牛乳房炎是乳牛乳房內的傳染病，通常會造成牛乳房紅腫硬化，並會降低牛乳的品質以及產量，是一種造成畜牧業年損失最大的疾病。目前檢測牛乳房炎的方式，例如：物理檢測、美國加州福尼亞檢測、電導度方法等等。然而近幾年來，紙基微流體裝置（µPAD）受到各界廣泛的關注，因為它具有簡單、可攜帶、可拋棄以及低成本的特性，可用於低成本的化學、生物及環境檢測等等。再者因為樣本與試劑不須使用機械或電子設備，單純以毛細現象即可進行輸送，降低了許多設備成本，而使其更容易作為檢測的裝置。目前已經發展出許多不同製備紙基微流體裝置的方法，例如：噴墨印刷法(inkjet printing)、紙張切割法(craft cutting)，以及光微影法(photolithography)等多種方法。在本研究中，我們透過結合酵素免疫分析法（ELISA）以及紙基微流體裝置研發一種新的紙基酵素免疫分析法（p-ELISA）來檢測牛乳房炎。這種方法首先需要在濾紙上壓出圖形，並塗上一層蠟，於加熱板加熱即可完成晶片，再利用酵素免疫分析法的間接法檢測牛乳房炎蛋白。實驗中將研究的操作條件，有抗體和抗原的濃度、用量，沖洗未標定抗體的用量及次數，以及檢測的色彩強度分析等。實驗結果顯示清洗次數及劑量會影響未專一性鍵結抗體所造成的訊號干擾，然而在抗體濃度方面，二抗濃度太高時，容易造成背景干擾，二抗濃度太低時，則會影響鍵接一抗的效果，而一抗使用濃度會影響抗原和二抗與其結合的數量。使用一抗濃度為2µg/mL，二抗濃度為0.05µg/mL，且每次清洗為10µL洗五次時，檢測PBS緩衝溶液中的Cathelicidin蛋白，在0.05~5µg/mL之間色彩強度會呈一線性關係，檢測最低濃度為0.05µg/mL。若使用牛乳直接進行檢測，則檢測最低濃度為0.5µg/mL。

Bovine mastitis is an intramammary infection disease in dairy cows, which usually causes udder swelling, redness, hardness, and even reduction in milk quantity. In recent years, microfluidic paper-based analytical device (µPAD) has been attracting attention because it is simple, inexpensive and power-free for fluid transport. In this study, we intended to establish a new approach to detect bovine mastitis by constructing a paper-based enzyme-linked immunosorbent assay (p-ELISA). The results showed that when using a primary antibody concentration of 2µg/mL, a secondary antibody concentration of 0.05µg/mL, and each wash with 10µL for five times, there existed a linear relationship between the color intensity and the concentration of Cathelicidin protein in PBS solution ranging from 0.05~5µg/mL. The detection limit was 0.05µg/mL. When commercial milk is used for detection, the detection limit was found out to be 0.5µg/mL.

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