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analytical currents Tracking single virus particles on membrane bilayers Interferometry has been used previously to detect biological entities such as microtubules and viruses and to measure particle scattering. However, time-resolved, or tracking, experiments by interferometry of biologically relevant parameters, such as the diffusion constant, haven’t been reported yet. So, Vahid Sandoghdar and colleagues at the Swiss Federal Institute of Technology in Zurich demonstrated that it’s possible to track individual, unlabeled viruslike particles on supported membrane bilayers under an aqueous buffer. Sandoghdar and colleagues analyzed the movements of a simian virus 40 (SV40) virion, which is a stripped-down version of the virus. The virion binds to the lipid GM1 on cell surfaces. This binding allows SV40 to enter cells and cause infection, but the mechanism isn’t well understood. So the SV40–GM1 in-
teraction is of interest in µm 2/s for the virions. medicine and biology. They then measured The investigators crethe diffusion of GM1 ated supported membrane by a fluorescence techbilayers with GM1 and nique while tracking introduced the virions. the virions by interferThey got a signal conometry and were surtrast of 3.02 ± 0.88% for prised to find that the SV40 virions and 0.75 ± free receptor was much 0.39% for SV40 viruslike more mobile (by >2 1 µm particles. As controls, orders of magnitude) they introduced cholera than when the virion toxin, which also binds Tracking of an unlabeled SV40 was bound to it. The GM1, and concanavalin virion that is bound to a support- investigators concluded A, which doesn’t, and got ed membrane bilayer with GM1. that the restricted the expected results. mobility was due to The trajectory was assembled Because the method from 50 frames acquired over 2.5 the highly multivalent is photostable, the inves- minutes. binding of the virion tigators monitored single to its receptor and the virions as they moved laterally on the correspondingly strong virus–memmembranes. The researchers calculated brane interaction. (Nano Lett. 2007, 7, a diffusion constant of 0.0088 ± 0.0004 2263–2266)
Tomographic phase microscopy sured with a laser light source.
sachusetts Institute of Technology and
The 3D tomographic phase-micros-
Harvard Medical School have developed
copy images paint a detailed picture of a
a new imaging technique, tomographic
cell’s interior without the need for dyes
phase microscopy, that measures the re-
or contrasting agents. The researchers
fractive index of a cell. The new method
could easily distinguish the nucleus and
is analogous to X-ray computed tomog-
nucleolus of a HeLa cell from the cyto-
raphy (CT), a technique often used to ob-
plasm by refractive index alone. The re-
tain 3D images of the human body. CT im-
searchers note that this technique also
ages are taken by an X-ray source as it
can be used to study the light-scatter-
rotates around the body, and these imag-
ing properties of cells and tissues and to
es are then combined into a detailed 3D
correct for sample-induced aberrations
composite. In tomographic phase micros-
in high-resolution light microscopy. (Nat.
copy, the refractive index is instead mea-
Methods 2007, 4, 717–719)
© 2007 American Chemical Societ y
Michael Feld
Michael Feld and co-workers at the Mas-
A 3D image of a HeLa cell constructed with tomographic phase microscopy. Nucleoli are colored green.
o c t o 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
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analytical currents Pushing (and pulling) the cellular envelope As a cell goes about its daily business of living, it pushes, pulls, stretches, and bends its proteins. At least that’s what most researchers thought, on the basis of in vitro and in silico experiments. But no one had shown that this was really true in a living cell. Dennis Discher and colleagues at the University of Pennsylvania and the Wistar Institute have now used a “shotgun” Cys-labeling approach to confirm that a living cell deforms its proteins when under force-induced stress. For these experiments, red blood cells (RBCs) served as a simple model system, because these cells commonly experience shearing forces in vivo as they flow through the body’s veins. The researchers reversibly lysed the cells to empty out nonmembrane-associated proteins and resealed the cells with a Cys-reactive fluorophore, IAEDANS, inside. They sub-
jected the RBCs to shearing forces before relysing them. The cells were solubilized, their proteins were denatured, and any remaining unlabeled Cys residues were alkylated with iodoacetamide. Upon separation by 1D SDS-PAGE and densiA molecular dynamics simulation of forced unfolding of tometry analysis, cells ex-spectrin and binding of a Cys-reactive dye. (Adapted posed to shearing forces with permission. Copyright 2007 American Association for had 50% more fluoresthe Advancement of Science.) cence in the bands corresponding to - and -spectrin, two chains The investigators also applied their of a membrane cytoskeleton protein shotgun Cys-labeling approach to mesthought to contribute to cell deformabilienchymal stem cells with similar results, ty. Other analytical analyses, such as LC/ and they note that the technique could be MS/MS, confirmed that at least six Cys extended to study conformational changsites are exposed when - and -spectrin es during signaling events. (Science 2007, are deformed under mechanical stress. 317, 663–666)
Electric field mapping in live cells with a nanosized voltmeter Raoul Kopelman and colleagues at the University of Michigan have developed a nanoscale voltmeter that permits researchers to carry out 3D electric field profiling throughout the entire volume of live cells. The voltmeter is a nanoparticle called an electro-PEBBLE (E-PEBBLE). Kopelman and colleagues derived the E-PEBBLE from the previously described PEBBLE technology that has been used to measure analytes, such as calcium, potassium, and nitric oxide, as well as physical properties such as viscosity in cultured cells. An E-PEBBLE consists of a 30 nm silane-capped mixed micelle shell. In its hydrophobic center, the micelle contains a fast-response, voltage-sensitive dye that shifts its fluorescence spectrum as the electric field changes. The shift is analyzed ratiometrically to reduce noise. 7230
E-PEBBLEs can be calibrated Hydrophobic core beforehand and then inserted Capping layer into any cell, cellular compartment, or external region. Blue light in Targeting layer Kopelman and colleagues inserted E-PEBBLEs into a Green/ 30 nm Electric field red light cell line of astrocytes with out well-characterized respiratory Silane shell profiles. They first measured Voltage dye Targeting molecule the mitochondrial electric fields and demonstrated that An E-PEBBLE has a voltage-sensitive dye encased in a they could follow the dissipamicelle. The nanoparticles can be calibrated externally tion of the fields as a particubefore delivery to cells. (Adapted with permission. lar chemical was added. Copyright 2007 Biophysical Society.) The investigators then analyzed the cytosolic electric fields can’t be thought of as a simple, electriand determined that the electric fields cally homogeneous solution. Instead, from the mitochondrial membranes it should be regarded as a complex, penetrated far deeper into the cytosol heterogeneous hydrogel with distinct than had been estimated previously. microdomains. (Biophys. J. 2007, 93, Their results suggest that the cytoplasm 1163–1174)
A n a ly t i c a l C h e m i s t r y / o c t o b e r 1 , 2 0 0 7
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Array of conjugated polymers detects proteins Quick, easy, and accurate protein sensing approaches are important for medical diagnostics and proteomics. Recent efforts have focused on protein detection by either fluorescence quenching or indicator displacement, but the results have had high limits of detection and have been applied to only four or five proteins. So Vincent Rotello at the University of Massachusetts Amherst, Uwe Bunz at the Georgia Institute of Technology, and colleagues have built a sensitive protein sensor array based on polymers that can discriminate among 17 proteins of varying properties. The investigators built the array with six functionalized, fluorescent poly(p-phenyleneethynylene) polymers (PPEs). The water-soluble conjugated
polymers bind protein surfaces through multivalent interactions, changing their fluorescence as they do so. The various charge and molecular characteristics of the polymers make them sensitive to a range of proteins. Of the 17 proteins selected as sensing targets, many had comparable molecular weights and isoelectric points, so the investigators could test the differentiation ability of the PPE-based sensor array. The investigators measured the fluorescence properties of the array before and after the addition of proteins. The six polymers displayed significant overlap in their absorption and emission spectra, so the same excitation and emission wavelength could be used for
all polymers to speed up analysis on a microplate reader. Of 68 samples that were randomly selected from the combination of 17 proteins, only 2 were misclassified. This meant the array had an identification accuracy of 97%. The protein concentrations were generally determined within ±5% deviation. But the investigators caution that more experiments are needed to demonstrate the robustness of the system, because cross-reactive arrays still are prone to errors. In particular, the arrays must be tested with complex mixtures to establish their ability to detect low-abundance species in the presence of large amounts of interferents. (J. Am. Chem. Soc. 2007, 129, 9856–9857)
Cell-based device detects oral-cancer biomarker Oral cancer afflicts more than 350,000 people annually, particularly in countries where risk factors such as tobacco use and alcohol intake are more pronounced. To help medical practitioners identify
Samples obtained from (a)
(b)
Coverslip
Waste outlet
Fluid inlet
Fluid traveled up to the
Membrane and support
adhesive layers, where a
PMMA base
2.5 µm
20 µm
the disease at preliminary stages, John McDevitt and colleagues at the University of Texas Austin have developed a sensor
sies entered the device through the base’s inlet.
(c)
Adhesives
tumor cell lines or biop-
narrow channel directed it to a membrane that captured the cells. Protein
(a) Schematic diagram of the layered device. (b) Cross section shows continuous fluid flow path for “on-membrane” assays. (c) Electron micrograph of a membrane before (left) and after (right) cell capture. (Adapted with permission. Copyright 2007 Royal Society of Chemistry.)
that detects an oral-cancer biomarker on cells. The Texas researchers focused their
and aggressive cancer phenotypes.
expression was measured by continuously flowing fluorescently labeled antibodies and other reagents
over the captured cells that sat in the
The multilayer-device was built on a PMMA base with a fluid inlet and outlet
pores of the membrane. McDevitt and colleagues identified
investigative efforts on tracking the ex-
port. A polycarbonate membrane with 0.4
cells with high levels of EGFR expression
pression of a protein called the epidermal
µm pores and an underlying support was
in
