First came microarrays of DNAs, thenproteins, and now living cells. This neweststrategy, developed by David Sabatini andJunaid Ziauddin of the Whitehead Insti-tute for Biomedical Research, allows thefunctional analysis of many gene productsin parallel and can be used to identifydrug targets or to discover gene prod-ucts that alter cellular physiology.

To begin, plasmid DNA that has beenencapsulated in gelatin is robotically plat-ed in a grid pattern on a slide. The slideis exposed to a lipid transfection reagent,placed in a culture dish, and coveredwith a medium containing cells. Whencells grow over a spot, they take up theplasmids and express the new DNA.

For example, the researchers platedmammalian cells on a slide containing aconstruct for green fluorescent protein(GFP). Cells next to the array spotswere transfected and expressed GFP,which was easily verified using fluores-cent light. The array spots were ~150-

µm diam, so theGFP-expressingclusters contained30–80 cells. Otherexperiments trans-fected cells withDNA for specificreceptors; thesecells were thenidentified with ap-propriate ligandsfor the receptors.

In a more so-phisticated experi-ment, microarrayscontaining 192 different complementa-ry DNAs cloned in expression vectorswere used to transfect mammalian cells.Screening these transfected cells identi-fied proteins involved in activities suchas tyrosine kinase signaling and cell deathand adhesion. Another analysis investi-gated subcellular distributions, such as aprotein that concentrated in the nucleus.

This new strategy works with variouscomplementary DNAs and detectionschemes and identifies cells that haveexpression levels only slightly abovebackground or that undergo transientchanges. The most difficult step, say theauthors, is making the DNA expressionconstructs for the microarrays. (Nature22000011,, 411, 107–110)

news

DNA/gelatin

Slide

Cells Lipid

Assay

(a) (b)

(a) The microarray protocol and (b) the laser scan of a microarray inwhich cells have taken up plasmid DNA and now express the greenfluorescent protein. (Adapted with permission. Copyright 2001 NaturePublishing Group.)

LLiivviinngg cceellll mmiiccrrooaarrrraayy

ANALYTICAL CURRENTS

J U LY 1 , 2 0 0 1 / A N A LY T I C A L C H E M I S T R Y 3 4 9 A

CCaattaallyyssiiss bbyy FFRREETTHigh-throughput screening has been so

well received in the pharmaceutical com-

munity that this powerful technique is mi-

grating into other areas of chemistry. In

this example, high-throughput methods

identify the optimal catalysts and catalytic

conditions for the formation of a-aryl

cyanoacetates. What makes this report in-

teresting is that

John Hartwig and

colleagues at Yale

University followed

the reactions by

fluorescence reso-

nance energy

transfer (FRET).

One of the start-

ing materials was tagged with a fluorophore

and the other with a quencher. When the

cyano acetate formed, the fluorescence

dropped. Thus, the FRET emission was in-

versely related to the reaction yield.

The formation of a-aryl cyanoacetates

was aided by the presence of a palladium

catalyst. Using a 96-well format and the

FRET data, the researchers investigated

the effects of various forms of the catalyst,

changing bases, and different solvents on

the reaction yield. (J. Am. Chem. Soc. 22000000,,

123, 4641–4642)

NMe2

O2S ON

O

CN

NMe2

O2S ONO

CNBocN

NN

Catalyst

Weak fluorescence

Strong fluorescenceBocN

N

Br

N

+

Reaction to form an a-aryl cyanoacetate, which was used to eval-uate catalysts.

3 5 0 A A N A LY T I C A L C H E M I S T R Y / J U LY 1 , 2 0 0 1

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Researchers know that capillary elec-trochromatography (CEC) often per-forms better than capillary HPLC. Thereason why has been less certain. ButUlrich Tallarek, Ernst Bayer, and col-leagues at Wageningen University (TheNetherlands) and Universität Tübingen(Germany) offer evidence that electroos-mosis through, rather than around, theporous media in the capillary is largelyresponsible for the improvement.

The researchers compared pressure-driven and electroosmotic flows througha fixed bed of electrically charged, porous

particles packed in a capillary under vari-ous conditions. By using NMR to tracethe fluid molecules, they separated theintra- and interparticle kinetics. Focusingon the intraparticle mass transfer kinetics,they determined that applying an electricfield significantly enhances the exchangeof fluid molecules, whereas applying pres-sure to the porous particles has little effect. This, along with other observa-tions, argues that an electroosomoticperfusion mechanism exists.

The researchers note that the inter-connectivity of the particles’ pores ismore important than their average di-ameter for electroosmotic perfusion.They suggest that attention to boththe electroosmotic and separation char-acteristics of CEC particles may enhanceanalyses. (Angew. Chem., Int. Ed. 22000011,,40, 1684–1687)

Gigapore

+

– – ––

–

++ +

+

+ ––

– ––

–

++ +

++

++

+

+

+ + + ++

+

–– –

––

– – ––

–

Macropore

Nominal particle

Subparticle

Dead-end poresFlow-through

pores

Pore flow

E

θ uax

ueo

uax ∝ E·cos2θ

Schematic showing the pore network in a hi-erarchically structured particle and the elec-troosmotic flow through a gigapore. (Adaptedwith permission. Copyright 2001 Wiley-VCHVerlag GmbH.)

EEvviiddeennccee ffoorr eelleeccttrroooossmmoottiicc ppeerrffuussiioonn

ANALYTICAL CURRENTS

Forensic toxicologists routinely test biolog-

ical materials for a wide array of pesti-

cides, therapeutic and illicit drugs, and

other xenobiotic substances. In previous

broad-scale screening techniques, access

to reference substances has limited the

availability and content of chromatograph-

ic or spectroscopic libraries, whether they

were generated in-house or purchased.

However, in drug discovery applica-

tions, time-of-flight MS (TOFMS) has

shown promise in determining the elemen-

tal composition of metabolites. Building on

this use, Merja Gergov and colleagues at

the University of Helsinki (Finland) and Ap-

plied Biosystems created an LC/TOFMS

method to qualitatively screen for toxico-

logically relevant drugs and their main

metabolites in urine samples without ref -

erence compounds. The researchers also

established an automated calibration ca-

pability and a post-run computer search

based on elemental formulas to help

process the data.

After solid-phase extraction and LC

separation of the sample, a full TOFMS

spectrum was recorded, automatically cal-

ibrated, and compared with the target li-

brary for positive identification. Entering

each substance’s elemental formula into a

program that calculated monoisotopic

masses created the target library, which

contains 433 drugs and metabolites rang-

ing from 105 to 734 Da. Final identification

was based on exact mass. Collection of

full spectra was a benefit because sam-

ples could be searched again for a par -

ticular compound without re-running.

The LC/TOFMS method was tested

with autopsy urine, and the 5- to 10-ppm

mass accuracy obtained for most com-

pounds compared well with GC and thin-

layer chromatography analyses. (Rapid

Commun. Mass Spectrom. 22000011, 15,

521–526.)

LLCC//TTOOFFMMSS ttooxxiiccoollooggyy ssccrreeeennss

J U LY 1 , 2 0 0 1 / A N A LY T I C A L C H E M I S T R Y 3 5 1 A

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On a macroscale, it’s possible to createpatterns in solutions containing paramag-netic particles by applying external mag-netic forces, which induce the formationof particle aggregates. Mark Hayes andcolleagues at Arizona State Universityand ThermoBiostar have been extendingthese observations to fluid-based micro -

devices as an alternative to traditionalmicrofabrication techniques.

The researchers demonstrate thatparamagnetic particles in microchannelsproduce unique, dynamic, and control-lable structures when interactions are in-duced and manipulated via an externalmagnetic field. Direct physical interfacewith a magnet is not required to see theeffect. These induced structures, referredto as supraparticle structures, are not at-tached to any channel wall and are notdistorted when rotated or when trans-ported by pressure or electrokinetic flow.Optical microscopy shows that the struc-tures respond to changes in externalmagnetic field strength and orientation,

and Brownian motion and electrostaticrepulsion quickly restore the original dis-array of the colloidal suspension oncethe magnetic field has been removed.

The formation of supraparticle struc-tures depends on the particle diameters,the volume fraction of the solution, themagnetic susceptibility, the intensity of

the magnetic field, the channel geometry,the system’s temperature, and the surfaceproperties of the microchannels. Thesefactors will influence possible on-chip ap-plications such as the fabrication of opticsor structural elements for separations, fil-tration, or biological interactions. (Lang-muir 22000011, 17, 2866–2871)

A

B

C

D

E

F

B

Behavior of paramagnetic particles in 20-µm-diam microchannels. (A) No applied externalmagnetic field, B. (B–E) Alignments of para-magnetic particles with different orientationsof B. (F) Upon removal of B, paramagneticparticles resume colloidal state.

AA lliinneeuupp iinn tthhee mmiiccrroocchhaannnneell

Imagine placing a single cell exactly

where you need it. For applications rang-

ing from building biosensor circuits, to

screening combinatorial libraries, to engi-

neering new tissues, such control would

be beneficial. George Whitesides and his

colleagues at Harvard University and Chil-

dren’s Hospital and Harvard Med-

ical School describe a method for

depositing proteins or cells selec-

tively into microwells and demon-

strate their approach by placing

mammalian cells into wells with

≤100-µm diams and ≤50-µm depths.

The microwells were fabricated

in the inert, transparent elastomer

poly(dimethylsiloxane). To keep the

cells off of surfaces other than the

microwells, the space between the

wells was coated with bovine serum

albumin (BSA). Air bubbles trapped in the

microwells kept any BSA from entering

these depressions. The microwells were

then coated with the protein fibronectin,

which promotes adhesion of cells.

The coated microwell plate was then

exposed to a suspension of bovine capil-

lary endothelial cells. In a typical experi-

ment with 50-µm-diam, 1.3-µm-deep mi-

crowells, the bovine cells adhered to

~70% of the wells. Narrower and deeper

wells had lower occupancies, and wider

and deeper depressions had enough

space for multiple cells in one well.

(Langmuir 22000011,, 17, 2828–2834)

Bovine capillary endothelial cells adhere selec-tively to wells coated with fibronectin. A layerof bovine serum albumin prevents adhesion be-tween the wells. (Inset) Two cells have spreadto entirely cover their wells.

CCeellllss iinn aa ppaatttteerrnn

3 5 2 A A N A LY T I C A L C H E M I S T R Y / J U LY 1 , 2 0 0 1

news

The scanning near-field microscope(SNOM) moved optical microscopy pastthe Rayleigh diffraction limit, which heldresolution to one-half of the optical wave-length. Instead, the SNOM’s resolutionincreases as its tip diameter decreases.Unfortunately, the intensity of the lightalso decreases.

A way around this limitation couldbe the scanning near-field exciton mi-croscope (SNEM), which creates imagesusing excitons—electronic excitations ina dielectric media—rather than an opti-

cal light field.Elisabeth Paule and Peter Reineker

of the Universität Ulm (Germany) in-vestigate the imaging properties of aSNEM and provide a theoretical frame-work for the optimization of its resolu-tion and contrast. They also model thedetection of single molecules on a sur-face, studying the intensity as a functionof the wavelength of the incident elec-tric field and the distance between thetip and sample. (J. Phys. Chem. B 22000011,,105, 4293–4304)

TThheeoorryy ffoorr aa SSNNEEMM

ANALYTICAL CURRENTS

Researchers have previously harnessedsingle-stranded DNA “hairpins” to serveas fluorescent probes, popularly knownas “molecular beacons”. Now, JocelynGrunwell and colleagues at the Universi-ty of California–Berkeley and LawrenceBerkeley National Laboratory use hair-pins as a model system for studying thekinetics of single molecules bound tosurfaces.

The researchers attached hairpins toglass surfaces using one of two biotin–streptavidin interactions or using a cova-lent immobilization strategy. They mon-itored the opening and closing of the

hairpins using fluorescence imaging andapplied a two-state model when analyz-ing the data.

On the basis of a previous study, theresearchers predicted that the averageopen-state lifetime for their hairpins wouldbe ~3 ms. Instead, the calculated lifetimeswere 133 ± 5.5 ms for a hairpin with a 7-base-pair (bp) stem and 142 ± 22 ms fora 9-bp stem. The difference between pre-dicted and actual was apparently due tovariations in the loops’ sequences. On theother hand, closed-state lifetimes were 45± 2.4 ms for a 7-bp stem and 103 ± 6.0ms for a 9-bp stem, and as expected, they

were independent of theloop characteristics.

The relatively longopen-state lifetimes sug-gest that being tetheredto a surface stabilizes theopen state of the hairpinand affects the overall ki-netics of the system. Thus,the researchers concludethat models derived frompolymer simulations andthermodynamic calcula-tions may not be generallyapplicable to hairpins andthat sequence informationshould be considered. (J.Am. Chem. Soc. 22000011,,123, 4295–4303)

Cy5

TMRBiotin or thiol forsurface attachment

Closed Open

kopen

kclosed

Even

ts

Even

ts

Energy transfer0 1

Energy transfer0 1

Schematic of the closed-to-open transition of a DNA hairpin.

IInnddiivviidduuaall hhaaiirrppiinn ddyynnaammiiccss

To improve the efficiency of DNA mi-

croarray analysis, researchers have

been working on ways to integrate on-

chip PCR. Sergei Tillib, Andrei Mirz-

abekov, and Boris Strizhkov at Ar-

gonne National Laboratory and the

Russian Academy of Sciences de-

scribe an approach in which on-chip

gel pads serve as reaction chambers

for PCR amplification.

Immobilized within each gel pad

are many copies of the two primers

needed for PCR. The sample DNA is

allowed to hybridize with the primers,

and the unhybridized sequences are

removed by washing. Then the chip is

soaked in a solution containing PCR

reagents and ribonuclease A, which

releases one type of PCR primer from

its tethers—a step that improves the

amplification efficiency. A third primer

can be used to test for particular sin-

gle-nucleotide mutations. After amplifi-

cation, the product is denatured and

allowed to interact with fluorescent

probes attached elsewhere on the

chip’s surface. The researchers tested

the method by analyzing mutations in

three antibiotic resistance genes in

Mycobacterium tuberculosis. (Anal.

Biochem. 22000011,, 292, 155–160)

PPoorroouuss ppaaddss ffoorr PPCCRR

J U LY 1 , 2 0 0 1 / A N A LY T I C A L C H E M I S T R Y 3 5 3 A

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For researchers, the complexity of can-cer continues to be vexing. So manythings can go wrong in a cell and leadto its transformation that it is difficultto pinpoint the specific mechanisms thatunderlie disease. Add to that the unfor-tunate fact that practically every knownvariety of cancer follows a different patho-logical course, and it is little wonder thatthe “war on cancer” has produced onlyspotty victories.

Even as the mutations and geneticcascades that lead to tumorigenesis be-come known, the contributions of envi-ronmental factors such as diet remainelusive. “There is a schoolof thought that some can-cers are initiated by a . . .modification of the DNAby some exogenous com-ponent,” says Paul Vourosof Northeastern Universi-ty. But so far, theory hasventured ahead of method-ology, and there is littleproof to support the hy-pothesis. Before research -ers can gauge exposurerisk accurately, they needtools to directly measuremolecular impact on cells.

In the current issue ofAnalytical Chemistry (pp2819–2827), Vouros,Robert Turesky, and their colleagues atNortheastern, the U.S. National Centerfor Toxicological Research, and Nestec,Ltd. (Switzerland) report a method thatthey hope will give researchers the powerto test exogenous influences on tumori-genesis. The method combines LC andmicroelectrospray ionization MS/MSto detect modified bases starting with~300 µg of DNA. That translates intofinding ~200–300 modifications percell, says Vouros. Although it’s a formi-dable task, it is close to the level thatis necessary to investigate the contribu-tions of diet to cancer risk.

The enhanced ability stems primarilyfrom an improvement in fluidics. A stan-

dard LC runs at 0.5–1.0 mL/min, butmore recent work has introduced flowrates of 10–20 nL/min. For these stud-ies, 10- to 20-nL flow rates couldn’tbe achieved routinely, so Vouros andhis colleagues settled on ~200 nL/min.“That provided a very dramatic improve-ment in sensitivity,” he says.

To demonstrate the method, theteam looked at the heterocyclic aromat-ic amine 2-amino-3-methylimidazo[4,5-f ]quinoline (IQ), which is a potentcarcinogen in rodents and nonhumanprimates. IQ is found in cooked meatand fish and in cigarette smoke conden-

sate, and it forms stable adducts withDNA. The researchers dosed rats with.05, .5, 1, and 10 mg/kg of IQ bybody weight and collected liver DNAsamples 24 h later. Even at the lowestdosage, the researchers could detect andquantify adducts with a S/N of 16:1,and the results were reproducible fromday to day.

The LC/MS approach has some ad-vantages over the most commonly usedmethod to measure DNA adducts: 32Ppost-labeling, in which adenosine triphos-phate—the cell’s fuel—is tagged withthe radiolabel, and the modified DNA isdetected by thin-layer chromatographyor HPLC. “[One] problem is that the

technique does not necessarily have ahigh resolving power,” Vouros explains.“You may see one spot, but it may actu-ally have two or three or more [kinds ofadducts] because the resolution is insuf-ficient, particularly for human samples,where a number of lesions may be de-tected.” In addition, the 32P post-label-ing does not provide structural informa-tion to identify the adduct. MS hasmore resolving and defining capabilitythan the radioactive method, he says.

However, the sensitivity of the capil-lary LC/MS technique (2 adducts in108 bases) hasn’t reached that of radio -

activity (1 adduct in 1010

bases). Still, the researchersare edging closer. “I thinkwe’re becoming competi-tive,” says Vouros. “The in-teresting thing is that you really cannot get by withone method alone, in myopinion . . . . [We are goingto] use those two techniquesin parallel.”

In time, Vouros hopes theMS technique’s sensitivityeventually will surpass thatof 32P post-labeling. Thework to date has been doneon a 10-year-old mass spec-trometer, he notes. “My ex-pectation is that, with better

instruments, people should be able toimprove on what we’re reporting nowby at least a factor of 10, if not better,in the very near future,” he says. Thereal test will be whether the methodcan detect DNA adducts from the lungtissue of, say, a chronic smoker.

Vouros thinks he’s close. “With a lit-tle bit better manipulation and improv-ing our know-how—and with a newgeneration of instruments—this canbe translated to humans in the near future,” he says. If he’s right, the oldquestion of Nature versus nurture, atleast as applied to cancer, might finallybe answered.

––JJiimm KKlliinngg

AAsssseessssiinngg ccaarrcciinnooggeenn eexxppoossuurree wwiitthh MMSS

Negative pressuresample introduction

Microbore valve with

0.5 µL internalsample loop

Waste

Packed capillarycolumn (75 µm I.D.)

Sheathless/liquid junctionµESI tip

400 µL/min. 200 nL/min. S

+–

Finnigan TSQ700

HV

Splitter

Schematic of the capillary LC/microelectrospray ionization MS system.

RESEARCH PROFILES

3 5 4 A A N A LY T I C A L C H E M I S T R Y / J U LY 1 , 2 0 0 1

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RESEARCH PROFILES

The protozoan parasite Cryptosporidiumparvum, usually found in water, can belife-threatening to people with impairedimmune systems and may cause seriousgastrointestinal illness in healthy people.The parasite’s oocysts are not killed bystandard disinfection methods such aschlorination and have caused major out-breaks of illness in the United States,United Kingdom, and Japan.

The standard proce-dure to detect C. parvumoocysts is staining withfluorescent antibodies andidentification under a mi-croscope. However, thisapproach often incorrectlyestimates the number oforganisms because it does-n’t distinguish betweenliving and dead parasites.In the current issue ofAnalytical Chemistry (pp3162–3167), RichardDurst and colleagues atCornell University de-scribe a quick, sensitive,test strip-based methodto detect small amountsof living oocysts via thebinding of amplified RNA to oligonu-cleotides immobilized on liposomes.

The method takes advantage of thefact that only living organisms produceRNA. “DNA stays around for a longtime,” Durst explains. “In contrast,messenger RNA is rapidly degraded.So if an organism is dead, [its RNA]won’t be around for very long.”

The procedure involves heating theorganisms to make them produce largeamounts of the RNA for heat shock pro-tein 70 (hsp70)—part of this sequenceappears to be unique to C. parvum—and extracting the RNA (Anal. Chem.2001, 73, 1176–1180). A segment ofthe hsp70 RNA is further amplifiedusing nucleic acid sequence-based am-plification (NASBA)—a method similarto PCR except that it does not requirethermal cycling—to produce a single-

stranded RNA target sequence. Lipo-somes bearing biotin and oligonu-cleotide reporter probes, which arecomplementary to the target sequence,are then added to the mixture. Theprobes will hybridize to any target se-quence that is present. The mixture isthen applied to a nitrocellulose teststrip.

The test strip contains two zones. The

first, called the capture zone, containsoligonucleotides that are complementa-ry to the reporter probe (and thus iden-tical to the target sequence), whereasthe second, called the antibody zone,contains anti-biotin antibodies. If notarget sequence is present, then the re-porter probes on the liposomes will bindto the oligonucleotides in the capturezone, providing an internal control. If,on the other hand, the target sequencewas present in the sample, then the re-porter probes on the liposomes will al-ready be bound to the target, leaving nosites available to bind to oligonucleo tidesin the capture zone. In this case, the li-posomes will migrate by capillary actionto the antibody zone, where their biotinlabels bind to the anti-biotin antibodies.Because the liposomes encapsulate a reddye, they are easily visualized in either

zone, indicating whether living parasitesare present in the test sample.

Once added to the strip, the test takesonly 8–10 min, says Durst, although ex-tracting the RNA from the organisms andperforming the NASBA takes an addition-al 2–3 h. “But even going through that,we still have an assay that can be donein less than a day,” he points out. Themethod can detect as little as 80 fmol of

C. parvum target sequence.The researchers devel-

oped the test-strip formatfor easy in situ use atwater treat ment plants.(Unlike PCR, NASBAcan be done with a simpleheating block rather than special thermal cyclingequipment.) Durst wouldalso like to see the testused in restaurants. “Ifyou’re washing food withcontaminated water,you’re taking somethingthat’s safe and contami-nating it,” he says. Anat-home test may evenbe possible in the future.

This approach is justone of many that Durst’s laboratory hasdeveloped using liposomes, and it couldbe easily adapted to other organisms bychanging the reporter probe and thecomplementary oligonucleotide, he says.For example, the researchers have useda similar RNA-based approach to detectShiga toxin produced in live E. coliO157:H7 or Shigella. In addition, anti-bodies or proteins can be attached tothe liposomes instead of the oligonu-cleotides. In collaboration with LaurieLocascio and Michael Tarlov at the Na-tional Institute of Standards and Tech-nology, the researchers also developeda micro fluidic C. parvum test, which iseven more sensitive (pp 2952–2958).“Sensitivity is one of the key advantageswe have with the liposome,” says Durst.“It’s almost a universal reagent.”

––AAllkkaa AAggrraawwaall

Side view

2

1

2

1

Side view

Antibiotin antibody

Biotin

Reporter probe andantisense reporterprobe

Targetoligonucleotide

Liposome

Schematic of the test strip assay. In a positive test, the liposome binds to (2) the antibody zone. In a negative test, it binds to (1) the capture zone.

DDeetteeccttiinngg lliivviinngg CCrryyppttoossppoorriiddiiuumm

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LABORATORY PROFILE

“Electrochemistry works!” was the radioreport from 2.5 km below the ocean’ssurface, 5 hours into the dive to studythe thermal vents off the west coast ofMexico. With this comment the star-board observer, Don Nuzzio, analyticalchemist and president of Analytical In-strument Systems, Inc. (AIS) of Flem-ington, NJ, marked the end of 2 yearsof hard work to get aboard the deepsubmergence vehicle (DSV)Alvin and signaled the sur-face support group to startcelebrating. That was oneyear ago.

Nuzzio founded AIS in1989 with the goal of makinglow-cost, portable, rugged,and reliable analytical instru-ments for use in the laborato-ry or the field. AIS receivedtwo Small Business Innova-tion Research grants to de-velop equipment to correlatethe chemistry at deep-seathermal vents with the distri-bution of life forms typicallypresent there. In one grant,the National Science Foun-dation funded the develop-ment of a probe to measuretemperature, pH, and electrochemistryin water columns and in the sedimentbeds below them. In the other grant,the National Aeronautics and Space Ad-ministration funded the adaptation ofthis instrument to the micromanipulatorson DSV Alvin, the small, maneuverable,underwater vehicle developed by Wood’sHole Oceanographic Institute (WHOI)in 1964. In a typical untethered dive,Alvin can transport a pilot and two ob-servers in relative comfort and safety toa depth of 4.5 km, where they can navi-gate the bottom, collect samples, takephotographs, and resurface the same day.

AIS built a submersible potentiostatto control several electrodes and performelectrochemical analyses. The electronicsinclude a microprocessor that can becontrolled by someone inside the per-

sonnel sphere via laptop computer. “TheAlvin instrument is capable of perform-ing most standard electrochemical tech-niques—dc, linear sweep, and cyclicvoltammetry; differential pulse, squarewave, and stripping analysis—all selec-table through the laptop,” says Nuzzio.

The electronic portion of the instru-ment weighs about one pound, but thealuminum casing to protect it from

the crushing pressures at the sea floorbrings the unit’s total weight to about100 pounds. This hardware was designedto operate with a special probe, madeby George Luther of the University ofDelaware, which includes voltammetricelectrodes, a temperature probe, and asyringe sampler to collect water samplesfor further analysis on the surface. Theprobe was inserted into the flowing wa-ters of the hydrothermal vents and en-dured extreme temperature shifts—from400 °C at the vents to 2 °C in theocean only a short distance away.

Entire ecosystems exist at black smok-ers—the thermal vents on the sea bot-tom that spew a hot solution of dissolvediron, hydrogen sulfide, and other com-plex ions into the cold dense brine at thesea floor. The origin and distribution of

the creatures that live there and survivein total darkness in a completely chemo -synthetic food chain has fascinated sci-entists since their discovery. In situ elec-trochemistry, pH, and temperature datawere combined with analyses performedaboard the surface support ship to helpunderstand the biology within the steeptemperature and concentration gradientsfound at the vents. The results were

published this year (Nature22000011,, 410, 813–816).

There are several compel -ling reasons to use voltam-metry in this setting. Samplescollected at the bottom andtransported to the surface foranalysis undergo a 200-foldchange in pressure. Sulfideequilibria, notoriously tem-perature- and pressure-sensi-tive, are difficult to duplicateand study at the surface.Measurements performed insitu should give much morereliable results, Nuzzio rea-soned. He chose electro-chemistry because it can sam-ple small volumes quicklyand reliably under extremeconditions. In addition,

voltammograms give information aboutthe identity and amount of the elec-troactive species present. “[Yet] the realpower of electrochemical measurementin situ is . . . that it can reveal informa-tion about species complexation—infor-mation not available with most metal-sensitive spectroscopies,” says Nuzzio.

“The Alvin crew generally has askeptical view of new equipment,” saysNuzzio. But the system worked so wellthat the same instrument—with a small-er housing and remote operating capa-bilities—was deployed from a platformoff the Knorr, another WHOI researchvessel, in late May to map out the watercolumn in the Black Sea. And Nuzziowas along to make sure everything wentsmoothly.

––ZZeellddaa ZZiieegglleerr

Nuzzio runs electrochemical analyses aboard the Alvin.

CO

UR

TES

Y O

F D

ON

NU

ZZIO

.

EExxttrreemmee eelleeccttrroocchheemmiissttrryy

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GOVERNMENT AND SOCIETY

At the August 2001 ACS National Meet-ing in Chicago, IL, board members areexpected to vote in favor of creating anew ACS division focused on laboratoryautomation. The new division will stemfrom a partnership with the Lab Robot-ics Interest Group (LRIG; http://lab-

robotics.org), a grassroots organizationbased out of Martinsville, NJ, whichbegan 14 years ago as a topical group ofthe North Jersey section of the ACS.

Dennis France of Novartis Oncology,executive chair of the LRIG Mid-AtlanticChapter, says the idea to create a new di-

vision sprang from the increasing growthof and demand for robotics in areas suchas high-throughput screening, drug dis-covery, and combinatorial chemistry.

This partnership between LRIG andthe ACS is not the first. “We would havejoint meetings with the ACS, and theturnout would be disappointing,” saysFrance. “We had all but given up on theACS because of those failed meetings.”But a renewed dedication by Les Mc-Quire of Novartis, former chair of theNorth Jersey section of the ACS, andother key players—namely Mark Hay-ward, chair-elect of the North Jersey MSgroup (Novartis); Bill Suits, current chairof the North Jersey section of the ACSdivision; and Alan Cooper (Schering-Plough)—persuaded France and his col-leagues at LRIG to give ACS another try.

Hayward, who pitched the renewedpartnership to Divisional Activities Com-mittee members at the 221st ACS Na-tional Meeting in San Diego, CA, saysthat the merger will address unmet needson both sides. “ACS had always hadsuccess with their national meetings butneeded a way to have the same successon a local or regional level, which theLRIG could provide,” says Hayward.“By the same token, a merger with ACSgives the LRIG the ability to provideorganized local service on a nationalplateau.” The LRIG currently has sevenchapters under its umbrella: Mid-Atlantic,New England, Southeast, Bay Area, SanDiego, Northwest, and European.

The long-term vision for the new ACSLaboratory Automation Division is torun more productive meetings, increasemember and vendor participation, andcontinue to provide researchers in thedrug discovery/high-throughput screen-ing market with cutting-edge instruments.“Since all of us are extremely busy sci-entists, we are trying to keep the infra-structure and processes simple,” saysFrance, “because the day that it becomescumbersome is the day that it may fallapart like a house of cards.”

––WWiillddeerr SSmmiitthh

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In May 2001, milk safety specialists atthe biennial National Conference on Interstate Milk Shipments (NCIMS) reviewed and approved a new multi-channel immunoassay as a method forscreening U.S. milk for the b-lactamfamily of antibiotics, reports NCIMSLaboratory Committee Chairman LeeJensen. The b-lactam family—namedfor the b-lactam ring in the structure—includes several heavily used antibi-otics, including penicillin,cephalosporin, and their deriv-atives. NCIMS acceptance ofany new test kit is the ulti-mate stamp of approval for using the assay in theU.S. National Milk DrugResidue Testing Program.

According to a 1992General Accounting Officereport to the U.S. Houseof Representatives, the Na-tional Milk Drug ResidueTesting Program was estab-lished after surveys conductedseveral years earlier by the WallStreet Journal and the Center for Sci-ence in the Public Interest indicatedthat 20% and 38%, respectively, of retailmilk samples tested may have containedresidues from drugs given to animals.Among those drugs were the suspectedhuman carcinogen sulfamethazine anddrugs not approved by the U.S. Foodand Drug Administration (FDA). Ac-cording to Rob Byrne of the NationalMilk Producers Federation, “The levelsof antibiotics in milk can be a consumerpublic health issue of antibiotic resist-

ance, [and] high levels of antibiotics inmilk [also] may reflect disease in thedairy cow.”

The maximum residue level allowedin milk for each of the six b-lactamsvaries between 5 and 50 ppb. The cur-rently approved tests are able to detectthe specified levels of four or five of thesix b-lactams approved for use in dairycattle, says a spokesperson from the

FDA’s Center for Veterinary Medi-cine (CVM). To test for all sixb-lactams, two or more testsmust be used. However, thenew test—an immunoassaycalled the Parallux systemfrom Idexx, Inc.— “pro-vides the means to detectall six of the currentlyused b-lactams and canalso be used to identifythe specific drug,” says theCVM’s Norris Alderson.

Other changes approvedat the NCIMS were aimed at

reducing false-positive results,which can waste many gallons of

milk and millions of dollars, Byrnesays. For example, any new test cannotbe >50% more sensitive than the regu-lated safety level of the targeted drugresidue. In addition, milk testing proto-cols were changed to further reducefalse-positives. Finally, he says, if a testkit is resubmitted to the FDA for evalu-ation because of changes in its ability todetect one drug residue, data will be ex-amined for all drugs that the kit claimsto detect.

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