Photochemical Coenzyme Regeneration in Sol Gel Matrices

For many enzymes, a cofactor (also referred to as a coenzyme) is required, and this is true of dehydrogenase enzymes, which require NAD or NADP as a cofactor (Eq. 4):

dehydrogenase

Reduced substrate + NAD(P)+< > Oxidized substrate + NADP(H) + H+ (4)

In optical sensing, enzyme activity in dehydrogenase enzymes can be measured conveniently because the reduced form of the coenzyme, NADH or NADPH, fluoresces. Sensors based on this reaction, however, cannot operate continuously without a renewable supply of the coenzyme. We have shown in recent work that a photooxidizer can be incorporated into the sol-gel matrix, along with the enzyme and cofactor, to regenerate by oxidation the reduced cofactor (25). The organic dye thionine was selected as the photooxidizing agent, because it is stable in the silica matrix, retains its excited-state properties, and is compatible and unreactive with the other components.

Thionine, thio+, absorbs light in the visible range with Xmax = 596 nm (Eq. 5). In solution, excited thionine, *thio+, oxidizes NADH (Eq. 6). As NADH is oxidized, the fluorescence emission of the excited thionine is quenched. Therefore, by exciting thionine (thio+) using visible light (596 nm), it is possible to regenerate NAD+.

The enzyme isocitrate dehydrogenase (ICDH) was used as a model dehydrogenase in this research because the Gibbs free energy of the ICDH reaction strongly favors the oxidation of isocitrate, thereby reducing experimental complications from back reactions (26). ICDH catalyzes the oxidation of isocitrate to a-ketoglutarate using NADP+ as the electron acceptor (Eq. 7):

ICDH

isocitrate + NADP+-> a-ketoglutarate + CO2 + NADPH + H+ (7)

The scheme for photochemical oxidation of NADPH by thionine coupled to the enzymatic reaction of ICDH is shown in Fig. 11.

Fig. 11. Enzyme cofactor regeneration for continuous isocitrate oxidation. The photochemical oxidation of NADPH by thionine is coupled to enzymatic oxidation of isocitrate by ICDH. When thionine is excited, it reacts with NADPH to reform NADP+. The regenerated NADP+ becomes available for another isocitrate oxidation. (Reproduced from Ref. 25 with permission.)

Fig. 11. Enzyme cofactor regeneration for continuous isocitrate oxidation. The photochemical oxidation of NADPH by thionine is coupled to enzymatic oxidation of isocitrate by ICDH. When thionine is excited, it reacts with NADPH to reform NADP+. The regenerated NADP+ becomes available for another isocitrate oxidation. (Reproduced from Ref. 25 with permission.)

We first established that NADPH undergoes a reaction similar to NADH with thionine (Eq. 6) in that the fluorescence of thionine is quenched as it oxidizes NADPH. The fluorescence emission spectra of thionine in buffer solution and encapsulated in wet silica gels showed essentially no difference, indicating that the optical properties of the thionine photooxidizer itself were not altered as a result of encapsulation. When NADPH-doped silica gels and NADPH buffer solutions were exposed to excited thionine, fluorescence quenching was observed in both the gels and solution. Figure 12 shows the NADPH concentration, expressed as a percentage of the initial NADPH concentration, over time in the presence of excited thionine (*thio+). The oxidation of NADPH can be described by a decay constant, koxidize, and the rate of oxidation of NADPH was about one order of magnitude slower in the sol-gel matrix. koxidize was 8.8(±1.0) x 10-4 s-1 in sol-gel silica, compared with 9.8(±2.9) x 10-3 s-1 for buffer solution. The slower observed rate in the sol-gel material may be explained by mass transport limitations. NADPH was immobilized in the gel, whereas thionine was added at time 0 and needed time to diffuse through the pores of the network.

The photochemical oxidation of NADPH can be coupled to the enzymatic oxidation of isocitrate by ICDH (Fig. 11). ICDH and thionine were coencapsu-lated in wet silica gels and incubated with NADP+, and isocitrate was then

Fig. 12. Disappearance of NADPH during exposure to excited thionine in buffer solution and in wet silica gels. The NADPH concentration is expressed as a percentage of the initial NADPH concentration at time 0. Open symbols show the decrease in NADPH in wet gels, and closed symbols show the decrease in NADPH in solution. Three starting NADPH concentrations were used for each sample type: (O) wet gel, 120 ||M NADPH; (□) wet gel, 80 |M NADPH; (A) wet gel, 40 ||M; (•) buffer solution, 120 |M NADPH; (■) buffer solution, 80 |M NADPH; (▲) buffer solution, 40 |M NADPH. The data for the six conditions were fit individually to a single exponential decay, y = 100e~bi. The decay parameter, b, for the wet gels and the solution samples was 8.8(±1.0) x 10-4 s-1 and 9.8(±2.9) x 10-3 s-1, respectively. (Reproduced from Ref. 25 with permission.)

Fig. 12. Disappearance of NADPH during exposure to excited thionine in buffer solution and in wet silica gels. The NADPH concentration is expressed as a percentage of the initial NADPH concentration at time 0. Open symbols show the decrease in NADPH in wet gels, and closed symbols show the decrease in NADPH in solution. Three starting NADPH concentrations were used for each sample type: (O) wet gel, 120 ||M NADPH; (□) wet gel, 80 |M NADPH; (A) wet gel, 40 ||M; (•) buffer solution, 120 |M NADPH; (■) buffer solution, 80 |M NADPH; (▲) buffer solution, 40 |M NADPH. The data for the six conditions were fit individually to a single exponential decay, y = 100e~bi. The decay parameter, b, for the wet gels and the solution samples was 8.8(±1.0) x 10-4 s-1 and 9.8(±2.9) x 10-3 s-1, respectively. (Reproduced from Ref. 25 with permission.)

injected into the sample at different times. As shown in Fig. 13, at each injection of enzyme substrate, the ICDH reaction was observed by the production of NADPH, as indicated by an increase in its fluorescence. When thionine was excited, NADPH was oxidized, resulting in a decrease in its fluorescence. Moreover, the enzymatic reduction of NADP+ to NADPH by ICDH followed by the photochemical oxidation of NADPH to NADP+ by thionine can be cycled. We generated a calibration curve to correlate the NADPH produced as a function of isocitrate concentration and the curve was essentially linear (Fig. 14). The data presented in Figs. 13 and 14 show that a photo-oxidizer can indeed regenerate the NADPH coenzyme, and that photochemical regeneration is possible in a bioactive solid-state silica glass (25). The collective results suggest that it is possible to utilize dehydrogenase enzymes as optical biosensors

time (seconds)

Fig. 13. Repeated ICDH reactions in a sol-gel silica monolith. ICDH, thionine, and NADP+ were encapsulated in a silica gel, and isocitrate was injected onto the gel at the times indicated by the arrows. To monitor the enzyme oxidation of isocitrate, the fluorescence at 460 nm was measured, which indicates generation of NADPH. Subsequently, thionine was excited (at the times indicated by the solid bars) to induce the oxidation of NADPH back to NADP+. This process could be repeated for at least four cycles. (Reproduced from Ref. 25 with permission.)

time (seconds)

Fig. 13. Repeated ICDH reactions in a sol-gel silica monolith. ICDH, thionine, and NADP+ were encapsulated in a silica gel, and isocitrate was injected onto the gel at the times indicated by the arrows. To monitor the enzyme oxidation of isocitrate, the fluorescence at 460 nm was measured, which indicates generation of NADPH. Subsequently, thionine was excited (at the times indicated by the solid bars) to induce the oxidation of NADPH back to NADP+. This process could be repeated for at least four cycles. (Reproduced from Ref. 25 with permission.)

Fig. 14. Calibration curve generated from repeated measurements using the same ICDH-doped gel. The maximum NADPH concentration produced during each cycle is plotted as a function of isocitrate concentration, and a linear curve was observed. (Reproduced from Ref. 25 with permission.)

without compromising the continuity of sensor function owing to depletion of coenzyme.

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