開發以多巴雙加氧酶為基礎的左旋多巴生物感測器

Abstract

左旋多巴是一種兒茶酚胺類的神經傳遞物質，為目前治療帕金森氏症最主要的藥物，因此監測血清和尿液中的左旋多巴對帕金森氏症患者非常重要。在這項研究中我們開發兩種方法檢測左旋多巴，在第一部分的研究中，提出了一種低成本的氧化鋅試紙檢測平台，提高檢測左旋多巴濃度的便利性，使用微波輔助水熱法在90分鐘內就可在試紙上合成氧化鋅奈米柱，氧化鋅可以放大綠色螢光訊號強度，增強對左旋多巴檢測的靈敏度，在475 nm的激發波長下測量537 nm放射波長的螢光訊號，與未經修飾的試紙相比，氧化鋅試紙生物感測器綠色螢光強度值增加了約3倍，檢測左旋多巴的線性區間為25-2000 nM，檢測極限值為24 nM，正常人血液中左旋多巴濃度約30-40 nM，氧化鋅試紙的檢測區間適合用於檢測正常人體中的左旋多巴濃度。在第二部分的研究中開發了一種新型的生物感測器，左旋多巴在SsDDO酵素催化下生成黃色的CHAPCA色素，在414 nm具有特徵吸收峰，只需10 min即可透過CHAPCA的生成對左旋多巴做劑量表現之吸收值測試，檢測區間為1-125 μM，偵測極限為2.3 μM，而帕金森氏症患者服藥後的濃度區間約1.26-12.27 μM，此檢測方法可以滿足監測帕金森病患者服藥後體內L-DOPA濃度的臨床要求。本實驗應用SsDDO酵素開發序列反應分析方法，對樣品中共存的多巴胺和左旋多巴做及時偵測。在這項研究中，所開發出的系統對左旋多巴檢測具有簡單、快速、靈敏且選擇性高等優點。我們也成功將酵素系統應用於胎牛血清樣品中偵測L-DOPA，證明其穩定性和適用性。L-DOPA is a precursor of catecholamine neurotransmitter dopamine, and is used to treat Parkinson's disease. Therefore, monitoring L-DOPA concentration in serum and urine is important for patients with Parkinson's disease. We proposed two approaches as part I and II of thesis. In the first part, ZnO nanorods (ZnO-NRs) were synthesized on paper-based device in less than 90 min using a microwave-assisted hydrothermal method. The ZnO-NRs amplify green fluorescence signals to enhance the detection and sensitivity of L-DOPA, best measured at excitation/emission wavelengths of 475/537 nm. We systematically characterized the effect of reaction conditions on the corresponding fluorescence enhancements. The proposed ZnO NRs-paper biosensor presented a 3-fold increase in green fluorescence compared to unmodified papers. The linear range of detection for L-DOPA was 25–2000 nM, with a limit of detection of 24 nM, which meets the clinical requirements for monitoring L-DOPA in normal persons ranges from 30 to 40 nM. The second part, L-DOPA was catalyzed by the SsDDO enzyme and converted into CHAPCA (λmax absorption at 414 nm). It only takes 10 min to detect the absorption value through the generation of CHAPCA. The linear range of detection for L-DOPA was 1–125 μM, with a limit of detection of 2.3 μM, which meets Parkinson's disease patients on L-DOPA therapy ranges from 1.26 to 12.27 μM. The SsDDO enzyme is used to develop a sequencial reaction analysis. It can be used to detect the coexisting of DA and L-DOPA in the samples. We also validate the performance of this system in fetal bovine serum, confirming its robustness and applicability.