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ANALYTICAL CURRENTS Monitoring nanostructures In materials science, how a product is constructed can often be as important to its function as what it is constructed from. Nowhere is this truer than in nanotechnology, where parameters such as crystal periodicity and shape can significantly affect nanomaterial behavior. Scientists typically use transmission electron microscopy (TEM) to monitor nanostructure formation, but this method provides 2-D images, which offer only partial information about 3-D objects. Scientists at the University of Cambridge (U.K.); the University of California, Davis; and Lawrence Berkeley National Laboratory, however, have developed a Z-contrast imaging method of scanning TEM (STEM) to study the 3-D growth of tin-rich quantum dots embedded in a silicon matrix. In the process, they gained new insights into how the tin and silicon interact to form dots of various shapes and sizes.
The scientists found that when (a) 001 projection (b) 110 projection tin is layered onto the matrix, silicon atoms diffuse into the tin layer, leaving voids in the matrix. These voids are selectively filled from one side by tin atoms, creating asymmetric quantum dots of 5 nm 5 nm irregular shape. Furthermore, void-filling follows an intermedi(c) 001 projection (d) 110 projection ate growth mode rather than a layer-forming or island-forming mode, offering insights on the surface and strain energies of the tin atoms on the silicon matrix. The researchers also noted 5 nm that some of the dots did not 5 nm form the expected octahedrons but instead formed rods. The STEM shows the formation of (a, b) well and (c, d) STEM experiments indicated that poorly structured quantum dots. (Adapted with perwithin a specific size range, the mission. Copyright 2005 AAAS.) quantum dots transform from an -Sn structure to the more energetically transition is reversed beyond this range. favorable -Sn structure but that this (Science 2005, 309, 2195–2198)
Dual ESI source for ion/ion reactions Conventional ion/ion techniques require
nately injected into the instrument, interfer-
types of ion/ion reactions. For example,
modifications to the mass spectrometer to
ences are minimized.
multiply protonated horse heart myoglobin
incorporate additional ionization sources.
With the new source, analyte mole-
was reduced in charge when it was react-
Although a single sonic spray source has
cules are ionized by a nano-ESI emitter,
ed with anions of perfluoro-1-octanol. Neg-
been developed for ion/ion reactions, its
whereas reagent molecules are ionized by
atively charged bradykinin was inverted to
ionization efficiency is low, and matrix ef-
a parallel ESI emitter. High potentials are
a positively charged species. The dual ESI
fects are possible when it is used to ana-
alternately applied to each sprayer to gen-
source was also used to produce gas-
lyze complex mixtures. Scott McLuckey
erate oppositely charged ions, which then
phase protein complexes of bovine ubiqui-
and co-workers at Purdue University have
migrate into a linear ion trap mass spec-
tin and bovine insulin. McLuckey and co-
developed a pulsed, dual ESI source that
trometer. Each sprayer can be optimized
workers say that additional sprayers could
can be coupled to any spray interface
independently.
be incorporated into the new source for
without modifying the mass spectrometer. Because the reagent and analyte are alter-
© 2005 AMERICAN CHEMICAL SOCIETY
The researchers demonstrate that the dual ESI source can be used for several
n MS experiments. (J. Am. Soc. Mass Spec-
trom. 2005, 16, 1750–1756)
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ANALYTICAL CURRENTS
Surfaces for protein sorting Stefan Diez and colleagues at the Max
crotubules, which are hollow
Planck Institute of Molecular Cell Biology
protein tubes of varying
and Genetics and the Leibniz Institute of
length. Diez and colleagues
Polymer Research Dresden (both in Ger-
demonstrated that the num-
many) have created polymeric gradient
ber of microtubules moving
surfaces that sort protein assemblies on
over the PEG–kinesin gradi-
the basis of size. According to the investi-
ent decreased linearly as
gators, the work is the first demonstration
the density of PEG increased
of self-organized sorting of proteins on a
along the silicon chip’s later-
surface.
al position. The velocity of
Diez and colleagues formed the surface
(a)
Kinesin PEG
Gradient of kinesin Gradient of PEG
Microtubule Substrate x location (mm) (b)
the microtubules was unaf-
gradients by adsorbing the motor protein
fected. Thus, the investiga-
kinesin to a silicon wafer precoated with
tors confirmed that the
10 µm x = 11 mm
poly(ethylene glycol) (PEG). The surface
PEG–kinesin surface was
coverage of PEG was precisely controlled
active and that the PEG did
by a temperature gradient. By varying
not harm the motor protein’s
the temperature, the investigators could
activity.
change the density of PEG and therefore
Diez and colleagues
generate a 1-D concentration gradient of
found that the densities of
10 µm x = 30 mm
10 µm x = 45 mm
(a) A schematic showing how the PEG–kinesin gradient surface sorts microtubules. (b) Fluorescence micrographs demonstrate that as the grafting density of kinesin decreases (and the number of microtubules in the field of view decreases), the average length of the microtubule increases.
the polymer. Because PEG prevents protein
PEG and kinesin on the sur-
adsorption, the incubation of kinesin on the
face could be used to sort microtubules
µm. The investigators suggested that the size sorting occurred because the longer
surface resulted in a concentration gradi-
on the basis of length. As the PEG grafting
ent that was inversely proportional to the
density increased from 0.5 to 2.2 mg/m
microtubules made contact with fewer ki-
polymer concentration gradient.
along the surface, the average length of
nesins when the PEG density increased.
the microtubules increased from 6 to 13
(Nano Lett. 2005, 5, 1910–1914)
Surface-bound kinesins can propel mi-
2
Dielectrophoretic trapping of single molecules Frank Bier and colleagues at the Fraunhofer Institute for Biomedical Engineering (Germany) and the Chalmers University of Technology (Sweden) have developed a method for reversibly trapping single molecules in aqueous solutions with an alternating electric field. The investigators suggest that their work could lead to the design of dielectrophoretic lab-on-a-chip systems for manipulating single molecules. The researchers fabricated planar gold electrodes on a silicon substrate by electron-beam lithography. The electrodes were triangular in shape to maxi448 A
mize the field gradient and minimize effects such as convection flow due to heating. Bier and colleagues studied the reversible trapping of a red algal autofluorescent protein called R-phycoerythrin in solution. When a 10-s electric field at 1 MHz and 10 V was applied, bright fluorescent spots appeared at the electrode tips. When the electric field was turned off, the fluorescent spots immediately disappeared. Similar results were obtained with a fluorescently labeled antibody. To determine whether the electrodes were trapping single molecules or ag-
A N A LY T I C A L C H E M I S T R Y / D E C E M B E R 1 , 2 0 0 5
gregates, the investigators quantitated the fluorescence intensity at the electrodes when the electric field was applied. The measurements from the intensity histograms indicated that single molecules of R-phycoerythrin were being trapped. The investigators also performed fluorescence correlation spectroscopy to confirm the trapping of single molecules. Finally, Bier and colleagues used a standard polarization model to calculate the forces acting on particles suspended in solution when the electric field was applied. (Phys. Rev. Lett. 2005, doi 10.1103/PhysRevLett.95.128102)
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Imaging single fusion events Manfred Lindau and colleagues at Cornell University have visualized single exocytotic events in neuronal cells by using electrochemical detector arrays. The release of individual vesicles containing certain neurotransmitters has been previously detected by carbonfiber electrodes as amperometric spikes. However, the new arrays permit highresolution time measurements, spatial localization of individual events, and quantitation of the flux of molecules. During exocytosis, membrane-bound secretory vesicles fuse with the cell’s plasma membrane to release their contents. For these studies, the researchers used chromaffin cells, which are derived from the adrenal gland and release catecholamines such as epinephrine, norepinephrine, and dopamine. The electrochemical detector array consists of four platinum electrodes patterned onto glass. The investigators used a patch pipette to place a chromaffin cell
in the same plane as the electrodes. As individual vesicles in the cells released catecholamines, oxidation currents were recorded by the electrodes. These currents permitted the investigators to determine the location of the fusion event and to follow the event’s dynamics with millisecond time resolution. As in amperometric measurements conducted with carbon-fiber electrodes, Lindau and colleagues measured “foot signals”, which are due to slow discharge of catecholamines through narrow fusion pores. The properties of the foot signals observed with the electrode array were similar to those measured by carbonfiber electrodes. The investigators also simultaneously imaged the release of the fluorescent dye acridine orange from the vesicles by optical microscopy and correlated the fluorescent microscopy data with the electro-
C D
B A
Vesicle fusion events from a chromaffin cell are monitored on an electrochemical detector array consisting of four platinum electrodes, labeled A–D. (Adapted with permission. Copyright 2005 National Academy of Sciences, U.S.A.)
chemical measurements of catecholamine release. (Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 13,879–13,884)
Missing phosphopeptides intensities did not.
By some estimates, ~30% of proteins are
test this possibility, the researchers loaded
phosphorylated at any given time. Howev-
a set of synthetic peptide–phosphopeptide
Finally, the researchers examined
er, the number of phosphorylation sites
pairs onto a C18 resin that is commonly used
whether phosphopeptides had lower ion-
identified experimentally is much smaller.
in proteomics experiments. To their sur-
ization/detection efficiencies than their un-
One reason for this discrepancy may be
prise, they found that a phosphopeptide
modified counterparts. Using a quadrupole
loss of phosphopeptides during MS analy-
often eluted after, not before, its peptide
TOF mass spectrometer, they generated
sis. To examine three possible causes of
partner. They speculated that this may be
ion chromatograms for each observed
loss, Marc Kirschner and colleagues at
due to charge neutralization between phos-
charge state of peptide–phosphopeptide
Harvard Medical School and Children’s
phorylated and basic residues, which actu-
pairs. The results indicated that more
Hospital Boston, MDS Sciex (Canada), the
ally reduced the peptide’s net hydrophilicity.
phosphopeptides showed better ionization/detection efficiencies.
University of Dundee (U.K.), and Cell Sig-
The researchers then tested whether
naling Technology performed qualitative
phosphopeptide signals were selectively
Given these results, they say that im-
and quantitative analyses of phosphopep-
suppressed in the presence of excess un-
provements in phosphoprotein enrichment,
tides and unmodified peptides by ESI MS.
modified peptides. They mixed peptide–
whether chromatographic or mass spectro-
Phosphopeptides may be lost when
phosphopeptide pairs with up to 100-fold
metric, may be necessary to identify more
samples are loaded onto reversed-phase
excesses of tryptic digests of various pro-
phosphorylation sites. (Mol. Cell. Proteo-
columns if the phosphopeptides are more
teins. Although the total signal intensity for
mics 2005, doi 10.1074/mcp.M500135-
hydrophilic than the unmodified peptides. To
the peptide pairs diminished, their relative
MCP200)
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