news
ANALYTICAL CURRENTS Detection of cocaine by an aptamer-based machine Itamar Willner and co-workers These strands are comple(a) at the Hebrew University of mentary to a reporter hairJerusalem (Israel) have depin DNA strand that has a signed an aptamer-based madonor–acceptor fluorescence chine to detect cocaine. To resonance energy transfer 5´ date, attempts to use DNA ap(FRET) pair attached. When Polymerization Nicking tamers as the recognition elethe single strand binds to (b) ment in both optical and electhe reporter, the hairpin trochemical sensors have been opens and disrupts the moderately successful, but FRET pair, and the signal 5´ most aptamers suffer from increases at the donor’s poor detection limits. In this emission wavelength. (c) case, the detection limit was The researchers note that improved by a signal amplificathe signal follows S-shaped hn tion method akin to PCR. kinetics over time because In the presence of cocaine, the single strand of DNA 5´ 3´ the DNA folds over onto itself must accumulate to a conto produce a seven-base duplex centration high enough to structure that serves as a open the hairpin at a reasonThe mechanism of the aptamer-based machine. (a) DNA polymerase primer for replication. DNA able rate. After an amplificapolymerase extends the double already has extended the second strand to reveal a recognition site for tion time of 60 minutes, the strand, revealing a nicking site an endonuclease. (b) The single strand is displaced and (c) binds to the investigators detected cofor the endonuclease Nt.BbvC reporter hairpin. When the hairpin opens, the FRET pair separates; this caine at a concentration of 5 3 10–6 M, which is a better is seen as an increase in the strength of the donor’s signal. I. After the DNA is nicked, detection limit than current displacement of a single strand methods offer. ( J. Am. Chem. Soc. 2007, reveals a new polymerase extension site. peated ad infinitum and produces mul129, 3814–3815) The polymerase extension step is retiple copies of the single-stranded DNA.
Watching single organic molecules move Imaging individual small molecules has
bon nanotubes. By transmission electron
tube, the molecule was observed to be im-
been difficult. But now, Eiichi Nakamura
microscopy, they observed whether the
mobilized in a fully stretched conformation.
and colleagues at the Japan Science and
molecule was moving or sitting still inside
When molecules were placed inside 1.2-
Technology Agency, the National Institute
the nanotube. The hydrocarbon chain
nm-diam nanotubes, some of them
of Advanced Industrial Science and Tech-
helped the investigators to distinguish be-
appeared to get their hydrocarbon tails
nology (Japan), and the University of Tokyo
tween the head and tail regions of the mol-
stuck to the wall of the nanotube, whereas
have come up with a way to observe them.
ecule and to correlate its motion relative to
others went through a series of conforma-
the nanotube.
tional changes in a stepwise fashion over a
The investigators trapped molecules with a characteristic long hydrocarbon chain individually inside single-walled car-
© 2007 AMERICAN CHEMICAL SOCIETY
When Nakamura and colleagues introduced a molecule into a 0.9-nm-diam nano-
period of several seconds. (Science 2007, doi 10.1126/science.1138690)
M A Y 1 , 2 0 0 7 / A N A LY T I C A L C H E M I S T R Y
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ANALYTICAL CURRENTS
Analyzing DNA by multinanopore force spectroscopy Clinical applications require new technolo-
cleotides at the opposite end that were
to the analyte-bound probes. Duplex DNA
gies for cheap DNA analysis. To meet the
complementary to an analyte’s sequence.
can’t pass through nanopores. As the in-
need, Andre Marziali and Carolina Tropini at
The investigators inserted the probes into
vestigators increased the applied force,
the University of British Columbia (Canada)
the pores by applying 200 mV. The avidin
the duplexes dissociated and the probe
have developed an array of nanopores that
anchors prevented the probes from com-
strands were pulled out of the nanopores.
quickly tests DNA duplexes for sequence
pletely translocating through the pores,
specificity. The detection is done electroni-
leaving the 14-nucleotide sequences stick-
Tropini correlated the applied force to the
cally, which means the approach could lead
ing out of the other side.
binding energy of the duplex. From their
to an inexpensive, disposable device for rapid clinical detection of polymorphisms. Marziali and Tropini incorporated hun-
Analyte DNA strands were allowed to interact with the protruding parts of the
By using a special model, Marziali and
calculations, they could distinguish analytes that differed from the probe sequence
probes. Marziali and Tropini next de-
with single-base resolution. To further im-
dreds of a-hemolysin protein molecules
creased the applied voltage so that any
prove the technique, the investigators sug-
into a lipid bilayer. The protein acted as the
unbound probes were released from the
gest replacing the a-hemolysin molecules
nanopore. Probe DNA strands had an
nanopores. They then reversed the polarity
with more robust synthetic pores. (Biophys.
avidin–biotin anchor on one end and 14 nu-
of the voltage so that a force was applied
J. 2007, 92, 1632–1637)
CMOS-compatible nanowires as detectors
TAREK FAHMY
curs by antibody-mediSemiconducting nano(a) (b) ated cross-linking of liwires can function as sen10 µm 200 nm gands on the cellular sursors for low concentraface. One consequence tions of species such as of the activation is the microorganisms, small release of acid. When molecules, and proteins. the investigators added Now, Mark Reed, Tarek antibodies against specifFahmy, and colleagues at ic T-lymphocyte ligands, Yale University have they detected changes in come up with a new appH, as a result of celluproach for building lar responses, within nanowires that’s compatible with complementary (a) Scanning electron micrograph and (b) optical micrograph of a semiconducting ~10 seconds. The investigators also metal oxide semiconduc- nanowire device. tackled the direct detector (CMOS) field effect tion of macromolecules without fluorestransistor technology. The method elim- etching kept the pattern defined by a cent, radioactive, or any other labels. inates the need for hybrid fabrication masking oxide layer and smoothed out With the streptavidin–biotin system, techniques and permits system-scale inedge imperfections. This resulted in they established that the nanowire sentegration of the sensors with signal pronanowires with trapezoidal cross secsors had a detection sensitivity of ~10 cessing and information systems. tions and an exposed silicon surface f M. Reed, Fahmy, and colleagues also The investigators fabricated semicon- amenable to functionalization. The infound they could discriminate between ducting nanowires with an anisotropic vestigators say the approach easily intelow concentrations of mouse immunowet etch. Tetramethylammonium hygrates with CMOS technology. globulin G and A proteins with comdroxide etched away particular planes Reed, Fahmy, and colleagues tested mercially available antibodies. (Nature in ultrathin silicon-on-insulator wafers the sensors by monitoring the real-time ~1003 more slowly than the rest. The activation of T lymphocytes, which oc2007, 445, 519–522) 3230
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news
Dynamically changing concentrations in droplets Daniel Chiu and colleagues at (a) lowered or turned off, water 0 s (b) 24 s (c) 71 s (d) 86 s (e) 231 s the University of Washington molecules that had left the Seattle have developed an opdroplet returned to it and intical vortex trap that dynamicreased its volume. cally changes the concentraBy switching the power 0 s (g) 72 s (h) 331 s (j) 247 s (i) 454 s of the trap, the investigators tions of aqueous droplets over (f) 4 orders of magnitude. They were able to perform consecsay that the manipulation of utive cycles of shrinkage and concentrations of nanoscale expansion of the droplets. A containers provides a new droplet’s volume could even Shrinkage and expansion of aqueous droplets dispersed in (a–e) acelevel of control that was preexpand beyond its original tophenone and (f–j) decanol. Scale bars = 5 µm. (Adapted with permisviously difficult to achieve. size if it accumulated the sion. Copyright 2007 Wiley-VCH.) The investigators focused water released from an adjaa laser through a microfabricent droplet. cated hologram to form a special type of terface was impenetrable to chemical The investigators suggest that the beam called a Laguerre–Gaussian beam. species inside the droplets. combination of this technique with conIt generated the optical vortex trap, Chiu and colleagues slowly “peeled” trolled single-droplet fusion would have which was then focused on individual off layers of water molecules at the applications in studying fundamental femtoliter-volume droplets. The dropaqueous–organic interface with the opchemical processes such as the effects of lets were surrounded by an immiscible tical vortex trap and concentrated the macromolecular crowding and protein continuous phase that had a slight solumolecules trapped inside the droplet. nucleation and crystallization. (Angew. Chem., Int. Ed. 2007, 46, 1326–1328) bility for water; the aqueous–organic in- When the power of the vortex trap was
Large-scale phosphoproteomics studies with ETD Electron transfer dissociation (ETD) has at-
human cells. They also compared ETD with
proteins are significantly more conserved
tracted a lot of attention from proteomics
collision-induced dissociation (CID) and re-
than other proteins. (Proc. Natl. Acad. Sci.
researchers because of its ability to frag-
ported 15 new phosphorylation motifs.
U.S.A. 2007, 104, 2193–2198)
ment posttranslationally modified peptides
Hunt and colleagues digested an ~600
Pandey and colleagues analyzed phos-
without the loss of fragile modifications.
pmol sample of total yeast protein and en-
phopeptides from human embryonic kidney
Now, two groups report the application of
riched for phosphopeptides with immobi-
293T cells. Proteins were digested, and
ETD to large-scale analyses of the phos-
lized metal-affinity chromatography. When
phosphopeptides were isolated with a TiO2
phoproteome. Donald Hunt and colleagues
ETD was performed with a linear ion trap
microcolumn and analyzed on a 3D ion trap
at the University of Virginia, Princeton Uni-
mass spectrometer, 1253 phosphorylation
mass spectrometer by ETD. A total of 1435
versity, the U.S. National Institutes of
sites were identified on 629 proteins. One
phosphorylation sites were identified, and
Health, and Thermo Electron identified sev-
of the phosphorylated proteins was Cdc10,
80% of those were novel. Because the c
eral phosphorylation sites in yeast, de-
which is involved in cytokinesis and mor-
and z fragment ions generated by ETD
scribed a novel motif for phosphorylation,
phogenesis. From the ETD spectrum, the
often were of low intensity, the researchers
and conducted a network analysis.
researchers deduced that one of the Cdc10
wondered whether ETD is actually better
peptides is phosphorylated on a histidine
than CID for phosphopeptide detection.
colleagues at the Johns Hopkins University
residue instead of serine, threonine, or ty-
When they compared the two methods,
School of Medicine, the University of South-
rosine. This unexpected finding suggests
ETD outperformed CID. However, the scien-
ern Denmark, Agilent Technologies, and the
that Cdc10 may be phosphorylated by Sln1,
tists note that the processes are comple-
Institute of Bioinformatics (India) discov-
the only known yeast histidine kinase. The
mentary. (Proc. Natl. Acad. Sci. U.S.A. 2007,
ered many novel phosphorylation sites in
team also reported that phosphorylated
104, 2199–2204)
In another study, Akhilesh Pandey and
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ANALYTICAL CURRENTS
Cantilevers measure solution-phase thermodynamics Eric Toone and co-workers at Duke Univer-
galectin-3 on quartz slides functionalized
lactose to gold-coated cantilevers. The tip
sity have developed an atomic force
2+ with Ni /nitrilotriacetic acid and attached
was brought into proximity with the surface and retracted 350–8003 per measure-
microscopy (AFM) method for con(a)
(b) 2
3
4
5
dissociate a bound complex, but this value cannot be directly related to
Applied force
thermodynamic properties such as free energy, because applying that
(d) 1
equilibrium state.
2
3
Toone and co-workers have circumvented this problem by using the probability of a binding event between
Applied force
two surface-functionalized moieties in the presence of various concentra-
as either a binding or nonbinding
3
event on the basis of its force–exten-
4
sion plot. As expected, the greater the
5
concentration of free ligand in solution, the smaller the probability that a
Extension (nm)
(c)
force removes the system from an
ment. Each retraction was classified
2
Force (pN)
iments measure the force required to
1
4
binding event occurred.
1
The researchers plotted the con-
2
5
centration of free ligand versus the
3
probability of a binding event and cal-
4
culated a solution-phase binding con-
5
stant. After correcting for the multiva-
Extension (nm)
ERIC TOONE
1
ic measurements. Typical AFM exper-
Force (pN)
ducting solution-phase thermodynam-
lency of the system, they determined
a value that is in good agreement (a) Binding interactions in the absence of free ligand. with values measured by isothermal (b) The associated force–extension plots all show bindtitration calorimetry. (Proc. Natl. ing events. (c) Binding interactions in the presence(a)of 70,000 R6G free ligand. (d) Only force–extension plots 1 and 3 show 60,000Acad. Sci. U.S.A. 2007, 104, 2579– FAM 50,000 a binding event. 2584)
tions of free ligand to calculate a solution-phase association constant. In proof-of-principle experiments, the
Intensity
researchers immobilized His-tagged
Dual-wavelength SERRS for multiplex DNA detection
40,000 30,000 20,000 10,000 0 –10,000 600
800
1000 1200 1400 1600 1800
Intensity
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Intensity
Intensity
Pity those haystack-crawling Raman shift (cm–1) (b) 25,000 (a) 70,000 R6G Cy 5.5 needle seekers. While they are 60,000 FAM Bodipy ROX 20,000 still out there searching, the 50,000 15,000 40,000 analogous problem of finding 10,000 30,000 and quantifying a specific DNA 20,000 5000 sequence in a mixture of oligos 10,000 0 has just gotten easier. Duncan 0 –10,000 –5000 Graham and co-workers at the 600 800 1000 1200 1400 1600 1800 600 800 1000 1200 1400 1600 1800 University of Strathclyde (U.K.) Raman shift (cm–1) Raman shift (cm–1) (b) 25,000 have shown that they can identiCy 5.5 ROX conjugates at excitation wavelengths of (a) 514.5 nm and 20,000 fy up to five individual DNA SERRS spectraBodipy of a mixture of five DNA–dye 15,000nm. (Adapted with permission. Copyright 2007 Wiley-VCH.) strands simultaneously at con(b) 632.8 centrations as low as 10–12 M in 10,000 intensity was linear down to ~10–12 M a heterogeneous mixture with dual5000gates and measured SERRS spectra of wavelength surface-enhanced resonance 0 each sample at two laser excitation for each conjugate. Raman scattering (SERRS). Now that the basic multiplex detec–5000wavelengths. At one or both wave600 800 each 1000chromophore 1200 1400 1600 lengths, had a1800 The researchers chose five dyes and tion has been shown, experiments are –1 Raman shift (cm ) that unique “fingerprint” spectrum attached them to the 5´ ends of DNA being carried out to demonstrate applicould still be distinguished in a mixture cations in molecular biology. (Angew. sequences. Then, they added silver colof all five samples. The fingerprint peak Chem., Int. Ed. 2007, 46, 1829–1831) loid particles to the DNA–dye conju-
