Introduction. Urea is the major end product of protein metabolism, which is excreted with urine through the kidneys. Determination of urea concentration in dialysis blood samples is necessary for the diagnosis of kidney disease as it has a significant impact on the health and mortality of patients . Modern methods of determining the concentration of urea in blood plasma and urine, such as spectrophotometry , gas chromatography , fluorimetry , variety of other chemical and physical approaches, have disadvantages associated with the complex and prolonged procedures of sample pretreatment, requirements for highly qualified staff and sophisticated expensive equipment, an inability of on-line measurements. Currently, the development of biosensors is relevant for medical diagnostics, environmental and food production control. This is due to the fact that they are easy-in- use, low-cost in mass production, prone to miniaturization, and can provide high-speed analysis with necessary level of sensitivity and selectivity .
In recent years, various biosensors are developed for analysis of urea in biological samples [5-8]. However, they are often characterized by a lack of sensitivity and stability. To improve these characteristics, semipermeable membranes of different types are often used . Sometimes, the bioselective elements of biosensors are based on recombinant enzymes [6,7]. Utilization of nanoscaled materials to develop biosensors with improved analytical characteristics has also been reported [8,9]. An application of cyclodextrins for immobilization can increase the biosensors productivity by providing favorable conditions for enzymes. It is due to unique cyclodextrin structure (the hydrophilic surface and hydrophobic intramolecular cavity) .
The aim. The development of a biosensor with improved analytical characteristics for urea determination in biological samples using cyclodextrins as a component of urease-based bioselective element.
Materials and methods. The potentiometric method of analysis was used in the work. pH-sensitive field effect transistors served as potentiometric transducers. The working transducers and Ag/AgCl reference electrode were connected to potentiostat. Urease was immobilized onto the transistors surface by covalent cross-linking of the enzyme with bovine serum albumin (BSA) and cyclodextrins using glutaraldehyde. The differential mode of work was used, the signals from the enzyme membrane were compared with those obtained from the reference membrane based on BSA only. Cyclodextrins were added to the enzyme membrane to improve the biosensor analytical characteristics.
Results and discussion. New potentiometric biosensors based on urease membrane with addition of cyclodextrins of different types were developed. The biosensor with 50% of в-HP-cyclodextrin in the membrane was shown to have the best analytical characteristics. The latter were compared with those of the biosensors based on urease only, without cyclodextrins. It was found that the use of cyclodextrin as a component of biomembranes resulted in an increase of biosensor sensitivity to urea by at least 60%. Additionally, the developed biosensors had good signal reproducibility (the error (RSD) did not exceed 3,5%) and high operational stability over 15 days.
Conclusion. A new biosensor for urea determination has been developed. The proposed biosensor is characterized by improved analytical characteristics due to the use of cyclodextrins as a component of the enzyme membrane. The obtained results are promising and the biosensor developed can be effectively applied in future in clinical diagnostics.
- Vineet Sharma et al. Spectrophotometric Determination of Urea in Urine Samples // Proc Indian natn Sci Acad 79, 2013, No. 1, pp. 51-56
- Kulika W., Oosterveldb M.J., Determination of 13C and 15N enrichments of urea in plasma by gas chromatography-combustion isotope ratio mass spectrometry and gas chromatography-mass spectrometry using the 2-methoxypyrimidine derivative // Journal of Chromatography B, 2003, vol.791, pp. 399-405.
- Shin-ichiro S., Jun'ichiro M.A. Fluorometric Determination of Urea With Urease // Analytical Letters, 1999, vol. 32 , Iss. 8, pp.1543-1551.
- Дзядевич С.В., Солдаткін О.П. Наукові та технологічні засади створення мініатюрних електрохімічних біосенсорів. - К.:Наукова думка. - 2006. - 255 с.
- Singh M, Verma N, Kumar Garg A, Redhu N. Urea biosensors // Sens. Actuators B 2008, 134:345-351.
- P. Velychko, О. О. Soldatkin, et al. A Novel Conductometric Urea Biosensor with Improved Analytical Characteristic Based on Recombinant Urease Adsorbed on Nanoparticle of Silicalite // Nanoscale Research Letters, 2016, 11:106.
- Soldatkin AP, Montoriol J, et al. A novel urea sensitive biosensor with extended dynamic range based on recombinant urease and ISFETs.// Biosens Bioelectron, 2003, 19:131-135
- Hamlaoui ML, Reybier K, et al. Development of a urea biosensor based on a polymeric membrane including zeolite. // Anal Chim Acta, 2002, 466:39-45
- O. Soldatkin, M. Shelyakina, V. Arkhypova, et al. Nano- and microsized zeolites as a perspective material for potentiometric biosensors creation.// Nanoscale Research Letters, 2015, 10:59.
- Cristiano P. da Silva a, Ana C. Franzoib Development of biosensor for phenolic compounds containing PPO in в-cyclodextrin modified support and iridium nanoparticles // Enzyme and Microbial Technology 52 (2013) 296- 301