Tri-enzyme electrode

E. Maestre, I. Katakis and E. Dominguez, Amperometric flow-injection determination of sucrose with a mediated tri-enzyme electrode based on sucrose phosphorylase and electrocatalytic oxidation of NADH, Biosens. Bioelectron., 16(1-2) (2001) 61-68. 

J. Diehl-Faxon, A.L. Ghindilis, P. Atanasov and E. Wilkins, Direct electron transfer-based tri-enzyme electrode for monitoring of organophos-phorus pesticides, Sens. Actuat. B. Chem., 36 (1996) 448-457. 

Table IV. Analysis of Milk Products with Tri-enzyme Electrode and Boehringer Mannheim (BM) Test-kit Methods...

Hamid JA, Moody GJ, Thomas JDR. Chemically immobilised tri-enzyme electrode for the determination of sucrose using fiow injection analysis. Analyst 1988 113 81. 

In a measurement ceU 20 mL of 0.1 Tris-buffer with pH 7.2 the respective sensor was incubated for 3 min with aflatoxin B1 in concentration range of 0.0 5-0.5 (xM previously diluted in dimethyl sulphoxide. After 3 min of incubation at 37°C 0.1 mL hnoleic acid was added to the samples so that the final concentration of the sample was 4 (xM. Based on the measurements, standard curves were constructed for the optical enzyme electrode (Fig. 6). 

Butyryl-choline detection in such a system can be realized by two different coupling approaches (i) co-immobilization of butyrylcholinesterase on a choline bienzyme electrode, which results in a tri-enzyme electrochemical sensing system (ii) use of solubilized butyrylcholinesterase in a coupled system with an electrode for choline determination, by addition of standard amounts of the dissolved enzyme to the measuring cell. In both cases butyrylcholinesterase activity affects the choline electrode response providing the presence of choline in the solution. Both coupling approaches are suitable for analysis of butyrylcholinesterase inhibitors such as organophosphorus compounds. 

Mizutani et al. (2003) determined the concentration of acetic acid in a manner similar to that of Mieliauskiene et al. (2006), the detection being performed using a combination of FIA with amperometric tri-enzyme biosensor detection. The biosensor was prepared by immobilizing AK, PK, and PyOx on a poly(dimethylsiloxane) (PDMS)-coated electrode. The oxygen consumption was monitored using the PDMS-coated electrode without interference from the PyOx reaction product (hydrogen peroxide). Thus, the biosensor-based system could be used for the determination of acetic acid from 0.05 to 20 mM with a sampling rate of 20 h and it remained stable for one month. This system was applied in the analysis of wine samples (Mizutani et al., 2003). 

Figure SJ Activity of the various states of the [NiFe] hydrogenase from A. vinosum as determined with a Pt electrode at 30°C.The reaction was performed in SOmM Tris/HCI (pH 8.0) in a volume of 2 ml. Oxygen was scavenged by adding glucose (90 mM) and glucose oxidase (2.5 mg/ml). Hydrogen peroxide was removed by catalase. When the system was anaerobic, an aliquot of H2-saturated water was added, and a little later enzyme (S-IOnM) was injected. Benzyl viologen (4.2mM) was used as electron acceptor.

In our experience with cultivated and wild soybean tissues, a discontinuous buffer system using 5 mM L-histidine (pH 7.0) as the gel buffer and 0.13 M Tris-40 mM citrate (pH 7.0) as the electrode buffer works well for the 23 enzymes we routinely assay. We employ this single buffer system whenever possible so that we can assay for as many enzymes as possible on a single gel. 

Co-immobilizing a second enzyme, NADH oxidase (280) or lactate dehydrogenase (288), permits the regeneration of NAD+. Measurements may be completed in Tris-HCl or carbonate buffers, but borate and glycine buffers inhibit L-alanine dehydrogenase (289). Highly selective L-histidine electrodes are available (290, 291) to determine histidine in urine (291). 

Figure 20. (A) The assembly of an integrated lactate dehydrogenase monolayer electrode by the cross-linking of an affinity complex formed between the enzyme and a PQQ-NAD monolayer-modified Au electrode. (B) Cyclic voltammograms of the integrated cross-linked PQQ-NAD / lactate dehydrogenase electrode (roughness factor ca. 15) (a) in the absence of lactate (b) with lactate, 20 mM. Recorded in 0.1 M Tris buffer, pH 8.0, in the presence of 10 mM CaCb, under Ar potential scan rate, 2 mV s . Inset amperometric responses of the integrated electrode at different concentrations of lactate upon application of potential 0.1 V vs. SCE.

Many approaches have been explored to overcome this problem, e.g., further addition of enzymes such as ascorbate oxidase [165,166], or peroxidase and catalase [167], that would be able to destroy the interferents. Alternatively, the coverage of the Pt working electrode with electropolymer-ized membranes such as 1,2-diaminobenzene [168] has been tried. However, this last procedure can cause a decrease in sensitivity. 

Finally, it should be noted that the recent development of so-called third generation biosensors to achieve direct electron transfer from redox enzyme, oxidoreductase to the electrode without mediators, but through a series of enzyme cofactors or conductive polymers to transfer electrons from the enzyme redox center to the electrode surface [161-164]. This concept with the current technology for preparing miniature sensors with nanotechnology is of great interest to many researchers trying to develop practical sensors in clinical, environmental and industrial analysis. Whether with mediators or without, research for optimum sensor development for various purposes will be intensive in the future. 

Most biosensors based on AChE have the enzymes immobilized on the surface of the sensor. The inhibition reaction being irreversible, the membrane with immobilized enzyme has to be replaced after several measurements or the biosensor can be use for only one determination. Due to this fact, the researchers tried to realize pesticide biosensors with a renewable surface or disposable biosensors based on screen-printed electrodes (SPE). The screen-printing technology provides a simple, fast and inexpensive method for mass production of disposable biosensors for different biomolecules starting with glucose, lactate and finishing with environmental contaminants as pesticides (Kulys et al., 1991) and herbicides (Skladal, 1992). 

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