Urea sensors

The way of enzyme entrapment has been described79 proposing the application of sol-gel matrices. The optodes of urea sensor were prepared by the sol-gel method and were stored in a refrigerator. As the pH sensitive dye the bromothymol blue was used. Since it is best acting in pH range 6 to 7.6, the pH of sol-gel bulks obtained in the experiment was chosen as pH 6. Before measurements, the optodes were incubated in the temperature 36.6°C. 

Okada T, Karube I, Suzuki S (1982) Hybrid urea sensor using nitrifying bacteria. Eur J Appl Microbiol Biotechn 14 149-154... 

Our work deals with only a few examples (i) the immunosensor for the forest-spring encephalitis diagnosis (a metal label is used for signal generation), (ii) the enzyme-free urea sensor and (iii) the platinum sensor for antioxidant activity determination. What they all have in common is a screen-printed transducer consisting of graphite or Pt nanoparticles. Transducer configuration is shown in Fig. 27.1. 

Enzyme-free urea sensor Nano carbon-containing transducer, covered with chemical catalytic system, is used as electrochemical enzyme-free sensor for urea determination in biological materials. 

The objective is to describe a new non-enzymatic urea sensor based on catalytic chemical reaction. The sensor consists of screen-printed transducer (IVA, Ekaterinburg, Russia) and catalytic system which is immobilized on the transducer surface as a mixture with carbon ink. The sensor is used for measuring concentration of urea in blood serum, dialysis liquid. Detection limit is 0.007 mM, while the correlation coefficient is 0.99. Some analysis data of serum samples using the proposed sensor and urease-containing sensor (Vitros BUN/UREA Slide, Johnson Johnson Clinical Diagnostics, Inc.) are presented. 

Urease-containing sensor3 Non-enzymatic urea sensor... 

Figure 16 shows the long-term stability of the FET using a 1.7 mM urea solution. The response amplitude of the sensor decreases rapidly after 40 measurements without adding EDTA (ethylendiamine tetraacetic acid) to buffer solutions (Fig. 16, —EDTA). Adding EDTA to the buffer increases the life of the urea sensor. The urea sensor is usable for more than 2000 assays, showing no decrease in the response amplitude (Fig. 16, +EDTA). The rapid decrease in the response amplitude in the absence of EDTA suggests that heavy metal ions inactivate urease. The effect of EDTA on the durability of a urea sensor has been previously reported (48).

Enzyme sensors can measure analytes that are the substrates of enzymatic reactions. Thermometric sensors can measure the heat produced by the enzyme reaction [31], while optical or electrochemical transducers measure a product produced or cofactor consumed in the reaction. For example, several urea sensors are based on the hydrolysis of urea by urease producing ammonia, which can be detected by an ammonium ion-selective ISE or ISFET [48] or a conductometric device [49]. Amperometric enzyme sensors are based on the measurement of an electroactive product or cofactor [50] an example is the glucose oxidase-based sensor for glucose, the most commercially successful biosensor. Enzymes are incorporated in amperometric sensors in functionalised monolayers [51], entrapped in polymers [52], carbon pastes [53] or zeolites [54]. Other catalytic biological systems such as micro-organisms, abzymes, organelles and tissue slices have also been combined with electrochemical transducers. 

The ammonia produced dissolves to form NH, which is sensed by the ISE. The signal generated by the NH4 produced is proportional to the logarithm of the concentration of urea in the sample. The response may be either steady state or transient. Typically, correction for background potassium is required, since the nonactin ionophore has limited selectivity for ammonium over potassium (Knh4/k = 0.1). Potassium is measured simultaneously with urea and used to correct the output of the urea sensor using the Nicolslcy-Eisenman equation. 

Garred LJ, Canaud B, Bose JY, Tetta C. Urea rebound and delivered Kt/V determination with a continuous urea sensor, Nephrol Dial Transplant 1997 12 535-42. 

Osaka T, Komaba S, Seyama M, Tanabe K (1996) High-sensitivity urea-sensor based on the composite fdm of electroinactive polypyrrole with polyion complex. Sens Actuators B 36 463 69... 

While the majority of enzyme electrodes fabricated have been rather large devices, there have been some recent reports concerning the development of miniaturized and even microsensors. For example, MeyerhoflF (M5) prepared an essentially disposable urea sensor (tip diameter 3 mm) by immobilizing urease at the surface of a new type of polymer-membrane electrode-based ammonia sensor (see Fig. 4). Alexander and Joseph (Al) have also prepared a new miniature urea sensor by immobilizing urease at the surface of pH-sensitive antimony wire. Similarly, lannello and Ycynych (II) immobilized urease on a pH-sensitive iridium dioxide electrode. In these latter investigations, ammonia liberated from the enzyme-catalyzed reaction alters the pH in the thin film of enzyme adjacent to the pH-sensitive wire. 

II. lanniello, R. M., and Yacynych, A. M., Urea sensor based on iridium dioxide electrodes with immobilized urease. Anal. Chhn. Acta 146, 249-253 (1983). 

Extended gate structures using iridium oxide layers have been used in an integrated glucose and urea sensor (Araki et al., 1985). 

Most urea sensors are based on potentiometry. The selectivity of the urea sensor described by Guilbault and Montalvo (1969) (see above) has been improved by replacing the glass electrode by an NH sensitive electrode based on nonactin (Guilbault and Nagy, 1973). The antibiotic was contained in a membrane of silicon rubber. 

Kobos et al. (1988) described the adsorption of urease on a fluorocarbon membrane for the construction of a urea sensor. The spontaneous adsorption was enhanced by a factor of 7 by perfluoroalkylation of the amino groups of the enzyme. The enzyme membrane was attached to an ammonia gas-sensing electrode. The urea sensor thus prepared exhibited a sensitivity of 50 mV per decade of urea concentration and a response time of 3 min. Only a small amount of enzyme could be adsorbed on the limited membrane surface, so the sensor was stable for only 7 days. 

To construct urea sensors based on pH electrodes urease has been immobilized by physical entrapment around the active sensor tip by a dialysis membrane (Nilsson et al., 1973) and by crosslinking with BSA and glutaraldehyde (Tran-Minh and Broun, 1975). For the latter, the sensor tip was dipped into the reaction mixture, allowed to dry in the air, and washed with glycine buffer to remove excess glutaraldehyde. The pH measured with this sensor was proportional to the logarithmic values of urea concentration in the range of 0.05 to 5 mmol/1. 

Hamann et al. (1988) developed a urea sensor based on a pH glass electrode (Forschungsinstitut Meinsberg, GDR) and urease from soy beans with a specific activity of 2 U/mg. The enzyme was immobilized by dipping the wetted sensor tip first into crystalline urease and then into a solution of methylene chloride containing 21-25 mg/ml of cellulose triacetate. After evaporation of the solvent an amount of enzyme corresponding to 3 U remained at the sensor. The layer thickness and the diffusion coefficient of urea in the layer were dermined to be 30-50 pm... 

The mechanically unstable glass electrodes in urea sensors have been replaced by antimony (Joseph, 1984), iridium (Ianiello and Yacynych, 1983), and miniature palladium electrodes (Szuminsky et al., 1984). Urease was included in a PVC sheath or crosslinked by glutaraldehyde directly on the electrode surface. Inactivation by heavy metals was diminished by addition of EDTA. 

Fitting into the trend towards improvement of the availability and simplification the preparation of biocatalytic layers for biosensors, the use of crude materials has been explored. Arnold and coworkers investigated the feasibility of employing Jack bean meal in a urea sensor (Arnold and Glaizer, 1984) and rabbit muscle acetone powder in a sensor for adenosine monophosphate (Fiocchi and Arnold, 1984). Both sensors turned out to be serious contenders with the appropriate enzyme electrodes with respect to lifetime and slope of the calibration curves. Other parameters, such as response time and linear range, were quite similar. 

An amperometric urea sensor based on the pH dependence of the anodic oxidation of hydrazine (Kirstein, 1987) has been utilized in the Glukometer GKM 02 for hemodialysis monitoring. For urea concentration in dialyzate the following correlation was obtained with the Ber-thelot method ... 

The determination of alanine aminopeptidase (AAP, EC 3.4.11.14) is of importance in the rapid diagnosis of liver and bile diseases. Common assays involve the coupling of alanine hydrazide cleavage with a chro-mogenic reaction. Kirstein (1987) proposed indicating the rate of hydrazine formation electrochemically. In contrast to an amperometric urea sensor based on this indication method (see Section 3.1.21), the pH value in the near-electrode space remains unchanged while the concentration of electrode-active hydrazine rises. The incubation period for AAP assay is lowered to half of that needed in the conventional method, the two correlating with r = 0.994. 

Anzai J, Tezuka S, Osa T, Nakajima H and Matsuo T 1987 Urea sensor based... 

G. Gauglitz, M. Reichert, Spectral investigation and optimization of pH and urea sensors. Sensors Actuators B 6 (1992) 83. 

Examples of enzymes used for biosensors include glucose oxidase for glucose sensors, alcohol oxidase for ethanol sensors, lactate oxidase for lactate sensors and urease for urea sensors. A typical enzyme reaction, as described by equation 5.2 might involve the transfer of an electron, a pH change, hydrolysis, esterification or bond cleavage. The type of enzymatic reaction that occurs determines the type of transducer that is used. 

---