Amperometric uric acid biosensor and two-dimensional liquid chromatography-mass spectrometer for urinary uric acid assay

• Main Authors: Chi-Ying Kao, 高啟瀛
• Other Authors: Cheanyeh Cheng
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/43634885607339651960

碩士 === 中原大學 === 化學研究所 === 104 === A new second generation amperometric uricase electrode has been developed by chemically binding both uricase and redox mediator ferrocenecarboxaldehyde to an inexpensive polyimide enamel taken off copper wire through the assistance of L-methionine functionalized gold surface which was formed by simple electrodeposition to improve the electrode stability and shelf life. The preparation of uricase electrode was characterized by FTIR and cyclic voltammetry (CV). During a 209-day testing period, the uricase electrode performance exhibits in average a low oxidation potential of 0.33 V, a response time of 5-15 s, a widest linear calibration concentration range (0-2.38 mM, r2 > 0.9952), a sensitivity of 50 μA mM–1, and a limit of detection (LOD) of 2.4 μM. The uricase electrode was still usable with good performance after 309 days. The uricase electrode has been applied for the determination of uric acid in human urine specimens using standard addition method and with the measurement accuracy and precision of 85.6-95.5% and 0.3-2.4%, respectively. Overall, the developed uricase electrode demonstrates a long-term stability and reusability that is potential for clinical application. In addition, we also developed a two dimensional high-performance liquid chromatography (2D-HPLC) which was formed by connecting a reversed phase C18 chromatographic column and a Pb2+ ion ion-exclusion chromatographic column. The liquid chromatography-electrospray ionization/ion trap-mass spectrometry(LC-ESI/IT-MS) for analysis of four common materials in human body fluids: ascorbic acid, uric acid, allantoin, and urea. The mobile phase for both columns was 0.005% (v/v) CH3COOH and at a flow rate of 1.0 mL min-1. The mass spectrometer detection signal was [M+H]+ positive ion. The external standard calibration curves for the four investigated analytes showed a linear concentration range of 0-10 μg L-1 with corresponding linear regression correlation coefficient in the range 0.9935-0.9982, The LOD of ascorbic acid, allantoin, urea and uric acid determined by the external standard calibration curve was 0.1, 0.2, 0.1 and 0.2 μg L-1, respectively. The LOD of uric acid obtained from the two dimensional liquid chromatography-mass spectrometry (2D LC-MS) was better than that obtained by using amperometric uricase sensor electrode about 2238-fold. The uric acid in human urine specimens has been quantitatively determined by standard addition method with measurement precision and accuracy of 13-61% and 92,9-95.0%, repectively.