Lab Profile: Collaboration pays off for NCSR - Analytical Chemistry

Lab Profile: Collaboration pays off for NCSR. David Bradley. Anal. Chem. , 2001, 73 (3), pp 70 A–72 A. DOI: 10.1021/ac0123882. Publication Date (Web...
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ANALYTICAL CURRENTS Bar codes for combinatorial libraries 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 Queensland (Australia) developed “colloidal bar coding”, in which the beads are encoded with 1–3-µm silica colloids bearing various 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 recombined into a single pool, and the

process repeats. In the end, the sequence of the compound attached to each bead is identified by the reporter particles on that bead. More recently, the researchers developed 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 attraction to the bead, and facilitate polymer bridging to the bead. The coating permanently adheres the reporter particles to the beads and reduces the likelihood that particles from one bead will contaminate other beads. Furthermore, by limiting the number of reporter particles that adhere to the bead during

each step, the researchers ensure that there is sufficient surface area available for later reporter particles. (Langmuir 2000, 16, 9709–9715)

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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 recombined into a single pool, and the process repeats.

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Zeroing in on cellular zinc Although Zn2+ seems to have important functions in the cell, much less is known about its

N N

H2N

HO

O

O

5 or 6-aminofluorescein

5 steps

COOH O O HO 5-substituted : ZnAF-1 6-substituted : ZnAF-2

Structure of the new Zn2+ sensor molecules, ZnAF-1 and ZnAF-2. 64 A

2+

which N,N,N´,N´-tetrakis(2-pyridyl-

cations. To study Zn better, Tetsuo Nagano

methyl)ethylenediamine is the Zn2+ acceptor.

and colleagues at the University of Tokyo

The Zn2+ complexes form immediately, and

(Japan) have developed highly selective Zn2+

the detection limits of these sensors are in

sensor molecules based on aminofluorescein,

the sub-nanometer range. In addition, the flu-

which are suitable for biological applications.

orescence intensities of the Zn2+ complexes

NH

N COOH

regulation than the regulation of many other

Although aminofluorescein alone does not

are nearly stable over the physiological

fluoresce, forming a cation complex can re-

range of pH values. Finally, the researchers

sult in fluorescence with a high quantum

note that ZnAF-1 is the first Zn2+ sensor mole-

yield. Thus, the researchers designed the

cule to distinguish between Cd2+ and Zn2+. (J.

Zn2+ sensor molecules ZnAF-1 and ZnAF-2, in

Am. Chem. Soc. 2000, 122, 12,399–12,400)

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ANALYTICAL CURRENTS Aggregating K+ sensor Here is a new strategy for selectively detecting ions—aggregating complexes. And, to enhance the spectral effects of aggregation, the complexes are linked to

Sweet new saccharide sensor Although amperometric sensors are widely used to monitor glucose, optical 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, concanavalin A (Con A), into a fluorescent sensor for saccharides. The first step in constructing the sensor is to synthesize a photoaffinitylabeling reagent, which is incorporated

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 observed 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 redshifted peak and diminished fluorescence. Swager and his collaborators attached the [15]crown-5 to various poly(p-phenylene ethylene) 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 interpolymer, ␲-stacked aggregates. The effect on the spectra was dramatic. With the most sensitive system they reported (a repeating unit consisting of a phenyl ring substituted with a methyl group followed by a phenyl linked to crown ether), a 1:0.5 crownether:K+ mole ratio (5.0 µM:2.5 µM) resulted in an 82% decrease in the fluorescence 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 intrapolymer complexes instead of the spectra-changing interpolymer aggregates. (Angew. Chem., Int. Ed. 2000, 39, 3868–3872)

into the sugar-binding pocket of Con A O

using photolabeling. The labeled Con

O

A is then treated to form a mercap-

O O O

tobenzyl site, which is subsequently

O

modified with a fluorescent iodoacety-

O O

O

K+

C10H21O O O O

C10H21O

O O

O O

O

O

R

IAEDANS-Con A is identical to that of

glucose derivatives. In addition, the

O O

C10H21O C10H21O

R

R

C10H21O C10H21O

O O O +O K O O O O O R O R R R

O C10H21O

native Con A, with mannose derivatives acting as stronger ligands than

O O O +O K O O O O O R O R

R

R

R

the final sensor, IAEDANS-Con A. The binding selectivity of

R

O

O

lated dansyl (IAEDANS) group, yielding

O

R

R C10H21O

R

All together now. A representation of what happens when K+ is added to a crown-ether-modified polymer. (Adapted with permission. Copyright 2000 Wiley-VCH Verlag GmbH.)

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 synthesis strategy may allow similar sensors to be developed using other saccharide-binding proteins. (J. Am. Chem.

Soc. 2000, 122, 12,065–12,066)

66 A

IMS combines with FT-ICR 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 spectrometer design is best? So far, IMS systems with quadrupole, magnetic-sector,

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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 transformion cyclotron resonance (FT-ICR) MS. FT-ICR has several advantages, such as high resolution and the absence of

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ANALYTICAL CURRENTS 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 differing by at least 5 orders of magnitude. The problem is made more difficult because the ICR and drift cells are inside the magnet bore. (On the other hand, placing the drift cell in the magnet reduces transverse diffusion and increases transmission efficiency.) Russell’s group solves the pressure

problem by dividing the instrument into cell pressure is determined to be a usefour regions and using differential pump- ful ~0.25 Torr. (Rev. Sci. Instrum. ing to create sections with different pres- 2000, 71, 4078–4085) sures. Aperture alignment is another problem they had to solve. The first studies with the new IMS/FT-ICR sys7 Tesla Superconducting Magnet tem are promisA B ing. Measured C D F H I TOF Region mobilities of Ar+ + Source Region and CO agree with literature valAnalyzer Region E G Drift Region ues, and the drift

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.)

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D

F

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J

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

databases. But sensitivity issues plague MALDI-TOF MS. Overcoming them requires manual steps with a method involving extensive incubation time for protein digestion. In the last issue of Analytical Chemistry (p 214), Thomas Laurell, György Marko-Varga, and colleagues at the University of Lund and AstraZeneca (both in Sweden) describe applications of a method they call “spot-on-a-chip”, which is 10–50 times more sensitive than current manual or robotic MALDI techniques. This method can perform analyses of 100 protein samples in 3.5 h. “It will be possible to analyze protein ex-

Automated MALDI-TOF

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Chip spotting. Microdispenser, MALDI target plate, and MALDITOF MS instrument.

pression at levels currently not possible, unless very careful and tedious enrichment procedures are employed,” says Laurell. The procedure begins with a sample digestion performed on a silicon microchip, 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 ~60-pL droplets into nanovials. 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 nozzle … can handle a wide range of fluids

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RESEARCH PROFILES with varying viscosities (up to 65 times the viscosity of water) and surface tensions…. This, in turn, means that we can handle virtually all fluids that we encounter 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 designed nanovials. After some experimentation, Laurell and his colleagues found that the best nanovial design is a shallow (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 solvent. Additional samples can be delivered to the chip to increase analyte con-

centration. “The rapid evaporation offers the very important feature of increasing the surface density of the analyte, 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 crystal 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” searching to find a good source in the well. “This is time-consuming and affects automation and throughput aspects negatively.” The team demonstrated spoton-a-chip enrichment by analyzing the

cytokine protein interleukin 8 from epithelial cells and the protein calumenin from adherent human fibroblasts. A 1-min digestion time was generally sufficient for the analysis, and the analysis 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-actuated 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. –James Kling

LAB PROFILE Collaboration pays off for NCSR Real technology transfer and commercialization are the products of collaboration between the National Centre for Sensor Research (NCSR; www.ncsr.ie) at Ireland’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 assurance, and environmental monitoring. It is based at DCU, where research involves everything from biomedical devices, biomaterials, and drug-delivery systems to gas sensors, automated monitoring equipment, and remote sensing. Director Brian MacCraith describes NCSR’s primary objective as “the development of a worldclass center of excellence in sensor research and the provision of significant resources to the sensor industry, both in Ireland and internationally.” The key is collaboration between academic research groups and industry. According to DCU’s Declan Raftery, the £9 million ($13.2 million) in fund70 A

ing from the Irish government to establish NCSR in July 1999 was part of a strategy to increase investment in Irish research and development (R&D). DCU’s track record with its 6-year-old Biomedical and Environmental Sensor Technology Centre and the DCU team’s multidisciplinary 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 repositioning of Irish science. “There has been a substantial reappraisal of the role of science, technology, and innovation in the Irish economy recently,” explains economist Aiden Kane of the National University 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 million] worth of state-of-the-art scientific equipment, fund a substantial expansion

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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 thinfilm and planar sensor fabrication laboratories; 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 gassensor testing and response testing systems, and they are using specially developed data-acquisition equipment for chemical, biochemical, and physical sensors. 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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LAB PROFILES represents the success of the Irish scientific community in persuading government 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 government has been running budget surpluses over the last number of years.”

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

“The Irish economy is performing extremely well,” adds Raftery. “In terms of [gross domestic product] growth in the last 6 years, Ireland has outperformed all other countries in the OECD [Organization for Economic Cooperation and Development].” As such, the Irish government has recognized that sustaining long-term growth means enabling multinational 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 endeavors by nurturing partnerships that could be crucial to the growth of smallscale 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 incentives, such as access to grants, in working 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 technologies and products. “Indeed,” adds Kane, “part of the attempt with this new funding is to link higher education research with the local economy.” Previously, there were few strong links between multinational companies and local economies in Ireland. “A vital part of the process is to increase significantly the investment in R&D in the universities, which it is hoped will help attract knowledge-intensive industries into Ireland 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. –David Bradley

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

GOVERNMENT AND SOCIETY British polymer scientists get enhanced service British academics supported by the government’s main funding body, the Engineering 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 polymers and provide “triple detection” gelpermeation chromatography (GPC) for obtaining more information about polymer branching and size, says Rapra principal consultant Steve Holding. Rapra, which specializes in the tech72 A

nology and business of rubber and plastics, 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 replacement and upgrading of equipment.” The most important development is the addition of triple detection to GPC, with organic solvents at near-ambient temperature. 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-

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hydrofuran and chloroform. This system gives measured molecular weights that are as close as possible to absolute, says Holding. The enhanced sensitivity could reveal subtle differences among samples that are not observed with conventional GPC. It will, for instance, provide information on branching and polymer size. Holding also says that aqueous GPC, which is much more difficult than GPC with organic solvents, will become available through the service. He adds that aqueous GPC will be phased in, concentrating initially on nonionic, processable, water-soluble polymers. –David Bradley