開發三維對焦平行流道於高效能流式細胞儀

Abstract

本研究開發一個快速、高通量(high-throughput)、且低價位之新型微米流式細胞儀(micro-fluidic cytometer)。流式細胞儀已有70餘年歷史，但缺點在於目前商轉的細胞儀不但昂貴、體積大、且檢測速度較慢。 爲了達到高通量的生物檢測，我們利用支狀串列微流道(cascade microchannels)，將樣本流道分爲 32條平行流道，可使檢測效率提高32倍；但實驗證明此構型會造成檢體在流道中位置不一，增加後端檢測困難。因此，我們再導入三維流體對焦方法改善，我們摒棄一般常用於微米流式細胞儀的幾何及純慣性流(inertial flow)對焦，原因爲幾何對焦有流速限制，無法達到高通量；純慣性流對焦須應用高速流場，細胞在長時間受力的情況下有不良影響，活性生物實驗皆不傾向此法。因此我們設計三維流道，利用檢體本身環境溶液流動對其進行流體對焦，利用一側向流(sheath flow)對檢體進行平面對焦(x-y平面)，再以一垂直流進行環狀對焦(y-z平面)，可使檢體在通過檢測端時固定位置，以利後端檢測。檢測端有32顆埋入PDMS的微球型透鏡陣列(micro-ball lens array)，分別對應到32條支流，形成的短焦距可使系統之N.A.値提升，縮減整體晶片體積。本實驗針對亮度(intensity)一致的螢光小球(flourescent microsphere)和標記螢光的老鼠巨噬細胞(Raw)進行細胞計數，計數方法爲利用激發光激發檢體，再以高速攝影機收集檢體螢光，最後對照片分析檢體通過檢測端時之亮度，以亮度判斷流體對焦程度。 結果顯示本系統可使螢光小球完全對焦，且針對密度爲5xl08 (cell/ml)之Raw可達到每秒140萬顆的高通量。

In this thesis, we have successfully developed a novel micro-fluidic cytometer, which has advantages of rapid, high-throughput and low cost. Cytometer has existed in the industry for more than 70 years, but the current cytometers are expensive, large and low efficiency. To reach high-throughput, we made 32 cascade sample channels to achieve 32 times efficiency. However, it will cause biopsy floating around the channel that makes following detection harder, therefore, we apply 3D fluid focusing to solve this problem. Our design is different from geometry cytometer which can not reach high-throughput; and the inertial cytometer which is an unfriendly-environment to cells. Thus, we design 3D channels filled with environmental solution of biopsy to fluid focus, which means that x-y plane focusing by sheath flow and y-z plane focusing by vertical flow encircling the biopsy, circumscribes the position of biopsy for following detection. In order to enhance N.A. of this system, 32 micro-ball lens was embedded under 32 channels, which can shorten the focus length for compressing the volume of chip. We demonstrate highly focusing by counting fluorescent microsphere which has identical intensity, and demonstrate high throughput by counting labeled cells (Raw). We count the biopsy by exciting it by laser and collecting its fluorescence when it passing through micro-ball lens array. Then analyze the intensity of fluorescence which determines the degree of fluid focusing. In summary, the system could absolutely confines fluorescent microsphere in the focusing area, and it could reach 140 million cell/s by detecting Raw cell of 5x108 cell/ml concentration.