以介電泳與微流方式分離生物微粒

Abstract

本文利用負介電泳力研究懸浮在流動液體中微粒的抓取及分離。介電泳力可藉對微流道底壁上兩片相距20μm的電極通上電壓相同但相位差180°的交流電產生。實驗顯示當微粒靠近電極狹縫 (稱DEP閘)時，會受到負介電泳力作用而抬升、且足夠的負介電泳力可抵抗載運流體對微粒的黏滯拖曳力(簡稱拖曳力)，將微粒攔截在DEP閘前。先前文獻主在尋求在一給定裝置下，可以攔截已知單一微粒(或稀薄狀况下)的臨界操作條件(給定電壓下的最大流速、或給定流速下的最小電壓)。但在實際情況中，隨著被攔截下來微粒量的增加，微粒間間隙縮小，相對地間隙間的流速增加，而提升了拖曳力；且先前被攔截下來的微粒也會承受後續微粒的撞擊。二者之綜合效應會使已被攔截的微粒脫離DEP閘，而使上述臨界操作條件失效。本文即針對此點提出承載量(定義為阻擋下的微粒達穩態時的數量)的觀念，以作為相關系統設計及性能量化比較的依據。 就直流道搭配直線型電極狹縫的設計，當頻率1MHz、 峰對峰電壓7Vpp、及流速156μm/s時，於乾淨溶液內每200μm的平均承載量可達15.7顆人體結直腸癌細胞(Colo205，約15~20μm)。利用此系統進行Colo205與大腸桿菌的分離實驗，結果顯示我們可有效地攔截Colo205、而同時讓大腸桿菌通過DEP閘。顯示若糞便檢體經此前處理後，將可有效地提升糞便DNA檢驗的準確率(未經處理約50%)。 在同樣的操作條件下，圓弧型電極狹縫系統的Colo205承載量較上述直線型電極狹縫者高(1MHz及7Vpp時可增至267%)；而直線型電極狹縫搭配雙層流道設計的系統則更可提升至367%。就圓弧型電極狹縫設計而言，其目的為提升負介電泳力的有效分量，以提升承載量；而在雙層流道的部分則是利用所產生的低速區和尖端電場集中效應來提升承載量。 就以介電泳作不同微粒分離的文獻中，因介電泳力與粒徑三次方成正比，故大都利用粒徑的差異來作分離。就相近大小的不同微粒，以介電泳作分離並不容易。對此我們利用柯莫氏因子的差異，以單層流道結合直線型電極狹縫的系統，成功地分離Colo205細胞與其尺寸相近(15±0.15μm)的聚乙烯微粒，其操作參數為1MHz、6Vpp、及流速468~624μm/s。

Trapping and separation of particles entrained in a moving liquid stream were studied in this thesis via negative dielectrophoresis (nDEP). The nDEP force can be generated by applying ac voltage with 180 degree phase shift at two electrodes (separated by a 20 μm gap) built on the bottom wall of a straight micro channel. When a particle approaches the location of the electrode gap (called the DEP gate), it will be lifted by the negative DEP force, and held before the DEP gate if the negative DEP force is suitably designed such that it can resist the fluid drag. Much effort in previous literature aims to determine the critical condition (the maximum background flow rate at a given applied voltage, or the minimum applied voltage at a given flow rate) for a single particle (or rare particles) in a given microfluidic system. In practical applications, the flow through area decreases, and thus the fluid drag increases, as more particles are trapped at the DEP gate. Furthermore, the succeeding particles may impact on the previously trapped particles. Sooner or later, the trapped particles may gather enough momentum to cross the DEP gate, and thus the critical condition for single particle (or rare particles) fails to apply. The present thesis thus introduce the concept of capacity (define as the average number of particles trapped at the DEP gate when the system reaches its quasi steady state), and propose that the capacity as a suitable indicator for quantifying the performance of the related micro system with DEP gates. The capacity of colorectal cancer cells (Colo205, 15~20 μm) was measured in a system with a straight electrode gap. It equals 15.7 cells per 200 μm channel width at 1 MHz frequency, 7 Vpp (peak-to-peak-voltage) and 156μm/s in flow rate. Separation between Colo205 cells and E. Coli was also performed using such a system: the Colo205 cells were blocked, with E. Coli past through the DEP gate. This suggests that the precision for the current Stool DNA test (less than 50% now) can be enhanced if the sample is pre-treated using the devices with DEP gate. It was found that the device with curved electrode gap performs better for particle trapping over straight electrode gap, and up to 267% times when it is operated at 1MHz and 7Vpp with an average background flow speed at 156μm/s. The capacity can be further increased up to 367% for a two-step channel design. The negative DEP force possesses a larger component resisting the background flow for the curved electrodes, and thus the capacity is increased. The capacity of the two-step channel is enhanced because of the physical blockage as well as the additional DEP force associated with the intensified electric field generated at the corner of the flow geometry. In literature, particle separation using DEP relies mainly on the particle size as the DEP force depends on the cubic power of its size. It is not easy to separate negative DEP particles with similar sizes. In this thesis, we have separated successfully the Colo205 cells and the polystyrene particles (at 15±0.15μm) based on their differences in Clausius-Mossotti factor using the straight electrode gap device. The operation parameters were 1MHz, 6V_pp and 468~624μm/s in flow speed.