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RESEARCH PROFILE Welcome to the microfluidic matrix Despite the promise of microfluidic devices for analyzing very small sample volumes and reducing instrument size, getting samples into the microfluidic chips has proven a difficult proposition. “People have shown chips that can analyze nanoliters, but they have to load microliters on those chips, and most of that gets wasted,” says Stephen Quake at the California Institute of Technology (Caltech). In the September 15 issue of Analytical Chemistry (pp 4718– 4723), Quake and his Caltech colleagues Jian Liu and Carl Hansen introduce a microfluidic device that performs PCR. Although other PCR microfluidic devices have been described, Quake says, “We have come up with a geometry and design that makes very effective use out of the entire couple of microliters that you load on the chip.” The fabrication process was described in earlier papers by Quake and colleagues (Science 2000, 288, 113– 116) and is also discussed in the current issue of Analytical Chemistry (pp 429 A–432 A). Briefly, two layers of poly(dimethylsiloxane) (PDMS), one containing fluid channels and the other containing pneumatic control channels, are fused together. A valve is created every time the control channel passes over the top of the fluid channel. If air is forced into the control channel, the thin layer of PDMS that separates the control channel from the fluid channel deforms and blocks the fluid channel, closing the valve. The researchers create a matrix of thousands of valves. The matrix is designed to have hundreds of individual reactors. In fact, if there are N sample delivery wells by N reagent delivery wells, N 2 reactors will result. In the PCR assay, a 20 20 matrix performed 400 PCR reactions. The samples and reagents are supplied sequentially and then mixed. The mixing in each reactor is conducted in a loop by opening and closing valves in
A B DNA in
Enzyme in
No-primer control out
Primers in
No-primer control in
Holes for pumps Primers out Holes for valves B&C DNA out
Enzyme out
Holes for valve A
Enter the matrix. The schematic diagram of a 20 20 matrix with one reactor shown in greater detail (expanded view). The colors in the diagram correspond to control lines (green), template sample (blue), DNA polymerase (yellow), primers (red), and rotary pump (white).
sequence, which peristaltically pumps the liquids within the chip. One important benefit of this matrix is that it reduces the amount of wasted reagent—in this case, polymerase. Although 20 DNA samples and 20 primers are pipetted onto the plate, only 2 µL of polymerase is pipetted only once and flows to all the reactors. Quake adds, “We have very carefully worked out and explained how much we are loading on the chip as a true measure of reagent consumption.” In addition, far fewer pipetting steps are needed than with traditional microwell PCR techniques. For 400 PCR reactions, this technique requires only 41 (or 2N +1) pipetting steps, whereas 1200 are needed for microwell assays. “It saves work, it saves time, [and] it reduces the possibility of errors,” he says.
The chip can also optimize primer selection. By supplying the same DNA sample to all of the reactors and using 20 different forward primers and 20 different reverse primers, the best ones for an application can be found. “You can be very clever and come up with many different ways to do different kinds of combinatorial experiments with this sort of geometry,” Quake says. “We would like to come up with ways to use this for gene expression, which is the next logical step,” continues Quake. “We think that this is going to be a very eye-opening demonstration for people of how you can do a large degree of integration, and you realize a really practical, immediate savings in reagents and reaction setup. …This is sort of a stake in the ground for people to say, ‘This is the state of the art right now.’” a –Michael J. Felton
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