An important goal for many areas of biological research and clinical testing is the ability to detect and quantitate thousands of DNA segments in an automated and inexpensive fashion. We are trying to combine the power of DNA amplification with the ability to detect thousands of different sequences at one time using tiny amounts of sample (DNA ?chip? technology). A major technical challenge is to develop a method for automatically subdividing a small liquid sample (around 0.1 ml) into thousands of smaller droplets that can be repeatedly heated to near boiling temperatures for DNA amplification without evaporating (?thermocycling?). This year we refined a method for doing this using a microscope slide with an array of highly wettable spots on an otherwise water-repelling surface. The slide is first wetted with a drop of sample solution and covered with a cover glass. The excess sample is then removed from regions between wetting spots by bringing the slide into contact with a second liquid that has a greater affinity for the spaces between the wetting spots than the original sample liquid. The second liquid is a pre-polymer that is subsequently cured to form a solid shell around each droplet. After thermocycling, the devices are ?read? by determining the amount of fluorescence in each droplet, which correlates with the amount of specific DNA product produced. Since not all liquids and surface coatings formed droplets using this procedure, we modeled the forces involved in droplet formation as a function of surface energies of the various materials. We used a surface energy-minimization computer program to solve the relevant mathematical equations and developed some approximate formulae that allow simplified calculation of the range of material properties and device dimensions for which droplets should form. We overcame a technical hurdle, which was that the acrylate based pre-polymer displacing fluid that we had been using inhibited DNA amplification when left in contact with sample solution for too long before curing. We found a different displacing fluid that cures by a different polymerization mechanism that does not inhibit DNA amplification. We experimented with devices that form droplets, not by virtue of surface energy differences but because of an array of cup- like indentations in an acrylate coating placed on the microscope slide. These devices were made by polymerizing thin layers of acrylate pre-polymer in specific regions by exposing coated slides to ultraviolet light through a mask. Finally, we investigated various methods of depositing DNA reagents (primers and probes) that distinguish different DNA amplification reactions in different ?cups? or wetting spots on the devices so that different DNA tests could be performed in different regions of a device. - PCR, DNA chip, surface tension, photopolymerization, encapsulation, micropatterning
| Silver, J; Mi, Z; Takamoto, K et al. (1999) Controlled Formation of Low-Volume Liquid Pillars between Plates with a Lattice of Wetting Patches by Use of a Second Immiscible Fluid. J Colloid Interface Sci 219:81-89 |
