Interaction between Room Temperature Ionic Liquid [bmim] BF4 and

Ionic liquids (ILs) as a kind of novel green solvent are being widely used in various researches related to the life sciences and chemistry, which dem...
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9864

J. Phys. Chem. B 2008, 112, 9864–9868

Interaction between Room Temperature Ionic Liquid [bmim]BF4 and DNA Investigated by Electrochemical Micromethod Ya-Ni Xie,† Sheng-Fu Wang,‡ Zhi-Ling Zhang,*,† and Dai-Wen Pang† College of Chemistry and Molecular Sciences and State Key Laboratory of Virology, Wuhan UniVersity, Wuhan 430072, P. R. China, and Faculty of Chemistry and Chemical Engineering, Hubei UniVersity, Wuhan 430062, P. R. China ReceiVed: April 26, 2008; ReVised Manuscript ReceiVed: May 21, 2008

Ionic liquids (ILs) as a kind of novel green solvent are being widely used in various researches related to the life sciences and chemistry, which demands the knowledge of interaction between ILs and biomacromolecules. However, the almost completely inert optical, electric, thermal properties of ILs make it difficult to directly obtain information about the interactions. Herein, by using a hydrophilic ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim]BF4) as a model, the electrostatic interaction between ILs and calf thymus DNA (ctDNA) was investigated by a surface electrochemical micromethod. A convenient and simple method was established to obtain the thermodynamic and kinetic information about the DNA-IL interaction only with microscale sample consumption. The quantitative thermodynamic and kinetic parameters about the interaction of [bmim]BF4 and ctDNA, such as the binding constant (K), the Gibbs energy of surface binding (∆Gb), and the dissociation rate constant (k), were obtained for the first time. Introduction Room-temperature ionic liquids (ILs), known as ambienttemperature molten salts, exhibit attractive properties including negligible vapor pressure, nonflammability, extraordinarily high chemical and thermal stability, good conductivity, and controllable hydrophobicity. A number of new applications of ILs in various fields have been reported in the past decades, including separation science,1,2 catalysis,3 electrochemistry,4 synthesis,5,6 and so on. Since Erbeldinger et al. first used anhydrous IL as an enzymatic reaction medium to improve the stability and activity of enzyme,7 ILs are being widely used in various research fields related to the life sciences. Ohno et al. successfully designed an IL-containing ion conductive DNA film, which opened a new field on the use of DNA as a biomass.8,9 An IL coated capillary was prepared for DNA separation and exhibited comparable long-term stability.10 Wang’s group used 1-butyl3-methylimidazolium hexafluorophosphate to quantitatively and fast extract trace amounts of DNA from aqueous phase.11 ILs have also played a role as ideal solvents or additives in enzymic catalytic reaction,7,13–17 immobilization18,19 and separation20 of enzyme, and electrochemistry21–24 of biomacromolecules. ILs show a great application potential in the separation of biomacromolecules, biosensing, and so on, which are based on the interaction between ILs and biomacromolecules. Therefore, to obtain knowledge about such interactions becomes more and more important and helpful. However, because ILs are almost completely inert species to produce optical, electric, thermal signals, it is difficult to acquire basic chemical and physical interacting information between ILs and biomacromolecules directly. Only relatively little data are reported on the interaction between ILs and DNA or protein. Li and his cooperator pointed that the DNA fragments could interact with a kind of dialky* To whom correspondence should be addressed. E-mail: zlzhang@ whu.edu.cn. Tel: +86 27 68756759. Fax: +86 27 68754067. † Wuhan University. ‡ Hubei University.

limidazolium-based ILs by electrostatic attraction, which was applied to design an IL coated capillary to separate DNA.10 With 31P NMR, FI-IR spectra, and resonance light scattering technique, Wang’s group verified the interactions between cationic 1-butyl-3-methylimidazolium and P-O bonds of phosphate groups in the DNA strands.11,12 However, those researches are rarely involved in thermodynamics and kinetics, and almost no quantitative thermodynamic and kinetic parameters about the interaction between biomacromolecules and ILs were obtained. The surface electrochemical micromethod based on DNAmodified electrodes is an effective research method to provide abundant thermodynamic and kinetic information. These electrodes are now of widespread application to study the interaction between biomacromolecules and other species, both electroactive and nonelectroactive.25–27 Because of the sensitive surface amplification effects, only DNA samples at the microscale level are needed. This method is simple, convenient, reliable, and reagent-saving and could possibly meet the needs for the difficulty in investigating the interaction beween biomacromolecules and ILs and provide valuable information about the thermodynamic and kinetic behavior. In this paper, the interaction between DNA and a nonelectroactive hydrophilic IL, [bmim]BF4, was investigated by the surface electrochemical micromethod by using Co(bpy)33+/2+ (bpy ) 2,2′-bipyripyl) as an electroactive indicator. Electrochemical studies suggested that [bmim]BF4 interacted with DNA electrostatically. The thermodynamic and kinetic parameters involved in the binding and dissociation process, such as the binding constant (K), the Gibbs energy of surface binding (∆Gb), and the dissociation rate constant (k), were obtained for the first time. This method was reagent-saving, and the microscale sample consumption could overcome the difficulties in the largescale preparation of biomacromolecules and ILs. It not only verified the electrostatic interacting mode of DNA and IL [bmim]BF4 but also provided various quantitative thermodynamic and kinetic parameters about this interaction. This knowledge will help us understand the thermodynamic and

10.1021/jp803655t CCC: $40.75  2008 American Chemical Society Published on Web 07/17/2008

Interaction between IL [bmim]BF4 and DNA

J. Phys. Chem. B, Vol. 112, No. 32, 2008 9865

kinetic behavior of the interaction between biomacromolecules and ILs and further benefit the development of the research field based on such interactions. The interaction between biomacromolecules and ILs could deeply affect the mechanisms and characters on the process applying ILs to fields such as biomolecule separation and electrochemistry, which showed an important fundamental significance in various research fields related to the life sciences and chemistry. Consequently, research in this field could provide prominent theoretic guidance and quickly promote the practical applications of ILs. Experimental Section Reagents. The IL 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim]BF4) was prepared as described in the literature, and the purity was checked by 1H NMR and FTIR spectroscopy.28 Tris-(2,2′-bipyripyl) cobalt perchlorate [Co(bpy)3(ClO4)3] was synthesized according to the literature.29 Double-stranded calf thymus DNA was obtained from Huamei Biotechnology Co. Ltd. and used without further purification. All other chemicals were of analytical grade. All solutions were prepared with ultrapure water (g18.0 MΩ cm) from a Water Pro Plus system (Labconco). Preparation of the DNA-Modified Gold Electrode. The working electrode was prepared as described previously:26 5 µL of 200 µg/mL ctDNA solution was pipetted onto the pretreated gold electrode surface and air-dried overnight at room temperature. It was then soaked in water for about 4 h to remove any unadsorbed ctDNA. The ctDNA-modified gold electrode obtained was denoted as ctDNA/Au. Apparatus. Cyclic voltammetry was performed on a CHI 617A electrochemical analyzer (CH Instruments, Shanghai, P. R. China). A conventional three-electrode system, including a ctDNA/Au as working electrode, a saturated calomel reference electrode (SCE), and a platinum counter electrode, was employed. The solutions were purged with high-purity nitrogen for 15 min before determination, and a nitrogen environment was then kept over the solution throughout all procedures at room temperature. All reported potentials were against a SCE independently of the liquid junction.

Figure 1. Cyclic voltammograms at a ctDNA/Au in PBS (10 mM, pH 7.0) (A) and [bmim]BF4 (B) containing (a) 0, (b) 10, (c) 15, (d) 20, (e) 25, (f) 30, (g) 40, (h) 50, (i) 60, and (j) 70 µM Co(bpy)33+/2+. The scan rate is 50 mV · s-1.

Results and Discussion Electrostatic Interaction between IL [bmim]BF4 and ctDNA. In 2003, Li’s group prepared an IL coated capillary and investigated for DNA separation.10 They indicated that the electrostatic DNA-IL interaction was the predominant factor for separation. Because the hydrophilic IL [bmim]BF4 was nonelectroactive over a wide potential range and Co(bpy)33+/2+ could interact electrostatically with ctDNA,30 Co(bpy)33+/2+ was used as an appropriate electroactive indicator to probe and characterize the interaction between [bmim]BF4 and ctDNA. Figure 1 shows electrochemical responses of Co(bpy)33+/2+ at a ctDNA/Au in 10 mM phosphate buffer solution (PBS, NaH2PO4/Na2HPO4, pH 7.0) and [bmim]BF4. A series of well defined reversible peaks were observed in cyclic voltammograms in PBS, and the peak currents increased along with concentrations of Co(bpy)33+/2+ because of the electrostatic interaction of Co(bpy)33+/2+ with ctDNA/Au (Figure 1A). However, in [bmim]BF4, no obvious redox peaks could be obtained under the same condition (Figure 1B). Also, there was no electrochemical response of Co(bpy)33+/2+ at a bare gold electrode (Figure SI-2 in the Supporting Information). The distinct difference of cyclic voltammetric (CV) responses illustrated that the existence of IL [bmim]BF4 could affect the electrostatic interaction between Co(bpy)33+/2+ and ctDNA prominently.

Figure 2. Cyclic voltammograms for 60 µM Co(bpy)33+/2+ at a ctDNA/ Au in PBS (10 mM, pH 7.0) containing different volume proportion (v/v) of [bmim]BF4: (a) 0%, (b) 0.3%, (c) 0.6%, (d) 0.9%, (e) 1.2%, and (f) 1.5%. The scan rate is 400 mV · s-1.

In PBS (10 mM, pH 7.0) containing 60 µM Co(bpy)33+/2+, a pair of obvious quasi reversible peaks for Co(bpy)33+/2+ with the formal potential 37.5mV was obtained at a ctDNA/Au. It was due to Co(bpy)33+/2+ binding predominantly by electrostatic means to negatively charged phosphates of the ctDNA backbone. When adding [bmim]BF4 into PBS, the charge currents increased rapid, whereas with the increase of content of [bmim]BF4 in PBS, the peak currents for Co(bpy)33+/2+ decreased, and the formal potential shifted back to positive gradually (Figure 2b-f). When the volume content of [bmim]BF4 in PBS (v/v) reached 1.5%, the formal potential for Co(bpy)33+/2+ shifted positively to 78.5 mV. Figure 3 shows

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Figure 3. E0′ of redox peaks for 60 µM Co(bpy)33+/2+ at a ctDNA/Au against the volume proportion (v/v) of [bmim]BF4 in PBS (10 mM, pH 7.0).

Figure 4. Cyclic voltammograms at a ctDNA/Au in PBS (10 mM, pH 7.0) containing (a) 60 µM Co(bpy)33+/2+, (b) 60 µM Co(bpy)33+/2+ and 1.5% [bmim]BF4 (v/v), and (c) 60 µM Co(bpy)33+/2+ after the ctDNA/Au in curve b washed with water. The scan rate is 400 mV · s-1.

the corresponding relationship of the formal potential for Co(bpy)33+/2+ with the volume content of [bmim]BF4 in PBS. The experiments revealed that both [bmim]BF4 and Co(bpy)33+/2+ interacted with ctDNA electrostatically. When adding [bmim]BF4 into the solution, Co(bpy)33+/2+ which had bound to ctDNA on the electrode surface was replaced gradually by [bmim]BF4; consequently, the surface concentration of Co(bpy)33+/2+ decreased, which resulted in the peak currents decreasing and the formal potential shifting positively. That is, there existed a competitive interaction between [bmim]BF4 and Co(bpy)33+/2+ with ctDNA, which showed us the possibility to study the interaction between [bmim]BF4 and ctDNA by this surface electrochemical micromethod. To research the dissociation of [bmim]BF4 bound with ctDNA/Au from the surface, a ctDNA/Au was immersed in PBS (10 mM, pH 7.0) containing 60 µM Co(bpy)33+/2+ and 1.5% [bmim]BF4 (v/v). The CV response is shown as curve b in Figure 4. Then, the electrode was rinsed with water and transferred to a solution containing 60 µM Co(bpy)33+/2+ (but no [bmim]BF4). The cyclic voltammogram showed that the peak currents for Co(bpy)33+/2+ increased dramatically, and the formal potential shifted negatively (Figure 4 curve c), which was similar to the CV response when the electrode was first immersed in PBS (10 mM, pH 7.0) containing 60 µM Co(bpy)33+/2+ only (Figure 4 curve a). The comparison of CV responses indicated that [bmim]BF4 bound to ctDNA on the electrode surface would dissociate partially from ctDNA when transferring back to PBS

Xie et al.

Figure 5. Plot of Γ-1 for Co(bpy)33+/2+ at a ctDNA/Au against the volume proportion (v/v) of [bmim]BF4 in PBS (10 mM, pH 7.0) containing (9)10, (0) 15, (2) 30, and (4) 45 µM Co(bpy)33+/2+.

containing only Co(bpy)33+/2+. Co(bpy)33+/2+ could reoccupy the sites and rebound to negatively charged phosphates of the ctDNA backbone, which were previously occupied by [bmim]BF4. It was clear that all of the binding/dissociation processes by both [bmim]BF4 and Co(bpy)33+/2+ were reversible; the interaction occurred competitively between [bmim]BF4 and Co(bpy)33+/2+ with ctDNA on the electrode surface. Thermodynamics of the Interaction between [bmim]BF4 and ctDNA. The fact that electrostatic interaction exists between [bmim]BF4 and ctDNA has been proved by CV responses above, which is consistent with Li and his co-worker’s research.10 However, no further work about the thermodynamic and kinetic behavior of such interaction is reported. The understanding of the thermodynamic and kinetic behavior would provide basic information about the DNA-IL interaction and would thus benefit the applications of ILs based on the interaction. Based on [bmim]BF4 and Co(bpy)33+/2+ competitive interaction with ctDNA, the surface electrochemical micromethod was used to investigate the thermodynamics and kinetics of DNA-IL interaction. According to the relationship of the fractional coverage (θ), the binding constant (K), the concentration (C), the surface coverage (Γ), and the saturation surface coverage (Γ∞), when a reversible, competitive interaction exists between two species A and B with ctDNA on ctDNA/Au and the interactions between bound molecules can be neglected, the following expression can be derived:27

ΓA-1 ) (KACAΓA∞)-1 + ΓA∞-1 + (KBKA-1CA-1ΓA∞-1)CB (1) That is, ΓA-1 should be directly proportional to CB, and the slope of the straight lines should be inversely proportional to the concentration (CA). The [bmim]BF4 and Co(bpy)33+/2+ competitive interaction with ctDNA was investigated by the above theory. According to Faraday’s laws.31 Γ ) Q/(nFA), where Q is the charge involved in the reaction, n is the number of electron transferred, F is Faraday’s constant, and A is the electrode area. The surface coverage of Co(bpy)33+/2+ can thus be obtained. Figure 5 shows plots of the reciprocal of surface coverage (Γ-1) for Co(bpy)33+/2+versus the volume content of [bmim]BF4 in PBS at various concentrations of Co(bpy)33+/2+ in solution. Clearly, under the condition of different concentrations of Co(bpy)33+/2+, the plots reveal the linear relationships which agrees with eq 1.

Interaction between IL [bmim]BF4 and DNA

Figure 6. Plot of slope of the straight line in Figure 5 against the corresponding concentration of Co(bpy)33+/2+.

It confirms that in the presence of both Co(bpy)33+/2+ and [bmim]BF4, the binding to ctDNA on a ctDNA/Au shows a reversible, competitive interaction. Figure 6 shows the inversely proportional relationship of the slope of the straight lines obtained in Figure 5 versus the concentration of Co(bpy)33+/2+. The higher the concentration of Co(bpy)33+/2+, the smaller the corresponding slope of the straight line. On the basis of eq 1, from further analysis of the slopes, a binding constant (K) of 4.26 × 104 mol-1 L for [bmim]BF4 bound to ctDNA can be obtained. According to the expression of ∆G b ) -RT ln K, the value of the Gibbs energy of surface binding (∆Gb) is determined to be -26.4 kJ mol-1 with a temperature of 298 K. Dissociation of [bmim]BF4 Bound to ctDNA/Au. Because there were competitive interaction between [bmim]BF4 and Co(bpy)33+/2+ with ctDNA, when adding [bmim]BF4 into solution, its interaction would influence the binding and dissociation of Co(bpy)33+/2+ bound at the competitive binding site. The dissociation of [bmim]BF4 from ctDNA on the electrode surface could be studied through comparing different dissociation of Co(bpy)33+/2+ from ctDNA with or without [bmim]BF4 in solution. A ctDNA/Au was immersed in PBS (10 mM, pH 7.0) containing 70 µM Co(bpy)33+/2+ or containing 70 µM Co(bpy)33+/2+ together with 1.5% [bmim]BF4 (v/v) for 15 min stirring until the CV responses were stable. The electrode was then rinsed with water and transferred to PBS (10 mM, pH 7.0). Then, the cyclic voltammograms recorded showed that the peak currents for Co(bpy)33+/2+ decreased gradually until they disappeared completely. It was due to the fact that Co(bpy)33+/2+ dissociated gradually from the ctDNA/Au electrode in the solution. Figure 7shows the time dependence of peak currents for Co(bpy)33+/2+ bound to ctDNA on the electrode surface. In the absence of [bmim]BF4, the dissociation of Co(bpy)33+/2+ from ctDNA was characteristic of first-order kinetics,27 and the plot of the logarithm of peak current (ln Ip) versus time (t) is linear. According to the slope, the dissociation rate constant of Co(bpy)33+/2+ from the ctDNA/Au could be calculated to be (4.8 ( 0.3) × 10-3 min-1, with a half-life time of 1.4 ( 0.03 min. On the other hand, the dissociation rate constant in the presence of 1.5% [bmim]BF4 (v/v) was determined to be (6.8 ( 0.3) × 10-3 min-1, with a halflife time of 1.0 ( 0.01 min (Figure 7 curve b). The difference between the two dissociation rate constants indicates that under the condition of containing [bmim]BF4 in solution, some effect exists to accelerate the dissociation of Co(bpy)33+/2+ from the ctDNA/Au, which is the result of competitive interactions between [bmim]BF4 and Co(bpy)33+/2+ with ctDNA on the electrode surface.

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Figure 7. Time dependence of peak currents for Co(bpy)33+/2+ bound to a ctDNA/Au in PBS (10 mM, pH 7.0) after the electrode was soaked in (a) 70 µM Co(bpy)33+/2+ solution for equilibrium and (b) 70 µM Co(bpy)33+/2+ and 1.5% [bmim]BF4 (v/v) solution for equilibrium.

Figure 8. Time dependence of ln ∆Γ for 70 µM Co(bpy)33+/2+ in PBS (10 mM, pH 7.0) at a ctDNA/Au bound with [bmim]BF4.

At any time t, when a ctDNA/Au onto which [bmim]BF4 was bound was transferred to a PBS (10 mM, pH 7.0) containing Co(bpy)33+/2+ only, an amount of Co(bpy)33+/2+ would bind to ctDNA on the electrode surface and occupy the sites made available by [bmim]BF4 upon dissociation. Then a derivate equation can be obtained

Γ[bmim]BF4-ctDNA(t) ) λ(Γ∞ - Γ(t)) ln Γ[bmim]BF4-ctDNA(t) ) ln(λ(Γ∞ - Γ(t))) )C + ln(Γ∞ - Γ(t)) )C + ln ∆Γ

(2)

where Γ[bmim]BF4-ctDNA(t) represents the surface coverage of [bmim]BF4 on the ctDNA/Au at time t, Γ∞ is the saturation surface coverage of Co(bpy)33+/2+ on the ctDNA/Au surface in the absence of [bmim]BF4 in solution, λ is a constant related to the binding site size of [bmim]BF4 to ctDNA, and C is a constant. As shown in Figure 8, the plot of ln ∆Γ versus t is linear and follows this equation:

ln ∆Γ ) -kt + A

(3)

Combining eqs 2 and 3 yields eq 4, which predicts that the plot of ln Γ[bmim]BF4-ctDNA versus t should be linear too.

ln Γ[bmim]BF4-ctDNA(t) ) C - kt + A ) -kt + constant (4) The linear relationship between ln Γ[bmim]BF4-ctDNA and t indicated that the dissociation of [bmim]BF4 from the surface obeyed first-order kinetics too. From Figure 8, the dissociation

9868 J. Phys. Chem. B, Vol. 112, No. 32, 2008 rate constant (k) was determined to be (9.6 ( 0.1) × 10-2 min-1 with a half-life time of 7.2 ( 0.07 min. Conclusions The interaction between a hydrophilic, nonelectroactive IL [bmim]BF4 and ctDNA was studied by a surface electrochemical micromethod, by using Co(bpy)33+/2+ as a redox indicator and diagnostic probe. The electrostatic interaction occurring between [bmim]BF4 and ctDNA immobilized onto the surface of gold electrode was proved. The thermodynamic and kinetic parameters of binding and dissociation such as the binding constant (K), the Gibbs energy of surface binding (∆Gb), and the dissociation rate constant (k) were obtained for the first time with microscale sample consumption only. Herein, a new method to investigate the interaction between DNA and almost nonelectroactive inert ILs was established. This method provides the possibility to accumulate data, consequently forming a basic thermodynamic and kinetic database on the biomacromolecule-IL interaction. These data are of great importance for understanding the thermodynamic and kinetic behavior of biomacromolecule-IL interaction and the applications of ILs based on the interaction, such as biomolecules separation and electrochemistry. Acknowledgment. This work was supported by the National Key Scientific Programs-Nanoscience and Nanotechnology (2006CB933100), the Science Fund for Creative Research Groups (20621502), the 863 Program (2006AA03Z320), the National Natural Science Foundation of China (30570490), and the Ministry of Education (306011 and IRT0543). Supporting Information Available: (1) 1H NMR spectra data of [bmim]BF4; (2) FTIR spectra of [bmim]BF4; (3) Cyclic voltammograms at a gold electrode in [bmim]BF4 containing different concentration of Co(bpy)33+/2+. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Shamsi, S. A.; Danielson, N. D. J. Sep. Sci. 2007, 30, 1729–1750. (2) Zhao, H.; Xia, S.; Ma, P. J. Chem. Technol. Biotechnol. 2005, 80, 1089–1096. (3) Welton, T. Coord. Chem. ReV. 2004, 248, 2459–2477.

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