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When synthesizing combinatorial libraries on beads, fabricating many compounds is the easy part. Plucking out a single bead and determining what’s attached is more difficult. Chemical tags can be covalently attached to the beads, but that requires extra reaction steps. So Matt Trau and colleagues at the University of Queens-land (Australia) developed “colloidal bar coding”, in which the beads are encoded with 1–3-µm silica colloids bearing vari-ous fluorescent “reporter” particles.

Using the bar coding technique, which has been reported previously, 6 fluorescent dyes can encode 16 million compounds during the “split-and-mix” synthesis of a combinatorial library. First, 6 fluorescent dyes are mixed and matched to create 64 types of reporter particles. Then, split-and-mix synthesis begins. The pool of beads is divided into groups. Each group is coated with a unique reporter particle, and a unique monomer—for example, a single amino acid—is attached. Thus, each reporter particle corresponds to a particular amino acid. Next, the groups are re-combined into a single pool, and the

process repeats. In the end, the se-quence of the compound attached to each bead is identified by the reporter particles on that bead.

More recently, the researchers devel-oped a method to robustly attach the reporter particles to the beads. Before library synthesis, the particles are coated with polyelectrolytes, which enhance the surface charge, promote electrostatic at-traction to the bead, and facilitate poly-mer bridging to the bead. The coating permanently adheres the reporter parti-cles to the beads and reduces the likeli-hood that particles from one bead will contaminate other beads. Furthermore, by limiting the number of reporter par-ticles that adhere to the bead during

each step, the researchers ensure that there is sufficient surface area available for later reporter particles. (Langmuir 2 2000000,,16,9709–9715)

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A B C D Colloidal bar coding during “split-and-mix”

synthesis of a combinatorial library. (a) The pool of beads is divided into groups. (b) Unique fluorescent “reporter” particles are attached to the beads in each group. (c) Unique monomers are attached to the beads in each group. (d) The groups are re-combined into a single pool, and the process repeats.

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ANALYTICAL CURRENTS

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Although Zn2+seems to have important func-tions in the cell, much less is known about its

regulation than the regulation of many other cations. To study Zn2+better, Tetsuo Nagano and colleagues at the University of Tokyo (Japan) have developed highly selective Zn2+ sensor molecules based on aminofluorescein, which are suitable for biological applications.

Although aminofluorescein alone does not fluoresce, forming a cation complex can re-sult in fluorescence with a high quantum yield. Thus, the researchers designed the Zn2+sensor molecules ZnAF-1 and ZnAF-2, in

which

N,N,N´,N´-tetrakis(2-pyridyl-methyl)ethylenediamine is the Zn2+acceptor. The Zn2+complexes form immediately, and the detection limits of these sensors are in the sub-nanometer range. In addition, the flu-orescence intensities of the Zn2+complexes are nearly stable over the physiological range of pH values. Finally, the researchers note that ZnAF-1 is the first Zn2+sensor mole-cule to distinguish between Cd2+and Zn2+. (J. Am. Chem. Soc.22000000,,122,12,399–12,400)

O O HO COOH H2N 5 steps O O HO COOH NH N N N

5 or 6-aminofluorescein 5-substituted : ZnAF-1 6-substituted : ZnAF-2

Structure of the new Zn2+sensor molecules, ZnAF-1 and ZnAF-2.

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6 66 AA A N A LY T I C A L C H E M I S T R Y / F E B R U A R Y 1 , 2 0 0 1

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Here is a new strategy for selectively de-tecting ions—aggregating complexes. And, to enhance the spectral effects of aggregation, the complexes are linked to

a polymer backbone. This novel approach is outlined by Timothy Swager and his colleagues at the Massachusetts Institute of Technology. According to the authors, the general principle behind this approach is similar to recognition events often ob-served in biological systems.

The complex used in this study is the crown ether, [15]crown-5. With Li+or Na+ions, the 15-member ether forms 1:1 complexes that show little change in UV–vis or fluorescent spectra. However, K+binds the crown ether in a 1:2 ratio, with the metal ion bridging two ligands. The resulting complex shows a new red-shifted peak and diminished fluorescence.

Swager and his collaborators attached the [15]crown-5 to various poly(pphen -ylene eth-ylene) backbones. The polymer backbones differed by the substituents attached to the phenyl unit and the spacing of the repeating crown ether. When K+was absent, the polymer chains were randomly oriented, but in

the presence of the metal ion and with the right polymer, the polymer chains were knitted together by K+to form in-terpolymer, _␲-stacked aggregates.

The effect on the spectra was dra-matic. With the most sensitive system they reported (a repeating unit consist-ing of a phenyl rconsist-ing substituted with a methyl group followed by a phenyl linked to crown ether), a 1:0.5 crown-ether:K+mole ratio (5.0 µM:2.5 µM) resulted in an 82% decrease in the fluo-rescence peak. Polymer length also had an effect, with longer chains being somewhat more sensitive.

Interestingly, the polymer system with crown ethers on every repeating phenyl group failed to respond to any of the alkali ions studied. The authors speculate that this polymer formed in-trapolymer complexes instead of the spectra-changing interpolymer aggre-gates. (Angew. Chem., Int. Ed. 22000000,, 39,3868–3872)

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Recent studies have shown that marrying ion mobility spectrometry (IMS) with a MS detector is a powerful combination for studying inter- and intramolecular interactions, such as protein folding and ligand binding. But which mass spec-trometer design is best? So far, IMS sys-tems with quadrupole, magnetic-sector,

and ion trap mass spectrometers have been developed. In this paper, David Russell and co-workers at Texas A&M University construct an instrument that combines IMS with Fourier transform-ion cyclotron resonance (FT-ICR) MS. FT-ICR has several advantages, such as high resolution and the absence of

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ANALYTICAL CURRENTS

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Although amperometric sensors are widely used to monitor glucose, opti-cal detection methods for saccharides are still desirable. Now, Itaru Hamachi and colleagues at Kyushu University (Japan) describe a general method for turning a lowly lectin protein, con-canavalin A (Con A), into a fluorescent sensor for saccharides.

The first step in constructing the sensor is to synthesize a photoaffinity-labeling reagent, which is incorporated into the sugar-binding pocket of Con A using photolabeling. The labeled Con A is then treated to form a mercap -tobenzyl site, which is subsequently modified with a fluorescent iodoacety-lated dansyl (IAEDANS) group, yielding the final sensor, IAEDANS-Con A.

The binding selectivity of IAEDANS-Con A is identical to that of native Con A, with mannose deriva-tives acting as stronger ligands than glucose derivatives. In addition, the binding affinities reported for IAEDANS-Con A are just slightly lower than those cited in the literature for native Con A. Finally, the researchers note that the generality of the synthe-sis strategy may allow similar sensors to be developed using other saccha-ride-binding proteins. (J. Am. Chem. Soc.22000000,,122,12,065–12,066)

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the radio frequency heating problems that often plague Paul trap systems. However, like all good marriages, this one also involves some compromises. Most important, the ion mobility region and the ICR cell require pressures dif-fering by at least 5 orders of magnitude. The problem is made more difficult be-cause the ICR and drift cells are inside the magnet bore. (On the other hand, placing the drift cell in the magnet re-duces transverse diffusion and increases transmission efficiency.)

Russell’s group solves the pressure

problem by dividing the instrument into four regions and using differential pump -ing to create sections with different pres-sures. Aperture alignment is another problem they had to solve.

The first stud-ies with the new IMS/FT-ICR sys-tem are promis-ing. Measured mobilities of Ar+ and CO+agree with literature val-ues, and the drift

cell pressure is determined to be a use-ful ~0.25 Torr. (Rev. Sci. Instrum. 2

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ANALYTICAL CURRENTS

7 Tesla Superconducting Magnet TOF Region B C D F H I E G Drift Region Analyzer Region Source Region D C F H E G J A

Instrument schematic. (A) Filament assembly, (B) gold seal flange, (C) two-section ICR cell, (D) analyzer trap plate, (E) ion gate, (F) drift cell, (G) tube, (H) Tyndall gate, (I) wire-ion guide, and (J) detector. (Adapted with permission. Copyright 2000 American Institute of Physics.)

RESEARCH PROFILE

MALDI time-of-flight (TOF) MS has become a common technique for pro-tein identification, allowing researchers to digest proteins with enzymes and then, with the MS data, generate a unique peptide map that can be com-pared with patterns in DNA and protein

databases. But sensitivity issues plague MALDI-TOF MS. Overcoming them requires manual steps with a method in-volving extensive incubation time for protein digestion.

In the last issue of Analytical

Chem-istry(p 214), Thomas Laurell, György

Marko-Varga, and colleagues at the University of Lund and AstraZeneca (both in Sweden) describe applica-tions of a method they call “spot-on-a-chip”, which is 10–50 times more sensitive than current manual or ro-botic MALDI techniques. This method can perform analyses of 100 protein sam-ples in 3.5 h. “It will be pos-sible to analyze protein

ex-pression at levels currently not possible, unless very careful and tedious enrich-ment procedures are employed,” says Laurell.

The procedure begins with a sample digestion performed on a silicon micro -chip, with etched channels and digestion enzymes immobilized on the surface of the silicon. From the digestion microchip, the sample is piped into a flow-through piezo-actuated microdispenser, which can deliver ~60pL droplets into nano -vials. Laurell originally developed the system for on-line picoliter sampling of a continuous-liquid-flow system.

The microdispenser contains a “push bar” linked to a piezoelement. When a pulse is applied to the piezoelement, the pushbar pushes into a flowchannel, and the resulting pressure forces a droplet into a micronozzle. The flow for any liquid can be optimized by varying the parameters of the pulse, says Laurell. “The well-defined microfabricated noz-zle … can handle a wide range of fluids

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Chip spotting. Microdispenser, MALDI target plate, and MALDI-TOF MS instrument.

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with varying viscosities (up to 65 times the viscosity of water) and surface ten-sions…. This, in turn, means that we can handle virtually all fluids that we en-counter in life science applications.” It also has the advantage of not heating the sample.

From the micronozzle, the sample is shot onto a chip containing specially de-signed nanovials. After some experimen-tation, Laurell and his colleagues found that the best nanovial design is a shal-low (20-µm deep) well with a 300 _⫻ 300-µm square surface and an inverted pyramid shape for the bottom.

The chip contains 100 vials, which are heated to 45 ºC to drive off the sol-vent. Additional samples can be deliv-ered to the chip to increase analyte

con-centration. “The rapid evaporation of-fers the very important feature of in-creasing the surface density of the ana-lyte, which, in turn, gives us the ability to analyze low abundant compounds and achieve automation at a higher throughput,” says Laurell.

Well-defined sample and matrix crys-tal layers form in the vial, which makes for reproducible spectra, says Laurell. Because the vial locations are fixed and the vial’s surface is identical to that of the laser spot, the MALDI instrument isn’t required to go “hot-spot” search-ing to find a good source in the well. “This is time-consuming and affects au-tomation and throughput aspects nega-tively.” The team demonstrated spot-on-a-chip enrichment by analyzing the

cytokine protein interleukin 8 from ep-ithelial cells and the protein calumenin from adherent human fibroblasts.

A 1-min digestion time was generally sufficient for the analysis, and the analy-sis time of 3.5 h for 100 samples could be shortened by running additional microchips in parallel, Laurell notes. Moreover, with its ability to handle a wide range of liquids, the piezo-actuat-ed microdispenser could also find wide application in automated systems. “Other microdispensing techniques can be automated, but it is very difficult to incorporate them as an on-line device in a flow system, which is the base for a vast variety of analytical methods,” says Laurell.

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–JJaammeess KKlliinngg

RESEARCH PROFILES

LAB PROFILE

Real technology transfer and commercial -ization are the products of collaboration between the National Centre for Sensor Research (NCSR; www.ncsr.ie) at Ire-land’s Dublin City University (DCU) and local industry.

NCSR is a one-and-a-half-year-old organization charged with developing chemical and biological sensors to solve problems of societal concern, including medical diagnostics, food quality assur-ance, and environmental monitoring. It is based at DCU, where research involves everything from biomedical devices, bio -materials, and drug-delivery systems to gas sensors, automated monitoring equip-ment, and remote sensing. Director Brian MacCraith describes NCSR’s primary objective as “the development of a world-class center of excellence in sensor re-search and the provision of significant resources to the sensor industry, both in Ireland and internationally.” The key is collaboration between academic re-search groups and industry.

According to DCU’s Declan Raftery, the £9 million ($13.2 million) in

fund-ing from the Irish government to estab-lish NCSR in July 1999 was part of a strategy to increase investment in Irish re-search and development (R&D). DCU’s track record with its 6-year-old Biomed-ical and Environmental Sensor Technol-ogy Centre and the DCU team’s multi-disciplinary strengths led to a successful bid for this massive funding.

Such investment, which is part of wider governmental funding of £1 billion ($1.47 billion), represents a reposition-ing of Irish science. “There has been a substantial reappraisal of the role of sci-ence, technology, and innovation in the Irish economy recently,” explains econ-omist Aiden Kane of the National Uni-versity of Ireland–Galway

(www.nuigalway.ie/ecn/staff/kane/ec3 01/ireland/sources). “In particular, in 1999, the government set up a number of funds to enhance the science base and R&D.”

“The NCSR money is being used to purchase over £4 million [$5.86 mil-lion] worth of state-of-the-art scientific equipment, fund a substantial expansion

of the research staff, and [construct] a new custom-designed research facility,” Raftery told Analytical Chemistry. “It is expected that, [in] 2001, we will have a team of 130 researchers working in the center,” he adds.

Among NCSR’s specialties are thin-film and planar sensor fabrication labora-tories; specialist equipment for surface, material, and interface characterization and analysis; NMR; electron, scanning tunneling, and atomic force microscopy; and ion chromatography. NCSR teams are also working on environmental gas-sensor testing and response testing sys-tems, and they are using specially devel-oped data-acquisition equipment for chemical, biochemical, and physical sen-sors. Optics technology and micro total analysis systems (µTAS) for water- and air-quality monitoring and biomedical applications are also being given the NCSR treatment.

So, is increased funding a sign of a booming Irish economy and a peace dividend on the Emerald Isle? Not quite, says Kane. “The funding mainly

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represents the success of the Irish scien-tific community in persuading govern-ment of the importance of investing in this area in order to assure continued economic success,” he says. “It is partly a consequence also of the very healthy state of the public finances: The govern-ment has been running budget surplus-es over the last number of years.”

“The Irish economy is performing ex-tremely well,” adds Raftery. “In terms of [gross domestic product] growth in the last 6 years, Ireland has outperformed all other countries in the OECD [Orga-nization for Economic Cooperation and Development].” As such, the Irish gov-ernment has recognized that sustaining long-term growth means enabling multi-national companies with a presence in Ireland to move into R&D instead of being simply footloose manufacturing facilities exploiting Irish tax breaks.

NCSR is perhaps helping to drive this economic upturn through its en-deavors by nurturing partnerships that could be crucial to the growth of small-scale industry. The teams work closely with a variety of local and international firms whose facilities in Ireland may not be set up to fully research and develop prototype processes for new devices and systems, or who may see other incen-tives, such as access to grants, in work-ing with an academic partner. Yorkshire Water, Analog Devices, Glanbia, and the Irish Sea Fisheries Board are among NCSR’s partners, and the center’s ClearCense water-quality (turbidity and

color) monitoring device, developed with Siemens Environmental (U.K.), is already commercially available.

Such links and technology transfer ultimately help manufacturers develop their own expertise in novel sensor tech-nologies and products. “Indeed,” adds Kane, “part of the attempt with this new funding is to link higher education research with the local economy.” Previ-ously, there were few strong links be-tween multinational companies and local economies in Ireland. “A vital part of the process is to increase significantly the investment in R&D in the universi-ties, which it is hoped will help attract knowledge-intensive industries into Ire-land and encourage existing facilities to develop R&D capabilities,” says Raftery. Funding initiatives, collaboration, and true innovation should help push the Irish economy further up the European league ladder.

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–DDaavviidd BBrraaddlleeyy

For additional information, see the Irish National Policy and Advisory Board for Enterprise, Trade, Science, Technology, and Innovation Web site

LAB PROFILES

GOVERNMENT AND SOCIETY

The ClearCense probe is a product of an NCSR and Siemens Environmental collaboration.

British academics supported by the gov-ernment’s main funding body, the Engi-neer ing and Physical Sciences Research Council (EPSRC), will have access to much- improved instrumentation and techniques for polymer analysis, thanks to the renewal of EPSRC’s contract with the polymer company Rapra Technology. The new techniques available through Rapra’s polymer characterization service will allow analyses of water-soluble poly-mers and provide “triple detection” gel-permeation chromatography (GPC) for obtaining more information about poly-mer branching and size, says Rapra prin-cipal consultant Steve Holding.

Rapra, which specializes in the

tech-nology and business of rubber and plas-tics, will provide polymer characterizations to U.K. academics (who have EPSRC grants) for at least the next 3 years, with a possible extension to 5 years. The main technique offered by Rapra is GPC.

According to Holding, “Part of the new agreement includes provision for re-placement and upgrading of equipment.” The most important development is the addition of triple detection to GPC, with organic solvents at near-ambient temper-ature. A combination of right-angle laser light scattering, viscosity, and refractive index detection, provided by a detector array, will be used to glean information from polymer samples analyzed in tetra

-hydrofuran and chloroform.

This system gives measured molecular weights that are as close as possible to abso lute, says Holding. The enhanced sensitivity could reveal subtle differences among samples that are not observed with conventional GPC. It will, for in-stance, provide information on branch-ing and polymer size.

Holding also says that aqueous GPC, which is much more difficult than GPC with organic solvents, will become avail-able through the service. He adds that aqueous GPC will be phased in, concen-trating initially on nonionic, processable, water-soluble polymers.

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–DDaavviidd BBrraaddlleeyy