Enzyme biosensors response time

In principle, there are two possible ways to measure this effect. First, there is the end-point measurement (steady-state mode), where the difference is calculated between the initial current of the endogenous respiration and the resulting current of the altered respiration, which is influenced by the tested substances. Second, by kinetic measurement the decrease or the acceleration, respectively, of the respiration with time is calculated from the first derivative of the currenttime curve. The first procedure has been most frequently used in microbial sensors. These biosensors with a relatively high concentration of biomass have a longer response time than that of enzyme sensors. Response times of comparable magnitude to those of enzyme sensors are reached only with kinetically controlled sensors. 

A bioelectrode functioning optimally has a short response time, which is often controlled by the thickness of the immmobilized enzyme layer rather than by the sensor as well as many other factors (see Table 7). The biosensor response time depends on (1) how rapidly the substrate diffuses through the solution to the membrane surface, (2) how rapidly the substrate diffuses through the membrane cmd reacts with the biocatalyst at the active site, and (3) how rapidly the products formed diffuse to the electrode surface where they are measured. Mathematical models describing this effea are thoroughly presented in the biosensor literature (5, 68). 

The dye is excited by light suppHed through the optical fiber (see Fiber optics), and its fluorescence monitored, also via the optical fiber. Because molecular oxygen, O2, quenches the fluorescence of the dyes employed, the iatensity of the fluorescence is related to the concentration of O2 at the surface of the optical fiber. Any glucose present ia the test solution reduces the local O2 concentration because of the immobilized enzyme resulting ia an iacrease ia fluorescence iatensity. This biosensor has a detection limit for glucose of approximately 100 ]lM , response times are on the order of a miaute. 

The method of enzyme immobilization constitutes a key factor in the construction of these systems as it is the biocatalytic membrane that largely determines sensitivity, stability and response-time characteristics of the biosensor. 

Use of microorganisms and plant and animal tissues as a biological component of biosensor are also described in the literature (9, 10). The principle is based on the use of the natural bio-reactive systems. They have several advantages over the isolated enzymes and receptors. Isolation of enzymes and receptors are often required to increase the response time. Enzymes and receptors retained in the cells are more stable and have longer lifetimes. Cell-based biosensors are also economical as no purification step is required. 

Stability, duration, sensitivity, interference, and availability of substrates to contact enzymes are the criteria for the success of an enzyme sensor. These criteria depend on sources of enzymes, immobilization techniques, and transducers used. Food matrices are much more complicated than the clinical samples, hence, these criteria become extremely important for the application of the enzyme sensor in food analysis. An extensive list of the response time, detection limits, and stability of biosensors was summarized by Wagner (59). 

Utilization of whole cells and tissues in biosensor has increasingly been used. Enzyme stability, availability of different enzymes and reaction systems, and characteristics of cell surface are the advantages of using cells and tissues in biosensor designs. Multi-step enzyme reactions in cells also provide mechanisms to amplify the reactions that result in an increase in the detectability of the analytes. The presence of cofactors such as NAD, NADP, and metals in the cells allows the cofactor-dependent reactions to occur in the absence of reagents. (34, 50, 69). However, the diffusion of analytes through cell wall or membrane imposes constraint to this type of biosensors and results in a longer response time compared to the enzyme biosensors. 

The stability of enzyme electrodes is difficult to define because an enzyme can lose some of its activity. Deterioration of immobilized enzyme in the potentiometric electrodes can be seen by three changes in the response characteristics (a) with age the upper limit will decrease (e.g., from 10-2 to 10 3 moll-1), (b) the slope of the analytical (calibration) curve of potential vs. log [analyte] decrease from 59.2 mV per decade (Nernstian response) to lower value, and (c) the response time of the biosensor will become longer as the enzyme ages [59]. The overall lifetime of the biosensor depends on the frequency with which the biosensor is used and the stability depends on the type of entrapment used, the concentration of enzyme in the tissue or crude extract, the optimum conditions of enzyme, the leaching out of loosely bound cofactor from the active site, a cofactor that is needed for the enzymatic activity and the stability of the base sensor. 

Acetylcholineesterase and choline oxidase Enzyme immobilized over tetra-thiafulvalene tetracyanoquinodi-methane crystals packed into a cavity at the tip of a carbon-fiber electrode. The immobilization matrix consisted of dialdehyde starch/glutaraldehyde, and the sensor was covered with an outer Nafion membrane. The ampero-metric performance of the sensor was studied with the use of FIA system. An applied potential of +100 mV versus SCE (Pt-wire auxiliary electrode) and a carrier flow rate of 1 mL/min. The Ch and ACh biosensors exhibited linear response upto 100 pM and 50 pM, respectively. Response times were 8.2 s. [97]... 

AChE was sandwiched with poly(diallyldimethyl-ammonium chloride) layers on the surface of CNTs. The OP biosensor thus developed was used to detect paraoxon (Figure 55.7) as low as 4 x 10 M with a 6 min response time in flow injection analysis. A high stability of the sensors was also a merit of this biosensor no deterioration in the response was observed after 1 week of eontinuous use of the sensor. Only a 15% deerease in the aetivity of the enzyme was observed after 3 weeks, though the authors... 

Alternatively and in contrast with the previous approaches targeting toxicity or specific parameters, the transduction of microbial metabolism can also be applied for the detection of bacteria. However, this sensing principle shows poor selectivity and long response times. This type of biosensor can only be used for well-defined samples because of the possible presence of enzymes from sources other than the tackled species [96]. 

Fig. 10.13. Schematic representation of the different methodologies for estimation of the enzyme inhibition, (a) Indirect determination the biosensor response is measured before and after its incubation with the inhibitor for a given time, (b) Direct determination the inhibitor is added during the steady-state reaction.

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