Terry C. Chilcott

Impedance spectroscopy of interfaces, membranes and ultrastructures

For the past century, impedance spectroscopy has provided a non-invasive means of characterizing the electrical properties of many systems. Even today, it often provides the only non-invasive method for detailed structural-functional studies of these systems. This is especially so of systems in which important processes occur at the molecular level, such as those processes associated with biological and synthetic membranes and interfaces that form between solutions and various solids (e.g. metals and colloid particles). The fundamental concepts of impedance spectroscopy are re-examined and a review is given of the role that impedance spectroscopy has played in the development of our understanding of cellular and synthetic membranes, cell biophysics and ionic systems in general. Special emphasis is given to the problems associated with solution-electrode interfaces, as well as unstirred layers, which can plague measurements on biological systems and have led to much confusion in the past. A description is given of a new computer-controlled, four-terminal digital impedance spectrometer, which provides resolutions in impedance magnitude and phase of 0.002% and 0.001 ° respectively over a frequency range of 10−2 to 105Hz and for impedances ranging from 10 to 109 Ω. We also describe impedance dispersions in terms of transfer functions which, when plotted along the negative frequency axis, yield “spectra” with distinct sharp peaks that identify fundamental frequency constants of the system. This “control engineering” form of presentation of impedance spectra demystifies the impedance analyses of these systems. The spectra and changes in these which occur as a result of perturbations to the system can be readily assessed and interpreted.

Electrical impedance spectroscopy characterisation of conducting membranes I. Theory

An electrical impedance spectroscopy (EIS) method for measuring changes in the electrical properties of synthetic membranes is investigated as a possible way of monitoring, in situ, the separation performance of these membranes including membrane fouling. Unlike other EIS methods, which require traditional electrodes in the feed and permeate solutions, alternating current is injected directly into the membrane via external electrical contacts with the edges of the membrane. A metal layer sputtered onto the surface of the membrane can be used to enhance its conduction properties. The impedance models of these systems is shown to be sensitive to membrane surface properties, including porosity, as well as electrical properties of solutions and the interfacial regions between the membrane surfaces and the solutions. The investigation indicates that fouling along the surface of the membrane might be more readily detectable than inside the pores.

Electrical impedance spectroscopy characterisation of conducting membranes: II. Experimental

An electrical impedance spectroscopy (EIS) method and apparatus that eliminates the need for electrodes in the feed and permeate solutions was evaluated as a means of characterising physical and performance properties of polysulphone ultrafiltration membranes in situ. The membranes were sputter-coated on one side with platinum before assembly in the apparatus. Alternating electrical current used for impedance measurements was injected directly into the coat via dry electrical contacts with the edges of the membrane. As the frequency of the EIS measurement was increased the current increasingly dispersed into the solution via the interfacial region (double layer) and/or fouling layers that the coat formed with the solution. These spatial dispersions manifested as characteristic dispersions with frequency of the impedance of the system. Water flux measurements, field emission scanning electron microscopy and atomic force microscopy were also used to quantify the important membrane performance parameters of porosity and surface roughness. These estimates were in good agreement with the impedance model for the in situ membrane system that was fitted to the measured impedance dispersions. The study shows that EIS measurements potentially can quantify membrane performance parameters in situ better than those techniques that require disruption of the membrane separation process. The method also has the potential for monitoring the deposition of particulate that can lead to fouling.

Differential effects of cholesterol and oxidised-cholesterol in egg lecithin bilayers.

Low frequency impedance measurements of pure egg lecithin (phosphatidylcholine) bilayers have revealed the presence of four layers which can be attributed to the acyl chain, carbonyl, glycerol bridge and phosphatidylcholine regions of the lecithin molecule. Measurements on bilayers formed in the presence of unoxidised-cholesterol revealed that cholesterol molecules were located in the hydrocarbon region of the bilayer with its hydroxyl groups aligned with the carbonyl region of the lecithin molecules. Measurements of oxidised-cholesterol lecithin bilayers revealed that these molecules protruded less into the hydrocarbon region and their polar hydroxyl group aligned with the glycerol bridge region of the lecithin molecule.

AC impedance of the bipolar membrane at low and high frequencies

The bipolar membrane or double fixed charge membrane (DFCM) consists of a central depletion layer of low conductivity surrounded by outer regions of high conductivity. The Nernst-Plank-Poisson equations for alternating currents are solved for each region separately with appropriate approximations. The two solutions are melded to give the solution for the membrane as a whole. The results are presented as the frequency dependence of the relevant capacitance and conductance. At the low and high frequency limits these parameters become independent of frequency, with a monotonic dispersion at intermediate frequencies in agreement with observation. The dispersion is interpreted in terms of the alternating electric field which is confined to the depletion layer at low frequency but is almost uniform through the entire membrane at high frequency. The dispersions are similar to those of a two element Maxwell-Wagner system, but the elements required for the latter may not be identified with the separate regions of the DFCM.

A novel method for the characterisation of the double fixed charge (bipolar) membrane using impedance spectroscopy

Abstract Double fixed charge membranes (DFCM) that is, bipolar membranes, are formed through the fusion of anion and cation exchange membranes. At the junction a depletion layer forms in which the mobile ion density is very low and the charge density is that of the fixed charges of the respective exchange membranes. The intrinsic properties of this region play a crucial role in determining the separation and electrical characteristics of DFCMs and can be modulated, in situ, through the application of an electrical potential across the membrane. An inspection of the molecular structures of ion transporters in biological membranes reveals that charged amino acids in these proteins also form junctions of juxtaposed fixed charges. We have developed electrodiffusion theories and derived expressions for the electrical impedance of the DFCM. These match those observed in biological membranes. Here we describe an experimental procedure in which impedance measurements can be used to monitor the aforementioned in vivo or in situ junction properties which cannot be otherwise readily determined from external measurements. Specifically, we show that the electrical transfer function of the DFCM, determined from expressions for the impedance, has only two frequency constants or “natural” frequencies. These frequency constants do not arise from Maxwell-Wagner layers within the DFCM but from an enhanced capacity of these membranes to store electric charge. The unique manner in which the intrinsic junction properties depend on the transfer function parameters, provides a means for their identification and, most importantly, a method of membrane characterisation.

Anomalous electrical behaviour of single-crystal glycine near room temperature

Abstract Electrical impedance measurements of single-crystal glycine reveal anomalous temperature dependence of the conductance and capacitance. Upon cooling the crystal from 50°C the conductance decreases smoothly from an initial value of 0.1 nS to about 0.02nS at 31°C. Further cooling, however, causes a dramatic increase in conductance with a magnitude approaching 100 nS at 21°C. Similar anomalous behaviour is exhibited by the concurrently measured capacitance; it is approximately temperature independent above 31°C but decreases precipitously below this temperature. This unusual electrical behaviour is not explained readily by the conduction mechanisms expected to apply to these materials but is consistent with the onset of pyroelectricity at 31°C.

Electrical characterizations of biomimetic molecular layers on gold and silicon substrates

Electrical impedance technology was used to characterize DNA recognition in a monolayer containing single-stranded DNA probes immobilized on a gold substrate using thiol self-assembly chemistry. Recognition of targeted complementary DNA was principally correlated with an eight-fold increase in the conductance of the monolayer and attributed to electron conduction through double helices formed upon the binding of the DNA targets to the probes. The high recognitive sensitivity was possible without the use of the redox labels or large bias voltages required for recognition using cyclic and Osteryoung square wave voltammetry. The impedance technology also provided atomic resolution of a hybrid bimolecular lipid membrane formed by deposition of a phospholipid:cholesterol monolayer onto a hydrophobic alkyl monolayer covalently attached to a silicon substrate via silicon-carbon bonds. Atomic resolution was achieved through preparation of membranes on surfaces approaching atomic flatness and the performance of impedance measurements over precisely defined areas of the surface in contact with solutions. Principally capacitive properties distinguished between the immobilized (octadecyl) and more fluidic (lipid:cholesterol) leaflets of the hybrid membrane. The lipid:cholesterol leaflets were structurally similar to those leaflets in free-standing bimolecular lipid membranes. The hybrid membrane therefore provides a highly stable and physiologically relevant surface for studying biomolecular interactions with membrane surfaces.

Electric field effects in proteins in membranes

Both the organization and function of protein nanostructures in membranes are related to the substructural properties of the lipid portion of the membrane. Potential differences that are established across the membrane and generate electric fields in these very thin portions are shown to modulate the organizational and functional properties of the protein modules. Many protein modules also have nonisotropic distributions of charged sites, including configurations in which there are regions containing predominantly positive fixed charges, juxtaposed with adjacent regions containing predominantly negative fixed charges. In these double fixed charge regions, very large electric fields can manifest in the ionic depletion layer at the junction of the two fixed charge regions. Consideration is also given to the manner in which the intense electric fields that are established in protein modules, such as proton ATPases, can modulate the chemical reactions that are associated with proton transport and dehydration reactions.

ELECTRICAL IMPEDANCE SPECTROSCOPY CHARACTERIZATIONS OF ALKYL-FUNCTIONALIZED SILICON(111)

This organic thin-film systems that are based on silicon-carbon covalent bonds have been shown to lead to densely packed alkyl monolayers that have potential bio-passivation or bio-sensing applications. Presented are electrical impedance spectroscopy (EIS) characterisations of a series of alkyl monolayers [CH3(CH2)mCH=CH2; m = 7, 9, 11, 13, 15] that were covalently linked to Si(111) wafers. The characterizations reveal capacitance, conductance and geometrical properties of the monolayers. The capacitance properties yield estimates of thicknesses for the monolayers that increase proportionally with each additional CH2 unit and are consistent with the known physical properties of these films such as dielectric constants and chain canting angles. This study illustrates that EIS charcterizations are able to probe immobilized surfaces on silicon with sub-atomic resolution which is so important in the development of practical bio-passivation or bio-sensing applications.