胡文聰臺灣大學：應用力學研究所賴彥勳Lai, Yen-HsunYen-HsunLai2007-11-292018-06-292007-11-292018-06-292004http://ntur.lib.ntu.edu.tw//handle/246246/62400本論文設計與製造的微細胞計數器，原理是利用細胞通過微孔洞時產生電阻抗變化，由量測訊號可得知細胞數目與大小。論文中提及兩種加工方法：第一種是利用微加工製程中的軟微影技術，與拉伸的玻璃毛細管，整合PDMS 和經裁切過後的毛細管尖端製作細胞計數平台；第二種方法為翻模的PDMS 流道與拉伸的玻璃毛細管前端整合。 此方式的好處是可利用較廉價的PDMS，和容易製作且具特定內徑的玻璃拉伸毛細管，製作出可以計數細胞的感測原件。 透過四點量測法量得電位差變化，透過適當的放大電路與數位濾波器計算，利用Labview 量測訊號波數與訊號強度並計算細胞數目與大小。 實驗包含了電訊號的校正，進樣品包含高分子微粒與紅、白血球的計數，並討論不同尺寸的微流道與細胞尺寸的訊號強度變化；在結果方面，對原始擷取訊號波形進行濾波與訊號處理，透過理論與數據分析反求進樣品粒徑大小，並透過統計圖來觀察樣品中的粒徑分佈。 兩種製作方式讓我們在一般設備下，提供了快速的計數器感測區製作流程，而不同尺寸的玻璃毛細管替換也更具製作彈性。實驗結果顯示成功計數了紅、白血球，而適當的接地與訊號解析將幫助我們得到更精確的結果；此平台提供了不錯的細胞數目與粒徑大小參考值，而此一簡易製成讓我們在生物微流體晶片的結合上將更方便。The thesis designs and fabricates a micro-coulter counter in sensing voltage pulse when a cell traveling through the aperture. And from signal, cell number and size information can be obtained. Two ways of fabrication are presented. First, using soft-lithography and pulled pipette, we integrate polydimethylsiloxane (PDMS) channel with only the tip section of a micro-pipette to perform cell/particle counting. Second, the whole pipette (tip and constant diameter regions) is inserted into a PDMS channel. The advantage of this integration is that one can use inexpensive PDMS to fabricate the overall device while the tip of pulled pipette, with a specific diameter, can be used as the aperture for a particular cell/particle population. By four-probe measurement, voltage potential differences will be get via pre-amplifier and filter. Using LabView® for data acquisition, we measured the changes of resistance to determine the count and the size of the particle. Experiments include the calibration of the electric signal, poly-spheres counting, red/white blood cell counting, and signal amplitude with different aperture size. Consequences show that the detected signals and waveforms of digital signal process. By the calculation, distribution of different mean diameter particles is shown with the histogram. Advantage of our count chip is that it can easily change the aperture diameter preparation via a molding process using common apparatus. The results provide the reference of cell counting and diameter distribution. And easy fabrication makes it convenient in integration with micro-fluidic chip. Results show that counting of red and white blood cells are successfully achieved. Proper grounding and signal conditioning are very critical in obtaining quality results.Chapter 1: Introduction ......9 1.1 Cell Counting and Motivation......9 Chapter 2：Detection Technology......11 2.1 Introduction to Coulter Counters ......11 2.2 A Brief Review in Miniaturize Coulter Counters......12 Chapter 3：Theory......16 3.1 Theoretical formulation for change in electrical resistance ......16 3.2 The Channel Geometry......19 Chapter 4：Experiments......20 4.1 Overall design......20 4.1.1 Manufacturing issue......20 4.1.2 Deep polymeric microchannel......20 4.1.2.1 SU-8 layer......20 4.1.2.2 Replica Modeling......23 4.1.3 Electrodes......24 4.1.4 Solution in channel (diluted RBC......25 4.1.5 Syringe Pump ......26 4.2 Design & Integration of PDMS and pipette......27 4.2.1 Pulling pipette......27 4.2.2 Integration of PDMS and pipette......28 4.3 Inlet/Outlet interface and filling ......29 4.3.1 Steps to assemble metal inlet/outlet tubing into PDMS without leaking from the junction......30 4.3.2 Steps to assemble glass inlet/outlet tubing into PDMS without leaking from the junction......31 4.4 Constant Current Circuitry......32 4.4.1 Four-Point Probe......32 4.4.2 Constant current biasing......34 4.4.3 Pre-Amplifier......35 4.4.4 Noise......36 4.5 Data Acquisition......37 Chapter 5：Results and Discussions......41 5.1 Inlet Module......41 5.2 Sensitivity range of the Coulter principle ......43 5.3 Sampling and dispersing particle ......45 5.3.1 Manual Cell Counting (by microscope).......45 5.3.2 Coulter Principle Cell Counting (by electric)......48 5.3.2.1 15µm Polysphere Samples ......48 5.3.2.2 Polysphere Samples (5µm)......52 5.3.3.3 Red Blood Cell (erythrocyte) Counting ......54 5.3.3.4 White Blood Cell Counting......56 Chapter 6：Conclusion and future work ......58 Appendix......60 References......732816751 bytesapplication/pdfen-US細胞計數電阻抗PDMScoulter counterthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/62400/1/ntu-93-R90543005-1.pdf