以微流體系統量測接合力之研究

Specific binding force measurement using microfluidic system

陳期宇(Chi-Yu Chen)

指導教授 ： 施文彬

國立臺灣大學/工學院/機械工程學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.00412

摘要

本論文以開發心臟病量測晶片為背景，量測專一性結合之蛋白質鍵結力。臨床上對於急性冠心症(acute coronary syndrome, ACS)的診斷為症狀評估、心電圖判斷以及心臟酵素濃度的檢測。為實現心臟病患之定點照護系統，本論文所屬計畫致力於開發一生物感測晶片，透過適體(Aptamer)捕捉血液中心臟酵素(Cardiac troponin I)以檢測血液所含酵素的濃度，由於本計畫所開發之生物晶片與微流道結合，透過真空所造成的壓差或毛細力將血液輸送至檢測區域，由於流體所產生的剪力有可能破壞蛋白質的專一性結合，因此本論文致力於量測此種專一性結合之鍵結力，以開發高精確度之簡易量測系統為目標。量測蛋白質間鍵結力之方法已有許多文獻
提出，然而多以原子力顯微鏡(Atomic force microscope)為主要量測方法，以此方法量測，需昂貴的量測儀器與精確控制的量測環境，因此本論文開發一微流體系統間接量測一級抗體與二級抗體之間專一性結合之鍵結力。在本論文中，首先將一級抗體(Rabbit IgG)共價接合於含有羧基(-COOH)的微球體上備用，接著將微流道之基板以氣象沉積之方式，沉積一層高分子層，此高分子層含有NHS-ester官能基，透過此官能基，二級抗體(Anti-rabbit IgG)共價接合於上，當含有一級抗體之微球體通入流到中，微球體上之一級抗體將被流道底部之二級抗體所捕捉而停留於流道中，接著通入緩衝液並逐漸加大流量，當流體所產生之剪力足以破壞鍵結時，微球體將被沖走。此外，利用螢光標記蛋白，單一微球體上所含蛋白質數量可透過螢光強度量化，透過上述實驗並佐以模型計算，一級抗體與二級抗體之間的鍵結力將能夠以計算之方式得知。在量測結果中，我們量測出此對抗體間的鍵結力約為118 pN，此結果與文獻所測出的Human IgG與Anti-human IgG相較之下較大，但已經非常接近。本論文所量測之結果，為凡德瓦力、靜電力與鍵結力的總和，若能以理論剪去靜電力與凡德瓦力的影響，單純由鍵結所提供之鍵結力即可得知。

關鍵字

鍵結力量測 ； 適體 ； 心肌蛋白 ； 急性冠心症 ； 一級抗體(rabbit IgG) ； 二級抗體(anti-rabbit IgG) ； 微流體 ； 微流體剪力 ； 微球體

並列摘要

We measured the specific binding force between proteins with the background of the development of heart disease detection biochips. In clinical, it is pathogonostic to have acute coronary syndrome (ACS) of three items that are clinical symptoms, electrocardiography (ECG) diagnosis and cardiac enzymes. To achieve the goal of constructing a point-of-care system for ACS patients, this thesis is dedicated to the development of biological sensing chip. Specifically, aptamers capture the heart blood enzyme (Cardiac troponin I) in order to detect the concentration of the enzyme contained in blood. Since the biochip was combined with the microfluidic channel, the blood was delivered into the sensing area through pressure difference by vacuum or the capillary force. The shear force generated by fluid could break the binding between proteins, so we are committed to measuring the binding force between specific bindings and developing a system with high accuracy to investigate different kinds of protein-protein interactions. The method of measuring the interaction force between proteins had been revealed by many literatures. However, most of them used atomic force microscope (AFM) to measure the force. This method of measurement need well controlled environment. Alternatively, we developed a microfluidic system to measure the binding force between primary antibody and secondary antibody indirectly. In our research, the primary antibody (Rabbit IgG) was covalently bonded to carboxylated (-COOH) microbeads. The substrate of the microchannel was deposited with a film of polymer containing NHS-ester functional group by chemical vapor deposition, and the secondary antibodies (Anti-rabbit IgG) can be covalently bonded on the substrate through these functional groups. The microbeads conjugated with the primary antibodies were injected into the microchannel and captured by the secondary antibodies on the substrate. Then the buffer was injected into the microchannel with gradually increased flow rate. When the shear force was large enough to break the bonds, the microbeads will be washed away. In addition, we quantified the numbers of proteins on one bead with the use of fluorescent labeled protein by fluorescence intensity measurement. With the above experiment and model calculations, the binding force between the primary antibody and the secondary antibody can be calculated. In the measurement results, the binding force between the antibodies was 118 pN. Though the result is greater than the binding force between human IgG and anti-human IgG measured from other literatures, it was still very close. The measured interaction force is attributed to the bond, van der Waals force and electrostatic force. If the van der Walls force and electrostatic force can be eliminated by calculation, the force only contributed by the bond can be investigated.

並列關鍵字

binding force measurement ； aptamer ； cardiac troponin I ； acute coronary syndrome ； rabbit IgG ； anti-rabbit IgG ； microfluidic ； shear force ； microbead

參考文獻

[12] C. C. Huang, “Development of Microfluidic Graphene-Aptamer Biosensors for Cardiac Troponin-I Detection,” Doctoral Dissertation, National Taiwan University, 2015.

[4] R. S. Wright, J. L. Anderson, C. D. Adams, C. R. Bridges, D. E. Casey, S. M. Ettinger, F. M. Fesmire, T. G. Ganiats, H. Jneid, A. M. Lincoff, E. D. Peterson, G. J. Philippides, P. Theroux, N. K. Wenger, and J. P. Zidar, “ACC/AHA practice guideline,” Circulation, vol. 123, pp. e426-e597, 2011.

[23] C. C. Roberts, J. A. Quinn, and D. A. Lauffenburger, “Receptor-mediated adhesion phenomena,” Biophys. J., vol. 58, pp. 107-125, 1990.

[1] Mayo Clinic: Heart disease, definition. http://www.mayoclinic.org/

[2] R. P. Giugliano and E. Braunwald, “The year in acute coronary syndrome,” J. Am. Coll. Cardiol., vol. 63, pp. 201-214, 2014.