Array Concept Offers New Way To Sort DNA Asymmetric flow cells etched on silicon chips show promise as size separation tool

Anovel technique for the continuous ι size separation of macromolecules I may provide an alternative to gel

electrophoresis methods used in hun­dreds of research laboratories worldwide to separate proteins and DNA. The tech­nique uses arrays of separation cells, etched by microlithography on silicon chips, to separate macromolecules in an electrophoretic flow stream.

The strategy was reported last month in Physical Review Letters [80, 1548 and 1552 (1998)] by two independent teams. Physicist Deniz Ertas of Harvard Universi­ty (now at Exxon Research & Engineer­ing, Clinton, N.J.) focused on its theoret­ical aspects, whereas physicist Thomas Duke of the University of Cambridge, En­gland, and physics professor Robert H. Austin of Princeton University proposed a practical device and evaluated its perfor­mance. Preliminary results on prototype arrays constructed in Austin's laboratory indicate that the idea works effectively.

In the past, Ertas says, "there was not a technological ability to do microarray sep­arations. You really need a very controlled geom­etry, and that wasn't available until the early '90s," when Austin and graduate student Wayne D. Volkmuth invented a method for manufactur­ing arrays by microlithog­raphy. "I guess that's why nobody came up with the idea before."

The microarrays con­sist of tiny, asymmetric flow cells. Each cell has a narrow entrance at the top and two exits at the bottom that connect to the next row of cells. A sample is introduced at the top of the array, and an applied electric field pulls sample molecules through the cells.

Inherent differences in the diffusion behavior of

large and small molecules cause the large ones to tend to hug the wall adjacent to the entry point of each flow cell. The smaller molecules tend to diffuse away from the wall. Sorting by size occurs when the molecules must flow around a barrier that leads to one or another of the next two cells in the array.

After the molecules have negotiated their way through the cells, they exit the device in size order at different locations, where they can be collected for subse­quent analysis or manipulation. The dy­namic range of an array—the range of molecular sizes it can separate—can be tuned simply by changing the applied electric field.

The design of the array can vary and has not yet been optimized. Ertas con­ceived of flow cells defined by diamond-shaped barriers, whereas Duke and Aus­tin successfully implemented the tech­nique using rectangular barriers. "We estimate that the cost is low—less than a dollar a chip—so the devices could be disposable," says Duke.

Arrays sort biomolecules by size

Injection

Small molecules

Large molecules Large

molecules Small

molecules Ε = applied potential

In an array proposed by Ertas (left), sample is injected at the top. Molecules of different size diffuse away from the left wall of each cell to a variable extent as they are pulled through the array by the applied electric field. Smaller molecules diffuse farther and thus tend to move to the right of the branching point at the base of each cell In a sorting device developed by Duke and Austin (right), a mix­ture is injected at the top left corner of the sieve and fractionated components are collected at the bottom right.

According to Ertas, "This technique may have major advantages over tradi­tional methods as an analytical separa­tion tool that can be easy to set up and automate, since no gel preparation or sample extraction is required. The main driving force for the work is separating DNA. The Human Genome Project and a lot of biotechnology rely on that process. It's a very expensive process, and it is still an art form, more or less."

Ertas adds: "Gel electrophoresis is a tricky business that requires very skilled labor. The advantage of something like this is that it can be easily automated. DNA separation has been very difficult and cumbersome. This is a way to make it a lot easier to deal with, and it would possibly require less finesse to be able to get consistent results."

Duke and Austin calculate that DNA molecules ranging in size from 100 to 20,000 base pairs can be separated by ar­rays with about the same resolution as is possible with gel electrophoresis, but much more rapidly. And the devices "are especially suitable for integrating molec­ular separation with subsequent analyti­cal steps," they note. "Hence, we believe that they will be useful in the develop­ment of lab-on-a-chip devices."

According to Duke and Austin, "The principal limitation of this type of sieve is that the overall throughput of molecules is low"—on the order of a femtomole— "since the mixture must be injected in a very localized region and the solution ^^^^^^mmm should be dilute enough

that steric intermolecular interactions are rare." However, "the problem with throughput is allevi­ated somewhat by the fact that the device can be operated continuous­ly. More molecules can be sorted simply by run­ning the device for a longer time."

Chemistry professor James W. Jorgenson of the University of North Carolina, Chapel Hill— who specializes in chemi­cal separations, including capillary electrophore­sis—says: "This is an inge­nious approach, and one which is very appealing from its fundamentally 'clean,' well-defined ge­ometry. It has some signif­icant underlying similari-

MARCH 9, 1998 C&EN 33

Sieve

s c i e n c e / t e c h n o l o g y

ties to field-flow fractionation, but none­theless it is different. It offers continuous mode operation, which helps improve the overall throughput of the device, which is otherwise low."

Ertas agrees the array technique is very similar to field-flow fractionation—a method in which a field (such as an elec­tric field) is imposed on a sample in a flat channel, causing component molecules to separate by size or other factors—but he believes it has some advantages. "It's a lot more efficient and the resolution is higher," he says.

Although Jorgenson doesn't dispute the estimate by Duke and Austin of the technique's potential speed advantage over conventional gel electrophoresis, he points out that its projected resolu­tion falls far short of what is achievable in capillary electrophoresis.

"Overall, I find these papers thought-provoking," Jorgenson says. "It would appear, however, that the [array tech­nique] may never be competitive, at least in terms of resolving power, with capil­lary electrophoresis. Of course, it may turn out to offer unique and useful capa­bilities that capillary electrophoresis can't offer—continuous versus batch op­eration, for instance. This is always hard to foresee with new technology."

Corporate fellow and group leader J. Michael Ramsey of the chemical and ana­lytical sciences division of Oak Ridge National Laboratory, who specializes in lab-on-a-chip development, says the asym­metric sieving concept "is a very intrigu­ing idea. I like the simplicity, in that fin­icky chemical preparations of sieving gels are not involved. Such simplistic ap­proaches fit well within the lab-on-a-chip concept that we have been working on for several years now."

Ramsey continues: "If you are going to mass produce microdevices for the elucidation of chemical and biochemical information, you don't want to have tricky steps at the manufacturing stage or at the point of use by the customer. This concept fits those requirements. It is po­tentially a very attractive functional ele­ment that could be added to the lab-on-a-chip toolbox." However, he adds, "it ap­pears that it is not the panacea for sieving biomolecules. For example, I don't think it is attractive for DNA se­quencing, given the theoretical resolving power stated in the articles."

Arrays are more appropriate as an ana­lytical tool than as a preparative one, say Duke and Austin. "They might, though, prove useful for recovering DNA from

scarce samples for subsequent amplifica­tion by the polymerase chain reaction."

In addition, they note that "sorting of globular, colloidal particles with sizes up to 10 urn would be feasible . . . by suit­ably enlarging the scale of the obstacles. Thus, microfabricated sieves could po­tentially be used for cell sorting, al­though the performance would be less effective than for small molecules."

On the other hand, says Ertas, the technique "probably wouldn't work for very small molecules—or not as well. The smaller the molecule the trickier it becomes, because your scale has to go down as you try to separate smaller and smaller things. But it should be useful for protein separations."

Duke agrees the method could work very well for proteins, "provided that electron-beam lithography is used to etch the fine detail required. It would be rather more expensive" to fabricate such chip arrays, he says.

Ertas adds, "It will be very interesting to see how this evolves, because I sus­pect there might be a lot of applications that nobody's actually thought of yet. This opens a whole new way of doing things."

Stu Borman

Self-organization of reagents yields high enantioselectivity

A collaboration of chemists at Cornell University and in the process research group at Merck Research Laboratories, Rahway, N.J., has yielded a commercially viable, highly enantioselective process for manufacturing a promising antiviral drug. The team also has proposed an ex­planation of the process' remarkable ste­reoselectivity based on spectroscopic and computational evidence [/. Am. Chem. Soc, 120, 2028 (1998)].

The drug candidate is a single-enantiomer benzoxazinone that inhibits viral reverse transcriptases. In clinical studies, it shows promise in treating HIV infections in combination with Merck's indinavir, a drug that inhibits viral pro­teases. Marketing of the reverse tran­scriptase inhibitor has been assigned to the joint-venture DuPont-Merck Pharma­ceutical, which is expected to file a new-drug application with the Food & Drug Administration this year.

The medicinal chemists who discov­

ered the reverse transcriptase inhibitor at Merck made it by resolving a racemate. That approach entails throwing away half of an expensive compound, however. So Andrew Thompson, Edward G. Corley, Martha F. Huntington, and Edward J. J. Grabowski of Merck process research sought an asymmetric synthesis.

Their solution turns out to be an enan­tioselective addition of a cyclopropyleth-ynyllithium reagent to a trifluoroacetophe-none to yield a single-isomer caibinol in about 97% enantiomeric excess. They next cyclize the carbinol to the drug itself. To provide the asymmetric induction for the addition, they add the lithium alkoxide salt of a single-isomer derivative of ephed-rine (2-pyrrolidino-l-phenyl-l-propanol) to the reaction mixture.

Such high stereoselectivity for addi­tion of a relatively small, highly sym­metrical group such as cyclopropyl-ethynyl is remarkable. And the course

Remarkably enantioselective addition . . .

OCHo

OCHo

. . . opens route to AIDS drug

A

ROLi s lithium salt of enantiomeric ephedrine derivative

34 MARCH 9, 1998 C&EN