Evaluation of Surface-Bound Membranes with Electrochemical

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Downloaded by CORNELL UNIV on July 20, 2012 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/ba-1994-0235.ch023

Evaluation of Surface-Bound Membranes with Electrochemical Impedance Spectroscopy Jianguo Li, N a n c y W . D o w n e r , a n d H. G i l b e r t S m i t h * T S I C o r p o r a t i o n , 57 U n i o n Street, Worcester, MA 01608

Membrane structures that contain the visual receptor protein rhodopsin were formed by detergent dialysis on platinum, silicon oxide, titanium oxide, and indium-tin oxide electrodes. Electrochemi-cal impedance spectroscopy was used to evaluate the biomembrane structures and their electncal properties. A model equivalent circuit is proposed to describe the membrane-electrode interface. The data suggest that the surface structure is a relatively complete single-mem-brane bilayer with a coverage of 0.97 and with long-term stability.’

C (OMMUNC IATO I N IN LIVING ORGANS IMS

is governed b y the c e l l bilayer membrane, w h i c h selectively recognizes specific c h e m i c a l messengers a n d responds accordingly. Receptor proteins located i n cellular membranes have evolved for highly specific recognition functions a n d are the natural sites o f action for a w i d e variety o f biologically active chemical components, i n c l u d i n g hormones, neurotransmitters, odorants, a n d many drugs. Biosensors based o n m e m b r a n e receptors require the p r o t e i n to be c o u p l e d both functionally a n d structurally w i t h electrical substrates. T h e interface between the biological recognition element a n d the solid substrate must allow electrical signal transduction and provide an environment con ducive to biological function. B o t h requirements are d e m a n d i n g . A significant barrier to p r o d u c t i o n o f this type o f sensor has b e e n the tremendous difficulty o f preparing functional a n d stable l i p i d - p r o t e i n bilayer membranes finked to * Corresponding author: 104 Foster Street, Littleton, MA 01460

0065-2393/94/0235-0491 $08.00/0 © 1994 American Chemical Society

In Biomembrane Electrochemistry; Blank, M., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1994.

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BIOMEMBRANE ELECTROCHEMISTRY

surfaces. S u c h membranes s h o u l d be functionally equivalent to free-standing membranes a n d must (1) p r o v i d e a " b l o c k i n g " interface to prevent u n i n t e n d e d permeabilities, (2) be chemically a n d mechanically stable o n solid substrates, a n d (3) incorporate specific p r o t e i n receptors.


Integral m e m b r a n e receptor proteins n o r m a l l y f u n c t i o n as part o f the l i p i d bilayer m e m b r a n e that separates intracellular a n d extracellular aqueous compartments. Proteins i n natural membranes are thus exposed to regions o f two different compositions: the high-dielectric aqueous m e d i a o n b o t h sides o f the membrane a n d the low-dielectric h y d r o p h o b i c hydrocarbon region at the core o f the l i p i d bilayer. Several procedures have b e e n developed to f o r m artificial membranes that retain properties o f natural biological membranes ( I , 2). These proce dures rely o n the self-assembly properties o f the m e m b r a n e components (lipids a n d proteins) to f o r m f u n c t i o n i n g m e m b r a n e structures. L i p i d bilayer structures have b e e n f o r m e d b y sonicating suspensions o f lipids to f o r m vesicles (3), spreading dissolved lipids across an orifice to f o r m planar bilayers (4), a n d by using L a n g m u i r - B l o d g e t t techniques to serially a d d monolayers to a surface ( 5 ) . A l t h o u g h L a n g m u i r - B l o d g e t t techniques offer good control i n deposition o f a l i p i d phase onto solid surfaces, the techniques for incorporating m e m b r a n e proteins are less w e l l developed. A n u m b e r o f workers have attached proteins to l i p i d monolayers a n d bilayers o n solid surfaces b y anchoring the p r o t e i n w i t h a h y d r o p h o b i c tail or b y covalently attaching the p r o t e i n to the l i p i d h e a d groups. O t h e r researchers have f o r m e d bilayers b y adsorbing vesicles containing proteins onto surfaces or b y i n c o r p o rating proteins into L a n g m u i r - B l o d g e t t films at an a i r - w a t e r interface a n d then depositing that film onto a surface (for reviews, see references 6 a n d 7). W e have reported the formation o f surface-bound l i p i d membranes that contain receptors o n planar electrode surfaces b y a m o d i f i e d detergent dialysis technique (8, 9). T h i s technique is an adaptation o f procedures previously used to incorporate m e m b r a n e proteins into free-standing l i p i d bilayer structures. Detergent dialysis has b e e n used to directly f o r m p r o t e i n containing bilayer m e m b r a n e vesicles f r o m solubilized p r o t e i n - l i p i d mixtures (10, 11). This approach allows the simultaneous incorporation o f l i p i d a n d protein, and it often retains p r o t e i n functionality. O t h e r approaches to f o r m i n g free-standing m e m b r a n e structures include the fusion o f p r o t e i n containing vesicles w i t h planar l i p i d bilayers (12) a n d the use o f detergent dilution techniques to insert proteins into unilamellar vesicles (13). T o assemble the surface-bound m e m b r a n e structure, w e first f o r m a hydrophobic monolayer b y u s i n g alkylsilanization to covalently attach l o n g chained hydrocarbon chains to hydroxyl groups i n the oxide layer o n the electrode surface. S u c h a h y d r o p h o b i c surface can be thought o f as one leaflet o f a m e m b r a n e bilayer. Alkylsilane-modified surfaces have b e e n w i d e l y used as substrates for l i p i d monolayers deposited b y L a n g m u i r - B l o d g e t t techniques. T h e lipids i n these monolayers have mobilities like those o f lipids


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i n natural membranes (14). H u a n g f o r m e d l i p i d multilayers o n such surfaces b y dialysis f r o m deoxycholate solutions o f l i p i d a n d f o u n d that w h e n p a l m i toyl-modified antibodies w e r e i n c l u d e d i n the detergent solution they also became associated w i t h the surface structures ( 1 5 ) . H u a n g f o u n d that the l i p i d diffused rapidly b u t that the antibody was relatively i m m o b i l e . W e simultaneously incorporate b o t h l i p i d a n d p r o t e i n b y using dialysis to remove detergent f r o m a s o l u b i l i z e d l i p i d - p r o t e i n mixture i n the presence o f the alkylsilanated substrate. U n d e r o u r conditions, f r o m the evidence i n this paper a n d elsewhere (9), the surface structures appear to b e single bilayer membranes. O u r hypothesis is that the h y d r o c a r b o n chains attached to the


surface serve as initiation sites f o r a l i p i d bilayer m e m b r a n e to f o r m as the detergent is slowly r e m o v e d . T h e m o d e l is o f a m e m b r a n e that is anchored to the surface b y h y d r o p h o b i c interactions w i t h the surface-bound h y d r o c a r b o n layer. Integral m e m b r a n e proteins are retained i n these structures b y their interaction w i t h the h y d r o p h o b i c core o f the m e m b r a n e

without being

directly attached to the electrode surface. M e m b r a n e s containing the visual p i g m e n t rhodopsin, a G - p r o t e i n - l i n k e d receptor, were chosen as a m o d e l system f o r this w o r k . R h o d o p s i n was one o f the first integral m e m b r a n e proteins whose amino acid sequence was deter m i n e d (16-18).

M o r e than 40 receptors have b e e n r e p o r t e d to have struc

tural a n d functional homologies w i t h r h o d o p s i n (19). T h i s chapter describes the use o f electrochemical i m p e d a n c e spectroscopy to evaluate l i p i d bilayer membranes containing r h o d o p s i n f o r m e d o n electrode surfaces.

Experimental Details Chemicals and Biochemicals. Acetonitrile ( A C N , 99%), tetrabutylammoniumtetrafluoroborate ( T B A F , 99%), tetracyanoethylene ( T C N E , 98%), and anhydrous n-hexadecane (99%) were used as received from Aldrieh. Chloroform (American Burdick & Jackson) and carbon tetrachloride (Mallinckrodt) were dried over aluminum oxide (Water Associates, Framingham, M A ) for at least 48 h before use. Octadecyltrichlorosilane (OTS) and dimethyloctadecylchlorosilane ( D M O C S ) were from Petrach Systems, Inc. (Bristol, P A ) . K C l , H S 0 , and H F (52%) of analytical reagent grade were obtained from Mallinckrodt. N-2-Hydroxyethylpiperazine-N'-ethanesulfonic acid ( H E P E S ) , ρ Κ 7.55) was from Sigma. Octyl^-D-glucopyranoside ( O G ) used as the detergent was from Calbiochem ( L a Jolla, C A ) . Disk membranes containing rhodopsin (Rh) were isolated from rod outer segments from bovine retinas (J. A . Lawson, Co., Lincoln, N E ) by flotation on 5 % polysucrose (Ficoll 400) (20). Platinum sheet (99.99%) and titanium rod (99.99%) were purchased from Johnson Matthey. p-Si ( 2 4 - 3 6 - Ω cm) and η-Si (100-Û cm) wafers with a silica layer thickness of 950 Â on the polished side and a gold film evaporated on the other side were provided by E G & G Reticon (Sunnyvale, C A ) . The indium-tin oxide (100-500Â-thick) coated glass substrates (ITO) were from Donnelly C o . (Holland, M I ) . 2

4

α

Electrode Preparation. Pt electrodes were polished with alumina polishing powder (1, 0.3, and 0.05 μηι) to a mirror finish and cleaned electrochemically by cycling the potential between hydrogen and oxygen evolution potentials i n 1 - M


494


H S 0 until the characteristic clean Pt wave pattern of hydrogen adsorption was observed. The electrode was next poised at 1.1 V versus a saturated calomel electrode (SCE) until the current decayed to less than 1 μΑ, which indicates complete oxidation of the electrode surface. The titanium electrodes were polished with diamond paste (1 and 0.3 μιη) in mineral oil and washed in acetone followed by deionized water wash. The T i 0 film was formed on titanium in 1 - M H S 0 while the potential was swept slowly in a positive direction at a sweep rate of 1 mV/s. The thickness of the anodic film was proportional to the applied growth voltage. Anodizing ratios for titanium at 1-mV/s sweep rate are reported to be 6 nm/V up to 2 V and 3.6 nm/V above 2 V (21). In this work, the growth potential was 31 V versus S C E , and the thickness was estimated to be 15 nm. The silicon oxide electrodes were 5- X 5-mm chips cut from a S i - S i 0 wafer. The ohmic contact was made by connection of a wire to the gold film on the rear side of the chip with silver conducting epoxy. The chip was then mounted on a glass tube and sealed along its edge with epoxy. A n etching process to reduce the S i 0 thickness was carried out in 5 % H F at room temperature. The I T O electrode was made of a 5- X 5-mm slice cut from I T O sheet. A copper wire was attached to the I T O film with silver conducting epoxy. The I T O slice was mounted on a glass tube with epoxy. A l l electrodes were washed thoroughly with deionized water and dried in a vacuum oven for 12 h at 100 °C before alkylsilanization. 2

4

2

2

4


2

2

Alkylsilanization. Alkylsilanization of electrode surfaces was carried out by a modification of the procedure of Sagiv (22). The anhydrous solvent was prepared with 80:12:8 hexadecane-chloroform-carbon tetrachloride under dried nitrogen in a glove bag (relative humidity < 4%). The dried electrodes were silanized by reaction in stirred 3 % (v/v) OTS solution for 3 h or 6% (v/v) D M O C S solution for 6 h. The silanized electrode surface was rinsed with dry solvent and then with chloroform and cured in a vacuum oven at 100 °C for 12 h. Detergent Dialysis. The technique of detergent dialysis, which is often used to form functional membrane vesicles (10, 11), was adopted to assemble membranemimetic structure onto electrode surfaces (9). The dialysis unit used in this work had two compartments separated by a dialysis membrane filter (Spectra/Pro 6 membrane, molecular weight cutoff 3500, Los Angeles, C A ) . The electrode was mounted in one compartment that contained detergent solubilized disk membranes. Dialysis was against a flowing stream of detergent-free buffer driven by a peristaltic pump (type 2232, Microperpex s, L K B , Sweden). Rod outer segment disk membranes were solubilized with 30-mM O G to a final rhodopsin concentration of 1 m g / m L . Care was taken to avoid bubbles in the dialysis compartments. The detergent dialysis was carried out at 4 °C in the dark or under red light filtered through a safe light filter (Kodak 2, Eastman Kodak Co.). The detergent dialysis was at a flow rate of 100 μΕ/πιπι for 20-30 h. Electrochemical Measurements. Cyclic voltammetry and alternating current (ac) impedance spectroscopy were performed using an ac impedance system ( E G & G Princeton Applied Research model 378) that included a potentiostat-galvanostat (model 273), a two-phase lock-in analyzer (model 5208), and an I B M PS/2 computer. For ac impedance measurements, a 5-mV sine wave was superimposed on an applied voltage bias from the potentiostat. The reference electrodes were saturated calomel electrodes ( S C E ; Fisher) for measurements in aqueous solution and silver electrodes


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for measurements in nonaqueous solution. A l l potentials are reported with respect to SCE.

Results and Discussion Surface Modification with Organosilanes. C h r o m a t o g r a p h i c (23) a n d other studies indicate that the order o f reactivity o f halosilanes w i t h silica is X S i R > X S i R > X S i R . This relationship was also observed w i t h Sn0 surfaces (24) based o n electron spectroscopy for chemical analysis ( E S C A ) . Organosilanes w i t h m o r e than one reactive group have the potential to b i n d to more than one surface site b u t also c a n cross-react to f o r m polymers. F o r instance, d u r i n g the i m m o b i l i z a t i o n o f diphenylphosphine groups o n silica, O s w a l d et a l . ( 2 5 ) demonstrated that the trichlorosilane reagent c l a i m e d slightly less than two sites p e r silane. T h e fate o f the remaining silane reactive group is important to the surface structure f o r m e d . A u e et al. (26, 27) claim that linear siloxane polymers f o r m that are b o u n d to the silica surface at only a f e w sites. A f t e r a dichloro- o r trichlorosilane forms one - S i - O - S i - surface link, p o l y m e r formation can occur i f a second S i - C l b o n d becomes h y d r o l y z e d b y w a t e r - f o r m i n g - S i - 0 ~ S i ( O H ) - . I f this event occurs i n the presence o f unreacted solution chlorosilane, a p o l y m e r chain can b e initiated at the S i ( O H ) site. S u c h p o l y m e r formation can occur o n most metal oxide surfaces. 3

2

2

3


2

P t electrode anodization i n sulfuric acid at 1.1 a n d 1.9 V versus S C E yields an approximate monolayer o f P t O a n d a surface layer o f predominantly P t 0 , respectively (28, 29). Reportedly, P t electrodes anodized at 1.1 V can be silanized u n d e r anhydrous conditions (30) to produce surface P t O - S i - bonds. W e have used the X S i R a n d X S I R silanes, O T S a n d D M O C S , to m o d i f y the surface o f our electrodes. X S i R silane was f o u n d to produce a more stable a n d reproducible silane layer o n electrode surfaces. Therefore, w e e m p l o y e d octadecyltrichlorosilane ( O T S ) to obtain h i g h sur face coverages for supporting the reconstituted m e m b r a n e structure. O T S has a C carbon chain w i t h a length o f 26.5 Â, w h i c h is approximately equivalent to a single l i p i d layer o f a m e m b r a n e bilayer structure. T o f o r m a single layer o f O T S o n the electrode surfaces w e attempted to m i n i m i z e p o l y m e r f o r m a tion b y utilizing d r y solvents a n d b y carefully a n d thoroughly washing excess silane f r o m the electrode w i t h fresh solvent before exposing the electrode to moisture. 2

3

3

3

1

8

Electrochemical Analysis of PtO-OTS Electrodes. O T S forms a neutral a n d l o w dielectric silane layer a n d is electrochemically inert. T h e O T S layer f o r m e d o n P t electrodes acts as a n insulating layer to block the active P t surface. E l e c t r o c h e m i c a l examination o f the P t O - O T S electrode provides information o n the structure o f the O T S layer o n w h i c h the l i p i d - p r o t e i n m e m b r a n e is to b e f o r m e d . W e examined the P t O - O T S


496


electrode surface i n a nonaqueous electrolyte (to avoid reduction o f P t O ) using tetracyanoethylene ( T C N E ) as the m o d e l redox species. C y c l i c voltammograms o f T C N E at P t oxide a n d P t O - O T S electrodes measured at the sweep rate o f 40 m V / s i n 0 . 1 - M T B A F i n acetonitrile are shown i n F i g u r e 1. T h e w e l l - d e f i n e d pair o f chemically reversible waves measured at the P t O electrode, w h i c h represents reduction o f T C N E to its anion a n d reoxidation o f the anion to T C N E ,

exhibits the 6 0 - m V peak

potential separation a n d wave symmetry expected f o r a reversible electro c h e m i c a l reaction. F o r the P t O - O T S electrode, the r e d u c t i o n a n d oxidation peak currents o f T C N E decrease dramatically. T h e peak potential separation


increases to 180 m V w h i c h indicates irreversibility o f T C N E at the P t O - O T S electrode. T h e P t electrode is still accessible to reduction a n d oxidation o f T C N E after O T S coating, b u t the effective (microscopic) electrode area is l o w e r e d b y the covalent b o n d i n g o f O T S to the P t surface. T h e smaller microscopic area gives rise to higher current densities, an increased charge transfer rate limitation, a n d irreversibility. T h i s series o f events suggests that a porous O T S layer is f o r m e d o n the P t O electrodes. T h e porous structure o f the O T S layer o n P t O was c o n f i r m e d b y the electrochemical impedance spectra o f T C N E . F i g u r e 2 shows the complex plane plots o f T C N E reduction at P t O a n d P t O - O T S electrodes at - 0 . 4 V , near the T C N E

reversible potential, w h e r e ΊΙ a n d Z " are the real a n d

Ι/μΑ

40 20 0 -20 -40 -60

-0.5

0 E/V vs SCE

Figure 1. Cyclic voltammetry of tetracyanoethylene (TCNE) on the PtO and PtO-OTS electrodes at 40 mV/s. 5-mM TCNE in acetonitrile containing 0.1-M TBAF.


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2000

Ε

Ε

Ο

Ο

c


497


2000

Γ

Γ

(Ohm)

(Ohm)

Figure 2. Complex plane plots of TCNE reduction on the PtO and PtO-OTS electrodes at 0.4 V in 5-mM TCNE in acetonitrile containing 0.1-M TBAF.

imaginary components o f the measured impedance, respectively. A complex plane plot w i t h a semicircle a n d a 45° tail was measured at the P t O electrode. T h i s plot is the n o r m a l plot expected for a charge transfer process at a smooth electrode surface (31-33). T h e P t O - O T S electrode, however, gave a sunken semicircle i n the measured complex plane plot. T h i s result may be indicative o f a porous electrode surface. D u e to the u n c o n t r o l l e d nature o f surface roughness, i t is difficult to describe a rough electrode surface mathematically. F r a c t a l analysis has b e e n used to describe diffusion at a r o u g h electrode surface, and sunken semicircular curves have b e e n simulated (34-36). T h e sunken semicircle measured w i t h the P t O - O T S electrode may suggest fractal surface features w i t h pores o f different sizes a n d shapes permeating through the O T S layer to the electrode surface. T h e effective area o f the O T S - c o a t e d P t O electrode can be d e r i v e d i f the charge transfer resistance ( K ) is k n o w n . R c t

ct

can be obtained f r o m impedance

data measured at a potential near t h e reversal potential (37, 38): R

ct

RT/(nFAI ), 0

=

w h e r e R is the universal gas constant, Τ is absolute tempera

ture, η is the n u m b e r o f electrons transferred p e r molecule o f T C N E , F is F a r a d a y s constant, I is the exchange current density, and A is the effective 0

surface area. Because the impedance spectra o f the P t O a n d P t O - O T S electrodes w e r e measured u n d e r the same conditions, the value o f R

ct

may

be assumed to be affected only by the effective surface area. I n F i g u r e 3, the impedance data are replotted as ΊΙ versus 1 / ω frequency ( 2 τ τ / ) .

K

extrapolation. T h e R

1 / 2

, w h e r e ω is the angular

t

is estimated f r o m the intercept o n the Z ' axis b y

c t

values are 95 a n d 980 Ω f o r P t O a n d P t O - O T S ,

c

respectively. A n O T S coverage factor, Θ, can t h e n b e estimated f r o m (1 — Θ)

2Lct(PtO)/^ct(PtO - OTS)* I n this case θ = 0.9.


498

BO IMEMBRANE ELECTROCHEMS ITRY 1800

PtO/OTS


PtO

(sec *) 1

Figure 3. Z' OS. ω plots for the impedance data from Figure 2. The uncompensated electrolyte resistance of 150 Ω is subtracted. 1 / 2

T h e electrochemical stability o f P t O - O T S i n aqueous saline solution was tested b y measuring cyclic voltammograms o f P t O a n d P t O - O T S electrodes i n 0 . 1 - M K G , p H 6.8 ( F i g u r e 4). T h e electrode capacitances that correspond to the cyclic voltammograms are shown i n F i g u r e 5 . These capacitances were measured at 1000 H z . T h e O T S layer c a n b e described as a planar capacitor i n series w i t h the P t O a n d double-layer capacitances. F o r m a t i o n o f the O T S layer decreased the capacitance; however, the P t O beneath the O T S layer was still r e d u c e d w h e n the potential was swept b e l o w 0.3 V . T h i s observation again is evidence that the O T S layer o n P t O is somewhat porous. T h e onset potential o f P t O reduction i n aqueous solution is p H - d e p e n d e n t . T h e O T S layer was chemically unstable at p H > 9. A t this p H , the capacitance immediately increased to the value o f the bare P t O electrode, w h i c h suggests that the O T S layer dissolves i n basic solutions. T h e hydrolysis o f silane o n S i 0 was previously observed i n 0 . 1 - M N a O H (24). T h e s i l a n e - P t O is reported to b e resistant to most solvents, i n c l u d i n g dilute aqueous a c i d (for a f e w minutes) ( 3 0 ) . O u r measurements c o n f i r m the stability o n P t O at neutral p H . T o characterize the membrane-coated P t O electrodes w e have chosen a potential w i n d o w f r o m 0.3 to 0.6 V w h e r e P t O is electrochemically stable a n d passive according to the cyclic voltammograms i n F i g u r e 4. T h e p H was 6.8. 2

Electrochemical Properties after Membrane Deposition. A rhodopsin-containing m e m b r a n e was deposited o n P t O - O T S electrodes b y dialysis, as described i n the experimental details section. A f t e r dialysis, the



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E/V vs S C E Figure 4. Cyclic voltammetry of the PtO, PtO-OTS, and PtO-OTS-Rh electrodes in 0.1-M KCl containing 20-mM HEPES, pH 6.8. The sweep rate is 20 mV/s.

electrodes w e r e gently rinsed w i t h saline b u f f e r to wash off excess vesicles o n the surface before electrochemical measurements. T h e changes i n the cyclic voltammogram a n d the capacitance

are illustrated i n Figures 4 a n d 5 .

D e p o s i t i o n o f the m e m b r a n e o n the electrode surface brought about further " b l o c k i n g " o f the effective area o f P t O , w h i c h resulted i n a further suppres sion o f the P t O reduction (see F i g u r e 4). T h e capacitance decreased further as the result o f a n additional capacitance d u e to the rhodopsin-containing membrane i n series w i t h the existing capacitance o f oxide a n d O T S layers (see F i g u r e 5). Impedance spectra w e r e r e c o r d e d i n 0 . 1 - M K C l , p H 6.8, at 0.4 V versus S C E . F i g u r e 6 shows the B o d e plots o f log|Z| versus l o g / a n d phase angle (Θ) versus l o g / , w h e r e |Z| is the impedance magnitude, / is the frequency, and phase angle θ is the arctangent o f the ratio o f the imaginary a n d real parts o f the measured impedance. T h e magnitude o f the impedance i n -



500

C/μΡ


PtO

PtO/OTS

PtO/ OTS/Rh

-0.4

0.4

0.8

E/V VS S C E Figure 5. Capacitances of the PtO, PtO-OTS, and PtO-OTS-Rh electrodes measured at 1000 Hz in 0.1-M KCl containing 20-mM HEPES, pH 6.8.

creased u p o n m e m b r a n e formation, a n d the phase angle was also sensitive to membrane deposition. A b r o a d phase-angle m a x i m u m plateau appeared i n the frequency range > 500 H z but disappeared after washing the electrode w i t h 30 m M O G to dissolve the deposited m e m b r a n e (see the half-opened dotted curve i n F i g u r e 6). These results are consistent w i t h the formation o f a p r o t e i n - l i p i d m e m b r a n e o n the O T S - t r e a t e d P t O electrode b y detergent dialysis.

M o d e l of Surface-Bound Membrane on Pt Electrode.

From

the foregoing discussion o f experimental results w e can develop a physical m o d e l o f the surface-bound m e m b r a n e that consists o f two layers, as schematically d e p i c t e d i n F i g u r e 7. T h e porous, h y d r o p h o b i c O T S layer provides a structure to anchor the reconstituted m e m b r a n e layer. P r o t e i n molecules w i t h b o u n d l i p i d may insert into the pores i n the O T S layer. T h e


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Ν

* S β· .

; !··

Ό)

Ο

Ο

ο

°

ο

Ο


PtO ο Ui

.· ·

Φ "Ο

Ο Ο

ο

Λ

°

ο

Ο

Ο

Ο Φ

ο

°

°

°

·

·

*

RO/OTS

0

Λ

·

Φ

ο

•

-80 •

t

#8

~

φ

"Ύ" °

Ο

PtO/OTS/Rh

9

«

Φ

log Frequency (Hz)

Figure 6. Bode ylots for the PtO, PtO-OTS, and PtO-OTS-Rh electrodes measured at 0.4 V in 0.1-M KCl containing 20-mM HEPES, pH 6.8.

Figure 7. Model of a single surface-bound membrane formed by detergent dialysis on an alkylsilanated electrode surface.

m o d e l assembly is essentially a bilayer m e m b r a n e i m m o b i l i z e d o n the elec trode surface. M o r e detailed i n f o r m a t i o n o n the physical structure a n d electrical p r o p erties c a n b e obtained b y analysis o f the impedance spectra, as presented i n F i g u r e 6. A n electrical equivalent circuit [resistance-capacitance ( R C ) circuit] was used b y F a r e ( 3 8 ) to interpret the capacitance a n d conductance data o f


502


l i p i d bilayers measured at l o w frequencies (e.g., 0.5 H z ) . Fare's equivalent circuit consists o f a parallel c o m b i n a t i o n o f conductances a n d capacitances f o r the p o r t i o n o f the electrode coated w i t h a L a n g m u i r - B l o d g e t t m e m b r a n e a n d for the uncoated p o r t i o n o f the electrode, b u t it does not i n c l u d e the polar region o f the bilayer. T h e concept o f a coverage factor was i n t r o d u c e d into the equivalent circuit b y Stelzel a n d S a c k m a n n ( 3 9 ) . Y o s h i d a et a l . (40) a d d e d elements f o r the polar region o f a l i p i d layer b y using a n equivalent circuit that consisted o f two pairs o f R C elements i n series f o r the hydrocar b o n core a n d the h y d r o p h i l i c surface region, respectively. A n equivalent circuit c a n be d e r i v e d f o r the surface-bound

membrane


f o r m e d i n this w o r k similar i n a m a n n e r to the approach taken f o r porous anodic films a n d porous electrodes (41-46). A n equivalent circuit network, p r o p o s e d i n F i g u r e 8a, corresponds to the m o d e l i n F i g u r e 7. T h i s network has three R C subnetworks that represent the oxide layer, the surface-bound m e m b r a n e layer, a n d the double layer. C resistance o f oxide.

and R

d l

o x

and R

o x

are the capacitance a n d

are the double-layer capacitance a n d the

polarization resistance, k n o w n as the charge transfer resistance at the m e m b r a n e - w a t e r interface. F o r the subnetwork o f the surface-bound m e m b r a n e layer, one b r a n c h represents a tightly p a c k e d alkylsilane a n d l i p i d bilayer i n series, a n d the other b r a n c h represents the pores a n d defects through the bilayer. C ^ , C

h

p

and R

(a)

a l k

,

R

l i p

are the capacitances

C ,k(8)

C„p(0)

R /9

Riip/θ

a

alk

a n d resistances o f

—WV—

CdKi-θ)

Rei/0-θ)

Rdi/(1^) oxide

—vwv— double layer

membrane

(b) Ox

oxide

Caik(e)

CH (6)

—II—

•HI—

p

membrane

double layer

Figure 8. (a) Proposed equivalent circuit for surface-bound membrane electrode interface, (b) Simplified equivalent circuit valid at higher frequency region.


23.

L l E T AL.

503


alkylsilane a n d l i p i d layers. R is the resistance o f electrolyte i n series w i t h the R C circuit o f the double layer at the o x i d e - w a t e r interface i n the pores. R is the series resistance o f the electrolyte, θ is defined as the coverage factor for the tightly p a c k e d bilayer o n the surface, a n d (1 — Θ) is the fractional area covered by pores a n d defects. e l

u


T h e simulation spectra o f the P t O electrodes w i t h a n d without the surface-bound m e m b r a n e are shown i n F i g u r e 9 for comparison w i t h the experimental data o f F i g u r e 6. T h e parameters used i n the simulation are listed i n T a b l e I. T h e first c o l u m n lists the values used for curve fitting the experimental spectra, and the second c o l u m n gives the corresponding values n o r m a l i z e d for unit area. This simulation reproduces the essential features o f the experimental data. A calculated capacitance for the tightly p a c k e d part o f the surface-bound membrane ( C ) can be obtained by treating C and C i n series. T h e resistance can be similarly calculated f r o m R a n d R . T h e calculated values o f 0.52 μΈ/cm a n d 1325 Ω c m are i n g o o d agreement w i t h literature values for natural membranes (47-48). T h e best curve fit for the coverage factor, Θ, was 0.97, w h i c h indicates formation o f a relatively c o m plete membrane b y the detergent dialysis approach. b l

a l k

dk

l i p

u

2

2

Capacitance values o f the P t O , C , f r o m the best curve fit is lower for the membrane-coated electrode than for the bare P t O electrode. A l s o the resistance, R , is higher for the membrane-coated electrode than for the o x

o x

5

log Frequency (Hz)

Figure 9. Theoretical simulation of impedance spectra from the proposed equivalent circuit in Figure 8a for curve fit to the spectra in Figure 6.


504


T a b l e I. Parameters f o r Best F i t to Impedance Spectra (Figure 6) Parameters PtO-Membrane C C d l

o x

C H

h p o x


flaik

% R& R fl i θ u

e

Bare P t O Electrode C C R& R K„ d l

o x

ox

Values for Model

Normalized Values

2.5 μ F 1.0 μ¥ 0.25 μ¥ 0.27 μ Ρ 800 Ω 3000 Ω 500 Ω 80,000 Ω 120 Ω 12 Ω 0.97

10 μ¥/οτη 4.0 μ¥/οχη 1.0 μ Ρ / c m 1.1 μ Ρ / c m 200 Ω c m 1200 Ω c m 125 Ω c m 20,000 Ω c m 30 Ω c m 3 Ω cm

6.0 μ¥ 3.0 n F 5000 Ω 40 Ω 80 Ω

2

2

2

2

2

2

2

2

2

2

24 12 1250 10 20

μ F/cm μΡ/cm Ω cm Ω cm Ω cm

2 2

2

2

2

bare P t O electrode. T h i s analysis suggests that the structure o f the oxide is altered b y alkylsilanization a n d m e m b r a n e deposition. It is likely that hydra tion o f the oxide layer differs b e t w e e n the bare electrode w h e r e the oxide has direct contact w i t h water a n d the membrane-coated electrode w h e r e the oxide is protected b y the alkylsilane a n d p r o t e i n - l i p i d layers. A t frequencies b e l o w 63 H z , the double-layer capacitance began to dominate the overall impedance o f the m e m b r a n e electrode. T h e electric potential profile o f a bilayer m e m b r a n e consists o f a h y d r o c a r b o n core layer and an electrical d o u b l e layer (49).

T h e dipolar potential, w h i c h originates

f r o m the l i p i d bilayer head-group zone a n d the incorporated p r o t e i n , partially controls transmembrane i o n transport. T h e m o d e l equivalent circuit p r e sented here accounts for the response as a f u n c t i o n o f frequency o f b o t h the hydrocarbon core layer a n d the d o u b l e layer at the m e m b r a n e - w a t e r inter face. T h e value o f C$

f r o m the best curve fit for the membrane-coated

electrode is lower than that for the bare P t O interface. F o r the m e m b r a n e coated electrode, the m o d e l gives a polarization resistance, R&, o f 80

kû

c o m p a r e d w i t h 5 k O for the bare P t O electrode. F o r m a t i o n o f the l i p i d m e m b r a n e creates a dipolar potential at the interface that results i n higher β . T h e incorporated r h o d o p s i n may also extend the double layer, w h i c h Λ

makes the layer m o r e diffuse and, therefore, decreases C ^ . T h e impedance response o f each layer o n the electrode surface can be attributed to elements of the spectrum i n terms o f frequency. A l t h o u g h the


23.

L i E T AL.

505


b r o a d phase-angle m a x i m u m plateau i n d u c e d b y t h e formation o f t h e surface-bound membrane o n the P t O electrode is attributed to several R C constants, the total impedance is d o m i n a t e d b y t h e admittance o f t h e surface-bound m e m b r a n e f o r frequencies > 5 0 0 H z . T y p i c a l double-layer capacitances are usually i n the range o f 1 0 - 4 0 μΈ/cm , w h i c h is at least 1 order o f magnitude greater than the membrane capacitance. T h u s at higher frequencies, the double-layer capacitance behaves like a short circuit a n d can be neglected. I n addition, R , R , R a n d R are usually i n the range o f 2 0 0 - 2 0 , 0 0 0 k f t c m , a n d are higher than t h e impedances o f the capacitance components i n t h e equivalent circuit. These resistances c a n b e treated as o p e n circuits at higher frequencies. R is small a n d c a n b e treated as a short circuit. R c a n b e either compensated experimentally o r subtracted i n t h e data analysis. T h e equivalent circuit i n F i g u r e 8a can therefore b e s i m p l i f i e d as shown i n F i g u r e 8 b . 2

o x

a l k

d l

h

2


e l

u

I n this s i m p l i f i e d f o r m , t h e m e m b r a n e capacitance, C , is i n series w i t h C . T h u s , C = C C / ( C — C ) , where C is the total capacitance o f the surface-bound m e m b r a n e electrode a n d C is the measured capacitance o f the electrode before m e m b r a n e formation. T h e m e m b r a n e capacitance, C , can thus b e estimated at a single frequency. F u r t h e r , t h e capacitance o f the tightly p a c k e d bilayer, C , can b e calculated f r o m C i f the coverage factor a n d the double-layer capacitance are k n o w n : C = ( C — (1 — 0 ) C ) / 9 . m

o x

m

o x

t

t

o x

t

o x

m

b l

m

b l

m

d l

B y using the imaginary component o f the measured impedance data f o r P t O - O T S a n d P t O - O T S - R h electrodes (Table II) at a frequency o f 1000 H z (after subtracting R ), the calculated C is 867 n F / c m a n d C is thus 584 n F / c m using = 10 μ Ρ / c m a n d θ = 0.97, w h i c h are close to t h e theoretical values d e r i v e d f r o m the best curve fit simulation. W e conclude that the simplified equivalent circuit m a y b e adequate f o r the surface-bound m e m b r a n e electrode. T h e thickness o f the tightly p a c k e d m e m b r a n e bilayer, d, can b e calculated f r o m d = e e / C , w h e r e e is the dielectric constant o f 2

m

u

2

b l

2

0

b l

Table II. Capacitances for Membranes Formed on Various Electrodes ^total

^latter ,

(nF/cm )°

(nF/6m )

2

Type of

2

OTS-

Electrode

Bare

PtO Ti0 ITO p-Si-Si0 n-Si-Si0

10,260 1,681 3,020 62.95 40.7

2

2

2

OTS 1100 1120 1423 60.1 39.7

memb. 800 607 710 57 39

b

OTSOTS 1232 3358 1490 1330 1627

memb. 867 950 926 610 793

CM (nF/cm )

Thickness

584 670 645 320 508

46.8 40.8 42.4 85.4 53.8

2

d

(A)

Capacitance measured at 1000 Hz for unmodified electrode (bare), after alkylsilanization |OTS), and after membrane deposition (OTS-memb.). Calculated capacitance for each surface layer. ^ Calculated capacitance of membrane using = 10 μ F / c m and θ = 0.97. Calculated using e = 3. a

2


506


the bilayer. T h e calculated thickness o f the m e m b r a n e bilayer o n the P t O electrode is 46.8 Â using a dielectric constant o f 3. Surface-bound membranes f o r m e d o n P t O electrodes w e r e chemically a n d mechanically stable. T h e P t O - O T S - R h electrodes were m o n i t o r e d b y measuring the capacitance w h i l e the electrodes w e r e kept i n b u f f e r at 4 °C for 11 days. A n y dissolution o f the surface-bound m e m b r a n e w o u l d result i n an increase i n capacitance. L i t t l e change i n capacitance was observed, w h i c h indicates that the membranes are stable.

Surface-Bound Membrane on Si0 , Ti0 , and ITO Electrodes. T h e detergent dialysis procedure was also u s e d to deposit m e m 2

branes o n S i 0 , T i 0 , a n d I T O electrodes. T h e planar S i - S i 0 electrode is a solid-state capacitor w i t h the n - o r p-type silicon substrate f o r m i n g one " p l a t e " o f the capacitor a n d the electrolyte at the surface f o r m i n g the other plate. T h e insulating S i 0 layer a n d surface-bound m e m b r a n e f o r m the dielectric. A l t h o u g h the planar S i - S i 0 electrode is a s i m p l e device, it has the same fundamental physical structure as other solid-state devices, such as field effect transistors ( F E T s ) , but it allows a s i m p l e r approach to analyze the device characteristics. F o r example, the F E T sensor relies o n changes i n its gate voltage caused b y a change i n the surface charge density u p o n exposure to an analyte. T h e gate o f the F E T is operated u n d e r inversion conditions a n d can be represented b y an equivalent circuit o f space-charge capacitance, silicon oxide layer capacitance, a n d m e m b r a n e capacitance i n series. A l l three capacitances must b e considered d u r i n g analysis o f the device response. I n contrast, the S i - S i 0 electrode can be operated u n d e r accumulation c o n d i tions a n d only the silicon oxide a n d m e m b r a n e capacitances n e e d to b e considered. S i 0 is extremely stable i n most biological solutions a n d suitable for alkylsilanization. T h e capacitance o f a 950-Â-thick S i 0 layer is typically 35 n F / c m , w h i c h is 1 order o f magnitude l o w e r than the capacitance o f the surface-bound m e m b r a n e . T h e total i m p e d a n c e o f the electrode is therefore d o m i n a t e d b y the S i 0 capacitance. 2


2

2

2

2

2

2

2

2

2

2

Ti0 f o r m e d b y electrochemical anodization o n a p o l i s h e d titanium surface is usually an η-type semiconductor. I n the potential w i n d o w w h e r e it is passive, T i 0 is i n a d e p l e t i o n c o n d i t i o n . T h e capacitance o f T i 0 is the space-charge capacitance described b y the M o t t - S c h o t t k y equation. 2

2

2

I T O electrodes behave electrochemically similar to P t electrodes, a n d the double-layer capacitance can be neglected at higher frequencies to allow easily calculation o f the m e m b r a n e capacitance. T h e simplified equivalent circuit i n F i g u r e 8b was u s e d to evaluate surface-bound membranes o n S i 0 , T i 0 , a n d I T O electrodes. Figures 10 a n d 11 present the capacitance curves for n - S i - S i 0 a n d T i 0 electrodes w i t h a n d without O T S - a n d rhodopsin-containing l i p i d membranes i n K C l buffer. A s w i t h the P t O electrodes, the capacitance decreases u p o n f o r m a t i o n o f an O T S layer a n d the m e m b r a n e o n the oxide surface. T a b l e II lists the 2

2

2

2


23.

L l E T AL.

507


C/nF 10

8

6


4

2

0

1.0

2.0

E/VvsSCE Figure 10. Capacitances of the n-Si-Si0 , n-Si-Si0 ~OTS and n-Si-Si0 OTS-Rh electrodes measured at 1000 Hz in 0.1-M KCl containing 20-mM HEPES, pH 6.8. 2

measured capacitances

(C

t o t a l

2

2

) for the u n m o d i f i e d electrodes a n d for the

electrodes after alkylsilanization a n d m e m b r a n e deposition. F r o m these mea sured values w e have used the s i m p l i f i e d equivalent circuit o f F i g u r e 8b to calculate the capacitance o f the O T S layer a n d the c o m b i n a t i o n o f the O T S layer a n d deposited m e m b r a n e . A d j u s t i n g the O T S - m e m b r a n e composite for the surface coverage, Θ, provides the capacitance o f the tightly p a c k e d bilayer, C , f r o m w h i c h the thickness o f the surface-bound m e m b r a n e is calculated. b l

These results are all consistent w i t h formation o f a single m e m b r a n e bilayer o n the electrode surfaces. Because the surface-bound m e m b r a n e capacitance is i n series w i t h the oxide layer capacitance, the change i n the total capacitance i n d u c e d b y the surface-bound m e m b r a n e is a f u n c t i o n o f b o t h the dielectric properties a n d the thickness o f the oxide layer. T h e dielectric constants for T i 0

2

and I T O

are 1 0 - 1 5 times higher than for S i 0 ; thus, a larger change i n capacitance is 2

observed w h e n the m e m b r a n e is f o r m e d o n T i 0 w i t h the S i 0

2

electrode (see

T a b l e II). T h e S i 0

2

2

a n d I T O electrodes than thickness o f the p - S i - S i 0

2

electrode was 550 Â (reduced f r o m 950 Â b y etching i n H F ) . T h e n - S i - S i 0

2

electrode h a d a 950-Â oxide layer. A s shown i n T a b l e II, the thinner the S i 0

2

o n the p-type silicon electrodes, the larger the change i n capacitance u p o n m e m b r a n e formation. T h e calculated capacitance o f the surface-bound m e m -


508



C/nF

TIO^OTS/Rh

ol

ι

,

,

0

03

1-0

1

E/VvsSCE

Figure 11. Capacitances of the Ti0 , Ti0 -OTS, and Ti0 ~OTS-Rh electrodes measured at 1000 Hz in 0.1-M KCl containing HEPES, pH 6.8. 2

2

2

brane itself o n the p - S i - S i 0 electrode is l o w e r than for the other electrodes. T h i s observation may indicate formation o f a tighter m e m b r a n e bilayer. T h e etching m a y provide a cleaner hydrated surface f o r better alkylsilanization a n d membrane deposition. 2

Summary and Conclusions E l e c t r o c h e m i c a l i m p e d a n c e spectroscopy provides a sensitive means f o r characterizing the structure a n d electrical properties o f the surface-bound membranes. T h e results f r o m i m p e d a n c e analysis are consistent w i t h a single b i o m e m b r a n e - m i m e t i c structure b e i n g assembled o n metal a n d semiconduc tor electrode surfaces. T h e structures f o r m e d b y detergent dialysis m a y consist o f a h y d r o p h o b i c alkyl layer as one leaflet o f a bilayer a n d the l i p i d deposited b y dialysis as the other. Proteins s u r r o u n d e d b y a b o u n d l i p i d layer may simultaneously incorporate into pores i n the alkylsilane layer b y h y drophobic interactions d u r i n g deposition o f the l i p i d layer. This m o d e l is further supported b y the composition o f the surface-bound membranes a n d b y F o u r i e r transform i n f r a r e d analyses ( 9 ) .


23.

L i E T AL.


509


These structures appear w e l l suited f o r investigations o f intramembrane charge redistributions associated w i t h receptor protein function a n d f o r applications that use receptors as the active element i n biosensor systems. T h e receptor protein is retained i n these structures b y its h y d r o p h o b i c interaction w i t h the core o f the membrane, a n d the environment a r o u n d the receptor m i m i c s that o f a natural m e m b r a n e . T h e receptor protein is thus free to undergo rotational a n d translational diffusion i n the plane o f the m e m b r a n e . T h i s s h o u l d a i d i n retention o f function relative to systems i n w h i c h the receptor is directly i m m o b i l i z e d o n a surface. T h e system is, o f course, not totally natural i n that one surface o f the membrane is b l o c k e d b y the electrode. Interactions m a y exist between the receptor a n d the electrode surface that inhibit functionality. W e have, h o w ever, f o u n d that aspects o f the function o f several receptors, i n c l u d i n g rhodopsin (8), the nicotinic acetylcholine receptor, a n d the C a - A T P a s e f r o m sarcoplasmic r e t i c u l u m can b e retained i n these systems (unpublished results).

Acknowledgments T h i s research was supported i n part b y the Office o f N a v a l Research.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

17. 18.

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RECEIVED f o r review F e b r u a r y 1992.

20, 1991.

ACCEPTED revised manuscript


July 10,

Evaluation of Surface-Bound Membranes with Electrochemical

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