of cytochrome c3 proteins (which are known to have three conformationally distinct electrochemical forms) adsorbed on Hg electrodes. The proteins studied were wild-type cytochrome c3 and two mutants (H70M and H70V) in which one of the heme axial ligands is replaced by methionine or valine, respectively. To probe the protein orientation and conformational change, all antibodies must bind to all non-native conformations induced by the adsorption on the electrode. An antibody that binds to a conformational epitope (a discontinuous epitope formed from residues adjacent in space but not necessarily in the sequence) will be most sensitive to the conformational changes. The authors determined that the antibodies 2A2 and 4H8 were sensitive to the potential dependent conformational changes of the absorbed cytochrome c3. The binding constants of the antibodies to cytochrome c3 were used to determine that the protein does indeed have three conformations at the various potentials. These results were confirmed by a separate radioimmunoassay. (J. Am. .hem. Soc. 1997,119, 5295-301)
BUSINESS
Andi protocols now under ASTM The responsibility for standardizing the instrumental reporting of analytical data, known as the Andi (analytical data interchange) Protocols, has been transferred from the Analytical Instrument Association (ALA) to the American Society for Testing and Materials (ASTM). According to Michael Duff, executive director of ALA, the association decided on the move because "it wanted the protocols to reside in an organization that can maintain them and test them." In addition Duff said that ASTM will bring software writers government scientists and instrument users into the protocol development process Andi was begun by AIA in efforts to make the analytical instrumentation as flexible as PCs in using software and equipment from various vendors (Anal. Chem. 1997,68,529 A). To od that, instruments needed a common language, similar to DOS or UNIX in the PC world. AIA had some success in developing data protocols for chromatography, MS, and
The image of copper In metal electrodeposition and etching applications, controlling the surface morphology is critical. Allhough organic additives are often used for control, their influence on the development of surfaces is poorly understood because few techniques can measure the interaction of the additive with the surface and simultaneously characterize the changes in deposit morphology. Dean S. Chung and Richard C. Alkire at the University of Illinois at Urbana-Champaign used confocal laser scanning microscopy in situ during copper electrodeposition and electrodissolution to track the adsorption of thefluorescentcarbocyanine dye DiOC while simultaneously monitoring the changing surface morphology Their data showed that, with and without copper, the dye adsorbed to polycrystalline gold and inhibited cathodic processes. Without copper, dye adsorption on gold remained unaffected by changes in cathodic potential up to -750 mV. During copper electrodeposition at -550 and -650 mV, the adsorbed dye restricted nucleation of the copper to a few active sites
IR. However, AIA was limited in how much it could develop the protocols. According to Duff, AIA voted to approach ASTM with the request to take over the development of the protocols. Details of how ASTM will handle the Andi protocols were not available at press time, but the protocols are expected to fall under Committee E-49 on Computerized Systems and Material and Chemical Information. ASTM expects to develop consensus documents on the protocols. Moreover, says Duff, ASTM will be able to sponsor round-robin testing and updates of the protocols as needed. Alan Newman
Varian buys Chemagnetics California-based Varian Associates has purchased Chemagnetics, manufacturer of solid-state NMR instruments, from its parent owner Otsuka Electronics. According to Varian, the purchase is a complementaryfitto its other lines of NMR systems. Varian plans to continue to operate Chemagnetics's Ft. Collins, CO, manufacturing facility as part of its overall NMR business.
Confocal laser scanning microscopy morphology map of the electrodeposition no copper on gold in a solution of DiOC6 dye end copper sulfate at -550 mV. .Adapted with permission from J. Electtochem. Soc. Copyright 1997 Thh Electrochemical Society.)
and dye adsorption was maintained across areas where nucleation had not occurred. Secondary ion MS indicated that the dye, or a derivative of it, was incorporated into the deposit during copper electrodeposition. (J. Electrochem. Soc. 1997,144, 1529-36)
MEETING NEWS
News from ASMS Celia Henry reports from Palm Springs, CA A sampling of research presented at the 1997 meeting of the American Society for Mass Spectrometry
MALDI on a chip First there was the "lab on a chip"; then there was "sequencing on a chip". Now, take low-nanoliter volumes of analytes, dispense them into a small well of a lessthan-one-inch-sized substrate, analyze them by MS, and—voila—you have "MALDI on a chip". The technique was described by Daniel P. Little of Sequenom (San Diego, CA). MALDI on a chip was used to analyze products based on a molecular biology technique known as primer oligo base extension, or PROBE reaction, for largescale DNA diagnostics. The polymerase chain reaction is first used to replicate a strand of DNA. The strand is placed on a solid support, and a short piece of DNA is annealed close to the mutation site. The
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News
LABORATORY PROFILE What's hot in the Manz lab this summer? Andreas Manz is a worried man. As the summer sunlight begins to creep over his desk, he knows that the glass roofs of his labs will soon let in enough light to boost the temperatures, leading to overheated students and technicians. The solution would be to install air conditioning, but although the budgetary means exist from the joint funding by SmithKline Beecham (SKB) and Zeneca of almost $16 million over five years, his building is only halfway through refurbishment. So plans to cool off will have to wait. As the temperature rises, though, so does Manz's enthusiasm for the work he and his team of 19 in the SKB/Zeneca Centre for Analytical Science at Imperial College, London (U.K.), are doing. And although the scale of his enthusiasm is enormous, the scale of their analytical devices is getting smaller. Manz became SKB Professor at Imperial College in November 1995 and brought with him from Ciba-Geigy (now Novartis) vast experience in compressing the scale of analytical equipment to submicroscopic proportions. His work is aided by lecturer Norman Smith, who wheels and deals to get "loan" equipment for the center. "We haven't paid for a single HPLC machine since we arrived," says Manz, "and we have more instruments for capillary electrochromatography in one lab than else in the world." Manz and colleagues, including Zeneca lecturer Andrew de Mello, concentrate their team's efforts on creating tiny channels in glass chips. These channels, they are finding, can be created at just a few micrometers diameter by etching a glass chip and laminat-
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ing it with a second layer of glass. This arrangement allows several thousand CE channels to be squeezed into a couple of square centimeters. Samples of just a few picoliters can be dripped into tiny wells. The potential theoretical plates are 100,000 in a 30-min run for standard GC, says Manz. "We reckon on a million plates in 10 minutes with our techniques, because we spend more time optimizing the miniaturization process in electrochromatography and on the chips." With these tiny sample volumes, Manz achieve separations in a hundredth the time used by conventional instruments but can retain the Scime resolution The chemist's dream would be to couple "lab-on-a-chip" devices or, as Manz prefers, "miniaturized total analysis systems" (u-TAS) to a mass spectrometer and a PC. Just 10,000 or so molecules of separated material could then be analyzed and identified. The ability to scale separation, which Manz perceives as perhaps the most important aspect of analytical science, to levels of picoliters and below could accelerate the development of areas of science such as medical diagnostics, including DNA analysis. Such developments could, for instance, allow analysis of components from just a single blood cell or microbe. Indeed, Manz will soon begin working with Rory Shaw at St Mary's Hospital in London, now part of Imperial College (IC), to accelerate patient assessment following antibiotic treatment for tuberculosis. "It can take several weeks to decide whether a patient is fully clear of infection," says Manz. "We hope to develop u-TAS technology that will allow separation of all microbial DNA from the patient so that the TB genome can be spotted much more readily." He predicts that the thriving field of u-TAS will soon become the technique of choice, allowing researchers—especially in pharmaceuticals—to manipulate minute samples to get the results they need without worrying about sample loss. He suggests that u-TAS might then be extended to environmental monitoring; for instance, tracking algal blooms in waterways and forecasting the data remotely via radio links on the chip. Manz believes u-TAS is not a limited technology, and he is forging links with other departments at IC such as plasma physics and electrical engineering. However, he is less interested in the commer-
Analytical Chemistry News & Features, August 1, 1997
Andreas Manz is thinking small.
cialization of his discoveries than in their creation. "We want to look at the fascinating leads and leave the commercialization to industry and others," he explains. Manz says that they are simply following nature's lead. The optimum scale for chemical reactions seems to be about 1-10 um. Enzymatic manipulation of DNA and neurotransmission takes place on this scale, for instance. "Some insect pheromones even act at the single-molecule level," points out Manz. 'Ten thousand molecules is probably a realistic lower limit for chemists, though!" Manz ponders the idea that industrial processes might be rendered safer and more efficient with u-TAS online monitoring Manz hopes that once academe and industry are routinely using u-TAS, the development of chemical chips will come to the fore in domestic use. "The Japanese are developing a chip that fits in the toilet bowl and analyzes urine for signs of disease," he points out. Similarly, a chip in a saucepan could spot pathogens and increase cooking time accordingly or instead recommend a takeout meal. Since his arrival at IC, Manz has succeeded in doubling die money the center has gained from industry and government sources other than SKB/Zeneca. But, as wiih many other academic analytical chemists, even doubling an initially small pot is simply not enough. As the summer heats up, the chemists will have to keep their cool as they shrink their instruments even without AC. (More information about Manz's work can be found at his Web site: http://www.ch.ic.ac.uk/manz/) David Bradley
primer is then extended by a number of bases that are characteristic of the particular mutation. The mass value of the predesigned products is far more analytically definitive than signals from fluorescence-based assays, which treat the hybridization characteristics as the "be all and end all" of detection. Also, because a particular mutation yields a known mass, problems with the reaction could easily be detected when an unexpected mass was encountered. Little says that they have not needed to use this particular advantage of MS because of the "high fidelity" of the polymerase enzymes used. In the experiments that Little described, a MALDI matrix was predispensed on the chip and nanoliter quantities of diagnostic material were placed on top using a piezoelectric pipettor (a tool that resembles a single element of an inkjet printer) or by a simple pin transfer. He says that the matrix material can also be mixed with the analyte solution or placed on the chip after the DNA is dispensed. Little presented data showing the reproducibility of the technique and its use in population studies. The same analyte solution was transferred with die piezoelectric pipettor into chip wells and analyzed reproducibly 100 times. When the samples do not necessitate washing the pipettor between transfers, the total time is only a few minutes. For parallel analysis of different samples, they used a 16-pin tool that was dipped into the samples and transferred to the chip. He showed PROBE reaction products for a 15-person population study using this method. Little described MALDI on a chip as an "enabling technology" for gene-hunting and pharmaceutical companies. Currently, the most powerful use of MALDI on a chip is in typing applications (which Little says will be particularly important for large-scale clinical studies) and microsatellite (sizing) analysis. "Diagnostic" or "signature" sequencing on a chip on a large scale is still further down the road.
The incredible shrinking mass spectrometers With field-portable instruments, the smaller the better, and portable mass spectrometers are no exception. Maria C. Prieto of Lawrence Livermore National Laboratory presented results on an ion cyclotron resonance mass spec-
trometer that fits on one side of an 18 x 12 x 3.5-in. briefcase. The instrument, which was developed by Dan Dietrich and Bob Keville, consists of an open-endcap cylindrical Penning trap, a piezoelectrically actuated inlet valve, and an ion-pump vacuum system, all of which are miniaturized. Power is provided by four 12-V laptop computer batteries. The analyzer cell is located in a 0.44-T permanent magnet. Prieto and co-workers characterized the performance of the analyzer cell by studying the variation of the ion cloud radius with applied rf voltage (needed for ion excitation). The ionization time needed to produce the same number of ions was documented for different trapping and compensation electrode voltages. The system currently achieves mass resolution of 500-100 at mid 10-7 torr pressures with a magnetic field homogeneity of ~ 1000 ppm. Another miniaturized instrument presented by Scott Ecelberger of The Johns Hopkins University Applied Physics Laboratory has been dubbed 'Tiny TOF". The instrument is designed for chemical and biological warfare agent detection, using biomarkers developed at the University of Maryland-Baltimore County and an analyzer developed at The Johns Hopkins University School of Medicine. Tiny TOF consists of a 9-cm coaxial TOF analyzer and a shoebox-sized vacuum chamber. A 600-ps pulsed N2 2aser is ssed for the UV MALDI experiments. The mass spectrometer, sensitive to the subpicomole level, has a mass range > 11,000 m/z znd resolution of 300-1000. Also seen at ASMS was the quadrupole array mass spectrometer developed by the Jet Propulsion Laboratory. (Anall Chem. 1997,69,335 A-36 A)
Using buffers with MS The conventional wisdom says that the buffered mobile phases used in HPLC are incompatible with MS analysis because they contaminate the ion source, create chemical noise, and reduce the analyte signal. Duncan Bryant and Sean Burnside of SmithKline Beecham (U.K.) demonstrated that the conventional wisdom might not always be right. They tested various nonvolatile and low-volatility buffers with the pharmaceutical compound eprosartan, including sodium dihydrogen phosphate, ammonium
dihydrogen phosphate, sodium acetate, and orthophosphoric acid. In each case, a 1- to 50-mM buffer gradient containing 1 ppm eprosartan entered the mass spectrometer at 100 uL/min. Sodium dihydrogen phosphate was usable up to 20 mM. At higher concentrations, the background chemical noise increased greatly, and the eprosartan signal decreased. The sodium acetate was completely unusable, and suppression occurred at concentrations as low as 5 mM. The orthophosphoric acid had the widest range, usable up to almost 50 mM. They also tested several flow rates, using sodium dihydrogen phosphate as the test buffer. They determined that the system is optimized in the range of 220 uL/min. At flow rates > 25 uL/min, they observed analyte signal suppression and a high level of chemical noise.
Low-pressure electrospray? Electrospray is considered an atmospheric pressure ionization technique, but does it have to be? According to results presented by Edward Sheehan and Ross Willoughby of Chem-Space Associates (Pittsburgh, PA), under the proper conditions electrospray ionization can be performed at pressures as low as 10"4 torr. They divided a plot of discharge onset voltage versus pressure into three regions. At intermediate pressures, the voltage was not high enough to overcome the threshold for cone-jet formation. However, at very low and atmospheric pressures, the discharge onset voltage was above the required minimum. At atmospheric pressure, there is enough heat to prevent the electrospray cone-jet from freezing. However, at reduced pressures, the entrance aperture needed to be heated to prevent freezing. They demonstrated low-pressure ESI with the protein myoglobin and caffeine. The ESI spectrum of myoglobin contained the characteristic multiply charged envelope, which was centered around the +11 ion. The spectrum of caffeine showed the predominant protonated molecular ion at m/z 195. By changing the voltage difference between the desolvation region and the exit aperture, they were able to fragment the m/z 195 ion. They said that several questions remain, including the optimum conditions, the ion-sampling efficiency, and the analytical use of the method.
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