Enzyme electrodes response time

Catalase was immobilized with gelatin by means of glutaraldehyde and fixed on a pretreated Teflon membrane served as enzyme electrode to determine hydrogen peroxide [248], The electrode response reached a maximum when 50mM phosphate buffer was used at pH 7.0 and at 35°C. Catalase enzyme electrode response depends linearly on hydrogen peroxide concentration between 1.0 X 10-5 and 3.0 X 10-3 M with response time 30 s. 

The rate of enzymatic reaction increases with substrate concentration for example, in the case of a cyanide electrode coupled with immobilized jS-gluco-sidase enzyme, a response time of 20 s for 10 moll amygdalin and Imin for 10 " moll amygdalin is obtained. Rather than waiting until an equilibrium potential is reached, the rate of potential change (AE/At) can be measured, the result being proportional to substrate concentration. 

An analogous urea sensor can be prepared using an ammonium selective glass membrane electrode as the transducer onto which the urease is immobilized. Using thin immobilized enzyme layers, response times for such devices can be 1 min or less at mM concentrations of urea. 

The linear region of the enzyme electrode response varies slightly from one amino acid to the next, but is generally situated between 10" and 10 2 M. The response time is less than one minute when the enzyme is coreticulated directly to the glass electrode [3], and several minutes when the enzyme is attached via other techniques [116, 117]. K, Na, and Li+ ions interfere with the electrode response (just like in the urea electrode with the pNH4 transducer), but this can be avoided with certain precautions. The buffer solution used to calibrate the electrode should not contain these ions, and the reference electrode should not use saturated KCl solution. 

The high specificity required for the analysis of physiological fluids often necessitates the incorporation of permselective membranes between the sample and the sensor. A typical configuration is presented in Fig. 7, where the membrane system comprises three distinct layers. The outer membrane. A, which encounters the sample solution is indicated by the dashed lines. It most commonly serves to eliminate high molecular weight interferences, such as other enzymes and proteins. The substrate, S, and other small molecules are allowed to enter the enzyme layer, B, which typically consist of a gelatinous material or a porous solid support. The immobilized enzyme catalyzes the conversion of substrate, S, to product, P. The substrate, product or a cofactor may be the species detected electrochemically. In many cases the electrochemical sensor may be prone to interferences and a permselective membrane, C, is required. The response time and sensitivity of the enzyme electrode will depend on the rate of permeation through layers A, B and C the kinetics of enzymatic conversion as well as the charac-... 

Most suitable would be the use of a perfectly NH4+ ion-selective glass electrode however, a disadvantage of this type of enzyme electrode is the time required for the establishment of equilibrium (several minutes) moreover, the normal Nernst response of 59 mV per decade (at 25° C) is practically never reached. Nevertheless, in biochemical investigations these electrodes offer special possibilities, especially because they can also be used in the reverse way as an enzyme-sensing electrode, i.e., by testing an enzyme with a substrate layer around the bulb of the glass electrode. 

Substrate determinations (Table 8.10) using enzyme electrodes must be performed under controlled conditions of temperature and pH and if standard solutions are used to calibrate the electrode they must be analysed at the same time. The response time of some enzyme electrodes may be several minutes and although for many the time is shorter this factor must be considered in the design of an assay method. 

Enzyme electrodes with amperometric indication have certain advantages over potentiometric sensors, chiefly because the product of the enzymic reaction is consumed at the electrode and thus the response time is decreased. For this reason, the potentiometric glucose enzyme electrode, based on reaction (8.1) followed by the reaction of HjO, with iodide ions sensed by an iodide ISE [39], has not found practical use. 

Figure 3.17 — Relationship between the micro-enzyme electrode diameter and its response time. The response times for single injections and repeated injections are represented by a solid and dotted lines, respectively.

Microbial sensors offer a number of assets, namely (a) they are less sensitive to inhibition by solutes and more tolerant to suboptimal pH and temperature values than are enzyme electrodes b) they have longer lifetimes than enzymes and (c) they are less expensive than enzyme electrodes as they require no active enzyme to be isolated. On the other hand, they lag behind enzyme electrodes in a few other respects thus, (a) some have longer response times than their enzyme counterparts b) baseline restoration after measurement typically takes longer and (c) cells contain many enzymes and due care must be exercised to ensure adequate selectivity e.g. by optimizing the storage conditions or using specific enzyme reactions) —some mutant microorganisms lack certain enzymes. 

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. 

Factors affecting the response time of an enzyme electrode [59,60]... 

The group of Ruhr pursued an approach where enzymes were only immobilized on specific areas of the electrode. The electrochemical detection was performed on the unmodified regions of the same electrode leading to faster response times of the sensor [58]. The authors used SECM to show the different kinetics at the modified and unmodified regions of the sensor surface [57]. SECM was (among other techniques [58,70]) used for microderivatization of the surface [63]. 

A miniaturized planar amperometric glucose sensor has been created on Sapphire substrates. Thin film titanium-gold electrodes are covered with an enzyme layer which is patterned by a lift-off technique [66]. This sensor exhibits a fast response time of 30 seconds but the linear measuring range is poor. 

Acetylcholineesterase Enzyme was covalently immobilized on a bovine serum albumin-modified H +-selective coated wire electrode. The sensor was used in 5mM phosphate buffer pH 7 at 30°C. The response time was 3-10 min for O.l-lOmM ACh. pH change of 6-8 had little effect. Coefficient of variation was 5.7 and 5.8%. [72]... 

---