The development of a point of care device for measuring blood ammonia

Abstract

Ammonia is produced in the body during the metabolism of amino acids. In the liver, it is converted to urea via the urea cycle and excreted by the kidneys as urine. Normal levels are between 11 to 50 µM, whereas a blood ammonia level of approximately 100 µM indicates pathology. Elevated blood ammonia is associated with a number of pathological conditions including liver and kidney dysfunction. Conditions such as these can affect brain function and can be fatal. Current blood ammonia analysis requires a laboratory blood test. Few, if any of the techniques used are suitable for point of care (POC) testing. The development of a reliable and simple method for blood ammonia determination is essential for clinical diagnosis and management of patient progress in order to prevent further debilitating illnesses developing, and extending life. This is particularly critical in many disorders such as hyperammonaemia of the newborn, inborn errors of metabolism including urea cycle defects, organic acidaemias, hyperinsulinism/hyperammonaemia, liver disease and other cause of hyperammonaemic encephalopathy. This thesis investigates the development of an electrochemical sensor for the measurement of ammonia in blood. Polyaniline has a known affinity for ammonia which operates on the deprotonation of the polyaniline backbone forming an ammonium ion. In this work, polyaniline nanoparticles were fabricated and inkjet-printed onto silver screen printed electrodes. The sensors were then incorporated into devices containing a gas-permeable membrane, which facilitated the measurement of gaseous ammonia from a liquid sample (blood) using electrochemical impedance spectroscopy. The combination of impedance spectroscopy with a gas-permeable membrane allowed the measurement of gaseous ammonia from solution. The ammonia device developed possessed refinements to enhance its sensitivity and included careful optimisation of other aspects of the measurement. For example, an air purge through the device gas chamber was employed to remove matrix interferences from the sensor and improve the specificity to ammonia. The pH of the sample to be analysed was modified in order to increase the mass of ammonia in solution, thus lowering the limit of detection (LOD) of the device. Finally, assay timings were optimised in order to increase the impedimetric response of ammonia. These optimisations resulted in the effective detection of ammonia in a liquid sample down to the lowest clinically relevant levels found in blood. The devices displayed an impedimetric baseline intra- and inter-variability of 25 and 6.9%, respectively for n = 15 over a period of 160 s. A calculated limit of LOD of 12 µM was achieved for human serum measurements. A coefficient of determination of 0.9984, slope of 0.0046 and an intercept of 1.1534 was obtained in human serum across the linear range of 25 to 200 μM ammonia (n = 3). The device was validated against a commercial spectrophotometric assay which resulted in excellent correlation (0.9699, p < 0.0001) with a slope of 1.4472 and an intercept of 0.5631 between both methods (n = 3). The devices could be stored in desiccant for up to five months and displayed minimal variation (0.64%) over time (n = 12)