結合開放式三明治免疫感測和螢光能量轉移技術偵測雙酚A

Abstract

近幾年雙酚A (Bisphenol A ,BPA) 受到世人非常大的關注，因為在我們的日常塑化用品中這個已知的化合物經確認會因使用不當導致刮痕、磨損，於後續高溫加熱、酸鹼、酒精、微波處理或強力清潔劑等作用下，即可能導致雙酚A釋出，而間接隨著食物或飲料進入人體。且已經確認會對人體造成不良影響。但對於雙酚A之定性及定量通常都需要像質譜儀之大型儀器才能準確的進行分析，但此類大型分析儀器昂貴一般民眾不可能購買，送測樣品也需要時間等待，因此我們希望能夠藉由抗體變異區 (scFv) 結合螢光共振能量轉移之技術建立快速篩檢之檢測方法。 實驗設計上我們對以 BPA 為抗原之 heavy-chain variable region (VH) 以及 light-chain variable region (VL) 做了兩種不一樣的修飾進行比較。一.在 VH 及 VL 上鍵結上有機螢光分子；二.在 VH 及 VL 上分別接上螢光蛋白，結合螢光共振能量轉移 (FRET) 之原理與三明治免疫方法來偵測 BPA。此兩種修飾各有其優缺點，一.有機螢光分子消光係數為螢光蛋白的 10 倍，因此發光強度與 FRET 之效率也較好，但在製備上較螢光蛋白繁瑣，二.螢光蛋白之修飾在表現完融合蛋白後即可直接進行檢測並且可以增加蛋白質的水溶性，但缺點為有二具體的現象產生。 檢測上有機螢光分子之修飾並沒有螢光共振能量轉移的現象產生，可能是因為螢光分子距離太遠或是螢光分子鍵結到了 VH 及 VL 的辨識區附近導致蛋白質的辨識能力下降所致，但在螢光融合蛋白的修飾上成功的觀察到螢光共振能量轉移之現象，並且可以偵測到 BPA 的濃度達 10-6 ~ 10-11 M，此濃度區間含括了 2016 年歐盟對於 BPA 之濃度不得高於 10-7 M之限制。

In recent years, bisphenol A (BPA) has received much attention from the world because it has been commercially applied in the production of variety of plastics present in common consumer goods. Under repeated heating, alcohol or microwave treatment, or strong cleaning agent, BPA may be released to groceries and indirectly delivered to the human body. It has been found that BPA can cause adverse effects to health. Unfortunately, the qualitative and quantitative analyzes of BPA usually requires an advanced instruments, expensive and time consuming methods. In this study we present a rapid and cost effective screening method for BPA analysis by the open sandwich immune assay. We performed two different modifications of two BPA -targeted proteins, the heavy-chain variable region (VH) and the light-chain variable region (VL). First, VH and VL were labeled with Rhodamine and Cy5, respectively. Next, we constructed two types of fusion protein, Cypet-fused VH and Ypet-fused VL. To compare these two modification, because of the high extinction coefficient of organic molecule which will have better FRET efficiency and the background value is relatively low but the material preparation is more cumbersome. The second modification of fusion protein has ten time lower extinction coefficient than fluorophore which may cause less efficient FRET and there are dimerization on fluorescence protein. Finally, by applying open sandwich immune assay, we didn’t observe FRET from organic label modification, maybe is because of the donor and acceptor fluorophore are too far to each others or organic molecule labelling to the surrounding of antigen-binding complementary determining regions. But we successfully observed the phenomenon of fluorescence resonance energy transfer on fusion protein modification, which enabled us to quantitatively analyze BPA. The mixtures of Cypet-VH, BPA and Ypet-VL with various concentration of BPA exhibited an emission spectroscopic change at maximum 527 nm. The sensitivity of BPA detection can be reasonably estimated to the level of 0.1 nM.