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ANALYTICAL CURRENTS Micropattern spectrophotometry George Whitesides and colleagues at Harvard University have developed an optical method that can collect continuous visible absorbance spectra at multiple locations in microsystems. Called micropattern spectrophotometry, the technique provides a novel way to simultaneously analyze the composition of multiple samples from a single stillcapture image. In the new approach, an unmodified bright-field optical microscope is combined with an array of microlenses and a diffraction grating, which disperses light transmitted by the sample into a continuous spectrum. The microscope objective is focused near the microlenses, so that the diffraction spectrum is observed in the primary image plane. (Normally, the diffraction spectrum is only observed in the rear focal plane.) The image is then captured with a CCD camera. The intensity of the first-order diffracted light is an-
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alyzed at each pixel, and the pixel number OG JG H2O BPB is converted to wavelength. Thus, for any +1 given location, the sample’s absorbance versus visible wave25 µm length can be readily obtained. Convex, Images of diffraction spectra (0 and +1 orders) obtained simultanecylindrical, and spher- ously for 3 dyes and water in 4 different microwells. (Adapted with ical microlenses were permission. Copyright 2005 National Academy of Sciences U.S.A.) evaluated. difference to the calibration procedure To demonstrate the procedure, the and scattered light. researchers simultaneously measured the Whitesides and colleagues also folabsorbance spectra of various dyes in lowed the dynamic switching of fluid multiple microwells. They obtained abstreams containing various dyes in lamisorbance maxima of 626, 436, and 601 nar flow. By capturing images of the difnm for bromophenol blue (BPB), orange green (OG), and Janus green (JG), fraction spectra while varying the flow rates, they obtained absorbance spectra respectively. The values were slightly difat a given location as a function of time. ferent than those expected from mea(Proc. Natl. Acad. Sci. U.S.A. 2005, surements with a benchtop spectropho102, 10,035–10,039) tometer. The researchers attribute this
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Ion/ion reactions for top-down proteomics With bottom-up MS-based proteomics
benchtop linear ion trap MS instrument.
react with benzoic acid anions during a
methods, posttranslational modifications
The addition of PTRs to the ETD fragmenta-
PTR to remove excess charge.
(PTMs) lose their context relative to one
tion process simplifies the spectra for easi-
another, and alternative splicing variants
er interpretation.
are often missed. However, the top-down
The researchers used ETD and PTRs to analyze several proteins, including ubiqui-
Hunt and colleagues modified a com-
tin and histones. They sequenced several
method requires an expensive FTMS in-
mercially available linear ion trap mass
N- and C-terminal residues for most of the
strument. To overcome these limitations,
spectrometer with a chemical ionization
proteins and identified PTMs on the termi-
Don Hunt and colleagues at the University
source that produces anions for ion/ion
nal amino acids. The sequences of the
of Virginia, Thermo Electron, and the Uni-
reactions. Proteins and large peptides are
middle sections of the proteins were not
versity of Victoria (Canada) have devel-
either directly infused into the ion trap or
determined, however, because these frag-
oped a new method that uses both elec-
separated by LC before MS analysis. Elec-
ments lay outside the mass range of the in-
tron transfer dissociation (ETD) and proton
trosprayed multiply protonated ions react
strument. The researchers believe that op-
transfer reactions (PTRs) to obtain N- and
with radical anions of fluoranthene or
timization of the instrument could resolve
C-terminal sequence information from
other polyaromatic hydrocarbons during
this problem. (Proc. Natl. Acad. Sci. U.S.A.
large peptides and whole proteins with a
the ETD process, and the product ions
2005, 102, 9463–9468)
© 2005 AMERICAN CHEMICAL SOCIETY
S E P T E M B E R 1 , 2 0 0 5 / A N A LY T I C A L C H E M I S T R Y
327 A
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ANALYTICAL CURRENTS Lectin arrays Microfluidic bioreactor for long-term monitoring Lloyd Smith and co-workers at the University of Wisconsin, Madison, have developed lectin arrays to determine carbohydrate patterns expressed on cell surfaces. Cell-surface carbohydrates affect many biological processes and are sometimes altered in tumor cells. To create the arrays, the researchers coated a gold thin-film substrate with a reactive selfassembled monolayer. A group of six lectins that bind to specific carbohydrates was then spotted onto the substrate. To verify that lectins were bound to the array surface, the researchers added biotinylated antibodies against two of the lectins to two different arrays. Both arrays were incubated with fluorescent streptavidin molecules and imaged. Only those spots corresponding to the targeted lectin were fluorescent on each array. When fluorescently labeled chicken ovalbumin was incubated with the array, the researchers observed that the immobilized lectins retained their abilities to bind specifically to certain carbohydrates on the protein. The lectin arrays were placed in the wells of a tissue culture plate and incubated with either fibroblast cells or adenocarcinoma cells. Each cell line showed a different lectin-binding pattern. Fibroblast cells only bound to three of the lectins, whereas the adenocarcinoma cells bound to five of the lectins. According to the researchers, the array could be expanded to include additional lectins for added information about the carbohydrates on cell surfaces. ( J. Am. Chem. Soc. 2005, 127, 9982–9983) (a)
(b)
Stephen Quake and colleagues at Stanford University have developed a chip with six independent nanoliter-scale bioreactors for monitoring bacterial growth. The microfluidic bioreactors provide a way to analyze the programmed behavior of bacterial populations over extremely long periods of time, something that is not possible with macroscopic reactors. The continuous operation of bioreactors is usually thwarted by the growth of microbial biofilms. For this reason, Quake and colleagues designed their bioreactors to operate in one of two states: continuous circulation or
A microfluidic chip with six independent microchemostats. The channels are filled with food dye for visualization. (Adapted with permission. Copyright 2005 American Association for the Advancement of Science.)
cleaning and cell dilution. The two states permit semicontinuous bacterial growth, which is interrupted by cleaning and dilution cycles to prevent the formation of biofilms. Each bioreactor, also called a “microchemostat”, consists of a growth chamber, an integrated peristaltic pump, and micromechanical valves. The valves are used to add media, remove waste, and recover cells. The bacterial cultures are studied by optical microscopy to provide real-time, noninvasive, automated measurements of cell density and morphology with single-cell resolution. Quake and colleagues studied the fluctuations in cell density and morphological changes in bacterial populations in each bioreactor under the control of a synthetic population-control circuit. The circuit regulated cell density by a negative feedback system based on a bacterial sensing mechanism. The investigators demonstrated that the chip can be used to monitor bacterial population dynamics over hundreds of hours. The bioreactor chip could have applications in high-throughput screening. The dynamic properties of bacterial populations, such as gene expression, could also be monitored in the bioreactors by fluorescence or luminescence.
(a) Fibroblast cells bind to three lectins and (b) adenocarcinoma cells bind to five lectins on a lectin array. 328 A
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 5
(Science 2005, 309, 137–140)
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Multifunctional pores as sensors Stefan Matile and colleagues at the University of Geneva (Switzerland) have developed synthetic multifunctional pores with optical readout that can be seen by the naked eye. According to the investigators, the pores could serve as “universal” sensors. To demonstrate that the pores are capable of distinguishing between adenosine diphosphate (ADP) and adenosine triphosphate (ATP), the investigators loaded vesicles with 5(6)-carboxyfluorescein (CF). The activity of the pores was measured as a function of the CF fluorescence, which was detectable by the naked eye. ATP blocked the pores, keeping the CF contained in the vesicles and quenched. ADP unblocked the pores, releasing CF from the vesicles and causing the dye to fluoresce. Matile and colleagues showed that the synthetic pores can also monitor the
reaction activity of the enzyme hexokinase as it uses ATP to phosphorylate D-glucose. They found that the velocity of the pore opening correlates with the enzyme concentration. The multifunctionality of the pores as universal sensors was demonstrated by pairing them with an enzyme as a cosensor. The investigators introduced the synthetic pores with hexokinase and ATP into soft drinks to detect the amounts of sugar present. Coca-Cola was treated with invertase to convert sucrose into glucose and fructose. Hexokinase used ATP to phosphorylate the glucose and fructose. The pores detected the amount of sugar in the soft drink by sensing the levels of ATP and ADP. Similar experiments to detect sugar content were also carried out with CocaCola Light, Red Bull, Fanta Orange, and Nestea Lemon. The sugar amounts
Coca-Cola Light
Coca-Cola
The amount of sugar in 1-mL aliquots of Coca-Cola Light and Coca-Cola was detected with synthetic multifunctional pores.
obtained by the synthetic pores closely matched the expected values. ( J. Am. Chem. Soc. 2005, 127, 9316–9317)
A force sensor with optical readout Jan Liphardt and colleagues at the Universi-
sors, in which the length of the ssDNA be-
ty of California, Berkeley, and the Lawrence
tween the dyes consisted of 10, 15, or 20
Berkeley National Laboratory have devel-
bases. The three sensors were able to dis-
oped a nanoscopic force sensor with opti-
criminate between high (15–20-pN) and low
cal readout. The sensor is capable of mea-
(0–5-pN) forces.
suring forces in experimental situations in
To determine whether the sensors were
which other force measurement methods
capable of measuring mechanical forces
don’t work well.
within nanoscale devices, such as DNA-
The sensor consists of single-stranded
FRET: 0.78 Force:
