Adsorption of adenine derivatives on a gold electrode studied by specular reflectivity measurement

J. Electroanal. Chem., 102 (1979) 109--116 © Elsevier Sequoia S.A., Lausanne -- Printed in The Netherlands

109

ADSORPTION OF ADENINE DERIVATIVES ON A GOLD ELECTRODE STUDIED BY SPECULAR REFLECTIVITY MEASUREMENT

KIYOKO TAKAMURA, ATSUKO MORI and FUMIYO WATANABE

Tokyo College of Pharmacy, Horinouchi 1432-1, Hachioji, Tokyo 192-03 (Japan) (Received 7th June 1978; in revised form l l t h December 1978)

ABSTRACT The adsorption of adenine, deoxyadenosine, deoxyadenosine-5r-monophosphate, -diphosphate and -triphosphate on a gold electrode has been studied by specular reflectivity measurement in 0.1 M NaClO4 solution. In the presence of these compounds, a marked decrease in reflectivity was found on reflectivity-potential curves in the potential region more positive than --0.8 V vs. Ag/AgC1, the decrease being ascribed to the adsorption of them. The magnitude of change in reflectivity was dependent on both the concentration and the electrode potential. The reflectivity change observed in the negative potential region was analyzed quantitatively according to the procedure previously described. The results were elucidated on the basis of the same isotherm as used by Green and Dahms in their adsorption study of aromatic hydrocarbons, and the number of solvent molecules being replaced through the adsorption of one organic molecule and the free energy change of adsorption were obtained. The former is suggestive of a flat orientation of the adsorbed molecule in contact with its adenine moiety on the electrode surface. It is also suggested from the latter that the presence of phosphate groups leads to a decrease in AGed resulting from their hydrophilic properties and a repulsive interaction between these groups and the negative charges on the surface.

INTRODUCTION

Interaction of biologically important substances with charged interfaces has b e c o m e of interest in recent years particularly in relation to the adsorption behaviour of such molecules at biological interfaces. A n u m b e r of studies have been made for DNA and related c o m p o u n d s from this viewpoint [1--3], however, the majority of the works have dealt with the electrochemical behaviour at the mercury electrode surface. For example, Krznari6 et al. [4,5] and Kinoshita et al. [6] studied the adsorption of adenine and its nucleoside and nucleotides on mercury electrodes and determined the adsorption parameters using capacitance data. Discussion was given on the orientation and the association of adsorbed molecules. Since such findings for the DNA components will provide useful information of the interfacial behaviour of their high polymers, an extension of the work to the adsorption on solid electrodes seems to be required. Recently optical techniques have been extensively applied to in situ examination of electrosorption phenomena because of their sensitivity to the surface state of electrodes [ 7,8]. Humphreys and Parsons employed an ellipsometric m e t h o d for the detection of the adsorption of DNA [9] and ~- and ~-quinolines [10] at a mercury electrode. Detailed observations were made for native and

110

denatured DNA, and the results were suggestive of an optical anisotropy in the adsorbed layer. In their adsorption study of quinolines, they confirmed the occurrence of reorientation of adsorbed molecules depending upon the electrode potential and the concentration of quinolines and proposed optical models of the adsorbed layer. Since the usefulness of the specular reflectance m e t h o d has been discussed b y McIntyre and Aspnes [11,12], Takamura et al. have successively applied the method to the detection of the adsorption of both inorganic and organic substances on solid electrodes [13--19]. Then it seems worthwhile to extend the m e t h o d to the electrosorption of some biological substances. The present paper is concerned with the adsorption of adenine and its derivatives on gold studied by the specular reflectance technique and the discussion of their adsorptivity by considering the effects of substituent groups. EXPERIMENTAL

Adenine obtained from Wako Pure Chemical Industries, Ltd., 2'-deoxyadenosine, 2'-deoxyadenosine-5'-phosphate (dAMP) and 2'-deoxyadenosine-5'-triphosphate (dATP) obtained from P-L Biochemicals, Inc., and 2'-deoxyadenosine-5'diphosphate (dADP) obtained from Sigma Chemicals, were used without further purification. As a base electrolyte solution, 0.1 M NaC104 was used, which was prepared by dissolving guaranteed grade material obtained from Kanto Chemical Co., Inc. A gold plate of 25 mm X 22 mm was used as a working electrode. The electrode potentials were measured relative to an Ag/AgC1 electrode. The counter electrode was also a gold plate. Pretreatment of the working electrode was made by the same procedure as described in the previous paper [ 18]. Electrolysis was carried o u t with a linear potential sweep and a potential step methods, the potential being applied from a HA101 potentiostat (Hokuto Denko Ltd.) in connection with a HB-107A function generator (Hokuto Denko Ltd.). Cyclic voltammetry was made by a conventional method. Optical measurements were made during the potentiostatic electrolysis according to the same procedure as described in the previous papers [18,19] with perpendicular polarization and at fixed wavelength of 500 nm using tungsten lamp. The light was directed on to the working electrode with an incidental angle of 15 ° . Current--potential (i--E), refiectivity--potential (R/Ro--E) and reflectivity-time (R/Ro--t) curves were recorded on a XY recorder Type 3077 (Yokogawa Ltd.). All the measurements were carried o u t at 26 -+ 1°C. Before each run to record a potential scan diagram, a sufficient amount of pure nitrogen gas was bubbled through the electrolytic solution to remove the dissolved oxygen. RESULTS AND DISCUSSION

i--E and R/Ro--E curves of gold measured simultaneously in the base electrolyte solution containing different concentrations of adenine are shown in Fig. 1. The potential scan was started from --1.1 V to the positive potential side and reversed at +0.4 V with a sweep rate of 100 mV s -l. As seen in Fig. 1, the base

111

.Lo O S m A c m -2

a

b

o

05% >'~ -1.0

-0.5

0

E/V vs Ag/AgCI Fig. 1. Current-potential and reflectivity-potential curves of Au in 0.1 M NaC104 in the absence and presence of adenine. A d e n i n e c o n c e n t r a t i o n : (a and c) 0, (b and f) 1.2 × 10 -4, (d) 9.0 × 10 -6, (e) 3.6 × 10 -s M. Wavelength: 500 nm. Potential sweep rate: 100 mV s -1.

electrolyte gives a fiat double layer region on the i--E curve over whole potential range investigated (curve a), but the R/Ro--E curve is characterized by two approximately linear portions intersecting at about --0.1 V for the sweep in either the anodic or cathodic direction (curve c). This potential value corresponds to the point of zero charge for gold in neutral solutions [20--22]. When adenine was added to the solution, no appreciable change was found on the i--E curve (curve b in Fig. 1), while the marked decrease in reflectivity was observed on the R/Ro--E curves in the potential region more positive than --0.8 V (curves d, e and f in Fig. 1). The reflectivity change was enhanced with the increase in the adenine concentration, but it tended to be saturated when the adenine concentration was more than 1 X 10 -4 M. Such a trend suggests that the adsorption of adenine takes place on the gold electrode surface, because the decrease in reflectivity was known to be produced by the adsorption of some organic compounds [ 17,18]. The hysteresis on the R/Ro--E curve, which is the most evident in the intermediate concentration, may be associated with the slow adsorption-desorption of adenine, as is the case of halide anions [14]. The R/Ro--t curves were measured by a potential step m e t h o d with the solutions containing different concentrations of adenine. The potential was first set at --1.0 V where no specific adsorption of adenine is expected and then stepped to a more positive value, and the subsequent decay of R/Ro was recorded until there was no further decrease in R/Ro. The same procedure was applied to the

112

.st 0 -1.0

-0.5 0 E / Vvs Ag/AgCI

Fig. 2. Potential d e p e n d e n c e of the reflectivity change due to the adsorption of adenine. Adenine c o n c e n t r a t i o n . (a) 1.8 x 10 -6 , (b) 3.6 x 10 -6, (c) 9.0 x 10 -6, (d) 1.8 x 10 -s, (e) 3.6 x 10 -s, (f) 1.2 x 10 -4 M. Wavelength: 500 nm.

base electrolyte solution as a reference. Then the difference between the magnitudes of reflectivity decrease at equilibrium in the presence and in the absence of adenine, denoted as AR/Ro, can be obtained by subtracting the latter from the former value obtained at the same potential. In Fig. 2, the values of AR/Ro are plotted against the electrode potential, in which the AR/Ro--E curves are of a quasi-bell-type having a maximum at --0.1 V. It is clearly seen that the adsorption of adenine occurs in more positive potential region than --0.8 V and attains its maximum at --0.1 V. This potential agrees with the point of zero charge observed in the base electrolyte solution. When the concentration of adenine is more than 1.0 X 10 -4 M, the AR/Ro at - 0 . 1 V tends to a limiting value. The reflectivity change due to the presence of an adsorbed layer formed on the electrode surface has been treated theoretically by McIntyre and Aspnes [11], who derived eqn. (1) as an expression of AR/Ro observed with perpendicularly polarized light when an optically uniform monolayer is present at an electrode-solution interface.

R0 1,0=1

X

\ns 2 --~MM

(1)

In eqn. (1), d is the thickness of monolayer, ¢ the incidental angle, k the wavelength of the incident light ray, ns the refractive index of the bulk electrolyte solution. ~ad and ~M are the complex dielectric constants of the adsorbed layer and the metal substrate, respectively. When the a m o u n t of adsorption is less than monolayer coverage, the change in reflectivity due to adsorption at surface coverage O, denoted as (AR/Ro)±.o,is expressed as AR

o)lo=( o)lo

o

(2)

provided that ~ad and ~M are not altered during adsorption [8,11,12]. The validity of eqn. (2) was actually justified in the adsorption studies of oxygen, some inorganic anions and organic c o m p o u n d s on various metal electrodes. For example, Koch [23] and Conway and Gottesfeld [24] proved the linearity between (AR/Ro)±and the charge required to form the oxide layer

113

during anodization of platinum. We obtained the adsorption isotherms of aromatic hydrocarbons on a gold electrode from the measurement of (AR/Ro)± by assuming that the relation given by eqn. (2) holds, and the data were suggestive of the flat orientation of these molecules on the electrode surface [18]. Such results agree very closely with those obtained by Green and Dahms [25] for the same adsorption systems with the radiotracer technique. Then an extension of the treatment based on eqn. (2) to the present case may be expected at least in the submonolayer region. On the basis of this assumption, an a t t e m p t was made to analyse the electrosorption of adenine quantitatively with the optical measurements at 500 nm, where a maximal value of --AR/Ro was obtained * Since the adsorption behaviour of adenine observed in Fig. 2 is very similar to that in the electrosorption of aromatic hydrocarbons on gold studied by Green and Dahms [25], application of the same isotherm as used by them to the present results was examined. The isotherm is represented by eqn. (3), 0/(1 - - 0 ) p = cK

(3)

where p is the number of water molecules being replaced by one molecule of adsorbed species, and c is the concentration in bulk solution. The constant K is the adsorption coefficient given by eqn. (4), K = e x p ( - - A G ° d / R T + c~FE/RT)

(4)

where AG°o is the standard free energy change of adsorption at the point of zero charge, a a constant, and E the potential difference between the point of zero charge and the potential at which the observation is made. Equation (3) is found to hold in the potential region where the orientation of the adsorbed solvent molecules is indepenent of potential. The use of eqn. (3) enables us to know the area occupied by the adsorbed adenine on the electrode surface in terms o f p . Combination of eqns. (3) and (4) leads to eqn. (5), 2.30 log(O/c) = 2 . 3 0 p log(1 -- 0) -- A G ° d / R T + c~FE/RT

(5)

Then the determination of p and AG°o was made using the data in Fig. 2 according to the following procedure. The potential range was restricted to the negatively polarized region (i.e., between --0.1 and --0.8 V) by considering that the point of zero charge remained unchanged t h r o u g h o u t the measurement. A linear relation between log c and E was obtained at constant AR/Ro, i.e., at constant 0, from which the value of oLF/2.3RT was calculated to be 11.2. Assuming that the limiting value of - - A R / R o = 1.7% in Fig. 2 corresponds to the reflectivity change at the monolayer coverage of adenine, the numerical values of p and AG°d can be obtained using eqn. (5). The parameter p can be determined from either log(O/c) vs. log(1 -- 0) plot at a constant potential or log 0 -- (aFE/2.3RT) vs. log (1 -- 0) plot in a given concentration. Examples of these two plots are shown in Figs. 3 and 4, respectively. Both the plots give straight lines and the values of p obtained from Figs. 3 and 4 are

* T h e t y p e of a d s o r p t i o n i s o t h e r m seems t o b e virtually i n v a r i a n t in t h e w a v e l e n g t h region o f 4 0 0 - - 5 2 0 nm.

I14

-5 r'l

~h r'q

~O

zs I OD

0

22

b

-0'8

-0'6 -O'h tog (1-8)

-0'.2

-1'0

-0'5 log (1-8)

0

Fig. 3. Plots o f l o g ( 8 / c ) vs. log(1 - - 8) f o r a d e n i n e a d s o r p t i o n . T h e values o f 8 were calculated using t h e values o f AR/Ro at (a) - - 0 . 3 , ( b ) - - 0 . 4 a n d (c) - - 0 . 5 V in Fig. 2. Fig. 4. Plot of log 8 - in Fig. 2.

(aFE/2.3RT)vs.

log(1 - - 8) for a d e n i n e a d s o r p t i o n using t h e curve (e)

4.7 and 4.8, respectively. The linearity of these plots, as well as the coincidence in p values obtained by the different methods, suggest that the present treatm e n t based on eqn. (3) is valid. The intercept of log (0/c) vs. log(1 -- 0) gives the value of AG°d to be --58.6 kJ mol -~ . From the value of p, the area occupied by one adenine molecule on the electrode surface can be calculated to be approximately 0.42 nm 2, assuming that of an adsorbed water to be 0.09 nm 2. This value is close to 0.40 nm 2 which is the estimated area of the plane molecule using a molecular model of adenine. Then it is quite likely that adenine is adsorbed in a flat orientation on the negatively charged electrode surface. This value is also close to that of the area occupied by adenine on a mercury electrode (reported by Kinoshita et al. [6] ). Reflectivity measurements of adenine derivatives, such as deoxyadenosine, dAMP, dADP and dATP, were also carried out in the same procedure. The presence of these derivatives caused no appreciable change on the i--E curves in the potential range from --1.1 V to + 0.4 V, however, it led to a remarkable reflectivity decrease on the R/Ro--E curves at more positive potentials than --0.8 V, indicating that the derivatives also adsorb on a gold electrode. Some examples are shown in Fig. 5. AR/Ro vs. E obtained for the derivatives by the same way as above are shown in Fig. 6a--d. Comparing to the case of adenine, none of the derivatives gives a simple bell-type AR/Ro vs. E relation, i.e., the decrease in reflectivity is n o t so significant at more positive potentials than --0.1 V. This fact seems an indication of the participation of deoxyribose and phosphate groups in the adsorption processes, especially in the positive potential region. The effect of HPO~- can be readily seen in more positive potential region, while that of deoxyribose is apparently very slight (Fig. 6e and f). The anodic oxidation of deoxyribose which

115

dATP

lo 05 DC

N O Oeoxyrl bose o

~ os 1G

dADP

NazHPO4

f

05

-1.0

o

-0'5 [) E / V vs Ag/AgC[

-05

0 -05 E / V vs Acj/AgCt

0

Fig. 5. R e f l e c t i v i t y - p o t e n t i a l curves o f A u in 0.1 M NaC1Oa in t h e a b s e n c e (a) a n d in t h e prese n c e o f (b) 1.4 X 10 -s M d e o x y a d e n o s i n e , (c) 1.4 × 10 -s M dAMP, (d) 1.1 × 10 -s M d A D P a n d (e) 1.1 × 10 -s M d A T P . W a v e l e n g t h : 500 n m . P o t e n t i a l s w e e p r a t e : 1 0 0 m V s -1 . Fig. 6. P o t e n t i a l d e p e n d e n c e o f t h e r e f l e c t i v i t y c h a n g e o b t a i n e d w i t h 0.1 M NaC104 s o l u t i o n s c o n t a i n i n g (a) 5.4 X 10 -s M d e o x y a d e n o s i n e , (b) 4.6 X 10 -s M dAMP, (c) 5.3 X 10 -s M d A D P , (d) 7.8 X 10 -s M d A T P , (e) 9.9 x 10 -s M d e o x y r i b o s e a n d (f) 1.0 × 10 -3 M Na2HPO4. Wavel e n g t h : 500 nm.

occurs at about --0.3 V prevents the detailed observation of the latter effect. However, the oxidation of deoxyadenosine, dAMP, dADP and dATP can n o t be recognized in the observed potential range, indicating the difficulty of oxidation of the deoxyribose moiety in these derivatives. Adsorptivity of adenine and its derivatives can be evaluated in terms of p and A G 0 d a s listed in Table 1. These values were obtained by the plots of log(O/c)vs. log(1 -- 0) and log 0 -- o~FE/2.3RT) vs. log(1 -- 0), in which the potential range was also restricted between --0.1 and --0.8 V. The values of p obtained for all TABLE 1 A d s o r p t i o n p a r a m e t e r s o f a d e n i n e a n d its derivatives o n A u f r o m 0.1 M NaCIO4 S t a n d a r d states 1 m o l 1-1 in s o l u t i o n a n d a value of 0 for w i t h 0 / ( 1 - - 0)P = 1 o n t h e surface. Compound

p

Adenine Deoxyadenosine dAMP dADP dATP

4.7 5.3 3.7 3.8 3.6

A G ° d / k J mo1-1 + 0.2 + 0.3 +- 0.3 + 0.3 + 0.4

--58.6 --56.1 --41.4 --42.3 --43.1

+ + + + +

1.6 0.8 1.2 2.4 2.0

116

the compounds are essentially equal, implying that the derivatives investigated are still adsorbed lying with the purin moiety fiat to the negatively charged electrode surface. The AG°d values of three deoxynucleotides are about 12 kJ mo1-1 smaller than those of adenine and deoxyadenosine. The lower adsorptivity of deoxynucleotides would be due to the hydrophilic property of phosphate groups. A repulsive interaction between phosphate groups of the adsorbed molecule and the negative charges on the electrode surface would also be responsible for such a tendency. REFERENCES 1 H. B e r g m G. Mflazzo (Ed.), T o p i c s in B l o e l e c t r o c h e m i s t r y a n d B i o e n e r g e t i c s , V o l . 1, J. Wiley, N e w York, 1976, pp. 39--104. 2 P.J. Elving in G. Milazzo (Ed.), T o p i c s m B i o e l e c t r o c h e m i s t r y a n d B l o e n e r g e t l c s , V o l . 1, J . Wiley, N e w York, 1976, pp. 179--286. 3 H. K l n o s h i t a , S.D. C h r i s t i a n , M.H. K i m , J . G . B a k e r a n d G. D r y h u r s t m D . T . S a w y e r (Ed.), E l e c t r o c h e m m a l S t u d i e s o f B i o l o g i c a l S y s t e m s , A m e r i c a n C h e m i c a l S o c i e t y , W a s h i n g t o n , D.C., 1 9 7 7 , p p . 113--142. 4 D. K r z n a r i g , P. V a l e n t a a n d H.W. N i l r u b e r g , J. E l e c t r o a n a l . C h e m . , 6 5 ( 1 9 7 5 ) 8 6 3 . 5 D. K r z n a r i ~ , P. Valenta0 H.W. N ~ i m b e r g a n d M. B r a n i c a , J. E l e c t r o a n a l . C h e m . , 9 3 ( 1 9 7 8 ) 4 1 . 6 H. K m o s h i t a , S.D. C h r i s t a n a n d G. D r y h u r s t ~ J. E l e c t r o a n a l . C h e m . , 8 3 ( 1 9 7 7 ) 1 5 1 . 7 W.N. H a n s e n , J . D . E . M c I n t y r e , R . H . M u l l e r , J. K r u g e r , V . S . S r i n i v a s a n a n d A . C . S i m o n in R . H . M u l l e r ( E d . ) , A d v a n c e s in E l e c t r o c h e m i s t r y a n d E l e c t r o c h e m i c a l E n g i n e e r i n g , V o l . 9, W i l e y , N e w Y o r k , 1 9 7 3 . 8 T. T a k a m u r a a n d K. T a k a m u r a i n T. T a k a m u r a a n d A. K o z a w a (Eds.), S u r f a c e E l e c t r o c h e m i s t r y , J a p a n S c i e n t i f i c S o c i e t i e s Press, T o k y o , 1 9 7 8 , p p . 1 7 9 - - 2 4 1 . 9 M.W. H u m p h r c y s a n d R . P a r s o n s , J. E l e c t r o a n a l . C h e m . , 7 5 ( 1 9 7 7 ) 4 2 7 . 1 0 M.W. H u m p h r e y s a n d R. P a r s o n s , J. E l e c t r o a n a l . C h e m . , 8 2 ( 1 9 7 7 ) 3 6 9 . 1 1 J . D . E . M c I n t y r e a n d D.E. A s p n e s , S u r f a c e Sci., 2 4 ( 1 9 7 1 ) 4 1 7 . 1 2 J . D . E . M c I n t y r e in R . H . Muller ( E d . ) , A d v a n c e s in E l e c t r o c h e m i s t r y a n d E l e c t r o c h e m i c a l E n g i n e e r i n g , Vol. 9, Wiley, N e w Y o r k , 1 9 7 3 , p p . 6 1 - - 1 6 6 . 1 3 T. T a k a m u r a , K. T a k a m u r a , W. N l p p e a n d E. Y e a g e r , J. E l e c t r o c h e m . S o c . , 1 1 7 ( 1 9 7 0 ) 6 2 6 . 1 4 T. T a k a m u r a , K. T a k a m u r a a n d E. Y e a g e r , J. E l e c t r o a n a l . C h e m . , 2 9 ( 1 9 7 1 ) 2 7 9 . 1 5 T. T a k a m u r a a n d K. T a k a m u r a , J. E l e c t r o a n a l . C h e m . , 3 9 ( 1 9 7 2 ) 4 7 8 . 1 6 T. T a k a m u r a , Y. S a t o a n d K. T a k a m u r a , J. E l e c t r o a n a l . C h e m . , 4 1 ( 1 9 7 3 ) 3 1 . 1 7 T. T a k a m u r a , K. T a k a m t t r a a n d Y. H a y a k a w a , D e n k i K a g a k u , 4 1 ( 1 9 7 3 ) 8 2 3 . 1 8 T. T a k a m u r a , K. T a k a m u r a a n d F. W a t a n a b e , S u r f a c e Sci., 4 4 ( 1 9 7 4 ) 9 3 . 1 9 F. W a t a n a b e , K. T a k a m u r a a n d T. T a k a m u r a , D e n k i K a g a k u , 4 3 ( 1 9 7 5 ) 4 6 9 . 2 0 E . K . V e n s t r e m , V.I. L i k h t m a n a n d P.A. R e b m d e r , D o k l . A k a d . N a u k S S S R , 1 0 7 ( 1 9 5 6 ) 1 0 5 . 21 M. P e t i t a n d J. Clavilier, C o m p t . R e n d . (Paris), C 2 6 5 ( 1 9 6 7 ) 1 4 5 . 2 2 J. C l a v i h e r a n d V . H . N g u y e n , C o m p t . R e n d . (Paris), C 2 6 7 ( 1 9 6 8 ) 2 0 7 . 23 D.F.A. Koch, Nature, 202 (1964) 387. 2 4 B.E. C o n w a y a n d S. G o t t e s f e l d , J. C h e m . Soc. F a r a d a y T r a n s . I, 6 9 ( 1 9 7 3 ) 1 0 9 0 . 2 5 M. G r e e n a n d H. D a h m s , J. E l e c t r o e h e m . S o c . , 1 1 0 ( 1 9 6 3 ) 1 0 7 5 .