SCIENCE
Looking for mosquito attractants What makes one person more attractive to mosquitoes than another? No one knows, but Ulnch R. Benner of the U.S. Department of Agriculture-Agricultural Research Service Center for Medical, Agricultural, and Veterinary Entomology and his co-workers at the University of Florida are developing methods that they hope will allow them to find the answer. They are working with the yellow fever mosquito (Aedes aegypti)) They describe their work in this issue of Analytical Chemistry (p. 1) The analysis of human emanations is complicated by the amount of water given off by the human body as perspiration, which dilutes the relevant compounds. 'The major problem is how to analyze the sample without the water getting in the way," says Bernier. "The problem is that when you start removing water, you start pulling some possibly important compounds from the sample, too." Bernier and his co-workers collect human skin emanations on glass beads by rolling them in people's hands The problem of losing attractants from the sample is alleviated but not elimi-
nated. "We knew we probably weren't getting all the attractants on the glass," says Bernier. "but we knew from previous experiments done here at LTSDA that at least some of the attractant compounds for this particular species of mosquito can be transferred to glass and evaporate back off." The beads are placed in an olfactometer to see how attractive they are to the mosquitos, and then the beads are analyzed by GC/MS. The compounds on the beads can be introduced into the chromatograph by either cryofocusing or purge and trap. In the cryofocusing technique, the beads are placed directly in the inlet of the gas chromatograph. The injection port is modified by inverting the fritted injection liner. The frit prevents the beads from dropping onto the column entrance. The size of tlie beads is important only for ease of handling and fitting in the insert. "These things are already hard to handle—2.9 mm, which is about the size of a BB—but they fit perfectly and leave a little bit of space inside the glass insert of a Varian 3400," says Bernier. With this particular setup, they could analyze a maximum of 12 beads at a time. "With that [bead] diameter, you can cram 12 in the insert without caus-
Modulated sensor A sensor that can see through cloudy solutions, including even skin, has been developed by a team at the University of Maryland School of Medicine. The device uses a mixture of fluorescent molecules with different decay lifetimes, only one of which is sensitive to the analyte of interest. The signal from the sensor is detected as a lowfrequency modulated signal, providing a measure of analyte concentration. The sensor is described in the Dec. 15,1998, issue of Analytical Chemistry (p. 5115). According to Joseph Lakowicz, director of the university's Center for Fluorescence Spectroscopy, fluorescence sensors generally suffer from a simple problem: to get a measurable signal the sample has to be fairly transparent, which limits the approach to relatively clear mixtures. In the
Schematic of the sensors with a nanosecond fluorophore and [Rufbpy)^*.
ing the frit to break, the beads to break, or beads to fall out the top." However, they found that for some compounds, particularly carboxylic acids, 12 beads overloaded the column. The purge-and-trap and cryofocusing techniques are suited to different compounds and are thus complementary. "The purge and trap did a wonderful job of looking at nonpolar to moderately polar volatile compounds. The cryofocusing did a good job of looking at the moderately volatile to relatively nonvolatile compounds," says Bernier. How well does the sampling method mimic the real-life attraction of people or animals for mosquitos? "That's more dependent on how well glass mimics it. 'Hie answer is that it's close but we're pretty sure it's not the total answer. It doesn't give us all the components. That goes back to the question of how many components are needed for mosquito attraction. We don't know the ultimate answer to that either. We know it's definitely more than three at this point." According to Bernier, they've identified more than 300 compounds on the beads Thcv think they're going to find that the ratios of compounds play a large role in determining to whom mosciuitos attracted Celia Henry
past few years, however, a solution has come into view. Rather than looking at fluorescence intensity, analysts are focusing on fluorescence lifetimes and correlating them with concentration. This approach neatly side-steps the opacity problem because the intensity decay is independent of the amount of light transmitted. Sometimes, however, lifetime measurements can be a hindrance, particularly if the decay time is on the nanosecond time scale, which requires fast and expensive electronics. In addition, fluorophores with nanosecond lifetimes often require excitation by a UV source with a light-amplitude modulation frequency of 100 MHz or more. "Lower frequencies and longer wavelengths would allow lower cost devices," explains Lakowicz. The researchers' answer is to use not one, but two, fluorophores in the same sen-
Analytical Chemistry News & Features, January 1, 1999 19 A
News sor. Onefluorophoreis sensitive to the analyte, and it has a nanosecond decay time. A second fluorophore, which does not respond to the analyte and does display a longer decay lifetime, is included. The mixture of the two lifetime fluorophore determines the modulation of the fluorescence emission at low frequencies—1-10 MHz, which are even lower than radio frequencies. "The intensity of the sensing fluorophore with a short decay time is revealed by the modulated signal at low frequency," explains Lakowicz. The researchers determine the modulation, which is equal to the fractional fluorescence intensity of the sensing molecule. The analyte concentration can be calculated from the fractional fluorescence intensity The sensor's modulation response is independent of the total fluorescence signal. The only requirement is a fluorophore that changes intensity in response to changes in the analyte concentration. However, fluctuations in the total signal don't affect the results, because the most important factor is the relative amplitude of the sample and reference fluorophores, which are immediately adjacent in the sensor.
The team has tested the idea using two mixtures: a system containing 6-carboxyfluorescein acting as a pH sensor and the organometallic fluorophore [ruthenium 2,2'(bipyridyl)3]2+ as the modulator. A calcium probe was made by replacing the fluorescein with Ca2+-sensitive Fluo-3. The most important advantage of modulated sensing, however, may be the range of analytes that can be measured. Numerous sensing fluorophores that change intensity in response to analytes are known. The extension to immunoassays is also possible, adds Lakowicz. Aside from the fact that modulated sensing with such a system can be used with less than ideal samples, including living tissue, it is also unaffected by disturbances to the equipment itself such as fiber optics being bent or the sample being moved. "The availability of numerous fluorophores, which are sensitive to a wide variety of analytes, makes this approach widely applicable," adds Lakowicz. Commercialization of modulated sensors for industrial and clinical applications is likely in the near future, he says. David Bradley
Rapid angle on wood stiffness
ter and charge-coupled device (CCD) detector have opened new possibilities in wood fiber diffraction. With the help of physicist Ian Cave, the team has developed protocols for rapid measurement of a key parameter related to timber quality. The powerful data-acquisition and handling capabilities of the detector allow the angle of crystalline microfibrils in wood cell walls to be determined at a rate of one sample every few minutes. Robinson emphasizes that the same measurement can be made withfilmdetectors, although it requires several hours per wood sample. Traditional X-ray diffraction methodology is unsuited to large numbers of samples. The advantage of the area detector is its speed, making possible for thefirsttime the mass screening of microfibril angles (mfa) in plantation seedlings and trees. The wood of softwood species is made up predominantly of tracheids, which are the skeletons of dead cells. Highly ordered bundles of cellulose chains (microfibrils) are embedded in the amorphous matrix of the tracheid cell wall, giving strength and stiffness to the cell wall. The microfibrils form a right-handed spiral that makes an angle (mfa) with the cell axis. Wood cell
Modern array detectors have revolutionized single-crystal structure analysis, and they are now indispensable for protein crystallography. At the Asian Crystallographic Association Conference in Bangi, Malaysia, crystallographer Ward Robinson and co-workers from the University of Canterbury (New Zealand) described how their X-ray diffractome-
Orientation of the microfibrils in the S2 layer. 20 A
Analytical Chemistry News & Features, January 1, 1999
walls consist of several layers of different compositions, and the mfa of the tracheid S2 layer (the middle layer of the secondary cell wall) has the greatest impact on the mechanical properties of wood. Low values of mfa tend to give the best mechanical properties (10-20° is considered optimum) . The mechanical properties of wood determine its use and economic value— hence the interest in mfa measurements. In Robinson's laboratory it takes about one minute to mount each microtomed wood sample on the goniometer and to record the wide-angle X-ray diffraction pattern. Sample thicknesses up to approximately 16 mm can be probed, although the overall sample dimensions are not critical. The data analysis provides a frequency distribution of mfa in the 1-mm diameter cylinder of material sampled by the X-rays. Robinson points out that a considerable amount of work lies behind the data-analysis procedure. Compared with single-crystal diffraction patterns the wood patterns are "dirty" with contaminant reflections and they require sophisticated mathematical methods for analysis The methods were developed by co-worker Cave and plied using commercial mathematical software packages Robinson became interested in applying modern X-ray diffraction instrumentation to mfa measurements when he was approached by a multidisciplinary research team of foresters, engineers, and plant scientists. The team is studying aspects of mfa in Pinus radiata, which accounts for more than 90% of plantation forest area in New Zealand. This pine grows rapidly; however, its wood has poor mechanical properties, compared with wood from other softwood species. Modest gains in wood stiffness can yield significant economic benefits; this advantage suggests focusing breeding and silviculture methods on reducing mfa. The possibility of rapid and low-cost measurement of mfa in many samples is exciting for the research team One study by the team has demonstrated that seedling clones that have the potential to produce stiffer wood can be identified by measuring the mfa oftfieirfirst annular growth rings. Of more immediate importance, mfa surveys can also be used to increase the economic yield of existing forest stands. Wood stiffness can vary widely within a plantation, and measuring the mfa of individual trees during harvest could dramatically reduce waste by tailoring the use of the log to die wood stiffness.
