9 Glucose oxidase (GOD)
Glucoseoxidase (GOD) catalyzes the oxidation of β‐D‐glucose to gluconic acid, by utilizing molecular oxygen as an electron acceptor with simultaneous production of hydrogen peroxide [71]. GOD is the most widely employed enzyme as analytical reagent, for the selective determination of glucose, an analyte of clinical as well as of industrial interest [72].
One of the practical aspects of assembling proteins in LbL films is their increased stability and retained activity [73]. GOD was chosen as a model enzyme by Onda et al. to investigate the effect of immobilization of enzymes on catalytic activity, storage stability, thermostability, and pH dependency [73]. GOD was assembled under conditions where it was negatively charged (pI= 4.2).
On a quartz slide, four precursor bilayers of PEI/PSS were assembled by alternate deposition, followed by the deposition of two bilayers of PEI/GOD plus one more PEI layer at the top, so that the whole assembly may be denoted as (PEI/PSS)4+(PEI/GOD)2+PEI.
The activity of immobilized GOD was assayed depending on the coupled enzymatic reaction of GOD and POD as illustrated in Figure 13. In principal, GOD converts D‐glucose and O2 to n‐glucono‐δ‐
lactone and hydrogen peroxide. Then, POD oxidizes DA67 (indicator) using H2O2 as oxidant. The reaction is easily followed by monitoring the absorbance at a wavelength of 665 nm using a spectrophotometer. The film was immersed in the upper space of a cuvette containing D‐glucose and indicator solution. The separation between the film and light beam was sufficient not to interfere with the light path [74].
(A)
(B) (C) (D) (E)
Figure 13: Sequential enzymatic process based on glucose oxidase (GOD) and peroxidase (POD).
To study long‐term storage stability of immobilized GOD in LbL films, 45 samples were prepared on quartz slides. Among them, 5 films were stored in water at 25°C, 20 films were stored in PIPES buffer (pH 7) at 4°C, and the others were exposed to air at 4°C. After given periods of time, each sample was washed with water and their activity was measured using the procedure mentioned previously. As depicted in Figure 14, the film samples stored in water at 25°C showed drastic decreases in activity, and approximately 70% of the activity was lost after 4 weeks. The authors mentioned that the reason for this deterioration was unclear; nevertheless they speculated that bacterial growth was the cause of deterioration. In contrast, the films kept in the buffer at 4°C did not show a significant decrease in enzymatic activity over 14 weeks. The films kept in air at 4°C showed 10% decrease in the first week but the activity was maintained during the following 13 weeks. The initial activity loss was probably due to air‐drying of the film. It was clearly shown that GOD activity was unaltered in the assembled films [73].
Figure 14: Storage stability of GOD immobilized in LbL films: (a) storage in pure water at 25°C; (b) storage in air at 4°C; (c) storage in PIPES buffer (PH 7) at 4°C [73]. “Reprinted from reference 73, Copyright 1999, with permission from Elsevier."
Proteins lose their activity upon denaturation by heat, thus investigating thermostabilization offered by LbL structures was of high interest. Immobilization may suppress structural deformations of enzymes, thus enhancing their stability. Before measuring the enzyme activity, the extent of release of GOD from LbL films caused by heat was examined and no GOD was released into water even after the film was immersed in hot water for 2 h. The GOD LbL films on quartz plates were incubated in water at a given temperature for 10 minutes. Immediately after the incubation period, measurement of enzymatic activity was performed at 25°C. The second measurement was carried out after keeping the film in air at room temperature for 30‐40 min. Separate samples were used for different temperature conditions. Aqueous GOD rapidly lost activity on incubation at 30‐40 °C (Figure 15a), becoming totally inactive at 50 °C. However, this loss in activity was partially recovered on returning the solution to room temperature (Figure 15b). The recovered activity decreased with the incubation temperature, and totally disappeared at 70 °C. It is reported that enzymes undergo destabilization by two different mechanisms: reversible unfolding due to conformational changes and permanent denaturation due to chemical changes [75]. The recovered
activity in the present case may be related to partial unfolding. On the other hand, GOD in alternately assembled films showed a remarkable improvement in thermostability (Figure 15c).
Significant reduction in the activity was not noted even after incubation at 50 °C, although incubation at above this temperature caused a rapid loss of activity. Interestingly, recovery such as found for aqueous GOD was not observed in the film sample at any incubation temperature (Figure 15d). The enhanced enzymatic activity and the absence of reversible changes (room‐temperature recovery) were attributed to suppression of conformational mobility of GOD by surrounding polymer chains [73].
Figure 15: Thermostability of GOD immobilized in LbL film and in aqueous solution. The samples were incubated at given temperatures for 10 min and their enzymatic activity was measured immediately after the incubation period and at 30‐40 min after keeping in air at room temperature (approximately 22oC). (a) relative activity of aqueous GOD immediately after the incubation at given temperatures. (b) relative activity of aqueous GOD 30 min after the incubation. (c) relative activity of GOD in the film immediately after the incubation. (d) relative activity of GOD in the film 30 min after the incubation. The activity relative to that at 22°C is plotted against incubation temperature[73]. “Reprinted from reference 73, Copyright 1999, with permission from Elsevier."
Nevertheless, deposition of enzyme multilayers on planar surfaces has a major drawback. When more enzyme multilayers are deposited to increase the total catalytic activity of the system, eventually the overall catalytic activity per enzyme layer decreases. This is mainly attributed to the hindered substrate diffusion due to the thickness of the deposited film. Schüler et al. used GOD LbL films to coat colloidal submicron polystyrene spheres rather than quartz slides, where GOD multilayers were deposited alternately with PEI. Microelectrophoresis and SPLS measurements revealed regular and step‐wise assembly of the multilayers on the colloids. The enzymatic activity was found to increase regularly with an increasing number of GOD layers (i.e. GOD amount) immobilized, indicating that the enzyme multilayer films were sufficiently permeable for substrate diffusion. Thus, unlike multilayer films on planar surfaces, substrate diffusion effects can be avoided with enzyme multilayer‐coated colloids since thick or dense protein films are not necessarily required to increase the enzymatic activity: the high surface area afforded by the particles, and control of the total number of particles in solution (wt %), allowed for the activity to be conveniently optimized [76].
In a different study [27], GOD was used as a model enzyme to investigate the effect of adding layers above the enzyme layer on enzymatic activity. Thus, one GOD layer was immobilized in the interior with multiple PAH/PSS polyelectrolyte layers deposited on top. The GOD activity was measured (as described before) as a function of increasing polyelectrolyte layer number (Figure 16). The decrease in activity most likely reflects diffusion phenomena; the more layers above the enzyme layer, the more it becomes difficult for the substrate molecule to diffuse into the enzyme layer. However, it is also possible that some of the enzyme catalytic centers may be blocked by deposition of additional polyelectrolyte layers. It is well‐known that polyelectrolytes within multilayer films interpenetrate one another and a layer can interpenetrate 3‐4 adjacent polymer layers [4]. Hence, immobilized enzyme coated with multiple polyelectrolyte layers may have significantly less enzymatically active sites exposed for catalysis reactions than enzyme that is covered by a single polymer layer [27].
Figure 16: Relative activity of PS particles coated with one layer of GOD as a function of additionally deposited PAH/PSS layers. The activity of the first GOD layer after adsorption of the first polyelectrolyte layer on top of it was normalized to 100% as the polymer adsorption step caused removal of some of the enzyme [27]. “Reprinted with permission from reference 27. Copyright 2000 American Chemical Society."
In an attempt to add more functionality, the group of Caruso et al. [27] also went for adding magnetic properties to the LbL enzyme coated particles. 200‐nm PS particles were pre‐coated with four layers of Fe3O4 nanoparticles and PDADMAC, followed by two additional polyelectrolyte layers (PSS/PAH) and an outer GOD layer. The particles were fabricated using the LbL approach, beginning with the magnetic nanoparticles and followed by the enzyme multilayers. The magnetic functionalized particles were drawn to the bottom of a reaction tube by a magnet. The activity of the GOD layer on the particles was measured and the particles were then separated with a magnet, after which they were washed several times with water and the cycle was repeated. The measured activity was within 15% for each cycle, showing that it was possible to recover the particles and that the immobilized enzyme remained active after cycling. The above strategy opened a promising pathway for the fabrication of tailored magnetic, biocatalytic, and reusable particles.
Moving from surface coating on particles to encapsulation in LbL shells, a novel LbL design for encapsulation of GOD was introduced by Zhu et al. [22], based on an interesting feature of the photosensitive material diazoresin, where the weak ionic interactions between diazoresin and PSS
are converted to stable covalent bonds upon irradiation with UV. The diazoresin attracted attention because it can improve the stability of the multilayer films by converting weak interactions between neighboring layers into covalent bonds. Furthermore, it may also be applied to a variety of materials containing sulfonate groups, carboxylic acid groups, or phenol groups [77‐79]. The procedure is simple and involves only two steps: first, ionic (for sulfonate groups) or H‐bonding (for carboxylic acid or phenol groups) self‐assembly, which forms the initial multilayer; second, UV irradiation, which converts the weak interaction between the neighboring layers to a covalent one (Figure 17).
Figure 17: Schematic representation of PSS/DAR structure changes upon UV irradiation [22]. “Reprinted with permission from reference 22. Copyright 2005 American Chemical Society.”
Efforts were made to prepare diazoresin‐based hollow polyelectrolyte microcapsules encapsulating GOD by LbL assembly on MnCO3 templates as a potential glucose biosensor. MnCO3 particles were coated with one bilayer of (PSS/PAH),and then 1‐5 bilayers of (PSS/DAR), followed by one bilayer of (PSS/PAH) as the outer layer. Hollow microcapsules were obtained by decomposing the MnCO3 cores with 0.1M HCl solution for 20 min. The microcapsule suspension was mixed with rhodamine labelled GOD, for 5 min. While still in the enzyme loading solution, the capsules were irradiated with a UV lamp for 5 min to cross‐link the multilayer wall, and then rinsed with deionized water.
The inclusion of one (PSS/PAH) bilayer as both the inner and the outer layers was applied to strengthen the microcapsule wall architecture prior to cross‐linking (inner layer) and prevent
particle aggregation upon UV irradiation (outer layer), which was previously observed to occur in cases where DAR was the terminal layer. UV‐vis and zeta potential measurements confirmed the alternate deposition of (PSS/DAR) multilayers on the micrometer‐sized dissolvable templates [22].
The reaction velocity of the enzyme‐catalyzed reaction was determined by an increase in absorbance at 500 nm resulting from the oxidation of o‐dianisidine through a peroxidase‐coupled system. Glucose oxidase catalyzes the oxidation of glucose to gluconic acid, during which the generation of H2O2 is indirectly measured by oxidation of o‐dianisidine in the presence of peroxidase. Encapsulated GOD inside the DAR‐based microcapsules effectively preserved 52.8% of the total activity per unit mass as compared to the free enzyme. This decrease can be attributed to either partial loss of enzyme structure, transport limitation due to the capsule walls and partitioned enzyme, or a combination of both factors [22].
Probably one of the most useful practical applications of immobilizing GOD on solid surfaces is the fabrication of glucose biosensors. GOD is a structurally rigid glycoprotein with two identical polypeptide chains, each containing a flavin‐adenine dinucleotide (FAD) redox center [80]. GOD is reported to be electroactive only in limited cases and that GOD electron‐transfer and enzyme activity are very sensitive to the environment [81, 82].
The direct electron‐transfer behavior of the GOD electrode in the absence of oxygen occurs as follows:
GOD‐FAD + 2e‐ + 2H+ GOD‐FADH2
In the presence of oxygen, the reduced enzyme is oxidized very quickly at the surface of the electrode:
GOD‐FADH2 + O2 GOD‐FAD + H2O2
The catalytic regeneration of the enzyme in its oxidized form causes the loss of reversibility and the increase in size of the reduction peak. Upon the addition of glucose, a competitive reaction occurs at the vicinity of the electrode surface, which leads to the decrease of the reduction peak and thus the sensitive determination of glucose.
Hodak et al. [83] reported LbL assembly of GOD with PAH‐ferrocene redox mediator, using a gold electrode modified by a thiol layer with negatively charged end groups on which electrostatically
layers of a positively charged redox polymer, PAH with ferrocene redox sites (Fc) attached along the backbone, and polyanionic GOD were built. The main drawback of this design was the need for electron‐transfer mediator to shuttle electrons between the redox centers of GOD and the electrode. However, the redox mediators used in conjunction with redox proteins are in no way selective but rather general, facilitating not only electron transfer between electrode and protein but also various interfering reactions.
Therefore, a mediator‐free glucose biosensor based on LbL immobilization of GOD was introduced by Zhang et al. [80]. The measurement cell consisted of a saturated calomel electrode as the reference electrode, a platinum wire electrode served as the counter electrode and pyrolytic graphite electrode with a modified surface by LbL deposition of GOD and PEI.
The immobilized GOD retained its catalytic activity and the electrode coated with single GOD‐PEI bilayer responded linearly to the glucose concentration ranging from 0.5 to 8.9 mM (r2=0.998), with a sensitivity of 0.66 µA mM‐1 and an estimated detection limit of 0.1 mM. Interestingly, the deposition of a second bilayer of PEI/GOD increased the sensitivity to 0.76 µA mM‐1 and lead to a detection limit of 50 µM. This increased sensitivity is believed to arise from higher enzyme loading. Noteworthy, the further deposition of PEI/GOD layers could not increase the sensitivity, suggesting that the enzymatic activity correlates with the electron‐transfer reactivity of GOD. The stability of the two PEI/GOD bilayers‐modified PGE was evaluated by examining the response current of the enzyme electrode. After the electrode was kept at 4°C in phosphate buffer (pH 7.0) for 1 month, 93% of the original current of reduction peak remained and its activity to glucose remained at about 90%. Thus, this enzyme electrode has good stability and might be used in glucose biosensor fabrications. Possible interference of substances, such as ascorbic acid and acetaminophen, was also tested with no significant changes in reduction peak current observed for ascorbic acid or acetaminophen at a concentration five‐fold as high as that of glucose, which showed the good selectivity of the enzyme electrode [80].
Based on the direct electron transfer between GOD and electrode surface, this “mediator‐free”
approach proves to be superior in its stability and convenience of electrode preparation compared to previously reported biosensors having an electron‐transfer mediator applied to shuttle the electrons between GOD and the electrode [80].