In situ surface-enhanced infrared spectroscopy study of adenine-thymine co-adsorption on gold electrodes as a function of the pH

1 “This is an Accepted Manuscript of an article published by ELSEVIER in JOURNAL OF ELECTROANALYTICAL CHEMISTRY on 2018, available at: https://doi.org/10.1016/j.jelechem.2017.11.054. It is deposited under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.” In situ Surface-Enhanced Infrared Spectroscopy Study of Adenine- Thymine co-adsorption on Gold Electrodes as a function of the pH Julia Álvarez-Malmagro, Manuela Rueda, Francisco Prieto* Department of Physical Chemistry.University of Seville. C/ Profesor GarcíaGonzález nº 2, 41012 Seville (Spain) e-mail: [email protected] ABSTRACT In situ attenuated total reflection surface enhanced infrared absorption spectroscopy (ATR-SEIRAS) has been used to study the co-adsorption of adenine and thymine on gold nanostructured electrodes from D2O and H2O solutions at acid, neutral and basic pH values. The comparison between the ATR-SEIRA band when adenine (A) and thymine(T) are co-adsorbed and the corresponding bands of both bases adsorbed individually has revealed the existence of interactions between the complementary bases at the goldelectrolyte interface, at acid, neutral and basic pH values. The most significant bands of adsorbed adenine undergo an enhancement in the presence of thymine. This can be explained if adenine adopts an orientation with the molecular plane more normal to the electrode surface and, at least at neutral pH, the surface concentrations of adenine increases in the presence of thymine. In the presence of adenine, the characteristic absorption bands of adsorbed thymine nearly disappear at potentials of the chemisorption region of thymine, suggesting the orientation of thymine molecular plane from normal to nearly parallel relative to the electrode surface, after the addition of adenine. The comparison of the spectral regions at 2900-3800 cm-1 of adsorbed adenine in H2O solutions in the absence and in the presence of thymine only reveals the enhancement 2 mentioned above. The bands corresponding to the NH2 stretching modes of adenine do not undergo any significant frequency shift, which permit us to discard the participation of adsorbed adenine in hydrogen bonding interactions (A-T) of the Watson-Crick or of the Hoogsteen types. Cooperative interactions including pi interactions A-T of the “edge to face” type and coplanar hydrogen bonding T-T are proposed to be responsible of the long range organization of the adlayer formed by the coadsorbed bases. KEYWORDS In situ ATR-SEIRAS; gold electrodes; adenine-thymine co-adsorption; complementary DNA bases; Watson-Crick interactions; Hoogsteen interactions 3 1.- INTRODUCTION The interactions between the complementary DNA bases, adenine and thymine are important in relation to genetic transcription and replication in living cell, among other biological processes [1,2]. Electrode interfaces can provide some analogies with the organized structures in living cell and the possibility to modify the electric field. Therefore, the study of the interactions of the DNA bases at electrode interfaces can be interesting to understand their biological activity under the influence of electric fields. The study can also derive into technological applications such as biosensor and platforms for drug delivery. Adenine and thymine are known to get adsorbed on gold electrodes. Recently we have obtained interesting microscopic information about adenine adsorption on single crystal and on nanostructured films gold electrodes by different “in situ” spectroelectrochemical infrared reflection-absorption spectroscopies, in a wide range of pH media [3–6]. These techniques are especially suitable to provide evidences about the molecular orientation, the coordination sites of the adsorbed molecule with the metal atoms as well as the intermolecular interactions. On the other hand, electrochemical techniques such as cyclic voltammetry, chronoamperometry and electrochemical impedance spectroscopy (EIS) have allowed us to characterise the thermodynamics and kinetics of adenine chemisorption on gold electrodes. Thus, it was concluded that the potential range at which the chemical adsorption takes place depends on the crystallographic orientation of the single crystal gold electrode and the reconstructed or unreconstructed state of the electrode surface [7]. In fact adenine adsorption on flame annealed reconstructed surfaces induces the lifting of the reconstruction, as was also proved by Scanning tunneling microscopy (STM) measurements [8]. The thermodynamic analysis performed on the base of the chronoamperometric data obtained in neutral pH mediums concluded about the weak nature of the chemisorptions process and suggested a tilted orientation of the molecular plane with respect to the electrode surface [7]. The kinetics of adenine adsorption was studied by EIS, taking advantage of the smoothness of the single crystal Au(111) electrode surface that permits to discard any effect of the surface heterogeneity on the 4 frequency dispersion of the impedance data, and the kinetic parameters were found to depend on the pH of the medium [9,10]. The effect of the pH value of the medium is particularly interesting because the biological activities of these compounds depend on their acid-base equilibriums that can change when the compound is located in organized environments [11–14]. That is the case of the first pKa of adenine that is 4.2 in solution (scheme 1) but it shifted to values lower than 1 when adenine is chemisorbed [4]. However, the same pKa2 (9.8 ) for adenine in solution was obtained for the chemisorbed adenine [6]. Therefore, only the neutral adenine (AdH3) and the anionic form (AdH2- ) have been reported to get chemically adsorbed. HN NN N NH2 H N NN N NH2 H N NN N NH2 pK1 = 4.2 AdH4+AdH3AdH2- H+ pK2 = 9.8 H+ 5 3 8 4 6 29 7 1 10 Scheme 1.- Acid-base equilibria of adenine. Thymine adsorption also depends on the pH of the medium. The pKa of thymine in solution is 9.6, however our “in situ” spectro-electrochemical infrared reflection absorption spectroscopy (IRRAS) analysis in a wide pH range (from pH 1 to 12) have shown that in the whole pH range the adsorption involves the deprotonation of thymine, yielding one of the tautomeric anionic thymine forms, the N3- or the N1- tautomer, depending on the intracyclic nitrogen atom that loses the H- atom (see scheme 2) [15]. Moreover, the detailed analysis of the FT-IR spectra on the base of Density functional theory (DFT) calculations allowed us to conclude that the preponderance of each tautomer at the interface is pH dependent, being the N3-tautomer the predominant component at pH 1 while at pH 12 the N1-tautomer is the preponderant one. In neutral media both tautomers get adsorbed, [15]. The orientations and reorientations with the electric field seem to be different for each tautomer. Thus, the N3 tautomer interacts with the surface by the N3 atom and the oxygen atom of the C2O carbonyl group at low chemisorption potentials and it rotates to interact also by the N3 atom and the oxygen 5 atoms of the two carbonyl groups as the potential is increased, while the N1-tautomer interacts with the metal in the whole chemisorption region by the N1 atom and the oxygen of the C4O group. HN N H CH3 OO N N H CH3 OO HN N CH3 OO 1 24 5 6 3 pKa=9.6 78 9 N1 tautomer N3 tautomer - H+ Scheme 2.- Acid-base equilibrium of thymine. In a previous paper we have studied the co-adsorption of adenine and thymine from acid solutions by surface-enhanced infrared absorption spectroscopy (ATR-SEIRAS). The high sensitivity and little interference from the solvent attained with this technique allow us to analyze the influence of small amount of one of the DNA base (below the sub-monolayer level) on the adsorption of the complementary base. It was observed that both bases remained adsorbed in the presence of each other in a wide potential range, but the analysis of the spectral signals in the 1300-1800 cm-1 region indicate that both molecules reorient on the surface to facilitate their intermolecular interactions. Adenine seems to adopt a more upright orientation while thymine adopts an almost flat orientation on the electrode. In nature the complementary bases are known to interact by two H-bonds either in the Watson-Crick (W-C) [1] or in the Hoogsteen (HG) [2] configurations (see Scheme 3). In both types of interactions, the molecules are using the same atoms that have been proposed as interaction sites with the gold atoms of the electrode surface. Thus, the adsorption model proposed for adenine adsorption on Au(111) surfaces from slightly basic solutions, on the basis of the results of quantitative subtractively normalized interfacial Fourier transform infrared reflection spectroscopy (SNIFTIRS) implies the interaction between the N10 and the N1 atom of adenine molecule with the gold atoms [5]. The tilt angles of adsorbed adenine molecule were also estimated by the technique and an angle of c.a. 400 with respect to the normal to the electrode surface [5]. On the other hand, the models for chemisorbed thymine propose the interaction of the molecule with the gold surface by the oxygen atoms and the 6 deprotonated N3 or N1 atoms with the molecular plane normal to the electrode surface[15,16]. Scheme 3.- Hydrogen bonding Watson-Crick (WC) and Hoogsteen (HG) interactions between adenine (A) and thymine (T). In this paper the study of the co-adsorption of adenine and thymine is extended to complete a wider pH range of conditions in order to analyse the co-adsorption of the adenine forms with different degree of protonation (the neutral or the anionic adenine molecules) as well as the different tautomeric forms of anionic thymine (N3- or N1- tautomers). The study of the interactions of the two thymine tautomers with adsorbed adenine is particularly interesting as it is known that “unstable” tautomers can induce unpaired bases interactions, which have been associated to some deseases, [17,18]. Experiments have been performed from solutions of pH 1, pH 7 and pH 11.5. The advantages of the ATR-SEIRAS techniques in the Kretschmann configuration have been explored to study the co-adsorption from solutions of very low concentrations of the bases. Experiments have been performed in H2O and in D2O solutions as both bases can get deuterated, thus changing the spectral characteristics. 2.- EXPERIMENTAL AT (WC) (HG) T A 7 Supporting electrolyte solutions were prepared in ultrapure water, freshly purified with a Millipore Direct-Q or a Millipore Milli-Q systems, or in deuterium oxide supplied by Sigma 99.99 %. The supporting electrolyte solutions were HClO4 0.1 M (pH 1) KClO4 0.1 M + NaOH (pH 7 and 11). HClO4, KClO4 and NaOH were from Merck Suprapur®. Adenine and thymine were obtained from Sigma-Aldhrich. Working solutions of thymine and of adenine of the desired concentration (usually 0.01 mM of adenine and 1 mM of thymine) were prepared by the addition to the electrochemical or to the spectroelectrochemical cell of the required amount of 10 mM stock solution of the DNA base prepared in the same supporting electrolyte.The pH of the solutions was measured with glass membrane combined electrode from Hamilton. The pH values obtained in D2O solutions were corrected according to [19]. All solutions were deareated by bubbling argon before to any measurement. In the electrochemical experiments a freshly flame annealed Au(111) electrode prepared according to the Clavilier method [20] was used as working electrode, a gold wire as counter electrode and as reference electrode a saturated mercury/mercurous sulphate reference electrode connected to the cell via a salt bridge filled with the same supporting electrolyte as in the working solution. All the potentials are referenced to the saturated calomel electrode (SCE). The voltammograms were recorded with an Autolab PGstat 30 from Ecochemie and with an IviumStat from Ivium Technologies. The spectro-electrochemcial cell used for the in-situ ATR-SEIRAS measurements was designed according to the Kretschmann configuration for internal reflection: The working electrode used was a gold film of 20-25 nm thickness deposited by sputtering (at c.a. 0.01 nm s-1 with a Leica EM SCD500 metalizer equipped with a quartz crystal microbalance) on one of the faces of a silicon prism bevelled at 60 degrees. The auxiliary electrode was a gold foil and the reference electrode was the same used in the electrochemical measurements. A Fourier transform infrared (FTIR) spectrophotometer NICOLET 6700, with a MCT- A detector and a VeeMaxTM -II accessory from PIKE Technologies for reflectance measurements was used to collect the ATR-SEIRA spectra, with a resolution of 4 cm-1. The reflectance spectra have been collected with p polarized radiation, by means of a ZnSe polarizer intercalated into the IR beam. 100 interferograms have been averaged to obtain one spectrum. Every ATR-SEIRA spectrum is represented as log⁡(𝑅0/𝑅) 8 (absorbance) where R0 is he reflectance spectrum taken as reference, collected in the supporting electrolyte in the absence of DNA base at the same potential than the sample spectrum and R is the sample reflectance spectrum collected in the measurement conditions , in the presence of the DNA base in solution. The spectra were collected under potentiostatic control by means of a CHI 1100A potentiostat from CH Instruments. The experimental procedure consists in the collection of the reflectance spectra of the supporting electrolyte at different potentials and then, the addition of the required dosage of stock solution of either adenine or thymine to get the desired concentration in cell, and after that the collection of the corresponding reflectance spectra. Successive additions of DNA base can be done over the same cell solution without modification of the optical properties of the experimental set-up. Some FT-IR absorption spectra of thymine and of adenine in solution have been obtained using an ATR cell equipped with a ZnSe prismatic window bevelled a 45º. 3.- RESULTS 3.1. Cyclic voltammetry of the coadsorption of thymine and adenine on Au(111) electrodes. In figure 1 are shown the cyclic voltammograms obtained with single crystal Au(111) electrodes in solutions containing either adenine (0.01 mM) or thymine (1 mM) or both bases (0.01M adenine + 1mM thymine) at different pH values. The selected concentrations of adenine and thymine correspond to one set that clearly reveals the effects of the coadsorption on the voltammetric results and, specially, on the ATRSEIRA spectra. The adsorption of adenine and that of thymine on Au(111) electrodes from solutions of different pH values has been previously studied by electrochemical and spectro-electrochemical methods [3,4,6,15,16,21]. The chemical adsorption/desorption of adenine on Au(111) originates a pair of voltammetric signals at potentials around 0 V, -0.3 V and -0.4 V at pH values of 1, 7 and 11.5, respectively. 9 Regarding the adsorption of thymine, two pairs of narrow voltammetric peaks at c.a. - 0.08 V and 0.28 V can be observed at pH 1, delimiting different thymine ad-layer organization as the potential increases[15,21,22]: at potentials lower than the first transitions peaks a layer of randomly physical adsorbed molecules is proposed, that originates a condensed phase at potentials between the two pairs of peaks and show the chemical adsorption/desorption wide voltammetric signals at potentials higher than the second pair of narrow transition peaks. As the pH increases, the adsorption/desorption peaks of thymine shift to lower potentials, diminishing the potential range of physisorbed condensed layer. At pH 11.5 no phase transition peaks are visible. The comparison of the adsorption/desorption voltammetric peaks corresponding to thymine in the absence and in the presence of adenine at the three pHs studied clearly show a change in the shape of the peaks corresponding to thymine adsorption. The changes depend on the pH value of the solution. At pH 1, the voltammetric signals corresponding to the chemical adsorption of thymine shift to lower potentials and change their shapes, especially the desorption peak. Moreover, the characteristic phase transition peaks of thymine adlayers on Au(111) electrodes from acid media[15,21,22] disappear in the presence of adenine. In neutral media the potentials of the voltammetric signals corresponding to the chemisorption of thymine do not shift in the presence of adenine; the shapes of the waves remain similar although the characteristic peaks of phase transitions in thymine adlayers also disappear in the presence of adenine and, in addition, a decrease in the charge associated to the chemical adsorption/desorption of thymine can be observed. In very basic media the voltammetric changes in the potential region corresponding to the adsorption of thymine in the absence and in the presence of adenine only reveal that the charge associated to the adsorption/desorption of thymine is lower in the presence of adenine. 16 medium. At pH 11.5 the ATR bands of the N1 tautomer of anionic thymine at 1560 and 1526 cm-1 totally disappear after the addition of adenine. a) pH 1 0.0 0.2 0.4 0.6 A / cm-1 0.0 0.2 0.4 0.6 E vs SCE / V -0.8 -0.4 0.0 0.4 0.8 0.0 0.2 T 1581 cm-1 T+A 1581 cm-1 b) pH 7 c) pH 11.5 T 1581 cm-1 T+A 1581 cm-1 T 1526 cm-1 x 5 T+A 1526 cm-1 Figure 5.- Integrated intensities of the thymine ATR-SEIRA band at c.a 1581 cm-1 (at pH 1 and pH 7) and at 1526 cm-1 (pH 11.5) in D2O solutions of 1 mM concentration as a function of the potential, recorded before (T - solid symbols), and after (T+A – open symbols) the addition of adenine to a final concentration 0.01 mM. In figure 6, the effect of the addition of thymine on the integrated intensity of the ATR-SEIRA band of adenine at c.a. 1640 cm-1 in D2O is analysed as a function of the potential. The enhancement of the absorption band of adenine in the presence of 17 thymine is clear at the three pHs considered, and it is significantly higher at pH 7 than at the other two pHs. A / cm-1 0.5 1.0 1.5 E vs SCE / V -0.8 -0.4 0.0 0.4 0.8 0.5 1.0 1.5 b) pH 7 c) pH 11.5 A A+T A A+T a) pH 1 0.5 1.0 1.5 A A+T Figure 6.-Integrated intensities of the adenine ATR-SEIRA band at c.a 1640 cm-1 in 0.01 mM adenine solutions in D2O as a function of the potential, recorded before (A – solid symbols) and after (A+T – open symbols) the addition of thymine to a final concentration 1 mM. 18 ATR-SEIRA spectra of adsorbed and co-adsorbed adenine and thymine have also been measured in H2O. The main differences that are noticed on the spectra obtained in protic media as compared to the spectra at the same conditions in deuterated media are due mainly to the NH and NH2 bending modes contributions to the absorption bands, especially in the band of adenine adlayer at c.a. 1650 cm-1 that in protic media is coupled to the scissoring mode of the NH2 group resulting in a broader band slightly shifted to higher frequencies. In the thymine spectra the hydrogen isotopic exchange has a weaker effect on the absorption bands, that are slightly blue shifted in the spectra collected in H2O as compared to the bands obtained in D2O. In figure 7 the spectra obtained in H2O for the adsorption and co-adsorption of thymine and adenine from pH 1 solutions are plotted at a potential higher than the onset of thymine adsorption. 19 wavenumber / cm-1 1450155016501750 log(R0/R) 0.5 V 0.01 a.u. T + A A A + T T a) b) 1679 1676 1678 1592 1658 1593 Figure 7.-In situ ATR-SEIRA spectra obtained for the adsorption of thymine in the absence and in the presence of adenine (a) and for the adsorption of adenine in the absence and in the presence ofthymine (b) from solutions at pH 1 in H2O on gold electrodes at the indicated potential. Lines as in figures 3 and 4. The addition of adenine originates a clear decrease (close to disappear) of the intensities of the bands corresponding to adsorbed thymine, in figure 7a. This effect is clear in the band of adsorbed thymine at 1592 cm-1, although a residual very small signal at this wavenumber can still be observed in the spectrum of both bases in figure 7a, (as the ATR band at 1580 cm-1 in D2O. In figure 7b it can be observed that the addition of thymine to the solution originates, as it happens in D2O, an enhancement of the bands of adsorbed adenine and the appearance of a small signal at 1592 cm-1. 20 wavenumber / cm-1 1400150016001700 log(R0/R) 0.5 V 1651 0.005 a.u. A + T 0.001 mM / A A + T 0.01 mM / A + T 0.001 mM A + T 0.1 mM / A + T 0.01 mM A + T 1 mM / A + T 0.1 mM 1577 Figure 8.-In situ ATR-SEIRA spectra obtained for the adsorption of 0.01 mM adenine/D2O at pH 1 in the presence of increasing concentrations of thymine (from 0 to 1 mM). The reference reflectance signal for each spectrum is the previous one recorded with the immediately lower thymine concentration. The influence of the concentration of the bases is illustrated in figure 8 in which some spectra are presented in a comparative way as examples of the changes exerted by successive additions of thymine on the adsorbed adenine ATR-SEIRA spectrum at a potential of the chemisorptions region of the two bases at pH 1 in D2O. It can be observed that the first addition of thymine, to a final concentration of 10-6 M involves a significant enhancement of the adsorbed adenine ATR band at c.a. 1650 cm-1 and that following dosages of thymine originate successively smaller enhancements. The blue shift of the adenine band is higher as the thymine concentration is increased, so that the effect at 1 mM thymine concentration shows up providing a bipolar band when 21 compared with the spectrum obtained after the previous thymine addition. The characteristic band around 1580 cm-1 corresponding to the N3 tautomer of anionic thymine is only detected at the higher thymine concentrations. 3.3.2. Spectral region 2800-3900 cm-1. The spectral region 2800-3900 cm-1 in H2O solutions contains information on the adenine NH2 stretching vibration bands that can be interesting in relation to the possible hydrogen bonding interactions between the co-adsorbed complementary bases. In figure 9 the comparisons between some spectra in this region obtained for individually adsorbed adenine and those obtained in the presence of thymine are presented at the three investigated pHs at potentials higher than the onset of the adsorption of both bases. Although generally the NH2 stretching modes do not provide intense bands and the frequencies are very close to OH stretching frequencies of the solvent, either in the absence and in the presence of thymine the signals corresponding to the symmetric and antisymmetric NH2 stretching modes of adsorbed adenine can be observed at c.a. 3220 cm-1and 3360 cm-1, respectively.[23] The addition of thymine originates an enhancement of these signals but not any significant change in their frequencies. The appearance of a shoulder at c.a. 3450 cm-1, can be associated to the stretching of hydrogen bonded OH from adsorbed solvent molecules at the three pHs in figure 9.[24,25] The spectra in acid media include a band at 3590 cm-1, assigned to the stretching of asymmetrically hydrogen bonded H2O co-adsorbed with perchlorate ions, that is also enhanced after the addition of thymine.[24,25] 22 300033003600 0.5 V a) A A + T 3590 3210 0.004 a.u. 3355 3342 3207 300033003600 0.5 V b) A A + T 3360 3211 300033003600 0.1 V c) A A + T 3383 3221 3450 3450 3450 3360 Wavenumber / cm-1 0.002 a.u. Figure 9.-In situ ATR-SEIRA spectra in the 2800-3900 cm-1 region obtained for chemisorbed adenine (red solid line) and after the addition of thymine from H2O solutions at a) pH 1, b) pH 6 and c) pH 11., at the potentials indicated in the figures. 4.- DISCUSSION Either for a FT-IRRA band and for an ATR-SEIRA band with p polarized radiation the integrated intensity (𝐴𝑝) is proportional to the surface excess of the absorbing species (Γ) and to the scalar product of the dipole vector of the vibrational transition (𝜇⁡) and the electric field intensity of the radiation (𝐸𝑝 󰇍 󰇍 󰇍 󰇍  )[26]: 𝐴𝑝⁡∝ ⁡Γ|𝜇⁡× 𝐸𝑝 󰇍 󰇍 󰇍 󰇍  |= ⁡Γ⁡|𝜇|2〈𝐸𝑝 2〉cos⁡ 2(𝜃) (1) Where 𝜃 is the angle between the dipole vector of the transition 𝜇 and the normal direction to the reflection surface (the surface of the metal grains of the nanostructured electrode in the case of a SEIRA band). Apart from any possible intensity transfer phenomenon that will be discussed below, the changes in the intensities of the signals corresponding to individually adsorbed thymine or adenine in the presence of the complementary base could have two causes: a change in the corresponding surface coverage and/or a change in the orientation of the molecular plane relative to the surface electrode. Then, the disappearance of the ATR- 23 SEIRA bands corresponding to thymine in the presence of adenine at high potentials could be ascribed either to a reorientation of the adsorbed thymine with the molecular plane parallel to the electrode surface or to its desorption caused by the adsorption of adenine. However, the voltammetric signals corresponding to thymine adsorption/desorption obtained in the presence of adenine indicate that thymine is also adsorbed in the presence of adenine. Therefore, it has to be concluded that the coadsorption of adenine originates a reorientation of the molecular plane of adsorbed thymine to a more parallel disposition over the electrode surface at all the pHs investigated. The remaining signal at 1581 cm-1 in D2O or at 1596 in H2O corresponding to the N3 tautomeric form of anionic thymine, that can be observed in the presence of adenine at pH 1 and pH 7, indicates that the molecular plane of this tautomer is slightly tilted relative to the electrode, in the presence of adenine. On the other hand, the absence of thymine ATR signals at pH 11.5 in the presence of adenine suggests that the N1 thymine tautomer in the presence of adenine adopts a full parallel orientation. The enhancement observed in a ATR-SEIRA band of adsorbed adenine in the presence of thymine can be quantified by the ratio between the corresponding integrated intensities of the bands obtained in the presence, 𝐴𝑝(𝐴𝑑+𝑇𝑚), and in the absence, 𝐴𝑝(𝐴𝑑), of thymine, that obey equation (1). The dipole vector of the transition can initially be considered the same in both cases, and if both spectra have been obtained in the same experiment the optical conditions must be also identical and so the mean quadratic electric field intensity of the radiation at the electrode surface, 〈𝐸𝑝 2〉. Then from equation (1) it is obtained: 24 𝐴𝑝(𝐴𝑑+𝑇𝑚) 𝐴𝑝(𝐴𝑑)=⁡Γ(𝐴𝑑+𝑇𝑚) Γ(𝐴𝑑)⁡×⁡cos⁡ 2(𝜃)(𝐴𝑑+𝑇𝑚) cos⁡ 2(𝜃)(𝐴𝑑) (2) Where Γ(𝐴𝑑+𝑇𝑚), Γ(𝐴𝑑), 𝜃(𝐴𝑑+𝑇𝑚)⁡and⁡⁡𝜃(𝐴𝑑) are the surface excess of adenine and angles between the normal direction to the electrode and the dipole vector of the vibrational transition of adenine in the presence (Ad+Tm) and in the absence (Ad) of thymine, respectively. Equation (2) indicates that under the assumed conditions the ratio between the intensities of the transition in the presence and in the absence of thymine can be due to a change in the surface concentration of adenine or/and to a change in the adenine molecular orientation. In figure 10 the ATR-SEIRA bands of adsorbed adenine at c.a. 1640-1650 cm-1 and 1320-1380 cm-1are represented in the presence and in the absence of thymine. These signals correspond to in-plane vibrational modes, but at each pH the corresponding transition dipole vectors of the labeled bands, obtained from DFT calculations reported previously [5], have different directions within the molecular plane, as can be observed in the right part of figure 10. Therefore, the comparison at each pH of the changes in both IR bands can provide information about possible rotations of the molecular plane of adenine as a consequence of the addition of thymine. 25 Figure 10.-In situ ATR-SEIRA bands of adsorbed adenine at c.a 1640-1650 cm-1 and 1320- 1390 cm-1 obtained from at each pH in 0.01mM adenine solutions before (red line) and after (blue line) the addition of thymine till 1 mM concentration in cell: a) pH 1 and 0.7 v, b) pH 7.5 and 0.5 V and c) pH 11.5 and 0.4 V The approximate directions of the transition dipoles vectors associated to the labelled bands are represented at the right of the corresponding graph. The bands at 1650 cm-1, 1639 cm-1 and 1633 cm -1 at pHs 1, 7.5 and 11 respectively correspond to a skeletal vibration including the stretching of C6N10 and C5C6 bonds[3,5,23]. The other bands labeled in figure 10 at 1315 cm-1, 1380 cm-1 and 1384 cm-1 correspond to other vibrational modes that include the of C5N7 and C4C5 bonds and the in-plane CH bending. Wavenumber /cm-1 1300140016001700 Adenine 0.01 mM Adenine 0.01 mM + Thymine 1 mM 1633 1384 0.02 a.u. x 5 1639 1380 0.01 a.u. x 5 1650 1315 log R0 / R) a) b) c) x 5 pH 7 pH 11 0.02 a.u. pH 1 N N N N H2N H N N N N H2N N N N N H2N H 1640 cm-1 1380 cm-1 1650 cm-1 1315 cm-1 1633 cm-1 1384 cm-1 Au Au Au 32 base induces us to consider other type of interactions, different from the Watson-Crick or Hoogsteen between co-adsorbed adenine and thymine. “Edge to face” pi-stacking interactions can originate a “T-shaped” geometry compatible with the spectral results in this paper for co-adsorbed adenine-thymine. 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