Microfabricated Devices for Rapid DNA Diagnostics (2000)

Science makes new technology and technology pushes science forward. For biological studies and hospital diagnoses, knowledge and techniques accumulated from other fields, such as semiconductors, optics, electronics, and chemistry, are generating huge impacts in almost every aspect and for almost everyone involved. Among these, tools for DNA diagnostics play a very important role. They are also essential to many genetic studies, drug discovery, and even forensic identifications. Working with Professor Stephen Quake, Professor Axel Scherer, and my colleagues in Caltech, I have developed several building blocks for rapid DNA sizing, cell sorting, molecular fingerprinting, and hybridization assays, based on those newly available technologies. First, a microfabricated flow-cell device was developed using 'soft lithography'. It offers a small, cheap, robust, and contamination-free alternative to the complicated glass-capillary structure used in a conventional flow cytometer. Based on this device, a highly sensitive single-molecule DNA sizing system was demonstrated. It is 100 times faster and requires a million times less sample than pulsed-field gel electrophoresis. For DNA molecules of 1-200 kbp, it has comparable resolution, which improves with increasing DNA length. To serve as a real substitute for a conventional flow cytometry, DNA and cell sorting has also been demonstrated under this system. Simple enclosed actuation schemes are implemented and system downtime due to capillary cleaning is totally eliminated because the device costs only pennies to make and thus becomes disposable. Therefore, there is no cross-contamination issue for both DNA sizing and cell sorting applications. Using this system, prototype work for rapid DNA molecular fingerprinting was devised as an alternative to the widely used Southern blot fingerprinting protocol. Molecular evolution, VNTR fingerprinting of human forensic samples, disease diagnosis based on restriction fragment length polymorphism (RFLP), and simple DNA genomic mapping can all be accomplished with this system. Because of the great flexibility of microfabrication, more complicated functions can also be designed and incorporated into these flow-cell devices. Therefore, this single-molecule sizing system can become a key component in the family of lab-on-a-chip devices. In addition, a multilayer soft lithography technique was invented, allowing monolithic microvalves and micropumps to be built into these flow-channel devices. Active microfluidic systems containing on-off valves, switching valves and pumps were made, entirely out of elastomer. The softness of these materials allows the device area to be reduced by more than two orders of magnitude compared with silicon-based devices. An actuation volume as small as about one picoliter is demonstrated. The other advantages of soft lithography, such as rapid prototyping, ease of fabrication, and biocompatibility, are retained. Based on these active components, an integrated diagnostic chip was built. More than two orders of magnitude improvement in terms of binding speed and efficiency over passive devices was shown. Selective surface patterning of DNA molecules, biotin, and avidin within the chips by elastomeric flow channels was also shown. With active pumping, we are able to make a rotary motion in these microfluidic devices and show fast inline mixing which overcomes the limitation of laminar flow in this low-Reynolds number regime. Moreover, the problem of buffer depletion due to electrolysis in electroosmotic or electrophoretic flow control does not exist in these devices. All of these serve as fast, cheap, and robust alternatives to many conventional techniques used widely in biological studies and hospital pathogenic diagnosis. They are all very simple to fabricate and easy to use. If desired, more complicated flow patterns and functions can also be incorporated with much less effort than their silicon counterparts. We anticipate that more applications and devices based on these systems and techniques will be developed rapidly in the near future.