Bienzyme sensors

SCHEME 55.3. Enzyme reactions in AChE-ChOx bienzyme sensors. 

Noguer T and Marty J-E. 1997. High sensitive bienzymic sensor for the detection of dithiocarbamate fungicides. Anal Chim Acta 347 63-70. 

The fact that the glucose dehydrogenase reaction can be driven backward by using a more acidic buffer and an excess of NADH has been utilized to recycle gluconolactone formed in the GOD-catalyzed reaction [321]. The signal was amplified eightfold. This bienzyme sensor... 

Misra SCK, Mathur P, Srivastava BK (2(X)4) Vacuum-deposited nanocrystalline polyaniline thin film sensors for detection of carbon monoxide. Sens Actuators A 114 30-35 Mulchandani AK, Wang CL (1996) Bienzyme sensors based modified electrodes. Electroanalysis 8 414-419 Muller JBA, Smith CE, Newton MI, Percival CJ (2011) Evaluation of coated QCM for the detection of atmospheric ozone. Analyst 136 2963-22968... 

Increasing the amount of enzyme attached to the surface will increase the amount of electrochemically detectable cofactor (e.g., NADH, H2O2) produced in the enzymatic reaction, up to a point. It has been shown for at least one system that the optimum maximum concentration of enzyme on the surface can clearly be reached [27]. In this work two enzymes, NADP -dependent L-malate dehydrogenase and p-hydroxybenzoate hydroxylase, were immobilized on a Clark electrode. The resulting bienzyme sensor behaved as if a single enzyme with a selectivity for L-malate was immobilized, and the optimal surface concentration for both enzymes was found to be approximately 5 U/cm. Furthermore, in order to fabricate a microelectrode with the fast time response necessary to measure neu-... 

C. J. McNeil, Determination of the herbicide chlorsulfuron by amperometric sensor based on separation-free bienzyme immunoassay. Sens. Actual. B 98, 254—261 (2004). 

Figure 18. Bienzyme multilayers for interferent-free glucose sensors.

N. Sato, K. Usui and H. Okuma, Development of a bienzyme reactor sensor system for the determination of ornithine, Anal. Chim. Acta, 456(2) (2002) 219-226. 

ChE-ChOx bienzyme-modified biosensors have been widely used mainly for detecting OP pesticides, which are summarized in Table 55.1. AChE and BChE are often used as ChE enzyme. This type of ChE-ChOx sensor can be used conveniently for detecting OP compounds, and detection time for qualitative analysis is reasonably fast. For quantitative determination of OP compounds, however, operation of the sensor is somewhat time consuming because multiple steps (a measurement of response to acetylcholine and inhibition) are required. 

FIGURE 55.3. A typical response of inhibition-mode AChE-ChOx bienzyme OP sensors, (a) Addition of acetylcholine and (b) addition of OP sample. —A/ corresponds to the output signal. (Reproduced from Anzai, 2006, with permission). 

Alternative ways to improve sensor sensitivity and stability of the bienzyme system reported use of redox polymers for bienzyme systems, specifically to interact with HRP instead of with oxidoreductases. 

For creatine determination the appropriate bienzyme electrode was used. Creatinine and creatine could be assayed in parallel by combining a trienzyme electrode and a bienzyme electrode. Both electrodes reached a steady signal 2 min after sample addition. With a 25 pi sample volume the linear range was 1-100 mg/1. The sensors were stable for more than 500 measurements. The CV of creatine determination in serum was 1.3% with 21.7 mg/1 and 11.7% with 8.2 mg/1, and a day-to-day CV of 8.4% was achieved. Comparison with the Jaffe method for 55 serum... 

The formation of low-molecular weight products of the a-amylase-cat-alyzed starch hydrolysis can be assayed by using a glucoamylase-GOD electrode (Pfeiffer et al., 1980). The sensor is covered by a dialysis membrane with a cutoff of 15 kDa which prevents starch from reaching the enzymes. The cleavage products can easily diffuse into the bienzyme membrane where they are successively degraded to glucose by glu-coamylase. As only the (B-anomer is formed, the sensitivity of the method... 

Since the sensor signal was linear over the normal range of 2.1-6.9 U/g hemoglobin, and only decreased activities are of clinical relevance, the bienzyme electrode is suitable for the measurement of any clinically possible value. 

In a sensor for lactate a bienzyme system composed of cytochrome 62 for lactate oxidation to pyruvate, and lactate dehydrogenase for conversion of pyruvate back to lactate has been used [321]. Hexacyanoferrate(III) served as electron acceptor for cytochrome b2- The reduced mediator was reoxidized at the electrode, thus giving a measuring signal depending on the analyte concentration. Attempts to determine both substrates of the recycling system have shown that, at tenfold amplification for lactate, the sensitivities for lactate and pyruvate are almost identical. The same recycling scheme has also been used in connection with Fe-EDTA as electron mediator in place of hexacyanoferrate(III) [336]. 

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