Application of voltammetric techniques to membrane studies

Journal of Biochemical and Biophysical Methods, 11 (1985) 317-326 Elsevier

317

BBM 00499

Application of voltammetric techniques to membrane studies Jan Kutnik * and H. Ti Tien ** Membrane Biophysics Lab, Department of Physiology, Michigan State University, East Lansing, MI 48824, U.S.A.

(Received 24 July 1984) (Received and accepted after revision 22 May 1985)

Summa~ The application of voltammetric metbods to planar bilayer lipid membranes (BLM) studies is described. BLM-compound interaction experiments lead to the measurement of the membrane current underlying transport phenomena. From measurements of current/voltage of BLM in unstirred solutions as a function of scan rates, it is possible to obtain both thermodynamic and kinetic information. In past years, a variety of techniques have been used to study the electrical properties of BLMs, but in terms of versatility, the cyclic voltammetric technique is outstanding. Cyclic voltammetry is the definitive means of characterizing the redox process of electroactive membranes. Key words: planar lipid bilayer; bilayer membranes; BLM; cyclic voltammetry; bioelectrochemistry; membrane biophysics.

Introduction

Artificial bilayer lipid membranes (planar BLMs and spherical bilayer liposomal membranes) have been a topic of increasingly active research since the early 1960s [1-4]. In addition to their use as models of biomembranes, planar BLMs and spherical liposomes are of interest for the separation of charges and charged species in solar energy conversion [4,5] and for their ability to sequester reagents in practical applications [3,6]. Substances such as antibiotics, drugs, poisons, dyes and detergents, which in many cases affect the membrane of living cells, also drastically alter the electrical characteristics, structure and mechanical properties of experimental bilayers. Sub* Permanent address: Institute of Physics, Maria Curie-Sklodowska University, Lublin, Poland. ** To whom all correspondence should be addressed. 0165-022X/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

318

stances which adsorb on the surface of the membrane or which become incorporated into it, or which penetrate through it are usually called modifiers. As has been shown by many authors [1-4], the BLM system is an excellent model membrane for the investigation of the interaction between modifier molecules and lipid membranes owing to easy access to both sides of the membrane. Thus, the BLM makes it possible to apply various voltammetric (or current-voltage) techniques in such investigations. Voltammetry is very useful in eliciting important information about the transport characteristics of the membrane and their dependence on lipid content, modifier concentration, pH and other parameters. The shape of a voltammogram ( I / V curve) may help in determining the way in which modifier molecules interact with the BLM, the rates of adsorption and desorption, the surface density of the adsorbed modifier and electron-transfer and redox reactions. Experimentally, the voltammetric technique is rather straightforward. However, it is important to realize that the processes occurring when the voltammogram is recorded are rather complex. For the proper interpretation of the results, many factors influencing the course of the I / V curve should be taken into account. Therefore, the aim of this paper is to provide some general information concerning the voltammetric techniques and show how they are applied to the BLM system.

Experimental section Materials To form BLMs the following lipids are commonly used: cholesterol (Ch), oxidized cholesterol (OC), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). These lipids are used in most experiments with BLMs, but other lipids may be used as well if they are of special interest to the experimenter. Usually, BLMs formed from mixtures of lipids are more durable than membranes formed from a single lipid. The most frequently used bathing solution is 0.1 M KC1. Other lipids and aqueous solutions have also been employed [4].

Formation of BLM In the so-called 'standard technique' [2,4], the BLM is formed, by use of a syringe, in a hole (0.3-3 mm in diameter) punctured in a Teflon cup. This technique is appropriate when a' modifier is added to the bathing solution. However, when the modifier is not water-soluble but soluble in organic solvents such as chloroform, hexane, octane, decane or butanol, it may be added to the forming solution and uniformly incorporated into the membrane. The disadvantage of the standard forming technique is that the membrane produced in this way contains a certain amount of the solvent. Due to this fact, the properties of such membranes differ somewhat from those of a pure lipid membrane. A 'solvent-free' membrane may be formed from two monolayers by use of the Takagi technique and its variations [4,7,8]. Some additional advantages of this technique are: (i) the possibility of forming the membrane with different lipids on the interfaces, (ii) the possibility of

319 G

Fig. 1. Schematic diagram showing an experimental arrangement: (a) simple arrangement; (b) voltageclamp arrangement. G = generator of increasing/decreasing voltage; V = millivoltmeter; Comp = compensating source of voltage; pA = picoammeter; X-Y Rec = x-y recorder; A = operational amplifier.

the modifier molecules placing in only one layer of the BLM. Obviously, membranes formed in this way show an inherent asymmetry and may be very useful in the I / V method. It should be mentioned that the Takagi technique (see ref. 4, p. 477) is more difficult than the standard technique. Another, rather sophisticated, technique of forming modified membranes which has been elaborated lately is the incorporation of .liposomes containing a modifier into a pure lipid membrane [3,4]. Liposomes prepared by the use of a sonicator are added to the bathing solution and then some of them spontaneously adsorb to the membrane interface. Subsequently, they are incorporated into the membrane. Although this technique seems to be easier than the Takagi technique, it is not possible to state whether the liposomes are totally adsorbed by the membrane or not. As a result, nothing can be said about the inherent asymmetry of the membrane. Further, another new technique of BLM formation on commercially available polycarbonate filters (Nucleopore membranes) has also been reported [9,10]. Finally, a number of papers have described other approaches to BLM studies [12,13].

Instrumentation The experimental arrangement consists of a double-compartment vessel with two or four nonpolarizing electrodes located in solutions on both sides of the membrane, a millivoltmeter, a picoammeter, an X-Y recorder and a source of linearly variable voltage (Fig. la). A voltage-clamp circuit is recommended (see Fig. lb) but not necessary if the input resistance of the picoammeter is much less than the membrane (108-101° $2) formed in a hole of 1.5 mm in diameter. A picoammeter with an input resistance of 10 6 $2 is sufficient; even if the measured current is 10 - 9 A, the potential drop caused by the input resistance of the picoammeter does not exceed 1 mV (10 -9 A × 106 $2 = 10 -3 V). Such a small potential drop causes negligible disturbance in voltage linearity. For voltage measurement any millivoltmeter may be used. A

320 a

i

J

~

v

Fig. 2. Voltammograms of BLMs: (a) a BLM under symmetric conditions, (b-g) BLMs under a variety of asymmetric conditions, (h,i) electron transfer and redox reactions are indicated (see text for details).

programmable voltage generator used in electrochemistry for voltammetry may be a very convenient source of voltage. An EC/225 Voltammetric Analyzer (IBM) is also satisfactory [14]. The X-Y recorder may be of any type provided its inputs match the outputs of the millivoltmeter and the picoammeter.

Procedure of I / V characteristic record (voltammograms) It is most convenient to start the I / V curve recording from the point of zero-current, which does not necessarily mean zero-voltage on the BLM. Usually a certain voltage exists on the membrane even if no voltage is applied from an external source. There may be a small potential difference between the electrodes. Also, a redox potential difference between the bathing solutions may produce some voltage due to the redox reaction across the BLM. Therefore, in order to attain the zero-current point it is necessary to apply an initial voltage to the membrane in order to compensate the inherent voltage in the system. In symmetrical conditions, the I / V curve should be symmetrical (see Fig. 2a). When these conditions are not maintained, other shapes of the I / V curve are registered (see Fig. 2b-g). Generally, the conductance of the membrane at a given point of the curve is defined as follows: d/ g = ~--~,

or g = tan a

(1)

where g = conductance, a = angle between the tangential and x-axis (see Fig. la), then, the current is described by the integral:

I= f g(V)dV

(2)

321 The I / V curve cannot be registered too fast because of the time constant of the electrical circuit owing to high membrane capacitance and resistance and because of some slow processes occurring in the system (chemical reaction, adsorption, desorption, diffusion). If the speed of scanning is too high, it results in hysteresis. Usually, the speed of 1-50 m V / s is satisfactory. Of course, in many cases the registered curves also show hysteresis at low scanning speeds, which provides desired information [14,16].

Theory BLM-modifier interaction Usually the BLM separating two aqueous solutions consists of a liquid hydrocarbon phase sandwiched between two layers of hydrophilic groups of lipids. An unmodified BLM (i.e., a BLM formed from common phospholipids or oxidized cholesterol dissolved in n-octane in 0.1 M KC1) will typically have the following electrical properties: membrane resistance (Rm) greater than 108 £ / c m 2, membrane capacitance (Cm) about 0.4 # F / c m 2, membrane potential (Era) about 0, breakdown voltage (Vb) 200 +_ 50 mV, and current/voltage ( I / V ) curves obeying Ohm's law [4]. With a few exceptions, the properties of BLM have been interpreted in terms of an ionic conductor connected in parallel with a capacitor. The molecular organization of the BLM is considered to be an ultrathin layer of liquid crystals in two dimensions having a fluid hydrocarbon phase of about 50 ~, thick [1]. As shown by R m, this liquid-crystalline structure of BLM is an excellent insulator, whose electrical properties, however, can be drastically altered by incorporating a variety of modifiers such as those discussed in this paper. It is generally recognized that biological membranes have the structure of a lipid liquid bilayer with proteins floating about or spaning across the bilayer [3,4]. The BLM is regarded as a good model whose main properties are very similar to those of biological membranes. Therefore, experiments with the BLM system have been instrumental in the characterization of the properties of biomembranes and in the conceptual development of modes of transport processes in ultrathin membrane separating two aqueous solutions [1-4]. Substances interacting with the BLM change its properties, which results in a change of the I / V characteristics of the membrane. In order to understand how these processes occur, it is necessary to understand what kind of mechanisms are involved in the interaction. If the modifier substance is water soluble, it may be added to the bathing solution so that its molecules can then approach the membrane interface and: (i) randomly adsorb on the surface (Fig. 3a, c), (ii) adsorb on the surface creating an ordered layer (Fig. 3b), (iii) penetrate the membrane to a certain depth (Fig. 3d, e), and (iv) penetrate across the membrane (Fig. 3f, g). Modifiers which remain in the membrane may alter its intrinsic structure by: (a) creating new channels (pores) for ions, (b) closing some channels, (c) closing or opening the channels depending on the electric field direction, (d) altering the permeability of channels, and (e) enabling a redox reaction to occur across the membrane.

322 BLM

BLM

BLM

(h)

D\ ~._%. /,-A0 (c)

{f)

\A

Io)

o:5

TCNQ.

Fig. 3. Different possibilities of modifier molecules location at the BLM/bathing solution interface and within the BLM: (a) adsorbed molecules with no charge; (b) adsorbed molecules (dipoles creating an ordered layer; (c) molecule closing the channel; (d) moveable dipole molecule gating the channel; (e) molecules driven into the channel; (f) molecule crossing the BLM through the channel; (g) molecule penetrating the membrane and creating a new channel; and (h) redox scheme.

Asymmetry of voltammograms There are three main reasons for the asymmetry of I / V curves: (i) inherent membrane asymmetry, (ii) the interaction between the membrane and the modifier, and (iii) the redox reaction across the membrane. As a measure of asymmetry, the characteristic voltage Vc pertinent to a certain characteristic conductance gc may be introduced [1]: g = g c e x p ( Vm~ ) -Vc

(3)

where g = conductance of the membrane, gc = characteristic conductance, Vm = applied potential difference/voltage, and Vc = potential difference/characteristic voltage at which the conductance reaches the characteristic value (gc). Eqn. (3), in general, is in good agreement with the commonly accepted expression for the membrane conductance: g =

go exp( neV]

kT ]

(4)

where: go = conductance at zero voltage (initial), n = constant, e = charge of the electron, V = voltage, k = Boltzman constant, and T = absolute temperature. However, the conductance expression may become more complicated if the conductance is examined in detail.

Ionic conductance of BLM The conductance mechanism in the BLM has been established over the past decade as the ionic transport across the membrane [1-4]. However, the electron flow due to redox reactions, also plays an important role in this mechanism [14]. In the ionic transport mechanism, ions move through pores or channels and the number of open or closed pores is governed by the Boltzman distribution from which eqn. (4) can be derived. In the case of symmerical conditions, the eqn. (4) has

323

to be written as following: g = go exp

ne I VI kT

(5)

where I V I = absolute value of V, and other symbols have the usual meaning. This is so because the conductance depends equally on positive and negative voltage. In other words, the I / V curve described by eqn. (5) has a symmetrical shape. In many cases, however, the shape of the I / V curve is not simply exponential but is almost linear at low voltages and is distinctly bent at higher voltages. In such cases the conductance of the membrane turns out to be a complex of two or more components. At a lower applied voltage, the component which is higher at zero voltage, but is weakly dependent on the voltage prevails. At higher voltage the component which is strongly dependent on the voltage increases more rapidly than the other one. Such complexity of conductance may result when two or more different transport mechanisms appear in the membrane, e.g., the pore and carrier transport or, in another case, the inherent and modifier-induced conductance. It is also possible that under a high voltage condition (the strong electric field), lipid molecules may reach their activation energy and 'flip-flop'. New pores are then created, which drastically change the conductance. In such cases the conductance equation has to be written as follows:

/ neV'~

g = ga exp I ~

l + g2

exp( meV] ~ }

(6)

where gl, g2 = initial conductances of the components, and n, m are constants. When the conductance is altered by a modifier, theoretical considerations as well as experimental observations [1-4] lead to the following formula:

(7)

gi = gio[M] s" [g] r" exp( nieV]kT]

where gi0, s, r, ni are all constants, [M] = modifier concentration in the solution, [K] = charge carrying ion concentration, and others have usual meaning. BLMs formed from different lipid monolayers may demonstrate an asymmetrical voltammogram resulting from different packing densities of molecules and from the inherent potential difference produced in the membrane because of differences in the dipole moments of the lipid molecules. A monolayer of tightly packed dipoles produces the potential difference:

6eo cos # VP

(8)

£0Cr

where G = number of dipoles on the unit surface, Pe = electrical dipole moment of the lipid molecule, /8 = mean angle between the dipole moment vector and the normal to the surface, c o = dielectric constant of vacuum, and ( r dielectric con=

324

stant of the medium. If we denote the potential differences produced by the two monolayers of the membrar~e as Vpi and Vp0, we obtain for the inherent potential difference: 3Vp = Vpi- Vp0,

(9)

This leads to the basic conductance equation:

g = go exp

[ ne(Iv+k T avp I) ]

(10)

Electronic conductance processes and cyclic voltammetry of B L M Electron transfer across the lipid bilayer has been shown to occur when coupled redox reactions (reduction and oxidation) take place on both sides of the BLM interposed between solutions having different redox potentials (for review and recent references, see refs. 1, 4, 14). The technique of choice for studying electronic processes in membranes is cyclic voltammetry (CV). This technique involves cycling the potential of a working electrode in an unstirred solution and measuring the resulting current. The potential of the working electrode, which is provided by a triangular potential waveform generator, is controlled relative to a reference electrode. Modern instrumentation provides a range of switching potentials (the potential of the electrode between two values) and scan rates (in mV/s). A potentiostat, which applies the potential to the system, forms the integral part of the device. Useful information, both thermodynamic and kinetic, on electron transfer and ensuing redox reactions may be gleaned from the resulting voltammogram [14]. Interpreting voltammograms based on electron transfer and subsequent redox reactions at the working electrode/solution interface has been well established and widely employed. However, cyclic voltammetry, a powerful technique for studying electron transfer and redox reactions, appears not to have been applied to the BLM and other membrane systems until recently [14,15]. As already mentioned, an unmodified BLM behaves essentially as an excellent insulator and does not function as a working electrode. It has been found that upon incorporation of certain organic semiconductors such as T C N Q (tetracyanoquinodimethane) into BLM, very exciting results are obtained [14]. T C N Q belongs to the class of molecular conductors, which have shown remarkably large conductivity at room temperature ( - 102-103 ~ - l c m - 1 ) . TCNQ is a planar, symmetric, 'electron-poor' molecule that can easily gain an electron to become an anion radical. In a study previously reported [15], on one side of the BLM electrons are donated to the membrane interface by electron donors, and on the other side of the membrane electrons are taken away from the membrane interface by electron acceptors. A modifier acting as an electron donor or electron acceptor incorporated into the membrane phase facilitates the redox reaction and enhances the electronic current produced in the system. Processes taking place in the case of the modified

325

BLM containing TCNQ are depicted in Fig. 3h as an example of such reaction [14]. The redox-BLM system acts similarly to the p-n junction of semiconductors. Apparently, the voltammogram of the system exhibits a characteristic shape. When it is recorded with a low speed of scanning, the voltammogram is similar to that of a silicon diode (Fig. 2b). However, when high scan speeds are employed the shape of the voltammogram may be more complicated (Fig. 2i). The electronic conductance of the BLM depends on the redox potential difference between solutions, on the applied voltage and on the modifier properties. It may be derived from the Volmer-Butler equation [17] and the conductance equation (Eqn. 1) and is expressed as follows:

ge = g~(0) exp

n'e ( V + A E )

kT

+ g'e~0)exp

kT

(11)

where: g~(0) and g~i0) denote initial conductances at 0 net current for cathodic and anodic components, respectively, n', n" are constants, and AE is the redox potential difference. Other symbols have usual meaning.

Remarks concerning voltammograms The total conductance is, of course, the sum of the induced conductance and all the other kinds of conductance. In some cases the voltammogram exhibits a complicated shape. Apart from asymmetry, sometimes there may appear hysteresis loops, minima or maxima, or saturation tendencies of the current (see Fig. 2d-h). Several factors in the system may be responsible for these effects. They are: (a) the electric field which drives modifier ions toward, and possibly into, the membrane or away from, and possibly out of it; (b) the speed of scanning the electric potential (voltage); (c) the RC-constant of the electric circuit (including the capacitance and resistance of the membrane); (d) the adsorption and desorption rates of the modifier; (e) the diffusion rates of modifier and charge carrying ions in the BLM; (f) the same diffusion rates in the unstirred layer adjacent to the membrane; and (g) the concentrations of the modifier and charge carrying ions. The electric field changes in time during the voltammogram recording and, if the scanning rate is greater than the rates of the diffusion a n d / o r adsorption (desorption) processes, this must result in delay of conductance changes with regard to the voltage change. As a result, the shape of the I / V curve is complex. If an appropriate formula for the description the I / V curve is found under given experimental conditions, the parameters (gi0, s, r, n, n i) describing some details of the interaction between the modifier and the membrane can be calculated. A computer program fitting the parameters to the experimental curve may be very useful.

Photoeffects When the modifier is a fight sensitive substance, changes in the BLM conductance induced by light usually can be observed [3,9]. If so, the following effects may be registered: (i) slope change of the I / V curve, and (ii) symmetry change of the curve. Depending on the character of the interaction, those changes are reversible or

326 i r r e v e r s i b l e w h e n the light is off. I n the c a s e of a r e d o x - B L M system, p h o t o s e n s i t i v e m o d i f i e r s can, u n d e r illumifaation, g e n e r a t e c h a r g e c a r r i e r s ( e l e c t r o n s , holes, ions) a n d a c c e l e r a t e t h e r e d o x r e a c t i o n across the m e m b r a n e [3]. S o m e d y e s u n d e r g o the p h o t o i s o m e r i z a t i o n process, t h e r e b y f a c i l i t a t i n g d y e m o l e c u l e p e n e t r a t i o n i n t o the B L M a n d c h a n g i n g its c o n d u c t a n c e [3,18]. O t h e r d y e s a n d p i g m e n t s (e.g. c h l o r o p h y l l ) in the B L M u n d e r g o p h o t o - o x i d a t i o n o r d e c o m p o s i t i o n , w h i c h is u s u a l l y an i r r e v e r s i b l e process, c a u s i n g a d e c r e a s e of p h o t o c o n d u c t a n c e [3,16].

Simplified description of the method and its application The use of voltammetric techniques to study the interaction of BLM-active substances is described. The simplicity of the method makes it very useful. However, it must be noted that success in eliciting significant information from the obtained results depends on the expeirmenter's knowledge and expertise in designing experiments. Nevertheless, the electrical properties and ultra-thinness at the molecular dimension of planar BLMs provide a unique opportunity to probe the problems associated with membrane bioenergetics and bioelectrochemistry. A list of up-to-date references to bilayer lipid membrane studies is included.

Acknowledgements M a j o r f u n d i n g o f this w o r k was p r o v i d e d b y an N I H r e s e a r c h g r a n t ( G M - 1 4 9 7 1 ) . W e t h a n k Ms. S h a r o n S h a f t for e x p e r t typing.

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