A coverslip method for controlled parallel sample introduction into arrays of (sub)nanoliter wells for quantitative...

We have developed a straightforward coverslip method that combines rapid sample introduction into arrays of 19.8-55.0-microm-deep microwells (prefilled with reactants) on a chip with complete sealing of these wells to avoid evaporation during optical detection. We used coverslips of 300-1500-microm-thick poly(methyl methacrylate) (PMMA) containing arrays of holes with diameters of 200 and 300 microm, and 800-microm-thick quartz slides containing arrays of holes with diameters of 100 and 200 microm. A coverslip was placed on top of a chip, thereby positioning the holes between the wells. A defined amount of aqueous sample solution was pulled between the cover and silicon nitride coated chip by means of capillary forces, resulting in entrapment of air in all wells. By partly or completely positioning the holes over the wells, air could escape through the holes, resulting in complete filling of the wells with the surrounding liquid within 0.1-0.2 s. Immediately after filling, the slide was rapidly shifted back to its original position and was pressed onto the chip for 5-30 s with a force of 1-2 kg cm(-2), resulting in complete sealing of the wells for minutes up to one-half hour. Because the well volumes of an array are identical and the wells are completely filled, reproducible assay volumes and flat menisci are obtained, making this method very suitable for quantitative analysis.