done some work with other materials. There's the ethylene glycol-terminated monolayer that [George] Whitesides [of Harvard University] made some time ago. We may be able to do the assay with that molecule as the monolayer in the grid and not need the surfactant." They are still tryAFM height images (40 \jm x 40 pm) of a rabbit IgG array before ing to determine how (left) and after (right) the addition of goat anti-rabbit IgG. quantitative the techsurfactant helps to solubilize nonspecific nique is. They can detect a single antigenbinding antibodies and prevent them from antibody binding event, but they haven't sticking to the grid. yet determined the dynamic range. For the They have used other grid materials, technique to work at usable levels, samples and it is unclear which will ultimately be might have to be diluted. "We hope that the best. "ODT was a good starting point. .I the technique will tell us about the concenmay work the best," says Porter. "We've tration of antibodies in solution until we
Sense and the single cell A new single-cell analytical technique promises to overturn theories about the time course of events during cellular damage during a heart attack. Jonathan Cooper of the Bioelectronics Research Centre in the Department of Electronics at the University of Glasgow (U.K.) and colleague Craig Bratten, working with cell biologist Peter Cobbold of the University of Liverpool (U.K.), have developed a photolithographically micromachined microtiter chamber (—600 pL) with an integrated electrochemical sensor. They designed the device to study the reactive events taking place in heart cells during cardiac ischemia (the sudden loss of blood supply to the heart usually caused by blockage of cirteries) one of the West's biggest killers. In the IVIarch 15 issue of Analytical Chemistry (p. 1164) the heam described the construction of the device and presented initial results from measurements of a single heart cell Their results challenge the accepted model for the release of purine during cardiac ischemia The UK researchers' method involved using a microscale device to electrochemically detect enzymatically generated hydrogen peroxide, measured at a platinized microelectrode that is held at an oxidizing potential of about 420 mV relative to a
saturate the surface," explains Porter. "We have to learn what's required to get that to work in our favor. Getting the dynamic range is one of the important questions for us to work on." Eventually, the grid will allow them to design multiple assays on a single wafer. The achievable density depends on the lithographic techniques and on the microscopy. "We may eventually be able to do 5 x 5 um or 10 x 11 0m array yizes," says Porter. "What we really need to do is make the size of the elements in the array much smaller or have the scan area of AFMs increase so that we can do more." They are investigating different ways of patterning the surface and novel AFM methods. The ultimate goal is to move this immunoassay method out of the lab and into the clinic. "We're interested in seeing if we can come up with a high-speed, low-cost approach for immunoassays," says Porter. Celia Henry
AglAgCl electrode. Signals from a single able to relate adenosine fluxes to observmyocyte (a muscle cell) were recorded as able changes in the cell [e.g., rigor contraccurrent versus time and integrated to give ture] ," explains Cooper. One hypothesis a total charge for the reaction. for cardiac injury is that once the cells lose their adenosine, they are unable to syntheCooper and his colleagues could then size sufficient ATP when the oxygenated calibrate the system for a given analyte problood flow is restored. "This idea no longer duced by the cell. They used a cascade of seems plausible," says Cooper, "as intact enzymes (adenosine deaminase, nucleotide phosphorylase, and xanthine oxidase) to see cells contain as much adenosine prior to lysis as they originally had ATP." what happens to the cell's energy metabolites after they simulate ischemia through metabolic poisoning. Using devices made at Glasgow and working in Liverpooll Bratten found that adenosine (but not the purine inosine) reached the extracellular space only after the cell had been damaged. So despite all of the cell's adenosine triphosphate (ATP), ~7 mM, being converted to adenosine, no detectable quantities are released until the cell is lysed. Their data challenge the current concept, obtained from whole heart and cell population studies that adenosine diffuses out of intact cells possibly to act vasodilator in response to the low concentrations of oxygen in the tissue The electroanalytical device with a circular platinized "The value of a single cell working electrode, encircled by a larger counter elecmeasurement is in eliminating trode. Rigor contracture is induced in the myocyte by tissue heterogeneity and in being injecting a medium that contains metabolic inhibitors. Analytical Chemistry News & Features, April 1, 1998 243 A
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Is there any possibility of using the discovery in drug design? Cobbold speculates that pharmacological intervention could be designed to release some of the accumulated adenosine from intact cells. He argues that if all the adenosine released by the ischemic heart has come from lysed cells, they are already irreversibly damaged. Cooper adds, "The point really is that the event of adenosine release appears to signal the end stage of cellular damage, and no amount of biochemical intervention afterwards will save the damaged tissue." There is some hope, though, says Cobbold. "Why not design a drug to promote adenosine efflux from healthy cells, with the aim of improving blood flow, and thereby prevent the cells reaching the point of irreversible injury?" Cooper points out that numerous pharmaceutical and biotechnology companies are currently developing single-cell techniques because they offer such advantages of insight. Indeed, Cooper and his colleagues worked on some aspects of the current research with GlaxoWellcome and Wellcome Trust support. However, there are still problems to face. For instance, the volumes involved are so small that local concentrations might be reached that are well above physiological conditions. He explains that in the present experiments this did not cause difficulties because the experimental time periods short. "The devices we have fabricated are still research tools, but in the future there is a chance of using them in array technologies, for screening compounds [drugs] as cell-based assays," Cooper adds. He also points out that ink-jet technology—of the kind more familiar in computer printers— can be used to dispense the required fluids. "We have just made a 4 x 4 array of these microanalytical devices," he says. Cooper believes the technology could be widely applicable to other cellular systems because the microlithographic techniques used are generic. This could also allow the researchers to reduce the dimensions of the titer vessel even further, making it more suited to smaller cell types (for example, the measurement of the release of the neurotransmitter glutamate from single brain cells). It should also be possible to incorporate more than one sensor into the device so that different analytes might be observed at the same time. David Bradley 244 A
NEWS FROM HPCE '98 Britt EricksonreportsfromOrlando, FL
Swiss go submicron Although several methods exist for surface elemental analysis with submicrometer resolution, molecular analysis on this scale is very difficult to achieve. Renato Zenobi and co-workers at the Swiss Federal Institute of Technology (ETH Zurich) have made it their goal to develop scanning near-field optical microscopy (SNOM) for chemical analysis on a scale of 100 nm or less. SNOM relies on a tapered fiber tip, traditionally made by heating and pulling, which is scanned in very close proximity (< 10 nm) over the sample surface. One practical limitation is the poor optical transmission of SNOM tips. Zenobi's group has developed an alternative way for fabricating such tips, using a chemical etching technique. Their tips are characterized by a large opening angle at the apex, leading to higher optical transmission (up to 0.1%-1%, depending on the aperture size) and a higher damage threshold, while preserving 50-150 nm apertures. Because of their high optical transmission, chemically etched SNOM tips open up new possibilities for analytical chemists, such as fluorescence imaging of biological tissue. For example, ,ctinfilamentsfrom the cytoskeleton have been imaged with better resolution than with state-of-the-art fluorescence microscopy. Fluorescence, however, does not usually give detailed chemical information, so the ETH researchers instead ex-
Quantifying HIV-1 RNA without amplification It's one thing to be able to detect the presence of the HIV-1 virus, known to cause acquired immunodeficiency syndrome (AIDS) in humans, but quantifying the degree of infection is a much different story. Quantitative measurement of the number of HIV-1 copies, referred to as viral load testing, is essential for monitoring the HIV-1 virus. Because more potent antiretroviral drugs are on the market today than ever before, the levels of HIV-1 RNA in infected individuals are often undetectable with currently available techniques. A method that offers improved sensitivity and reproducibility is desirable, particularly for
Analytical Chemistry News & Features, April 1, 1998
All aglow: micrograph of a near-field optical tip with — 100 nm opening.
plored Raman spectroscopy through their SNOMtips(Anall Chem. 1997, 69, 749). They demonstrated surface-enhanced Raman spectroscopy (SERS) with a spatial resolution
