news
ANALYTICAL CURRENTS Biosensing arrays based on RNA Often shunned as unstable or annoyingly prone to forming secondary structures, RNA may finally earn some respect, thanks to a new array of RNA-based biosensors developed by Ronald Breaker and colleagues at Yale University.
The pixels in this new array are “RNA switches” made from ribozymes—folded RNA strands that can catalyze RNA cleavage at specific sequences—and arranged on a gold surface. Each ribozyme remains inactive until it encounters an effector molecule (which Analyte 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 might be an oligoA A A CO B B cGMP B nucleotide, an enC C cCMP C D D cAMP D zyme cofactor, a E E FMN E Reaction F F Theo F protein, or a pharG G FMN + Theo G H H All H maceutical agent) I I None I for which it has Positive Negative RNA array been tailored. A prototype RNA array. (Left) Initially, all pixels bear 32P tags. (Middle) Then the riboThe 32P tags have been cleaved off wherever the RNA switch was zyme springs into matched with the correct effector molecule. (Right) A “negative” action—in this plate, which shows the cleaved fragments. (Adapted with permission. case, cleaving off a Copyright 2001 Nature America.) segment of itself 2+
that has been labeled with 32P. The researchers monitor the loss of radioactivity at each address in the array to determine which pixels react to various effector molecules. In one experiment, an array that included seven types of RNA switches— one of which requires two effector molecules—was tested by incubation with various analyte solutions. In another experiment, strains of E. coli were phenotyped based on the accumulation of cyclic adenosine monophosphate (cAMP) in the culture media. This array included an RNA switch that recognizes cAMP. Strains were differentiated on the basis of the absence or relative abundance of cAMP, which was deduced from the extent of the ribozyme reaction. (Nat. Biotechnol. 2001, 19, 336–341)
Golden optical sensors Here is a handy way to follow the binding of
sembly of the disulfide on the gold surface,
selective monolayers onto gold film and mon-
molecules onto a gold surface. Israel Rubin-
they saw increasing absorption for the three
itoring molecular binding to the monolayer.
stein and colleagues at the Weizmann Insti-
pyrene UV bands and an increase and red
(J. Am. Chem. Soc. 2001, 123, 3177–3178)
tute of Science (Israel) find that ultrathin is-
shift for the gold surface plasmon absorption
land-type gold films act as optical sensors
band at ~600 nm.
when evaporated onto transparent sub-
The researchers observed a linear corre-
strates. Changes in the surface plasmon in-
lation between the plasmon intensity change
tensity obtained directly from difference
(PIC) and the pyrene UV band, which indicat-
(transmission UV–vis) spectra show a linear
ed that the PIC was related to the number of
correlation with the number of molecules
molecules bound to gold.
binding to gold and therefore measure the amount of molecular deposition. In one experiment, researchers bound a
Further experiments established the generality of the method for different types of molecules and adsorption modes in both liq-
cyclic disulfide with two pyrene residues to
uid and gas phases. The researchers envi-
gold and collected a series of transmission
sion creating a variety of chemically and bio-
UV–vis difference spectra. During self-as-
logically selective sensors by adsorbing
Noncontact atomic force microscope topographic image of an island-type, unannealed ultrathin gold film evaporated on quartz.
J U N E 1 , 2 0 0 1 / A N A LY T I C A L C H E M I S T R Y
301 A
news
ANALYTICAL CURRENTS Unsafe air at home The suspected human carcinogen formaldehyde can cause itchy skin, nausea, and difficult breathing at concentrations as low as 100 ppbv. To date, however, studies attempting to link exposure to formaldehyde in indoor air and health problems have had only some success. Now, Janusz Pawliszyn and colleagues at the University of Waterloo (Canada) describe a solid-phase microextraction
(SPME) sampling method for determining formaldehyde levels in indoor air. The SPME fiber is loaded with the derivatizing agent o-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine (PFBHA) hydrochloride. Formaldehyde reacts with the PFBHA to form a stable formaldehyde– PFBHA oxime, and the amount of oxime on the fiber is directly proportional to the formaldehyde level in the air. Analysis is conducted by GC with flame ionization detection, F F F F H H and the researchers comO pare this method to the F CH2 F C + C standard absorbent-tube O N C H O NH H H 2 F F F F H method employed by the PFBHA Oxime Formaldehyde National Institute for Occupational Safety and Health (NIOSH). Reaction between formaldehyde and the derivatizing agent Indoor air was sampled o-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine that occurs on from three rooms at each the solid-phase microextraction fiber. of six locations, both occu-
pational and nonoccupational. For the NIOSH method, the sampling time was 8 h. For SPME, both 10-min grab sampling and 8-h time-weighted averages were used. The SPME analyses revealed formaldehyde levels of 10–380 ppbv, which correlated well with the NIOSHbased concentrations. Levels of 103 and 380 ppbv—both higher than NIOSHacceptable occupational standards of 16–100 ppbv—were measured in two rooms at one residential location; whereas average concentrations at the workplaces surveyed were between 10 and 50 ppbv. On the basis of these results, the researchers conclude that SPME is an accurate, cost-efficient method that reduces analysis time by an order of magnitude and could be extended to the field testing of formaldehyde levels in drinking water. (Environ. Sci. Technol. 2001, 35, 1481– 1486)
Quantifying chirality The role of chirality is probably one of the
ecule, the now-larger complex will presum-
pound was 0.1132, whereas the value for
most difficult factors to determine in bio-
ably have a slower rotational correlation
the S compound was 0.0991. Taking this
molecular interactions with drugs or other
time and a higher anisotropy value. This is
one step further, the researchers intro-
ligands. Isiah Warner and his colleagues at
great for studying binding interactions in
duced a variable based on the ratio of
Louisiana State University and Southern
general.
anisotropy values for R and S compounds.
Illinois University introduce a clever method
However, Warner and his colleagues
This new fluorescence chiral selectivity
for measuring the contribution of chirality
realized that if the target molecule has chi-
term was measured for a series of com-
to a binding interaction. The technique,
ral centers, then enantiomeric ligands
pounds that interacted with a host com-
which is based on fluorescence anisotropy,
should have slightly different binding inter-
pound. These values were compared with
could have broad applications in such areas
actions, which will be reflected in their
chiral selectivity values determined by
as drug development, enzyme–inhibitor
anisotropy values. Moreover, all the other
micellar CE for the same system, yielding
studies, and studies of chiral stationary
nonenantioselective factors that affect anis-
a near-linear relationship. Finally, the re-
phases.
otropy should be the same for enantiomers,
searchers related the fluorescence chiral
and therefore, the measured values will re-
selectivity term to the difference in free
flect just the difference due to chirality.
energy of association for the enantiomeric
Among the many factors that affect fluorescence anisotropy is the rotational motion of the fluorescing molecule. Thus, if a fluorescing molecule binds to another mol302 A
For example, the anisotropy value for an R enantiomer binding to a naphthyl com-
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 1
pair. (J. Am. Chem. Soc. 2001, 123, 3173– 3174)
news
Spin labels for “thrifty” NMR screening Biomolecular NMR spectrometry has been receiving considerable attention because it can detect the molecular interaction between a protein target and a ligand, even at very low levels. However, NMR screening requires large amounts of concentrated protein, which limits it to proteins that express well and are soluble to high-micromolar concentrations. Now, Wolfgang Jahnke and colleagues at Novartis Pharma AG (Switzerland) reduce the amount of protein needed for primary NMR screening. The work is an extension of a previous study using spin-labeling for second-site NMR screening, in which the spin label— a paramagnetic moiety with an unpaired electron—is attached to a ligand that is known to bind to the target protein. In primary screening, on the other hand, the protein target is labeled. These labels influence the relaxation of the bound compounds. The result is that, upon dissociation, those compounds are easily identified by their broadened and weakened resonances. The researchers use the SLAPSTIC (spin labels attached to protein side chains as a tool to identify interacting compounds) method to label samples for a primary screen to identify possible ligands for the FK506-binding protein, FKBP. The relaxation effects observed with 1-µM spin-labeled FKBP were similar to those observed using 60-µM nonmodified FKBP, clearly demonstrating a drastic reduction in required protein concentration and a high ligand solubility. The SLAPSTIC method is not applicable to strongly binding ligands with slow dissociation rates, and it does not provide detailed information on the binding site. Nevertheless, the researchers note that the general reduction in required protein concentration and the ease of automating the new method open NMR screening to a more diverse group of compounds. (J. Am. Chem. Soc. 2001, 123, 3149–3150)
One handy mixing machine! Bernhard Lendl and co-workers at the Vi-
of the short residence time inside the
enna University of Technology (Austria)
mixer, the scientists say. This allowed the
present a new way to study chemical
researchers to record a reference spec-
reactions in solution using a microma-
trum before the reaction. When rapid
chined mixing unit, which is incorporated
switching of an injection valve stopped
in a flow cell. The micromixing unit is fast
the flow, highly reproducible diffusion-
and yields highly reproducible results.
controlled mixing took place inside the
The team tested the mixer using three
flow cell, and the spectral changes result-
model reactions: an acid–base neutraliza-
ing from the reaction could be followed by
tion, the addition of sulfite to formaldehyde,
time-resolved FT-IR.
and the basic hydrolysis of methyl mono-
The researchers report that the total
chloroacetate. They also studied the com-
volume for one experiment was ~5 µL, and
plete mixing process in the system via
the mixing times achieved were in the mil-
computational fluid dynamics simulations.
lisecond range. Possible applications for
When the flow in both of the mixer’s
the new approach include the study of
feeding channels was maintained, almost
protein reaction dynamics. (Appl. Spec-
no mixing of the liquids occurred because
trosc. 2001, 55, 241–251)
Waste
Flow on
To mixer (2)
(2) Micromixer
From mixer (3)
Reactant A inlet
(3)
Reactant B inlet
SP
Streamlines, no mixing
SP (1)
To mixer (1) Waste
Waste
Stopped flow
To mixer (2)
(2) Micromixer
From mixer (3)
Reactant A inlet
Reactant B inlet
SP
Diffusion based mixing
SP To mixer (1) Waste
(3)
(1)
Micromixer in the liquid handling system. (a) In the “flow” mode, there is little mixing of the liquids. (b) In the “stopped-flow” mode, diffusion-based mixing occurs. (Adapted with permission. Copyright 2001 Society for Applied Spectroscopy.) J U N E 1 , 2 0 0 1 / A N A LY T I C A L C H E M I S T R Y
303 A
news
ANALYTICAL CURRENTS (M-24H+)24–
MS for genotyping STRs Short tandem repeats (STRs)—stretches of DNA in which 1–5 base pair units are repeated many times—are best known for their role in DNA fingerprinting. STR analyses are usually performed using electrophoresis, but David Muddiman and James Harris at Virginia Commonwealth University show that MS—like the ice in Robert Frost’s poem “Fire and Ice”—is also great and will suffice. To prove the concept, the researchers generated two PCR products from an STR locus, or region, of the human tyrosine hydroxylase gene by employing either the Pfu or Taq polymerase. The products were identical except that the one made by Pfu polymerase was bluntended whereas the one made by Taq polymerase was di-adenylated. (A very small amount of the Taq product was mono-adenylated.) Thus, the m/z ratios
Blunt ended
of the products differed slightly. The samples were fed into the mass spectrometer via flow injection using a manual valve. Analysis with electrospray ionization MS distinguished between the two products and even revealed small peaks for the monoadenylated product. The researchers note that the throughput during these experiments was equivalent to ~300 genotypes/ day. (Rapid Comm. Mass Spectrom. 2001, 15, 348–350)
Pfu
Spectrum 10
di-adenylated
(M-24H+)24–
Mono-adenylated
Taq
Spectrum 29
Blunt ended
(M-24H+)24–
Pfu
Spectrum 50
di-adenylated Spectrum 72
Mono-adenylated
2080
(M-24H+)24– Taq
m/z
2160
MS distinguishes between a reference DNA sequence (bluntended) and a di-adenylated product. A small peak representing mono-adenylated product is also present. (Adapted with permission. Copyright 2001 John Wiley & Sons, Ltd.)
No medals for these bacteria Margot A.-S. Vigeant and colleagues at the
can help explain events preceding the per-
scientists measured the distance between
University of Virginia are working to curtail
manent attachment of potentially harmful
an E. coli bacterium and a clean quartz
“nasty” swimming bacteria from reaching
microorganisms to surfaces. Such studies
surface as the bacterium was swimming
the surface and “getting the Olympic gold.”
are important because bacterial motility has
laterally along the surface using a high-
been implicated as a factor in virulence;
speed, high-resolution charge-coupled de-
motile bacteria reach surfaces
vice camera with the TIRAF apparatus. The
faster than nonmotile bacteria and
microscope was capable of capturing im-
may adhere more rapidly. By un-
ages of a bacteria field twice per second
derstanding the initial steps of ad-
for >1 min.
The researchers want to help control
hesion, the researchers propose
(Left to right) Time series showing movement of a smooth-swimming bacterium along a quartz surface during a ~2-s interval. The bar at the top is ~5 µm.
The images were translated into quanti-
that a strategy to prevent adhe-
tative distance measurements with nano-
sion can be found.
meter-scale resolution. Both motile and
The researchers note that TIRAF
nonmotile bacteria were observed within
is different from related approach-
100 nm of a clean quartz surface. The team
es such as atomic force micro-
is working on another paper that will focus
infectious bacteria with the aid of a total
scopy and TIR fluorescent microscopy be-
on conditions and cell types to determine
internal reflection aqueous fluorescence
cause it does not require the immobilization
the forces responsible for adhesion. (Lang-
(TIRAF) microscope. The TIRAF, they say,
of cells on a surface for measurement. The
muir 2001, 17, 2235–2242)
304 A
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 1
news
RESEARCH PROFILES ECL for near-field imaging must be thick enough to eliminate pincomplex then oxidizes tri-n-propylamine, Even physical laws can have loopholes. holes, yet the tip must remain uncoatConsider the law against using light to producing the chemiluminescence with a resolve an object that is smaller than the ed.” Another difficulty with NSOM is maximum wavelength of 645 nm. This the control of the probe-to-sample diswavelength of the light. Known as the reaction occurs in a thin layer (~20 nm) tance. “The optical near-field is available at the electrode tip, creating a tiny light classical diffraction barrier, this law is only within a distance of ~20 nm from obeyed almost without exception. source. the tip,” he explains. “If the tip is furYes, diffraction can be defeated. The The resulting ECL light source has ther away, the resolution is lost.” loophole is that, as light passes through many of the same characteristics as an Bard says that many NSOM systems an optical fiber, some of the light’s enNSOM source. The two sources are apuse the “shear force mode” for surface ergy is available in a thin layer (~20 nm proximately the same size; the probe tip detection and probe positioning. The thick) on the outer surface of the fiber. can be scanned near the sample surface This is the region of the evanescent field probe is attached to a tuning fork that and inside the near-field, creating a highvibrates the tip laterally. As the tip apor the optical near-field. Microscopy resolution image; and finally, the image proaches the sample surface, the amplitechniques using the near-field can reresolution depends primarily on the tip’s solve objects much smaller diameter. than the wavelength of With the exception that x,y,z inchworms the laser light. This phethe laser is replaced by an nomenon has led to excitelectrochemical workstaing new applications, one tion, the block diagram of of which is near-field scanthe ECL scanning probe HV ning optical microscopy microscope (see figure) is WE Approaching, scanning controller (NSOM). identical to an NSOM diElectrochemical In the May 15 issue of agram. Using this system HV workstation Analytical Chemistry (pp and a probe with a 155-nm RE z-Pusher 2153–2156), Yanbing Zu, tip, the Texas researchers CE Dither oscillation Zhifeng Ding, Junfeng produced an ECL nearPiezo driver Zhou, Youngmi Lee, and field test sample image Current Allen Bard at the Univerthat demonstrated a resoTuning fork sity of Texas–Austin delution of 230 nm. Unlike Water solution Oscillation Input Sample output scribe a new scanning laser-based probes, Bard ECL intensity PMT probe microscope that notes, ECL light sources Feedback Lock-in amplifier further defies the diffraccan produce broadband tion barrier. In lieu of a radiation at several waveControl signal laser and optical fiber, this lengths. In addition, ECL microscope uses electroBlock diagram of electrogenerated chemiluminescence imaging apparatus. does not heat the sample generated chemiluminesas laser sources tend to do. cence (ECL). This apWhen the researchers tude of the fork/probe vibration is proach avoids the problems inherent in began this project, they thought that changed, and the amplitude variation is making NSOM probes, yet the instruthe ECL tip might replace the tuning used to adjust the probe distance. “The ment’s resolution remains high. fork assembly, Bard says, “but we have In NSOM, images are created by scan- problem with this technique,” he notes, not yet accomplished this.” The team “is that the probe can extend only a mil- also wanted to generate a stable ECL ning a probe over the specimen while limeter or so from the tuning fork withkeeping the tip close to the surface. The light source at the tip. “Here we sucprobe is a tiny optical fiber, drawn down out damping the oscillations; therefore, ceeded,” he notes. “We were able to any sample solution must be very thin.” to 50–100 nm and coated with metal, generate a perfectly stable light source In the new instrument, light is genexcept at the tip. Laser light passing that could last for hours.” erated at the tip of a metal electrode by through the optical fiber forms an optical And because of that, we have another ECL instead. The ECL reaction chosen near-field region at the tip. The resoluloophole, based on electrochemistry, which for this work involves the electrogenera- defeats the classical diffraction barrier. tion of NSOM is usually 50–150 nm. –James P. Smith and tion of Ru(2,2´-bipyridine)3+ “Making these probes is no trivial 3 from Vicki Hinson-Smith task,” says Bard. “The metal coating Ru(2,2´-bipyridine) 32+. The Ru(III) J U N E 1 , 2 0 0 1 / A N A LY T I C A L C H E M I S T R Y
305 A
news
RESEARCH PROFILES Multipurpose nanopore sensors nel blockages. An interaction between to the mean lifetime of the complex. The use of cell-like membrane channels Furthermore, “you can measure the to detect analytes in solution is appealing polymer and analyte can have one of concentration [of analyte] by measuring because such a scheme could, in principle, two effects. It may add enough bulk to the polymer to prevent the polymer how long it takes the complex to bind allow ready analysis with minimal samfrom entering the pore at all, which will to the [channel],” explains Kasianowicz. ple preparation. Several research teams register as long current plateaus. Never“If you have a lot of polymer that’s free have put together systems that rely on and only a few bound polymers, it might modifying naturally occurring, membrane- theless, there will still be some valleys take a minute to get the polymer to clog bound ion channels. Such approaches typ- because there will still be some free polymer, and the frequency of the valthe pore.” Such kinetic data also helps ically measure changes in the ionic curleys will indicate the concentration of ensure accuracy. “It should help distinrent through the nanometer-scale pore. free polymer—and by inference, the guish between the analyte and interferThe problem is that such systems are concentration of the analyte. ing molecules,” he adds. difficult to engineer. The structural eleThe other possibility is that the polyBecause the polymers can have unique ments of ion channels are not well unsignatures, the simultaneous measurederstood, making attempts to alter them mer will still enter the channel, though its added bulk will change the depth ment of multiple analytes is possible, even tricky at best. and frequency of the valleys. The durawith a single channel, says Kasianowicz. In the May 15 issue of Analytical tion of the current valley will correspond In one experiment, the researchers Chemistry (pp 2268–2272), John Kasfound that a bare poly(dT) polymer ianowicz and colleagues at the U.S. can be distinguished from a poly(dC) National Institute of Standards and Analyte-free or poly(dA) polymer because each Technology describe an advance that yields a distinctive pattern of plateaus should make such systems simpler to Current vs. time – + and valleys. (Poly[dG] was omitted build and more powerful in scope. because of solubility problems.) In The approach is deceptively simple. another experiment, the team detectRather than fusing a binding site died two analytes, avidin and an ␣-brorectly to the ion channel, the researchPolymer-induced blockades modeoxyuridine antibody, using a ers decided to fuse it to a polymer single ion channel. “Hopefully, that could thread freely through the + Analyte [we’ll] be able to use the same pore channel. They chose that “lovable to detect the concentration of many standby”, DNA, as the polymer, in analytes simultaneously,” Kasianowicz large part because it is easy to synadds. “Now the challenge is: Can we thesize and its properties have been design many different polymers to exhaustively studied. provide a large number of unique The approach offers an immediate Fewer blockades/time signatures?” dividend. “A much larger class of anModel I In time, Kasianowicz and others alytes can bind to a site on the polyhope to use channels composed of mer than to one in a very narrow materials other than ␣-hemolysin, nanoscale channel,” says Kasianowicz. which suffers from the usual frailties The simplicity carries with it surof proteins—instability at extremes prising power. Rather than the anaof temperature and pH and a distresslyte interacting with a modified bioing tendency to get chewed up by logical channel, the system is now Pore occluded Model II passing proteases. made up of the channel (in this case, for t ~ 1/koff ␣-hemolysin), the polymer, and the “Our goal [now] is to develop a analyte—and their interactions yield more robust system that minimizes Analytical sensor based on nanometer-scale pores useful diagnostic information. false-positive artifacts,” says Kasianoand polymers. (Top) In the absence of analyte, polyThe bare polymers thread in and wicz. “The methods we demonstrated mers thread freely through the pore, causing shortout of the channel slowly enough to here should be transferable to such lived dips in the ionic current. When an analyte is provide readily measurable “on/off” a new system and might ultimately bound to the polymer (middle), the polymer may be current measurements, in which prove useful in [various] sensing prevented from entering the pore or (bottom) may plateaus correspond to open channels applications.” occlude the pore for a period equal to the mean –Jim Kling and valleys indicate short-lived chantime that the analyte is bound to the polymer. 306 A
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 1
news
Putting a fine point on ESI High throughput and miniaturization are two fields that go hand-in-hand as researchers try to squeeze more assays from less material faster. Have we reached the limit, or are there still technological developments that can push the envelope? In the May 15 issue of Analytical Chemistry (pp 2183–2189), researchers at Delft University of Technology (The Netherlands) describe a method for dotting enzymes into arrays while preserving enzymatic activity. The researchers abandon the current methods of creating protein microarrays—“spotting” with pins, stamping, or spraying from piezoelectric dispensers. Instead, using electrospray ionization (ESI) in a novel way, this group, led by Gijs van Dedem, arrays biomaterials in microspots precisely and accurately. These results open the door for the miniaturization of a myriad of assays and could reduce the costs associated with biochemical and environmental screening. Previous work from the group at Delft and elsewhere had shown that ESI can be used to deposit biological materials onto substrates, rather than sending them flying through the air, as when ESI is used for MS. For example, other researchers have electrosprayed biological materials onto conductive surfaces using dielectric masks to create a desired pattern of spots (Anal. Chem. 1999, 71, 3110–3117). The Delft group takes a different approach. Using 150-µm-diam capillaries, they bring an ionized solution very close (within 400 µm) to a silicon nitride substrate. The electric field created by the proximity of the ionized fluid in the capillary to the surface causes the fluid to spray. Similarly, moving the substrate further from the tip stops the spraying instantly. Then the substrate is shifted sideways and lifted up again to deposit the next spot. Using this setup, the researchers vary the size of the spot by adjusting the flow rate—we’re talking pL/s here—and the
spray distance. They report spot sizes in the 130- to 350-µm range, with spacing of 400 µm. The spacing is tightly controlled by moving the substrate platform with an xyz stage.
Parallel spraying setup using three capillaries placed 600 µm apart. Dispensing multiple solutions simultaneously could eliminate some artifacts in enzyme assays.
Graduate student Robert Moerman, the paper’s first author, points out that this method provides reproducibility and accuracy not attainable with other methods. “The novelty is that we reduce the distance between the capillary tip and surface drastically, and therefore, obtain small spots,” he says. “It is indeed surprising that the liquid at the tip of the capillary is changed into a cone instantaneously, which is necessary to obtain reproducible spots.” In contrast, stabilizing the cone and spray takes 0.5–1 s when using a 600-µm-o.d. capillary, he adds. Another advantage of this approach, the authors say, is that it can be used to array multiple substances simultaneously. Many methods for creating arrays require that different solutions be deposited sequentially, which is fine in some cases. But if the array is to be used in enzymatic assays, leaving spots on the surface for different lengths of time could cause artifacts. Multiplexing can be done simply by
attaching groups of capillaries to the system (see figure) and spraying different solutions into different wells. “We think that a future spraying device will consist of a row or array of small nozzles [50- to 200-µm i.d] connected to a channel reservoir,” says Moerman. In earlier work, Moerman and colleagues arrayed relatively robust enzymes such as glucose oxidase and horseradish peroxidase. In the current work, they tackled some labile, metabolically important enzymes: lactate dehydrogenase, glucose-6-phosphate dehydrogenase, and pyruvate kinase. Initially, spraying these enzymes onto a liquid layer preserved all their activities, whereas spraying them onto a dry surface was damaging, presumably due to high salt concentrations and charge density as the liquid evaporated. However, the researchers found that by lowering the conductivity of the solution and adding mild detergent and the sugar trehalose, enzyme activity could be fully preserved. The investigators were surprised that certain surfactant–trehalose combinations preserved enzyme activity, Moerman says. “It appeared that low to moderate currents using low buffer concentrations allow the enzymes to survive upon landing,” he explains. “Combined with the very low speed of the liquid through the capillary, this is likely to be a very powerful tool for enzyme dispensing.” The Delft group is gearing up to measure glycolytic enzymes simultaneously on a subnanoliter scale, which will require accurately spraying 15 metabolites into 15 different wells. The researchers have developed a new method for adding a sample containing 15 different enzymes simultaneously while preventing evaporation during sample addition and kinetic measurements. In the works is luminescence detection technology for simultaneously and quantitatively measuring kinetics from the 15 different reactions. –Laura DeFrancesco
J U N E 1 , 2 0 0 1 / A N A LY T I C A L C H E M I S T R Y
307 A
news
MEETING NEWS News from the
221st ACS National Meeting—Elizabeth Zubritsky reports from San Diego, CA.
Flexible immunoassays on a chip
}
tether various antibodies, helping to orient them for binding with labeled antigens. Fluids on the chip were driven electroPutting immunoassays on microchips kinetically, which gave the assay considreduces reagent consumption and speeds erable flexibility, says Verpoorte. In parup analyses. Arash Dodge, Elisabeth ticular, the amount of sample could be Verpoorte, and colleagues at the Unicontrolled by varying the duration of versity of Neuchâtel (Switzerland) show the applied voltage from 30 to 300 s. that microchips also offer flexibility. The researchers miniaturized a hetero- Such control might be especially useful for competitive assays, because an overgeneous immunoassay, the most familiar example being the home pregnancy test. abundance of sample can swamp the binding sites and destroy the competitive quality of the assay. The incubation times, which ranged from
