Application of potentiometric biosensor based on recombinant urease for urea determination in blood serum and hemodialyzate

摘要

Potentiometric biosensor based on two identical pH-sensitive field-effect transistors for urea determination was developed. Recombinant urease with low affinity to urea (K_m=200 mM) was immobilized via entrapment in PVA/SbQ photopolymer on one transistor and served as a biorecognition element of the biosensor, while bovine serum albumin in PVA/SbQ photopolymer placed on the second transistor was used for reference. Biosensor was characterized by a wide range of urea determination: 0.5-15 mM linear range (operational detection range - 0.5-40 mM) and quick response time (1-2 min). Samples of blood serum and hemodialysate without urea caused no biosensor response. All this allowed analysis of blood serum samples that were diluted 10 times, what led to smaller measurement error in comparison with 100-500 times dilution in case of biosensors based on non-modified urease from soy beans. Urea concentrations in 10 samples of serum were determined; biosensor results correlated well with two control methods of urea measurement. Furthermore, kinetics of decreasing urea concentration in dialysate during hemodialysis was demonstrated. Biosensor measurements of dialysate samples were verified by traditional colorimetric determination of urea. Proposed biosensor can be effectively used for analysis of samples with different concentrations of urea and for hemodialysis control. What is more, characteristics of the biosensor make possible real-time measurements of urea concentration during hemodialysis by pumping dialysis fluid through the working cell. Biosensor showed no significant decrease of responses during 5 month of storage.