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Optics and chips GC, LC, CE, and other rnalytical techniques usually take up acres of bench space, but tesearchers working in the area of micro-total analysis systems (uTAS) hope to remedy this situation and provide miniature chemical equipment analogous to microelectronics. This emerging technology wiil provide inexpensive, portable, and cheap-to-run equipment that can handle tiny sample volumes and use minimal reagents. Although miniature gas and liquid chromatographs are already showing great promise, a basic need exists to integrate them at the chip level with similarly shrunken detector systems. Andreas Manz and Jan Eijkel of the Zeneca/SmithKline Beecham Centre for Analytical Sciences at Imperial College (U.K.), collaborating with Herbert Stoeri of the Technical University of Vienna (Austria), have built the first optical emission detector on a tiny sliver of glass in another step toward the ultimate "lab on a chip". They demonstrated that their device works the same as its bench-top counterparts but on a much smaller 50 nL scale. They hope to easily integrate the miniature detector on a chip-based gas chromatograph, such as that developed by Steve Terry of Stanford University. Optical emission usually involves desol-
vation of the analyte and atomization and excitation of the molecules using a flame or a plasma. In the July 15 issue of Analytical Chemistry (p 2600), the team reported d new device based on plasma excitation, which, they say, avoids the problems associated with the small exposed flame that was used in a previously developed micromachined flame detector. The device is a 14 x 30-mm HF--eched glass plate. It features a gas inlet and outlet, a pressure sensor connection, and various electrodes (see the schematic in the accompanying figure). This forms the top plate below which the team attached a second glass plate (20 x 30 mm) with h plasma chamber, inlet and outlet channels, and electrode chambers. To test the setup, the researchers fed helium gas with different concentrations of methane into the chip. The optical emission from the plasma in the chamber was then picked up externally by a fiber and sured by a photomultiplier tube. They obtained a detection limit of 3 x 10~12 g methane/s in a volume of 50 nL using excitation of the CH diatomic band. "This compares well with the values achieved by everyday plasma-based detectors," says Eijkel. However, the concentration-based detection limit was 500 ppm, which, because of the small volume of the
Schematic of the chip layout. Features of the 14 x 30-mm top plate: (1) gas inlet, (2) gas outlet, (3) pressure sensor connection, (4) electrodes, and (6) electrode connection pads. Etched in the 20 x 30-mm bottom plate are (5) a plasma chamber, (7) an inlet channel, (8) an outlet channel, and the electrode chambers, which are not indicated.
plasma, is higher than for conventional plasma detectors. The researchers do not consider this problem serious because they anticipate using the device for fast throughput of small samples with high concentrations and not for very high-sensitivity analyses. However, the lifetime of the cathode is rather low at 2 h, because of sputtering, which, they say, must be improved. David Bradley
NEWS FROM THE INTERNATIONAL SYMPOSIUM ON ENVIRONMENTAL ANALYTICAL CHEMISTRY Celia Henry reportsfromJekyllIsland, GA.pathogenic bacteria are sensitive but slow, with analysis times measured in days, or even weeks. A typical detection limit is 1 Detecting critters colony-forming unit in 25 g of food, but as many as 14 days might be required before in food the colonies can be seen. Millions of cases of food-borne illness Therefore, the search is on for a method strike in the United States each year, as sensitive as, but much faster than, tradiresulting in thousands of deaths and tional bacterial cultures. The ideal method billions of dollars in medical care costs, will be sensitive, fast, inexpensive, low--ech, according to the Centers for Disease and capable of being performed on-site. ImControl and Prevention. Methods that munoelectrochemistry is a promising techrapidly detect pathogenic bacteria, such as Salmonelll, E. coli 0157:H7, and List- nique that involves labeling bacteria with eria monocytogenes, could reduce the enzyme-antibody conjugates and then detecting the electroactive product of the eneconomic and public health toll these organisms exact. Jeffrey Brewster of the zyme reaction. In Brewster's method, the antibody is organism-specific, but alkaline U.S. Department of Agriculture hopes phosphatase is used as the enzyme label that immunoelectrochemistry will be regardless of the target organism. one such method. Traditional methods for detecting The method of capturing the bacteria can
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make or break the technique. Brewster has used several methods for capturing the bacteria, including antibody-coated electrode surfaces, antibody-coated magnetic particles, and membrane filtration. The coated electrode is incubated with the sample for 30 min before analysis. Unfortunately, because bacteria diffuse slowly, only a small number of the cells exposed to the electrode is actually detected. The capture efficiency was only 20%, and the detection limit for Salmonella waa s aisappointing ~10 5 cells exposed/mL. With the magnetic particles, a 30min incubation period is also required. The electrode is a thin layer of graphite silk-screened on a thin piece of plastic. A magnet under the electrode pulls the
particles to the electrode. The detection against a bare eleclimit is improved over the coated electrode to detect the entrode (5000 cells/mL), but it still does zyme reaction. Coating not approach the necessary sensitivity. the filter prevents the According to Brewster, the method is label from sticking to plagued by high background levels and it, which would hinder physical interference. distinguishing the bacteria from the filter These two methods inherently have and decrease the detwo levels of selectivity. .Two events tection sensitivity. have to occur. [The bacteria] have to bind [to the antibody], and then the label A more recent twist has to stick to [the bacteria] as well," on the filtration Brewster notes. "Say you're trying to de- method involves backtect E. coli. You may get 1% of oflmonellaflushing thefirstmemthat will stick to the capturing antibody. brane onto a second There's a certain level of interference membrane after the there. If only 1% of the label sticks to the bacteria have been Salmonella, you've decreased the inter- labeled. Because the ference by a factor of 100." second membrane never contacts the laIf the capture efficiency were better, bel, the interference the antibody-coated electrode would problem is eliminated. probably have the best detection limit, "We've only shown says Brewster. However, as an overall that the method works technique,filtrationis the best of the and that get better three capture methods. It is 99% efficient; takes only seconds; and is simple, signal to background" says Brewster "We cheap, and scalable. The sample is run haven't done the studthrough a filter, and then the bacteria ies to really nail down are labeled with the alkaline phosthe detection limit" phatase. The filter is then pressed
Selecting the selectors A chiral separation is only as good as its chiral selector. Unfortunately, there's no way to know ahead of time what the best chiral selector will be, and the process of choosing the selector for a particular separation is still an empirical exercise of trial and error. Two methods for screening chiral selectors, described by Tingyu Li of Vanderbilt University, may change that. In thefirstmethod, Li uses an iterative strategy to determine the chiral selectors within a mixture library. He synthesized a 16-member library of the form L-[module l]-[module 2]-Gly-NHCH2CH2CH3. In any given peptide, module 1 contained an aromatic group dinitrobenzoyl (Dnb), benzoyl, 2-naphthoyl or 9-anthroyl—and module 2 contained an amino acid—leucine alanine glycine or proline. The llbrary was separated on a column with a stationary phase composed of L-(l-naphthyl)leucine ester immobilized on silica gel Tofindchiral selectors the corresponding D-peptide library was synthesized and separated on the
Schematic of how the microorganisms are detected using immunoelectrochemistry.
same column. Because the two chromatograms differed, the mixture must contain at least one compound whose enantiomers interact differently with L-(l-naphthyl) leucine ester. Synthesizing and separating sublibraries helped determine the chiral selectors by process of elimination. The libraries were successively divided and analyzed until it became apparent that the selectors contained Dnb in module 1. A similar iterative process was followed to identify the appropriate amino acid. The molecules Dnb-Ala-Gly-NHCH2CH2CH3 and DnbLeu-Gly-NHCH2CH2CH3 were pinpointed as potential chiral selectors. To test whether they really could separate the enantiomers of (1-naphthyl) leucine ester, the potential selectors were immobilized on silica gel and used as the stationary phase for a chromatographic separation. Both selectors effectively resolved racemic (l-naphthyl)leucine ester, with separation factors of 6.9 for the Ala-Gly column and 8.0 for the Leu-Gly column. The second method involved the parallel synthesis and screening of the chiral
selectors. The success of this method depends on the efficiency of both the synthesis and the screening procedures. The library contained the same modules as the previous library, but the components were synthesized on an Abu-PS resin, where Abu is 4-aminobutyric acid, and PS is polystyrene. The resins were placed in individual microwells and incubated with racemic (l-naphthyl)leucine ester. The list of potential chiral selectors was whittled down by analyzing the enantiomeric ratio of the (1-naphthyl) leucine ester in the supernatant with circular dichroism. The ellipticities of the supernatant of only two wells were significantly above the noise level, identifying Dnb-L-Ala and DnbL-Leu as possible chiral selectors. The candidates were then immobilized onto silica gel and used as stationary phases for the separation of racemic (1-naphthyl) leucine ester. In this C3.sc the separation factors were 4.7 for the Dnb-L-Ala column and 12 for Dnb-L-Leu. Similar experiments using tentagel resin resulted in lower ellipticities demonstrating that the solid support can affect the screening outcome
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