Electrochemical properties of niosomes modified Au electrode and DNA recognition

Colloids and Surfaces B: Biointerfaces 67 (2008) 179–182

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Electrochemical properties of niosomes modified Au electrode and DNA recognition Yang Chang-Ying ∗ , Yin Chong, Zhou Yi-Yong, Dai Zhong-Xu Hubei Key Laboratory of Natural Products Research and Development, Three Gorges University, 8 University Avenue, Yichang 443002, PR China

a r t i c l e

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Article history: Received 11 March 2008 Received in revised form 7 June 2008 Accepted 11 August 2008 Available online 19 August 2008 Keywords: Niosomes Herring sperm DNA Au electrode Electron transfer resistance (Ret )

a b s t r a c t Non-ionic surfactant vesicles (NSVs), also referred to as niosomes, have been studied as an alternative to conventional liposomes. In this paper, electrochemical inspection of the interaction between Herring sperm DNA and niosomes has been investigated after a simple and novel method for the formation of niosomes on Au electrode. Each step of electrode modification has been confirmed with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The assembly of octadecanethiol (ODT) layer on the electrode surface generates a packed film that introduces a barrier to the interfacial electron transfer (Ret ), and the subsequent immobilization of niosomes onto the self-assembled monolayer (SAM) layer results in a further increase of Ret , due to the formed bilayer almost blocked the redox probe to the electrode surface. When Herring sperm DNA was added, the Ret value decreased, indicating that the barrier of the redox probe to the surface was disrupted. The addition of DNA caused the formation of some transmembrane channels for the redox probe across the niosomes. A good linear relationship between Ret value and DNA concentration was found over the 0–0.05 mg mL−1 concentration range. © 2008 Elsevier B.V. All rights reserved.

1. Introduction The interactions in mixed solutions of DNA with multilamellar vesicles have attracted a great interest from the biomedical sciences, not only because of its direct biological implications, but also for a number of applications concerning separation, purification and transfection of DNA. Mixed systems with surfactants/lipids can be used as packaging agents for delivery of nucleic acids to cells. These are interested in understanding interactions and studying and controlling microstructures formed, both in bulk and at interfaces [1–4]. In contrast to naked DNA, DNA-entrapped within multilamellar vesicles is protected from nuclease attack in biological milieum. Liposome-entrapped DNA [5] has been shown to promote greater humoural and cell-mediated immune responses against the encoded antigen in immunised mice [6] than naked DNA or DNA complexed to identical preformed liposomes. Similar observations have been made with plasmid DNA-entrapped within non-ionic surfactant based vesicles prepared by the same method [7–9]. In recent years, non-ionic surfactant vesicles (NSVs), also referred to as niosomes, have been studied as an alternative to conventional liposomes. In fact, if compared to phospholipids vesi-

cles, niosomes formed with synthesized polymers are thought to have the potential for repeated administration, lower costs, and large-scale production. They offer higher chemical stability, and great availability of surfactant classes [10–12]. They are widely used as the modeling cell membranes, but also as microreactors and as targeted drug carriers to decrease the drug toxicity [13–17]. So, properties and applications of the niosomes are one of the most noticeable researches in the amphiphilic molecular organized assemblies. In this work, we have developed a simple and novel method for the formation of niosomes, composed of several non-ionic surfactants, on Au to prepare modified Au electrode. The interfacial properties of the electrode, e.g. electron transfer resistance were investigated in the presence of a redox probe by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). With the fabrication of an electrochemical sensor based on the niosome modified Au electrode, the interaction between Herring sperm DNA and niosomes was demonstrated, and some preliminary results were presented here. 2. Experimental 2.1. Materials

∗ Corresponding author. Tel.: +86 717 6395676; fax: +86 717 6395580. E-mail address: [email protected] (Y. Chang-Ying). 0927-7765/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2008.08.005

Tween 80, Span 80 from Shanghai Commonage Pharmacy Co. (99%), PEG 6000 from Sinopharm Chemical Reagent Co. Ltd. (99%).

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Herring sperm DNA was obtained from Sigma and octadecanethiol (ODT) was obtained from Fluka. All other chemicals were of analytical reagent grade and double distilled and deionized water was used for the preparation of the systems. 2.2. Preparation of niosomes [18] Tween 80, PEG 6000, Span 80 and water were vortex mixed at mass ratio of Tween 80/PEG 6000/Span 80/H2 O = 1.000/ 0.025/0.30/0.500. The samples were mixed with water (v/v = 1:1) and sonicated for 40 min to prepare niosomes in Sonicator (KQ-100 Sonicator, Jiangsu Kunshan Sonicator Co. Ltd.). 2.3. Modification of Au electrode with niosomes The Au electrode was first polished carefully with alumina slurries (1, 0.3 and 0.05 ␮m) and washed ultrasonically with double distilled water. Then it was electrochemically cleaned in 1 M H2 SO4 by cyclic potential scanning between +0.20 and 1.60 V until a standard cyclic voltammogram was obtained. After the electrode was rinsed with large amount of double distilled water and absolute ethanol, it was immersed into a solution of 1 mM octadecanethiol in absolute ethanol for a minimum of 12 h for the formation of the self-assembled ODT monolayer (SAM). Subsequently, the electrode was rinsed with absolute ethanol and water to remove non-chemisorbed species. After the electrochemical characterization of the self-assembled layer, 1 ␮L of niosomes was dropped onto the surface of SAM followed by drying in air. 2.4. Electrochemical measurements Electrochemical measurements were carried out with an Autolab PGSTAT 12 electrochemical impedance analyzer and potentiostat/galvanostat (Eco Chemie, The Netherlands) connected to a computer (Eco Chemie software: Frequency Response Analyzer (FRA 4.9) and General Purpose Electrochemical System (GPES 4.9) for impedance and cyclic voltammetry, respectively) (ECO Chemie BV, The Netherlands). A conventional three-electrode system was used throughout. A Au electrode was used as working electrode, a saturated calomel electrode (SCE) as the reference electrode, and a Pt plate was used as counter electrode. Five millimolars of K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] (1:1) mixture containing 0.1 M KCl was used as a redox probe in the electrochemical measurements. In CV measurements, the potential swept from −0.2 to 0.6 V with a scan rate of 50 mV s−1 , and the electrolyte solutions were thoroughly degassed with N2 and kept under a N2 blanket. The impedance spectra were performed in the frequency range from 100 mHz to 68 kHz, with a voltage amplitude of 10 mV. The frequency interval was divided into 60 logarithmically equidistant measure points. All measurements were carried out at room temperature.

Fig. 1. Cyclic voltammograms of 5 mM K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] containing 0.1 M KCl at the bare Au electrode (a), electrode modified with ODT (b), and then coated with niosomes (c). Scan rate: 50 mV s−1 .

effect of ODT on the electron transfer of Fe(CN)6 3−/4− . With the niosomes were deposited on the thiol modified-electrode, the cyclic voltammogram presented less Faradaic currents, partly due to the increase of the modification layers. It suggested that the multiimmobilization layers could greatly decrease the electron-transfer rate of Fe(CN)6 3−/4− within the applied potential. CV experiments confirmed that the thiol layer and niosomes were successfully assembled on the Au electrode. This phenomenon was further confirmed by the results of EIS measurements. 3.2. Electrochemical impedance spectroscopy The each steps of Au electrode modification were controlled also with electrochemical impedance spectroscopy . This method is very useful for studying the molecular interactions occurring at the interface [20,21]. Fig. 2 shows the impedance spectroscopy as Nyquist plot (−Z vs Z) of a bare Au electrode, after deposition of ODT, and continue modified by niosomes, using 5 mM [Fe(CN)6 ]4−/3− as marker ions in 0.1 M KCl at frequency from 0.1 Hz to 68 kHz. It can be seen that impedance spectra of a bare Au electrode exhibits almost the straight line, which is characteristic for

3. Results and discussion 3.1. Cyclic voltammetry of niosomes modified Au electrode The cyclic voltammetric study was carried out to probe the packing structure of the modified electrode through the redox behavior of a reversible Fe(CN)6 3−/4− couple [19]. Fig. 1 shows the CV responses of 5 mM Fe(CN)6 3−/4− at bare Au and niosomes modified Au electrode, respectively. Fe(CN)6 3−/4− produces a couple of well-defined redox waves at bare Au with a peak-to-peak separation (Ep ) of 90 mV at 50 mV s−1 . After the self-assembly of ODT, the shape of CV changes dramatically, no redox reaction of Fe(CN)6 3−/4− was apparently observed because of the blocking

Fig. 2. EIS of bare Au electrode (a), electrode modified with ODT (b), and then coated with niosomes (c). Solution composition: 5 mM K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ], 0.1 M KCl; the frequency range: from 0.1 Hz to 68 kHz.

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Fig. 3. Randle’s equivalent circuit for the electrochemical impedance spectroscopy measurements.

a limiting step of the electrochemical process. The SAM of ODT on Au shows a large semicircle formation in the range of frequency (line b), insulating layer on the electrode introduced a barrier to the interfacial electron transfer. It can be seen that the semi-circular diameter enlarged dramatically with continued modification of niosomes (line c), and the diffusion-controlled part of impedance spectroscopy did not appear at lower frequency, implying a high electron-transfer resistance, and very slow electron transfer kinetics of Fe(CN)6 ]3−/4− probe on Au electrode due to formation of hydrophobic, more insulating layer of niosomes on the electrode surface. To obtain data about the impedance of modified electrodes, the Randle’s equivalent circuit (shown as Fig. 3) was selected to fit the measured results. In EIS, the total impedance depends on several parameters such as: electrolyte resistance (Rs ), double layer capacitance (Cd ), electron-transfer resitance (Ret ) and Wartburg element (Zw ). Electrolyte resistance (Rs ) and Warburg (Zw ) element are the parameters, which represent bulk properties of the electrolyte solution and diffusion of the applied redox probe, respectively. Table 1 summarizes the extracted values resulted from fitting the measured electrochemical impedance spectra with the above equivalent circuits for the bare Au, Au with immobilization, which are extracted from the respective computer simulated spectra. The value of the electron transfer resistance (Ret , semicircle diameter) depends on the dielectric and insulating features at the electrode/electrolyte interface [22]. The Ret for the bare Au electrode is 0.64 k, and the assembly of ODT layer on the electrode surface generates a packed film that introduces a barrier to the interfacial electron transfer (Ret = 9.77 k), and the subsequent immobilization of niosomes onto the SAM layer results in a further increase of Ret (61.7 k), due to the rather insulating nature of membrane on the SAM-electrode surface. It could be also seen from Table 1 that Rs remain unchanged after the modification of Au electrode. Our experiments also showed that Cd increased 100 times after the SAM layer formed, and the capacitance was quite large when niosomes modified on the electrode (0.001 → 0.102 → 0.132 ␮F), which reflects the forming process of the supported bilayer [23]. There was a space charge capacitance of double layer with the capacitance of the dielectric film. From the above, it could be confirmed that the niosomes was successfully

Table 1 The values of Au, Au–ODT, Au–ODT-niosomes electrodes, and after DNA addition in electrolyte resulted from fitting the experimental data with the equivalent circuit shown in Fig. 3

Bare electrode Au–ODT Au–ODT–niosomes 0.01 mg/mL DNA addition 0.02 mg/mL DNA addition 0.03 mg/mL DNA addition 0.04 mg/mL DNA addition 0.05 mg/mL DNA addition

Rs (k)

Cd (␮F)

Ret (k)

Zw (k)

0.150 0.146 0.170 0.164 0.159 0.166 0.169 0.166

0.001 0.102 0.132 0.117 0.112 0.109 0.105 0.103

0.64 9.77 61.7 55.6 52.2 47.4 41.0 37.9

4.72 0.48 2.14 2.43 2.91 3.32 4.14 4.62

Fig. 4. EIS on ODT–niosomes modified electrode in 5 mM K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ], 0.1 M KCl, and in presence of 0 (a), 0.01 g L−1 (b), 0.02 g L−1 (c), 0.03 g L−1 (d), 0.04 g L−1 (e), 0.05 g L−1 (f), and 0.06 g L−1 (g) DNA in solution. The frequency range: from 0.1 Hz to 68 kHz.

modified on the Au surface and formed supported bilayer through ODT assembly. 3.3. Electrochemical response of the interaction between DNA and niosomes Surface-immobilized method is facile to directly evaluate the interaction between molecules occurring at the interface. EIS is a sensitive and non-destructive technique and widely used for characterizing the biological binding, which always used multilayer films [24–27]. The niosomes modified electrode was used for study the interaction of the niosomes and DNA, existing at the interface. The differences in resistance before and after DNA binding were taken as the signal produced by the affinity reaction between immobilized niosomes and DNA. The Nyquist plots for modified electrode before (a) and after interacting with different concentration DNA (b–g) in 5 mM Fe(CN)6 4−/3− solution with 0.1 M KCl as supporting electrolyte were shown in Fig. 4. As can be seen, the diameter of the semicircle significant decreases, implying lower electron-transfer resistance, due to the stepwise addition of DNA to the electrolyte solution. But our experiment showed that the resistance of bare electrode increased with DNA added, and same phenomenon was observed to ODT assembled electrode. It was indicated that DNA interacted with niosomes but not inner ODT layer. In the absence of DNA, the niosomes served as a rather effective barrier against electron tunneling. The addition of DNA caused the formation of some transmembrane channels for the redox probe across the niosomes. It was consistent with the result obtained from the cyclic voltammetry measurements (Fig. 5). The cyclic voltammetry behavior significantly changed after DNA addition. The increase in the peak-to-peak separation indicated that the integrity of niosomes might have been disturbed. The results also demonstrated that the more DNA bound on the electrode surface was, the lower the impedance value, and the increase of the permeability of the bilayer was more remarkable. The Ret value decreased when DNA was added (Table 1), indicating that the barrier of the redox probe to the surface was partly disrupted. A good linear relationship between Ret value and DNA concentration was found over the 0–0.05 mg mL−1 concentration

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4. Conclusions

Fig. 5. Cyclic voltammgrams of the niosomes/ODT-coated gold electrode in 5 mM K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] containing 0.1 M KCl (a), and with the addition of 0.03 mg mL−1 DNA (b).

The assembly of ODT layer on the electrode surface generated a tightly packed film that introduced a barrier to the interfacial electron transfer, and the continued immobilization of niosomes onto the SAM layer resulted in a further increase of electron-transfer resistance, due to the rather insulating nature of membrane on the SAM-electrode surface. This work proposed an interesting method to build novel sensors for determination of the interaction between molecules occurring at the interface, which acted as the modeling cell membranes. The cyclic voltammetry and electrochemical impedance spectroscopy were used to evaluate the DNA–niosome interaction on the surface of the electrode. The electron-transfer resistance decreased linearly with the concentration of the DNA in the range from 0 to 0.05 mg mL−1 , indicated that the film had a fractal surface with defects permeating through membrane to the electrode after it interacted with DNA. The method combining the electrochemical impedance spectroscopy and self-assembled bilayers may provide an alternative for the in situ detection of biomolecules such as DNA. This measuring system is relatively simple in the comparison to other spectroscopic one and can provide an initial assessment of membrane permeability and serve as a sensitive probe of structural changes in membrane. Acknowledgement We gratefully acknowledge financial support of National Natural Science Foundation of China (Grant No. 30570015). References

Fig. 6. The linear relationship between the concentration of DNA and the electrontransfer resistance.

range with a correlation coefficient of R = 0.995 (Fig. 6). After the addition of DNA with 0.04 mg mL−1 , the Ret value decreased from 61.7 to 41.0 k with a decrease of 33.5%, originating from the specific interaction between DNA and niosomes. It was indicative that Ret is more effective to the detection of the strong bonding between DNA and niosomes. From the result of CV and EIS, for one case, DNA might just disrupt the membrane and then leave from the membrane; for another case, DNA could bind with the membrane, and insert at least partly into the membrane or lead to some defect or lesion in the membrane, resulting in the increased permeability of the membrane. The assembled bilayer had a fractal surface with defects permeating through membrane to the electrode after it interacted with DNA.

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