Analytical Currents: DNA arrays without amplification

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ANALYTICAL CURRENTS DNA arrays without amplification In DNA arrays, PCR is increasingly seen as an undesirable complexity. Now, SoJung Park, T. Andrew Taton, and Chad Mirkin at Northwestern University describe a DNA array assay that does not require amplification or temperaturedependent hybridization to identify mismatched targets. The new assay relies on Mirkin’s strategy of labeling one end of the target DNA with gold nanoparticles. When the target DNA binds to probe DNA immobilized between two electrodes, the gold nanoparticles fill the gap between the electrodes. In principle, the amount of target DNA can be determined by capacitance or conductivity measurements, but in practice, the sensitivity is improved if the gold nanoparticles are enhanced with a silver coating. The coating is applied by immersing the device in a silver-containing solution after the targets have hybridized to the probes.

Ag+ hydroquinone

e–

Illustration of the electrical detection of DNA, beginning with probe molecules immobilized between two electrodes and finishing with the detection of target DNA molecules labeled with gold nanoparticles enhanced with silver. (Adapted with permission. Copyright 2002 American Association for the Advancement of Science.)

Analyses were conducted on arrays of four electrode pairs. In one example, the four corresponding probes represented the correct complementary sequence and three possible mismatches for a target derived from the sequence for the anthrax lethal factor. The typical strategy for distinguishing matched from mismatched targets is to raise the temperature during hybridization, because perfectly matched targets are more stable at higher temperatures. A perfectly matched strand was shown to

Revealing fragile ganglioside structures Gangliosides, which are found in eukaryot-

skaya, and Catherine Costello of the

ic cells, serve crucial functions as anti-

Boston University School of Medicine

gens, receptors, and intercellular signaling

use a high-pressure (1–10 mbar) bath

have a signal 105 times stronger than the mismatched targets—significantly better than the selectivity ratio obtained with a comparable fluorescence-based assay (2.6:1) or Mirkin’s “scanometric” approach, which uses the same nanoparticle labeling scheme (11:1). The researchers also found that they got better mismatch discrimination if they varied the Na+ concentration instead of the temperature. They achieved detection limits as low as 500 fmol of target DNA. (Science 2002, 295, 1503–1506)

1305.80

(a) 1277.77 1000 1250

1552.90 1500

1750

2000

(b)

2250

molecules. Small structural variations in

gas under MALDI-FTMS desorption

these complex lipid–oligosaccharide mole-

to generate ions with a low average

cules are responsible for specific biologi-

vibrational energy. Metastable frag-

–2Neu5NAc(Na) 1618.88 –Neu5NAc(Na) –3Neu5NAc(Na) 1931.96 1305.80 LH

cal activities, and mixtures of gangliosides

mentation is minimized by the ex-

1000 1250

are usually found in biological systems. But

change of hydrogen for sodium ion

glycosidic bonds, particularly the fragile

and collisional cooling. Unfragment-

1500

1750 2000 m/z

2500

2750

[M(3Na)+Na]+ 2245.02

2250

2500

2750

bonds linking sialic acid residues, fragment

ed, sodiated molecular ions with up

extensively under electrospray and MALDI

to five sialic acid residues become

Positive-ion MALDI-FTMS spectra of ganglioside GT1b (a) without and (b) with high-pressure collisional cooling gas. (Adapted with permission. Copyright 2002 Elsevier Science.)

ionization, and so these mixtures have

the base peak in both positive- and

matography assays to correlate specific bi-

been difficult to analyze—until now. Peter O’Connor, Ekaterina Mirgorod-

negative-ion modes. The researchers an-

ological functions with structures. (J. Am.

ticipate coupling MS with thin-layer chro-

Soc. Mass Spectrom. 2002,13, 402–407) M A Y 1 , 2 0 0 2 / A N A LY T I C A L C H E M I S T R Y

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ANALYTICAL CURRENTS Is it a fossil or not? sion involves geochemistry, a major aspect of the debate is conflicting interpretations of the Raman spectra of these structures. The debate is reminiscent of the controversy over the 1500 1750 2000 1250 fossil-like structures found in a Martian meteorite. According to Schopf’s team, Raman bands at ~1350 cm–1 and ~1660 cm–1 are characteristic of carbonaceous material. Confocal imaging with a vertical resolution of 1–3 500 1000 1500 2000 2600 2800 3000 3200 3400 µm shows that the carbon Wavenumber (cm–1) is in a high concentration, Biology or geology? Raman spectra of the structures shown which suggests a biogenic in the upper right corners. (Adapted with permission. Copyorigin. right 2002 Nature Publishing Group.) The Brasier group also sees the Raman spectrum, but they find that there is little absorption of the incident laser intensities, they say it is a ~200-nmlight, which indicates to them that the sized graphite particle. (Nature 2002, carbon is dilute. Based on relative mode 416, 73–81) Intensity

Ladies and gentlemen . . . in this corner, weighing in at ~3500 million years . . . J. William Schopf and colleagues from the University of California–Los Angeles and the University of Alabama– Birmingham use optical methods and laser-Raman microprobe spectroscopy to argue that tiny fossil-like structures are indeed remains of ancient, Precambrian prokaryotic microbes. And, in the opposite corner . . . also weighing in at ~3500 million years and using similar analytical methods . . . Martin Brasier and colleagues from the University of Oxford, University of Portsmouth, Royal Holloway University of London (all in the United Kingdom), the Geological Survey of Western Australia, and the Australian National University say “not true.” The “fossils” are artifacts formed from amorphous graphite. In dueling papers, the researchers debate whether fossil-like, micrometer-sized structures seen in very old geological formations are the result of biology or geology. Although much of the discus-

Dip-pen is mighty for protein arrays Demonstrating the power of dip-pen nanolithography, Chad Mirkin, Milan Mrksich, and colleagues at Northwest-

ern University and the University of Chicago construct protein arrays with 100- to 350-nm features. The arrays exhibit little nonspecific bindMHA-coated ing, which can obscure the AFM tip difference between features Passivation and inactive areas and lead to higher background signals. In dip-pen nanolithograProtein adsorption Au surface phy, an atomic force microscopy tip is coated with an lgG Lysozyme “ink”—in this case, 16-mercaptohexadecanoic acid (MHA). If the environment Anti-lgG is humid enough, a water binding droplet forms when the tip Illustration of the dip-pen fabrication of protein arrays. Either touches a substrate surface, IgG or lysozyme is adsorbed to the substrate. The binding of and the ink is transferred. The researchers validated antibody to IgG indicates that the adsorbed protein remains this fabrication method by aractive. (Adapted with permission. Copyright 2002 American raying dots or lines of MHA, Association for the Advancement of Science.) 240 A

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

passivating the surrounding areas, and immersing the arrays in protein solutions. Monolayers of protein adhered to the MHA-coated regions but not the intervening spaces. In initial experiments, rabbit antibody bound to immunoglobulin G (IgG) arrays, indicating that IgG retained its activity. In later experiments, the researchers fabricated an array of Retronectin, a commercial recombinant protein, to study cell adhesion. In particular, they wanted to investigate the importance of the size and distribution of the cellular anchor points known as focal adhesions. Previous studies were limited to 1-µm resolution, but the new work demonstrates that cells can adhere to 200-nmdiam protein spots that are separated by 700 nm. (Science 2002, 295, 1702– 1705)

news

Sites with proximity to potential human,

New research shows that compounds such as antibiotics, hormones, and detergent

industrial, and agricultural wastewater

metabolites are more widespread in the

were sampled using standard USGS meth-

environment than initially anticipated. Using

ods. Five new analysis methods were de-

five new analytical methods, Dana Kolpin

veloped for the various classes of com-

and colleagues from six U.S. Geological

pounds. Three of the methods targeted

Survey (USGS) offices found that 80% of

antibiotics and prescription and over-the-

the 139 streams tested in 30 states during

counter drugs, and essentially required

1999 and 2000 contained organic waste-

solid-phase extraction and LC/MS with

water contaminants. Of the 95 contami-

positive electrospray ionization to detect

nants targeted, 82 were present. Because

them. The other two methods used con-

many organic wastewater contaminants

tinuous liquid–liquid extraction and GC/

such as pharmaceuticals and household

MS to detect 46 organic wastewater con-

chemicals pass through wastewater treat-

taminants (including insecticides, plasti-

ment processes, the compounds aren’t

cizers, and detergent metabolites) and 14

necessarily new to waterways. However,

steroids. Selective ion monitoring was in-

for the first time in the United States, these

corporated into all methods to increase

researchers’ techniques quantified trace

sensitivity, but only target detection was

levels and took hydrogeologic, climatic,

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

and land-use settings into consideration.

1202–1211)

4

Total concentration Frequency of detection

5

Percent

80 60

1

5

3

7

2 22

40

7

5

11 14

5

2

1

20

Ste ro i ds rip tion d In s rug e ct De s ter r e gen pel l ent tm eta bol ites Dis infe cta nts Pla stic ize Fire rs ret ard ant s An tibi otic Ins s ect ic id Re es pro duc P AH t iv e Oth s er hor p re mo scr nes ipti on d ru An gs tiox ida nts Fra gra nce s So lve nt

0

ADAPTED FROM KOLPIN ET AL.

100

npr esc

Christina Rudzinski, Albert Young, and Daniel Nocera at the Massachusetts Institute of Technology introduce the first supramolecular microfluidic optical chemosensor, which glows green when it detects polyaromatic hydrocarbons. According to the authors, the sensor demonstrates that the intricate signal transduction mechanisms and functional requirements of supramolecular optical sensors can be preserved in a miniaturized system. To date, direct spectroscopic methods have been used for optical sensing on microfluidic platforms, but these methods made it difficult to distinguish between analytes. Here, the “3R” sensing strategy—which is characteristic of supramolecular chemosensors—recognizes, relays, and reports interactions between polyaromatic hydrocarbons in solution and a cyclodextrin supramolecule receptor site that has been modified with a terbium(III)-enclosed macrocycle. Such interactions trigger an adsorptionenergy transfer-emission mechanism, which generates green luminescence. Submicromolar quantities of polyaromatics can be detected without any amplification because even such tiny signals are clearly visible against the background, which remains dark. The researchers immobilized the chemosensor using various organic and inorganic matrixes and found that sol–gel procedures gave the most even distributions of material. The microfluidic device was fabricated in polymer on a quartz substrate, and 1-mm sol–gel squares were photolithographically patterned within a serpentine microchannel. The researchers found that the luminescence increased monotonically with the concentration of biphenyl, and they detected this analyte at a concentration of 5 µM. On the basis of such results, the researchers project that reliable detection will be possible with 700-nm-thick films. (J. Am. Chem. Soc. 2002, 124, 1723–1727)

Clear, but not clean water

No

Green light for microfluidic sensor

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

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ANALYTICAL CURRENTS Palladium replaces silver in SNOM/SERS Watching chemical reactions as they progress under actual conditions is a tricky enough business. But Volker Deckert and Christian Fokas of the Eidgenössische Technische Hochschüle Zürich (Switzerland) want to do more than uncover a reaction mechanism. They aim to discover simultaneously the morphology of the reaction sites. Their goal is to corre-

late information derived from a model system—the heterogeneous hydrogenation of benzene over palladium—so that they can determine a method for optimizing the catalytic substrate. In previous work, scanning near-field optical microscopy (SNOM) combined with surface-enhanced Raman scattering (SERS) achieved 100-nm spatial resolu-

H/D exchange comes to proteomics With protein databases growing by leaps

in conjunction with peptide mass finger-

and bounds, identifying a particular pro-

printing using MALDI MS to analyze a pro-

tein by MS requires an increasingly accu-

tein digest. They find that it takes three

rate mass determination. Willy Bienvenut

repetitive treatments to optimize H/D ex-

and colleagues from Geneva University

change with peptide fragments from the

Hospital, the Swiss Institute of Bioinfor-

enzyme digest. H/D exchanges of 95% or

matics, GeneProt (all in Switzerland), and

higher are observed.

Institut National de la Santé et de la

The exchange method is used to identify

Rechere Médicale in Lyon (France) bor-

a protein from virus-infected HeLa cells and

row a method already beloved by protein

validate the presence of a second protein.

chemists—hydrogen/deuterium (H/D) ex-

Limitations of the method include the need

change—and ask whether this technique

to manually retrieve the data from the spec-

can improve protein mass accuracy.

tra and lower sensitivity. (Rapid Commun.

1000

1200

1400

1600

2492.08

2044.6

1880.93 1907.92 2045.03

1724.84 1731.86

45 nm

2058

1800 Mass (m/z)

2528.79

2075.63

1912.46

2075.19

0 nm

1755.31

1183.97

944.766

(b)

2045 Mass (m/z)

1593.21

1506.26

1639.95

1399.68 1439.82

1163.63

927.493

(a)

Mass Spectrom. 2002,16, 616–626)

1567.76

1479.82

The authors investigate H/D exchange

2070

2000

2075 2080 Mass (m/z)

2200

2400

2600

MALDI MS spectra of bovine serum albumin digest with (a) no treatment and (b) following H/D exchange. Zoom boxes show the isotopic distributions. (Adapted with permission. Copyright 2002 John Wiley & Sons.) 242 A

tion of a sample on a silver surface. In the current paper, the researchers extend the combined techniques to a palladium catalyst. The nanofabricated catalyst is described as a silver “island”—because it looks like one when viewed with atomic force microscopy—coated with palladium. The SERS signal is enhanced with an atomic force microscopy tip instead of a surface-enhancing substrate. Comparing the spectra of reagents and projected products using far-field SERS showed one anomaly. The spectrum for 1,3-cyclohexadiene on an island in the absence of palladium was similar to that of benzene adsorbed on a palladium-coated island under reactive conditions. This result was surprising because silver is usually inactive under these conditions, and it led the authors to conclude that the same intermediate was formed in oxidative and reductive pathways. Although the group has not yet obtained in situ morphological images, nearfield SERS under nonreactive conditions shows that Raman enhancement for an analyte occurs only within the vicinity of an island. This demonstrates the capability of the combined techniques to identify a specific reactive site by correlating morphology with spectra. (Appl. Spectrosc. 2002, 56, 192–199)


200 nm

Noncontact atomic force microscopy image of the silver/palladium island catalyst within a chemically etched pit. (Adapted with permission. Copyright 2002 Society for Applied Spectroscopy.)

news

CE finds true variety among cells Relative fluorescence

Han and Lillard ana28 S lyzed individual cells at 155 530 multiple stages of the cell (a) cycle and identified multi(b) LMM ple RNA peaks that most 18 S 14 16 18 20 22 24 12 likely corresponded to Time (min) transfer RNA (tRNA) and (a) various subunits of riboso2000 4000 LMM 500 mal RNA (rRNA). Mes1500 5000 senger RNA is unlikely to have been observed. (b) The total RNA increased as the cells progressed from 12 14 16 18 20 22 24 G1 to the S, G2, and M Migration time (min) phases, with the biggest inRNA peaks from single cells in (a) G and (b) S phase. The 18S crease coming between S and 28S ribosomal RNA peaks are indicated. The low-molecularand G2. However, there was considerable variation mass peaks probably include transfer and 5S ribosomal RNA. (Adapted with permission. Copyright 2002 Elsevier Science.) among the types of RNA. When the levels of the various RNAs were normalized with respect to the total RNA, the fraction doubled between the G2 and M phases. attributed to low-molecular-mass RNAs— The variations in 18S and 28S RNA synthose 500 nm. And it’s relativetichannel ATR spectra were obtained tools aren’t good enough, then you have ly easy to make numerous sol–gel wavewithout using a monochromator because to build a new hammer,” says Saavedra. guides with varying broadband responses. outcoupling at the integral grating spec“We developed it specifically because our The researchers are applying the techtrally disperses the entrapped light. The toolbox is missing something.” nique to observe how proteins interact device was used to measure broadband In the April 15 issue of Analytical with proteolipid films being developed ATR spectra over a visible wavelength Chemistry (pp 1751–1759), Saavedra’s range of 390 nm to >645 nm. Because in Saavedra’s lab. Saavedra says the new group explains how the new instrumenmethod might become an accessory to a tation works. The waveguide was fabricat- the pathlength in a waveguide ATR geometry is wavelength-dependent, the visible/near-IR adsorbance spectrometer. ed by dip-coating a glass substrate with a researchers had to calculate and apply a ~300-nm-thick, sol–gel composite layer. “The applications are in spectroscopy of pathlength correction before the ATR They used a commercially available prism interfacial molecules in any field,” he says. —Cheryl M. Harris spectra could be quantitatively compared as the incoupler, with an integral holo-

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RESEARCH PROFILES Multiplex MALDI method for gene expression containing a common nucleic acid adduct In nucleic acids analysis, PCR is the star. quite lie flat, and a little flap of probe might be mistaken for a longer flap. hangs off at the overlap site. The DNA For parallel gene expression assays, it’s To prove the principle of the assay, hard to beat microarrays. Yet, in the April repair enzyme recognizes such overlaps and cleaves the flaps, generating the sig- the researchers created probes for four 15 issue of Analytical Chemistry (pp nal that indicates successful detection. If common RNA targets. Each probe had 1745–1750), W. Travis Berggren, Lloyd a different flap—TTG, TTTT, TTTTT, the probe and Invader don’t hybridize, Smith, and colleagues at the University or TTTTTT—and the molecular weights there’s no overlap, no cleavage, and thus, of Wisconsin–Madison and Third Wave of the cleaved flaps ranged from 1281 no signal. Technologies describe a new multiplexed to 2168. With this add-a-nucleotide apAfter the flap is cleaved, the rest of the gene expression assay that eliminates probe “falls off” the target, so it’s possible proach, the researchers project that it PCR and the solid-phase chemistry of to amplify the signal if multiple probes hy- should be possible to multiplex ~30–50 microarrays. bridize and get cleaved in turn. The likeli- targets per MALDI spectrum before enWhy would they want to compete hood of such turnover is increased by run- countering practical limitations, such as with such established technologies? Beexceeding the optimal mass cause one size doesn’t fit all range on the mass spectromnucleic acids assays, says Site of 5’ nuclease cleavage eter. But Berggren notes Berggren, a graduate stuProbe oligonucleotide Invader oligonucleotide 5’ that adding single nucleodent in Smith’s lab. For exNonhybridizing sequence Target-specific sequence 3’ RNA target 5’ tides provides “100–300 ample, not everybody needs times more [peak] separaexpression data for thoution than we need. If one sands of genes at once, as Isothermal reaction Excess of probe were willing to redesign offered by microarrays. And near probe Tm the chemistry of the cleavPCR seems to be shifting 5’ 5’ 5’ 5’ 5’ Accumulation of age product to make smallfrom a technique that 5’ cleavage product er mass tags . . . much higheveryone is grateful to have er levels of multiplexing Schematic of the Invader assay. The cleaved flaps from the probe to one that people somecould be achieved.” oligonucleotide, which constitute the signal indicating successful times want to avoid. “It’s Generally speaking, hybridization, are detected by MALDI time-of-flight MS. sort of like a marriage,” exMALDI is also amenable plains Smith. “Although to automation and highPCR is still very, very powerthroughput analysis, Berggren says. Howning the reaction very near the probe’s ful, it has been around close to 20 years ever, because MS requires relatively clean melting temperature. (The Invader oligo now, and people have gotten to know all samples, the new method has a clean-up has a higher melting temperature, so it the ‘ins and outs’—all the shortcomings.” step between the Invader reaction and remains bound to the target most of the One difficulty with PCR is getting the MS analysis, which limits the throughtime.) Because the amplification is linear, quantitative information. Because PCR put. “This is probably the last step that not exponential, it is easy to quantify the exponentially amplifies the amount of needs to be worked out,” he concedes. target DNA, small variations in the initial results, Berggren notes. To simplify cleanup, Berggren tagged Although the Invader assay has used conditions can lead to large differences the flaps with biotin, making it easy to fluorescence detection to date, Smith and in the amount of product. To avoid this capture the cleaved flaps on streptavidinBerggren switched to MALDI time-ofproblem, Berggren, Smith, and colcoated beads. But a more permanent soleagues switched to a signal amplification flight detection. Berggren says that they lution probably would be a solid-phase “recognized early on that this Invader technique—Third Wave’s proprietary purification step, he adds. technology is an ideal candidate for mass “Invader” assay. Now that the core technology has spec detection” because it’s easy to design The Invader assay is a hybridization been demonstrated, Berggren and Smith flaps of various lengths and, thus, various technique that uses two oligonucleotides hope that others will explore its advanmasses. (Tailoring the flaps this way does(oligos)—a probe and an Invader—both n’t interfere with hybridization.) Thus, tages for multiplexing and throughput. of which hybridize to the target DNA multiplexing is straightforward. For exam- They don’t envision it replacing the bigor RNA. The two oligos overlap beple, adding a single nucleotide to the name methods, just complementing them. cause the Invader extends into the reflap increases the mass by ~300 Da, which But you never know when you might hit gion where the probe sits, thus “invadgives ample separation between peaks. It on the next PCR. ing” its territory. Because the Invader —Elizabeth Zubritsky also avoids the possibility that a short flap essentially binds first, the probe can’t

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New strategy for zeptomole detection minish the need for polymerase chain weight, pharmacologically active protein. Walt explains that by choosing only reaction (PCR) in certain cases because 3 candidates instead of 25 as in the pre- high levels of amplification would be unnecessary. “If you have a few hundred vious work, “We stacked the deck in molecules, then you would only have our favor with respect to making sure that we didn’t have sequences that were to run [PCR] a few times, and that too closely related.” Now, the researchers would save time,” he adds. Second, Epstein sees the low deteccan very quickly detect zeptomoles of tion limit as a plus for DNA, which translates examining gene expresto ~600 molecules Fiber-optic bundle sion because the sensor using only 10 micromay pick up genes that sensors. Although S/N are expressed at very increases as the square P low levels and, thus, are root of the number of missed by less sensitive microspheres, it was methods. Third, going important to limit to microscale and lookthe number of microing at smaller volumes spheres to keep the sigimproves both sensinal from spreading out tivity and uncertainty. and becoming unde“When we bind a limittectable, says Walt. This ed number of molecules array maximized the to a small volume we ratio of DNA molecules generate a high local to microspheres and concentration on the maintained a good sigmicrosphere, making nal average. Microspheres and wells it easy to detect even The researchers purthough the absolute chased pure target DNA (Top) Schematic of a 500 µmnumber of molecules of known concentration diam fiber-optic bundle. is small,” Walt says. He with attached fluorescent (Bottom) Atomic force microadds that the principles labels and simply perscope image of the 3.1-µmin this paper should formed serial dilutions etched wells containing funcapply to any kind of to keep track of the tionalized microspheres receptor interacting number of molecules. to detect DNA. with an analyte, potenWalt explains that they tially including some simulated a real-world of his other work, such as detecting biexperiment, which would contain high ological agents. background levels of nontarget DNA, At this point, the researchers are by adding a billion-fold higher concen“trying to reduce the size of the beads tration of salmon sperm DNA to the from microscale to nanoscale, which we targets. The researchers detected the hope will enable us to improve our detargets among these inhibitors, but tection limit even more.” Just how low it took longer than it did with pure can they go? “We would like to go to samples. single molecules,” says Walt. Maybe it’s Having reliable, low detection limits could have various implications. First, not so crazy. After all, no one imagined a low detection limit may reduce the making phone calls without operators need for sample manipulation, and “the when Alexander Graham Bell presented less you have to manipulate your samthe first telephone, and now we have ple, the more quantitative you can be,” digital cell phones. —Rachel Petkewich says Walt. For example, it could diJASON EPSTEIN/DAVID WALT

Phones made fiber-optic technology famous, but the filaments can do more in the lab than help ring up a pizza delivery. Fiber optics also can serve as chemical sensors. In the April 15 issue of Analytical Chemistry (pp 1836–1840), researchers at Tufts University report zeptomole (10–21) detection limits with improved S/N using functionalized microspheres in a fiber-optic array. The project was started by Myoyong Lee, a postdoctoral fellow in David Walt’s group at Tufts before she was hired at the National Institutes of Health, and continued by Jason Epstein, currently a doctoral candidate in Walt’s lab. In earlier work, the researchers could not go below a detection limit of 10 f M (Anal. Chem. 2000, 72, 5618–5624). But the new detection limit is 2 orders of magnitude better. “We are demonstrating . . . the ability to improve the detection limit by reducing the uncertainty of where the molecules are,” says Walt. The researchers create their arrays by etching wells into the distal face of a 500-µm-diam optical imaging fiber bundle composed of ~6000 fibers. After applying a microsphere suspension to the fiber tip and evaporating the loading solution, the 3.1-µm-diam microspheres sit in these wells. To capture specific DNA sequences, the researchers attach probe oligonucleotides to the microspheres via polymer linkers. When target DNA carrying a fluorescent label hybridizes to the probes, a custom imaging system records the probe sequences lighting up. According to Walt, “you get better hybridization” and more molecules per bead by using the intervening polymer rather than by attaching the DNA directly to the bead. In the current paper, the researchers focused on pushing down the detection limits, using three DNA sequences— two from the cystic fibrosis gene and one from a cytokine, a low-molecular-


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RESEARCH PROFILES Taking some of the “guesswork” out of protein identification

Relative error [ppm]

Intensity/relative units

Linear, systematic errors are High-throughput identification 100 (a) caused by differences in the enerof proteins takes another step PO2570 gy and flight paths of ions generforward with a new algorithm 50 ated from different sample posifrom a team at the Max Planck tions on a target grid. The result Institute (Germany). Most data0 is a different m/z calculated for base search engines confidently equivalent ions from different identify proteins by the number 1,000 2,000 3,000 m/z samples. Because the difference of “hits” (peptide mass values 500 (b) in masses is linear, it can be used derived from experiments that to correct the error, says Egelmatch the peptide masses from 0 hofer. “Then the standard deviaknown proteins in a database) tion . . . can be put in the scortogether with the researcher’s ing formula to help decide if educated judgment. The new –500 proteins are identified or not.” statistical scoring algorithm for 1,000 2,000 3,000 500 The strategy begins by searchMALDI time-of-flight mass (c) ing a protein database for a minispectra, described in the April mum of five matching peptides 15 issue of Analytical Chemistry Y’ = 0.0218X–238.2 0 within a defined range. MSA cal(pp 1760–1771), automates the culates the relative errors of the judgment phase and more accu–500 peptide masses and the linear rerately identifies tryptic digests of 1,000 2,000 3,000 500 gression on the errors for each plant and mammalian proteins, (d) sequence. The standard deviation says Volker Egelhofer. (SD) then identifies the false When a database search reY’ positives that are excluded from turns few hits, the protein may the succeeding regression calcube difficult to identify because lation on the remaining masses. there are so many candidates. –500 The scoring equation incorpo“The bigger [databases] are, the 1,000 2,000 3,000 500 (e) rates the percentage of the promore difficult the search betein’s sequence covered by the cause they have more similar Y’ = 0.0232X–239.6 matched peptides and the data proteins,” explains Egelhofer. 0 from three rounds of regression, However, in testing the new SD, and removal of outliers. scoring system with several hun–500 All the factors in the scoring aldred common proteins, which 1,000 2,000 3,000 500 gorithm interact dynamically, says were independently confirmed (f) Egelhofer. “If you have a lot of by DNA sequencing, the group hits (n), you have found a lot of obtained accurate identifications Y peptides, and you have a much with scores of 99%. “The prohigher SD because the error tein was always the correct one,” grows with more values.” Includhe says. “In some cases, you –500 1,000 2,000 3,000 ing the sequence coverage helps don’t find a protein, but then m/z to avoid false positives in such you don’t find a false positive cases. On the other hand, a small[either], and that’s the great ad(a) MALDI time of flight mass spectrum from a tryptic digest er SD means that you can have vantage of this algorithm.” of recombinant human ␤-actin and (b) a plot of relative mass fewer hits or lower sequence covThe new strategy was motivat- deviations. The mean of the relative deviations is calculated, erage and still find the correct ed by the team’s discontent with and dashed red lines indicate the standard deviation to this protein, he explains. the number of false positives remean. Outliers are excluded. Successive linear fits (c, e) reThe dynamic quality of the turned by an earlier version of sult in (d, f) decreasing standard deviations. algorithm provides adaptability, their sequence database search our data and saw that they always have program, the mass spectrometry analyzand a “fudge factor” (F ) helps control a linear error,” he says, “and we just ing (MSA) algorithm (Anal. Chem. the stringency of the search. The rethought about how to improve this.” 2000, 72, 2741–2750). “We looked at searchers never observed false positives

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using the default setting (F = 1.0), but a user might want to reduce the value in searching a small database where there may be fewer hits. Because this lowers the stringency of the search and, consequently, increases the chance of false positives, corresponding changes in other variables are needed to counteract the effect, thus maintaining the overall stringency of the scoring function. In addition, adaptability to the quality of mass spectra is inherent in the SD, which depends on factors such as calibration, tuning the mass spectrome-

ter, and S/N. The group hasn’t yet found a general strategy for including modifications of amino acid residues in the scoring algorithm, but they suggest an empirical change in n that is based on the number of modified peptides detected. Another advantage of the new algorithm, which also enhances throughput, is the need for only one external calibrant. Egelhofer says that external calibrants formerly had to be placed near each sample in the target, so there might be as many calibrants as samples. But with the new

algorithm, he says, “you can put [the calibrant] somewhere in the target, measure it one time, and measure all the peptides that you want to know.” This also eliminates the need for an internal calibrant and, thus, avoids the problem of internal calibrant and analyte peaks overlapping. The researchers have transformed the SwissProt and NCBI databases to forms compatible with MSA. Access to the new version of MSA is free on the Internet at www.scienion.de/msa. —Judith Handley

MEETINGS News from the Atlanta, Ga.

54th American Academy of Forensic Sciences Meeting—Wilder D. Smith reports from

Big trouble for money launderers? In July of 2000, then-U.S. Attorney General Janet Reno took offense at a Federal Bureau of Investigation (FBI) finding that money launderers were so confident in their ability to successfully smuggle U.S. currency into and out of the country, they were shipping cash through U.S. ports by the crate. Thomas Jourdan, a supervisory special agent with the FBI who holds a Ph.D. in chemistry, was told by then-FBI laboratory Assistant Director and Division Head Donald Kerr to work on a solution that the U.S. Customs Agency could use to screen cargo at ports. “I was already working on measuring the amount of cocaine on money in normal U.S. circulation,” says Jourdan. Finding crates of money amid the cargo at the > 300 ports of entry that span the United States provided a much different challenge. “The problem was how to tell if a crate marked ‘engine parts’ actually had those parts in it, or if it contained U.S. currency,” he says. Opening each and every crate that comes through a port is an unreasonable and time-consuming task, he says. Jourdan considered several approaches. “However, the thing that screamed out at

me was that if you’ve ever been around a large amount of money . . . it has a distinct odor to it,” he says. “It seemed to me that a portable instrument that could detect the volatile organic compounds [VOCs] in money was what was needed.” Jourdan initally considered using micropower impulse radar instruments. To decide which VOCs to look for, he teamed up with Donald Blake at the University of California–Irvine and Trang Vu of the U.S. Customs Service, who had done work on detecting VOCs from money using GC/MS. “We decided to first pick a set number of VOCs that were associated with currency and then . . . program an ion mobility spectrometer [IMS] to look for these compounds,” says Jourdan. The group settled on a list of 10 VOCs considered to be common to all new and circulated U.S. currency: hexanal, heptanal, octanal, 2-hexenal, 2-heptenal, 2-pentyl furan, 1-heptanol, pentanoic acid, and two volatile breakdown products of illicit drugs—methylbenzoate from cocaine and acetic acid from heroin. In the meantime, Kevin Linker at Sandia National Laboratories–Albuquerque (SNL) was working on the necessary instrument—a portable, six-inch, cubelike instrument called the “Hound” that, cou-

pled with an IMS, would detect explosives, drugs, and other chemicals. The idea for the portable Hound system was born from larger, walk-through explosives detection portals that were developed by SNL. To collect vapors, the Hound uses portable solid-phase microextraction technology in which a device that looks like a blower inhales a large volume of air and corrals the scent of explosives and other chemicals onto a metalized screen. The unit then vaporizes the compounds into a more concentrated sample of air, which is delivered to a detector. Although the Hound can detect explosives down to the parts-per-trillion level with several cubic feet of air, detecting VOCs on money is still in the preliminary stages. “We have only worked in the lab with solutions that represent VOCs from money,” says Linker. Having a portable system with the same type of sensitivity for detecting VOCs in money would greatly improve the detection efforts and efficiency of U.S. Customs agents, says Jourdan. “Essentially [Customs agents] could drill a small hole in the crate, take a 10-second air sample, do the analysis, and get a readout in about 4 seconds,” he says.


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A typical crime scene has some sort of “fingerprints”—blood, semen, a single strand of hair, or even a used cigarette butt. Using PCR to collect DNA from such samples is very effective and nothing new to the forensic community. But for forensic scientists Ray Wickenheiser and Richard Jobin at the Royal Canadian Mounted Police in Regina, Saskachewan (Canada), an unheard-of application of the technique would change the possibilities for crime scene analysis as far as the eye could see. For four hours, a victim was brutally beaten, raped, and held in an apartment. During the ordeal, one of her contact lenses was dislodged. After being released, the victim waited three days to report the incident because she feared further retaliation—a delay that gave the assailant enough time to thoroughly clean the crime scene. In fact, the only physical evidence collected from the crime scene were 19 shattered fragments of a light blue contact lens found in a vacuum cleaner in the apartment. Cells from the corneal epithelium (eyeball) and the bulbar epithelium (interior edges of the eyelids) regenerate every 6–24 hours. Every time a person wearing a contact lens blinks, thin slices of epithelial cells are sheared from the interior eyelid and collect on the surface of the contact lens, providing a source of DNA. To retrieve the biological material

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REPRINTED WITH PERMISSION OF RAY WICKENHEISER

PCR clears up a blurry crime

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STR 3, containing loci Amelogenin (sex typing - blue), D5S818 D13S317 and (green), D7S317 STRSystem System 3, containing loci Amelogenin (sex-typing blue),(blue), D5S818 (blue),(green), D13S317 (blue) from right to left from respectively. variation in peakagain, height from loci toin loci is evident, and D7S317 (blue), left to Once right,again, respectively. Once variation peak heightalong fromwith n-4 peaks. Note also a small Amelogenin peak at 112, which corresponds to the Y chromosome, indicating a loci to locicomponent. is evident,Given along n–4 peaks. Note also abag small Amelogenin peak 112, which trace male thewith nature of the vacuum cleaner source of the contact lensatfragments, this is not surprising.to the Y chromosome, indicating a trace male component. Given the nature of corresponds

the vacuum cleaner bag source of the contact lens fragments, this is not surprising.

from the lens, Wickenheiser and his colleagues relied on standard techniques. They began with a procedure very similar to cleaning a common contact lens. Usually, the lens is immersed in a saline solution, and an enzyme is added to digest the proteins, releasing them into the solution and leaving a clean contact lens. “This was very similar to what we were doing, except we were using a protease solution to break up the proteins, which breaks up the cells and releases the DNA into the solution,” says Wickenheiser. Then they extracted the

DNA using a protocol that they had previously perfected. Finally, the DNA was purified with the common phenol– chloroform method. This application of PCR was significant because “it was the first time it was ever used on a contact lens,” says Wickenheiser. The victim’s DNA was found on the lens, placing her at the crime scene, which was all Wickenheiser and his team needed to prove. The assailant eventually confessed to the crime, and a new world of applications for PCR became a little clearer to forensic scientists.

PEOPLE Analytical chemists receive awards Jonathan V. Sweedler, professor at the University of Illinois at Urbana–Champaign and the Neuronal Pattern Analysis and Biological Sensor groups at

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the Beckman Institute, will receive the 2002 Heinrich-Emanual-Merck prize for Analytical Chemistry. The award is given by Merck KGaA, Darmstadt (Germany), and recognizes chemists whose work provides solutions to analytical problems in the areas of life sciences, material sciences, or the environment. Sweedler’s work currently involves scaling spectroscopic methods to the


nano- and attoliter-volume regimes. His group has also developed detection systems for CE; MALDI time-of-flight MS systems to profile peptides in individual neurons and cellular processes; and highresolution, nanoliter-volume NMR spectroscopy for liquid-phase samples. The award will be presented in April to Sweedler in Munich at the Analytica 2002 meeting.

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Thomas C. Werner, professor at Union College, received the 2002 ACS Award for Research at an Undergraduate Institution, sponsored by Research Corp. The award was announced at the ACS National Meeting in April in Orlando, Fla. Werner received his B.S. in chemistry from Juniata College (1964) and his doctorate in analytical chemistry from the Massachusetts Institute of Technology (1969). He held postdoctoral positions at Harvard Medical School and Massachusetts General Hospital (1969– 70) and was a research associate at Tufts Medical School (1970–71). Werner served as a professor and the chair of chemistry at Union College (1985–91), where he is currently the Florence B. Sherwood Professor of Physical Sciences.

Michael J. Weaver (1947–2002) Michael J. Weaver, professor of chemistry at Purdue University, died unexpectedly on March 21, 2002. Weaver received his B.Sc. from Birkbeck College, University of London, in 1968 and his Ph.D. from Imperial College of Science, Technology, and Medicine, University of London, in 1972. He served as a postdoctoral research associate at the California Institute of Technology from 1972 to 1975. In 1975, he began his academic career as an assistant professor at Michigan State University; in 1982, he came to Purdue University as an associate professor and was promoted to professor in 1985. An electrochemist by definition, Weaver also explored surface chemistry in the gas phase; vacuum and nanomolecular environments; photon-based vibrational tech-

niques; and analytical, physical, and materials chemistry. Weaver was best known for his pioneering work on surface-enhanced Raman scattering and IR spectroscopy at electrochemical interfaces. Weaver’s list of accolades and nominations is considerable. His most recent honor was being recognized as one of the country’s top 100 chemists from 1981 to 2000, according to a report by the International Science Institute. He received the 1989 David C. Graham Award from the Electrochemical Society; the 1995 Faraday Electrochemistry Medal from the Royal Society of Chemistry; the 1997 Carl Wagner Memorial Award from the Electrochemical Society; and the 1999 Electrochemistry Award from the American Chemical Society. He was recently nominated for a Purdue Distinguished Professorship award. His colleagues say that his unexpected death is a major loss to Purdue and the scientific community. “It was a big blow,” says Hilkka Kenttämaa, professor and head of Purdue’s analytical chemistry division. “This tragic event is an astronomical loss to fundamental electrochemistry, which may never be fully restored,” says Andrzej Wieckowski, professor of chemistry at the University of Illinois and a personal friend of Weaver’s. “His loss will set the community a decade back, and major efforts will be needed to restore the equilibrium.” “[Mike] always got excited about our work,” says graduate student Sally Wasileski, a member of the Weaver group. “He would say to us that having good graduate students and watching them evolve [into] scientists was one of his favorite things about being an academic.” In some ways, Weaver seemed to be a step behind the world outside of chemistry. “He still wrote scientific papers on a legal pad with a number-two pencil,” says Wasileski. “Every e-mail he ever wrote, he would sign, ‘Cheers Mike,’ which he bragged he could type with two fingers.” She adds, “We senior students will continue to do the work Mike would have wanted us to do and try our best to meet up to Mike’s very high standards.” —Wilder D. Smith


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Analytical Currents: DNA arrays without amplification

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