開發檢測赭麴毒素A的分析方法

Abstract

由青黴菌與麴菌所分泌的二次代謝物-赭麴毒素A (Ochratoxin A, OTA)常存在於穀製食品中，當人體食入後有可能會造成器官、組織異常、腎病變等問題。由於傳統的分析方法需要耗費大量的時間及成本在檢測以及樣品的前處理上，因此本論文發展出一種簡單方便且成本低廉的快速分析OTA的方法。即是利用人類血清蛋白(Human serum albumin, HSA)與OTA具有非常好的結合能力，將其修飾在磁性奈米粒子及光纖表面做為濃縮OTA的探針。 本論文的第一部份是將HSA以共價鍵結方式修飾於磁性奈米粒子表面上，利用此官能化的磁性奈米粒子萃取水溶液中的OTA，從實驗結果發現直接進樣質譜分析的OTA (10-6 M)在m/z 402的分子離子訊號峰強度很弱，但是經由磁性奈米粒子探針萃取OTA後可觀察到增強的OTA分子離子峰，證實此探針具有萃取濃縮OTA的效果。實驗中也加入Bradykinin做為定量之內標準品，透過本實驗室自行開發的無鞘流界面可將已被HSA官能化磁性奈米粒子所濃縮的OTA樣品溶液導入離子阱質譜儀進行分析，可藉由選擇離子監測模式觀察到OTA(m/z 402)以及Bradykinin(m/z 1059)之圖譜，將選擇的此二離子峰積分面積相除得一比值，在不同濃度OTA的分析下所得到的比值對OTA濃度做圖可以得到一檢量線，可應用於酒樣中OTA之定量分析，偵測極限約在10-8 M左右。 而本論文的第二部份是利用修飾有HSA的光纖做為萃取OTA的探針，並且結合OTA具有螢光放光的特性，可以在濃縮OTA至光纖探針後直接以螢光光譜法進行分析。實驗中我們以三種與OTA結構類似的胺基酸進行測試，結果發現此光纖探針可以專一辨識OTA，並且可透過微波加熱加速萃取的方法縮短萃取時間至20 秒，而偵測極限為10-8 M左右，實驗也證明此方法可應用於酒樣中OTA之檢測。 本論文發展出兩種可進行親和濃縮OTA的探針，並且分別結合電噴灑質譜法以及螢光光譜法為偵測方法，這兩種方法具有快速、簡單方便、且成本低廉等優點。

Ochratoxin A (OTA), which is toxic and commonly found in grain, is a secondary metabolite generated from several strains of Penicillium and Aspergillus. Conventional analytical methods for OTA are time-consuming and expensive. In this dissertation, simplified and cost-effective methods for detection of OTA were proposed. It has been demonstrated that human serum albumin (HSA) has good affinity with OTA. Thus, this work demonstrated the feasibility of using HSA immobilized magnetic nanoparticles and optical fibers as the affinity probes for selectively trapping OTA from aqueous solutions. In the first part of this dissertation, HSA was covalently bound onto the surfaces of magnetic nanoparticles. The generated nanoparticles were then used to trap OTA, followed by direct introduction of the samples into an electrospray ionization (ESI) mass spectrometer. The results showed that the intensity of the deprotonated OTA molecular ion at m/z 402 was greatly improved comparing with that was performed prior to the concentration steps. The results shows that this affinity probes have the capacity to trap OTA from aqueous samples. When performing quantitative analysis, bradykinin ([M-H]－=1059) was added in the sample for being used as the internal standard. A calibration curve was obtained by plotting the ratio of the peak area at m/z 402 to m/z 1059 obtained from selected ion monitoring (SIM) results as a function of the concentraiton of OTA. The concentraion of OTA in alcoholic samples can be estimated based on the calibration curve. The detection limit for OTA using this appraoch is as low as 10-8 M. Additionally, HSA modified optical fibers were used as the probes for selectively trapping OTA from aquesous samples, followed by directly placing the HSA-fiber conjugated with target species into a fluorophotometer for luminescence detection. The results show that the HSA-fiber has specific-trapping capacity for OTA. Under microwave-heating for only 20 s, OTA in sufficient amounts for lumiescence detection can be enriched onto the fiber. Furthermore, the detection limit is as low as 10-8 M. This approach also has been empolyed to determine OTA in alcoholic samples. This work has demonstrated that the two affintiy approaches combined with either ESI MS analysis or luminescence detection have the advantages including short-analysis time, simplicity, and low cost. These two apparoches may have potential to be used for rapidly screening OTA in a real-world application after further improvement.