Enzyme Sequence Sensors

In potentiometric enzyme electrodes lyases producing carbon dioxide or ammonia are used as terminal enzymes of sequences. In fact, the term enzyme sequence electrode was introduced on the occasion of the design of a potentiometric D-gluconate sensor containing gluconate kinase (EC 2.7.1.12) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) (Jensen and Rechnitz, 1979). The authors found that for such a sensor to function the optimal pH values of the enzymes and the transducer should be close to each other. Furthermore, cofactors, if necessary, must not react with one another nor with constituents of the sample. It was concluded that the rate of substance conversion in multiple steps cannot exceed that of the terminal enzyme reaction. A linear concentration dependence is obtained when an excess of all enzymes of the sequence is provided, i.e. complete conversion occurs of all substrates within the enzyme membrane. Different permeabilities of the different substrates results in different sensitivities. This is particularly important with combinations of disaccharidases and oxidases, where the substrate is cleaved to two monosaccharides of approximately the same molecular size. The above 

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The combination of the creatinine-converting enzymes with sensors indicating primary reaction products, such as ion sensitive electrodes, NH3 gas sensors, or thermistors, is an effective alternative to enzyme sequence sensors (see Section 3.2.1). Enzyme reactors as well as true biosensors for creatinine have been described. 

Enzyme Sequence Sensors for Phosphatidylcholines and Acety-choline... 

The enzyme sequence sensor approach will be illustrated for the determination of citrate. Here three enzymes are immobilized on an oxygen sensor and the linear reaction sequence is employed ... 

Cell components or metabolites capable of recognizing individual and specific molecules can be used as the sensory elements in molecular sensors [11]. The sensors may be enzymes, sequences of nucleic acids (RNA or DNA), antibodies, polysaccharides, or other reporter molecules. Antibodies, specific for a microorganism used in the biotreatment, can be coupled to fluorochromes to increase sensitivity of detection. Such antibodies are useful in monitoring the fate of bacteria released into the environment for the treatment of a polluted site. Fluorescent or enzyme-linked immunoassays have been derived and can be used for a variety of contaminants, including pesticides and chlorinated polycyclic hydrocarbons. Enzymes specific for pollutants and attached to matrices detecting interactions between enzyme and pollutant are used in online biosensors of water and gas biotreatment [20,21]. 

This line has been initiated in the late 70s by Rechnitz group (1), who introduced enzyme sequences into enzyme electrodes. The field has been considerably widened by introducing the competitive (parallel), recycling and accumulation sensors. The following main merits of such sensors shall be pointed out ... 

The development of analytically useful enzyme electrodes is limited by the availability of purified and stable enzyme preparations. In an effort to extend the range of measurable species using ISE devices further, Rechnitz and co-workers (Rl) recently introduced bacterial- and tissue-based bio-selective electrode systems. These sensors are prepared in much the same manner as the enzyme probes except that whole intact cells are utilized as the immobilized reagents. There are several potential advantages to this novel approach, including (1) no need to extract and purify the enzymes involved, i. e., low cost (2) enzymes which are unstable when extracted from the cell may be used in situ to maximize and preserve their activity (3) if desired enzyme reactions require cofactors, these co ctors need not be added to the assay mixture because they are already present in the intact cell and (4) analytical reactions involving multistep enzyme sequences already present in the cells may be used to detect given analytes. 

Matsumoto et al. (1985) developed a sensor with the same enzyme sequence by immobilizing the enzymes covalently to silanized glassy carbon by glutaraldehyde. The sensor had a half-life of 7 weeks. Electrochemical interferences were compensated for by use of an additional, enzyme-free electrode. 

Wollenberger et al. (1983) combined COD and CEH immobilized on Spheron particles in a sensor for total cholesterol. The sensor showed no response when separately fixed enzymes were used, but was active with coimmobilized enzymes. In terms of bound activity CEH was 6 times less active than COD. The dependences of the current signal of the enzyme sequence electrode for free and esterified cholesterol were equal for aqueous standard solutions and serum samples (Fig. 90). This indicates a sufficient CEH activity in the immobilized preparation. 

After calibration and measurement the currents were evaluated using a computer. In this manner, four measurement values from a complex sample could be obtained within 5 min without the performance of any separation step. The AMP sensor has been extended to the recognition of free fatty acids having a 6-10 carbon atom chain by coupling of acyl-CoA synthetase. In the reaction catalyzed by this enzyme acyl-CoA and AMP are liberated from fatty acids in the presence of ATP, CoA and Mg2+. AMP enters the four-enzyme sequence incorporated in the sensor. 

All biosensors exploit a close harmony between a selective biorecognition system and a physicochemical transducer (Figure 12.lA and Figure 12.1B). The biorecognition system is typically an enzyme, sequence of enzymes, lectin, antibody, membrane receptor protein, organelle, bacterial, plant or animal cell, or whole slice of plant or mammalian tissue. This component of the sensor is responsible for the... 

When the three-enzyme sequence based on creatinine amidohydrolase is used, any creatine present can interfere with the determination of creatinine, so two sensors are used one to determine the total creatine plus creatinine and one to determine just creatine (by only using creatine amidinohydrolase and sarcosine oxidase). Creatinine is determined by difference. Amperometric sensors are generally based on this sequence and do not suffer from interferences. They are usually designed to respond to peroxide, though some have used oxygen electrodes. Typically, Pt electrodes are used. A sensor for just creatine only requires the creatine amidinohydrolase and sarcosine oxidase sequence. 

One of the pitfalls of microbial sensors, viz. their low selectivity, can be overcome by combining cells with an immobilized enzyme. Thus, creatinine deaminase (CDA, EC 3.5.4.21) hydrolyses creatinine to N-methylhydantoin and ammonium ion, the ammonia produced being successively oxidized to nitrite and nitrate ion by nitrifying bacteria. These bacteria have not yet been characterized but are known to be a mixed culture of Nitrosomonas sp. and Nitrobacter sp. The reaction sequence involved is as follows ... 

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