建構產生定量包覆式液滴之通用數位微流體平台與其應用於蛋白質結晶與液態萃取之研究

Abstract

包覆式液滴的好處有改善流體操控性、降低生物體的沾黏、減少揮發與 簡化封裝，然而，以傳統的滴定方式來產生可調控油水比例體積的包覆式 液滴是困難的，甚至於將包覆在外層的油殼去除，也是難以實現。在此， 我們提出一個可以分別操控水與油的平行板元件來達成三個目標，首先， 利用介電濕潤與介電泳分別產生25 nl 水滴與 2.5 nl 油滴，其體積可以精準 的控制，並使兩不相溶的液體接觸自然低形成包覆式液滴，在此使用的油 滴為10,100,1000 cSt 的矽油，且水與油的比例保持在10 : 1 與2 : 1。第二目 標為包覆式液滴的驅動電壓、揮發測試與蛋白質防沾黏的量測，比較水在 空氣的環境與在油的環境中，我們發現黏滯係數較低的矽油包覆著水有較 小的驅動電壓。對於動態揮發的測試，在溫度 20 ± 1 ℃與相對濕度60 ± 3%的環境中且包覆式液滴的移動速率為25 mm s-1，當水油比例為10 : 1 時， 在22 分鐘後，其水的體積損失了16.6%。而水油比例為2 : 1，35 分鐘後， 水的體積損失了17.5%；而對於沒有油包覆的水滴，6 分鐘內，其體積損失 則為11.4%。而在靜態揮發實驗中，若精準的控制水在油殼中的位置，則揮 發速率可以降低到每分鐘0.04%。在蛋白質沾黏的實驗中，在5 □g ml-1 有 螢光標記的牛血清蛋白的水溶液，當外層包覆著一層油殼時，則可以大幅 地降低沾黏的現象。第三目標為去除油殼，我們在晶片上利用毛細力控制 正己烷，使其形成一個洗滌槽，藉由己烷溶矽油而不溶水的特性，達到去 除油殼的目的。除此之外，藉由產生可調控油水比例的包覆式液滴且控制 揮發速率的特性，我們提供了兩種生醫與化學的應用在此平台上，在蛋白 質結晶應用上，將250 nl 含有6% 溶解酶蛋白的液滴包覆著一層250 nl 的 油殼，藉由控制揮發，晶體從成核到成長，約需要80 分鐘，晶體的尺寸即 可超過100 □m。在萃取應用上，利用調整油水比例的特性可提供更有彈性 且可程式化的數位微流體之液態萃取，並以環狀路線來移動包覆式液滴與 將核心液滴分散在油殼的方式來增加萃取後的濃度。對於0.1 mM Rhodamince 6G 在25 nl 水滴，外層包覆著25 nl 的正辛醇，萃取後，經過200 秒，其將水滴分散到油殼的方式之辛醇濃度相較於移動包覆式液滴的方 式提高兩倍。而萃取後，其濃度可以藉由不同的油水比例來控制。另外， 萃取後的包覆式液滴則可以利用介電濕潤操控介電泳，使核心水與油殼完 全的分開。

A water-core and oil-shell encapsulated droplet exhibits several advantages including enhanced fluidic manipulation, reduced biofouling, decreased evaporation, and simplified device packaging. However, obtaining the encapsulated droplet with an adjustable water-to-oil volume ratio and a further removable oil shell is not possible by reported techniques using manual pipetting or droplet splitting. We report a parallel-plate device capable of generation, encapsulation, rinsing, and emersion of water and/or oil droplets to achieve three major aims. The first aim of our experiments was to form encapsulated droplets by merging electrowetting-driven water droplets and dielectrophoresis-actuated oil droplets whose volumes were precisely controlled. 25 nL water droplets and 2.5 nL non-volatile silicone oil droplets with various viscosities (10, 100, and 1000 cSt) were individually created from their reservoirs to form encapsulated droplets holding different water-to-oil volume ratios of 10 : 1 and 2 : 1. Secondly, the driving voltages, evaporation rates, and biofouling of the precise encapsulated droplets were measured. Compared with the bare and immersed droplets, we found the encapsulated droplets (oil shells with lower viscosities and larger volumes) were driven at a smaller voltage or for a wider velocity range. In the dynamic evaporation tests, at a temperature of 20 ± 1 ℃ and relative humidity of 60 ± 3%, 10 cSt 10 : 1 and 2 : 1 encapsulated droplets were moved at the velocity of 0.25 mm s-1 for 22 and 35 min until losing 16.6 and 17.5% water, respectively, while bare droplets followed the driving signal for only 6 min when 11.4% water was lost. Evaporation was further diminished at the rate of 0.04% min-1 for a carefully positioned stationary encapsulated droplet. Biofouling of 5 mg ml-1 FITC-BSA solution was found to be eliminated by the encapsulated droplet from the fluorescent images. The third aim of our research was to remove the oil shell by dissolving it in an on-chip rinsing reservoir containing hexane. After emersion from the rinsing reservoir, the bare droplet was restored as hexane rapidly evaporated. Removal of the oil shell would not only increase the evaporation of the core droplet when necessary, but also enhance the signal-to-noise ratio in the following detection steps. In addition, we demostrated biomedical and chemical of this platform. In the application of protein crystallization, a 250 nl aqueous droplet containing 6% lysozyme protein was encapsulated by a 250 nl 10 cSt silicone oil. With tunable evaporation rates of the core aqueous droplet, 100 um lysozyme crystals were obtained in 80 min. In the extraction application, various liquid manipulating schemes and adjustable water-to-oil volume ratios offer flexible and programble digital microfluidic liquid-liquid extraction. By looping and dispersing encapsulated droplets containing a 25 nl water core and a 25 nl octanol shell, we also investigated extraction efficiency. A 25 nl water core whose original concentration of Rhoamine 6G was 0.1 mM. Within a (200 s) extraction time, the concentration of extracted Rhodamine 6G in the 25 nl octanol shell through dispersing method was two times higher than that through looping method. The R6G concentration in octanol after extraction was tunable by changing different water-to-oil volume ratios. In addition, the core and shell droplets would be separated after the extraction process.