Membranes enzyme-based biosensors

In recent years the electrochemistry of the enzyme membrane has been a subject of great interest due to its significance in both theories and practical applications to biosensors (i-5). Since the enzyme electrode was first proposed and prepared by Clark et al. (6) and Updike et al. (7), enzyme-based biosensors have become a widely interested research field. Research efforts have been directed toward improved designs of the electrode and the necessary membrane materials required for the proper operation of sensors. Different methods have been developed for immobilizing the enzyme on the electrode surface, such as covalent and adsorptive couplings (8-12) of the enzymes to the electrode surface, entrapment of the enzymes in the carbon paste mixture (13 etc. The entrapment of the enzyme into a conducting polymer has become an attractive method (14-22) because of the conducting nature of the polymer matrix and of the easy preparation procedure of the enzyme electrode. The entrapment of enzymes in the polypyrrole film provides a simple way of enzyme immobilization for the construction of a biosensor. It is known that the PPy-... 

Despite their potential importance, there are few analytical models of whole cell biosensing devices—particularly when compared to the plethora of models describing enzyme based biosensors [62]. Although aspects of cellular biochemistry are similar to those of isolated enzymes [63], problems arise in modelling the physicochemistry of whole cells due to their complex nature they are large (typically 0.2-10 jxm) they may contain a variety of biological structures (membranes, organelles, etc.) they incorporate a diversity of biochemical pathways and they may contain many types of active site. 

Table 11.19 Liquid Membrane Electrodes (LME), Gas-Sensing Electrodes (GSME), and Enzyme-Based Biosensors (EBB) [56]...

Figure 7.10 Concentration profiles in an enzyme-based biosensor where transport through the membrane layers is rate limiting.

Time response. In most situations enzyme kinetics have very little effect on the response time of enzyme-based biosensors. From the analysis given above, it is clear that one should operate these devices under conditions where the analyte concentration within the sensor is much less than Km- For sensors which are in the membrane diffusion limiting regime (section 7.3.1.1 above), the response characteristics of the membrane material will be governing. These depend on the thickness of the membrane and the diffusivity of the analyte in the membrane material. An approximate estimate of the membrane lag time is... 

Schematic diagram of an enzyme-based potentiometric biosensor for urea in which urease is trapped between two membranes.

CNTs and other nano-sized carbon structures are promising materials for bioapplications, which was predicted even previous to their discovery. These nanoparticles have been applied in bioimaging and drag delivery, as implant materials and scaffolds for tissue growth, to modulate neuronal development and for lipid bilayer membranes. Considerable research has been done in the field of biosensors. Novel optical properties of CNTs have made them potential quantum dot sensors, as well as light emitters. Electrical conductance of CNTs has been exploited for field transistor based biosensors. CNTs and other nano-sized carbon structures are considered third generation amperometric biosensors, where direct electron transfer between the enzyme active center and the transducer takes place. Nanoparticle functionalization is required to achieve their full potential in many fields, including bio-applications. 

Cellular biosensors have been widely described [11-55]. In many cases, the cells have been used in a manner analogous to that of enzyme based devices simply because they contain substantial quantities of particular enzymes. There are, of course, advantages to this approach since the enzymes do not have to be isolated and so may be cheaper but also more active and more stable than the purified components. However, the reproducibility, speed of response and selectivity of the cell based devices will, in general, be less favorable than their enzyme based counterparts. This is because of the relatively large physical size of the cells, the presence of membranes that hinder diffusion and the presence of enzymes other than the one(s) of particular interest. Nevertheless, a range of approaches has been adopted to improve the selectivity and other characteristics of whole cell biosensor devices. These were reviewed by Racek [11] and include ... 

Chen, Q., Kobayashi, Y., Tekeshita, H., Hoshi, T., Anzai, J. (1998). Avidin-biotin system-based enzyme multilayer membranes for biosensor applications optimization of loading of choline esterase and choline oxidase in the hienzyme membrane for acetylcholine biosensors. Electroanalysis 10 94-7. 

The above approach for measurement of urea using an enzyme-based potentiometric biosensor assumes that the turnover of urea to ammonium at steady state provides a constant ratio of ammonium ions to urea, independent of concentration. This is rarely the case, especially at higher substrate concentrations, resuitmg in a nonlinear sensor response. The hnearity of the sensor is also limited by the fact tiiat hydrolysis of urea produces a local alkaline pH in the vicinity of the ammonium-sensing membrane, partially converting NH to NH3 (pKa = 9.3). Ammonia (NH3) is not sensed by the ISE. The degree of nonlinearity may be reduced by placement of a semipermeable membrane between enzyme and sample to restrict diffusion of urea to the immobilized enzyme layer. 

One of the primary applications of entrapment immobilization has been to prepare enzyme-electrode-based biosensors [27], and one of the first functional enzyme electrodes utilized urease entrapped in an acrylamide film to detect urea using an ammonium ion selective electrode [28]. Highly hydrophobic bilayer lipid membranes and liposomes have also been used to entrap highly labile biomolecules (see chapter 9). Such films and layers are, however, inherently unstable themselves and are useful primarily as research tools. 

Matsue et al. [27] were the first to explore an enzyme-based OECT biosensor. They used Diaphorase as the entrapped enzyme in a polypyrrole transducing layer for the detection of NADH via a redox mediator (the sodium salt of anthraquinone-2-sulfonic acid). The net result was the conversion of polypyrrole from its conducting state to its insulating state in the presence of NADH. The device showed a response time of 15--20 min in the presence of NADH. Later Nishizawa et al. [26] exploited the pH sensitivity of the polypyrrole film for the design and fabrication of OECT sensors for pH and for pencillin. The Penicillinase enzyme was entrapped in a membrane which was coated with a polypyrrole film, in which a decrease in pH was observed in the presence of penicillin due to the hydrolysis of penicillin by Penicillinase. 

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