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ANALYTICAL CURRENTS Nanoswitch is a biosensor turn-on
(a) P2
AATGCGG Atomic force P2 -S-S-TTACGCCCTAG microscopy confirmed that P1 T2 and P2 formed stable nanoassemblies with average P1/P2 P1/P2 + BamHl sizes of 300–400 (b) (c) nm. After 1 h of incubation with BamHI, the T2 of the MRS pair returned to baseline levels (~59.4 ms), (a) Diagram of a magnetic relaxation switch nanoassembly called the nanoassemP1/P2, and phase-contrast atomic force microscopy images of P1/P2 blies were no (b) before and (c) after BamHI treatment. longer present, and monodisperse nanoparticles (50–60 nm) were oband noted that recent developments in served instead. Additional experiments high-throughput NMR might further using two methylation-sensitive restricexpand the throughput of the assay. Antion enzymes (BamHI is not methylaother advantage, they add, is that contion-sensitive) confirmed that the nanoventional NMR spectrometers could be assemblies could be methylated normally. adapted to measure water T2 relaxation times. (J. Am. Chem. Soc. 2002, 124, The researchers conducted some of 2856–2857) the experiments in 384-well microplates
(i)
(ii)
(iii)
(iv)
(v)
β1 NTR1 D1
16000
RFU
printed. The supported membranes were characterized thoroughly, and tests confirmed the specificity of ligand–receptor interactions. More comprehensive tests verified that the microarrays preserve activity well enough to discriminate among three adrenergic receptors and that it is possible to estimate the binding affinities of the ligands from the data collected. (J. Am. Chem. Soc. 2002, 124, 2394–2395)
(a)
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v
(b) (i)
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(iii)
β1 β2 α2A
24000
RFU
Membrane protein microarrays Because approximately half of the current molecular targets are membrane-bound proteins, Joydeep Lahiri and colleagues at Corning, Inc., decided to develop a microarray specifically for such proteins. The trick was to develop surface chemistry that could immobilize the lipids associated with membrane proteins on surfaces. The researchers settled on microscope slides modified with ␥-aminopropylsilane, and single-bilayer microspots were fabricated by depositing vesicular solutions of either gel- or liquid-phase lipids using a quill-pin printer. Then, arrays containing three G protein-coupled receptors—an adrenergic, a neurotensin, and a dopamine receptor—were
GATCCTACGAG-S-S-P1 GATCCTC
BamHl
12000 0
(c) 0
[Neurotensin] (nM) 1 2 4 8
16
RFU 65000
ii
iii
3000 0
0
i
6000
RFU
J. Manuel Perez and colleagues at Harvard Medical School have designed magnetic relaxation switches (MRS) that, when used in pairs, can detect enzymes that cleave or methylate DNA. The researchers say these MRS pairs—called nanoassemblies or clusters when they self-assemble—can act as biosensors for recognizing and monitoring DNA-cleaving agents in real time without radioactive probes or electrophoresis. The MRS clusters are magnetic nanoparticles conjugated to synthetic oligonucleotides (oligos). When the nanoparticles hybridize to a target, they assemble into highly stable nanoassemblies and cause a decrease in the spin–spin relaxation time (T2) of adjacent water protons. The researchers designed an MRS pair, P1 and P2, which hybridize to one another to form the recognition site for the BamHI restriction enzyme. P1 and P2 each had a T2 of ~60 ms, whereas allowing P1 and P2 to hybridize reduced the T2 to ~32 ms.
AATGCGGGATCCTACGAG-S-S-P1 -S-STTACGCCCTAGGATCCTC
0
4 8 12 16 20 [Neurotensin] (nM)
Ligand binding to microarrays of G protein-coupled receptors. Ligand binding is specific (a) when three receptor families (1, NTR1, and D1) are mixed or (b) when three adrenergic receptors (1, 2, and ␣2A) are used. (c) The binding affinity can be estimated from the microarray data. J U N E 1 , 2 0 0 2 / A N A LY T I C A L C H E M I S T R Y
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ANALYTICAL CURRENTS Quantum dots at the ends of their ropes
1.8 1.7 Water
LBL/ ILBL
570 nm and luminesce at 600 nm. The layered film’s emission couples with the absorption of the tethered particles, which makes energy transfer between the two quite efficient, and the particles significantly quench the film’s luminescence. Now, here is the interesting part. Changing the dielectric coefficient of the solvent makes the “ropes” wind and unwind, and the separation distance between the linked nanoparticle and the film changes. The researchers demonstrate this by varying the composition of a water–ethanol mixture and watching the changing spectra. The higher the ethanol content, the more intense the luminescence. As the researchers point out, this is an example of an organized nanoparticle system with gradual and straightforward tunable optical
1.6
INP +
For years, chemists have been tethering compounds to surfaces with long molecular chains and looking at their properties. But what if you could attach something to a surface and reel it in and out, like a fish caught on a hook? That is exactly the system that Sebastian Westenhoff of Hamburg University (Germany) and Nicholas Kotov of Oklahoma State University call a “quantum dot on a rope.” In this system, CdTe nanoparticles are coupled to a thin-film surface via poly(ethylene glycol) chains. The film surface consists of alternating layers of an anionic polyelectrolyte with a poly(pphenylene ethynylene) backbone and poly(allylamine hydrochloride). The thin film alone is strongly luminescent and has a 460-nm emission. The CdTe particles, on the other hand, absorb at
1.5 1.4 Ethanol 1.3
100
75
50 25 % of H2O
0
Effect of solvent composition on the 460-nm luminescence intensity of the layered film with tethered nanoparticles.
coupling. (J. Am. Chem. Soc. 2002, 124, 2448–2449)
CE purifies carbon nanotubes Stephen Doorn and co-workers at Los
than the current options, size-exclusion
tection window located 75 cm from the
Alamos National Laboratory and the Uni-
chromatography or field-flow fractionation.
sample intake.
CE separations are based on the charge-
versity of Kentucky show the first use of CE
The researchers performed real-time
and size-dependent mobility of solution-
Raman spectroscopy of the separation
phase species under an applied electric
process and single-wavelength UV–vis de-
for separating carbon nanotubes by size
field. The researchers report that the rapid,
tection to confirm that CE provides high-
(a)
high-resolution separations available with
resolution separations of nanotube frac-
Total intensity
for the purification of single-walled carbon nanotubes. They say the method is better
8000
CE have the potential to separate a nan-
tions with baseline separation. Atomic
6000
otube sample into discrete fractions of uni-
force microscopy analysis of the nano-
4000
formly sized tubes. The team performed CE
tubes showed that the sample contained
2000 0
Raman intensity
(b)
4
6 Time (min)
8
2800
10
a mixture of tubes ranging from ~75 nm to 2 µm long, whereas the collected fractions
1-m-long fused-silica capillary, with the de-
were grouped by length. Short tubes eluted before longer tubes.
c a
1800
b
800 –200
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2
with 360-nm absorbance detection on a commercial instrument using a 75-µm-i.d.,
2
4
6 Time (min)
8
10
Capillary electropherograms of singlewalled carbon nanotubes using Raman detection. (a) Electropherogram showing total intensity (background fluorescence plus Raman scattering) collected at 560.37 nm. (b) Electropherogram showing scattered Raman intensity at 1591 cm–1.
A N A LY T I C A L C H E M I S T R Y / J U N E 1 , 2 0 0 2
Doorn and his colleagues say that because total tube charge and charge density ultimately depend on the tube diameter, the method also should be effective at separating tubes on the basis of diameter. (J. Am. Chem. Soc. 2002,124, 3169–3174)
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Pairs identify binding sites Titanium gives NOA a good image duced contrast when NOA is performed on living cells. The researchers attached
first use of titanium substrates for near-
unlabeled human endothelial cells to pol-
field optical analysis (NOA) of living cells.
ished titanium disks and looked at their
The researchers say their observations
optical structure and topography with hy-
show that nanoscale, label-free, nonde-
drophobically coated optical biosensors
structive imaging of cells living in a com-
mounted on a near-field scanning optical
plex milieu could be accomplished
microscope. The NOA image discriminat-
through NOA and interpreted with the
ed between living and dead cells and
help of atomic force microscopy (AFM).
showed optical contrast on the nanoscale.
NOA via near-field scanning optical microscopy currently has the most potential to be a noninvasive imaging tool, say
The cell organelles, for instance, were visRange 2,11 µm
(a)
cause of its high resolution
performed a detailed in-
and sensitivity for observ-
spection of the AFM/NOA profiles of the nuclear
129.4
tween key biomolecules. But they point out that
(b)
adapting NOA technology
129.8
to cellular imaging is no
128.6 (c)
(d)
20 µm
0
Range 4,378 V
20 µm
129.0
indicated high optical activSommer and Franke
0
(b)
ible as dark spots, which ity, in the living cell.
Sommer and Franke, be-
ing the interactions be-
128.6 (a)
15N
Andrei Sommer and Ralf-Peter Franke at the University of Ulm (Germany) report the
20 µm
Add this to the growing toolbox of methods for identifying drug binding sites. Johan Weigelt and co-workers at Biovitrum (Sweden) show that by isotopically labeling two amino acids and using NMR spectroscopy, they can easily screen for binding to a selected site, even in large biomolecules. The approach is demonstrated with the human adipocyte fatty acid binding protein FABP-4, which has been implicated in insulin resistance. In this case, the challenge is that the very similar FABP-3 is expressed in heart and muscle tissue, and thus a potential drug would have to be specific to FABP-4. As it turns out, valine 114 and valine 115 in FAB-4 are replaced with isoleucine and leucine, respectively, in FAB-3, and that provides an obvious analytical marker.
area, which revealed the potential of NOA to image intracellular structures across apical cell membranes. The researchers
simple feat because it re-
129.0
quires precisely localizing
say that combining titanium
129.4
and imaging low-contrast,
substrates with adequate
9.7
9.6
9.5
9.7
9.6
9.5
1H
Valine 115 cross-peak spectra. FABP-4 (a) alone (blue) and (b–d) in various mixtures. The cocktail of candidate compounds (b) includes a compound that binds to the protein (red) or (c) omits the binding compound. (d) A 1:1 mixture of the ligand and FABP-4.
The authors were able to produce FABP-4 from prototrophic E. coli cells in which the valines were labeled with 13C and 15N. This was confirmed by NMR spectroscopy and MS. The labeled fatty acid binding protein was then exposed to a cocktail of five compounds, including one known to bind to FABP-4, and the
particularly interfaces in a liquid environment. Sommer found that polished titanium, an extensively biocompatible material, enhances the irradiation-in-
20 µm
0
shape-variant, soft surfaces,
129.8
0
(a) Topography and (b) related near-field optical analysis (NOA) of human endothelial cells. The NOA image allows discrimination between the living and dead cells.
two-dimensional 1H–15N correlation NMR spectrum for valine 115 alone (rather than the entire protein) was monitored for chemical shifts in the cross-peak arising from the binding of a compound. The authors warn that this method may lead to false positives related to various secondary nonbonding effects or false negatives arising from phenomena that counteract the chemical shift. For
optical biosensors may open new opportunities in designing high-throughput array technologies based on NOA principles. (J. Proteome Res. 2002,1, 111–114)
the former problem, care should be taken when numerous chemical shifts are observed, and for the latter, labeling more than one amino acid pair should reduce the chances of a false positive. Because the spectra are much simpler, this method can be used with larger proteins than other conventional methods. (J. Am. Chem. Soc. 2002, 124, 2446–2447)
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ANALYTICAL CURRENTS Characterizing porous silicon sensors Porous silicon (PS) films are well suited In the new optical setups, either the mined that pore size, porosity, and the to sensing applications because they have intensity of reflected light at a single thickness of the PS layer are all imporlarge surface areas that can adsorb gas or wavelength was measured using a lowtant. In particular, sensor response can vapor species. Even better, a thin film of power red diode, or the spectrum of rebe maximized by using a low etching PS generates a characteristic reflectance flected light from = 400–1000 nm current density to create a larger numspectrum—a series of concentric light was measured using a white light (tung- ber of smaller pores. When a singleand dark rings, known as Fabry-Pérot sten) source. In exploring the relationwavelength source is used, thicker samfringes—which shift in wavelength when ship between sensor performance and ples appear to yield larger changes in an analyte adsorbs to the inner walls of PS morphology, the researchers deterthe reflected signal. The researchers cite the silicon pores. To a 13.4-µm-thick PS sensor demonstrate that such that has a detection limit Gas mixer measurements need not of 250 ppb of ethanol IR cell Air or N2 at 1 Atm require expensive and vapor. They also confirm Diode complex instrumentation, the assumption that capilBeam splitter laser Michael Sailor and collary condensation is reAir or N2 at 1 Atm PS sample leagues at the University sponsible for the high senof California–San Diego sitivity of PS vapor sensors studied PS sensors using and explain that the miEthanol Gas out two relatively inexpensive croporous structure of the Amplified Computer Low-temperature bath setups and describe the film tends to concentrate photodiode (acetonitrile/dry ice, –52 ∞C) physical characteristics that the analyte vapors in nanoExperimental setup for testing and calibrating porous silicon laser interferogovern the performance domains. (Langmuir, metric vapor sensors. of the sensors. 2002, 18, 2229–2233)
FRETting over SNPs Joydeep Lahiri and colleagues from Corn-
probes are labeled with the fluorophore
transfer (FRET) between the donor–acceptor
ing, Inc., unveil a strategy for detecting sin-
donor Cy3 at the 5´ end and attached to the
pair leads to a decrease in fluorescence for
gle-base mismatches via DNA microarrays
surface of the microarray. A complementa-
the Cy3 channel (570 nm) and an increase
that offers high discrimination factors. The
ry 15-mer sequence, which contains a sin-
in the Rox channel (614 nm).
approach builds on the molecular beacon
gle artificial mismatch and is labeled with
concept in which binding to an oligonucleo-
the rhodamine acceptor Rox at the 3´ end,
Now, an unlabeled complementary sequence, which matches the X probe per-
tide (oligo) moves a donor and
fectly, is introduced. The new seA
acceptor pair away from each other to generate a fluores-
quence kicks out the mismatched D
D
cent signal. In this example, SBM
the signal is used to identify a single-nucleotide polymorphism (SNP) that could be a marker for genetic disease.
X
A
Bind FRET
AM
acceptor Y
A
X
Bind unlabeled
A
D
on the Cy3 channel. If the total
complement to Y Y
Rox oligo, which boosts the signal discrimination factor for the bind-
X
Y
SNP, SNP: Schematic for single-base mismatch DNA microarray. The single-base mismatch (SBM), FRET donor (D), FRET acceptor (A), and artificial mismatch (AM) are indicated.
Their prototype DNA array
ing is defined as the product of the factors for the Cy3 and Rox channels, the authors argue that they achieved discrimination fac-
was constructed with two 15-mer probe se-
is then introduced and hybridized to the at-
tors as high as 8.5, depending on the particu-
quences that differ at a single nucleotide—
tached probe. With the DNA sequence hy-
lar SNP and artificial mismatch. (J. Am.
in effect, creating “X” and “Y” probes. Both
bridized, fluorescence resonance energy
Chem. Soc. 2002,124, 2396–2397)
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RESEARCH PROFILES Raising the bar (code) in multiplexed analyses backing is removed, and the template is dissolved—releasing billions of identically striped cylinders. Depending on the size and shape of the pores in the template, the cylinders can be 30–300 nm in diameter and 0.4- to 15-µm long. Although the idea of depositing metal into the pores of a filter (or other template) was first described by other researchers, making striped particles was
COURTESY OF NANOPLEX TECHNOLOGIES.
Organization is the key to many of the n-dimensional challenges that scientists will face in the next decade. The need to track millions of molecular interactions as assays acheive ever-higher throughput is a prime example. Some approach this task armed only with the sheer force of meticulous record keeping. Others attempt to cut the problem down to a more manageable size by capitalizing on easily distinguishable molecular labels, which enable tests to be combined and run concurrently. In this way, the size of the tracking problem can be divided by the number of unique identifiers. Fluorescent labels have long served this purpose well. But a new approach, which uses miniscule striped cylindrical particles that can be read using reflectance microscopy, stands ready to dramatically increase the number of unique identifiers and interleave gracefully with many current record-keeping strategies. In the May 15 issue of Analytical Chemistry (pp 2240–2247), Michael Natan, Griffith Freeman, and a team of colleagues at SurroMed, Inc., and its spin-off company, Nanoplex Technologies (both in Mountain View, Calif.), demonstrate that a large number of these easily read particles can be identified quickly with an optical microscope. The researchers also suggest that other uses for these particles, affectionately known as Nanobarcodes. The researchers produce a single “flavor” of striped nanoparticles by starting with a template, usually an alumina filter. They vapor-deposit a silver cathode onto the back of the template and use that as the working cathode in an electrochemical cell. Then, they introduce a solution containing a metal ion to the other side of the template and electrically deposit the metal onto the cathode in the shape of the pores in the filter. By controlling the time, current, and electroplating solutions, particles are built up “stripe by stripe,” says Freeman. After forming the encoded particles in the template, the cathode
A tightly arranged layer of one flavor of striped cylindrical particles reflects encoded information in this optical reflectance image.
the work of Natan’s research group when he was at Pennsylvania State University. “And the idea of releasing those particles and then using them in a multiplexed bioassay is something that we developed here at Nanoplex Technologies,” says Natan. The researchers also developed software that automatically reads the encoded particles from microscopic images captured under illumination at strategic wavelengths. The software reports critical parameters, such as particle length (in pixels), for every particle found in the microscopic visual field and interprets the code. Because the accuracy of the interpretation may be affected by the size of each stripe and the properties of
the microscope’s imaging system, a correlation coefficient indicates the certainty of the assignment. Rejecting particles that have questionable assignments improves the accuracy of the count. The researchers demonstrated the use of these striped cylindrical particles in a molecular-recognition assay. In the standard assay, fluorescent tags distinguish among the receptors or adducts present. Having multiple tags means using multiple excitation or emission filters, which may make the assay more expensive or exclusive and reduce the sensitivity of detection by reducing the amount of available light. In the new system, fluorescent tags still signal that specific molecular interactions occur, but the task of identification is shifted to the barcodes. Each antibody or receptor is covalently bound to its own flavor of striped cylinder and, thus, is identified with a specific code. Using a microscope in reflectance mode, the identity of the receptor is revealed by reading the code of the microscopic bar to which it is bound. “One of the most attractive features is that the instrumentation used to do the readout here is an optical microscope,” says Natan. In addition to tagging a molecule or a class of molecules, Natan sees wider applicability for the striped nanoparticles, including nonbiological tagging. Says Natan, “You could also consider using these particles for traceability or security applications, such as authenticating a document or a small part for, say, an airplane.” Using only gold and silver in an eight-stripe format, the researchers made 102 members of a library of uniquely identifiable barcodes. If more metals are used, the dimensionality of the code increases, and larger numbers of unique identifiers can be made with fewer stripes. According to Natan, “It’s clear that we’ve only scratched the surface of the number of unique identifier particles that can be made.” —Zelda Ziegler
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MEETINGS 223rd ACS National Meeting—Elizabeth Zubritsky reports from Orlando, Fla.
Analyzing individual ultrafine air particles Airborne particles
