Lactate cytochrome 82 electrode

The activity of the enzyme is also strongly affected by the presence of inhibitors. Fluoride ions inhibit urease (173) and oxalate ions inhibit lactate oxidase (174), but the major inhibitors are heavy-metal ions, such as Ag+, Hg +, Cu " ", organophosphates, and sulfhydryl reagents (/i-chloromercuribenzoate and phenylmercury(II) acetate), which block the free thiol groups of many enzyme active centers, especially oxidase (69). Inhibiting the enzyme electrodes makes it possible to quantify the inhibitors themselves (69), for example, H2S and HCN detection using a cytochrome oxidase immobilized electrode (176). 

The direct anodic oxidation of cytochrome c at a bipyridyl-modified electrode has already been incorporated in enzyme electrodes for lactate, carbon monoxide, and hydrogen peroxide. Here, cytochrome c is reduced by cytochrome b2, CO oxidoreductase, or horseradish peroxidase and anodically reoxidized. Cytochrome c has also been applied to couple mitochondria and chloroplasts to redox electrodes (Albery et al. 1987). Although no practically applicable sensor has been constructed as yet, this principle offers a new avenue to the determination of inhibitors of photosynthesis or respiration (Cardosi and Turner, 1987). 

In most amperometric cytochrome b2 electrodes the reaction is followed by anodic oxidation of ferrocyanide at a potential of +0.25 V or above. The first of such sensors was assembled by Williams et al. (1970), who immobilized the enzyme (from baker s yeast) physically at the tip of a platinum electrode within a nylon net of 0.15 mm thickness. The large layer thickness resulted in a response time of 3-10 min. Owing to the low specific enzyme activity used, the sensor was kinetically controlled. Therefore the linear measuring range extended only up to 0.1 Km-A similar sensor has been applied by Durliat et al. (1979) to continuous lactate analysis. The enzyme was contained in a reaction chamber of 1 pi volume in front of the electrode. This principle has also been employed in the first commercial lactate analyzer using an enzyme electrode (Roche LA 640, see Section 5.2.3.3X With a sensor stability of 30 days and a C V below 5%, 20-30 samples/h can be processed with this device. 

In order to fabricate reagentless sensors, Kulys and Svirmickas (1980b) coimmobilized cytochrome b2 together with semiconductive organic metal complexes (NMP+TCNQ ) by physical entrapment on the tip of a Pt probe. The charge transfer complex acts as a mediator and permits lactate to be measured at a potential between -0.03 and +0.4 V. Oxygen decreased the electrode sensitivity by 50% by spontaneous reaction with the reduced organometallic compounds. 

Shinbo et al. (1979) developed a potentiometric cytochrome b2 electrode. The change of the redox ratio of ferrocyanide/ferricyanide was indicated. The plot of the potential change versus the logarithm of lactate concentration yielded an S-shaped curve. 

Attempts to construct lactate sensors by biochemical modification of electrode surfaces have been made using LDH as well as cytochrome b2. 

Yao and Musha (1979) modified an n-octaldehyde-containing carbon paste electrode with NAD+. LDH was added to the measuring solution. The NADH formed upon lactate injection was oxidized electrochemically, giving a well-defined, linear-sweep voltammetric peak. The peak area was linearly related to the concentration of lactate in the range 0.025-1.0 pmolyl. Durliat and Comtat (1978) adsorbed cytochrome b2 on... 

An enzyme electrode based on coimmobilized cytochrome b2 and laccase (Scheller et al., 1987b) allows an explanation of the principle of substrate recycling in enzyme electrodes in greater detail (Fig. 100). The advantage of this system is that the cosubstrate, oxygen, as well as the analytes, hydroquinone and benzoquinone, are electrochemically active. This permits one to study different parts of the recycling process. Recycling of the analyte in the presence of the substrate of cytochrome b2, lactate, results in an increase in the sensitivity by a factor of 500 as compared with lactate-free operation. Under conditions that are optimal for laccase the analyte is almost completely in the oxidized state, i.e. it... 

The first enzyme electrode-based lactate analyzer was developed in 1976 by La Roche (Switzerland) (see Table 23). It uses cytochrome b2 in a tiny reaction chamber on top of a platinum electrode polarized at +0.25-0.40V. The solution for blood sample pretreatment recommended by the manufacturer has been improved by Soutter et al. (1978) by addition of cetyltrimethylammonium bromide. This compound hemo-lyzes the sample, stabilizes the lactate content, and leads to a good correlation with the spectrophotometric reference method using deproteinized blood ... 

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]. 

Since mitochondrial cytochrome c was available commercially (horse heart muscle being the most common source) and could readily be purified to a high level, it formed the basic subject for most of the pioneering studies. Many ideas concerning the electrochemical mechanism, in particular, the mode of interaction with the electrode, have developed around the considerable wealth of information that is available [14, 18] on the structure and properties of the protein molecule. The extent to which the metal centre is buried is illustrated well in Fig. 1 which shows the 3D structure [19] of yeast (iso-1) cytochrome c and a view of the exposed active site. The major function of cytochrome c is as electron donor to cytochrome c oxidase (Complex IV), the membrane-bound enzyme that is the terminus of the aerobic respiratory chain and a site for proton translocation. Another physiological oxidant of cytochrome c (in yeasts) is cytochrome c peroxidase, a soluble enzyme whose crystal structure is known (see Sect. 7). The most important reduc-tant of cytochrome c is the cytochrome Cj component of the membrane-bound hcj complex (Complex III), but others (see Sect. 6, Scheme 5) include cytochrome b, sulfite oxidase, and flavocytochrome (lactate dehydrogenase, found in yeasts). 

Lactate/pyruvate Cytochrome b2/lactate dehydrogenase Pt-electrode... 

Fig. 16 Schematic coupling model of the photo-switchable interactions between cytochrome c and the mixed SAM of spiropyran/merocyanine-terminated and 4-pyridine thiol with (a) the reduction of O2 by COx and (c) the oxidation of lactate by lactate dehydrogenase (LDH). (b) When the electrode is in the cationic merocyanine state, repulsive interactions disallow the functioning of the bioelectrocatalytic processes [130].

Lactate is a small biological molecule that functions as metabolite in the mitochondria and a precursor to pyruvate in the citric acid cycle [87]. In 1997, Bardea et al. developed a lactate BFC using NAD -dependent lactate dehydrogenase (LDH) [88]. They introduced a new method for enzyme immobilization that enabled better oxidation of the substrate and allowed the enzyme to have eleetrieal eontact with the electrode. Covalently linked PQQ and native NAD" form a monolayer on gold electrodes to induce affinity interactions with cross-linked NAD -dependent LDH. In 2001, Katz et al. further improved on the concept of this anode, coupling it with a cytochrome c oxidase cathode to produce a self-powered biosensor that is active only in the presence of the anode s substrate, lactate [89]. With this addition, the cell is completely dependent on substrate for voltage and current output, which is ideal for BFCs. 

In another recycling system we co-immobolized cytochrome bz and laccase in a gelatin layer in front of an oxygen electrode. In the presence of the cytochrome bz substrate, lactate, BQ is reduced to H2Q. Therefore, this enzyme substitutes the cathode of the previous cycling system (Figure 6). However, the substrate recyling is not restricted to the phase boundary (as with electrochemical recyling) but it proceeds in the total volume of the enzyme layer. At the optimum pH of cytochrome b2 (pH 6.5) and lactate saturation a maximum amplification of 500 has been obtained. 

Figure 7. Influence of the lactate concentration on the H2Q converting capacity of the laccase/cytochrome b2 electrode/phosphate buffer pH 6.5, E = +400 mV vs SCE.

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