news
analytical currents Counting rare tumor cells in vivo Quantitating the levels of circulating tumor cells is difficult, but this information helps physicians determine whether a patient requires chemotherapy after tumor surgery. Now, Philip Low and colleagues at Purdue University and the Mayo Clinic Rochester have developed an in vivo method that noninvasively counts the tumor cells in the bloodstream. The researchers labeled cells with folate–dye conjugates instead of antibodies, because many human carcinomas are thought to express folate receptors. In addition, antibodies cause problems with background fluorescence and are cleared out by phagocytosis; this causes an underestimation of the number of circulating tumor cells. The conjugates specifically labeled circulating tumor cells, leaving normal cells untouched.
Low and colleagues imaged the labeled cells by multiphoton microscopy. By opting for a 1D line-scanning method rather than the conventional 2D scanning approach, they increased the rate of data acquisition 250-fold. The researchers detected leukemia cells circulating in the bloodstream of mice. And when The specificity of in vivo labeling of leukemia they implanted lung cancer cells cells in the bloodstream of mice. (Adapted with in mice to mimic cancer papermission. Copyright 2007 National Academy of tients, they found that the num- Sciences, U.S.A.) ber of circulating tumor cells increased exponentially over time at very early stages, all the patients with tumor growth. had circulating tumor cell counts Next, the investigators tested their greater than background. Similar method on blood samples obtained samples from healthy donors didn’t from 12 ovarian cancer patients at contain the marker cells. (Proc. various stages of the disease. ExNatl. Acad. Sci. U.S.A. 2007, 104, cept for those patients who were 11,760–11,765)
Nano-optode sensors are brighter than QDs and biocompatible Heather Clark and colleagues at the Charles Stark Draper Laboratory incorporated quantum dots (QDs) into an ion-selective
Biocompatible coating Ion-selective polymer Quantum dot
polymer to form enhanced Na+ nano-optode sensors. The sensors also have a biocompatible coating that allows them to measure intracellular ion concentrations. The polymer matrix contains a light-ab-
moionophore absorption decreases, and the QD’s fluorescence is not absorbed, so it can be seen by the naked eye. The researchers found that the dynam-
An idealized schematic of an ion-selective nano-optode. The QD in the nano-optode is chosen
ic range of their sensor was 1 mM to 1 M, and they observed minimal photobleaching. After adjusting the components of the
carefully so that its emission wavelength
nano-optode, they could tune the resolu-
sorbing pH indicator (chromoionophore)
overlaps the absorption wavelength of the
tion of their sensors to 80 µM at the typical
and an ion-binding molecule (ionophore).
ion-selective polymer. At low Na + concen-
intracellular Na + concentration of 17 mM.
As a positive ion binds to the matrix, a hy-
trations, chromoionophore absorption at
They also note that a different combina-
drogen ion is released, and the pH of the
this wavelength is high, and the QD’s emis-
tion of QD and ion-selective polymer could
polymer changes; this, in turn, changes the
sion fluorescence is absorbed, leading
lead to sensors for other ions. (J. Am.
properties of the chromoionophore.
© 2007 American Chemical Societ y
+
to little visible light. As Na binds, chro-
Chem. Soc. 2007, 129, 8418–8419)
S e p t e m b e r 1 , 2 0 0 7 / A n a ly t i c a l C h e m i s t r y s
6427
news
analytical currents
Optical microcavities for detecting single molecules Kerry Vahala and colleagues at the Cal-
surfaces of the micro-
system activation, and
ifornia Institute of Technology have de-
cavities with either bi-
determined that the
veloped a single-molecule detection ap-
otin or antibodies, and
sensor had a working
proach based on optical microcavities in
when target molecules
range of 10 –6 to 10 –18
which the target molecule doesn’t have to
were captured, the
M. When the investiga-
be labeled.
circulating light sam-
tors tested serum solu-
Planar arrays of microtoroid resona-
pled them many times.
tors were fabricated in silica. The mi-
The binding inter-
crotoroids were coupled to a tunable la-
action produced red
ser and a tapered optical-fiber waveguide
shifts of the resonant
and immersed in water. Light inside the
wavelength that were
microcavities circulated constantly, al-
monitored in real time.
though a portion of the optical field evanesced into the surrounding liquid. The investigators functionalized the
A scanning electron micrograph of a microtoroid optical resonator. (Adapted with permission. Copyright 2007 American Association for the Advancement of Science.)
With this approach, Vahala and col-
tions with 300, 600, or 900 aM of IL-2, the total resonant wavelength shift increased proportionally to the concentration of IL-2, and
the individual binding events were re-
leagues tracked single molecules of in-
solved. (Science 2007, DOI 10.1126/sci-
terleukin-2 (IL-2), a marker for immune-
ence.1145002)
Protein folding by fast 2D NMR spectroscopy
Tex (s)
NMR can provide a wealth the pH from 2 to 8 quickly of structural information and then recorded a series V III about stable protein forms of SOFAST–heteronuclear 71 41 but is often too slow to give multiple quantum coheruseful data about dynamic ence spectra at a rate of 0.1 II I 70 42 IV processes, such as folding per second. They quantified 11 7 69 43 50 and unfolding. Conventional refolding kinetics for 92 of 12 6 68 44 49 2D NMR experiments re121 backbone amide sites in 13 5 67 45 48 200 quire ~100 scans and can the protein and calculated 14 4 66 100 record only very slow kinetic time constants for folding 15 3 65 10 events with time constants that agreed well with previ16 2 5 of minutes to hours. Now, ously measured values. 17 1 1 Bernhard Brutscher and colTo further demonstrate leagues at the Institut de the potential applications Biologie Structurale JeanColor-coded H/D exchange rates of human ubiquitin at pH 11.95 deof SOFAST, the researchers Pierre Ebel and the Institut rived from SOFAST data. (Adapted with permission. Copyright 2007 measured the unfolding kide Recherches en TechnoloNational Academy of Sciences, U.S.A.) netics of ubiquitin by H/D gies et Sciences pour le Viexchange methods. H/D vant (both in France) have developed the SOFAST method. The folding of exchange rates of solvent-accessible sites band-selective optimized flip-angle this protein has been studied in the past at high pH are typically too fast to meashort-transient (SOFAST) real-time 2D by line-shape analysis of individual 2D sure by conventional 2D NMR methods. NMR spectroscopy to study protein NMR cross-peaks in a single spectrum, With SOFAST, the investigators quanti folding and unfolding on the timescale but the technique is limited to slow fied H/D exchange kinetics at sites of seconds to minutes. kinetics and is prone to experimental that were not previously detectable by The researchers first used apo--lact- errors of up to 25%. The team initiNMR. (Proc. Natl. Acad. Sci. U.S.A. albumin as a model system to illustrate ated a refolding event by increasing 2007, 104, 11,257–11,262) 6428
A n a ly t i c a l C h e m i s t r y / S e p t e m b e r 1 , 2 0 0 7
news
Separation by solid-supported bilayer electrophoresis It’s tough to separate, purify, resolved into two distinct No cholesterol 25 mol % cholesterol and detect lipids and memchromatographic features Initial Initial brane-bound proteins, because with an area ratio of ~70:30. it’s easy to inadvertently change After testing the migration the native structure of the molof each pure isoform, the 30 min 30 min ecules or even lose them. So, investigators confirmed that Paul Cremer and colleagues at they were electrophoretically Texas A&M University have separating the two isomers devised a new way to separate (which exist in a 70:30 mole A comparison of the band broadening in epifluorescence microcomponents of lipid memratio) from each other. graphs of fluorescently labeled lipids migrating in pure POPC branes that doesn’t harm the (left) and in POPC with 25 mol % of cholesterol (right). A mixture of lipids, molecules. tagged with two different The investigators created a solid-supvironment can be introduced into the fluorescent labels, was next separated ported lipid bilayer out of 1-palmitoylsolid-supported lipid bilayer and migrate in the POPC–cholesterol bilayer to 2-oleoyl-sn-glycero-3-phosphocholine via electrophoresis for separation. prove that complex mixtures could be (POPC) and cholesterol. The lipid After comparing the migration of a resolved. Cremer and colleagues sugbilayer acts as a separation medium for fluorescently labeled lipid in pure POPC gest that the method can be extended electrophoresis, much like the way cross- or POPC with cholesterol, Cremer and to the separation and purification of linked acrylamide or agarose works as colleagues found that the cholesterol membrane-bound proteins as well as a gel for separations in conventional was necessary to limit band broadenlabel-free imaging of freeze-dried lipid electrophoresis. Molecules that would ing during the electrophoresis. In the bilayers. (J. Am. Chem. Soc. 2007, 129, otherwise disintegrate in a nonlipid enPOPC–cholesterol bilayer, the band 8072–8073)
Raman microspectroscopy of mitochondria Max Diem and colleagues at Northeast-
protein peaks are abundant, and used hi-
ern University have used confocal nonres-
erarchical clustering to divide the spec-
onant Raman microspectroscopy to track
tra into five clusters. Each cluster cor-
mitochondrial distribution in cells. Mito-
responded to a biochemically distinct
chondria typically are visualized by elec-
region and was pseudocolored to create
tron microscopy or fluorescence-labeling
an image of the cell.
methods. Both of these techniques are at
After collecting Raman spectra, the
least somewhat invasive, and researchers
investigators stained the cells with a flu-
wanted to find a method that could track
orescent dye specific for mitochondria.
mitochondria yet would minimally disrupt
When they compared the fluorescence
the intracellular environment.
signal from the dye to their Raman imag-
In Raman spectra, the changes be-
(a)
(b)
(a) A fluorescence image of a HeLa cell after staining with green, mitochondriaspecific fluorescent dye. (b) A five-cluster Raman map of the same cell. (Adapted with permission. Copyright 2007 Biophysical Society.)
es, they found a distinct overlap of one
to be carried out on live, unlabeled cells
tween regions of a cell are subtle and
cluster with locations of high mitochon-
noninvasively and in their native envi-
usually not easily discernible by the na-
drial distribution.
ronment. Diem and colleagues believe
ked eye, so the investigators relied on
The researchers note that although
that their method could eventually be
multivariate methods to analyze their
their study was carried out on fixed
used to track mitochondrial processes
spectra. They focused on the spectral
cells, the use of a water-immersion ob-
such as migration in real time. (Biophys. J.
jective would allow the same procedure
2007, 93, 668–673)
–1
range of 1200–1800 cm , a region where
S e p t e m b e r 1 , 2 0 0 7 / A n a ly t i c a l C h e m i s t r y
6429
