Enzyme electrodes creatinine

The demand for monitoring common metabolites of diagnostic utility such as glucose, urea and creatinine continue to provide the impetus for a staggering research effort towards more perfect enzyme electrodes. The inherent specificity of an enzyme for a given substrate, coupled with the ability to electrochemically detect many of the products of enzymatic reactions initiated the search for molecule-selective electrodes. 

In an early application, an enzyme electrode system was reported for the determination of creatinine and creatine, using a combination of creatinine amidohy-drolase, creatine amidinohydrolase and sarcosine oxidase, co-immobilized on an asymmetric cellulose acetate membrane. Thus, the hydrogen peroxide produced was detected to give a quantitative measure of creatine and creatinine in biological fluids [70]. 

Davis, G., Green, M. J. c Hill, H. A. O. (1986) Detection of ATP and creatinine kinase using an enzyme electrode. EnzymeMicrob Tech, 8,349-352. 

The concentrations of a number of analytically relevant compounds, such as creatinine, pyruvate, hormones, and drugs, are often so low that their assay with enzyme electrodes requires large sample volumes or is totally impossible. The detection limit of usual enzyme electrodes is... 

Yamato, H., M. Ohawa, and W. Wernet. 1995. A polypyrrole/three enzyme electrode for creatinine detection. Anal Chem 67 2776. 

A three-enzyme electrode system, such as needed for creatinine measurement, poses a more difficult enzyme-immobilisation problem, in that different enzymes have different immobilisation requirements and their microenvironmental interrelationships need to be optimised. For one creatine sensor, the requisite creatine amidinohydrolase and sarcosine oxidase were immobihsed in polyurethane pre-polymer and PEG-hnked creatinine amidohydrolase was attached via diisocyanate pre-polymer to create a polyurethane adduct [14]. The likelihood of enzyme inactivation with chemical immobih-sation is high, but provided an enzyme preparation survives this, long-term stability is feasible. In the case of these three particular enzymes, a loss of activity resulted from silver ions diffusing from the reference electrode the material solution was to protect the enzyme layer with a diffusion-resisting cellulose acetate membrane. 

The future of ISEs in the clinical chemistry instrumentation is quite exciting. As described in subsequent sections of this article, the coupling of enzyme and immunological reagents to ISE detectors to form bioelectrode systems appears to offer manufacturers a new approach toward the detection of metabolites such as creatinine and urea directly in blood and urine samples. Ultimately, such biosensors will be placed into complete electrode-based automated clinical analyzers. In addition, continued research on new membrane formulations, particularly liquid membrane ionophore systems, will result in the development of addition electrodes which can be incorporated into current analyzer systems to expand the electrolyte menu. Indeed, recent efforts have indicated that membranes selective fi)r bicarbonate (F5) and lithium (Z2) are likely additions in the near future. 

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. 

The very same enzymes have been combined by Mascini et al. (1985a) in an FIA system. Creatinine iminohydrolase was immobilized on the inner wall of nylon tubing (diameter 1 mm, length 1 m) and the ammonia liberated in the enzymatic reaction was measured with an NH3 electrode. Owing to the low sensitivity of the indicator electrode, the linear range was only 0.01-0.2 mmolA. 

Meyerhoff and Rechnitz (1976) developed a potentiometric creatinine sensor by inclusion of creatinine iminohydrolase between the gas-permeable membrane of an ammonia electrode and a dialysis membrane. Since the specific activity of the enzyme used was very low, 0.1 U/mg, only 43 mU could be entrapped at the electrode. Therefore the sensor was kinetically controlled and reacted to addition of the enzyme activator tripolyphosphate by an increase in sensitivity from 44 mV to 49 mV per concentration decade and a corresponding decrease of the detection limit. These effects agree with theoretical considerations of reaction-transport coupling. The samples were treated with a cation exchanger to remove endogenous serum ammonia. 

Microbial hybrid electrodes using nitrifying bacteria fixed together with NH3-producing enzymes on an oxygen probe have been developed for the determination of urea (Okada et al., 1982) and creatinine (Kubo et al., 1983a). For urea assay a membrane containing immobilized... 

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. 

It has been reported [48] that the detection limit can be lowered significantly by the addition of approximately 5% of doped PPA into the enzyme containing layer. This effect was demonstrated on enzyme potentiometric sensors for urea and creatinine based on field-effect transistors and on ion-selective electrodes. The origin of this effect is not obvious and no explanation has been offered in the original paper. 

Many potentiometric en2yme electrodes for widely different analyte species can be prepared merely by choosing the appropriate ISE transducer and immobilized enzymatic reagent. Table 5 summarizes the enzymes and ISEs/gas sensors used to construct biosensors for a number of important biomolecules, ranging from urea and creatinine to amino acids, nucleotides, and even glucose and penicillin. In the... 

An inverse process of enzyme-catalyzed dissolution of a polymer coating [550, 551] leads to an easily detected increase in electrode capacitance. Such a process was studied for urea and immunoglobulin G (IgG) [550] and urea and creatinine in serum [551]. 

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