SCIENCE
Artificial Membrane Probes Electrochemistry of the Living Cell Incorporation of conductor TCNQinbilayerUpid membrane lets researchers perform cyclic voltammetry experiments A recently developed electronconducting artificial membrane will lead the study of bioenergetics in new directions, according to H. Ti Tien, a professor of biophysics and physiology at Michigan State University. The artificial membrane, which can serve as a working electrode in cyclic voltammetry experiments, may help scientists simulate more accurately the electrochemistry that goes on in living cell membranes, Tien says. In bioenergetics, Tien explains, electron-transfer and redox reactions play central roles. These processes are mediated by specialized components. In green plants, for example, the conversion of solar energy into chemical free energy—photosynthesis—takes place in membranebound subcellular organelles called chloroplasts. Similarly, the oxidative phosphorylation of cell respiration takes place in mitochondrial membranes. In both instances, the energy-transducing membranes consist of redox protein components intercalated among lipid molecules, which are organized in the form of a lipid bilayer. To gain some quantitative understanding of these membrane-bound reactions, experimenters, using ordinary electrochemical techniques, have measured the redox potentials (E 0 / ) of the components of the electron-transfer chains. Extensive tables of redox potentials of biological compounds have been compiled,
Tien notes. However, he adds, these measurements typically have been made in simple electrochemical cells using platinum electrodes, with no participation by the membrane. One approach to the study of these organelle membranes has been to resort to membrane reconstitution, using artificial lipid bilayers in either spherical or planar configuration (C&EN, Jan. 2, page 25). For the application of electrochemical or electrooptical methods, the planar bilayer lipid membrane (BLM) is preferable, Tien says. One way to make a planar BLM is to use a brush to spread a mixture of lipids, dissolved in a volatile solvent, across a small orifice between two compartments containing aqueous solutions. As the solvent evaporates, the membrane forms spontaneously: The lipid molecules align themselves in two layers, with their hydrophilic "heads" pointing toward the aqueous media in the two compartments and their hydrophobic "tails" pointing inward. Typically, the membrane is less than 100 Â thick. The aqueous solution/BLM/aqueous solution system is of great help in characterizing the properties of biomembranes and understanding the transport processes that go on, Tien says. The arrangement allows easy access to both sides of the membrane. Highly sensitive electrical techniques can be used to measure the membrane's resistance, capacitance, potential, dielectric breakdown voltage, and current/ voltage characteristics. The BLM can be considered to be a bimolecular liquid crystal in two dimensions having a fluid hydrocarbon core about 50 Â thick. It's worth noting, Tien says, that because of the membrane's extreme thinness, an electric field gradient
Tien: novel approach to bioenergetics of 100,000 volts per cm can easily be developed across it. He points out that this sort of membrane normally is an excellent insulator; however, its electrical properties can be altered by adding appropriate compounds to it. For example, pigmented BLMs have beeen studied by photoelectrospectrometry and found to be capable of light-induced electron-transfer reactions. In contrast, there's been little success in demonstrating that similar electronic processes can take place in BLMs in the absence of light. The main reason for failure has been the lack of suitably modified membranes. Now, Tien and his coworkers, postdoctoral associate Zenobia K. Lojewska and Jan Kutnik (of the Institute of Physics at the University of Curie-Sklodowska in Poland) have prepared BLMs that can conduct electrons even in the dark [/. Phys. Chem., 88, 3172 (1984)]. They have been using an established electrochemical technique, cyclic voltAugust 6, 1984 C&EN
19
oOf6ifC6
Membrane doped with TCNQ functions as redox electrode Reference electrode r
Auxiliary electrode^
Reference electrode
Working electrode
Inner aqueous solution
Outer aqueous solution
TCNQ
For cyclic voltammetry experiments, TCNQ-doped bilayer lipid membrane is used as working electrode in combina tion with saturated calomel reference and auxiliary electrodes
ammetry, to study the redox reac tions that take place in these mem branes. Tien notes that the work has been supported by a grant from the National Institutes of Health. The Michigan State team's inno vation was to saturate the lipid solu tion used to form the membrane (a mixture of lecithin and oxidized cho lesterol in ^-octane) with 7,7',8,8'-tetracyano - ρ - quinodimethane (TCNQ). Then they used the result ing TCNQ-BLM as part of a cyclic voltammetry system. TCNQ is one of a class of molecular conductors sometimes referred to as "organic metals." Its electronic properties— including its ease of reduction and its large conjugated 7r-electron sys tem—are known, Tien says, adding that it retains these properties when incorporated into the BLM. In conventional cyclic voltam metry practice, a stationary work ing electrode in a quiescent solu tion is used with a reference elec trode (usually a standard calomel electrode) and an auxiliary electrode. A repetitive potential of triangular waveform is applied between the reference and working electrodes. The current-voltage (I/V) relation ships are plotted to give a voltammogram. The data from the voltammogram yield useful information, both thermodynamic and kinetic, on electron transfer and redox reac tions at the electrode-solution inter face. To apply cyclic voltammetry to the BLM system, one must make 20
August 6, 1984 C&EN
a "conceptual effort," Tien says: Consider one side of the membrane as the working electrode and the other side as the connection to the external circuit. As noted, an unmodified BLM is essentially an insulator. With the usual potassium chloride bathing solution, so is the TCNQ-BLM. But in experiments with ascorbic acid in one bathing solution and a mix ture of potassium ferro- and ferricyanides in the other, the TCNQ-BLM behaved quite differently. Accord ing to Tien, the changes in electri cal properties—decreased resistance, increased capacitance and membrane potential;' and asymmetrical I/V curves—left little doubt that trans membrane redox reactions were tak ing place at the BLM/solution interfaces, with electrons moving across the BLM. Recently, Tien's group has ob tained a voltammogram of the benzoquinone/hydroquinone (BQ/H2Q) redox couple, using the TCNQBLM. Tien notes that the reaction, BQ + 2H+ + 2e- - H 2 Q, is one of the elements of the electron-transfer chain in both the thylakoid mem brane of the chloroplast and the cristae membrane of the mitochon drion. Also, BQ/H2Q, the simplest quinonoid couple known, has been well-characterized electrochemically. The BQ/H2Q voltammogram pro vides the first compelling evidence, Tien says, that the TCNQ-modified BLM must function as a "working" redox electrode, just as a platinum electrode does in conventional electrochemistry. That fact is significant, according to Tien. He points out that E 0/ val ues for electron-transfer chain com ponents, when known, usually have been determined with platinum electrodes. But it's known that these components are closely associated with the lipid bilayer; the values of E 0 / measured with platinum elec trodes may be quite different from the actual values in the membrane. Thus, Tien says, the cyclic voltam metry technique, using TCNQmodified BLMs as working elec trodes, affords a new approach to determining E 0 / values for cyto chromes and other membrane-bound molecules. Ward Worthy, Chicago
New metallocene defies electron shell theory Researchers at the University of Oklahoma have a mystery on their hands. They've created a cyclopentadienyl sandwich compound of tin(II)—a molecule that, if earlier experience with related compounds is any guide, ought to have a bent structure and a protruding lone pair of electrons. Except that this struc ture, to their astonishment, is com pletely symmetrical, and the lone pair is nowhere to be found. "It is the first molecule to violate decisively the valence-shell electron pair repulsion (VSEPR) theory," as serts inorganic chemistry professor Jerold J. Zuckerman, who made the discovery with staff crystallographer Mary Jane Heeg and Christof Janiak, a visiting graduate student from Technical University of Berlin [/. Am. Chem. Soc, 106, 4259 (1984)]. Their work was supported by the Office of Naval Research. "The VSEPR model is such a beau tiful thing," ^Zuckermanexplains, because it can predict, for instance, the structure of molecules contain ing subvalent atoms of the group IV elements germanium, tin, and lead. These atoms, like carbon and silicon, usually form molecules in which they bind to four other groups. But in the divalent state, the atom connects to only two groups, leaving a lone pair of elec trons to occupy its own place in the coordination sphere. VSEPR theory predicts, for exam ple, that in the cyclopentadienyl compounds of germanium(II), tin(II), and lead(II), the rings will be in clined towards each other substanti ally. This deviation from the sym metric, parallel-ring sandwich struc ture characteristic of ferrocene is due to the space-filling effect of the lone pair, which, like a corpulent diner at a busy lunch counter, crowds the diners next to him to make more room for himself. All the metallocene compounds of germanium(II), tin(II), and lead(II) whose structures have been eluci dated have been shown to possess the severely bent structure predict ed by VSEPR theory. Until now. Zuckerman's rule-breaking find is
TECHNOLOGY decaphenylstannocene, [r75-(C6H5)5Cs]2Sn(II), the first main-group sand wich compound with a symmetri The first product to be made in space cal structure. It differs from earlier is about to be offered commercially. examples by the sheer bulk of its The National Aeronautics & Space ligands: Both cyclopentadienyl rings Administration has turned over to carry a phenyl substituent on each the National Bureau of Standards ring carbon. The rings are planar, 15 g of 10-μπι latex particles that staggered, and exactly parallel. The NBS will make into a small-particle 10 phenyl groups, according to the Standard Reference Material (SRM). researchers, "are canted to each NBS expects to have the SRM units cyclopentadienyl ring oppositely in ready for sale early in 1985. a double-opposed paddlewheel fash The latex particles are monodision to give molecules of Sio sym perse (identical size) polystyrene metry." But where's the lone pair? spheres. They were produced in "It's got to be somewhere!" Zucker- space during 1983 aboard the space man declares. shuttle in a monodisperse latex A plausible explanation is sug reactor developed for NASA by a gested by literature data on inorgan team headed by principal investi ic compounds of subvalent group gator John W. Vanderhoff, a chem IV elements, as well as by the in istry professor at Lehigh University. tensely bright yellow color of deca NBS will divide the latex parti phenylstannocene: The "missing" cles into about 600 SRM units of electrons might be delocalized throughout the vast electronic sys tem formed by the 10 phenyl rings. Such derealization could produce the compound's intense color, Zuckerman believes. However, he's quick to add, this is just speculation. What is clear, though, is decaphenylstannocene's "fantastic sta bility," in the Oklahoma professor's words. Other stannocenes are mar ginally stable, decomposing around 100 °C. But his compound can be heated reversibly past 300 °C. This enhanced thermal stability suggests that pentaphenylcyclopentadienyl groups could be used to stabilize other metal systems, includ ing some pesky lanthanide and actinide complexes. Zuckerman and others already are actively engaged in synthesizing other metallic ana logs of decaphenylstannocene. He tells C&EN that the germanium analog, for instance, appears to be "a lot more stable" than the unsubstituted parent molecule, germanocene. If this effect proves general, it might open up the subvalent organ ic chemistry of a variety of metals. Apparently, the symmetry of decaphenylstannocene is a conse quence of the presence of both bulky coordinating groups. The related stannocene in which only one of Latex spheres made in space ( top) are the five-membered rings has phe uniform and precise, compared to nyl substituents on it also has been those made under the influence of made by Zuckerman's group and gravity (bottom) using the same found to be bent. D recipe and experimental hardware
Space-made latex particle standard to be sold about 0.5% by weight in water. Each unit will consist of a 5-mL vial con taining about 15 million spheres. A unit will sell for about $400, with proceeds from the sale shared by NASA and NBS to recover the costs of producing and certifying the material. The experiment package that pro duced the spheres consists of four, 1-foot-high chemical reactors, each containing 100 mL of a latex-forming recipe, housed in a 2-foot-high met al cylinder. The latex reactor experi ment has been operated on five shuttle missions beginning in March 1982 and was last flown in February. It will continue to be flown on fu ture flights to provide particles in larger sizes up to 100 μπι. Spherical uniformity and preci sion mark the space-grown particles. Because of the influence of gravity, ground-based processes for making spheres larger than about 3 μπι haven't yielded a material uniform enough in shape for use as a reli able reference material. Gravity tends to distort the spheres so that they become egg-shaped. Also, they stick together in clusters. In space, particles in a variety of sizes from 5 to 30 μπι have been produced. The new SRM has a wide range of uses as a calibration tool for man ufacturers and users of instruments that measure small particles. Among potential users are technologists who monitor environmental particulate pollution from industrial plants; pro ducers of finely ground products such as paint pigments, inks, toners, cements, explosives, chemicals, and other powder materials; and medi cal researchers who calibrate instru ments to count blood cells and per form other diagnostic measurements. To determine the average diame ter of the spheres in a small-particle SRM, NBS examines the spheres us ing a scanning electron microscope to view individual particles, an opti cal microscope to study group pat terns of the particles, and a laser light-scattering technique to mea sure the particles suspended in water. It is only after average parti cle diameter is established by these techniques that NBS will certify a reference material. D Auguste, 1984 C&EN
21
