Science: What's downstream?

Branch was made part of the Center for Medical ... application of genetics to what we call operational ... Projects Agency, has found its home at the ...
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The ATOFMS samples particles at the beach.

Before the particles enter the mass spectrometer, they travel through a particle sizing region, with two continuous-wave laser beams for aerodynamic sizing. The laser beams are directed to the system by fiber optics, eliminating the need for aligning the laser. On average, about 10% of the sized particles are chemically analyzed, or "hit". The histograms of hit versus missed particles are well matched in terms of aerodynamic diameter. Therefore, when the system is properly aligned, no size bias is apparent. Prather says that —90% of the sized particles are missed because the Nd:YAG laser used for desorption and ionization has relatively low power and a small probe volume. The instrument is being used with more established atmospheric monitoring techniques to determine scaling factors that can be used to convert the number distribution to a real distribution of what is present in the atmosphere. For the ATOFMS data to be used with historical

conventional data, the number distribution must be scaled to a mass distribution. Prather says, "As part of developing a new technique, you have to show that you can match existing technology before you go on." She comments that a strength of doing continuous monitoring in conjunction with conventional offline sampling is that the continuous monitoring allows more judicious offline sampling, saving both time and money. The instrument travels well. Allhough it hasn't actually been operated during transport, Prather says that because the instrument works before transport and can be fully operational within 10 min. of arrivall ,i is reasonable to assume that the instrument could work en route. She says that one of the goals is to use the instrument aboard aircraft to monitor air quality. In the recently published work, the performance of the ATOFMS was characterized with wood smoke particles, because they are easily generated and form a broad distribution centered at relatively small sizes. Now, however, the instruments are being used to study real atmospheric problems. The mass spectra are being used to broadly characterize particles according to origin, rather than to provide a definitive chemical identification. "We do really well at identifying the inorganic f ("in-

tent The organic content is more difficult because you can get fragmentation We would like to as far as the organic content goes have one or two oeaks stand out as tracer Deaks If we see that peak

Schematic of the particle sizing and mass spectrometer regions of the instruments.

ethyl sulfoxide (DMSO) to quantitatively generate a methyl radical. A fluorescaminederivatized nitroxide then reacts with the methyl radical, forming a stable product, Identifying free radicals is a difficult task, Omethylhydroxylamine, which is sepaparticularly in complicated biological and rated by reversed-phase HPLC and quantienvironmental matrices. In many cases, fied fluorometrically. The method detects radical concentrations are too low to be nanomolar levels of OH in complicated maquantified by traditional spin trapping trices. In contrast to aromatic hydroxylation methods. Neil Blough and co-workers at techniques involving benzoic acid or salithe University of Maryland-College Park cylic acid, this method is fairly innocuous to and the University of Maryland School of cells andtissues.Both methods require Medicine, Baltimore, have developed a high reagent concentrations to trap radicals new, highly sensitive method for detectlike OH quantitatively. With aromatic hying and quantifying trace levels of the hydroxyl radical (OH) in biological matrices. droxylation however you can run into solubility and toxicity problems says Blough The method which is described in this issue of Analytical Chemistry (p. 4295) in- High concentrations of DMSO do not pose much of a problem volves the reaction between OH and dim-

What's downstream?

combination of peaks, we can be reasonably sure that it came from a particular source or had a particular reaction occur on it. We're trying to learn about the chemistry of these particles as well as their origins." The next step for the instruments is more of what they've already been doing—monitoring atmospheric phenomena. Prather's group, in collaboration with Glen Cass's group at the California Institute of Technology, used the ATOFMS to study marine aerosol chemistry. She says, "The experiments went extremely well in that we directly monitored aerosol processes as they were occurring on the particles. We plan to perform similar field measurements as part of SCOS97. In addition, when the field studies are completed we plan to interface the ATOFMS instruments to reaction chambers so we can run laboratory studies on aerosol reactions to obtain complementary information on tropospheric processes" Celia Henry

According to Blough, the initial goal was to lower free radical detection limits to levels typically found in natural waters. Spin trapping with electron paramagnetic resonance (EPR) detection is not sufficiently sensitive or reliable to investigate radical processes in many environmental situations. Blough and his co-workers have been developing a more sensitive alternative to spin trapping for about 10 years. "Originally, the idea was to have a pure opttcal lensor," says Blough. When a niiroxide is covalently attached to a fluorophore, the fluorescence is efficiently quenched. However if the nitroxide reacts with a free radical the quenching is eliminated and the fluorescence increases thus providing a sensitive means for measuring radical scaveng-

Analytical Chemistry News & Features, November 1, 1997 6 5 3 A

News

LABORATORY PROFILE Laying the groundwork for DNA-based clinical methods DNA research at the Armed Forces Institute of Pathology (AFIP) is moving in a new direction. In February, the DNA Technology Development Branch was made part of the Center for Medical and Molecular Genetics (CMMG). As a result, the analytical focus shifted to include genetic disease in addition to human identification (i.e.,forensics). Col. Barry H. Thompson, director of CMMG, says, "CMMG is the coordination point for a Department of Defensewide effort in medical genetics that is intended to do two things: increase the application of genetics to what we call operational medicine—medicine in the field—and address the variability and availability of clinical genetics services for active duty forces and their family members. We're trying to bring together the existing assets in genetics in the system such that we can improve to the clinical side of things and apply of the recent advances in technology to operational medicine " The center is concentrating on four areas of DNA technology: robotics MS CE and microchips The goal is to develop a completely automated DNA assay. For now, however, robotics are primarily confined to sample preparation. The system is based on a robotic arm capable of x-y-z manipulation that connects two pipetting stations, a thermal cycler, and a plate reader. "We've developed a robotic system that automates the purification of DNA from blood and couples that to polymerase chain reaction (PCR) amplification " says Capt Phillip

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Belgrader, chief of the Advanced DNA Technologies Development Branch. 'We're in the process of integrating a detection system to that platform. Right now, it's going to be a plate reader for TaqMan assays. It's quite feasible to integrate those with the MS systems, but that will be down the road." A portable, miniaturized, microchipbased thermal cycler, designed at Lawrence Livermore National Laboratory and funded by the Defense Advanced Research Projects Agency, has found its home at the center, and the CMMG researchers have put tremendous effort into developing new assays for the instrument. Belgrader says, "I've been collaborating with people at USAMRIID (U.S. Army Medical Research Institute for Infectious Diseases). We've shown that the instrument can be used for detecting biological warfare agents such as anthrax and plague. We used it to detect about 13 copies of anthrax in 28 minutes" They have also used the instrument to distinguish single nucleotide polymorphisms In a blind studv they selected the correct species of orthopoxvirus (the viral genus that includes smallpox) from a STOUD of five species

are more traditional areas in biology and pathology. There must be some level of importance to this technology." Thompson adds, "I'm particularly intrigued with the MS, in that the detection of a single base-pair change in the DNA is a diagnostic capability for a genetic disorder or many genetic disorders." Separations are also important. "We use HPLC as a sample prep for cleaning material," says Michael Marino. "One problem our sister group, the service branch of the Armed Forces DNA Identification Laboratory, has is that they get a lot of denatured DNA, basically from bones. When you do the amplification, you get more than one product. When you do sequencing, you want to be specific to just one [product]. We use HPLC to demonstrate that you can actually isolate [individual PCR products]." "Everything we do is with real DNA, rather than starting with synthetic DNA targets," says Ross. "The issue of management of genetic information as it pertains to clinical samples is a delicate one, which people here are taking very seriously. We have a base of 'blinded' samples [samples without personal identification] available to us that is sufficient to explore the various technologies at our disposal and demonstrate feasibility for certain assays. Actual testing programs would follow only when appropriate legal and ethical policies are in place " Although the move to operational medicine is an ultimate goal, the technology development group isn't currently under pressure in that area. "Right now, there's not a big push because we're a pretty new establishment," says Ross, "but that will come. We have an interim period during which we can develop some very useful technology that can be directly applied in that sense." Celia Henry

Assays are also being developed to apply MS to DNA diagnostics using peptide nucleic acid (PNA) probes rather than DNA probes. PNAs are DNA mimics that have a peptide backbone instead of the sugar phosphate backbone found in DNA. Philip Ross, who is primarily responsible for the MS work, says, "In general PNAs work better than DNA because they have better hybridization characteristics. PNA has better recognition of a complementary sequence and is more destabilizing in the presence of a single-base mismatch." Belgrader describes the PNA assays: "We use [streptavidin-coated] magnetic beads to capture our biotinylated PCR product. We denature the PCR product and add the PNA probe. Each probe is specific for a particular allele, which may differ by only a single nucleotide. We take the magnetic bead-PCR productPNA complex and directly subject it to the MALDI process. Any PNAs that are bound to the DNA are stripped off and detected. The DNA and magnetic beads stay behind." The possibilities for DNA analysis with MS are generating excitement at CMMG. Ross says, "At a place like Automated DNA processing—extraction, AFIP, investing in the latest in off-theamplification and product analysis—at the shelf MS is a big thing, because there Center for Medical and Molecular Genetics.

Analytical Chemistry News & Features, November 1, 1997

generate lipid radicals; it can attack DNA and form DNA radicals. "You can have a situation in which you have small amounts of OH being generated, but that can initiate a radical chain reaction," explains Blough. Therefore, various products are likely to form downstream from the OH generation site. Blough does not intend to abandon EPR methods altogether. He believes that EPR and fluorescence techniques can complement each Neil Blough and Beibei Li trapping OH. other. "EPR is another way of looking. "This tells you the reactton occurred," ing at your loss of probe as well as the says Blough. "The problem is that ti doesn'' environment in which your probe retell you exactly what you trapped." Identifi- sides," explains Blough. The nitroxide radical has a distinctive EPR signal. When cation of these radical adducts requires a it reacts with a free radical, it becomes powerful separation technique such as diamagnetic, and therefore the EPR signal HPLC. drops. With the new fluorescence techLC/MS could be a powerful approach nique, information about product formafor the separation and identification of tion can be obtained. The primary advanradical adducts in biological systems. Blough has previously used the method to tage of the fluorescence method is greater sensitivity. "Viewing product formation is identify photochemically generated radisensitive than detecting reactant cals in natural waters (Anal. Chem. 1196, far 68,867). "That's our ultimate goal," says loss We can in 104-105 Blough. "OH is an initiator, but ultimately product conversions from the fluoreswe want to see what the downstream reac- cence signal coming out whereas with tion products are." The process is compli- EPR we have to look at v'ery small signal cated because OH is very oxidizing and losses on too of a very large background" relatively indiscriminate it will react with says Blough just about anything that it comes into conThe fluorescence method is used to tact with. When OH reacts with biomoleinvestigate the rate of OH production in a cell system, following the addition of the cules it can generate carbon-centered radicals; in the presence of lipids OH CHXI anticancer compound diaziquone. "Normal-

A light flash from DNA (not RNA) Imagine that you want to know whether DNA is present in your sample. The test must be simple, reliable, fast (you do not want to wait for PCR amplification), sensitive, selective (you are not interested in RNA or any other compounds), and cheap. Marta Elena Diaz-Garcia and her co-workers at the University of Oviedo (Spain) describe a strategy for detecting double-stranded DNA (dsDNA) with room-temperature phosphorescence (RTP) even in the presence of RNA (Anal Chem. 1199 69,2406-10). Often, a researcher is not interested in the total amount of nucleic acids in a sample and wants quantitative information about only one type of nucleic acid. A possible approach is to remove the unwanted nucleic acids and use a less specific probe to detect what's left. However, such additional steps in an analytical protocol are

ly, cells are pretty tightly controlled in their redox reactions," says Blough. The diaziquone basically goes into the cell, accepts electrons from electron transport chains or redox enzymes, and then passes these electrons to oxygen to form superoxide. "It short-circuits the system, thus producing superoxide and hydrogen peroxide, which can be toxic to the cell," explains Blough. .I trace metals are present, OH can be generated via the Fenton reaction. The end result is the death of the cancer cell. The ultimate challenge is to go into a cell system and discover where these radicals are formed. However, it is not certain whether OH is being detected inside or outside the cell, or both. 'We have to know what the exchange rates are between the exterior and interior of the cell," emphasizes Blough. To measure internal OH production, the probe must be inside the cell. It is unclear at this time whether the cells have ever actually been "cracked open", admits Blough. "Right now, we are trying to get at this issue of exchange kinetics using both EPR and fluorescence spectroscopy. We are looking to see how rapidly the probe and the product partition in and out It is a critical issue " If the OH is exterior to the cell ,i is not as important a situation because other constituents will react with the OH before it gets to the cell On the other hand if the OH is generated inside the cell it could cause irreoarable damage and ultimately kill the cell Britt Erickson

undesirable. Compared with the methods for DNA determination that exist today, our RT P approach has a number of remarkable advantages," Diaz-Garcia says. In contrast to radioactive and fluorescent probes for DNA, a method that uses phosphorescence has a unique and specifictimewindow. Time-resolved spectroscopy is necessary to find the signals of interest and to remove background noise and the spectra of interfering lumophores—an easier task on the millisecond (i.e., with phosphorescence) than on the nanosecond (fluorescence) timescale. Moreover, the RTP emission wavelengths are in the visible part of the spectrum, allowing the use of cheap optical fibers An RTP complex intercalates with dsDNA and photodiode technology. The main and allows detection. problem wasfindinga probe that allows the detection of DNA with this technique. detection of D-glucose via a glucose oxiRTP sensing systems developed so far dase minireactor. The oxygen-sensitive by the Oviedo group include various complex is immobilized on an ionmetal chelates or erythrosine B for the exchange resin, which is packed in a conquantitative analysis of oxygen and for the ventional flow-through cell. This arrangeAnalytical Chemistry News & Features, November 1, 1997 6 5 5 A