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ANALYTICAL CURRENTS
Chemometric analysis of tumors by Raman microscopy Ahcene Taleb and colleagues at Queen’s
the profile of the one that’s causing the ma-
ly classify 100% of the cells into benign and
University Belfast (Northern Ireland, U.K.)
lignancy. The challenge is to characterize
malignant groups. PLSDA also correctly
have applied Raman microscopy and che-
the Raman spectra of individual cells and
classified a greater proportion of individual
mometric analysis to tumor cells. They
understand how they contribute to spectra
spectra from each cell. PCA was used to
probed the biochemical differences between
obtained from tissue samples.
image the distribution of nucleic acids,
a malignant and a normal prostate cell line
Taleb and colleagues probed cells from
lipids, and proteins inside individual cells
and analyzed how well different multivari-
an immortalized normal prostate cell line
and confirm the differences in composition
ate analysis techniques classified the two.
and a malignant cell line (derived from
and distribution of these biomolecules be-
prostate metastases) with a 633-nm laser.
tween the benign and malignant cell lines.
chemical properties of cancerous tissues
They compared several spectral prepro-
The investigators suggest that Raman
with Raman techniques. But the analysis of
cessing methods: partial least-squares dis-
microscopy could potentially be used for
gross tissue samples is problematic be-
criminant analyses (PLSDAs), principal
biomarker identification for diagnostic
cause tissues contain multiple cell types.
component analyses (PCAs), and adjacent
pathology and to probe changes in cellular
Each cell type has a different biochemical
band ratios (ABRs).
biochemistry without tags. (J. Phys. Chem.
Previous studies have probed the bio-
profile, which makes it difficult to discern
PLSDA and ABR were able to accurate-
B 2006, 110, 19,625–19,631)
A high-speed, multichannel confocal microscope A novel type of confocal microscope has been developed that has high spatial and temporal (in the nanosecond range) resolution. Stuart Yin and his colleagues at Pennsylvania State University, General Opto Solutions, and the U.S. Army Aviation and Missile Command have demonstrated that the frequency-division multiplexed multichannel fluorescence confocal microscope can monitor dynamic events inside living cells. In the microscope, the exciting laser beam is first split into multiple beams, each of which is modulated at a different frequency. The beams are focused at different locations on the sample to form multiple focal points. The focal points each produce a fluorescent emission spot—the emissions are also modulated at different frequencies. The modulated emissions are collected together on a highly sensitive, large-dynamic© 2006 AMERICAN CHEMICAL SOCIETY
range photomultiplier tube (PMT). When the signal from the PMT is demodulated by FT, the fluorescence emitted from the different regions of the sample can be distinguished by the corresponding carrier frequency. Yin and colleagues used the microscope to study a living rat cardiac myocyte. They loaded a fluorescent calcium-ion indicator into the cell to track the dynamics of calcium signaling during cardiac excitation–contraction coupling. The investigators first recorded the signal from the cell at rest. Then, as they stimulated the cell to contract by applying an electrical field, Yin and colleagues simultaneously recorded the changes in calcium concentration in bulk cytosol and around the membrane region over 5–10 s. They showed that the calcium-ion concentration does indeed change during cell contraction and
10 µm
Myocyte
Focusing spots
Two beams of light are focused on a living rat cardiac myocyte. (Adapted with permission. Copyright 2006 Biophysical Society.)
that the concentration was 5–6 higher around the membrane region than in the cytosol. (Biophys. J. 2006, 91, 2290–2296)
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ANALYTICAL CURRENTS
Rapid, sensitive virus detection by SERS The rapid detection of viral pathogens is critical for applications such as health care and the prevention of pandemics and bioterrorism. But it’s challenging to discriminate among different types of viruses while maintaining reasonable sample throughput and detection sensitivity. Ralph Tripp and colleagues at the University of Georgia have now developed a spectroscopic assay to detect trace levels of viruses within 30–50 s by surface-enhanced Raman scattering (SERS). The assay has high sensitivity and specificity and doesn’t require the viruses to be tagged or changed in any way. Tripp and colleagues used a silver nanorod array as the SERS substrate. The technique was capable of distinguishing different RNA viruses, such as the adenovirus, rhinovirus, and HIV. It was possible to assign Raman bands in the viral spectra to components such as the nucleic acid
bases, amino acids, and proteins. The variations in the Raman bands help to identify the viruses. The investigators found that the detection properties of the array were not hindered by the biological media that contained the viruses. Next, Tripp and colleagues demonstrated that different strains of a virus could be distinguished. They took three strains of the influenza virus A and found that the Scanning electron micrograph of the silver nanoSERS spectra were sufficiently difrod array. The rods are 868 nm long. ferent to identify the individual plateau or even decreased.) Values as low strains in a mixture. The differences in inas 100 PFU/mL were detected. Tripp and tensities and frequencies were most obvi–1 colleagues suggest that SERS spectra of ous in the 900–700-cm region. various viruses and viral strains could be The investigators found that the SERS collected in a reference library of vibraintensity decreased linearly as the viral 3 tional Raman fingerprints and used to concentration decreased from 10 PFU/mL quickly and accurately identify viruses. over 2 orders of magnitude. (At amounts 3 (Nano Lett. 2006 doi 10.1021/nl061666f) >10 PFU/mL, the intensity reached a
Potentiometric sensing of proteins with picomolar detection limits hydrogen peroxide, and Eric Bakker, Joseph Wang, Au Au Au Au the resulting silver ions Ernö Pretsch, and colleagues at Purdue University, Arizona ISE were detected with a polymer membrane silver-ionState University, and the Au selective microelectrode. Swiss Federal Institute of On the basis of other Technology Zurich have deOH OHOH OH OH OHOH OH OH OHOH OH OH OHOH OH HO Ag / Red veloped a potentiometry+ recent work, the investigas s s s s s s s s s s s s s s s s s s s s s s s Ag Au Au Au Au (b) (a) (c) (d) tors expect further reducbased detection protocol for tion of the final measureultrasensitive, nanoparticleThe detection scheme illustrates (a) addition of mouse IgG antigen, ment sample volume to based immunoassays in mi(b) capture of antimouse IgG antibody labeled with gold nanopartiresult in even lower deteccrovolume samples. The cles, (c) catalytic deposition of silver ions, and (d) silver dissolution tion limits, below the curtechnique builds on recent and potentiometric detection with a silver-ion-selective electrode. rently demonstrated level advances with ion-selective electrodes based on polymer membranes system, primary antimouse immunoglob- of ~12.5 pmol of IgG in a 50-µL sample. And, because potentiometric deteculin G (IgG) antibodies on a gold subthat contain ionophores as selective retion is less dependent on sample-volume strate captured target mouse IgG anticeptors. These electrodes can directly changes than other electroanalytical gens. Gold nanoparticle tags conjugated measure target ions in the subnanomotechniques, they suggest that the new to a secondary antimouse IgG antibody lar concentration range. protocol will be attractive in ultraminiaThe new detection protocol was based were added, followed by timed catalytic turized configurations. ( J. Am. Chem. silver deposition onto the gold labels. on a sandwich immunoassay coupled to Soc. 2006, 128, 13,676–13,677) nanoparticle amplification labels. In a test The deposited silver was dissolved with +
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Evaluation of de novo sequencing algorithms 100 spectral peak. NovoHMM is 80 based on a novel, generative, hidden Markov statistical 60 model, and AUDENS uses a 40 QSTAR dynamic programming ap20 proach to construct and eval0 uate sequence paths through 20 LCQ the spectrum data. 40 The investigators based 60 evaluations on relative se80 quence distances between de 100 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 novo and known sequences, Relative sequence distance as well as algorithm sensitivity and dependence on specComparison of de novo sequencing algorithm perfortrum quality. mance for QSTAR (above line) and LCQ (below line) data, The researchers found that showing percent of correctly identified peptides at various PEAKS outperformed the sequence lengths. Dark blue: AUDENS; blue: NovoHMM; other algorithms for QSTAR green: PEAKS; orange: Lutefisk; and brown: PepNovo. data, followed by Lutefisk showing a slight advantage over PEAKS and PepNovo; all three algorithms deand PepNovo. But none of the algopended strongly on spectrum quality. rithms managed to exceed 50% of exact Even though NovoHMM and AUpeptide sequence identification for both DENS were not quality-dependent, QSTAR and LCQ data sets, leaving the PEAKS still performed better throughfield open for improved de novo seout the tested range of spectrum qualiquencing programs. ( J. Proteome Res. ty. Performance in analyzing LCQ data 2006, doi 10.1021/pr060222h) was lower overall, with NovoHMM
Discriminating between leucine and isoleucine in MS In mass spectrometers that use low collision energies to fragment peptides, leucine and isoleucine are often indistinguishable. Switching to higher collision energies can solve the problem, but many instruments that perform routine proteomic analyses operate at lower fragmentation energies. Andrea Armirotti and colleagues at the University of Genoa (Italy) addressed this problem by performing consecutive MS steps. They determined that the protonated molecular ion of isoleucine generates a 69-Da ion, whereas leucine does not. The researchers exploited this characteristic gas-phase fragmentation to distinguish between leucine and isoleucine in various peptides and in a tryptic digest of myoglobin. The researchers note that their method worked on a 10-year-old instrument that has low resolution. Thus, they argue that the approach will work on a wide range of mass spectrometers, not just the latest and greatest models. (J. Am. Soc. Mass Spectrom. 2006, doi 10.1016/j.jasms.2006.08.011)
Identified peptides (%)
Identification of new and modified proteins from tandem mass spectra requires de novo sequencing—determining peptide sequences without the aid of a protein database. But how do you choose among the various de novo sequencing algorithms that have emerged? Xiang Zhang and colleagues of Purdue University and Lomonosov Moscow State University evaluated the performance of five algorithms. They demonstrated significant quality differences among the approaches and found that the algorithms generally performed better when analyzing data from a QSTAR instrument rather than an LCQ instrument. Zhang and colleagues selected five popular and readily available algorithms for analysis: Lutefisk, PepNovo, PEAKS, NovoHMM, and AUDENS. The first algorithm converts ions into “evidence lists”. The second algorithim follows a graph-theoretical strategy. The third extends that approach by assigning a reward/penalty score to every possible mass value, not just to those that correspond to an observed
Correction to “High-Resolution and Accurate Mass Analysis of Xenobiotics in Food” by E. Michael Thurman, Imma Ferrer, and Jerry A. Zweigenbaum Because of an editing error in the October 1 issue, two recent papers on MS-based methods for detecting unauthorized substances in food were mistakenly presented in an unfavorable light in this feature article. The second paragraph on p 6704 said that current methods would miss chloramphenicol in shrimp and malachite green in salmon. However, the text should have read: “But what if forbidden pesticides or drugs are applied, and the relevant ions are not selected? Then these compounds may very well be missed by current monitoring techniques. Recently, however, MS has been used to identify two forbidden substances in seafood: chloramphenicol (an antibiotic) in shrimp (7 ) and malachite green (a parasitoid) in salmon (8 ).” The editors regret the error.
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ANALYTICAL CURRENTS Near-field microscopy with a superlens Rainer Hillenbrand and colleagues at the Max Planck Institute for Biochemistry (Germany) and the University of Texas at Austin have combined a scanning near-field optical microscope (SNOM) with a superlens. A limitation of subdiffraction near-field microscopy is that the probe has to be very close to the surface of the object being investigated. With the new approach, however, the investigators demonstrated that they could image buried objects at subwavelengthscale resolution. A superlens is a thin slab of a material that has a negative permittivity. A highresolution optical image is formed on the side of the slab opposite to the one that is being investigated by a probe. Hillenbrand and colleagues used a 440nm-thick SiC membrane as a superlens. Each side of the membrane was coated with a 220-nm-thick layer of SiO2. One of the SiO2 layers was covered with an Au layer patterned with holes of various diameters and separations. The investigators could directly map the Au image plane by recording both the amplitude and phase of the optical field distribution from the probe of a scattering SNOM. Superlensing was ex-
pected to occur at mid-IR wavelengths. When the wavelength was close to 11 µm, the IR amplitude and phase images resolved the 1200-nm and 860-nm holes in the Au layer. Even the 540-nm holes showed sufficient optical contrast. But when the wavelength was 9.25 µm, the superlensing condition wasn’t present, and the pattern in the Au layer couldn’t be resolved. Hillenbrand et al. point out that near-field microscopy with a superlens isn’t limited to SiC lenses or IR wavelengths. Visible to terahertz frequencies can be used, and materials such as flat metal films and thin slabs of polar materials can work as superlenses. The approach could permit high-resolution imaging of structures, including biological ones, that cannot be directly investigated by a near-field probe. (Science 2006, 313, 1595)
(a)
SiO2
220 nm
SiC superlens
440 nm
SiO2
220 nm 60 nm
Au
(b)
(c)
(a) Experimental setup for near-field microscopy through an 880-nm-thick superlens. (b) IR phase contrast image vs (c) a control image at a wavelength where superlensing wasn’t expected. (Adapted with permission. Copyright 2006 American Association for the Advancement of Science.)
Why ionic liquids have low melting temperatures A new quantitative model developed by Ingo Krossing and colleagues of the Albert-
large entropy changes that favor melting. The method predicted the melting
method can predict the IL’s dielectric constant, which is often a more difficult pa-
Ludwigs-Universität Freiburg (Germany),
points and dielectric constants of 9 ILs with
École Polytechnique Féderale de Lausanne
a standard error of the estimate of 8 °C and
(Switzerland), and Ruhr University of
2.5 units, respectively. The researchers
might be helpful in designing new ILs. After
Bochum (Germany) explains why ionic liq-
suggest that the model also can estimate
one identifies an ion pair of interest, the
uids (ILs) have low melting temperatures. In
the melting temperatures of unknown salts
calculations that indicate the pair’s poten-
short, ILs are liquid because the large size
and of known ILs for which no melting
tial to serve as an IL can be conducted
and conformational flexibility of the ions in-
point has been observed. Alternatively, if
within 2–4 h. (J. Am. Chem. Soc. 2006, 128,
volved lead to small lattice enthalpies and
the melting point is known, then the
13,427–13,434)
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rameter to measure. Finally, the authors say that the method
