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  • 學位論文

電流式尿酸生物感測電極開發及二維液相層析質譜儀於尿液中尿酸之分析應用

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

指導教授 : 鄭建業

摘要


本研究發展一種新型第二代電流式尿酸生物感測電極,使用市售的聚酰亞胺漆包銅線作為電極基材,透過電鍍金於銅線表面及甲硫胺酸功能化之輔助,將氧化還原媒介物二茂鐵甲醛及尿酸酶鍵結於銅線上。此尿酸感測電極製作過程各步驟的表面化學結構特徵,已藉由傅立葉轉換紅外線光譜儀鑑定。透過循環伏安法及標準品添加電流-時間方法測試209天,平均而言,電極使用電壓維持在0.33 V,電極表現出寬廣的尿酸檢量線線性範圍(0-2.38 mM, r2 > 0.9952),靈敏度為50 μA mM–1,感測時間為5至15秒,偵測極限值則為2.4 μM (403 μg L-1)等良好的性能。此尿酸感測電極以標準品添加法應用於人體尿液樣品中尿酸測定,其量測準確度為85.6-95.5 %,而精確度為0.3-2.4 %。顯示此電極為一堅固且可重複使用之精確、準確及方便量測的新型第二代電流式尿酸生物感測電極。 此外,發展聯結逆相C18層析管柱與鉛離子型離子排斥層析管柱之二維高效液相層析電噴灑離子阱質譜儀系統,分析人體體液中四種常見物質:抗壞血酸、尿酸、尿囊素及尿素。兩支分離管柱的分析動相與流速為0.005% (v/v)乙酸及每分鐘 1.0 毫升,其質譜儀偵測離子峰訊號為[M+H]+。四種分析物的外部標準品檢量線的線性範圍為0-10 μg L-1,其線性迴歸相關係數為0.9935-0.9982,而抗壞血酸、尿囊素、尿素與尿酸之偵測極限值分別為0.1、0.2、0.1及0.2 μg L-1。又二維高效液相層析質譜儀的尿酸偵測極限值較電流式尿酸酵素感測電極偵測極限值要好2238倍。以標準品添加檢量線分析人體尿液樣品中之尿酸量測精確度為13-61%,準確度為92.9-95.0%。

並列摘要


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.

參考文獻


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被引用紀錄


張書瑜(2016)。應用於腎結石復發預防之多參數尿液檢測SoC設計〔碩士論文,中原大學〕。華藝線上圖書館。https://doi.org/10.6840%2fcycu201600577

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