RESEARCH PROFILE Big step toward lab on a chip Will analytical chemistry follow in the footsteps of the electronics industry and miniaturize instruments into the laboratory equivalent of an integrated circuit? In the Dec. 1,1996, issue of Analytical Chemistry (p. 4081), researchers from the University of California-Berkeley and the Lawrence Livermore National Laboratory took a big step in that direction by reporting on the microfabrication of a polymerase chain reaction (PCR) amplification system coupled to CE on a single chip. "This is the first integration of two fundamentally different processes on these chips," says Richard Mathies of the University of California-Berkeley. "Without moving macroscopic fluids, we can extract DNA from the PCR reactor and get a good CE analysis." The integrated chip consists of a PCR reactor fabricated in silicon with internal heaters formed by chemical vapor deposition and is connected to a CE system etched in glass. Using approximately 20-uL solutions, the researchers successfully amplified and analyzed a (3-globin target in less than 20 min and Salmonella DNA in under 45 min. "The nice thing was that we gained the predicted increase in speed," says M. Allen Northrup of LLNL. In addition, Northrup points out, the system is "hands-off" and potentially disposable and inexpensive. A key development in making the chip work is the "electrophoretic valve" filled with hydroxyethylcellulose (HEC) that connects the two devices but keeps reagents segregated. "There are reagents in the typical electrophoresis buffer that will kill the polymerase chain reaction, and the salt concentration in the typical PCR reaction inhibits the injection and separation in CE analysis," says Mathies.
Both Mathies and Northrup admit being initially uncertain that the valve would work. "We were worried that, during [PCR] thermal cycling, unamplified liquid was going to leak out into the CE channels; but it didn't happen because the small channel (100 um wide, 8 um deep) acts as a fluidic resistor and because of the HEC gel," says Northrup. Other unique features of the system include a disposable polypropylene liner in the PCR reactor to prevent DNA from sticking to the sides; integrated thin-film heaters that speed up thermal cycling, using silicon in the PCR reactor for its good thermal properties; and building the CE from glass that can stand up to the required high voltages. Although the scientific details were challenging, constructing the chips turns out to be fairly easy. By micromachining standards, the micrometer-sized dimensions of this chip are large, and the other steps, such as etching, bonding, and vapor deposition, are straightforward. "You could contract this to be made by commercial micromachining houses," says Northrup. One benefit of this system is that it allows optimization of the PCR reaction through real-time monitoring by CE of the products. "Run [the PCR reaction] for 25 cycles, and look at what you've got—if it's great, stop; otherwise, run it for five more cycles," explains Mathies. As a result, efficiency could improve, and precious samples would be conserved. Mathies and Northrup credit the Department of Commerce's Advanced Technology Program, which has been attacked by some as "corporate welfare", for initiating the collaboration. However, with the proof of concept now completed, the two groups are moving in somewhat different research directions.
Mathies's group is targeting applications to DNA sequencing under the Human Genome Project. "[In an earlier paper (Woolley, A. T ; Mathies, R. A. Anal. Chem. 1995, 67, 367680.) ] we showed that you can do sequencing on a chip, but the performance isn't as good as the state-of-theart slab gels and capillaries," says Mathies. They are now testing systems that work in the 100-nL range and looking at other designs for valves. Northrup's group, with funding from the Defense Advanced Research Projects Agency, is using this new technology as the basis of clinical instrumentation. The LLNL scientists just delivered to the military what is believed to be the first portable battery-operated (1.5 W) DNA analysis system. The device fits into a small suitcase and could be used for identifying battlefield remains, as well as food and water pathogens in remote locations. They also have used the system to detect RNA from the AIDS virus and from the hepatitis C virus in human tissues. The Northrup group is now building arrays of microfabricated PCR chambers and tackling the complex question of miniaturized sample preparation. Wherever this research goes, the major advance, says Mathies, is demonstrating that the integrated circuit analogy can apply to analytical chemistry. "Now we have sufficient confidence in our abilities to transfer various technologies onto a chip and then integrate them, fabricating chips to do processes you can't do out in the lab," he says. "My vision is that a student will be able to create a mask to design their whole experiment on a chip." Alan Newman
The PCR/CE microdevice showing the experimental setup (left, courtesy of Mathies) and a schematic (right).
Analytical Chemistry News & Features, January 1, 1997 17 A
