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ANALYTICAL CURRENTS We’ll drink to this method Although there is nothing wrong with judging the age of scotch whiskeys by taste, Sheryl Wiskur and Eric Anslyn at the University of Texas–Austin introduce a more modern means based on a UV–vis technique. The key is a synthetic receptor that binds a class of molecules, which are formed as scotch ages, rather than a single analyte. Traditionally, the age of a scotch was linked to the concentration of gallic acid, which appears as a result of tannin hydrolysis. However, the wooden casks in which the liquor is stored add other “gallate-like” compounds—how much and which kinds depend on the type, age, and previous exposure of the wood. The receptor was designed to bind gallate and a broad range of related compounds. The colorimetric indicator pyrocatchol violet was chosen for this ap-
plication because of its structural similarity to gallic acid. When a gallate-like molecule is captured by the receptor, the indicator is displaced and changes in color from yellow to maroon. The resultant UV–vis spectra are used to generate a “response number” that is characteristic of age. Because the sensor targets the gallate analogues, it demonstrates better age correlation than simply quantifying the gallate by UV and LC. The researchers note that, in this case, the low selectivity of the syntheic receptor is an advantage, not a flaw. (J. Am. Chem. Soc. 2001, 123, 10,109–10,110)
Studying supercritical solvents by spectrometry Applications of supercritical fluids are in-
University (both in Japan) have designed a
without protection from the heat, a water-
creasing, but few ways exist to study the
cell for a general-purpose spectrometer
cooled jacket protects the double beam UV
mechanisms that make them unique be-
that handles supercritical fluids at tempera-
spectrometer in three places when the cell
cause requisite conditions ruin standard an-
tures up to 600 ºC and pressure up to 600
is heated with a small electric furnace. The
alytical equipment.
MPa. Acquiring a successful UV–vis ab-
cell is made from Hastelloy-X and has syn-
Okitsugu Kajimoto
sorption spectrum under supercritical
thetic sapphire windows to withstand heat
and colleagues at
water conditions proved the high-tempera-
and pressure.
the Japan Science
ture, high-pressure optical cell works well.
11 10 9
The researchers collected UV spectra
3 4
and Technology
2
Corp. and Kyoto
1
Because general instruments cannot accommodate a high-temperature cell
8
6 7
5
12
Block diagram of the high-temperature, high-pressure optical system. (1) Conventional spectrometer, (2) cooling box for the optical cell, (3) furnace of the optical cell, (4) optical cell, (5) sample injection tube, (6) heater, (7) preheating unit, (8 and 9) cooling jackets, (10) sample exhaust tube, (11) back-pressure regulator, and (12) HPLC pump. (Adapted with permission. Copyright 2001 American Institute of Physics.)
of NiBr2 at temperatures ranging from ambient temperature to 430 ºC at 39.5 MPa. They intend to use the cell to collect information about local density changes around solutes in supercritical reactions, which are believed to control the reactions. (Rev. Sci. Instrum. 2001, 72, 3605–3609)
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ANALYTICAL CURRENTS Profiling membrane-bound proteins Because many membrane-bound proteins serve as receptors, pores, and pumps, they are prime targets for new diagnostic procedures and therapies. Unfortunately, membrane-bound proteins are also particularly difficult to isolate and study. One approach that is now being applied to this problem is the isotope-coded affinity tag (ICAT) method for quantitative proteome analysis, developed by Ruedi Aebersold, who is now with the Institute for Systems Biology, and colleagues. The ICAT technique uses isotopic labeling to internally calibrate proteins detected by MS. The labeling replaces the standard blocking procedure for preventing cysteines from reacting nonspecifically with their neighbors. Instead, the cysteines are isolated with a biotin affinity tag connected to a linker, which can be made “heavy” (incorporates deuterium) or “light” (incorporates hydrogen). Samples with light and heavy tags are combined and cleaved, and the fragments are separated using multidimensional chromatography and analyzed with MS. The ratios of the labeled peaks give quantitative information, and MS/MS combined with database searching identifies the peptides. Aebersold, David Han at the University of Connecticut Health Center, and
colleagues applied this method to the fraction of proteins from the microsomes (intracellular vesicles) of a human myeloid leukemia cell line. One sample was exposed to 12-phorbol 13-myristate acetate to induce differentiation, whereas the control sample remained untreated. The relative abundances of 491 proteins were determined. The predominance of transmembrane and membrane-associated proteins was confirmed, and some moderate- to low-abundance proteins, including membrane receptors, channels,
kinases, and phosphatases, were identified. Furthermore, the method showed changes in the levels of some of the membrane-associated signal transduction proteins following the 12-phorbol 13-myristate acetate treatment. (Nat. Biotechnol. 2001, 19, 946–951) In a related paper, researchers working with Aebersold systematically evaluated the experimental conditions that result in robust, reproducible results using the ICAT method. (Anal. Biochem. 2001, 297, 25–31)
Cell surface antigens, receptors and membrane proteins (11.2%) Functionally uncharacterized proteins (22%)
Channel proteins (4.9%)
Kinases and phosphatases (5%)
GTPases and GTP binding proteins (4.7%)
Possible transmembrane proteins
Metabolic enzymes (15%)
Ribosomal proteins (8.8%)
Possible transmembrane proteins
Mitochondrial proteins (5.1%) Cytoskeletal proteins (4.5%)
Hypothetical proteins (14.1%)
Golgi /ER proteins (4.7%)
Categories of proteins identified in the study of the microsomal fraction of human myeloid leukemia cells. (Adapted with permission. Copyright 2001 Nature America.)
A protein to stick with It is a natural combination—electrodes coated with hydrophobin, a small, naturally occurring sulfur-rich protein that has the
electroactive molecules. The hydrophobin studied was derived from the fungus Pisolithus tinctorius. It
dense and prone to blocking as, for example, a covering of alkanethiols. In the presence of a long, hydrocarbon
remarkable property of adhering to almost
quickly self-assembles on thin-film mercu-
chain ubiquinone, the hydrophobin-modi-
any surface. Renata Bilewicz, Ewa Rogals-
ry electrodes and, with more time, on
fied electrodes absorbed the big molecule
ka, and colleagues at the University of
glassy carbon and gold electrodes, effec-
on the surface, but the smaller quinone
Warsaw (Poland) and the Université Henri
tively covering the entire surface. The lay-
and azobenzene molecules penetrated into
Poincaré (France) investigate the proper-
ers are stable over time, in various pH so-
the pores of the self-assembled layer.
ties of various electrodes covered with hy-
lutions, and at a range of electrical
These findings suggest the hydrophobins
drophobins and find that the sticky protein
potentials. Capacity currents did not de-
could be the basis of a new class of modi-
holds fast to surfaces, limits access to the
crease when an electrode was coated,
fied electrodes. (J. Phys. Chem. B 2001,
electrode, and acts as a hosting matrix for
suggesting that the protein layer is not as
105, 9772–9777)
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The next nose A hallmark of the olfactory system, and the electronic “noses” that imitate it, is that odors are identified by the overall pattern of responses coming from an array of nonspecific sensors. This contrasts with receptor–ligand binding, in which the receptor is precisely tailored to the ligand. Now, David Walt and colleagues at Tufts University describe an array that combines both approaches to sensing. To achieve this, the researchers created an array of nine esterases, all of which catalyzed the hydrolysis of esters to carboxylic acids. Acid formation, in turn, was monitored by the fluorescent pH indicator fluorescein. Some of the
enzymes worked on various esters, whereas others were selective enough to distinguish between the D- and L-forms of a single analyte. In all, 23 esters, ranging from simple aliphatics to multifunctional chiral esters, were tested with this system. The array’s output—the slope and standard deviation from a plot of fluorescence intensity over time—were fed into a principal component analysis algorithm. Although four esters were consistently misidentified, the researchers suggest that the approach seems to be capable of distinguishing analytes in simple mixtures. (J. Am. Chem. Soc. 2001, 123, 9443–9444)
Molecular thermometer rises to the occasion Call it the latest prescription to detect environmental changes on the molecular level. Nirmala Chandrasekharan and Lisa Kelly at the University of Maryland–Baltimore County have developed a “molecular thermometer” by successfully synthesizing a polymer film that changes luminescence at temperatures ranging from 25 to 85 ºC. Such sensors could be useful in mapping temperature changes on two-dimensional surfaces. This dual-fluorescence temperature sensor uses luminescent “re-
Breaking up isn’t hard to do
porter” molecules incorporated into a
for applications such as decorative coatDon’t like your nanoparticles clumped too closely to each other? Researchers in ings, catalysis, optical filters, and nonlinear optical materials. Spain have found a way to keep them Liz-Marzán’s group first deposited a 5apart when they are incorporated into a to 7-nm-thick shell of silica on the nanosol–gel using silica gel. Luis Liz-Marzán particles to preserve their colloidal stability and colleagues at the Universidade de during the incorporation into the sol–gel. Vigo demonstrate that coating the Two different methods are used to prenanoparticles with a thin silica shell pare silica gels loaded with nanoparticles— helps control the particles’ morphology one starting with sodium silicate and the and properties, avoids aggregation durother beginning with tetramethoxysilane. ing the gelation process, and allows UV–vis absorption spectra and transthe colloid to be cleaned by standard mission electron microscopy showed no purification techniques. Immobilizing aggregation of the metal particles durnanoparticles within gels is important ing the sol–gel transition. Moreover, the optical properties of the starting colloid are fully retained in the gel. Although the researchers specifically chose gold nanoparticles because of their sensitivity to aggregation and to the environment in which they are embedded, they say the same procedures Transition electron micrographs of silica gels loaded with can be used for the incorAu@SiO2 nanoparticles prepared according to (a) the sodium poration of other types of silicate method and (b) the tetramethoxysilane method. A nanoparticles. (Langmuir 2001, 17, 6375–6379) larger porosity is observed for the sodium silicate method.
searchers encapsulated and anchored
transparent polymer matrix. The reperylene and N-allyl-N-methylaniline (NA), respectively, in a soft elastic polystyrene matrix. By itself, NA does not fluoresce. However, in the presence of perylene, a temperature-dependent equilibrium develops between an exciplex of the two and the monomers, resulting in a “two-color” material with a ratiometric change in the visible region of the spectrum. With an excitation wavelength in the UV, the researchers found strong blue emission under “hot” conditions and green emission when the sample was “cool”. The smallest temperature change that could be measured was 2 °C, given the spectrometer and scan rate used. The accuracy was determined by the fluctuations in the measured temperature (±1 ºC). Tuning the plasticity of the polystyrene matrix could extend the temperature range, say the researchers. (J. Am. Chem. Soc. 2001, 123, 9898–9899)
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RESEARCH PROFILES Tasty plastic beats glass surfaces are inherently more hydrophobic than glass. “The spray generated from our plastic prototype was very stable,” says Rossier. A quiescent, charged ESI plume emerges directly from the edge of the substrate and precludes the need for a separate tip. In addition, says Rohner, “Proteins tend to adsorb onto glass.” And, treatments for making the glass surface more hydrophobic tend to degrade over time. However, proteins do not adsorb to the plastics from which the chip is made, say the researchers. Once the spray was on, there was no background signal from the plastic. Even with repeated use and with different analytes, there was no evidence of “carry-over” or contamination. Using myoglobin as the analyte, Girault’s group found that the MS signals were quite stable over time at flow rates of ~200 nL/min and with protein solutions of ~5 µM. They demonstrate sensitivities at the picomolar level and a linear dynamic range spanning nine decades of concentration. A requisite for ESI is some means of charging the analyte solution to a high voltage. Gold-coated glass tips tend to
HUBERT GIRAULT, ECOLE POLYTECHNIQUE FÉDÉRALE DE LAUSANNE
Lausanne, Switzerland, is known for the specialty dessert mille feuille, literally “thousand layers”, which is made from dozens of alternating layers of pastry and cream, then topped with pink icing. Now, another intricate laminated structure from this region is making news—a miniaturized electrospray ionization (ESI) source made from plastic. In the November 15th issue of Analytical Chemistry (pp 5353–5357), Hubert Girault, Tatiana Rohner, and Joël Rossier at the Ecole Polytechnique Fédérale de Lausanne (Switzerland) describe how they made a plastic nanospray ESI source and ran it through its paces. The chip has a flow channel with a constant cross-sectional area and an integrated microelectrode for precise control of ESI spray current. The miniaturized source can be made with a minimum of manual steps and overcomes many of the drawbacks of using glass ESI sources for the analysis of proteins by HPLC/MS. Most problems with traditional glass ESI sources arise because glass is naturally hydrophilic. Spray solution is attracted to the spray nozzle and tends to form drops that disrupt a stable spray. Plastic
The “delicious” sheathless polymer-based ESI device coupled to a capillary. 656 A
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degrade at all potentials and very rapidly at potentials >3 kV. Wires inserted into the nozzle have a longer life, but tend to perturb the flow and provide a surface for bubble formation. The difficulties associated with incorporating a metal electrode into a glass ESI source get even worse as the size decreases. The integrated microelectrode in Girault’s chip charges the solution inside the flowing cell near the spray tip with no flow disruptions, and it can be run at potentials of up to 7–8 kV without degrading, say the scientists. The plastic device can be used repeatedly at these high voltages with no loss of performance. Fabrication of the new nozzle starts with a Mylar film substrate ~100-µm thick. On the side that will become the bottom, they use excimer laser ablation to cut a channel into the substrate. Then, they fill this “L”-shaped, flat-bottomed groove with carbon ink—the same material used to make glassy carbon electrodes. After curing, the electrode is sealed in place with a sheet of polyethylene backed with poly(ethylene terephthalate) (PE/PET). Another groove is ablated into the top, which crosses the electrode channel at a right angle. This channel is cut just deep enough so that its intersection with the electrode channel forms one face of the flow channel, creating a minute electrical liquid junction of controllable area. The top is laminated with another layer of PE/PET to form the top wall of the fluid channel, and connection pads and inlet holes are added. Finally, the whole sandwich assembly is cut to form the spray tip and establish the distance between the liquid junction electrode and the end of the nozzle. Girault’s group has been making prototype chip ESI tips using a computercontrolled excimer laser and photomasks for some time now. “When we chose to make plastic chips specific for protein analysis, we chose a more difficult road,” says Rossier. But this group makes it look like a piece of cake. a —Zelda Ziegler
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Beyond the cellular “cookbook” whereas the newer experiments in Tan’s the molecular beacon—a short, fluoresThe sequence of the human genome lab monitored the dynamics of molecutells us as much about how cells operate cently tagged oligo that is folded like a lar beacon–mRNA hybridization in indihairpin. While the beacon remains foldas a cookbook describes what people ed, the fluorophore at the 5´ end is very vidual cells. eat. We know which ingredients are Tan and Perlette targeted the mRNA used and how they are grouped, but the close to a quencher at the 3´ end, so the of the -actin gene in kangaroo rat kidfluorophore can’t generate a signal. But book doesn’t tell us which recipes are ney cells. A molecular beacon at a conwhen another oligo, complementary to popular, when the meals are served, or centration of 10–6 M was injected into the sequence in the beacon’s loop, is how much is eaten. the cytoplasm of the cells, and the fluoIn the November 15 issue of Analyt- present, the beacon unfolds and hybridizes to it. In the process, the rescent signal intensity was measured ical Chemistry (pp 5544–5550), Weievery 3 min for 15 min. The hong Tan and John Perlette researchers observed reproat the University of Florida ducible increases in fluoresdescribe their approach for cence, suggesting that the going beyond the genomic molecular beacons were un“recipes”. You might say that folding and hybridizing to the researchers are peering the mRNA. Control experiinto the “kitchen” to watch ments verified that the beathe “food” preparation: They cons were selecting the corare monitoring the expresrect target. The same sion of messenger RNA experiments were performed (mRNA), the template for for -1 andrenergic receptor making proteins, in living mRNA with similar results. cells. More advanced applicaResearchers have monitions include comparing gene tored mRNA expression for expression in normal versus years, using time-consuming diseased cells and before verNorthern blots—in which sus after treatment with the oligonucleotides (oligos) drugs, and monitoring the siof interest are tagged and multaneous expression of separated by gel electromultiple genes. Tan’s lab is phoresis—or, more recently, One cell was chosen from a group (top left) of kangaroo rat kidney also working with gene thermicroarrays that are studded cells and injected with molecular beacons complementary to -actin apy researchers at the Uniwith row after row of oligos. mRNA. The color change represents the increase in fluorescent sigThese methods cannot be versity of Florida who want nal intensity as the molecular beacons hybridize to their targets over used to monitor living samto monitor gene expression ples because the cells must a 15-min period. once a new DNA segment be destroyed to extract the has been delivered to cells. mRNA, says Tan. “The clear “Our ultimate goal is not quencher and fluorophore separate, and difference is that our method is able to only to monitor gene expression but the fluorophore can generate a signal monitor living cells—even single cells,” protein levels,” says Tan. “It is really the when excited by the proper wavelength he says. Ultimately, this approach may protein [that] does the job.” The reof light. reveal the nuances of mRNA producsearchers have already developed some Molecular beacons are already used tion, such as how it varies from cell to tools toward that end, including molecfor applications such as monitoring the cell or over time. ular beacons that recognize DNA-bindaccumulation of product in polymerase Tan says that this kind of work is ing proteins and molecular beacon apchain reactions and identifying mutasimilar to neuroscience experiments in tamers for real-time protein studies. tions in genes. And other researchers which researchers track cellular messenThere’s still a lot for us to learn, but have detected mRNA in living cells gers in real time. “But people could not every day we know a little more about using molecular beacons. However, in do this [with mRNA] because they did these cellular “kitchens” and the subthose experiments, data was collected not have the molecular probe,” he says. tleties of their “cuisines”. a —Elizabeth Zubritsky just once for a group of cells, says Tan, The probe that he and Perlette use is
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MEETING NEWS News from the Federation of Analytical Chemistry Meeting—Cheryl M. Harris reports from Detroit, MI
Researchers turn up the heat in ICPMS method Diane Beauchemin at Queen’s University (Canada) got an idea from an undergraduate analytical chemistry course and did something quite original. She and graduate student Heidi Chen heated a knotted reactor and filter system to enhance the flow-injection, on-line precipitation, and dissolution of trace metals for analysis by inductively coupled plasma (ICP) MS. They also used an organic precipitant without any coprecipitation carrier. Their objective was to selectively determine trace amounts of chromium, manganese, iron, cobalt, nickel, and copper in saline water, and the results were more than encouraging. But why hadn’t the knotted reactor and filter system been heated before? “Your guess is as good as mine as to why nobody had tried heating the system!” says Beauchemin. “I thought that it was logical since, in the undergraduate analytical laboratories that I instruct, heating is used to enhance the precipitation process in gravimetry experiments.” Chen says she wasn’t surprised that researchers
& Spectroscopy Societies (FACSS)
debated the work during her presentation, “because we are kind of … challenging the current published papers,” she explains. “We believe that our technique provided a more complete separation of the analyte from the matrix.” Although ICPMS has become widely used for multielement analysis, its application to saline waters remains problematic because of its low tolerance for dissolved solids (
