The fabrication of hydrogel microstructures based upon poly(ethylene glycol) diacrylates, dimethacrylates, and tetraacrylates patterned photolithographically on silicon or glass substrates is described. A silicon/silicon dioxide surface was treated with 3-(trichlorosilyl)propyl methacrylate to form a self-assembled monolayer (SAM) with pendant acrylate groups. The SAM presence on the surface was verified using ellipsometry and time-of-flight secondary ion mass spectrometry. A solution containing an acrylated or methacrylated poly(ethylene glycol) derivative and a photoinitiator (2,2-dimethoxy-2-phenylacetophenone) was spin-coated onto the treated substrate, exposed to 365 nm ultraviolet light through a photomask, and developed with either toluene, water, or supercritical CO2. As a result of this process, three-dimensional, cross-linked PEG hydrogel microstructures were immobilized on the surface. Diameters of cylindrical array members were varied from 600 to 7 micrometers by the use of different photomasks, while height varied from 3 to 12 micrometers, depending on the molecular weight of the PEG macromer. In the case of 7 micrometers diameter elements, as many as 400 elements were reproducibly generated in a 1 mm2 square pattern. The resultant hydrogel patterns were hydrated for as long as 3 weeks without delamination from the substrate. In addition, micropatterning of different molecular weights of PEG was demonstrated. Arrays of hydrogel disks containing an immobilized protein conjugated to a pH sensitive fluorophore were also prepared. The pH sensitivity of the gel-immobilized dye was similar to that in an aqueous buffer, and no leaching of the dye-labeled protein from the hydrogel microstructure was observed over a 1 week period. Changes in fluorescence were also observed for immobilized fluorophore labeled acetylcholine esterase upon the addition of acetyl acholine.

A fluorescence biosensor is described that is based on a photopolymerized poly(ethylene glycol) (PEG) hydrogel incorporating fluorescein isothiocyanate dextran (FITC-dextran) and tetramethylrhodamine isothiocyanate concanavalin A (TRITC-Con A) chemically conjugated into the hydrogel network using an alpha-acryloyl, omega-N-hydroxysuccinimidyl ester of PEG-propionic acid. In the absence of glucose, TRITC-Con A binds with FITC-dextran, and the FITC fluorescence is quenched through fluorescence resonance energy transfer. Competitive glucose binding to TRITC-Con A liberates FITC-dextran, resulting in increased FITC fluorescence proportional to the glucose concentration. In vitro experiments of hydrogel spheres in a solution of 0.1 M phosphate-buffered saline (pH 7.2) and glucose were conducted for multiple TRITC-Con A/FITC-dextran ratios. Hydrogels were characterized on the basis of the percent change in fluorescence intensity when FITC-dextran was liberated by increasing glucose concentrations. The optimum fluorescent change between 0 and 800 mg/dL was obtained with a TRITC-Con A/FITC-dextran mass ratio of 500:5 micrograms/mL PEG. Fluorescent response was linear up to 600 mg/dL. At higher concentrations, the response saturated due to the displacement of the majority of the FITC-dextran and to concentration quenching by free FITC-dextran. Dynamic fluorescent change upon glucose addition was approximately 10 min for a glucose concentration step change from 0 to 200 mg/dL.

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A simple approach is described for preparing poly-(ethylene glycol) hydrogel materials with encapsulated seminapthofluorescein (SNAFL)-organophosphorus hydrolase enzyme conjugates. Direct determination of enzyme-catalyzed neurotoxin hydrolysis is provided by the self-referencing, pH-sensitive dye SNAFL-1, whose emission spectrum changes at lambda = 550 in response to pH. Using spectrofluorimetry and paraoxon as a model organophosphate, paraoxon concentrations as low as 8 x 10(-7) M could be readily detected. On the basis of the signal-to-noise ratio, a detection limit of 16 nM was determined. The materials demonstrated high stability against enzyme-denaturing, leaching, and photobleaching when stored under ambient conditions.

In this paper we show that hyperbranched polymers can be used as a host matrix for electrostatic entrapment of enzymes. Specifically, amine-functionalized glucose oxidase (GOx+) and horseradish peroxidase, as well as poly(amidoamine) dendrimer-modified horseradish peroxidase, reversibly sorb into polyanionic, hyperbranched poly(sodium acrylate) (PAA-) films that are on the order of a few hundred angstroms thick. The quantity of GOx+ entrapped within the PAA- films depends on the nature of film preparation but is typically on the order of 0.06 unit/cm2. The extent to which entrapped GOx+ retains its activity depends on the film history, but for PAA-/GOx+ composites not exposed to glucose and stored at 4 degrees C, the original activity is retained for up to 68 days and perhaps longer.