開發微流體紙基檢測片以分析水中的鈣及鎂離子濃度

現今科技日新月異，隨著人們生活品質的提升，工業化程度亦逐漸提高，水汙染儼然成為需要正視的課題。而水源的好壞除影響農作物生長及糧食生產外，雜質存在水中與否也會影響工業生產效率。飲用水中存在的礦物質以鈣與鎂離子為主，常造成水質硬化，而硬度高的水除了對人體健康產生影響外，亦會影響到食物的品質。根據行政院環境保護署公告之飲用水水質標準，總硬度最大限值為300 mg/L。國外文獻指出，歐盟提出飲用水中礦物質推薦濃度，鈣離子為40-80 mg/L，鎂離子為20-40 mg/L，因此針對不同地區飲用水礦物質含量檢測及風險評估非常重要。傳統儀器檢驗設備不利於現地飲用水水質檢測，故開發迅速、高效率、能源損耗率低的方法為現今迫切需改善的課題。目前常見的水質快速檢測方法多僅能檢測水的總硬度，無法分別測定鈣或鎂離子的含量。利用不同濃度之鈣及鎂離子與呈色試劑在鹼性下具有化學呈色反應的原理，以分光光度法進行定量檢測，並分析試劑與樣品呈色之最適化濃度，以利後續開發微流體紙基檢測片，達成快速分析的目的。透過紫外-可見光光譜 (Ultraviolet-visible spectroscopy, UV-Vis) 於波長473-509 nm間，不同濃度 (6-200 ppm) 的鈣離子在微鹼性下與試劑反應後可得到良好線性及迴歸方程式，y = 0.0006x + 0.0302，R² = 0.998，偵測極限可達6 ppm；另於波長560-574 nm間，結合兩種偶氮化合物與不同濃度 (3-20 ppm及20-200 ppm) 的鎂離子，亦獲得相當好的線性及迴歸方程式，y = 0.001x + 0.0024，R² = 0.9967及y = 0.0004x + 0.0163，R² = 0.9922，偵測極限可達3 ppm。再利用此呈色方法針對水中其他常見離子進行檢測，發現可以排除其他離子的干擾。紙基檢測片之開發中，選用擴散性及滲透性較佳的Whatman NO.41濾紙做為檢測片的基底材料，設計微流體通道、試劑反應區及樣品注入區，使用3D列印技術，在濾紙上藉由光固化樹脂以建構疏水性屏障，製備單層紙基檢測片。將1.5 μl的呈色劑及掩蔽劑事先滴入於檢測區，再滴入4 μl之待測標準液或樣品，經化學反應後掃描紀錄呈色情形，再利用Image J軟體調整最適化之閾值及分析像素面積值，繪製標準檢量線。鈣離子標準檢液於6-200 ppm的迴歸方程式為y = 641.41ln(x) + 7423.1，R² = 0.9827，偵測極限可達6 ppm；而鎂離子標準檢液於3-200 ppm的迴歸方程式為y = 612.94ln(x) + 8484，R² = 0.9946，偵測極限為3 ppm。配製混合標準檢液並代入上述迴歸方程式，驗證鈣及鎂離子可同時分析的潛力。將鈣及鎂離子之檢量線所運用的配製方法應用於感應耦合電漿發射光譜儀檢測，回收率均可達90 % 以上。真實水樣品的檢測與驗證方面，分別採用分光光度法、紙基檢測法，與委託屏科大檢驗中心及中央畜產會檢驗中心採用公告檢驗方法比對。結果發現分光光度法檢測結果不穩定，易產生較大的誤差；而紙基檢測法與公告檢驗方法比較具有較高的相符度，顯示相對誤差落於10 % 以下，且具有半定量的效果。由以上研究結果可得知，鈣及鎂離子運用紙基檢測，經由提高重複試驗的次數，可使絡合呈色反應穩定進行，且能降低試劑與樣品的添加量，以及縮短檢測時間。採用3D列印技術印製之晶片具有客製化、高精度及成本相較儀器檢驗方法便宜的優點，因此即時性與便捷性提高，未來可發展作為環境或食品分析領域中樣品的快篩檢測的一項便捷工具。

關鍵字

微流體紙基裝置；水質；鈣離子；鎂離子；比色法

並列摘要

With the rapid advancement of technology and the improvement of life quality, the industrialization has also gradually increased, hence water pollution has become an issue that needs to be addressed. Water source not only affect the growth of crops and food production, the presence of impurities also affects the efficiency of industrial production. The minerals present in drinking water are mainly calcium and magnesium ions, which often cause water quality hardening. Water with high hardness will not only affect human health but also affect the quality of food. According to the drinking water quality standard announced by the Environmental Protection Agency of the Executive Yuan, the maximum limit of total hardness is 300 mg/L. Literature pointed out that the European Union proposes the recommended concentration of minerals in drinking water, calcium ion is 40-80 mg/L, magnesium ion is 20-40 mg/L, so it is very important to detect the mineral contents and assess risks of the drinking water in different regions. Traditional instrument detection equipment is not suitable for on-site drinking water quality detection, so the development of rapid, high-efficiency and low-energy loss method is an urgent subject that needs to be improved today. At present, most of the common water quality rapid detection methods can only detect the total hardness of water, and cannot determine the content of calcium or magnesium ions respectively. Using the principle that different concentrations of calcium and magnesium ions, would have a chemical color reaction under alkaline conditions, which allows to perform quantitative detection by spectrophotometry. The optimal concentration of reagent and sample of color reaction were investigated to facilitate the subsequent development of microfluidics paper-based chips, and achieving the purpose of rapid analysis. Through ultraviolet-visible spectroscopy (UV-Vis) between 473-509 nm, different concentrations (6-200 ppm) of calcium ions reacting with the reagent under sub alkaline condition, can obtain a good linear and regression equation, y = 0.0006x + 0.0302, R² = 0.998, and the detection limit can reach 6 ppm. For magnesium ions with different concentrations (3-20 ppm and 20-200 ppm), exhibited absorbance at 560-574 nm wavelengths, after reacting with two kinds of diazo compound, also obtain good linearity and regression equations, y = 0.001x + 0.0024, R² = 0.9967 and y = 0.0004x + 0.0163, R² = 0.9922, respectively. The detection limit for magnesium is down to 3 ppm. This coloration method is then used to detect other common ions in water, and it is found that the interference of other ions can be excluded. In the development of the paper-based chips, Whatman NO.41 filter paper that has better diffusivity and permeability is selected as the base material of the paper-based chips. The microfluidic channel, the reagent reaction zone, and the sample injection zone were all designed on this chip. By utilizing 3D printing technology, a single-layer paper-based chip is prepared by constructing a hydrophobic barrier on the filter paper with light-curing resin. First, dropped 1.5 μl of the color agents and masking agents onto the detection zone, then 4 μl of the standard solution or sample to be tested were added. After the chemical reaction, scanned and recorded the color rendering condition, then use the Image J software to adjust the optimal threshold and analyze area intensity in pixels, finally draw standard calibration curves. The regression equation of the calcium ion standard solution at 6-200 ppm is y = 641.41ln(x) + 7423.1, R² = 0.9827, and the detection limit can reach 6 ppm. The regression equation of the magnesium ion standard solution at 3-200 ppm is y = 612.94ln(x) + 8484, R² = 0.9946, and the detection limit is 3 ppm. Prepare mixed standard solution and bring it into the above regression equation to verify the potential of simultaneous analysis of calcium and magnesium ions. The preparation method used in the calibration curves of calcium and magnesium ions is applied to the detection by inductively couple plasma optical emission spectrometer (ICP-OES), and the recovery rate can reach higher than 90%. In term of detection and verification of real water samples, spectrophotometric method and paper-based detection method are utilized respectively, also compared with the results from authentic inspection method conducted by Center for Agricultural and Apuarcultural Product Inspection and Certification (CAAPIC) and accredited laboratory in National animal industry foundation (NAIF). The results showed that the detection results of the spectrophotometry are unstable and prone to large errors; while the paper-based detection method has a higher degree of conformity with the authentic inspection method, indicating relative error value below 10 % and satisfactory semi-quantitative results. From the above results, it is concluded that the paper-based detection of calcium and magnesium ions, could be stabilized and realized by increasing the number of the repeated tests, and could reduce the amount of reagents and samples consumed, and shorten the detection time. Paper-based chips printed with 3D printing technology have the advantages of customization, high precision, and lower cost than instrument inspection methods, so the immediacy and convenience are thus improved. In the future, it can be a convenient tool and developed as a rapid screening tool for samples in the field of environmental or food analysis.