Spatial confinement of avidin domains in microwell arrays

Microwell arrays were chemically etched across the distal faces of coherent fiber-optic bundles. A typical 1.6 mm diameter array comprised ∼3000 individual microwells that were ∼1–14 μm deep and ∼22 μm wide. A methodology involving organosilane functionalized microwell surfaces and site-selective photobiotin chemistry was developed to partially fill microwells with a thin avidin layer. Avidin microwell arrays were characterized using charge coupled device optical microscopy and scanning electron microscopy. The avidin microwell arrays had individual well volumes that were six orders of magnitude smaller and up to 30-fold more numerous than commercially available avidin-coated microtiter plates. Preliminary results indicated that individual avidin microwells were ideally suited to house single biological cells. Using standard epifluorescence microscope optics and a mercury-arc lamp, an individual 22 μm wide microwell could be optically addressed and selectively filled with avidin without the use of a photolithographic mask. The ability to control both the size and position of avidin domains on the microwell array surface demonstrates the utility of this methodology towards fabricating a single microwell array with multianalyte sensing capabilities.