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 ∼3000 individual optical fibers that were etched chemically. Individual microwells were ∼1 to 14-μm deep with ∼22-μm widths and were filled partially with a chemical sensing (polymer + dye) layer to produce a microwell array sensor (MWAS). MWASs were fabricated using a technically expedient, photoinitiated polymerization reaction whereby a ∼2 to 10-μm thick pH-sensitive or O2-sensitive sensing layer was immobilized inside each microwell. The pH-sensing layer comprised fluorescein isothiocyanate−dextran conjugate immobilized in a photopolymerizable poly(vinyl alcohol) membrane. The O2-sensing layer comprised a ruthenium metal complex entrapped in a gas-permeable photopolymerizable siloxane membrane. pH and PO2 were quantitated by acquiring luminescence images using an epifluorescence microscope/charge-coupled device imaging system. The pH-sensitive MWAS displayed a pK a of ∼6.4 and a response time of ∼2.5 s. The O2-sensitive MWAS behaved according to a nonlinear Stern−Volmer model with a maximum I 0/I of ∼4 and a response time of ∼2.5 s. MWASs are advantageous in that suitably sized samples such as single biological cells can be co-localized with the sensing matrix in individual microwells.