Anal. Chem. 2001, 73, 2484-2490
Fabrication and Characterization of Microwell Array Chemical Sensors David D. Bernhard, Shalini Mall, and Paul Pantano*
Department of Chemistry, The University of Texas at Dallas, Richardson, Texas 75083
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 14µm 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 O2sensing 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 pKa 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 I0/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. Combined imaging and chemical sensing (CICS) is a new technique that utilizes imaging fiber chemical sensors (IFCSs) and charge-coupled device (CCD)-based epifluorescence microscopy to concurrently view a remote sample’s morphology and interfacial chemistry.1,2 Examples of interfacial chemistries studied recently with pH-sensitive IFCSs include localized corrosion at metal surfaces3 and fertilization biochemistry at sea urchin egg surfaces.4 The traditional IFCS sensing layer has been fabricated by spin coating a (semi)transparent, planar polymer layer across a polished imaging fiber’s distal face (Figure 1, top left). In practice,
