pH-Sensitive phosphate lipid self-assembled monolayers on gold

COLLOIDS AND Colloids and Surfaces

ELSEVIER

A

SURFACES

A: Physicochemical and Engineering Aspects 103 ( 1995 ) 159-165

pH-Sensitive phosphate lipid self-assembled monolayers on gold Naotoshi Nakashima *, Toshiyuki Taguchi Department of Applied Chemistry, Faculty of Engineering, Nagasaki University, Nagasaki 852, Japan Received 7 February 1995; accepted 24 April 1995

Abstract

Self-assembled monolayers (SAMs) of a phosphate lipid, bis(11-mercaptoundecyl)phosphoric acid, were formed on gold electrodes via chemisorption from ethanol solutions by altering the immersion time. The electrochemistry of the lipid monolayers on gold electrodes was examined by using methylviologen and dipropanesulphonate-4,4'-bipyridinium as a cationic and a zwitterionic redox-active marker, respectively. SAMs of n-dodecylmercaptan, 11-mercaptoundecane1-ol and 11-mercaptoundecane-l-oic acid on gold electrodes which were fabricated by 24 h adsorption from ethanolic solutions of each thiol compound, could not block the redox reaction of methylviologen. However, SAMs of the phosphoric acid lipid on gold which were prepared by adsorption over 6 h exhibited pH-responsive ion-gating phenomena; at lower pHs (
Keywords: Gold electrodes; Monolayers; Self-assembled monolayers 1. Introduction

The design and characterization of modifying electrodes by assembled organic monolayer, bilayer and Langmuir-Blodgett (LB) films are currently being studied by many research groups. Selfassembled monolayers (SAMs) of alkanethiols and alkyl-disulfides built-up on gold are the subjects of considerable interest especially, because the strong gold-sulfur coordination bonding enables the formation of stable monolayers even in solution [ 1 ]. Excellent versatility in introducing various functional groups at the terminal position of the monolayers produces sophisticated strategies for making * Corresponding author. 0927-7757/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 9 2 7 - 7 7 5 7 ( 9 5 ) 0 3 2 3 6 - 3

monolayer systems with specific functions I-2-11]. Our interest in this field is to design and characterize SAMs of lipids on metal electrodes. From the aspect of practical application and utilization of biomembrane functions, developments of molecular membrane electrodes having biomembranemimetic structures and functions should be highly attractive and important. Preliminary accounts of the formation of SAMs of the mercaptancontaining lipid, 1, (see Scheme 1) on gold and their pH-responsive electrochemical properties have been given in the literature [10,11]. Characteristics of molecular bilayer membranes of acidic phospholipids in aqueous solution have been investigated extensively in relation to membrane functions such as ion recognition, ion transport,

N. Nakashima, T. Taguchi/Colloids Surfaces A: Physicochem. Eng. Aspects 103 (1995) 159-165

160

HS(CH2)110,, ~zO sF HS(CH2)110 "OH

HS(CH2)11- CH3 2

1

HS(CH2)ll-OH 3

HS(CH2)lo-C "~O OH 4

phase separation, morphology and cell fusion [12-15]. Self-assembled monolayers serve as ultimately thin barrier layers against the approach of solutionphase species to an electrode surface. In this paper we describe the pH-responsive gate properties and ion responses of SAMs of 1 on gold with methylviologen (MeV) and l,l'-dipropanesulphonate-4,4'bipyridinium (SO3V) as electroactive markers. A comparison of the blocking ability of SAMs of 1 with those of thiol compounds, 2-4 is also reported. 2. Experimental The synthesis of bis(11-mercaptoundecyl)phosphoric acid, 1, is as follows [11]. 11-Bromoundecanol (22 g, Jansen Chem. Co.) and phosphorus oxychloride (4.5 g) were dissolved in benzene (70 ml) and the mixture was refluxed for 24 h. After evaporating the solvent, 20 ml of water was added to the residue followed by stirring overnight at room temperature. Chloroform (25 ml) extraction and solvent evaporation gave a viscous oil, which was recrystallized twice from ethyl acetate to yield bis(ll-bromoundecyl)phosphoric acid. Bis(11-bromoundecyl) phosphoric acid (2.0 g), thiobenzoic acid (2.0 g) and triethylamine (1.5g) were dissolved in ethanol (50ml) and the mixture was refluxed for 48 h under a nitrogen atmosphere. Addition of ethyl acetate (40 ml) to the reaction mixture at ambient temperature gave a precipitate, which was filtered off. Evaporating the solvent gave an oily product which was dissolved in ethanol (2.0 ml) followed by the addition of IN HC1 (15 ml) to produce a precipitate, which was recrystallized (20ml) twice from ethyl acetate. The obtained bis(llbromoundecyl)phosphoric acid (0.5 g) was reacted

with hydrazine hydrate (1.0 g) in methanol (30 ml) for 4 h under a nitrogen atmosphere. The reaction mixture was allowed to cool to room temperature, and 1N HC1 was added to produce a precipitate, which was separated and recrystallized twice from ethyl acetate (20 ml). (yield 0.2 g, mp 67-68°C). The product was identified with IR and 1H-NMR spectroscopy, TLC and elemental analysis. Calculated analysis for C22HavO4PS2: 56.14% C, 10.06% H; found: 55.85 % C, 9.87% H; N was not detected. n-Dodecylmercaptan, 2, was obtained from Tokyo Kasei and was used without further purification, i l-Mercaptoundecane-l-ol, 3, was prepared by the reaction of ll-bromoundecane-l-ol and thiobenzoic acid followed by reduction with hydrazine. ll-Mercaptoundecane-l-oic acid, 4, was available from a previous study [9]. MeV dichloride was prepared by the conventional method. 1,1'-SO3 V was synthesized from 4,4'-bipyridine and 1,3-propane sultone (Tokyo Kasei Co.). The final synthetic products were identified with 1HNMR and IR spectroscopy, TLC, and elemental analysis. The experimental procedure for making monolayer electrodes using 1 is as follows. A gold disk electrode (Bioanalytical Systems, diameter 1.6 ram) was polished with alumina (Beuhler Co., particle sizes: 1, 0.3 and finally 0.025 p.m) and was rinsed with acetone, Millipore water (Milli-Q Plus) and then ethanol, followed by immersion in an ethanolic solution of 1 (10 -3 M) for a given time at 25°C. The modified electrodes were rinsed twice with pure ethanol and then allowed to air dry. Cyclic voltammetry was conducted with a potentiostat (Toho Giken PS-06 or Bioanalytical Systems, BAS100B) in 0.1 M KC1 aqueous solution at 25°C in the presence of a given electroactive compound. A saturated calomel electrode (SCE) and a platinum wire were used as the reference electrode and the counter electrode, respectively. The pH was adjusted with HC1 and NaOH aqueous solutions. 3. Results and discussion

3.1. Ion-gate monolayers Fig. 1 shows cyclic voltammograms (CVs) of MeV for SAMs of alkanethiols 2-4 on gold, pre-

161

Nakashima, T. Taguchi/Colloids Surfaces A: Physicochem. Eng. Aspects 103 (1995) 159-165

I/%, l i -0.~

I5#A

-0.950

c/ I

i

1

i -0.950

~

EN

-0.950

pH 8

vs. SCE

.

5

~

pH 6 . 2 ~

T 1

10 /.zA

pH 4.38

f

I

-1.0 E/V vs.SCE

Fig. 1. Cyclic voltammograms (CVs) of SAMs of 2 (curve a), 3 (curve b) and 4 (curve c) on gold electrodes which were prepared by 24 h adsorption from 10 mM ethanolic solution of each thiol compound. Dotted lines show CVs at a bare electrode. The solutions were 2mMMeV in 0.1 M KC! (pH 7.0). Scan rate, 200 mV s -1.

pared by 24 h immersion of polished gold-disk electrodes in a 10 m M ethanolic solution of each thiol compound. Despite a significant dipping time, the blocking ability of these three coated electrodes for MeV is very small. Similar behavior was obtained at lower ( p H 3.5) and higher ( p H 11.6) pHs. MeV is water soluble but it is evident that it permeates easily into the SAMs of 2 - 4 on gold over a wide p H range. However, SAMs of 1 on gold showed high blocking ability against MeV and the barrier was p H responsive. Typical CVs for the modified electrode prepared by 6- and 24-h electrode immersion are shown in Fig. 2. The plots of anodic currents of the v o l t a m m o g r a m s as a function of p H for both electrodes give breaks near p H 4 and p H 10 (Fig. 3). A contribution of hydrogen bonding between phosphoric acid and phosphate anion at the monolayer surface may be

v

I

l

i

,

I

-0.4

E/V vs. SCE

]

-1.0

I

I

i

i

=

I

-0.4

E/V vs. SCE

Fig. 2. CVs of SAMs of I on gold electrodes at 25°C. Dipping time in preparing the SAMs were 6 h (left) and 24 h (right) in 1 mM ethanolic solution of 1. The solutions were 2 mM MeV in 0.1 M KC1. Scan rate, 200 mV s 1.

suggested for the bimodal p H profiles (Fig. 4). The pH-responsive electrochemistry required higher surface coverage because self-assembled monolayers of 1 on gold (with a soaking time was less than 1 h) could not act as a barrier against the electron transfer reaction of MeV at all measured p H values (pH 3-12). Fig. 5 shows CVs of S O 3 V on a 1/gold electrode in Which the electrode was prepared by 40 min adsorption from an ethanolic solution of 1. Although the change in the intensity of the voltammograms was not large, the anodic currents gave a p H dependence (Fig. 6). Monolayers of 1 on Au prepared by a long dipping time (longer than 6 h) blocked the permeation of the marker into the monolayers at all measured p H values (Fig. 6). As noted above, when MeV was used as the electroactive marker, the redox couple between MeV and the MeV cation radical (Eq. 1) was inhibited at the lower p H region where the

N, Nakashima, T. Taguchi/Colloids Surfaces A: Physicochem. Eng. Aspects 103 (1995) 159-165

162

12

a

b

10

I0

8

2

0

w

0

v

3

5

7

9

11

5

3

7

g

11

pH

pH

Fig. 3. Plots of anodic currents (ir~) of the CVs (shown in Fig. 2) as a function of pH. Dipping time for preparing the SAMs of 1 were 6 h (a) and 24 h (b). Open and closed circles are pH lowering and pH increasing cycles, respectively.

-S-'v'~O..

0

-S - ~ ' ~

O

-S"vvv~" 0

OH

i-S

O

,P.

-S " - ' ' . ~ '

0

.0

~P"

- S ~ O

Au

.S .~..-v-.~. O

"OH

~ ~ ' ~

0

-S-~A~"

O

-S-"~'~

O..0

;p:

~ ~ ' -

O~ .0

O)ot H

~-'~'-

0

0/

~-~-~

O..0

,S~'-'vv~ 0

"OH

.P.

O"

sp"

-S-'v'~- 0

Au

;p".0

•S ~

0

. S ~ O ,

P"

-S -~'A~-~- 0

.O

;-~A~-

"OH

0

~p". 0

•S - ' ~ - ' v ' - O

"O)ot

~ ~-'v~'- 0

.O/H

~ ~ ' ~

0

-S"v~

0

,.P.

OH

"0"

~p".0 "0" .0

7, O"

Fig. 4. Possible models for the structure of the monolayer of l on gold and for the intermolecular hydrogen bonding of 1.

CHs-N

*'CH3

(++)

~

Red

Ox

CH3--

N ~ (+)

(1) terminal group of the lipid was neutral (phosphoric acid). At higher pH values, where the monolayer possessed anionic charge, a clear redox behavior of MeV was evident. This shows that by lowering the pH, the monolayer closes a gate allowing MeV into the membrane and opens it at higher pH values (see Fig. 7). Both MeV and the MeV cation radical are positively charged. Coulombic interaction between the negatively charged monolayers and the positively charged marker is thought to

cause the marker to become incorporated into the monolayers. A cationic marker, 1-trimethylammonium methyl ferrocene, also gave a similar pH-responsive gating behavior for the SAM of 1 with a rather lower surface coverage [11]. In contrast, when SO3V was the marker, the gate was opened at lower pH values and closed at higher pH values (see Fig. 6). In this case, however, the gating property was observed only for the anodic current. This can also be explained by the coulombic interaction; i.e., SO3V (oxidized form) is a zwitterion and therefore is not pH responsive. The net charge of the reduced form of SO~-V is anionic (Eq. 2), which causes electrostatic repulsion with the anionic lipid monolayer in the higher

N. Nakashima, T. Taguchi/Colloids Surfaces A." Physicochem. Eng. Aspects 103 (1995) 159-165

163

Red

"O3S(CH2)3- N

+- (CH2)3SO 3"

Ox

"O3S (CH2)3-- N

~

(0) pH region. A possible model for the interfacial structures of the monolayer membranes for the pH-sensitive electrochemistry is shown in Fig. 7. 3.2. Ion response

Umezawa and co-workers have described the voltammetric response of LB films (multilayers or monolayers) of dodecylphosphoric acid and dioctadecyldimethylammonium bromide toward the alkaline earth metal ions and the perchlorate anion [16], and of a long-alkyl-chain derivative of cyclodextrin for electroactive compounds such as [Co(phen)3] 2+, [Mo(CN)8] 4- and p-quinone

i' t f t

,,/bare

t i

phlo.4

pH 7

i

]

.

a

*

- (CH2)3SO 3-

(2)

(-) [17]. LB films, in general, are not stable for long in aqueous solutions. Stability is one of the most important requirements for ion sensors. SAMs provide stable monolayers even in aqueous solutions because the mercaptan group is thought to form coordination linkages with gold. Steinberg et al. reported ion-selective voltammetric responses of SAMs with a tetradentate ligand on gold [3]. We have described voltammetric responses of SAMs of 1 for alkaline earth metal ions [ 11 ]. In this study, MeV was used as an electroactive marker. Both SAMs of 1 on gold electrodes prepared by 6 h and 12 h soaking in ethanol solution of 1 exhibited voltammetric response. Fig. 8 shows the intensity of anodic currents of the voltammograms as a function of the concentration of CaC12. The sensitivity was found to be one order higher than the experiment with the SAM of 1 having more disperse surface coverage [11]. Structural defects in the monolayer may cause the decrease in the sensitivity. Note that surface coverages are related to the sensitivity of ion recognition. The

~

pH 5 . 2 ~ _ / / ~ pH 4 . 2 ~ / / / I

-0.9

!

1 2 /z A ,i

!

!

i

I

-0.4

EN vs. SCE

Fig. 5. CVs for a SAM of 1 on a gold electrode. Dipping time in preparing the monolayer was 40rain in 1 m M ethanolic solution of 1. The dotted line shows a CV at a bare electrode. The solutions were 2 m M SO~-V in 0.I M KC1. Scan rate, 200 mV s - I.

0

m

2

m

4

6

8

10

12

pH Fig. 6. Plots of ipa of the voltammograms for l/Au monolayers as a function ofpH. Dipping times for preparing the monolayers were 40rain (O, 0 ) and 6 h (A). The solutions were 2 m M SO3V in 0.1 M KC1.

164

N. Nakashima, T. Taguchi/Colloids Surfaces A. Physicochem. Eng, Aspects 103 (1995) 159-165

lower pH

higher pH

S~O, ~0 S~o'P'oN S~O, =0 S ~ O "P"OH S~O, ~0 S~o'P'oH S~O~ ~0 S~o'P'oH S~O. ~0 S~o'P'oH S~O. ~0 S ~ O "P"OH

~0~ .0 ~O'P,O -

M.v

2" "o ~0...0 ~ 0 "P"0"

MeV

~-,,,",.-~o

~ o ~0~

"SOs'-V

~0~ ~ 0

"P"o~0 "P"0" .0 "p"0

~ 0 ~ 0

.0 "P"0"

~ 0 ~ 0

..0 "P"0"

~ 0

SOs'-V

~ 0 ~o'P'o

..0 -

~

~

SOs'-V

S03"-V

~ 0 , ,0 ~o'P'o ~ 0 ~o'P'o

.0 ,

Fig. 7. Schematic illustration for the pH-responsive gates of the SAMs of lipid 1.

10

8

ja 6

2

response time in the voltammogramic change was very fast (less than 5 s). The added Ca 2÷ is believed to chelate with phosphate groups. However, the binding energy of the linkage on this twodimensional monolayer system is expected to be rather small, because simple rinsing in pure water can remove the binding ion. A possible molecular mechanism for the ion-responsive change in the voltammograms is shown in Fig. 9. Chelation of Ca 2+ with the phosphate at the terminal of the monolayer decreases the negative charge-density of the surface, which leads to the decrease in electrostatic attraction between MeV and the monolayer,

4. S u m m a r y and conclusions

(1) Phosphoric-acid-terminated alkanethiol provided organized self-assembled lipid monolayers on gold electrodes which acted as barriers against the permeation of MeV into the monolayers. SAMs of dodecylmercaptan, ll-mercaptoundecane-l-ol and l l-mercaptoundecane-l-oic acid were unable to block the electrochemistry of MeV. (2) SAMs of the phosphate lipid showed pH-responsive permeation for MeV and SO3V. The gate of the monolayer for MeV was opened at higher p H values and closed at lower p H values. In contrast, for S O j V , the gate was closed at higher p H values and opened at lower p H values. This gating behavior could be explained by coulombic interaction between the terminal functional group of the monolayer and the redox-active species in solution, p H changes may cause a structural change in the monolayers which causes the

_S~o'P'o

.S~o'P"o C a 2* ~

1

0

-6

-5

I -4

*

'

t

-3

-2

-1

Iog[CaCl2] / M

Fig. 8. ip, of CVs (scan rate, 200 mV s -1) of the monolayer of 1 vs, Ca2÷ concentration: curve a, electrode dipping for 6 h; curve b, electrode dipping for 15h. The solutions were 2 mM MeV in 0.1 M KCI at pH 9.0.

S~o'P-o -S~O, .0 ~ S . , ~ o ' P "0

~.P".0

-S~O

"0),, 2*

~O.p.O

-S~O..0 S~o-P'~o

-S~O

•

Fig, 9. Possible molecular mechanism for the ion-induced voltamrnetric response at the lipid monolayer/electrode interface.

N. Nakashima, T. Taguchi/Colloids Surfaces A, Physicochem. Eng. Aspects 103 (1995) 159-165 p H - r e s p o n s i v e p e r m e a t i o n . H o w e v e r , the d e t e c t i o n a n d c h a r a c t e r i z a t i o n o f the expected small conform a t i o n a l c h a n g e s a c c o m p a n y i n g the p H c h a n g e require further study. (3) S A M s of the p h o s p h a t e lipid r e s p o n d e d to the C a 2÷ ion, which was d e t e c t e d by the change in the v o l t a m m o g r a m s o f MeV. H i g h e r surface c o v e r a g e with the m o n o l a y e r gave h i g h e r ion sensitivity. Defects in the m o n o l a y e r are likely r e l a t e d to the sensitivity. Finally, it is w o r t h y n o t i n g a g a i n t h a t selfa s s e m b l e d lipid m o n o l a y e r s with o r g a n i z e d surface structures have b e e n successively f o r m e d via the simple c h e m i s o r p t i o n m e t h o d from o r g a n i c solution. D e l i c a t e m o d u l a t i o n of lipid m o n o l a y e r s m a k e s possible the design of novel interfaces with specific functions.

[2] [.3] [4] [5] [6] [7] [.8] [9] [10]

Acknowledgments

[11]

This w o r k was s u p p o r t e d , in part, b y a G r a n t i n - A i d for Science R e s e a r c h from the M i n i s t r y of E d u c a t i o n , Science a n d Culture, J a p a n a n d the T o r a y Science F o u n d a t i o n .

[12] [,13] [,14] [,15]

References [ 1] For a review see: A, Ulman, An Introduction to Ultrathin Organic Films: From Langmuir-Blodgett to Self-

[16] [,17]

165

Assembly, Academic Press, Boston, MA, 1991, pp. 237-304. M. Mfiller, H. Ringsdorf, E. Rump, G. Wildburg, X. Zhang, L. Angermaier, W. Knoll, M. Liley and J. Spinke, Science, 262 (1993) 1706. S. Steinberg, Y. Tor, E. Sabatini, I. Rubinstein, J. Am. Chem. Soc., 113 (1991) 5176. K.D. Schierbaum, T. Weiss, E.U.T. Velzen, J.F.J. Engbersen, D.N. Reinhoudt and W. G6pel, Science, 265 (1994) 1413. N.L. Abbott and G.M. Whitesides, Langmuir, 10 (1994) 1493. B.R. Herr and C.A. Mirkin, J. Am. Chem. Soc., 116 (1994) 1157. C. Miller, P. Cuendet and M. Gr/itzel, J. Phys. Chem., 95 (1991) 877, E.W. Wollman, F.C. Doris, C.D. Frisbie, I.M. Lorkovic and M.S. Wrighton, J. Am. Chem. Soc., 116 (1994) 4395. M. Kunitake, Y. Deguchi, K. Kawatana, O. Manabe and N. Nakashima, J. Chem. Soc., Chem. Commun., (1994) 563. N. Nakashima, T. Taguchi, Y. Takada, K. Fujio, M. Kunitake, O. Manabe, J. Chem. Soc., Chem. Commun., (1991) 232. N. Nakashima and T. Taguchi, in T.E. Mallouk and D.J. Harrison (Eds), Interfacial Design and Chemical Sensing, ACS Symp. Ser. 561, American Chemical Soc., Washington DC, 1994, pp. 145 154. S. Ohnishi, Adv. Biophys., 8 (1975) 35. R. Nayar, L.D. Mayer, M.J. Hope and P.R. Cullis, Biochem. Biophys. Acta, 771 (1984) 343. N. Nakashima, R. Ando, H. Fukushima, T. Kunitake, Chem. Lett., (1985) 1503. H. Trauble, M. Teubner, P. Wooley and H. Eibl, Biophys. Chem., 4 (1976) 319. M. Sugawara, K. Kojima, H. Sazawa and Y. Umezawa, Anal. Chem., 59 (1987) 2842. K. Odashima, M. Kotato, M. Sugawara and Y. Umezawa, Anal. Chem., 65 (1993) 927.