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. thefilterprevents 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 Salmonellaflushing 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 you 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 lHmodule 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 library 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 case, 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.
Analytical Chemistry News & Features, August 1, 1999 5 1 7 A
