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研究生(外文):Po-Jen Chien
論文名稱(外文):The Development of D-lactate Biosensor
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目前D-lactate檢測方式的相關文獻有很多種,許多方法已有高靈敏度、偵測極限低等優點,但大多需要耗費大量的時間且檢測成本昂貴。本實驗旨在開發一新型D-乳酸生物感測器,能較快速地檢測檢品中D-lactate的濃度並且同時具有良好的靈敏度與偵測極限等檢測能力。在本研究中,我們利用紫外光波段之發光二極體(UV-LED)、光譜儀以及D-乳酸去氫酶(D-lactate dehydrogenase)來建立此D-乳酸生物感測器。當環境中存在氧化態菸鹼醯胺腺嘌呤二核苷酸(NAD+)時,D-乳酸去氫酶會將D-乳酸催化成丙酮酸,並同時將NAD+還原為NADH。NADH 具有吸收340 nm紫外光,並且放出491 nm螢光的光學特性,藉由測量螢光強度,即可得知檢品中所含之D-乳酸濃度。
實驗結果顯示,此D-乳酸生物感測器在濃度5 μM -150 μM 之間有良好的線性關係(R2 = 0.9964),其同日間測量之準確度介於103.96~109.09%,精密度介於4.28~6.82%,異日間測量之準確度介於102.84~104.59%,精密度介於4.04~12.40%。本研究所使用之D-乳酸去氫酶最適當反應條件為溫度25℃及pH 8.5,在90分鐘內,此D-乳酸生物感測器可測量約10~15個樣品,而用高效液相層析儀(HPLC)則只能測定1個檢品。與現有其他測量D-乳酸方法相比,此D-乳酸生物感測器提供了一個快速、方便且準確的測量新選擇。
Previous studies had reported that D-lactate concentration in serum was related to several diseases, including diabetes, short bowel syndrome, trauma and ischemic sepsis. In our lab, we also found that D-lactate abnormally raise in serum and urine from kidney injured rats, such as chronic diabetes kidney injury and aristolochic acid induced acute nephropathy. It seems that D-lactate might have potential to be a new biomarker of kidney injure. However, the methods for such as D-lactate detect HPLC fluorescence or GC/MS are time-consuming. These methods can’t be applied to deal with large samples. Thus, purpose of this study was to establish a biosensor which can detect D-lactate concentration in a short time.
In our study, we used UV-LED, spectrometer and D-lactate dehydrogenase (D-LDH) to construct this biosensor. D-LDH is the enzyme which can catalyze D-lactate and NAD+ to pyruvate and NADH. NADH has fluorescent characteristic that absorbs excitation light at 340 nm and it releases fluoresce at 491 nm. By detecting the fluorescence intensity, we can find out D-lactate concentration.
Our results show that our D-lactate biosensor has good linearity (R2 = 0.9964) and calibration range (5 μM -150 μM). Accuracy of intra-assay between are 103.96 ~ 109.09%, precision are between 4.28 ~ 6.82%. Accuracy of inter-assay between are 102.84 ~ 104.59%, precision are between 4.04 ~ 12.40%. The appropriate pH and temperature for D-LDH in this experiment is pH 8.5 and 25℃, and the time of reaching steady state is about 30 minutes. It can detect about 10~15 samples in 90 minutes, but the HPLC just can detect 1 sample in 90 minutes. Compared to other detection methods, this new biosensor provides a rapid, accurate and convenient choice.
目 錄 I
附圖目錄 IV
附表目錄 V
中文摘要 VI
英文摘要 VIII
縮寫表 IX
第一章 緒論 1
第一節 乳酸 1
1.1.1乳酸簡介 1
1.1.2生物體內D-lactate來源 2
1.1.3 D-lactate相關疾病 4
第二節 D-Lactate 檢測方法 7
第三節 菸鹼醯胺腺嘌呤二核苷酸 10
第四節 生物感測器簡介 12
第五節 研究目的 13
第二章 實驗材料與方法 14
第一節 實驗試藥及儀器 14
2.1.1實驗試藥 14
2.1.2實驗儀器 14
第二節 實驗方法 16
2.2.1 D-lactate生物感測器的設計 16
2.2.2 實驗條件 20
第三節 實驗參數分析 21
2.3.1 NADH螢光及激發光光譜 21
2.3.2酵素反應時序圖 21
2.3.3溫度差異實驗 21
2.3.4 pH值差異實驗 22
第四節 分析方法之確效 23
2.4.1檢量線線性(linearity) 23
2.4.2準確度試驗(Accuracy) 24
2.4.3精密度試驗 24
2.4.4血清去蛋白處理方法 24
2.4.5大鼠血清測試 25
第三章 實驗結果 26
第一節 實驗參數分析結果 26
3.1.1 NADH激發光、螢光光譜 26
3.1.2 NADH檢量線 28
3.1.3酵素反應時序圖 30
3.1.4溫度差異實驗結果 32
3.1.5 pH值差異實驗結果 34
第二節 分析方法確效之結果 35
3.2.1 檢量線線性(linearity) 35
3.2.2準確度(Accuracy)和精密度(Precision) 37
第三節 大鼠血清測試 39
第四章 討論 41
第一節 D-lactate生物感測器之建立 41
第二節 D-LDH相關探討 42
第三節 D-lactate生物感測器之優點及限制 44
第五章 結論 46
參考文獻 47
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