Lactate dehydrogenase sensors

Reliable measurements of L-lactate are of great interest in clinical chemistry, the dairy and vine industry, biotechnology, or sport medicine. In particular, blood lactate levels are indicative of various pathological states, including shock, respiratory insufficiencies, and heart and liver diseases. Silica sol-gel encapsulation of the lactate dehydrogenase and its cofactor was employed as a disposable sensor for L-lactate51. The sensor utilized the changes in absorbance or fluorescence from reduced cofactor nicotinamide adenine dinucleotide (NADH) upon exposure to L-lactate. 

Gorton et al. reported carbon paste electrodes based on Toluidine Blue O (TBO)-methacrylate co-polymers or ethylenediamine polymer derivative and NAD" " with yeast alcohol dehydrogenase for the analysis of ethanol [152,153] and with D-lactate dehydrogenase for the analysis of D-lactic acid [154]. Use of electrodes prepared with dye-modified polymeric electron transfer systems and NAD+/NADH to detect vitamin K and pyruvic acid has also been reported by Okamoto et al. [153]. Although these sensors showed acceptable performances, insensitivity to ambient oxygen concentration, sensor stability and lifetime still need to be improved to obtain optimal dehydrogenase based enzyme biosensors. 

Resistance to vancomycin is via a sensor histidine kinase (VanS) and a response regulator (VanR). VanH encodes a D-lactate dehydrogenase/a-keto acid reductase and generates D-lactate, which is the substrate for VanA, a D-Ala-D-Lac ligase. The result is cell wall precursors terminating in D-Ala-D-... 

Fig. 102. Schematic representation and measuring curves of a recycling sensor for ADP and ATP with pyruvate measurement via the sequence lactate dehydrogenase-lactate monoxygenase. PEP = phosphoenolpyruvate. (Redrawn from Wollenberger et al., 1987a).

Weigelt et al. (1987b) attempted the measurement of the lactate/py-ruvate ratio in plasma by using a lactate dehydrogenase-LMO sequence electrode. The sensor was connected to a pC>2 meter and was equally sensitive for lactate and pyruvate. Determination of concentrations of both substrates in a sample requires a time period of about 3 min. 

Figure 17.6 (a) The principle of an accumulation biosensor, (b) Current-time curves for a sensor operated without and with intermediate accumulation. The difference in the steady state and peak currents represents the amplification of the sensor response, (c) Glycerol measurement without and with NADH accumulation on an enzyme electrode containing immobilized glycerol dehydrogenase, lactate dehydrogenase, and lactate monooxygenase. NADH is stripped by pyruvate addition after 6 min accumulation (reproduced with the permission of Elsevier Science Publishers BV). 

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]. 

The measurement of the lactate/pyruvate ratio in plasma is possible by using a lactate dehydrogenase-lactate monooxygenase sequence electrode [373]. The sensor is equally sensitive to lactate and pyruvate (Figure 14-34), because of the high enzyme loading and the... 

Figure 14-34. Calibration graph of a lactate dehydrogenase/lactate monooxygenase electrode for ( ) lactate and (X ) pyruvate. (See also Figure 14-36 for sensor conflguration.) Reproduced from [423] with permission from Springer-Verlag.

Schematic representation of a lactate dehydrogenase (LDH) - lactate monooxygenase (LMO) sensor for the determination of pyruvate kinase activity. PEP = phosphoenolpyruvate.

Response times for the lactate dehydrogenase biosensor in either the lactate or pyruvate sensing mode range from 6 to 12 minutes. Faster response is obtained with higher concentrations. In addition, the lifetime of this sensor is from 3 to 7 days depending on the extent of enzyme loading and the storage conditions. 

Urea biosensors containing urease are based on the detection of NH and HCO [7,204, 205]. Lactate dehydrogenase immobilized in PANI was used for lactate measurements [7]. Cholesterol sensors have been fabricated using choles-... 

A similar system has been used in construction of a lactate sensor where lactate oxidase (LOD) and lactate dehydrogenase (LDH) were coupled. Using a very thin layer of coimmobilized LOD and LDH a very high amplification effect was reached, about 4100 times [159]. 

The determination of lactate is important in medicine, because certain disorders are related to lactic acidosis, and in industry, for the control of milk and other food products. A lactate sensor can be constructed by coupling amperometric transducers with various enzymes, such as lactate dehydrogenase, cytochrome b2, lactate oxidase and lactate monooxygenase. Lactate dehydrogenase requires NAD as a cofactor, whereas cytochrome b2 uses potassium ferricyanide, which is much cheaper [173]. Cytochrome b2 catalyses the following reaction ... 

The creation of an optical sensor that detects the cofactors on which many enzymes dqiend is a much massociated with many enzymes, particularly dehydrogenases. This cofactor has a maximal absorption at 340 nm and a maximal fluorescence emission at 400 nm, which is easily detectable with a photomultiplier. NADH also has the advantage that it can be immobilized on the same support as the enzyme (see 3.3.1.e). nber-optic biosensors based on the fluorimetric detection of NADH have been constructed for the determination of lactate and pyruvate [208]. These sensors use inunobilized lactate dehydrogenase AD) to catalyse the following equilibrium reaction ... 

Matsumoto et al (41) prepared a multi-enzyme electrode using glucose oxidase, invertase, mutarotase, fructose-5-dehydrogenase, and catalase to simultaneously detect glucose, fructose, and sucrose in fruit juices and soft drinks. Detection of multi-components by enzyme sensors was also reported in analysis of sucrose and glucose in honey (42) and drinks (43), and L-malate and L-lactate in wines (44). 

Albareda-Sirvent and Hart [65] L-Malate, l-lactate Wines Lactate oxidase or malate dehydrogenase/in sol-gel matrix Sol-gel thick-film printed graphite electrode (contained NAD+ for malate sensors)/+ 350 mV vs. Ag/AgCl for lactate sensor and -125 mV vs. Ag/ AgCl for malate Meldola s Blue (mediator for the dehydrogenase)... 

---