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ANALYTICAL CURRENTS Absolute configuration of a barely chiral molecule
(10 –21 Å2/sr/cm)
gy by moving to the (a) other end of the molec3.0 ular size spectrum. To do this, they synthesized 0 2 2 2 [ H1, H2, H3]-neopentane. –3.0 The researchers first computed ROA spectra (b) for each of the nine po4.0 tential rotamers for both the (R) and (S) configu0 rations of the molecule. The spectra for each –4.0 enantiomer were combined to give an average 1300 1200 1100 1000 900 800 predicted spectrum; it –1 was then compared to Wavenumber (cm ) the experimental spectrum of their sample. In (a) Average ROA spectra for (R )- and (S)-[2H1, 2H2, 2H3]-neopentane. (b) Comparison between calculated (black) and exthe end, the molecule was determined to be in perimental (blue) spectra for the (R ) configuration. the (R) configuration. the stereochemistry of just about any The authors note that now that the ab2 2 molecule should be accessible by ROA. solute configuration of (R)-[ H1, H2, 2 H3]-neopentane has been determined, (Nature 2007, 446, 526 –529)
Discerning terminal glycosylation with the naked eye Glycosylation can be one of the most diffi-
The authors demonstrate their method
cubated with a boronic acid–dye conjugate
cult posttranslational modifications to char-
by detecting terminal galactose residues.
that results in a red color only if galactose
acterize because of the diversity of struc-
They performed parallel experiments on
is present.
tures that may be attached to the protein.
three proteins: asialo-fetuin (a-fet), which
Typical experiments to analyze glycosyla-
has six glycosylation sites that terminate
treated with b-galactosidase turned red
tion patterns require complex instrumenta-
with galactose; fetuin (fet), the same pro-
upon treatment with this method, and the
tion and are best performed by someone
tein but with the glycan chains terminating
red color became less intense as the
highly skilled in glycoanalysis. Now, Ole
in sialic acid instead; and BSA, which is
galactose concentration decreased. The
Hindsgaul and colleagues at Carlsberg
not glycosylated. The researchers incubat-
authors note that their methodology can be
Laboratory (Denmark) have introduced a
ed the proteins with b-galactosidase to re-
extended to detect any terminal glycosyla-
general method for identifying the terminal
lease any terminal galactose residues,
tion moiety as long as a specific glycosi-
glycosylation state of a glycoprotein that
which were then captured as the acyclic
dase is available. (Angew. Chem., Int. Ed.
can be read by the naked eye.
oxime on glass beads. The beads were in-
2007, 46, 2403 –2407)
© 2007 AMERICAN CHEMICAL SOCIETY
As expected, only a-fet that had been
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JACQUES HAESLER
Arbitrary units
Werner Hug and colleagues at the University of Fribourg (Switzerland) have tested the limits of their newly designed Raman optical activity (ROA) spectrometer by determining the absolute configuration of (R)-[2H1, 2H2, 2H3]-neopentane. This molecule has a very subtle chirality, which is determined solely by substitution of deuterium at three of its five carbons. ROA is akin to CD spectroscopy in that it compares how beams of oppositely polarized light react with a molecule. The difference is that CD measures the absorption of that light, whereas ROA assesses the Raman scattering. Both techniques give information about molecular structure, but an ROA spectrum contains more bands and thus more information about fine structural details, such as stereochemistry. ROA has been used to study proteins from intact viruses, but Hug and coworkers wanted to push the boundary of what was possible with this technolo-
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ANALYTICAL CURRENTS Breath test for lung cancer Early diagnostic tests for lung cancer are sorely needed because the disease usually is not caught until it has reached an advanced stage, when treatment is less effective. Now, Peter Mazzone and colleagues at the Cleveland Clinic have demonstrated that a colorimetric sensor array recognizes a signature set of volatile organic compounds (VOCs) in the breath of lung cancer patients. The sensor could lead to a cheap, early, and noninvasive test for lung cancer. Mazzone and colleagues used an array that consisted of 36 chemically sensitive compounds embedded as dots in a disposable cartridge. This type of system had been previously shown to be sensitive at the lower parts-per-million to upper parts-perbillion range for specific VOCs. The investigators asked individuals with lung cancer or other lung diseases and healthy subjects to breathe into a device that drew their exhalations across the array. The color changes in the 36 compounds were periodically recorded on a flatbed scanner during the sample collection, converted into numerical vectors, and statistically analyzed. Mazzone and colleagues found that lung cancer patients had a unique VOC signature. With data from 70% of the subjects, they drew up a prediction model and applied it to the remaining 30% of the subjects. The model predicted lung cancer with 73% sensitivity and 72% specificity. The investigators acknowledge that gaseous chemical sensors are criticized for their inability to identify specific chemicals and because they lack the sensitivity to identify all potentially important VOCs. However, they point out their goal isn’t to identify breath constituents but to detect VOC patterns that act as signatures. Their study indicates that these chemical array systems have the potential to be developed into a useful clinical test. (Thorax 2007, doi 10.1136/thx.2006.072892)
Ultrasonic wave drives nanowires to generate current Devices with nanowires and nanotubes usually receive power from an external source, such as a battery. But for some systems, the external source may be a hindrance. Zhong Lin Wang and colleagues at the Georgia Institute of Technology have developed a nanowire-based generator to produce continuous direct current. It could be a portable and cost-effective way to power nanoscale devices.
1 µm
300 nm
Cross-sectional electron micrograph of aligned nanowires and the zigzag electrode. (Inset) The electrode forces a nanowire to bend. (Adapted with permission. Copyright 2007 American Association for the Advancement of Science.) The investigators had previously demonstrated the conversion of mechanical energy into electric energy when piezoelectric nanowires were deflected by an atomic force microscope tip. In the new paper, Wang and colleagues replaced the tip with an ultrasonic wave so that all the nanowires were actuated simultaneously to produce continuous current. When excited by an ultrasonic wave, the electrode moved down and first pushed on the nanowires close to it, creating a piezoelectric potential across each nanowire. However, current did not flow. Nanowires that were not in contact with the electrode’s teeth vibrated from the ultrasonic wave’s stimulation. Once the electrode came farther down to contact all the nanowires, piezoelectric discharge occurred and current flowed. The output power of a nanowire was 1–4 f W. The investigators suggested if a nanowire were optimized to produce 10 f W, the output power per unit of area would 2 2 be 10 mW/cm . Thus, a generator with nanowires on a 1-cm
area could operate up to 1000 nanodevices. (Science 2007, The colorimetric sensor array consists of 36 chemically sensitive dots in a disposable cartridge. (Adapted with permission. Copyright 2007 Thorax.) 3954
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MicroRNA detection by nanowire formation
A tumor-targeting fluorescent probe Hisataka Kobayashi, Yasuteru Urano, and colleagues at the U.S. National Institutes of Health, the University of Tokyo (Japan), the Japan Science and Technology Agency, and
Zhiqiang Gao and colleagues at the
Science Applications International Corp. have designed a fluorescent probe that can be
Institute of Microelectronics (Singa-
targeted to cancer cells. Their probe, AM-TG-b-gal, exhibits a dramatic increase in
pore) have devised an electrical
fluorescence upon cleavage by b-galactosidase. Further hydrolysis by intracellular es-
biosensor for microRNAs (miRNAs)
terases creates a hydrophilic molecule that is well retained by cells.
that is based on the formation of
The authors demonstrate the utility of their probe by implanting mice with a common
polyaniline (PAn) nanowires in
ovarian cancer cell line, then targeting an avidin–b-galactosidase conjugate to the lectins common on the surface of these cells. AM-TG-b-gal was administered and pro-
nanogaps. Their system consists of a
vided a long-lived, tumor-specific fluorescent signal. The authors hope that eventually
chip with a peptide nucleic acid
their molecule could be used to assist surgeons in identifying tumors during laparo-
(PNA) that is complementary to the
scopic surgery. (J. Am. Chem. Soc. 2007, 129, 3918– 3929)
miRNA of interest and displayed between two microelectrodes.
Visualizing neurons in the brain in 3D
When the target miRNA forms a complex with the PNA, the phosphate groups on the miRNA backbone provide a negatively charged surface on HANS-ULRICH DODT
Seeing neuronal networks in (a) (b) brains has been impossible until now. Hans-Ulrich Dodt and colleagues at the Max Planck Institute of Psychiatry, the Max Planck Institute of Neurobiology (both in Germany), and the Vienna University of Technology (Austria), have come up Autofluorescence images (a) the surface and (b) the with a way to image neurons blood vessels of a mouse embryo. Scale bar = 2 mm. in whole, fixed mouse brains. Dodt and colleagues modified the 100-year-old idea of light sheet illumination, also known as ultramicroscopy, so that optical sections were generated by stepping the sample through a thin illumination plane. But ultramicroscopy was developed for optically transparent objects—mouse brains are opaque. So the investigators applied a special clearing procedure, also nearly a century old, in which the sample was immersed in a medium with the same refractive index as protein; the light moved through the specimen and wasn’t scattered. In this manner, Dodt and colleagues were able to clear specimens as large as 2 cm. The investigators imaged neurons expressing green fluorescent protein (GFP) in whole brains of transgenic mice and detected single GFP-labeled neurons in excised mouse hippocampi. They went on to reconstruct 3D images of dendritic trees and spines of a certain class of neurons in the hippocampi. Dodt and colleagues also could visualize small details inside whole mouse embryos. The circulatory system, including capillaries, was especially obvious, because the blood remaining in the vessels strongly autofluoresced. The team also imaged the entire bodies of fruit flies, including muscles and the optic lobes. The investigators say their approach will help with high-throughput phenotype screening of transgenic mice and benefit the study of disease models. (Nat. Methods 2007, 4, 331–336)
which protonated aniline molecules are deposited. Gao and colleagues add a combination of horseradish peroxidase and H2O2 to polymerize the aniline into PAn nanowires that bridge the gap between the two electrodes and provide an electrical signal corresponding to the presence of miRNA. The authors indicate that multiple PNA capture probes could potentially be displayed on the same chip for multiplex analysis of miRNAs and that the system could lead to a low-cost electrical array for miRNA expression profiling. (J. Am. Chem. Soc. 2007, 129, 5437–5443)
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(a)
Orientation indicators
ANALYTICAL CURRENTS
Coding elements Analyte detection region
Multifunctional particles for high-throughput analysis Patrick Doyle and colleagues at the Massachusetts Institute of Technology have developed a way to make multifunctional particles that carry more than a million codes for analyte tagging. These particles can be useful for highthroughput screening applications, such as clinical diagnostics or combinatorial chemistry, in which several analytes must be detected simultaneously. Doyle and colleagues exploited laminar flows by running two streams, one with a fluorescent dye and the other with an acrylate-modified probe, down a single channel. The investigators polymerized the particles with bursts of UV light across the streams; a photomask determined the particles’ 2D morphology. In this manner, particles distinct in regions—a fluorescently patterned region for encoding and a probe-loaded region for target capture—were synthesized in one step. The particles were designed so they were “read” along lanes that ran down their lengths. Alignment indicators helped to orient the particles and identify the code’s position and the read “direction”. The flat, long shape of the
(a)
Reading lanes (b)
Orientation indicators Coding elements Analyte detection region Reading lanes
(a) (b) Schematic of particle features for encoding and analyte detection; encoding scheme 20 allows the generation of 2 (1,048,576) unique codes. (b) Differential interference contrast image of particles. Scale bar = 100 µm. (Adapted with permission. Copyright 2007 American Association for the Advancement of Science.)
particles helped to align them for scanning in a flow-through device. The spatial separation of various chemistries on the particles allowed decoding and target detection to be done with a single fluorophore. Doyle and colleagues demonstrated a multiplexed, single-fluorescence detection of DNA oligomers that could be scanned rapidly in a flow-through microfluidic channel. With commercially available acrylate-modified oligonu-
cleotide probes, they synthesized three batches of particles for DNA sequence detection. Two batches were loaded with specific probes; the third one (the control) didn’t have a probe. Fluorescently labeled oligonucleotides with complementary sequences served as the targets. The investigators showed that the assays were reproducible and that they could detect DNA oligomers easily at 500 amol. (Science 2007, 315, 1393– 1396)
Modeling chiral separations on surfaces Chiral separations can be easier to perform
an achiral phase into two enantiomeric do-
clusters that have been observed experi-
on surfaces than in solution, but the effects
mains—induced by a surface.
mentally. The team also noted that increas-
that dictate the final pattern of adsorbed
The researchers applied parallel tem-
pattern are complex and have not been
pering Monte Carlo simulations to study the
change the dynamics to favor the formation
teased out. Now, Irina Paci and Mark Rat-
importance of molecular geometry and en-
of heterochiral pairs, in which different
ner at Northwestern University and Igal
ergetics in promoting chiral separations
types of chiral molecules alternate.
Szleifer at Purdue University have explored
over a range of temperatures. By control-
The researchers suggest that future
the effect of restricting the configurational
ling the balance between steric and elec-
simulations should consider the effects of
space available to the molecules to two di-
trostatic interactions, they could encour-
solvents, the geometric and energetic
mensions. They asked whether this condi-
age the assembly of small aggregates of
makeup of the substrate, and dynamic ef-
tion alone could be the basis for chiral
identical chiral molecules. These self-as-
fects such as diffusion. (J. Am. Chem. Soc.
symmetry breaking—i.e., the resolution of
sembled structures resembled the rosette
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ing the complexity of the molecules can
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Refined theory of gradient elution chromatography Georges Guiochon and Fabrice Gritti of the
ly by H. Poppe. In the earlier model, the
locity than that of the mobile phase and,
University of Tennessee Knoxville and Oak
equations derived assumed that the gradi-
therefore, is distorted as it moves along
Ridge National Laboratory present a gen-
ent front remained unchanged and propa-
the column. Their refined model does not
eral equation to predict the band variances
gated along the column at the same speed
account for changes in the front’s profile
of compounds during gradient elution
as the mobile phase.
but does assume that the front moves at
chromatography. The team’s efforts are an
However, Guiochon and Gritti note that
addendum to a model developed previous-
the gradient front moves at a different ve-
a lower velocity than the mobile phase. (J. Chromatogr., A 2007, 1145, 67–82)
Probes for the metabolome Metabolites are tricky molecules to study. Whereas proteins and transcripts can be predicted to a certain extent from the genome sequence, metabolites are synthesized by enzymes and have no direct connection to the genome of an organism. In addition, metabolites are structurally more diverse than genes and proteins and can exhibit a wide range of
metabolite enrichment by tagging and proteolytic release (METPR), the researchers captured and studied metabolites with amines, thiols, ketones/aldehydes, and carboxylic acids. A set of reactive groups targeting specific metabolite classes was synthesized and bound to resins with a small, arginine-containing peptide. In the
Metabolome
Trypsin
Metabolite LC/MS
BENJAMIN CRAVATT
C-terminal cleavage
Mass ion intensity
Metabolite
Retention time
Schematic of the METPR method. Various capture agents are added to a resin to target specific classes of metabolites. Captured metabolites are released by trypsin cleavage and can be identified by their amine tags.
properties. Only a few enrichment methods exist for the isolation of metabolites from complex mixtures, and these target a small number of metabolite classes. Now, Benjamin Cravatt and Erin Carlson at the Scripps Research Institute developed a general tagging strategy to enrich and analyze many types of metabolites. With the method, called
METPR protocol, trypsin is added after metabolites are captured on the functionalized resins. Trypsin cleaves at the arginine residue; this event results in the release of the metabolite with a small tag. The rest of the peptide, including the arginine, remains on the resin. Finally, tagged metabolites are analyzed by LC/MS.
Cravatt and Carlson tested the METPR strategy by adding a simple mixture of small molecules to the capture probes. Each probe isolated the expected type of molecule. In addition, molecules that were present at low abundances were preferentially isolated with METPR. The researchers could detect some small molecules at a con7 centration of 100 pmol/10 cells, which is ~5–20-fold less than that detected with typical nonenrichment LC/MS metabolomics workflows. The efficiencies were up to ~50% for the various probes. In another series of experiments, METPR performance was demonstrated with human breast cancer cell lines. Again, the expected classes of metabolites were captured. When the scientists performed MS/MS to determine the structures of the captured molecules, they discovered that the tag did not interfere with this process. In addition, the researchers treated breast cancer cells with an antioxidant and compared the metabolomes of these cells with those of untreated cells. Differential profiles were observed for metabolites of all four classes with METPR. Cravatt and Carlson say that because different metabolites may be detected with METPR and conventional techniques, these methods are complementary. (Nat. Methods 2007, 4, 429–435)
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