Photoswitching enzyme electrode

Scheme 8 Assembly of a photoisomerizable glucose oxidase monolayer electrode and the reversible photoswitchable activa-tion/deactivation of the bioelectrocatalytic functions of the enzyme electrode.

Table 2 summarizes different possible applications of photoswitchable biomaterials, while detailing the nature of the biomaterial, the area of application, and, when possible, specific examples. Reversible light-induced activation and deactivation of redox proteins (enzymes) corresponds to write - read - erase functions. The photonic activation of the biomaterial corresponds to the write function, whereas the amperometric transduction of the recorded optical information represents the read function of the systems. Switching off of the redox functions of the proteins erases the stored photonic information and regenerates the photosensory biomaterial. These integrated, photoswitchable redox enzyme electrode assemblies mimic logic functions of computers, and may be considered as first step into the era of biocomputers. 

Redox enzymes are the active component in many electrochemical enzyme electrode biosensor devices.1821 The integration of two different redox enzymes with an electrode support, in which one of the biocatalysts is photoswitchable between ON and OFF states, can establish a composite multisensor array. The biomaterial interface that includes the photoswitchable enzyme in the OFF state electrochemi-cally transduces the sensing event of the substrate corresponding to the nonphoto-switchable enzyme. Photochemical activation of the light-active enzyme leads to the full electrochemical response, corresponding to the analysis of the substrates of the two enzymes. As a result, the processing of the signals transduced by the composite biomaterial interface in the presence of the two substrates permits the assay of the... 

Photoswitchable enzymes could have an important role in controlling biochemical transformations in bioreactors. Various biotechnological processes generate an inhibitor, or alter the environmental conditions (pH, for example) of the reaction medium. Photochemical activation of enzymes that adjust environmental conditions or deplete the inhibitor to a low concentration may maintain the bioreactor at optimal performance. More specifically, integration of the photoswitchable biocataly-tic matrix with a sensory electrode might yield a feedback mechanism in which the sensor element triggers the light-induced activation/deactivation of the photosensitive biocatalyst. 

The photoisomerizable enzyme monolayer electrode also revealed photoswitchable bioelectrocatalytic activity (Figure 7.10). In the presence of ferrocene carboxylic acid (5) as a diffusional electron transfer mediator, the nitrospiropyran-tethered GOx (4a) revealed a high bioelectrocatalytic activity, reflected by a high electrocatalytic anodic current. The protonated nitromerocyanine-GOx (4b) exhibited a two-fold lower activity, as reflected by the decreased bioelectrocatalytic current. By the reversible photoisomerization of the enzyme electrode between the 4a- and 4b-states, the current responses are cycled between high and low values (Figure 7.10, inset). 

FIG. 7.9 Ass mbix of a photoisomerizabie glucose ackfase monofayer elecirophotoswitchable activation/deactivation of the Uoelectrocatalyttc functions of the enzyme electrode. 

Figure 3-30. Organization of a photoswitchable glucose oxidase electrode for the bioelectrocatalyzed oxidation of glucose (A) The synthesis of the photoisomerizable nitrospiropyran-FAD cofactor. (B) The reconstitution of apo-glucose oxidase, apo-GOx, with the photoisomerizable FAD-cofactor (20a). (C) The assembly of the reconstituted photoisomerizable GOx on an electrode surface and the photoswitching of the bioelectrocatalytic function of the enzyme electrode in the presence of ferrocene carboxylic acid (21) as mediator.

Another application for photoswitchable enzymes attached to gold surfaces is the cytochrome c-mediated biocatalysed reduction of O2 by cytochrome oxidase, using a functional pyridine-nitrospiropyran photoiso-merisable mixed monolayer electrode (see Sect. 6.1, Fig. 39) [8,16,17]. 

Photoswitchable electrical communication between enzymes and electrodes has also been achieved by the application of photoisomerizable electron-transfer mediators [195, 199]. DilTusional electron mediators (viologen or ferrocene derivatives) were functionalized with photoisomerizable spiropyran/merocyanine units. These mediators can be reversibly photoisomerized from the spiropyran state to the merocyanine state (360 < A < 380 nm) and back (A > 475 nm). An enzyme multilayer array composed of glutathione reductase or glucose oxidase was electrically contacted only when the photoactive group linked to the redox relay (viologen or ferrocene derivative, respectively) was in the spiropyran state. 

FIG. 7.8 Electronk transduction of photoswitchable bioeiectrocatal)rtic functions of redax enzymes by the tethering of photoisomerizable units to the protein. (R is a dtffusional electron mediator that electrically contacts the redox site of the protein with the electrode support.)... 

Figure 3-31. Cyclic voltammograms corresponding to the photoswitchable bioelectrocatalyzed oxidation of glucose, 50 mM, in the presence of ferrocene carboxylic acid, (21), 5x 0 M, as diffusional electron mediator (a) and (c) In the presence of the SP-GOx monolayer electrode generated by the irradiation of the electrode A, > 475 run. (b) and (d) In the presence of the MRlT-GOx monolayer electrode generated by the illumination of the electrode with filtered light 320 nm < A < 380 nm. Inset cychc photoswitchable ON and OFF amperometric responses of the functionalized enzyme monolayer upon the light-induced isomerization of the interface between the SP GOx and MRI I GOx, respectively. Reproduced with permission from ref. 88. Copyright 1997 American Chemical Society.

The bioelectrocatalyzed oxidation of glucose in this system originates from the primary oxidation of the ferrocene carboxylic acid, (21), to the respective ferrocenylium cation that mediates the oxidation of the enzyme s redox center and its activation towards the oxidation of glucose. Photoisomerization of the enzyme monolayer to the MRH-GO state switched-off the bioelectrocatalytic functions of the protein monolayer, and only the electrical response of the diffusional electron mediator was observed, Fig. 3-31, curves (b) and (d). By the cyclic photoisomerization of the enzyme-monolayer electrode between the SP-GOx and MRlT-GOx states, the reversible photoswitching of the enzyme activity between ON and OFF states was demonstrated, Fig. 3-31 (inset). 

Fig. 31a). The native FAD cofactor was extracted from GOx and the semisynthetic FAD cofactor was reconstituted into the apo-GOx (apo-GOx) (Fig. 31b). This reconstituted enzyme includes a photoisomerizable unit directly attached to the redox center of the enzyme, and hence, the enzyme is predisposed for optimized photoswitchable bioelectrocatalytic properties. The photoisomerizable enzyme was assembled on an Au-electrode as described in Fig. 31(c). The bioelectrocatalytic oxidation of glucose was stimulated in the presence of ferrocene carboxylic acid as a diffusional electron-transfer mediator. The (28a)-state of the reconstituted GOx was inactive for the bioelectrocatalytic transformation, whereas photoisomerization of the enzyme to the (28b)-state activated the system (Fig. 32). By the cyclic photoisomerization of the enzyme mono-layer between (28a) and (28b) states, the bioelectrocatalyzed oxidation of glucose was cycled between the off and on states, respectively (Fig. 32, inset). It was also found that the direction of the photo-bioelectrocatalytic switch of the (28a/28b)-FAD-reconstituted GOx is controlled by the electrical properties of the diffusional electron-transfer mediator [385]. With ferrocene dicarboxylic acid as a diffusional electron-transfer mediator, the enzyme in the (28a)-state was found to correspond to the switched off biocatalyst, while the (28b)-state exhibits switched on behavior. In the presence of the protonated 1-[1-(dimethylamino)ethyl]ferrocene, the direction of the photobioelectrocatalytic switch is reversed. This control of the photoswitch direction of the photoisomerizable GOx was attributed to electrostatic interactions between the diffusional electron-transfer mediator and the photoisomerizable unit linked to the FAD. The (28b)-state attracts the oxidized negatively charged... 

Photostimulation of redox enzymes could transduce recorded optical signals as an amperometric response by their electrical interaction with electrode interfaces. For example, amperometric transduction of recorded optical signals was accomphshed using nitrospiropyran-modified glucose oxidase as photoswitchable material (Fig. 44) [14]. 

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