利用酵素連續催化顯色反應偵測酪胺酸及酪胺酸酶活性

Abstract

酪胺酸是一種芳香族胺基酸，是合成兒茶酚胺類和黑色素的重要前驅物。酪胺酸的代謝紊亂可能會導致生物流體中的酪胺酸濃度水平異常，故酪胺酸為檢測疾病的重要生物標記物。酪胺酸酶是一種含銅氧化酶，在酪胺酸代謝生成黑色素的路徑中，酪胺酸酶是重要的限速酶，而酪胺酸酶的異常表達，也可能造成黑色素瘤癌等疾病，因此能利用簡單的方法偵測酪胺酸及酪胺酸酶活性，具有重要意義。在這項研究中，我們開發了酵素連續催化之比色法生物感測器，利用酪胺酸在酪胺酸酶和4,5-多巴雙加氧酶 (DOD) 的催化反應下，所生成的黃色甜菜醛胺酸和具有紅螢光的甜菜色素作為輸出訊號，選擇性偵測酪胺酸及酪胺酸酶活性。本實驗所開發出的系統對酪胺酸及酪胺酸酶濃度具有良好的線性關係 (酪胺酸：R=0.998；酪胺酸酶：R=0.994) 及偵測極限 (酪胺酸：1.102 μM；酪胺酸酶：0.072 U/mL)，也成功利用標準添加法應用於偵測真實樣品胎牛血清及人工尿液中的酪胺酸和酪胺酸酶活性，且得到良好的準確性。此外，透過麴酸作為抑制劑模型測試，證實此感測器也可應用於篩選出酪胺酸酶的潛在抑制劑。Tyrosine, an aromatic amino acid, is a vital precursor for the synthesis of melanin and catecholamines, including L-DOPA and dopamine. Metabolic disorders associated with tyrosine could lead to abnormal tyrosine levels in biological fluids. As a result, tyrosine is also an essential biomarker for the detection of some diseases such as alkaptonuria and tyrosinemia. Tyrosinase, a copper-containing oxidase, is the critical rate-limiting enzyme for the synthesis of melanin. It is reported that melanoma cancer is related to the aberrant expression of tyrosinase. Thus, it is important and necessary to develop an easy quantification assay of tyrosine and tyrosinase activity. Tyrosinase could hydroxylate tyrosine to L-DOPA, which is the substrate of 4,5-dopa dioxygenase (DOD). DOD could convert L-DOPA to betalmic acid, the precursor of betalains including betaxanthins and betacyanins. In this study, we have developed the enzyme cascade-triggered colorimetric biosensor based on the formation of yellow betalamic acid and red fluorescent betaxanthin for the detection of tyrosine and tyrosinase activity. The performance of the proposed biosensor responds good specificity and sensitivity to tyrosine and tyrosinase activity and even the various concentrations of tyrosine spiked in fetal bovine serum and artificial urine. In addition, this biosensor can also be used to screen the inhibitor of tyrosinase.