SCIENCE
What's DNA's electrochemical sine? Electrochemical methods for DNA detection usually rely on the electroactivity of the nucleobase, but this approach is plagued by high backgrounds and irreversible adsorption of the molecule on the electrode surface. Werner Kuhr and his group at the University of California-Riverside take a previously developed method—sinusoidal voltammetry for the oxidation of carbohydrates (1) and nucleotides (2) at copper electrodes—and extend their work to oligonucleotides and DNA They detect these analytes by monitoring the oxidation of the nucleic acid's sugar backbone. Their work appears in the current issue of Analytical Chemistry (3) The sinusoidal waveform allows the analysis to be shifted from the time domain to the frequency domain via a Fourier transform. Noise is primarily confined to the fundamental frequency, whereas the Faradaic current is shifted to higher harmonics. "The advantage of the sine wave is that the background stays essentially a linear function of the excitation wave," explains Kuhr. "If we start with a pure sine wave, we keep the background at the fundamental frequency; all the good information goes to higher harmonics; and we can isolate the signal from the background." In these experiments they monitored the first six harmonics "Essentially what we have now is an efficient way of doing background subtraction," Kuhr comments. "That's why we can work with copper, which is really difficult in the time domain. By using this sinusoidal method, we can be efficient with how we subtract off our background. And that lets us work with these more complex systems." The basic reaction is the oxidation of the sugar backbone. But at the copper electrode, the bases contribute to the signal because amine and hydroxyl functional groups are oxidized as well. "Even though we don't have any idea of the mechanism yet, the end result is that adenine and cytosine (that is, purines and pyrimidines) have very different frequency spectra. We can distinguish or essentially call the bases based on these frequency spectra. We get the qualitative information that we can use for identification as well as the quantitative information"
Because the method relies on the oxidation of the sugar moieties in DNA the twofold increase in the number of easily accessible sugar units makes the technique more sensitive to double-stranded DNA (dsDNA) than to single-stranded DNA (ssDNA). That is the reverse of electrochemical techniques that depend on the electroactivity of the bases, because the bases are shielded inside the helix of dsDNA At equal concentrations, the signal is proportional to the length of the DNA strand. They haven't yet estimated the resolution for length measurements. "If we have 100 versus 99 bases, I don't know if we could tell the difference," Kuhr says. Of course, researchers are usually more interested in sequencing DNA than simply detecting it. If two almost identical strands of DNA differed by only one additional base, this method would be hard pressed to tell the difference between the two strands. However, the method by itself need not sequence DNA. "In traditional sequencing, what you would do is add dideoxy-terminated bases with different colored tags," says Kuhr. "Certainly, you should be able to do the same kind of thing with electrochemical tags." However, Kuhr isn't racing off to look for new tags. "We haven't even looked at the existing dyes. A lot of these dyes are aromatic compounds that might have fairly good electron transfer properties. It would be interesting to take a look at those and see if we can use the traditional fluorescent dyes." The sensitivity of this new method is equivalent to fluorescence techniques. Therefore, the advantage for the electrochemical method is its simplicity and low cost. 'You don't need lasers. You can essentially put an array of detectors on a chip or on a credit card-type device. You could do an analysis or a hybridization assay. If you wanted to do the whole sequencing reaction, that gets more complicated," Kuhr says. Because the method is more sensitive to dsDNAthan ssDNA, it is an ideal candidate for working with hybridization probes. Kuhr has worked on derivatizing other electrodes with biotin-avidin but says that it's tricky because the copper layer is continually coming off. The group is now taking a slightly different approach. They immobilize the hybridization probe on the wall of a fused-
silica capillary and wash in the target, which hybridizes with the probe. The strand is then removed from the capillary wall and washed past the detector. So far, they've only worked with synthetic oligomers, but they're looking at a target for an oligomer commonly used in tuberculosis assays. DNA wasn't Kuhr's original target when he began investigating sinusoidal voltammetry. 'We started working in the area with the idea of getting sensors that worked better for neurotransmitter dynamics. It gave only a marginal—maybe an order of magnitude—enhancement for things like dopamine, so it wasn't as interesting." Kuhr is now excited about more than just detecting DNA. "The really neat aspect we're exploiting is that not only can you get better sensitivity but you can extend the range of measurement or the range of applicability of electrochemical methods." Celia Henry
References (1) Singhal, P.; Kawagoe, K. T.; Chrrstian, C. N.; Kuhr, W. G. Anal. Chem. 1997, 69, 1662-68. (2) Singhal, P.; Kuhr, W. G. Anal. Chem. 1997, 69, 3552-57. (3) Singhal, P.; Kuhr, W. G. Anal. .hem. 1997, 69,4828-32.
BUSINESS
Bruker acquires Siemens Bruker Instruments has acquired the analytical X-ray instrument business of Siemens Analytical X-ray Systems and has established a new subsidiary, Bruker AXS, to handle the new business. Bruker AXS produces X-ray fluorescence, powder X-ray diffraction, and single-crystal diffraction instruments. Worldwide sales are approximately $50 million. According to Bruker, AXS will operate as an autonomous business with sites in Madison WI and Karlsruhe Germany. A demonstration lab will be established in Tsukuba Japan All of the 260 former Siemens X-ray Systems ployees have been hired by Bruker The company says it has no plans for downsizing or relocations
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