ECS Electrochemistry Letters, 1 (2) G1-G3 (2012) G1 2162-8726/2012/1(2)/G1/3/$28.00 © The Electrochemical Society DNA Conformational Transitions at Different Concentrations and Temperatures Monitored by EIS L. M. Bravo-Anaya, a E. R. Mac´ ıas, a F. Carvajal Ramos, b V. V. A. Fern´ andez, c N. Casillas, d, ∗ J. F. A. Soltero, a and E. R. Larios-Dur ´ an a, z a Universidad de Guadalajara, Departamento de Ingenier´ ıa Qu´ ımica, C. P. 44430 Guadalajara, Jalisco, Mexico b Centro Universitario UTEG, Departamento de Investigaci´ on, C. P. 44460 Guadalajara, Jalisco, Mexico c Universidad de Guadalajara, Departamento de Ciencias Tecnol´ ogicas, C. P. 47819 Ocotl´ an, Jalisco, Mexico d Universidad de Guadalajara, Departamento de Qu´ ımica, C. P. 44430 Guadalajara, Jalisco, Mexico We present an EIS study and its analysis to determine the conformational behavior of DNA calf-thymus (13 kbp). The study is carried out at open circuit potential at a pH of 7.3. The results are interpreted in terms of the adsorption impedance theory by using an equivalent circuit approach similar to the one proposed by Frumkin-Melik-Gaikazyan-Randles. All equivalent circuit parameters are inspected as a function of DNA concentrations where it is possible to identify two transitions on the double-layer structure at DNA ca. 0.33 ± 0.07 and 1.50 mg/mL related to the overlap, C*, and entanglement, C e , concentrations. © 2012 The Electrochemical Society. [DOI: 10.1149/2.014202eel] All rights reserved. Manuscript submitted April 3, 2012; revised manuscript received May 29, 2012. Published July 20, 2012. Electrochemical Impedance Spectroscopy (EIS) is an outstand- ing and non-destructive technique suitable for investigating charged surfaces interacting with molecules such as DNA. Recent reports in the literature have demonstrated its potential to investigate DNA hy- bridization events that allow the development of DNA biossensing platforms, inmunosensors, and gene therapy. 1–3 The last one requires a full understanding of DNA conformational changes before attempt- ing a transfection process, which involves the collapsing of extended DNA chains into a more compact, arranged particles containing one or a few molecules before entering a cell. 4,5 Since DNA is a complex charged molecule, it becomes evident that most of the conformational changes perceived for DNA strains in solution or interacting with charged electrode surfaces may depend on several parameters, such as concentration, pH, temperature and polarization potential. 6 One of the alternatives currently applied to investigate transitions in polymer science consists of measuring rheological properties at a frequency range (0.0016–16 Hz). 7–9 Since DNA is a biopolymer it can be analyzed in a reometer with a cone-plate geometry, apply- ing an oscillatory mode, while measuring the viscoelastic properties. 8 Thus, it is possible by this technique to derive two critical parameters sensible to DNA conformation, i.e. the overlap (C*) and the entan- glement concentration (C e ). The first one consists of a dilute polymer solution without any interaction between the molecules until reaching C*, where molecules start interacting each other and getting packed, then reaching the overlap concentration C e , where polymer chains are entangled. 7,8 By applying rheological techniques a calf-thymus DNA solutions at a pH 8.0 have been analyzed identifying both transitions at 0.35 and 2.0 mg/mL for C* and C e respectively. 8 Although rheological methods provide macroscopic properties of DNA chains in solution a microscopic overview of the systems is still needed. 7,8 In this direction a method that has contributed to the understanding of DNA conformations at a microscopic scale is di- electric relaxation. 10 By applying this technique it has been possible to investigate the dynamics and structure of water bound to DNA molecules at high frequency domain (10 6 –10 −2 Hz). 11,12 A different frequency window can be also explored by EIS (10 3 -10 −3 Hz), in con- junction with an analysis of double-layer capacitance as a function of the polarization potential. These techniques open new possibilities to further investigate the adsorption phenomena and conformational studies at the electrode-DNA interface, whose results can be compared directly with rheological measurements. 11,13 At the EIS frequency range the results can be tentatively interpreted in terms of a sudden loss of the ability of the DNA molecule to respond to the alternating voltage applied to the electrodes, likely due to changes in the DNA conformation. 11,12,14 ∗ Electrochemical Society Active Member. z E-mail: roxana.larios@red.cucei.udg.mx In this paper we present an EIS investigation of the interface formed by a platinum electrode and calf-thymus DNA molecule. It can be described as a new alternative method for detecting C * and C e con- centrations. EIS measurements are performed at open circuit potential and different concentrations and temperatures. EIS results are trans- formed into complex capacitance data and are interpreted in terms of the adsorption impedance theory by using an equivalent circuit similar to the one proposed by Frumkin-Melik-Gaikazyan-Randles (FMGR). 15–18 An evaluation of each equivalent circuit parameter, as a function of DNA concentrations and temperatures, is proposed to obtain information concerning to the double-layer structure. This eval- uation allows to readily detect the overlap and the entanglement con- centrations, C* and C e , of DNA molecules, similar to the ones detected by macroscopic rheological measurements. 8 Both transitions are as- sociated to a double-layer structure rearrangement as a function of the DNA molecule conformations at the interface. Experimental DNA solutions preparation.— DNA solutions were prepared by weighting appropriated amounts of DNA extracted calf-thymus (13 kbp) using a 9:1 ratio of HPLC water and EDTA/Tris-HCl buffer previously prepared. Buffer’s pH was adjusted to 7.3 by adding NaOH solution (3 M). Fifteen DNA solutions were prepared in a concentra- tion range from 0.01 to 2.5 mg/mL. All the chemical reagents come from Sigma-Aldrich (99% purity) and were used as received. In or- der to prevent the solution degradation and water evaporation, DNA solutions were refrigerated at 4 ◦ C. Electrochemical impedance spectroscopy (EIS) measurements.— A two-platinum electrode cell from Fisher Scientific was used to perform EIS measurements at open circuit potential (OCP). Both platinum electrodes surfaces were 0.25 cm 2 . Impedance spectra were measured with a 1260 FRA instrument coupled to a potentiostat model 1287 both from Solartron, Inc. The amplitude of potential perturba- tion was 10 mV, with a frequency range of 1 kHz-1mHz and 7 points per decade were recorded. The impedance data were collected by the commercial software Zplot, and the theoretical parameters were calculated from an equivalent circuit and fitted to the experimental impedance data using the commercial software Zview. All the mea- surements were carried out at three different temperatures (i.e., 20, 30 and 40 ◦ C). Before each EIS experiment routinely a cyclic voltamme- try in 0.5 M H 2 SO 4 at 50 mV/s was applied to guaranty the cleanliness of the electrodes. Results and Discussion Equivalent circuit.— Adsorption studies performed by EIS and their interpretation by the impedance adsorption theory have been ) unless CC License in place (see abstract). ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see 207.241.231.82 Downloaded on 2018-07-21 to IP