Fabrication and characterization of microwell array chemical sensors

Microwell arrays have been fabricated on the distal face of coherent fiber- optic bundles. A typical microwell array comprises similar to 3000 individu al optical fibers that were etched chemically, Individual microwells were s imilar to1 to 14-mum deep with similar to 22-mum widths and were filled par tially with a chemical sensing (polymer + dye) layer to produce a microwell array sensor (MMAS). MWASs were fabricated using a technically expedient, photoinitiated polymerization reaction whereby a similar to2 to 10-mum thic k pH-sensitive or O-2-sensitive sensing layer was immobilized inside each m icrowell. The pH-sensing layer comprised fluorescein isothiocyanate-dextran conjugate immobilized in a photopolymerizable poly(vinyl alcohol) membrane . The O-2-sensing layer comprised a ruthenium metal complex entrapped in a gas-permeable photopolymerizable siloxane membrane. pH and PO2 were quantit ated by acquiring luminescence images using an epifluorescence microscope/c harge-coupled device imaging system, The pH-sensitive MWAS displayed a pK(a ) of similar to6.4 and a response time of similar to2.5 s. The O-2-sensitiv e MWAS behaved according to a nonlinear Stern-Volmer model with a maximum I -0/I of similar to4 and a response time of similar to2.5 s, MWASs are advan tageous in that suitably sized samples such as single biological cells can be co-localized with the sensing matrix in individual microwells.