Urea biosensor

Urea is the most important end product of protein degradation in the body. Its concentration in blood depends on the protein catabolism and nutritive protein intake and is regulated by renal excretion. Thus the estimation of blood urea nitrogen is important in the assessment of kidney failure. The normal level of urea ranges from 3.6 mM to 8.9 mM. All enzymatic methods for urea determination are based on the principle of urea hydrolysis by urease  

Most biosensors described in the literature for the determination of urea are potentiometric based on NH4 or HCOj sensitive electrodes [181, 182]. Osaka and co-workers constructed a highly sensitive and rapid flow injection system for urea analysis with a composite film of electropolymerised inactive PPy and a polyion complex [183]. Pandey and co-workers fabricated a urea biosensor based on immobilised urease on the tip of an ammonia gas electrode (diameter 10 pm) made from a PPy film coated onto a platinum wire [170]. The enzymatic response was achieved in the wide range of 0.001-0.05 M with a stability of more than 32 days. Cho and co-workers [184] developed a procedure for urea determination by crosslinking urease onto PANI-Nafion composite electrodes, which could sense the ammonium ions efficiently. Such a urea biosensor has a detection limit of about 0.5 pM and a response time of 40 seconds. 

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Construction of the optode for urea biosensor requires immobilisation of protein (and pH indicator) in the host matrix. There are several methods enabling protein entrapment. One can use gels, polymers, saccharose, various meshes and membranes78. 

The same research group described a small-scale electronic urea biosensor based on a urease-covered conductivity sensor (Watson et al., 1987/88). The conductometric sensor was prepared on a silicon wafer by applying the following sequence of steps thermal oxidation, deposition... 

Barhoumi et al. discussed a ZnAl-based enzyme nanohybrid system for a urea biosensor, in which ureases were entrapped within layered double hydrides [157]. Tsai et al. employed a sol-gel method to fabricate an enzymatic optical biosensor array for the analysis of multiple samples [152]. 

Sheppard NF Jr, Mears D, Guiseppi-Elie A (1996) Model of an immobilized enzyme conductimetric urea biosensor. Biosens Bioelectron 11 967-979... 

Figure 10.9 Response curve of a conductometric urea biosensor...

Figure 10.13 Lineweaver-Burke plot for the urea biosensor based on PPy/PVS films...

Guilbault and Montalvo were the first, in 1969, to detail a potentiometric enzyme electrode. They described a urea biosensor based on urease immobilized at an ammonium-selective liquid membrane electrode. Since then, over hundreds of different applications have appeared in the literature, due to the significant development of ion-selective electrodes (ISEs) observed during the last 30 years. The electrodes used to assemble a potentiometric biosensor include glass electrodes for the measurement of pH or monovalent ions, ISEs sensitive to anions or cations, gas electrodes such as the CO2 or the NH3 probes, and metal electrodes able to detect redox species some of these electrodes useful in the construction of potentiometric enzyme electrodes are listed in Table 1. 

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-... 

Chen, J.C., Chou, J.C., Sun, T.P., Hsiung, S.K. Portable urea biosensor based on the extended-gate field effect transistor. Sens. Actuat. B 91, 180-186 (2003)... 

Immimodiagnostics and enzyme biosensors are two of the leading technologies that have a greatest impact on the food industry. The use of these two systems has reduced the time for detection of pathogens such as Salmonella to 24 hr and has provided detection of biological compounds such as cholesterol or chymotrypsin [15]. Biosensors analyses Beta lactams in milk and presence of urea in milk that lead to production of synthetic milk, the biocomponent part of the urea biosensor is an immobilized urease yielding bacterial cell biomass isolated from soil and is coupled to the ammonium ions selective electrodes of a potentiometric transducer. The membrane potential of all types of potentiometric cell based probes is related to the activity of electrochemically-detected product [16]. 

Analyte glucose biosensor, urea biosensor, glutamine selective electrode... 

Later, the same idea was applied to determine urea in blood serum [19]. The system was formed by two urea biosensors, obtained by immobilization of urease enzyme (EC 3.5.1.45) onto ammonium-selective membranes, plus ISEs for... 

In a second system reported by the same laboratory, a sensor array incorporating a urea biosensor was proposed for the determination of urea in blood serum [19]. Similarly to the previous case, the urease enzyme was immobilized employing BS A and glutaraldehyde onto an ammonium ISE, taking profit of the appearance of ammonium product after the enzyme hydrolysis of urea. As before, the array was complemented by ISEs for K", Na and with the idea of counterbalancing any interference by alkaline ions. Authors achieved correct prediction of urea and potassium, not sodium or ammonium initially present. Correlation and prediction... 

Jha SK, Kanungo M, Nath A, D Souza SF (2009) Entrapment of live microbial cells in electropolymerized polyaniline and their use as urea biosensor. Biosens Bioelectron 24... 

The pH electrode is another suitable transducer for the construction of a urea biosensor. The classical glass electrode is sensitive to H ions and urease is attached in a gel of either polyacrylamide [107] or methacrylamide-aoylamide cqxdymer [108]. me metallic electrodes are also sensitive to H ions (for example, the antimony electrode) and can also be used in conjunction with a urease membrane [109]. The enzyme pH electrode detects a very weak variation in proton concentration arising from an enzymatic reaction, and the signal amplitude is determined by the buffering capacity of the solution. Both the nature of the buffer solution and the working pH value of the biosensor can reduce its practical use. A differential measurement at different pH values can be used to correct for any variation. 

There are several technologies proposed for urea detection through use of immobilized urease. Most of them offer a narrow detection limit in either low or high concentrations of urea. Srivastava et al. [12], described a urea biosensor utilizing... 

Srivastava, P.K., Kayastha, A.M., and Srinivasan (2001). Characterization of gelatin-immobilized pigeonpea urease and preparation of a new urea biosensor. Biotechnol. Appl. Biochem. 34 55-62. 

Castillo-Ortega, M.M., Rodriguez, D.E., Encinas J.C., Plascencia M., Mendez-Velarde A., and Olayo R. (2002). Conductometric uric acid and urea biosensor prepared from electroconductive polyanibne-poly (n-butyl methacry methacrylate) composites. Sens. Actual. B. Chem. 85(l-2) 19-25. 

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