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Spring 2004 Crooks Group Research Highlight

Biosensors Based on Hydrogel-Entrapped Enzyme Arrays

We recently reported that photopolymerized hydrogel micropatches can be used for immobilizing enzymes and E. coli within microfluidic devices (Anal. Chem. 2002, 74, 4647-4652; Anal. Chem. 2003, 75, 22-26). Such proteins and cells are physically entrapped within the photocrosslinked microgel matrix, but analytes (e.g., small molecules) are able to freely diffuse through nanopores within the gel. We demonstrated that entrapped enzymes and E. coil retain their activity within the gels, which means that the composite gel/biomaterial can be used as a sensor unit or microbioreactor.

This general approach has now been expanded upon by graduate student Jinseok Heo and former postdoctoral associate Dr. Gi Hun Seong (now at Hanyang University in Korea), who have shown that an array of hydrogels can be used to simultaneous detect multiple analytes. The hydrogel microarray consists of enzymes entrapped with hydrogel micropatches, which themselves are photolithographically defined within microfluidic channels. An array of PDMS channels (Figure 1) was used in this work to separate individual enzyme reactions and thereby prevent diffusional mixing. The microfluidic system was prepared as follows. First, the PDMS microchannel array was prepared using an established micromolding method. Second, the PDMS structure was reversibly attached to acrylate-functionalized glass. Third, hydrogel precursor (polyethylene glycol diacrylate) solutions were mixed with different combinations of enzymes, and then these solutions were pumped into each of the microfluidic channels. Fourth, hydrogel/enzyme composite micropatches were fabricated by UV photopolymerization of the gel precursor solutions through a photomask. Following gel formation, the PDMS mold was removed from the glass and the remaining unpolymerized hydrogel precursor solution was washed away. Finally, a second PDMS channels, having a greater height and width than the gel, was irreversibly affixed to the glass supporting the microgel array.

Figure 2(A) shows an optical micrograph of an array of hydrogel/enzyme composite micropatches. All of these microstructures contained both glucose oxidase (GOx) and horseradish peroxidase (HRP) enzymes. Figure 2(B) is a fluorescence micrograph obtained 5 min after amplex red and a glucose-containing cocktail was pumped into the channels and the pumping was stopped. The glucose concentrations in the three channels were, from left to right, 10, 1, and 0.1 mM. The final fluorescence product, resorufin, is formed as a result of the two consecutive enzyme reactions shown in Figure 3. The fluorescence observed from microgels in Figure 2(B) confirms that two consecutive enzyme reactions were conducted by the co-immobilized enzymes in the gel plugs. Another important finding was that the fluorescence intensity obtained from the microgels (the solid white line in Figure 2(B)) was strongly dependent on the glucose concentration. Thus, a calibration curve for an analyte can be easily obtained from the simultaneous enzyme reactions occurring in the different channels.

This hydrogel-entrapped enzyme array can also be used for simultaneous sensing of multiple analytes. To demonstrate this principle, glucose and galactose, which have very similar chemical structures, were examined. In the presence of GaOx and HRP, galactose and amplex red follow an analogous reaction pathway to that shown in Figure 3. Three different combinations of enzymes (GOx/HRP, HRP only, and GaOx/HRP) were immobilized in the gel arrays. In Figure 4, the gels in the first channel hosted GOx/HRP, the second hosted HRP only, and in the third GaOx/HRP were present. Figure 4(A) shows that only the gels in the first channel respond to the glucose/amplex red solution and fluoresce. In contrast, when a galactose/amplex red solution was introduced into the three channels, only the gels residing in the far right channel fluoresced (Figure 4B). Finally, when a glucose/galactose/amplex red solution was used, the gels in the first and third channels both showed light emission.

This hydrogel-entrapped enzyme array approach can be extended to other analytes if an appropriate signal transducer, either optical or electrochemical, is chosen, and it will provide a fast screening method for detecting a small volume of sample solution containing multiple analytes.