News
Understanding chemisorbed CO The chemisorption and subsequent reaction of CO on transition metal surfaces is a topic of fundamental interest in heterogeneous catalysis. NMR spectroscopy of 13 CO has proven to be a powerful technique for probing the diatomic's structure, dynamics, and electronic structure on a metal surface. However, the relationship between the observed NMR values and the electronic properties of the metal surface are still being unraveled (Anal. Chem. 1998, 70, 518 A527 A). This type of information can lead to new ways of probing catalyst and electrocatalyst surface structures. Andrzej Wieckowski, Eric Oldfield, and their research groups at the University of Illinois at Urbana-Champaign
Lions, tigers, and bears Chemists are usually not the first people who come to mind for determining species of birds, reptiles, and mammals; however, recent work by Edgard 0. Espinoza and co-workers at the National Fish and Wildlife Forensics Laboratory and Southern Oregon University could make that a thing of the past. The researchers analyzed unpurified whole blood and dried blood samples from 980 animals, representing 62 species, looking for a- and B-protein pairs in hemoglobin whose molecular weights could serve as markers for species identification. Using electrospray ionization (ESI) MS, they were able to differentiate one species from another based on distinctive oc/B-pairs in 86% of the cases despite hemoglobin variability observed in many species. The authors believe that the 14% of cases with overlapping a/S-pair molecular weight values could be differentiated by implementing a separation such as HPLC before MS analysis. In some cases, more than one signal appeared in the mass spectrum around the regions associated with the molecular weights of the a- and B-protein chains in hemoglobin. The secondary signals might provide some information useful for species determination. However, the authors chose to use only the most abundant signals. Although the technique is unable to dif372 A
demonstrate a quantitative relationship between ligand NMR shut and the surface electronic state. To achieve that goal, they used 3 C and ' 'Pt NMR to collect Knight shift, relaxation, and/-coupling data for CO, which was chemisorbed on a platinum electrocatalyst in an electrochemical environment. This was combined with quantum chemical calculations based on density functional theory. They report a linear correlation between the 13C Knight shift of chemisorbed 13CO and the clean surface Fermi level local density of states of platinum catalysts, which amounts to —11 ppm/Ry^-atom"\ In addition, they investigated the li,5Pt NMR line shape, the 195Pt nuclear spin-lattice and the spin-spin relaxation behavior for a platinum electrocatalyst. (/. Am. Chem. Soc. 1999, 121, 2996-3003)
ferentiate all species, it offers some advantages over other techniques currently used for species identification, such as electrophoresis. The high resolving power of ESI-MS makes the technique more discriminating, allowing for the identification of polymorphic forms of hemoglobin, which go undetected by electrophoresis. The technique is also useful for corroborating hemoglobin-sequencing data. Although some of the authors' data agreed with the reported sequences, in many cases it did not, suggesting errors in the reported sequences. (Anal. Biochem. 1999 268 252-61)
MIP for estrogenic compounds Stilbene estrogenic compounds, such as hexestrol, 17B-estradiol, and fra«s-diethylstilbestrol, have been in the news as potential endocrine-disrupting compounds, which are thought to be linked to reproductive abnormalities in wildlife and increasing cases of breast cancer. Although they have been banned from use in the European Union, they continue to be used elsewhere as growth promoters by the meat industry. Jonathan A Tarbin and Matthew Sharman of CSL Food Science Laboratory (Norwich, U.K) have developed a selective method, which uses a molecularly imprinted polymer (MIP), prepared with a hexestrol template, to determine phenolic estrogenic compounds in animal tissues. The MIP was prepared using a noncovalent imprinting technique, in which 2-(diethylamino)ethyl methacrylate served as the functional monomer and trimethylolpropane trimethacrylate served as the cross-linking monomer. A blank polymer was also prepared without the hexestrol template using an analogous protocol Both polymers were packed in 150 x 4 6-mm i d stainless steel HPLC columns for evaluation The polymer synthesized with the hexestrol template showed selectivity towards hexestrol, as well as compounds containing the same carbon backbone as hexestrol (fraHS-diethylstilbestrol and dienestrol). Some recognition was shown for zeranol and the estradiols, but much less than that for hexestrol. (Anal. Commun. 1999,36, 105-07)
SCIENCE
Just say NO Dubbed by Science magazine as the 1992 "Molecule of the Year," nitric oxide (NO) continues to be one of the most versatile and important chemicals known. NO is a common air pollutant that forms when nitrogen is burned; however, in the body, it takes on various roles—from regulating blood pressure to triggering memories to killing off pathogens. Because of its biological importance, several attempts have been made to create fast, selective methods for detecting NO. In this issue (p 2071-75), Raoul Kopelman and co-workers at the University of Michigan describe a new type of NO biosensor based on a fluorescent dye-
Analytical Chemistry News & Features, June 1, 1999
labeled heme domain of soluble guanylate cyclase (sGC). The sGC-based sensor offers improved sensitivity (as much as an order of magnitude) over previously developed NO biosensors based on the hemoprotein cytochrome c' (Anal. Chem. 1999, 71,1767-72). "We believe the improved sensitivity is because of a difference in the binding constants of the proteins," says Susan Barker, a graduate student in Kopelman's lab. "They are both proteins that are well known to selectively bind nitric oxide. We expected the guanylate cyclase to bind more tightiy to nitric oxide because it is found in the human body; whereas the cytochrome c' was extracted from exotic bacteria" adds Kopelman.
Rather than using native sGC heme, which is difficult to isolate in good yields and pure form, the researchers collaborated with Michael Marietta and Yunde Zhao (also at the University of Michigan) to produce a high yield of heme domain by overexpressing the N-terminal fragment of the heme-binding subunit of sGC in Escherichiiaoll "We needed to be able to produce a suitable quantity of [sGC] to actually make the sensors with. We took only the important part of the molecule—the nitric oxide binding part and lost the peripheral protein," explains Barker. Because the heme domain is much smaller (44 kDa) than native heterodimeric sGC (150 kDa) overexpression of the heme domain yielded a greater number of NO binding sites per unit mass The heme domain was labeled with the fluorescent reporter dye Oregon Green 488 carboxylic acid, succinimidyl ester. Oregon Green responds to changes in the protein's conformation after the protein has bound to NO. Gold-coated optical fibers (100-um core diameter) were immersed in the dyelabeled heme domain solution, allowing the peptide to adsorb to the surface of the gold.
Shaping up The physical and chemical properties of nanoparticles are influenced by both the size and the shape of the particles. Size exclusion chromatography (SEC) has already been used to separate nanospheres of different sizes. Although the ability to make nanoparticles of specific shapes has improved, most techniques produce a mixture of shapes. Now GuorTzo Wei, C. R. Chrrs Wang, and du-Ken Iiu of the National Chung-Cheng University (Taiwan) hope to use SEC to separate nanoparticles of different shapes (Anall Chem. 1999 71, 2085-91). One of the problems with trying to analyze nanoparticles by SEC is the irreversible adsorption of the particles on the stationary phase caused by the large surface area of the packing material. The authors had previously found that the addition of the surfactant sodium dodecyl sulfate (SDS) to the mobile phase decreased the amount of adsorption. When they used water as the eluent to separate a mixture of differently shaped nanoparticles (rods and spheres) by SEC, the adsorption was so severe that no sig-
Detection of nitric oxide produced by macrophages with an optical sensor based on the heme domain of soluble guanylate cyclase.
Fluorescent polystyrene reference spheres coated with a hydrophilic polymer, which contained carboxylic acid groups, were then added to the fiber. The sensor response is based on the ratio of the fluorescence intensity of the dye to the fluorescence intensity of the reference spheres. By using this ratiometric approach rather than measuring absolute intensities, signal changes due to optical variations or light scattering can be distinguished from changes in analyte concen-
tration. "Changes in the alignment of the laser or the light setup of the microscope don't matter, because they affect the reference signal and the analyte signal equally," explains Kopelman. The sensors were used to measure extracellular NO released by BALB/c mouse macrophages. In essence, these white blood cells use NO as a chemical weapon against pathogens, says Kopelman. Minimal NO was released from untreated cells; whereas high levels of NO (111 uM) were
nal was observed at all, indicattng that even nanoparticles that have been stabilized by the addition of cationic surfactant can encounter problems with irreversible adsorption. Although adding SDS to the eluent reduced the adsorption problem, it had little effect on the separation. The authors found that adding the nonionic surfactant Brij-35 with the SDS allowed nanoparticles of different shapes to be observed, but the resolution was very low.
'With the addition of Brij-35, we apparently induce surface adsorption [to a certain extent], depending on the concentration of Brij-35 being added," says Wei. "With SDS and Brij-35, the separation is a combination of steric effects and adsorption effects. SDS is required to eliminate the surface adsorption and Brij-35 is added to improve the shape separation by introducing an adsorption factor that is shape-dependent in the separation." The separation can be optimized byfixingthe SDS concentration at 40 mM nmd sdjustinff the Brij-35 conhigh as possible without causing baseline deterioration With a mixture of 40 mM SDS and 30 mM Brij-35, the difference in the retention times was 0.50 min. The rods and spheres were not baseline-resolved, but the detector was a UV-vis diode array, which allowed a spectrum to be collected that assisted in interpreting the chromatogram. Wei suggests that the SEC was inadequate because the gross size difference between the rods and spheres was insufficient for them to be separated effectively. Celia Henry
A transmission electron micrograph of a mixture of nanorods and nanospheres.
Analytical Chemistry News & Features, June 1, 1999 3 7 3 A
News releasedfromcells activated with recombinant mouse interferon-y and lipopolysaccharide. The sensor's limit of detection is 1 uM NO. The sGC-based sensor does not respond to common interferents, including nitrate, nitrite, and oxygen. However, the presence of glutathione was found to decrease its sensitivity. "The [glutathione interference] problem is unique to guanylate cyclase," says Barker. Cytochrome c'-based NO sensors are not affected by glutathione. Because glutathione is produced by macrophages, the sGC-based sensors had to be calibrated in a phosphate buffer containing the same amount of glutathione present in the cellular environment of interest. The heme domain and sGC are both relatively unstable proteins and, therefore, denature at high temperatures. "That is where the cytochrome c'-based sensors have an advantage—they are much more stable," says Barker. The sGC-based sensors can be used at room temperature for several hours, but they cannot be stored at room temperature overnight. However, sensors stored at 0-4 °C remain stable for several days. "They're like a lot of biological materials; you'd better keep them in the fridge," remarks Kopelman. Britt Erickson
that serves as an initial screen is performed with the porogen solvent, which, in this case, is methylene chloride. A MIP is most likely to bind to a template in the porogen solvent. Therefore, complete release of the template into the porogen solvent is a good indicator that the MIP is no good. "A rapid and quantitative release of template when washing the polymers in the porogenic solvent is a good indicator for absence of high affinity imprinted sites," says Sellergren. "On the other hand, retention of template is no evidence for their presence. However, a quantitative release ecn be used aa s ariterion for discarding materials, thus saving time in the further screening for selectivity." To test their assumption that release of the template into the porogen solvent means that the MIP is not selective, Sellergren and Lanza screened all of the MIPs— even the ones that had completely released the template in the initial screen— for selectivity for rebinding of terbutylazine (a triazine herbicide that had been used as the template molecule). The initial release test indicated that only polymers made with (trifluoromethyl) acrylic acid (TFM) and methacrylic acid (MAA) should be expected to show an enhanced affinity for the template molecule in methylene chloride. The results of the rebinding experiments were as they expected. "Only the polymers that significantly retained the Rapid MIPs template in the release test showed selective rebinding proDerties " savs Sellergren screening "Thus a complete release in the porogenic Selectivity is the word for molecularly imsolvent seems to be a valid criteri[on] to printed polymers (MIPs). Unfortunately, exclude materials" many factors involved in the synthesis of MIPs ultimately affect their recognition Polymers made with MAA as the funcproperties—the type and concentration of tional monomer rhowed the highest selecthe functional monomer, the cross-linking tivity for rhe template. Interestingly, , fullmonomer, the solvent (porogen), and the scale version of the MAA polymer, which template, as well as the experimental condi- was used as a chromatographic stationary tions. Synthesizing new MIPs or optimizing phase, displayed the highest capacity faca given MIP requires systematically varytor for atrazine in acetonitrile (k' = 18) that ing these factors and analyzing the resulthas been reported under similar condiing materials. tions. The retention of atrazine waa ssgnificantly higher with a polymer imprinted That's where Borje Sellergren and with terbutylazine than with a polymer Francesca Lanza of Johannes Gutenberg imprinted with atrazine. Sellergren susUniversity Mainz (Germany) come in. like pects that this is due to the difference in Toshifumi Takeuchi and his co-workers at basicity and hydrophobicity of the temHiroshima City University Qapan) (Anal plates and because the terbutylazine site Chem. 1999, 71, 285-90), Sellergren nnd can be expected to be sterically capable of Lanza have developed a method for rapidly accommodating atrazine. synthesizing and screening "mini" MIPs, which have been produced in a scaledAccording to Sellergren, the most imdown synthetic process (Anal. Chem. portant outcome of this work is the 1999, 71,2092-96)) However, ,n Selleramount oftimeit will save in synthesizing gren and Lanza's method, the washing step and optimizing MIPs. Although only six 374 A
Analytical Chemistry News & Features, June 1, 1999
MIPs were synthesized in this work, the method should be applicable to screening large numbers of MIPs. "There is no doubt that the screening methods developed by us and [Takeuchi's group] will bring significant savings in terms of time and reagents in the search for MIPs with desired performance," says Sellergren. Sellergren says they still have important steps to consider. "It is important to demonstrate the usefulness of the method in the imprinting of difficult templates where the present established protocol fails to produce MIPs with the desired recognition properties," he says. "Furthermore, because the consumption of template is typically a few milligrams, the imprinting of more precious templates can now be systematically assessed." In addition, he says it is important, in many cases, to find MIPs that recognize analytes in water or to find materials with higher sample load capacities or improved mass transfer properties. "In view of the large number of options it be of interest to find ways to further miniaturize the process" he says. Celia Henry NEWS FROM ABRF '99
The Association of Biomolecular Resource Facilities Elizabeth Zubritsky reports from Durham, NC.
N e w biological standards considered Despite the enormous growth in bioanalytical techniques, the sole biological standard is still bovine serum albumin (BSA). However, the quality and compliance committee of the Association of Biomolecuiar Resource Facilities (ABRF) hopes to change that. The committee is working on plans for additional biological standards—peptides, proteins, and possibly other molecules—according to a report given by Alan J. Smith, chairman of the committee's working group for peptide standards. At this point, the committee has spoken to the National Institute of Standards and Technology (NIST) about developing new biolocical standards, but no formal ar-
