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fbtwitterlinkedinvimeoflicker grey 14rssslideshare1
Languages: English
Types: Doctoral thesis
Subjects:
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. \ud 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. \ud 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. \ud 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).
  • The results below are discovered through our pilot algorithms. Let us know how we are doing!

    • 1.1.1. The role of the liver in ammonia metabolism ................................................6
    • 1.1.2. The role of the kidneys in nitrogen metabolism...........................................11
    • 1.1.3. The effects of ammonia on the central nervous system ...............................12
    • 1.1.4. The digestive system ....................................................................................13
    • 1.1.5. The relationship between the lungs and ammonia .......................................15
    • 1.1.6. Muscle metabolism and exercise and their association with ammonia........16
    • 1.2.1. Test kits and POC devices............................................................................20
    • 1.2.2. Conducting polymers such as polyaniline used as ammonia sensing
    • materials .................................................................................................................22
    • 1.2.3. The reaction mechanism of ammonia and polyaniline.................................25
    • 2.4.2. Electrode fabrication via screen printing......................................................45
    • 2.4.3. Polyaniline nanoparticle synthesis ...............................................................46
    • 2.4.4. Inkjet printing of polyaniline nanoparticles .................................................46
    • 2.4.5. Assembly of the aqueous ammonia sensing device .....................................47
    • 2.4.6. Characterisation techniques..........................................................................49
    • 2.4.7. Cyclic voltammetric analysis of polyaniline dispersions .............................49
    • 2.4.8. Electrochemical impedance spectroscopic measurement of ammonia ........50
    • 2.4.8.1. Ratiometric method...............................................................................50
    • 2.4.9. Spectrophotometric measurement of ammonia ............................................50
    • 2.4.10. Bradford protein assay................................................................................50
    • 2.4.11. Oil red O analysis for cellular lipids ..........................................................51
    • 3.2.1. Fabrication and characterisation of polyaniline nanoparticles .....................57
    • 3.2.2. Screen printed silver interdigitated electrode design as part of the inkjet-
    • printed polyaniline sensor.......................................................................................61
    • 3.2.3. Impedimetric assessment of polyaniline sensors..........................................63
    • 3.2.4. Impact of the synthesis method on particle size and its effect on sensor
    • impedance...............................................................................................................66
    • 3.2.5. Impact of the synthesis method on zeta-potential and its effect on sensor
    • impedance...............................................................................................................68
    • 3.2.6. Characterisation of the ammonia sensor in response to liquid samples .......71
    • 3.2.6.1. The use of a hydrophobic membrane for ammonia gas measurement
    • from a liquid sample...........................................................................................72
    • 3.2.6.2. Ammonia sensor reproducibility and drift ............................................74
    • 3.2.6.3. Investigation of the polyaniline nanoparticle inkjet printing process on
    • the characterisation and performance of the ammonia sensor ...........................75
    • 3.2.7. Electrochemical characterisation of inkjet-printed polyaniline films ..........81
    • 4.2.2. Strategies to eliminate solvent interferents ..................................................97
    • 4.2.2.1. Investigation of the effect of membrane composition...........................97
    • 4.2.2.2. Ammonia sensor recovery ....................................................................99
    • 4.2.2.3. Investigation of sample exposure time in the sampling chamber .......103
    • 4.2.3. Time course analysis of the ammonia measurement process.....................110
    • 4.2.4. Sensor pre-calibration.................................................................................112
    • 5.1.1. Blood buffering capacity ............................................................................122
    • 5.1.2. Consideration of sample interferences .......................................................123
    • 5.2.1. Spectrophotometric analysis of ammonia in solution using the Berthelot
    • reaction .................................................................................................................124
    • 5.2.2. Validation of the device using the Abcam® spectrophotometric assay.....125
    • 5.2.3. Interference study .......................................................................................131
    • 5.2.4. Characterisation of sample matrix effects on ammonia measurement.......134
    • 5.2.4.1. Determination of ammonia in a protein sample matrix ......................134
    • 5.2.4.2. Ammonia analysis in serum ................................................................136
    • 5.2.4.3. Spectrophotometric assessment of protein and lipid assay interference
    • 5.2.4.4. Delipidated and deproteinated serum as a matrix for ammonia
    • determination using the ammonia device.........................................................142
    • 5.2.5. Lifetime study of the blood ammonia device .............................................149
    • CHAPTER 7 ............................................................................................................169
    • OVERALL CONCLUSIONS ..................................................................................169
    • 7.1. CONCLUSIONS...............................................................................................170
    • 7.2. CLOSING STATEMENT.................................................................................171
    • LIST OF PUBLICATIONS AND PRESENTATIONS...........................................173 1. SCIENTIFIC PUBLICATIONS.......................................................................174 2. ORAL PRESENTATIONS ..............................................................................174 3. POSTER PRESENTATIONS ..........................................................................175
    • APPENDIX ..............................................................................................................178
    • Windmiller, J.R. and Wang, J. (2013) Wearable Electrochemical Sensors and
    • Biosensors: A Review. Electroanalysis. 25 (1), pp.29-46.
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    The results below are discovered through our pilot algorithms. Let us know how we are doing!

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