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Microscale separat i o n s go o n l i n e w i t h MS
from a 150-um inner-diameter fused-silica capillary with a 5-10-um pulled-needle tip. They loaded a mixture of styrene, divinylbenzene, 1-dodecanol, and toluene into the needle with a catalyst and allowed it Neither packing chromatographic mateto polymerize. The resulting monolithic rial into needles nor miniaturizing support structure has electrospray sources similar properties to is a particularly new commercial polymeric idea. The novelty is in chromatographic mathe combination. In terials and can be used this issue of Analytical for reversed-phase Chemistry (p. 4879), separations. Terry D. Lee and his co-workers at the BeckLee and his coman Research Institute workers performed of The City of Hope online LC/MS analysis (Duarte CA) describe of a standard mixture of a microscale electroribonuclease A, cytointerface that chrome c, holotransincludes a polymeric ferrin, and apomyoglosuDoort inside the bin with the needles electrospray needle and a quadrupole iontrap mass spectrome"We've combined ter. The test mixture the column with the Chromatograms for the LC/MS was analyzed at flow electrospray interface analysis of a protein standard rates of 700 nL/min at as a single piece," says mixture using (A) a needle packed 50 psi. Compared with Lee. "We eliminate a with the PS/DVB monolith, (B) a a commercial polylot of the plumbing, needle packed with C18 support, meric stationary phase and we use the small and (C) a needle packed with a and a C stationary dimensions of an ideal commercial polymeric support. phase the monolith electrospray source." cave higher resolution separations of the The device is approximately the same nrntein mixture which the authors atsize as the emitter tips used for infusion nanoelectrospray but it allows a chrotritiute to the selprtivity of the nnlystyrene/ matographic separation prior to the mass divinvlbenzene stationary phase and the spectrometric analysis physiral nature of thp nnre structure In The researchers made the device
addition the interfarp was uspri for the
LC/MS/MS analysis of the peptide mixture from an in-gel tryptic digestion of a silver-stained two-dimensional gel spot. "Our ultimate goal is to do these kinds of separations on chip-based systems. We always had a quandary with how we were going to pack columns on a chip with a chromatographic support," says Lee. "This gives us a way to do that because you can polymerize it directly into a channel and have a shot at doing on-chip LC separations. This is important to us because we've had better success with our applications using LC rather tiian CE." A chip-based LC system provides the means to scale down the analysis to take full advantage of the sensitivity of modern electrospray mass spectrometers. 'We've been involved for the last two or three years in building an effective electrospray source on a chip. We feel we have that now," says Lee. However, interfacing the chips to the mass spectrometer is still difficult. 'We think we have solutions to the problems, but more development is needed before we can convince people," says Lee. Lee feels that one of the best aspects of the new interface is the relative ease with which it can be made. "Anyone can make them. We're using them routinely in our lab, and we don't really have any special equipment." He hopes to see people start using this technique. "There's an opportunity for a company to start building them because of the simplicity of putting them together. They could be mass-produced if anybody had a notion to do it." Celia Henry
DNA-cation binding at micropipets Micropipet electrodes were introduced more than a decade ago by G. Taylor and H. Girault at the University of Edinburgh (United Kingdom) to study ion-transfer reactions across the interface of two immiscible electrolyte solutions. However, several technical problems, including low-quality voltammograms, large background currents, and interferences from parallel charge-transfer processes, prevented their widespread use. Recently Michael V. Mirkin and Yuanhua Shao of Queens College772 A
CUNY showed that many of these problems could be resolved by the independent silanization of the inner and outer walls of the pipet, affording reproducible quantitative measurements (Anal. Chem. 1998, 70, 3155-61). In the November 15 issue of Analytical Chemistry (p. 4653-60), Mirkin and his co-worker B. R Horrocks of the University of Newcastle-upon-Tyne (United Kingdom) apply ion-transfer voltammetry at micropipets to the measurement of cation binding toDNA
Diagrams of unfacilitated (A) and facilitated (B) ion-transfer processes at micropipets.
Analytical Chemistry News & Features, December 1, 1998
Two methods—one based on nonfacilitated and the other on facilitated ion-transfer voltammetry at micropipets—are described
for determining DNA-cation binding constants. In both methods the organic phase (1,2-dichloroethane, DCE) is contained inside the micropipet. In the nonfacilitated approach, the cation of interest (/V-methylphenanthroline, MP+) is present in the aqueous phase and transferred to the organic phase by applying sufficiently negative voltage. When DNA is added to the aqueous phase, a sharp decrease in diffusion current is observed. The binding constant is calculated based on the normalized steady-state current as a function of DNA concentration. Because the experiments are performed under diffusion-limiting conditions and at steady state the binding constants can be determined without knowing the micropipet size binding kinetics or diffusion coefficient values Measurements of DNA binding to electrochemically active ions are common, says Mirkin. "What is new here is that the technique is done with micropipets based on ion transfer rather than on oxidation/reduction," he says. In the facilitated approach the cation is present in the organic phase and is transferred to the aqueous phase containing DNA "To my knowledge, there have been no previous reports on facilitated ion transfer from an organic phase to water," says Mirkin, "and there have been no publications on facilitated transfer involving DNA" In principle, the ion can be transferred from the organic phase into water in the absence of a complexing agent; however, it requires significant voltage. "We do not observe the transfer of M P without adding DNA to the aqueous phase because it is outside our potential window," says Mirkin. The DNA acts as a facilitating agent, lowering the potential necessary for the ion transfer to occur. Binding constants are estimated based on the difference between the half-wave potentials with and without DNA Because M P is a known DNA intercalator, the mechanism of ion transfer is believed to involve insertion of the cation's planar aromatic rings into DNA duplexes at the DCE/water interface. Stripping of the cation from the DNA complex produced a symmetrical surface wave. Further work is underway to investigate the nature of the interfacial film. The possibility of developing a method for stripping DNA duplexes that may lead to detection of hybridization schemes is also being explored. Britt Erickson
Microfabrication without a cleanroom
trench." CSuCP is best suited for patterns requiring narrow (down to 1 um) but Standard methods of designing and generat- widely separated (several microns) ing microscale patterns on surfaces are slow features. and expensive. Chrome masks are typically Masters were prepared by spin coating made by commercial suppliers, and the pho- a silicon wafer with a mixture of poly (methtolithography must be carried out in a clean- yl methacrylate) and rhodamine B in chloroom. For researchers whose needs aren't roform. The desired patterns were ablated particularly complex, such hurdles to trying into the film with a laser operating at new simple patterns may be disappearing. .n 532 nm. Rhodamine B was selected bethe November 15 issue of Analytical Chemis- cause its maximum absorption is near the try (p. 4645)) George M. Whitesides and co- laser wavelength. They tried other polyworkers at Harvard University describe a mers, including polystyrene and polyuremethod for rapid generation of elastomeric thane, and organic solvents, such as dichlomasters for microcontact printing (uCP) and romethane and ethyl acetate, but found the a new variant of uCP called controlled sagcombination of PMMA and chloroform to ging microcontact printing (CSuCP). Neibe the best. Whitesides says, "The ther of these methods needs to be carried PMMA/CHC1 system was used because out in a cleanroom. both the dye and the polymer dissolve well in chloroform and the resultant spincoated surface of the dye/polymer matrix is flat This choice was largely empirical" Stamps are made by casting PDMS on the master Devices were created by wetting the stamps with a solution of hexadecanethiol in ethanol and pressing them to the gold substrate. As examples, they fabricated a minielectrode array, which was used for cyclic voltammetry of a hexamineruthenium (III) chloride and lithium perchlorate redox/electrolyte solution. They also created a stamp with an array of microwells that were used as microreactors for the crystallization of KN03. The quality of the devices depends on three factors how fresh the gold surface is, the concentration of the thiol solution, and the etching proceSteps involved in using CSyCP for dure. The stamps are hardy some have qeneratinq 1 -fum features. survived than 200 uses over a period of four months. Both techniques use an elastomeric They have been able to make features master to transfer a pattern to a substrate. as small as 5 um with conventional uCP In conventional uCP, the pattern is in the and 1 um with CSuCP. Whiiesides says, raised portion of the poly(dimethylsilox'We believe it might be possible to go to ane) (PDMS) stamp; the pattern is trans1 um [with uCP] and 0.5 um [for CSuCP] ferred to the surface when the stamp is with more elaborate optical instrumentabrought into contact with the substrate. In tion [that provides] better focusing of the contrast, the pattern for CSuCP is a series laser beam and beam profile correction." of trenches on the stamp. Most of the surThese techniques hold the promise of face of the stamp touches the substrate— bringing micropatterning within reach of only the trenches don't touch the substrate. the average chemistry laboratory. "For According to Whitesides, the major differchemical laboratories that do not need ence between the two patterning techcomplex patterns or multilayer fabrication, niques is that the latter results in features and that do not have access to a cleanroom, on the surface that are smaller than the this method provides an alternative," says features on the stamp. "These trenches Whitesides. "All that is really needed for tend to 'shrink'" says Whitesides "so that simple patterns is an x-y stage and a low the unprinted portion of the substrate is power laser." narrower than the original width of the Celia Henry Analytical Chemistry News & Features, December 1, 1998 773 A
