates in dsDNA, an organic solvent such as n-butanol or isoamyl alcohol and the addition of poly-cationic substances such as protamine or polylysine are needed to release EtBr from dsDNA. In fact, washing the dsDAFE with Tris buffer alone was not effective for removing the EtBr from the dsDNA. Therefore, the effect of applied potential to release the EtBr from the dsDNA in dsDAF was examined. This was examined by immersing the dsDAFE in a Tris buffer solution and applying a removal potential with stirring for a constant time after steps 1 and 2. The relationship between the applied removal potential and the peak current of EtBr in the dsDAF is shown in Figure 6. By applying a more positive potential than +700 mV in the Tris buffer solution for a constant time, the peak current of EtBr in the dsDAF

decreased substantially, and the magnitude of the decrease was dependent on the applied removal potential. In the case of applying a potential of +1000 mV, 10 min was required to eliminate the electrode response of EtBr completely, while, in the case of applying a potential of +700 mV for 10 min, the electrode response of EtBr in the dsDAF was 40% of the original peak current. Gherghi et al. in their study of interactions between EtBr and DNA using carbon paste electrode reported that the peak current of EtBr intercalated in DNA was reduced by applying higher potentials (17). Thus, it can be concluded that EtBr intercalated in dsDAF was released from the electrode into the buffer solution by applying a more positive potential than +700 mV. Figure 7A shows CVs after the intercalative accumulation of EtBr, followed by the application of a positive potential of +1000 mV for 10 min. Furthermore, the peak current of EtBr obtained using the recycled electrochemically treated dsDAFE corresponded with that measured with a newly prepared dsDAFE, as shown in Figure 7B. The possibility of the repeated utilization of the dsDAF was confirmed by five repetitive experiments of accumulation and release of EtBr on the dsDAF. Therefore, it can be concluded that the electrochemical release of EtBr from DAFE is possible and that the DAF on the electrode as a removal agent for harmful chemical substances can be regenerated. Application to Environmental Waters Spiked with EtBr. To evaluate the possibility of using the dsDAFE in applications to environmental samples, the accumulation and determination of EtBr in river waters spiked with 10 µM EtBr were attempted using the dsDAFE. River water samples were collected from the Toyohira, Sosei, and Shin Rivers in Sapporo City (Hokkaido, Japan) in November 2005, and the samples were spiked with 10 µM EtBr. The recoveries of EtBr obtained from measurements of the Toyohira, Sosei, and Shin River water samples of 97.6, 98.2, and 102%, respectively, were in good agreement with the electrode response for 10 µM EtBr in a 0.1 M Tris buffer solution. In addition, the accumulation VOL. 40, NO. 13, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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protamine. These results suggest the possibility of the repeated utilization of a DNA-alginate complex film on the electrode as a removal agent for harmful chemical substances if a dsDAFE having a sufficiently large surface area could be prepared.

Acknowledgments This work was supported, in part, by the 21st Century Center of Excellence Program (E-01) funded by the Ministry of Education (Culture, Sports, Science and Technology).

Literature Cited FIGURE 8. Cyclic voltammograms of 10 µM DM in Tris buffer solution with a washing process. (a) dsDAFE, (b) AFE, and (c) GCE. The scan rate was 100 mV s-1. and release of EtBr based on negative and positive electrochemical control were also applicable to the environmental water samples. These results indicate the possibility that the proposed method using dsDAFE can be applied to the accumulation and determination of dyes such as EtBr in actual environmental water samples. Electrochemical Behavior of Daunomycin at dsDAFE. Daunomycin (DM) is a clinically useful anthracycline antibiotic that controls the multiplication of neoplasm cells. The antitumor activity has been attributed to the intercalation of its planar aromatic ring between DNA base pairs. It is known that the DM core lies between the base pairs, the aminosugar that is attached to the ring lies in the minor groove, and another ring protrudes into the major groove (18). Since DM is utilized in the treatment of leukemia, it is often discharged into the urine of patients. Therefore, daunomycin (DM) as a real environmental material was selected, and the electrochemical behavior of DM at the dsDAFE was investigated to evaluate the validity of DAFE. Figure 8 shows CVs measured for the three types of electrodes (dsDAFE, AFE, and GCE) after washing these electrodes by immersion in 10 µM DM in Tris buffer solution with stirring for 60 min. Although the voltammetric responses of DM at the AFE and GCE were very small, that of DM at the dsDAFE was largely maintained even after the washing process. This indicates that DM can also be accumulated by the DAFE by intercalating into the DNA in the film and therefore can be detected. DAFE can be applied to not only dye compounds such as EtBr but also electroactive antitumor agents such as DM in the environmental field. In this study, a DNA-alginate complex film (DAF) was modified on the surface of a GCE by using a dialysis membrane. By using the DAF electrode, the electrode responses of electroactive EtBr and DM after their interaction with DAF were observed. The accumulative behaviors of EtBr and DM with dsDNA in the film were also confirmed by measuring the electrode response based on the intercalation of EtBr after washing. A more negative potential than -200 mV at the DAFE enhanced the accumulation of EtBr. This electrochemically enhanced effect of EtBr may be due to the electrostatic attraction between positive charge of EtBr and negative charges of the dsDAFE. Furthermore, by immersing the dsDAFE in a Tris buffer solution and applying a positive potential above +700 mV to the DAFE after the accumulation of EtBr for a constant time with stirring, EtBr that had intercalated in the dsDAF was electrochemically released from the electrode into the buffer solution without the need for an organic solvent or a high basic poly-cation such as a

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Received for review January 16, 2006. Revised manuscript received April 24, 2006. Accepted April 25, 2006. ES060084X