Analytical Currents: Clear, but not clean water

In DNA arrays, PCR is increasingly seenas an undesirable complexity. Now, So-Jung Park, T. Andrew Taton, and ChadMirkin at Northwestern University de-scribe a DNA array assay that does notrequire amplification or temperature-dependent hybridization to identifymismatched targets.

The new assay relies on Mirkin’sstrategy of labeling one end of thetarget DNA with gold nanoparticles.When the target DNA binds to probeDNA immobilized between two elec-trodes, the gold nanoparticles fill thegap between the electrodes. In princi-ple, the amount of target DNA can bedetermined by capacitance or conduc-tivity measurements, but in practice,the sensitivity is improved if the goldnanoparticles are enhanced with a silvercoating. The coating is applied by im-mersing the device in a silver-contain-ing solution after the targets have hy-bridized to the probes.

Analyses were conducted on arrays offour electrode pairs. In one example, thefour corresponding probes representedthe correct complementary sequence andthree possible mismatches for a target de-rived from the sequence for the anthraxlethal factor.

The typical strategy for distinguishingmatched from mismatched targets is toraise the temperature during hybridiza-tion, because perfectly matched targetsare more stable at higher temperatures.A perfectly matched strand was shown to

have a signal 105 times stronger than themismatched targets—significantly betterthan the selectivity ratio obtained witha comparable fluorescence-based assay(2.6:1) or Mirkin’s “scanometric” ap-proach, which uses the same nanoparticlelabeling scheme (11:1). The researchersalso found that they got better mismatchdiscrimination if they varied the Na+ con-centration instead of the temperature.They achieved detection limits as low as500 fmol of target DNA. (Science 2002,295, 1503–1506)

news

e–Ag+

hydroquinone

Illustration of the electrical detection of DNA, beginning with probe molecules immobilized be-tween two electrodes and finishing with the detection of target DNA molecules labeled withgold nanoparticles enhanced with silver. (Adapted with permission. Copyright 2002 AmericanAssociation for the Advancement of Science.)

DDNNAA aarrrraayyss wwiitthhoouutt aammpplliiffiiccaattiioonn

ANALYTICAL CURRENTS

RReevveeaalliinngg ffrraaggiillee ggaanngglliioossiiddee ssttrruuccttuurreessGangliosides, which are found in eukaryot-

ic cells, serve crucial functions as anti-

gens, receptors, and intercellular signaling

molecules. Small structural variations in

these complex lipid–oligosaccharide mole-

cules are responsible for specific biologi-

cal activities, and mixtures of gangliosides

are usually found in biological systems. But

glycosidic bonds, particularly the fragile

bonds linking sialic acid residues, fragment

extensively under electrospray and MALDI

ionization, and so these mixtures have

been difficult to analyze—until now.

Peter O’Connor, Ekaterina Mirgorod-

skaya, and Catherine Costello of the

Boston University School of Medicine

use a high-pressure (1–10 mbar) bath

gas under MALDI-FTMS desorption

to generate ions with a low average

vibrational energy. Metastable frag-

mentation is minimized by the ex-

change of hydrogen for sodium ion

and collisional cooling. Unfragment-

ed, sodiated molecular ions with up

to five sialic acid residues become

the base peak in both positive- and

negative-ion modes. The researchers an-

ticipate coupling MS with thin-layer chro-

matography assays to correlate specific bi-

ological functions with structures. (J. Am.

Soc. Mass Spectrom. 2002,13, 402–407)

M AY 1 , 2 0 0 2 / A N A LY T I C A L C H E M I S T R Y 2 3 9 A

1305.80

1277.77 1552.90

1000 1250 1500 1750 22502000 2500 2750

m/z

[M(3Na)+Na]+2245.02

1000 1250 1500 1750 22502000 2500 2750

–Neu5NAc(Na)1931.96

LH

–2Neu5NAc(Na)1618.88

–3Neu5NAc(Na)1305.80

(a)

(b)

Positive-ion MALDI-FTMS spectra of gangliosideGT1b (a) without and (b) with high-pressure colli-sional cooling gas. (Adapted with permission.Copyright 2002 Elsevier Science.)

2 4 0 A A N A LY T I C A L C H E M I S T R Y / M AY 1 , 2 0 0 2

news

ANALYTICAL CURRENTS

Ladies and gentlemen . . . in this corner,weighing in at ~3500 million years . . .J. William Schopf and colleagues fromthe University of California–Los Ange-les and the University of Alabama–Birmingham use optical methods andlaser-Raman microprobe spectroscopyto argue that tiny fossil-like structuresare indeed remains of ancient, Precam-brian prokaryotic microbes. And, in theopposite corner . . . also weighing in at~3500 million years and using similaranalytical methods . . . Martin Brasierand colleagues from the Universityof Oxford, University of Portsmouth,Royal Holloway University of London(all in the United Kingdom), the Geo-logical Survey of Western Australia, andthe Australian National University say“not true.” The “fossils” are artifactsformed from amorphous graphite.

In dueling papers, the researchers de-bate whether fossil-like, micrometer-sizedstructures seen in very old geologicalformations are the result of biology orgeology. Although much of the discus-

sion involves geochemistry,a major aspect of the de-bate is conflicting interpre-tations of the Raman spec-tra of these structures. Thedebate is reminiscent ofthe controversy over thefossil-like structures foundin a Martian meteorite.

According to Schopf’steam, Raman bands at~1350 cm–1 and ~1660cm–1 are characteristic ofcarbonaceous material.Confocal imaging with avertical resolution of 1–3µm shows that the carbonis in a high concentration,which suggests a biogenicorigin.

The Brasier group alsosees the Raman spectrum,but they find that there islittle absorption of the incident laserlight, which indicates to them that thecarbon is dilute. Based on relative mode

intensities, they say it is a ~200-nm-sized graphite particle. (Nature 2002,416, 73–81)

Inte

nsity

Wavenumber (cm–1)

1250 1500 1750 2000

500 1000 1500 2000 2600 2800 3000 3200 3400

Biology or geology? Raman spectra of the structures shownin the upper right corners. (Adapted with permission. Copy-right 2002 Nature Publishing Group.)

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Demonstrating the power of dip-pennanolithography, Chad Mirkin, MilanMrksich, and colleagues at Northwest-

ern University and the University ofChicago construct protein arrays with100- to 350-nm features. The arrays ex-

hibit little nonspecific bind-ing, which can obscure thedifference between featuresand inactive areas and lead tohigher background signals.

In dip-pen nanolithogra-phy, an atomic force micros -copy tip is coated with an“ink”—in this case, 16-mer-captohexadecanoic acid(MHA). If the environment is humid enough, a waterdroplet forms when the tiptouches a substrate surface,and the ink is transferred.

The researchers validatedthis fabrication method by ar-raying dots or lines of MHA,

passivating the surrounding areas, andimmersing the arrays in protein solutions.Monolayers of protein adhered to theMHA-coated regions but not the inter-vening spaces. In initial experiments,rabbit antibody bound to immunoglob-ulin G (IgG) arrays, indicating that IgGretained its activity.

In later experiments, the researchersfabricated an array of Retronectin, acommercial recombinant protein, tostudy cell adhesion. In particular, theywanted to investigate the importance ofthe size and distribution of the cellularanchor points known as focal adhesions.Previous studies were limited to 1-µmresolution, but the new work demon-strates that cells can adhere to 200-nm-diam protein spots that are separatedby 700 nm. (Science 2002, 295, 1702–1705)

MHA-coatedAFM tip

Au surface

Passivation

Protein adsorption

Anti-lgGbinding

lgG Lysozyme

Illustration of the dip-pen fabrication of protein arrays. EitherIgG or lysozyme is adsorbed to the substrate. The binding ofantibody to IgG indicates that the adsorbed protein remainsactive. (Adapted with permission. Copyright 2002 AmericanAssociation for the Advancement of Science.)

DDiipp--ppeenn iiss mmiigghhttyy ffoorr pprrootteeiinn aarrrraayyss


n ews

New research shows that compounds such

as antibiotics, hormones, and detergent

metabolites are more widespread in the

environment than initially anticipated. Using

five new analytical methods, Dana Kolpin

and colleagues from six U.S. Geological

Survey (USGS) offices found that 80% of

the 139 streams tested in 30 states during

1999 and 2000 contained organic waste-

water contaminants. Of the 95 contami-

nants targeted, 82 were present. Because

many organic wastewater contaminants

such as pharmaceuticals and household

chemicals pass through wastewater treat-

ment processes, the compounds aren’t

necessarily new to waterways. However,

for the first time in the United States, these

researchers’ techniques quantified trace

levels and took hydrogeologic, climatic,

and land-use settings into consideration.

Sites with proximity to potential human,

industrial, and agricultural wastewater

were sampled using standard USGS meth-

ods. Five new analysis methods were de-

veloped for the various classes of com-

pounds. Three of the methods targeted

antibiotics and prescription and over-the-

counter drugs, and essentially required

solid-phase extraction and LC/MS with

positive electrospray ionization to detect

them. The other two methods used con-

tinuous liquid–liquid extraction and GC/

MS to detect 46 organic wastewater con-

taminants (including insecticides, plasti-

cizers, and detergent metabolites) and 14

steroids. Selective ion monitoring was in-

corporated into all methods to increase

sensitivity, but only target detection was

reported. (Environ. Sci. Technol. 2002,36,

1202–1211)

Ster

oids

100

0

Perc

ent

Total concentrationFrequency of detection

80

60

40

20

45

Nonpr

escr

iptio

n dr

ugs

Inse

ct re

pelle

nt

Dete

rgen

t met

abol

ites

Disin

fect

ants

Plas

ticize

rsFir

e re

tard

ants

Antib

iotic

sIn

sect

icid

esPA

Hs

Repr

oduc

tive

horm

ones

Othe

r pre

scrip

tion

drug

sAn

tioxid

ants

Frag

ranc

esSo

lvent

15 3 7

2

22 7 511

14 5 2 1

Emerging organic contaminants in U.S. streams reported by the USGS are broken down into 15 categories. The number of compounds in each category is shown above the bars.

CClleeaarr,, bbuutt nnoott cclleeaann wwaatteerr

Christina Rudzinski, Albert Young, andDaniel Nocera at the Massachusetts In-stitute of Technology introduce the firstsupramolecular microfluidic opticalchemosensor, which glows green whenit detects polyaromatic hydrocarbons.According to the authors, the sensordemonstrates that the intricate signaltransduction mechanisms and functionalrequirements of supramolecular opticalsensors can be preserved in a miniatur-ized system.

To date, direct spectroscopic meth-ods have been used for optical sensingon microfluidic platforms, but thesemethods made it difficult to distinguishbetween analytes. Here, the “3R” sens-ing strategy—which is characteristic ofsupramolecular chemosensors—recog-nizes, relays, and reports interactionsbetween polyaromatic hydrocarbons insolution and a cyclodextrin supramole-cule receptor site that has been modifiedwith a terbium(III)-enclosed macrocycle.Such interactions trigger an adsorption-energy transfer-emission mechanism,which generates green luminescence.Submicromolar quantities of polyaromat-ics can be detected without any amplifi-cation because even such tiny signals areclearly visible against the background,which remains dark.

The researchers immobilized thechemosensor using various organic andinorganic matrixes and found thatsol–gel procedures gave the most evendistributions of material. The microflu-idic device was fabricated in polymer ona quartz substrate, and 1-mm sol–gelsquares were photolithographically pat-terned within a serpentine microchan-nel. The researchers found that the lu-minescence increased monotonicallywith the concentration of biphenyl, andthey detected this analyte at a concen-tration of 5 µM. On the basis of suchresults, the researchers project that re -liable detection will be possible with700-nm-thick films. (J. Am. Chem. Soc.2002, 124, 1723–1727)

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AD

AP

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FR

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KO

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ET

AL.


news

ANALYTICAL CURRENTS

Watching chemical reactions as they pro -gress under actual conditions is a trickyenough business. But Volker Deckert andChristian Fokas of the EidgenössischeTechnische Hochschüle Zürich (Switzer-land) want to do more than uncover areaction mechanism. They aim to dis-cover simultaneously the morphology ofthe reaction sites. Their goal is to corre-

late information derived from a modelsystem—the heterogeneous hydrogena-tion of benzene over palladium—so thatthey can determine a method for opti-mizing the catalytic substrate.

In previous work, scanning near-fieldoptical microscopy (SNOM) combinedwith surface-enhanced Raman scattering(SERS) achieved 100-nm spatial resolu-

tion of a sample on a silver surface. Inthe current paper, the researchers extendthe combined techniques to a palladiumcatalyst. The nanofabricated catalyst isdescribed as a silver “island”—because itlooks like one when viewed with atomicforce microscopy—coated with palladi-um. The SERS signal is enhanced withan atomic force microscopy tip insteadof a surface-enhancing substrate.

Comparing the spectra of reagentsand projected products using far-fieldSERS showed one anomaly. The spec-trum for 1,3-cyclohexadiene on an islandin the absence of palladium was similarto that of benzene adsorbed on a palla-dium-coated island under reactive con-ditions. This result was surprising be-cause silver is usually inactive underthese conditions, and it led the authorsto conclude that the same intermediatewas formed in oxidative and reductivepathways.

Although the group has not yet ob-tained in situ morphological images, near-field SERS under nonreactive conditionsshows that Raman enhancement for ananalyte occurs only within the vicinityof an island. This dem onstrates the ca-pability of the combined techniques toidentify a specific reactive site by corre-lating morphology with spectra. (Appl.Spectrosc. 2002, 56, 192–199)

45 nm

0 nm

200 nm

Noncontact atomic force microscopy imageof the silver/palladium island catalyst withina chemically etched pit. (Adapted with per-mission. Copyright 2002 Society for AppliedSpectroscopy.)

PPaallllaaddiiuumm rreeppllaacceess ssiillvveerr iinn SSNNOOMM//SSEERRSS

With protein databases growing by leaps

and bounds, identifying a particular pro-

tein by MS requires an increasingly accu-

rate mass determination. Willy Bienvenut

and colleagues from Geneva University

Hospital, the Swiss Institute of Bioinfor-

matics, GeneProt (all in Switzerland), and

Institut National de la Santé et de la

Rechere Médicale in Lyon (France) bor-

row a method already beloved by protein

chemists—hydrogen/deuterium (H/D) ex-

change—and ask whether this technique

can improve protein mass accuracy.

The authors investigate H/D exchange

in conjunction with peptide mass finger-

printing using MALDI MS to analyze a pro-

tein digest. They find that it takes three

repetitive treatments to optimize H/D ex-

change with peptide fragments from the

enzyme digest. H/D exchanges of 95% or

higher are observed.

The exchange method is used to identify

a protein from virus-infected HeLa cells and

validate the presence of a second protein.

Limitations of the method include the need

to manually retrieve the data from the spec-

tra and lower sensitivity. (Rapid Commun.

Mass Spectrom. 2002, 16, 616–626)

(a)

Mass (m/z)

(b)

1000 1200 1400 1600 1800 2000 2200 2400 2600

927.

493

1163

.63

1399

.68

1439

.82

1479

.82

1567

.76

1639

.95 17

24.8

417

31.8

6 1880

.93

1907

.92

2045

.03

2044

.6

2045Mass (m/z)

2058

2492

.08

944.

766

1183

.97

1506

.26

1593

.21

1755

.31

1912

.46

2075

.63

Mass (m/z)

2528

.79

2075

.19

2070 2075 2080

MALDI MS spectra of bovine serum albumin digest with (a) no treatment and (b) follow-ing H/D exchange. Zoom boxes show the isotopic distributions. (Adapted with permis-sion. Copyright 2002 John Wiley & Sons.)

HH//DD eexxcchhaannggee ccoommeess ttoo pprrootteeoommiiccss


n ews

RNA may be synthesized at various timesduring the cell cycle, and the details ofits production and how it differs from onecell to the next are still sketchy. FutianHan and Sheri Lillard at the Universityof California–Riverside address this prob-lem by using CE to measure the levelsof RNA synthesis in individual cells atvarious stages.

The researchers borrow a trick fromtissue-culture protocols to select cellsthat are in the same stage of the cellcycle. They use a modified version ofthe “shake-off” method, which takesadvantage of the fact that many culturedcells round up during mitosis and beginto pull away from the surface wherethey are growing. Shaking the flask re-leases the mitotic cells into the culturemedium but leaves the other cells at-tached. The culture medium is siphonedoff, providing a population of synchro-nized cells. After starting in synchrony,the cells will reach the G1, S, and G2stages more or less together.

Han and Lillard ana-lyzed individual cells atmultiple stages of the cellcycle and identified multi-ple RNA peaks that mostlikely corresponded totransfer RNA (tRNA) andvarious subunits of riboso-mal RNA (rRNA). Mes-senger RNA is unlikely tohave been observed.

The total RNA increasedas the cells progressed fromG1 to the S, G2, and Mphases, with the biggest in-crease coming between Sand G2. However, therewas considerable variationamong the types of RNA.When the levels of the vari-ous RNAs were normalizedwith respect to the total RNA, the fractionattributed to low-molecular-mass RNAs—those <155 bases long, which tentativelyincludes tRNA and 5S rRNA—nearly

doubled between the G2 and M phases.The variations in 18S and 28S RNA syn-thesis were more subtle. (Anal. Biochem.2002, 302, 136–143)

LMM

Rela

tive

fluor

esce

nce

Migration time (min)

(a)

(b)

500 15002000 4000 5000

18 S

28 S

12 14 16 18 20 22 24

Rela

tive

fluor

esce

nce

Time (min)12 14 16 18 20 22 24

(a)

(b) LMM

155530

RNA peaks from single cells in (a) G and (b) S phase. The 18Sand 28S ribosomal RNA peaks are indicated. The low-molecular-mass peaks probably include transfer and 5S ribosomal RNA.(Adapted with permission. Copyright 2002 Elsevier Science.)

CCEE ffiinnddss ttrruuee vvaarriieettyy aammoonngg cceellllss

Thomas Jovin and colleagues at the MaxPlanck Institute for Biophysical Chem-istry (Germany) have designed a system

for collecting spectrally resolved fluores-cent lifetime images. To get their spec-troscopic fluorescence lifetime imaging

microscopy (sFLIM) system,the researchers combined fre-quency-domain lifetime im-aging with two-dimensionalspectral imaging using a Hada -mard transform programma-ble array microscope. Theyreport a resolution of ~50(�/∅�) and a wavelengthrange of ~430–750 nm andsay that their system shouldbenefit lifetime or fluores-cence resonance energy trans-fer (FRET) studies based onmicroscopy.

FRET techniques are oftenused in experiments to clarifythe temporal– spatial relation-ships among multiple chemi-

cal pathways in intact biological systems.The researchers studied FRET between a histone protein, which was fused withan enhanced green fluorescent protein,and the DNA intercalation dye propidi-um iodide. Studies were conducted invitro and in tissue from Drosophila sali-vary glands.

The sFLIM setup allows the lifetimeand intensity spectra of the donor andacceptor to be observed simultaneously.The instrumentation is also readily adapt-able to multifrequency studies. And insome cases, sFLIM made it possible todetermine whether a fluorophore underinvestigation was in the cellular compart-ment of interest. Also, by observing thelifetime spectra using their instrument,the researchers were able to optimizethe wavelength range of a conventionalFLIM experiment. (Appl. Spectrosc.2002, 56, 155–166)

Microchannel plateintensifierRelay optics

CCD camera Imagingspectrograph

Anamorphicoptics

Spatial lightmodulator

Tube lens

ObjectiveOrder selection iris

AOMAr+ laser

Signal generator 2

Signal generator 1

f/2 = 28.84 MHz

f = 57.68 MHzf

f/2

∆Φ

f

Diagram of the spectroscopic fluorescence lifetime imag-ing microscopy system. (Adapted with permission. Copy-right 2002 Society for Applied Spectroscopy.)

SSppeeccttrraallllyy rreessoollvveedd fflluuoorreesscceennccee lliiffeettiimmee iimmaaggiinngg


news

ANALYTICAL CURRENTS

Japanese researchers attempt to overcomethe current limitations in investigatingprotein phosphorylation by developingcustom-made, genetically encoded fluo-rescent indicators for protein phosphory-lations. Yoshio Umezawa and colleaguesat the University of Tokyo and the JapanScience and Technology Corp. introducethese “phocuses”—an abbreviation of thewords “phosphorylation” and “custom-made”—to visualize protein phosphory-lation by the insulin receptor.

Protein phosphorylation plays one ofthe most important roles in cell signal-ing. Scientists have used electrophoresis,immunocytochemistry, and in vitro as-says to study protein phosphorylation.But Umezawa and his team point outthat those methods do not provideenough information about the spatialand temporal dynamics of the criticalprocesses of protein phos-phorylation and dephos-phorylation in living cells.

To make the phocuses,Umezawa’s group joinedtwo mutants of green fluo-rescent protein—cyan fluo-rescent protein (CFP) andyellow fluorescent protein(YFP)—using a tandem fu-sion domain. The fusiondomain had three parts: asubstrate domain for thekinase of interest, a flexiblelinker sequence, and aphosphorylation recog -nition domain.

If the kinase of inter-est—in this case, the in-sulin receptor—was pres-ent, it phosphorylated atyrosine in the substrate,which made it possible for the phosphorylationrecognition domain toidentify the substrate andbind to it. This bindingchanged the conformationof the phocus from a lin-ear molecule to a folded

one. CFP and YFP—initially at oppositeends of the linear molecule—werebrought near one another, which in-creased the fluorescence resonance energy transfer (FRET) between them.Conversely, if a phosphatase was added,it dephosphorylated the substrate do-main, and the phocus unfolded, sepa -rating CFP and YFP and decreasing theFRET signal.

The researchers note that it may bepossible to make this approach evenmore general by replacing the phospho-rylation-recognition domain with single-chain antibodies directed against thesubstrate domain. They suggest that,with further improvements, phocusesmay be used for high throughput toscreen pharmaceuticals that regulate kinase and phosphatase activities. (Nat. Biotechnol. 2002, 20, 287–294)

YFP

CFP

Flexible linker(GNNGGNNNGGS)

CFP YFP

OH

P

Phosphatase Protein kinase

Tyr (Ser, Thr)480 nm

440 nm

Phosphorylationrecognition domain

(SH2, ScFv, etc.)

Substrate domain

440 nm

535 nm

FRET

The new “phocus” fluorescent indicators for protein phos-phorylation in living cells. When the substrate is phosphory-lated, CFP and YFP—cyan and yellow mutants of green fluo-rescent protein—are near one another, which increases theFRET between them. Dephosphorylation returns the indica-tor to a linear conformation and reduces the FRET signal.(Adapted with permission. Copyright 2002 Nature PublishingGroup.)

RReesseeaarrcchheerrss ““pphhooccuuss”” oonn nneeww fflluuoorreesscceenntt iinnddiiccaattoorrss

RESEARCH PROFILES


n ews

What do you do when the hardware storejust doesn’t have that special tool you’relooking for? The answer, if you’re ScottSaavedra, is to build your own.

Saavedra, Sergio Mendes, and JohnBradshaw at the University of Arizonahave designed a new single-mode, sol–gel planar integrated optical waveguide(IOW) spectrometer that can measurebroadband attenuated total reflection(ATR) spectra of thinmolecular films over theentire visible wavelengthrange. The performanceof the platform, whichcan transmit light downto at least 400 nm, isunprecedented and of-fers potential bioanalyti-cal sensing applicationsin the medical field.

What sparked theinnovation was Saave-dra’s quest to studyproteins immobilizedat the interface betweena liquid and solid.Rather than measureabsorbance at a partic-ular wavelength, Saave-dra wanted to measurespectra of adsorbedprotein films, thus get-ting more information on their proper-ties. Because no instrumentation had yetbeen developed to do that, necessity be-came the mother of his invention. “Ifyou want to know something about asample, and the existing measurementtools aren’t good enough, then you haveto build a new hammer,” says Saavedra.“We developed it specifically because ourtoolbox is missing something.”

In the April 15 issue of AnalyticalChemistry (pp 1751–1759), Saavedra’sgroup explains how the new instrumen-tation works. The waveguide was fabricat-ed by dip-coating a glass substrate with a~300-nm-thick, sol–gel composite layer.They used a commercially available prismas the incoupler, with an integral holo-

graphic diffraction grating acting as thedispersive outcoupling element. Theycompared the waveguide spectrometer’sresponse with that of a conventional, trans -mission-based UV– vis spectrometer. Theteam measured the full width at half maxi-mum of various laser lines, ranging from0.5 to 1.3 nm, to assess spectral resolu-tion. The measured limits of detection forthe waveguide spectrometer at 400– 600

nm were 8.0 and 10.1 milliabsor banceunits for transverse electric and trans versemagnetic polarizations, respectively.

The researchers also acquired visibleATR spectra of an adsorbed submono-layer of horse heart cytochrome c. Mul-tichannel ATR spectra were obtainedwithout using a monochromator becauseoutcoupling at the integral grating spec-trally disperses the entrapped light. Thedevice was used to measure broadbandATR spectra over a visible wavelengthrange of 390 nm to >645 nm. Becausethe pathlength in a waveguide ATRgeometry is wavelength-dependent, theresearchers had to calculate and apply apathlength correction before the ATRspectra could be quantitatively compared

with the spectra of the native protein.Saavedra began working on the wave-

guide instrument when he came to Ari-zona in 1991. He knew that typical single-mode planar IOWs are useful for studyinginterfaces, but they have a characteristi-cally narrow spectral bandwidth. Differ-ent wavelengths of light must propagateat different internal reflection angles toproduce a guided mode for each wave-

length. To solve thatproblem, the group ini-tially used an achro-matic coupler designedspecifically to match aparticular waveguidestructure (Langmuir1996, 12, 3374– 3376).“We came up with anelegant approach thatbasically ‘tricked’ thewaveguide [into] ac-cepting light whereevery wavelength constituent was incou-pled and resonant atits respective couplingangle,” says Saavedra.Using the achromaticcoupler guided them intheir current research.Their new design is notachromatic, which makes

it much easier to use on a daily basis.Unlike the waveguide used in their

previous spectrometer, the sol–gel wave-guide can be cleaned with oxidizing acidsand reused. They also emphasize that pre-vious designs were limited to measuringwavelengths of >500 nm. And it’s relative-ly easy to make numerous sol–gel wave-guides with varying broadband responses.

The researchers are applying the tech-nique to observe how proteins interactwith proteolipid films being developedin Saavedra’s lab. Saavedra says the newmethod might become an accessory to avisible/near-IR adsorbance spectrometer.“The applications are in spectroscopy ofinterfacial molecules in any field,” he says.

——CChheerryyll MM.. HHaarrrriiss

Incident white light(from 150-W xenon arc lamp)

Adsorbedmolecular film

~300- to 400-nm thicksol–gel glass

waveguide film

Gratingcoupler

(λ ≅ 400 nm)SF6 coupling prism

Outcoupled, dispersed light

Glass substrate(microscope slide)

Schematic of broadband coupling from a xenon lamp into a single-mode, planar integrated optical waveguide.

NNeeww wwaavveegguuiiddee mmeetthhoodd aaddddss ttoo ssppeeccttrroossccooppyy ttoooollbbooxx


news

In nucleic acids analysis, PCR is the star.For parallel gene expression assays, it’shard to beat microarrays. Yet, in the April15 issue of Analytical Chemistry (pp1745–1750), W. Travis Berggren, LloydSmith, and colleagues at the Universityof Wisconsin–Madison and Third WaveTechnologies describe a new multiplexedgene expression assay that eliminatesPCR and the solid-phase chemistry ofmicroarrays.

Why would they want to competewith such established technologies? Be-cause one size doesn’t fit allnucleic acids assays, saysBerggren, a graduate stu-dent in Smith’s lab. For ex-ample, not everybody needsexpression data for thou-sands of genes at once, asoffered by microarrays. AndPCR seems to be shiftingfrom a technique thateveryone is grateful to haveto one that people some-times want to avoid. “It’ssort of like a marriage,” ex-plains Smith. “AlthoughPCR is still very, very power-ful, it has been around close to 20 yearsnow, and people have gotten to know allthe ‘ins and outs’—all the shortcomings.”

One difficulty with PCR is gettingquantitative information. Because PCRexponentially amplifies the amount oftarget DNA, small variations in the initialconditions can lead to large differencesin the amount of product. To avoid thisproblem, Berggren, Smith, and col-leagues switched to a signal amplificationtechnique—Third Wave’s proprietary“Invader” assay.

The Invader assay is a hybridizationtechnique that uses two oligonucleotides(oligos)—a probe and an Invader—bothof which hybridize to the target DNAor RNA. The two oligos overlap be-cause the Invader extends into the re-gion where the probe sits, thus “invad-ing” its territory. Because the Invaderessentially binds first, the probe can’t

quite lie flat, and a little flap of probehangs off at the overlap site. The DNArepair enzyme recognizes such overlapsand cleaves the flaps, generating the sig-nal that indicates successful detection. Ifthe probe and Invader don’t hybridize,there’s no overlap, no cleavage, and thus,no signal.

After the flap is cleaved, the rest of theprobe “falls off” the target, so it’s possibleto amplify the signal if multiple probes hy-bridize and get cleaved in turn. The likeli-hood of such turnover is increased by run-

ning the reaction very near the probe’smelting temperature. (The Invader oligohas a higher melting temperature, so itremains bound to the target most of thetime.) Because the amplification is linear,not exponential, it is easy to quantify theresults, Berggren notes.

Although the Invader assay has usedfluorescence detection to date, Smith andBerggren switched to MALDI time-of-flight detection. Berggren says that they“recognized early on that this Invadertechnology is an ideal candidate for massspec detection” because it’s easy to designflaps of various lengths and, thus, variousmasses. (Tailoring the flaps this way does-n’t interfere with hybridization.) Thus,multiplexing is straightforward. For exam-ple, adding a single nucleotide to theflap increases the mass by ~300 Da, whichgives ample separation between peaks. Italso avoids the possibility that a short flap

containing a common nucleic acid adductmight be mistaken for a longer flap.

To prove the principle of the assay,the researchers created probes for fourcommon RNA targets. Each probe hada different flap—TTG, TTTT, TTTTT,or TTTTTT—and the molecular weightsof the cleaved flaps ranged from 1281to 2168. With this add-a-nucleotide ap-proach, the researchers project that itshould be possible to multiplex ~30–50targets per MALDI spectrum before en-countering practical limitations, such as

exceeding the optimal massrange on the mass spectrom-eter. But Berggren notesthat adding single nucleo -tides provides “100–300times more [peak] separa-tion than we need. If onewere willing to redesignthe chemistry of the cleav-age product to make small-er mass tags . . . much high-er levels of multiplexingcould be achieved.”

Generally speaking,MALDI is also amenableto automation and high-

throughput analysis, Berggren says. How-ever, because MS requires relatively cleansamples, the new method has a clean-upstep between the Invader reaction andthe MS analysis, which limits the through-put. “This is probably the last step thatneeds to be worked out,” he concedes.To simplify cleanup, Berggren taggedthe flaps with biotin, making it easy tocapture the cleaved flaps on streptavidin-coated beads. But a more permanent so-lution probably would be a solid-phasepurification step, he adds.

Now that the core technology hasbeen demonstrated, Berggren and Smithhope that others will explore its advan-tages for multiplexing and throughput.They don’t envision it replacing the big-name methods, just complementing them.But you never know when you might hiton the next PCR.

——EElliizzaabbeetthh ZZuubbrriittsskkyy

MMuullttiipplleexx MMAALLDDII mmeetthhoodd ffoorr ggeennee eexxpprreessssiioonn

5’Invader oligonucleotide

RNA target

Site of 5’ nuclease cleavage

Accumulation of5’ cleavage product

Probe oligonucleotide

Isothermal reactionnear probe Tm

Excess of probe

Nonhybridizing sequenceTarget-specific sequence5’ 3’

5’ 5’ 5’ 5’ 5’

Schematic of the Invader assay. The cleaved flaps from the probeoligonucleotide, which constitute the signal indicating successful hybridization, are detected by MALDI time-of-flight MS.

RESEARCH PROFILES


n ews

NNeeww ssttrraatteeggyy ffoorr zzeeppttoommoollee ddeetteeccttiioonn

Phones made fiber-optic technology fa-mous, but the filaments can do more inthe lab than help ring up a pizza deliv-ery. Fiber optics also can serve as chem-ical sensors.

In the April 15 issue of AnalyticalChemistry (pp 1836– 1840), researchersat Tufts University report zeptomole(10–21) detection limits with improvedS/N using functionalized microspheresin a fiber-optic array. The project wasstarted by Myoyong Lee, a postdoctoralfellow in David Walt’s group at Tuftsbefore she was hired at the National In-stitutes of Health, and continued byJason Epstein, currently a doctoral can-didate in Walt’s lab.

In earlier work, the researchers couldnot go below a detection limit of 10 fM(Anal. Chem. 2000, 72, 5618–5624).But the new detection limit is 2 ordersof magnitude better. “We are demon-strating . . . the ability to improve thedetection limit by reducing the uncer-tainty of where the molecules are,” saysWalt.

The researchers create their arraysby etching wells into the distal face ofa 500-µm-diam optical imaging fiberbundle composed of ~6000 fibers. Afterapplying a microsphere suspension tothe fiber tip and evaporating the load-ing solution, the 3.1-µm-diam micro -spheres sit in these wells.

To capture specific DNA sequences,the researchers attach probe oligonu -cleo tides to the microspheres via poly-mer linkers. When target DNA carryinga fluorescent label hybridizes to theprobes, a custom imaging system recordsthe probe sequences lighting up. Ac-cording to Walt, “you get better hy-bridization” and more molecules perbead by using the intervening polymerrather than by attaching the DNA di-rectly to the bead.

In the current paper, the researchersfocused on pushing down the detectionlimits, using three DNA sequences—two from the cystic fibrosis gene andone from a cytokine, a low-molecular-

weight, pharmacologically active pro-tein. Walt explains that by choosing only3 candidates instead of 25 as in the pre-vious work, “We stacked the deck inour favor with respect to making surethat we didn’t have sequences that weretoo closely related.” Now, the researcherscan very quickly detect zeptomoles ofDNA, which translatesto ~600 moleculesusing only 10 micro -sensors. Although S/Nincreases as the squareroot of the number ofmicrospheres, it was important to limit the number of micro -spheres to keep the sig-nal from spreading outand becoming unde-tectable, says Walt. Thisarray maximized theratio of DNA moleculesto microspheres andmaintained a good sig-nal average.

The researchers pur-chased pure target DNAof known concentrationwith attached fluorescentlabels and simply per-formed serial dilutionsto keep track of thenumber of molecules.Walt explains that theysimulated a real-worldexperiment, which would contain highbackground levels of nontarget DNA,by adding a billion-fold higher concen-tration of salmon sperm DNA to thetargets. The researchers detected thetargets among these inhibitors, but it took longer than it did with puresamples.

Having reliable, low detection limitscould have various implications. First,a low detection limit may reduce theneed for sample manipulation, and “theless you have to manipulate your sam-ple, the more quantitative you can be,”says Walt. For example, it could di -

minish the need for polymerase chainreaction (PCR) in certain cases becausehigh levels of amplification would beunnecessary. “If you have a few hundredmolecules, then you would only haveto run [PCR] a few times, and thatwould save time,” he adds.

Second, Epstein sees the low detec-tion limit as a plus forexamining gene expres-sion because the sensormay pick up genes thatare expressed at verylow levels and, thus, aremissed by less sensitivemethods. Third, goingto microscale and look-ing at smaller volumesimproves both sensi -tivity and uncertainty.“When we bind a limit-ed number of moleculesto a small volume wegenerate a high localconcentration on themicrosphere, makingit easy to detect eventhough the absolutenumber of moleculesis small,” Walt says. Headds that the principlesin this paper shouldapply to any kind of receptor interactingwith an analyte, poten-tially including some

of his other work, such as detecting bi-olog ical agents.

At this point, the researchers are“trying to reduce the size of the beadsfrom microscale to nanoscale, which wehope will enable us to improve our de-tection limit even more.” Just how lowcan they go? “We would like to go tosingle molecules,” says Walt. Maybe it’snot so crazy. After all, no one imaginedmaking phone calls without operatorswhen Alexan der Graham Bell presentedthe first telephone, and now we havedigital cell phones.

——RRaacchheell PPeettkkeewwiicchh

Fiber-optic bundle

Microspheres and wells

P

(Top) Schematic of a 500 µm-diam fiber-optic bundle. (Bottom) Atomic force micro-scope image of the 3.1-µm-etched wells containing func-tional ized microspheres to detect DNA.

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RESEARCH PROFILES

High-throughput identificationof proteins takes another stepforward with a new algorithmfrom a team at the Max PlanckInstitute (Germany). Most data-base search engines confidentlyidentify proteins by the numberof “hits” (peptide mass valuesderived from experiments thatmatch the peptide masses fromknown proteins in a database)together with the researcher’seducated judgment. The newstatistical scoring algorithm forMALDI time-of-flight massspectra, described in the April15 issue of Analytical Chemistry(pp 1760–1771), automates thejudgment phase and more accu-rately identifies tryptic digests ofplant and mammalian proteins,says Volker Egelhofer.

When a database search re-turns few hits, the protein maybe difficult to identify becausethere are so many candidates.“The bigger [databases] are, themore difficult the search be-cause they have more similarproteins,” explains Egelhofer.However, in testing the newscoring system with several hun-dred common proteins, whichwere independently confirmedby DNA sequencing, the groupobtained accurate identificationswith scores of 99%. “The pro-tein was always the correct one,”he says. “In some cases, youdon’t find a protein, but thenyou don’t find a false positive[either], and that’s the great ad-vantage of this algorithm.”

The new strategy was motivat-ed by the team’s discontent withthe number of false positives re-turned by an earlier version oftheir sequence database searchprogram, the mass spectrometry analyz-ing (MSA) algorithm (Anal. Chem.2000, 72, 2741–2750). “We looked at

our data and saw that they always havea linear error,” he says, “and we justthought about how to improve this.”

Linear, systematic errors arecaused by differences in the ener-gy and flight paths of ions gener-ated from different sample posi-tions on a target grid. The resultis a different m/z calculated forequivalent ions from differentsamples. Because the differencein masses is linear, it can be usedto correct the error, says Egel-hofer. “Then the standard devia-tion . . . can be put in the scor-ing formula to help decide ifproteins are identified or not.”

The strategy begins by search-ing a protein database for a mini-mum of five matching peptideswithin a defined range. MSA cal-culates the relative errors of thepeptide masses and the linear re-gression on the errors for eachsequence. The standard deviation(SD) then identifies the falsepositives that are excluded fromthe succeeding regression calcu-lation on the remaining masses.The scoring equation incorpo-rates the percentage of the pro-tein’s sequence covered by thematched peptides and the datafrom three rounds of regression,SD, and removal of outliers.

All the factors in the scoring al-gorithm interact dynamically, saysEgelhofer. “If you have a lot ofhits (n), you have found a lot ofpeptides, and you have a muchhigher SD because the errorgrows with more values.” Includ-ing the sequence coverage helpsto avoid false positives in suchcases. On the other hand, a small-er SD means that you can havefewer hits or lower sequence cov-erage and still find the correctprotein, he explains.

The dynamic quality of thealgorithm provides adaptability,

and a “fudge factor” (F ) helps controlthe stringency of the search. The re-searchers never observed false positives

Inte

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/rela

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m]

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–5001,000 2,000 3,000500

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(a)

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Y’ = 0.0218X–238.2

PO2570

Y’ = 0.0232X–239.6

m/z

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0

(b)

(a) MALDI time of flight mass spectrum from a tryptic digestof recombinant human �-actin and (b) a plot of relative massdeviations. The mean of the relative deviations is calculated,and dashed red lines indicate the standard deviation to thismean. Outliers are excluded. Successive linear fits (c, e) re-sult in (d, f) decreasing standard deviations.

TTaakkiinngg ssoommee ooff tthhee ““gguueesssswwoorrkk”” oouutt ooff pprrootteeiinn iiddeennttiiffiiccaattiioonn


n ews

MEETINGS

News from the 5544tthh AAmmeerriiccaann AAccaaddeemmyy ooff FFoorreennssiicc SScciieenncceess MMeeeettiinngg—Wilder D. Smith reports fromAtlanta, Ga.

BBiigg ttrroouubbllee ffoorr mmoonneeyyllaauunnddeerreerrss??In July of 2000, then-U.S. Attorney Gen-eral Janet Reno took offense at a FederalBureau of Investigation (FBI) finding thatmoney launderers were so confident intheir ability to successfully smuggle U.S.currency into and out of the country, theywere shipping cash through U.S. ports bythe crate. Thomas Jourdan, a supervisoryspecial agent with the FBI who holds aPh.D. in chemistry, was told by then-FBIlaboratory Assistant Director and DivisionHead Donald Kerr to work on a solutionthat the U.S. Customs Agency could useto screen cargo at ports. “I was alreadyworking on measuring the amount ofcocaine on money in normal U.S. circu-lation,” says Jourdan.

Finding crates of money amid thecargo at the > 300 ports of entry thatspan the United States provided a muchdifferent challenge. “The problem washow to tell if a crate marked ‘engine parts’actually had those parts in it, or if it con-tained U.S. currency,” he says. Openingeach and every crate that comes through aport is an unreasonable and time-consum-ing task, he says.

Jourdan considered several approaches.“However, the thing that screamed out at

me was that if you’ve ever been around alarge amount of money . . . it has a dis-tinct odor to it,” he says. “It seemed tome that a portable instrument that coulddetect the volatile organic compounds[VOCs] in money was what was needed.”Jourdan initally considered using mi-cropower impulse radar instruments.

To decide which VOCs to look for, heteamed up with Donald Blake at the Uni-versity of California–Irvine and Trang Vuof the U.S. Customs Service, who haddone work on detecting VOCs frommoney using GC/MS. “We decided tofirst pick a set number of VOCs that wereassociated with currency and then . . .program an ion mobility spectrometer[IMS] to look for these compounds,” saysJourdan. The group settled on a list of 10VOCs considered to be common to allnew and circulated U.S. currency: hexa-nal, heptanal, octanal, 2-hexenal, 2-hepte-nal, 2-pentyl furan, 1-heptanol, pentanoicacid, and two volatile breakdown productsof illicit drugs—methylbenzoate from co-caine and acetic acid from heroin.

In the meantime, Kevin Linker at San-dia National Laboratories–Albuquerque(SNL) was working on the necessary in-strument—a portable, six-inch, cubelikeinstrument called the “Hound” that, cou-

pled with an IMS, would detect explosives,drugs, and other chemicals. The idea forthe portable Hound system was born fromlarger, walk-through explosives detectionportals that were developed by SNL.

To collect vapors, the Hound usesportable solid-phase microextractiontechnology in which a device that lookslike a blower inhales a large volume ofair and corrals the scent of explosivesand other chemicals onto a metalizedscreen. The unit then vaporizes thecompounds into a more concentratedsample of air, which is delivered to a detector.

Although the Hound can detect explo-sives down to the parts-per-trillion levelwith several cubic feet of air, detectingVOCs on money is still in the preliminarystages. “We have only worked in the labwith solutions that represent VOCs frommoney,” says Linker.

Having a portable system with thesame type of sensitivity for detectingVOCs in money would greatly improvethe detection efforts and efficiency ofU.S. Customs agents, says Jourdan. “Es-sentially [Customs agents] could drill asmall hole in the crate, take a 10-secondair sample, do the analysis, and get areadout in about 4 seconds,” he says.

using the default setting (F = 1.0), but auser might want to reduce the value insearching a small database where theremay be fewer hits. Because this lowers thestringency of the search and, consequent-ly, increases the chance of false positives,corresponding changes in other variablesare needed to counteract the effect, thusmaintaining the overall stringency of thescoring function. In addition, adaptabilityto the quality of mass spectra is inherentin the SD, which depends on factors suchas calibration, tuning the mass spectrome-

ter, and S/N. The group hasn’t yet founda general strategy for including modifica-tions of amino acid residues in the scor-ing algorithm, but they suggest an empir-ical change in n that is based on thenumber of modified peptides detected.

Another advantage of the new algo-rithm, which also enhances throughput, isthe need for only one external calibrant.Egelhofer says that external calibrants for-merly had to be placed near each samplein the target, so there might be as manycalibrants as samples. But with the new

algorithm, he says, “you can put [the cali-brant] somewhere in the target, measureit one time, and measure all the peptidesthat you want to know.” This also elimi-nates the need for an internal calibrantand, thus, avoids the problem of internalcalibrant and analyte peaks overlapping.

The researchers have transformed theSwissProt and NCBI databases to formscompatible with MSA. Access to the newversion of MSA is free on the Internet atwww.scienion.de/msa.

——JJuuddiitthh HHaannddlleeyy


news

PPCCRR cclleeaarrss uupp aa bblluurrrryyccrriimmeeA typical crime scene has some sort of“fingerprints”—blood, semen, a singlestrand of hair, or even a used cigarettebutt. Using PCR to collect DNA fromsuch samples is very effective and noth-ing new to the forensic community. Butfor forensic scientists Ray Wickenheiserand Richard Jobin at the Royal Canadi-an Mounted Police in Regina, Saskache -wan (Canada), an unheard-of applica-tion of the technique would change thepossibilities for crime scene analysis asfar as the eye could see.

For four hours, a victim was brutallybeaten, raped, and held in an apartment.During the ordeal, one of her contactlenses was dislodged. After being re-leased, the victim waited three days toreport the incident because she fearedfurther retaliation—a delay that gavethe assailant enough time to thoroughlyclean the crime scene. In fact, the onlyphysical evidence collected from the crimescene were 19 shat tered fragments of alight blue contact lens found in a vacu-um cleaner in the apartment.

Cells from the corneal epithelium(eyeball) and the bulbar epithelium (inte-rior edges of the eyelids) regenerate every6–24 hours. Every time a person wearinga contact lens blinks, thin slices of epithe-lial cells are sheared from the interior eye-lid and collect on the surface of the con-tact lens, providing a source of DNA.

To retrieve the biological material

from the lens, Wickenheiser and his col-leagues relied on standard techniques.They began with a procedure very simi-lar to cleaning a common contact lens.Usually, the lens is immersed in a salinesolution, and an enzyme is added to di-gest the proteins, releasing them intothe solution and leaving a clean contactlens. “This was very similar to what wewere doing, except we were using aprotease solution to break up the pro-teins, which breaks up the cells and re-leases the DNA into the solution,” saysWickenheiser. Then they extracted the

DNA using a protocol that they hadpreviously perfected. Finally, the DNAwas purified with the common phenol–chloroform method. This application ofPCR was significant because “it was thefirst time it was ever used on a contactlens,” says Wickenheiser.

The victim’s DNA was found on thelens, placing her at the crime scene, whichwas all Wickenheiser and his team need-ed to prove. The assailant eventually con-fessed to the crime, and a new world ofapplications for PCR became a little clear-er to forensic scientists.

MEETINGSBand sizes

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Contact lens—scene

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1200800400

0Known sample—complainant

STR System 3, containing loci Amelogenin (sex typing - blue), D5S818 (blue), D13S317 (green), and D7S317(blue) from right to left respectively. Once again, variation in peak height from loci to loci is evident, along withn-4 peaks. Note also a small Amelogenin peak at 112, which corresponds to the Y chromosome, indicating atrace male component. Given the nature of the vacuum cleaner bag source of the contact lens fragments, thisis not surprising.

STR System 3, containing loci Amelogenin (sex-typing blue), D5S818 (blue), D13S317 (green),and D7S317 (blue), from left to right, respectively. Once again, variation in peak height fromloci to loci is evident, along with n–4 peaks. Note also a small Amelogenin peak at 112, whichcorresponds to the Y chromosome, indicating a trace male component. Given the nature ofthe vacuum cleaner bag source of the contact lens fragments, this is not surprising.

PEOPLE

AAnnaallyyttiiccaall cchheemmiissttss rreecceeiivvee aawwaarrddssJonathan V.Sweedler, profes-sor at the Univer-sity of Illinois atUr-bana–Champaignand the NeuronalPattern Analysisand BiologicalSensor groups at

the Beckman Institute, will receive the2002 Heinrich-Emanual-Merck prize forAnalytical Chemistry. The award is givenby Merck KGaA, Darmstadt (Germany),and recognizes chemists whose work pro-vides solutions to analytical problems inthe areas of life sciences, material sci-ences, or the environment.

Sweedler’s work currently involvesscaling spectroscopic methods to the

nano- and attoliter-volume regimes. Hisgroup has also developed detection sys-tems for CE; MALDI time-of-flight MSsystems to profile peptides in individualneurons and cellular processes; and high-resolution, nanoliter-volume NMR spec-troscopy for liquid-phase samples. Theaward will be presented in April toSweedler in Munich at the Analytica2002 meeting.

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Thomas C. Wer -ner, professor atUnion College,received the 2002ACS Award forResearch at anUndergraduate Institution, spon-sored by ResearchCorp. The award

was announced at the ACS NationalMeeting in April in Orlando, Fla.

Werner received his B.S. in chemistryfrom Juniata College (1964) and hisdoctorate in analytical chemistry fromthe Massachusetts Institute of Technol-ogy (1969). He held postdoctoral posi-tions at Harvard Medical School andMassachusetts General Hospital (1969–70) and was a research associate at TuftsMedical School (1970–71). Wernerserved as a professor and the chair ofchemistry at Union College (1985–91),where he is currently the Florence B.Sherwood Professor of Physical Sciences.

MMiicchhaaeell JJ.. WWeeaavveerr((11994477––22000022))

Michael J. Weaver,professor of chem-istry at PurdueUniversity, diedunexpectedly onMarch 21, 2002.

Weaver re-ceived his B.Sc.from BirkbeckCollege, Universi-

ty of London, in 1968 and his Ph.D. fromImperial College of Science, Technology,and Medicine, University of London, in1972. He served as a postdoctoral re-search associate at the California Instituteof Technology from 1972 to 1975. In1975, he began his academic career as anassistant professor at Michigan State Uni-versity; in 1982, he came to Purdue Uni-versity as an associate professor and waspromoted to professor in 1985.

An electrochemist by definition, Weaveralso explored surface chemistry in the gasphase; vacuum and nanomolecular envi-ronments; photon-based vibrational tech-

niques; and analytical, physical, and mate-rials chemistry. Weaver was best knownfor his pioneering work on surface-en-hanced Raman scattering and IR spec-troscopy at electrochemical interfaces.

Weaver’s list of accolades and nomina-tions is considerable. His most recenthonor was being recognized as one of thecountry’s top 100 chemists from 1981 to2000, according to a report by the Inter-national Science Institute. He receivedthe 1989 David C. Graham Award fromthe Electrochemical Society; the 1995Faraday Electrochemistry Medal from theRoyal Society of Chemistry; the 1997Carl Wagner Memorial Award from theElectrochemical Society; and the 1999Electrochemistry Award from the Ameri-can Chemical Society. He was recentlynominated for a Purdue DistinguishedProfessorship award.

His colleagues say that his unexpecteddeath is a major loss to Purdue and the sci-entific community. “It was a big blow,”says Hilkka Kenttämaa, professor and headof Purdue’s analytical chemistry division.“This tragic event is an astronomical lossto fundamental electrochemistry, whichmay never be fully restored,” says AndrzejWieckowski, professor of chemistry at theUniversity of Illinois and a personal friendof Weaver’s. “His loss will set the commu-nity a decade back, and major efforts willbe needed to restore the equilibrium.”

“[Mike] always got excited about ourwork,” says graduate student Sally Wasiles-ki, a member of the Weaver group. “Hewould say to us that having good grad-uate students and watching them evolve[into] scientists was one of his favoritethings about being an academic.”

In some ways, Weaver seemed to be astep behind the world outside of chem-istry. “He still wrote scientific papers on alegal pad with a number-two pencil,” saysWasileski. “Every e-mail he ever wrote, hewould sign, ‘Cheers Mike,’ which hebragged he could type with two fingers.”She adds, “We senior students will contin-ue to do the work Mike would have want-ed us to do and try our best to meet upto Mike’s very high standards.”

——WWiillddeerr DD.. SSmmiitthh

Documents

Analytical Currents: Clear, but not clean water