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ANALYTICAL CURRENTS A better glucose mousetrap Taking a cue from nature, Sylvia Daunert and her colleagues at the University of Kentucky describe a new approach to a glucose sensor that, unlike other detection schemes, uses no reagents. The key is a protein from E. coli, which is modified to undergo a change in fluorescence when the protein captures glucose. This protein could become the basis of a sensor for monitoring the health of diabetics. The E. coli galactose/glucose-binding protein (GBP) is normally involved in active transport and chemotaxis—the process by which bacteria move from or to a concentration of chemicals. GBP’s 309 amino acids take on an overall ellipsoidal shape that forms a sort of “mousetrap” for glucose. Three peptide strands act as a flexible hinge, connecting two different but similarly folded domains within the ellipsoid. Buried deep within the cleft formed by the two domains is a site where glucose binds.
This triggers the hinge, closing the protein trap as solvent molecules are expelled and causing the sugar to bind more tightly. To turn GBP into a sensing molecule, a single cysteine was incorporated near the mouth of the trap where the conformational changes would be greatest. A fluorophore that is sensitive to the local environment was attached to the cysteine site. Thus, when GBP trapped a glucose molecule, the fluorescence would change. The Kentucky researchers investigated three different cysteine locations and three environment-sensitive fluorophores. The best system uses a coumarin-based flurophore, which has the greatest signal change, 30% fluorescence quenching upon glucose binding, and a detection limit of 1 ⫻ 10–6 M. Response time is 5 min, but by sacrificing signal intensity, shorter times can be used. (Anal. Biochem. 2001, 294, 19–26)
Structure of GBP showing the three sites modified with cysteines, glucose (G) in the binding pocket, and a calcium ion (C). (Adapted with permission. Copyright 2001 Academic Press.)
Fractals add dimension to gold DNA probes Variations in fractal patterns are infinitely
modified oligonucleotide. These aggregate
as the number of bases in the oligo target
fascinating, and the principles have been
into a network as the probe attaches to a
string becomes longer. An increase in den-
applied to problems with variable condi-
complementary single-strand target oligo-
sity is reflected by an increase in Df as in-
tions. Now, J. Houston Miller and Glauco R.
nucleotide. And the interference between
traparticle cross-linking increases at higher
Souza of George Washington University
scattered electromagnetic waves from the
oligo-target concentrations. The researchers
apply angle-dependent light scattering and
particles within the fractal aggregate is a
believe that their method gives a better
fractal dimension (ADLS/FD) analysis to de-
factor in calculating the fractal dimension
picture of aggregate structure in solution
tect small structural variations as Au–DNA
(Df) of the aggregate, a value that is simul-
and is advantageous because it avoids
nanoparticles assemble into aggregates.
taneously sensitive to both the concentra-
harmful waste and is faster than previous
tion and length of an oligo target.
methods. (J. Am. Chem. Soc. 2001, 123,
The Au–DNA probe is a 100-nm gold nanoparticle covalently bonded to a thiol-
The researchers find that Df decreases
6734–6735)
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ANALYTICAL CURRENTS Silver nanoparticles look good in gold Gold nanoparticles are a hit. They can be used for sensitive, selective diagnostic methods, in part, because the gold particle surfaces are easily modified with various recognition elements such as DNA oligonucleotides. When the oligonucleotides bind a complementary DNA, A
B
I
II
I
II
the particles change color. On the other hand, silver nanoparticles modified with oligonucleotides tend to degrade under conditions needed to effect DNA hybridization. Too bad, because silver nanoparticles have a surface plasmon band with an extinction coefficient ~4 times greater than gold particles of the same size; moreover, III the plasmon band for silver falls into a different spectral region than the one for gold. Now, Chad Mirkin and his co-workers at Northwestern University describe III a synthetic procedure for
the best of both worlds—silver nanoparticles covered with a monolayer of gold. Their core–shell Ag/Au nanoparticles have the optical properties of the silver core but the surface chemistry of gold. Modifying the surface with alkylthiolcapped oligonucleotides resulted in stable particles under conditions of DNA hybridization. Mixing the core–shell nanoparticles with gold particles raises the possibility of two-color assay systems. In addition, the new synthetic approach may be applicable to other particles such as platinum, opening the door to wide-ranging combinations of optical properties. (J. Am. Chem. Soc. 2001, 123, 7961–7962)
(A) Core–shell Ag/Au nanoparticles (B) compared with 13-nm gold nanoparticles under conditions of (I) no surface modification, (II) with surface oligonucleotide, and (III) at 58 °C—above the melting temperature at which the DNA duplex comes apart.
Pollutant monitor—An environmental bargain What’s cheap, sensitive, and always on the
(DOAS)—to measure the elusive pollutant,
three separate units: the collection unit,
job? For these researchers, it’s their new
Wiesen’s group says it knows of no one
where the HONO from the sample gas
pollutant monitor. Peter Wiesen and col-
else who has introduced a sensitive, cheap,
flow is collected; the azo dye unit, in which
leagues at Bergische Universität-Gesam-
and compact on-line device. Each of the
a 0.8-mM n-(1-naphthyl)ethylenediamine-
thochschule Wuppertal (Germany) have
two channels of the LOPAP instrument has
dihydrochloride solution is injected to form
designed a new instrument that measures gaseous nitrous acid (HONO) in
the final azo dye; and the detection Gas inlet
Gas outlet
situ using wet chemical sampling and
tube as an absorption cell. R1
photometric detection. They say the
The group validated the instrument’s Stripping coils, debubbler
long path absorption photometer (LOPAP) is a compact, continuously working HONO monitor for ambient air
R2
Channel 2
phy and for 0.1- to 20-ppbV concentrations in a large outdoor smog chamber
Mixing volume
Mixing volume
for measurements of higher concen-
UV/VIS lamp
UV/VIS lamp
gases, and indoor environments. Although researchers have used
Teflon AF tubing
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Teflon AF tubing Waste
Waste Spectrometer
various techniques—such as differential optical absorption spectrometry
performance for HONO concentrations of 3 and 30 ppbV using ion chromatogra-
Channel 1
measurements in the troposphere or trations in smog chambers, exhaust
unit, which consists of a long Teflon
Schematic setup of the LOPAP instrument.
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using DOAS. They reached detection limits ranging from ~3 to 50 pptV, with response times from 4 to 1.5 min, respectively. Accuracy was in the 10–15% range. (Environ. Sci. Technol. 2001, 35, 3207–3212)
news
Wellpat a shape Patterning biomolecules on planar substrates is important for developing molecular and cellular biosensors and proteomic arrays. Previous methods often resulted in a low surface density of the immobilized biomolecule or could not easily create isolated structures. The new Wellpat method from Ashutosh Chilkoti and Jinho Hyun of Duke University overcomes these problems. Using poly(dimethylsiloxane) (PDMS), this group patterned several different shapes of elastomeric microwells and channels as small as 30 µm. These structures feature hydrophobic regions between hydrophilic wells, which encourages aqueous biomolecule solutions to enter and remain in the microwells. Forming patterns from aqueous solutions is only one advantage of the Wellpat method. Substrates as variable as polymers, gold, or glass can be used as
pattern supports. Varying the contact time between the biomolecule and the surface, unlike the microcontact printing technique, can change the concentration of the biomolecule. In contrast to the PDMS flow channel method,
3-in-1 Ion manipulator Many ion systems, like time-
+1000V
Solution
breaking down ion beams into small packets to function ef-
Ion beam
fectively. Edward Roberts and
Macor spacers
colleagues at the Australian National University under33Ω
stand the need for high-cur-
µwell
Master
Vbunch – 0 tp 100V +
spectrometers, so they develneously makes packets with a new kind of ion gating, com-
Substrate
presses the packets to higher density via bunching, and re-
µwell PDMS
Air HDT plasma adsorption
µwell
µwell
(b)
100µm
duces the voltage of the
Input
Sectional view of combined gating, bunching, and potential re-referencing unit. The chain of resistors, which set up the bunching field, and the re-referencing switch pairs are shown, but circuit details have been omitted for clarity. The variable bunching voltage, Vbunch, fluctuates the compression. (Adapted with permission. Copyright 2001 American Institute of Physics.)
packet to ground by potential µwell
Figure 1 (a)
33Ω
rent, low-divergence ion
oped one device that simulta-
PDMS
150kΩ 100Ω 100Ω 100Ω 100Ω 100Ω 100Ω
of-flight spectrometry, require
packets for photofragment Scheme 1
Wellpat can construct numerous isolated structures of different shapes, including circles and squares. Pattern materials include biotinylated peptides as well as many other types of biomolecules. (J. Am. Chem. Soc. 2001, 123, 6943–6944)
100µm
Scheme 1: Construction of elastomeric microwells. Figure 1: Optical micrographs of PDMS molds filled with an aqueous solution: (a) square microwells, (b) microchannels.
re-referencing, thus simplifying the design
A single field effect transistor is the main
of the necessary optics.
fast, high-voltage switch that reduces
Traditionally, ion-beam gating is
voltage from 1 kV to ground within a few
achieved when plates deflect a beam
nanoseconds and is ultimately responsi-
across an opening or through grids of op-
ble for the combined operation. Other
posing potentials. By deflecting an ion
photofragment spectrometers had sepa-
beam with two spaced, cylindrical lenses
rate sources for each function, but this
with small openings at different poten-
new design incorporates only one voltage
tials, these researchers successfully per-
supply, one digital signal, and has higher
formed ion gating with lensing and did not
flux density from a compact design. Pre-
introduce any divergence into the beam.
liminary experimental results of the simul-
The packets are compressed after
taneous functions agreed with modeled
passing through a series of charged alu-
data, but the instrument will require some
minum rings linked by resistors to de-
optimization. (Rev. Sci. Instrum. 2001, 72,
crease the number of rings needed.
2915–2922)
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ANALYTICAL CURRENTS New techniques to FRET over Jerker Widengren, Claus A. M. Seidel, and co-workers at the Max Planck Institute for Biophysical Chemistry (Ger(a)
...
trans δ+
µ δ+
N
N – SO3
– O3S
...
cis
– SO3
δ+ N
N δ+
– O3S (b)
many) show two new ways that fluorescence correlation spectroscopy (FCS) can characterize fluorescence resonance energy transfer (FRET). They also present a theory of the two approaches to measure FRET efficiencies. In the first method, the researchers determined FRET efficiencies from the detected acceptor fluorescence rate per molecule. They took advantage of FCS’s ability to determine the mean absolute number of fluorophores. In the second method, the group used FRET-dependent fluorescence fluctuations generated by the trans–cis isomerization of an acceptor dye to calculate effiencies. They demonstrated their technique by collect-
trans CH2
δ+ δ+
δ+
cis
δ+
(a) Charge distributions for acceptor fluorophore Cy5 in a trans and cis conformation, with µ being the net electric dipole moment in the trans form. (b) Possible spatial arrangement of the Cy5 molecule in a trans and cis conformation with respect to the linker arm and DNA molecule.
ing FCS data on a series of double-stranded DNA molecules, with the number of base pairs between the donor and acceptor fluorophores varying from 4 to 22. Other researchers had theorized as far back as 1984 that FCS could be used to monitor intramolecular dynamics by changes in FRET efficiencies. But the research team says its method to measure FRET efficiencies via the trans–cis isomerization kinetics is completely new. The rate of interchange between the trans and cis states, as measured by FCS, was found to be proportional to the excitation rate and can be used as a direct measure of the FRET efficiency. An important advantage in using trans–cis fluctuations to monitor FRET is that the relative population of the cis state remains more or less constant over the whole range of excitation rates, say the researchers. The measured isomerization rate depends only on the fluctuations in the acceptor fluorescence. (J. Phys. Chem. A 2001, 105, 6851– 6866)
A word of caution In electrospray (ES) ionization experiments, the geometric arrangement and composition of an electrode apparently have little effect on the subsequent mass spectrum of many analytes. However, Gary Van Berkel and his colleagues at Oak Ridge National Laboratory propose that the oxidation reactions that supply positively charged ES droplets can influence the spectrum. Using an ES emitter fitted with a wire working electrode submerged in a sample solution, the group found that the pH of an aqueous solution decreased with the oxidation of water at platinum or stainless steel electrodes. And the hydrogen ions generated could interfere with the formation of analyte ions and their abundance in a mass spectrum, especially for compounds with acidic or basic sites. The metal electrodes also corroded.
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Iron, stainless steel, platinum, and copper were oxidized, forming metal ion complexes with 1,10-phenanthroline in acetonitrile, as shown by MS. This could be an advantage for studies of metal ligand chemistry or as a method of ionizing molecules by forming a metal ion complex. Neutral nickel(II) octaethylporphyrin generated an increasingly complex product spectrum during a 25-min oxidation. The good news is that ionizing a neutral molecule makes it detectable. The time that reaction products diffuse through the solution and mix with the analyte influences the extent of oxidation effects on the mass spectrum. Other conditions that contribute to the effect are the solvent, ES current, flow rate, electrode material, and chemical properties of the analyte. The result is a timedependent change in the mass spectrum.
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Time-lapsed photographs of a nano-ES capillary containing the 1,10-phenanthroline iron(II) indicator after applying 900 V to the iron electrode in solution. (Adapted with permission. Copyright 2001 Elsevier Science.)
The authors conclude that the changes in solution composition cannot be determined by a simple calculation from ES current and solution flow rate. (J. Am. Soc. Mass Spectrom. 2001, 12, 853–862)
news
RESEARCH PROFILES Internet algorithm tracks microorganisms Besides ferreting out tiny disease-causing “beasts” for food safety and medical diagnostics, foiling terrorists is also a major goal behind developing a rapid, easy, and accurate system to identify microorganisms. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS offers the best prospect for an autonomous, suitcasesize, portable system that can rapidly tell people in the field if they are exposed to biological warfare agents. In the current issue of Analytical Chemistry (pp 4566–4573), Plamen Demirev and Catherine Fenselau of the University of Maryland, along with Jeffrey Lin and Fernando Pineda of Johns Hopkins University, improve the accuracy of their MALDI-TOF analysis and database search algorithm to identify microorganisms. Many microorganism identifications follow the traditional path of cell lysis, proteolysis, peptide analysis, and a fingerprint mass spectrum. But Demirev says that mass spectra for the same organism can be different with different sample preparations, matrixes, and spectrometers. He and his colleagues avoid these complications and the need for a matching fingerprint pattern by finding masses of protein biomarkers in intact unknown organisms. They then apply their software algorithm, with a significance testing component, to search Internet protein databases. “In our case, we’re not interested in individual proteins,” says Demirev. “Instead of taking hours or days to identify proteins, for us it’s sufficient to assume that we’re dealing with a single organism.” A prerequisite for their approach, says Demirev, “is that all potentially expressible proteins should be in the database. I think that a few months ago the number of [microorganisms whose genomes are sequenced in public databases] was around 80, and it’s increasing constantly. So at some point, we believe that the approach will be actually practical.” The researchers chose to test their Web algorithm on Helicobacter pylori
(strain 26695) as a model organism. H. pylori is a gram-negative bacteria, estimated to be present in half the world’s population and implicated in causing gastric ulcers and cancer. Also, its genome, unique set of biomarkers, and amino acid sequences are well established. Comparing positive and negative ion mode spectra for the same sample provided additional information. For H. pylori, the authors concluded that most positive Query database with observed mass
No
Is there a hit?
Yes Match observed mass to a database protein Check N-terminal AA sequence of matched protein Are PTM rules fulfilled?
Yes
Count the match and continue search
No ADD 131 to observed mass for “new” observed mass
Tentative flowchart for including N-terminal methionine cleavage in a proteome database search algorithm.
mode peaks are singly charged individual protein biomarkers. Another doublemode advantage comes from recalibrating. First, the researchers choose two intense peaks within 2 kDa of each other in one mode. From the second mode, they choose an equivalent doublet of peaks that are close in mass to the first pair. Recalibrating with sets of doublets together with averaging masses from both modes improves mass accuracy by a factor of 2. Demirev says that analysis only from molecular weight “is rather risky,” but the ability to discriminate between false and true matches for a microorganism increases with better mass accuracy. The search algorithm ranks possible organisms by the number of “hits”—the
number of protein biomarker masses that match biomarkers for the unknown sample. However, the team found that ranking alone sometimes leads to misidentification because larger microorganisms have more expressible proteins and tend to produce more hits. Significance level testing, the probability of random hits, levels the playing ground and is influenced by factors such as the proteome size, the number of detected and matched biomarker peaks, and the mass accuracy. A higher value for the significance level indicates a greater probability of mismatches rather than matches, and a lower value indicates a better match. Even if there are fewer hits, on a scale of 0–1, a significance level closer to zero means that the hits are less likely to be random—thus, a more probable identification of the correct microorganism. Their research on H. pylori and E. coli illustrates these principles. But there is yet another refinement that improves the significance level. In matching biomarkers, Demirev says they noticed that post-translational modifications (PTM) were not always included. They then “looked one step further into the database,” he says. The simplest PTM for bacteria and other prokaryotes is “chopping off the N-terminal methionine,” he says. Other researchers have defined rules for this cleavage in relation to amino acid sequences, so Demirev and his colleagues incorporated an iterative step into their search algorithm. They adjust the mass of the biomarker by 131 Da for methionine and repeat the search. This decreased the significance level for H. pylori from 0.036 to 0.002, upping the chances for accuracy. Possibilities for the future include incorporating rules for other PTMs as information becomes available, or using tandem MS to get sequence tags for intact proteins. Demirev notes that his colleagues are looking at the “other side of the mirror,” the statistical probability of true, rather than false, matches. a —Judith Handley
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RESEARCH PROFILES PEBBLEs measure O2 in living cells About five years ago over a steak dinner at home, Raoul Kopelman talked about his new optical nanosenors with his then 20-something-year-old son, Leeron, but his tongue kept tripping over the words describing them. So, Leeron told his dad to just call them “PEBBLEs.” Today, PEBBLE (probes encapsulated by biologically localized embedding) continues to have a cute ring to it, but it also has serious possibilities in biology and medicine. In the September 1 issue of Analytical Chemistry (pp 4124–4133), Kopelman and colleagues at the University of Michigan–Ann Arbor, present the first sol–gel-based, ratiometric PEBBLEs to do real-time measurements of oxygen in living cells—rat C6 glioma cells. The researchers also describe the fabrication of the sensors using a custom method in which the sensor size distribution and other properties are modified by adding
poly(ethylene glycol) (PEG). “As long as the cell is alive, it will use oxygen,” says Koppelman. “So, the amount of oxygen in a cell is an indicator of what is happening.” For example, if there is a cancer cell that’s growing too fast, it may be using more oxygen than the other cells. The group used a “gene gun” to blast tiny 50- to 300-nm PEBBLE sensors into the cell. “Because everything is so small, it’s easy for the membrane to heal and close back for the cell to live,” Kopelman says. Unlike free dyes, the sol–gel PEBBLE matrix significantly reduces the toxicity of the indicator and reference dyes to the cells so that a variety of dyes can be used. The matrix also protects the fluorescent dyes from interference by proteins in the cell, which allows for reliable in vivo chemical analysis, says Kopelman’s team. The probes (a) Nomarski illumination of sol– contain an oxygel PEBBLEs in C6 glioma cells. gen-sensitive fluo(b) Oregon Green fluorescence rescent indicator, of PEBBLEs. (c) [Ru(dpp)3]2+ fluoRu(II)–tris(4,7rescence of PEBBLEs. (d) Overdiphenyl-1,10laid images of (a) and (b). (e) phenanthroline) Overlaid images of (a) and (c). ([Ru(dpp)3]2+), and a reference oxygen-insensitive fluorescent dye, Oregon Green 488-dextran, for ratiometric intensity measurements. They have excellent reversibility, dynamic range, and stability to leaching and photobleaching. The group also says the PEBBLEs have a response time in the millisecond range due to their spherical shape. In the begin-
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ning, the research went smoothly, but problems making the sol–gel PEBBLEs started a year ago when graduate student Hao Xu tried using the Stöber method. The process often resulted in clumps of particles. “Instead of those little nanoparticles being nice little spheres, they started aggregating, playing games,” recalls Kopelman. “You get a big stone instead of a lot of little particles.” Desperate, Kopelman added PEG MW 5000 monomethyl ether, a steric stabilizer, which reduced the particle diameter to the 0.1- to 0.6-µm range. The PEG also improved the sensor performance in oxygenated water. The sol–gel PEBBLEs are just the most recent version of PEBBLE sensors from Kopelman’s lab. His group has fabricated PEBBLEs for measuring calcium, magnesium, and pH levels. Kopelman began working on his PEBBLE concept in 1996 during a brainstorming session as he, along with neurotoxicologist Martin Philbert and graduate students, began writing a U.S. Army grant proposal. Then graduate student Heather Clark, now a postdoc at the University of Connecticut, started the project. At first, Kopelman had suggested they prepare the PEBBLEs—which are made of polymers—by making big chunks of the material and grinding them up. Clark, however, proposed preparing the PEBBLEs through microemulsion polymerization, which worked much better, he says. The team’s goal is to make very selective chemical indicators for cells, and they’re looking at putting a protein or enzyme into the sol–gel PEBBLEs, says Kopelman. The nice part about the research is that it’s “soft chemistry”, all done at room temperature and essentially in a test tube. Although there’s “no heroic chemistry” involved, one has to do things right, Kopelman says. “It’s like baking a cake. If you don’t have some experience, just using the recipe may not be enough.” a —Cheryl M. Harris
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MEETINGS News from the 4th Annual Foodborne —Laura Ruth reports from St. Pete Beach, FL
Counting bugs
CDC/PEGGY S. HAYES
This past September, the U.S. Department of Agriculture’s Agricultural Marketing Service (AMS) Microbiological Data Program (MDP) began collecting food samples in 10 states to analyze pathogens and indicator organisms on fresh fruits and vegetables, according to Anita Okrend, the branch chief for AMS. The
Pathogen Analysis Conference
data will establish benchmarks to assess potentially harmful foodborne microorganisms; provide uniform procedures for sampling, testing, and reporting; and provide data for decision-making purposes to federal agencies such as the Centers for Disease Control and Prevention and the Food and Drug Administration, state public health agencies, private industry, and other interested parties. The MDP began as a pilot program in January 2001 and is associated with the better-known Pesticide Data Program (PDP), which collects 600–700 samples from 10 states with populations representing ~50% of the United States. Several of the test states serve as food distribution centers. The MDP can be run at a reasonable cost, says Okrend, because it uses the same samples collected for the PDP. According to Okrend, routine and easy-to-use laboratory testing methods are necessary for the MDP samples. This year, the MDP will be testing for generic E. coli and Salmonella. Culturing is being used, together with the most probable number method, to count E. coli, which is believed to be an indicator of fecal contamination. Salmonella will initially be identified by immunoassay and then confirmed by culturing. Testing for
Shigella, naturally found in the human intestine and another indicator of contamination, is expected to begin in 2002. At that time, the AMS expects an easyto-use polymerase chain reaction kit to be complete. The MDP baseline data is published online at http://www.ams. usda.gov/science/mdp.
Corrections The August 1, 2001 news story on division awards (p 415 A) contained two errors. The award for Spectrochemical Analysis was presented to M. Bonner Denton at the National ACS Meeting in Chicago, IL. Stanley Crouch’s biography should have read: Stanley Crouch, emeritus
professor at Michigan State University and adjunct professor at Arizona State University, will receive the Award in Chemical Instrumentation sponsored by the Dow Chemical Foundation.
2002 Pittsburgh Conference Memorial National College Grant Program The Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy and its cosponsoring technical societies, the Spectroscopy Society of Pittsburgh and the Society for Analytical Chemists of Pittsburgh, announce the 2002 Pittsburgh Conference Memorial National College Grants (PCMNCG) Program. Grants, with a maximum of $9000 each, will be awarded to the science departments of 12 small colleges. The grants are intended for the purchase of
Crouch retired from MSU in 2000, but
scientific equipment, audiovisual or other teaching aids, and/or library materials for use in the teaching of science at the undergraduate level. Announcement of the awards will be made by February 2002. For more information on the grants and submission deadlines, contact Hyman Schultz, The Pittsburgh Conference, PCMNCG, 300 Penn Center Blvd., Ste. 332, Pittsburgh, PA 15235-5503 (412-825-3220, ext. 189, fax 412-8253224, schultzh©pittcon.org).
did research and published in the areas of kinetic methods of analysis, flow analysis methods, computerbased chemical instrumentation, and analytical spectroscopy. Crouch continues to write textbooks and related technical materials. a
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