Urea sensors based on electrodes in direct contact with the medium have limited long-term stability when exposed to complex media. Here, we present a urea biosensor based on urease immobilized in an alginate polymer, buffered at pH 6, and placed in front of a newly developed fast and sensitive CO2 microsensor, where the electrodes are shielded by a gas-permeable membrane. The CO2 produced by the urease in the presence of urea diffuses into the microsensor and is reduced at a Ag cathode. Oxygen interference is prevented by a Cr2+ trap. The 95% response time to changes in urea concentration was 120 s with a linear calibration curve in the range 0-1000 μM and a detection limit of 1 μM. The Ni2+ cofactor to improve sensor performance was continuously supplied from a reservoir behind the sensor tip. The stability of the urea sensor was optimized by the addition of bovine serum albumin as a stabilizer to the urease/alginate mixture that was cross-linked with glutaraldehyde and Ca2+ ions. This immobilization strategy resulted in about 70% of the initial urea sensor sensitivity after two weeks of continuous operation. The sensor was successfully tested in blood serum.
© 2020 American Chemical Society.