Micrometer- and nanometer-scale structures (collectively, “microstructures”) are useful for making micro-sensors, micro-actuators, micro-instruments, micro-optics, microfluidic devices, and micro-reactors. Previous methods for making such structures have focussed on their production within solid substrates through, for example, the use of photoresists and chemical etching techniques. Since there are a limited number of solid substrates that can be used to produce microstructures, the range of chemical and physical properties exhibited by known structures also is limited.
One approach to altering the chemical and physical properties of the surfaces of microstructures is to apply a thin film of a polymeric material to the surface of a microstructure etched into a substrate. For example, Yan et al. (U.S. Pat. No. 5,830,539) describes applying a polymeric material coating to the interior of a micro-well etched in a silicon wafer.
Another approach is to construct microstructures entirely from polymeric materials. Husemann et al. (J. Am. Chem. Soc., 122: 1844-45, 2000) recently reported the generation of polymer array brushes using a combination of surface-initiated polymerization and photoacid-based chemical transformation of the polymer chains. However, because this technique is based upon a tert-butyl acrylate polymer, the range of chemical and physical properties of the resulting microstructures is limited.
Chip-based microwell arrays have greatly increased the capability to perform analytical and biochemical reactions and assays. These microwell arrays allow a large number of reactions to be carried out simultaneously, at much higher speed and more efficiently than conventional analyses. The small volume of the microwells dramatically reduces the amount of reagents needed, resulting in reduced waste and lower costs. Most of the microwells are silica-based microwells that are fabricated in silicon wafers or glass slides using photolithography and etching. See, for example, Mensinger et al., in Micro Total Analysis Systems; van den Berg and Bergveld Eds., Kluwer Academic Publishers, London,; p. 237 (1995); Kricka and Wilding, “Micromechanics and Nanotechnology” in Handbook of Clinical Automation, Robotics, and Optimization, Kost and Welsh Eds., John Wiley and Sons: New York, p 45 (1996); and Kovacs et al., “Silicon Micromachining: Sensors to Systems,” Anal. Chem., 68: 407A (1996).
Conventional methods for manufacturing microwells are relatively expensive and the necessary facilities are not routinely accessible to most chemists. Furthermore, as the surface-to-volume ratio increases dramatically for the microfabricated wells as compared to conventional reaction tubes, it is important that the devices are chemically compatible with the reactions taking place inside them. However, silicon chip-based devices may not be compatible with biochemical assays or reactions. For example, native silicon is an inhibitor of polymerase chain reaction (PCR) and amplification. Thus, PCR reactions performed in silicon chip-based nanowells show poor reproducibility.
Therefore, recent investigations have focused on polymer-based microwells. For example, microwells have been generated in ordered arrays by etching with a bundle of optical fibers (Pantano and Walt, “Ordered Nanowell Arrays,” Chem. Mater., 8: 2832, 1996). Ewing and co-workers fabricated picoliter microvials in PS by an embossing technique using structures formed by a photolithographic patterning technique (Clark, et al., “Electrochemical Analysis in Picoliter Microvials,” Anal. Chem. 69: 259 (1997)). Recent work by Whitesides and Chilkoti demonstrated large arrays of microwells in poly(dimethylsiloxane) (PDMS) (Jackman et al., “Fabricating Large Arrays of Nanowells with Arbitrary Dimensions and Filling Them Using Discontinuous Dewetting,” Anal. Chem., 70: 2280 (1998). These microwell arrays were fabricated by casting PDMS elastomer against a master that was prepared by conventional photolithography.
Equally important for the successful implementation of microwell arrays for analytical and biochemical applications is the delivery system that controls the precise placement of the reagents into the wells. Systems and processes that deliver reagents in an array format include a pin tool that loads the reagents mechanically, ink jet printing that dispatches microscopic drops of liquids on active surfaces, and electrically polarizing the array sites. An attractive alternative is to create functional microwells that can perform surface-induced placement of reagents into the wells. In a recent report, Whitesides et al. selectively deposited proteins into microwells made of PDMS by first trapping air bubbles in the well bottoms and then coating the well-top with one protein (Ostuni et al., “Selective Deposition of Proteins and Cells in Arrays of Nanowells,” Langmuir 17: 2828 (2001)). The air bubbles were then removed and a second protein was delivered into the wells.