具反饋機制的磁鉗系統架設與DNA單分子共價錨定開發

Construction of magnetic tweezers system with feedback control and development of covalently DNA single molecule anchoring

陳昱銘(Yu-Ming Chen)

指導教授 ： 許志楧 ； 陳之碩

國立清華大學/原子科學院/生醫工程與環境科學系/碩士(2019年)

摘要

近年來單分子操縱系統廣泛運用在生物領域相關研究，其可觀察單一分子的特性可看到以往無法觀察到的細微現象，其中磁鉗系統為具有奈米等級解析度且可以觀察多個生物分子的單分子操縱系統，以磁鐵及磁珠施予外力於欲觀察物體上，利用磁珠繞射圖譜影像進行比對以得到磁珠高度或角度，並由磁珠布朗運動得到磁力等資訊，在DNA、蛋白質酵素及細胞實驗中皆有廣泛應用。本實驗中以顯微鏡為基礎，將磁鉗系統架設於倒立式顯微鏡上，並先以pH錨定DNA進行系統校調及軟體測試，為了進行影像比對，先利用迭代法及邊界填充法定出磁珠中心，並將磁珠資訊轉換成徑向矢量，以影像比對方式得到磁珠高度，實驗中也撰寫軟體以布朗運動為基礎評估磁力大小。另外實驗中以馬來酰亞胺與硫醇於特定pH下的專一性設計一套具低非特異黏附及高可靠度的共價錨定方法，達到單一視野下多條DNA的目標，並以此錨定方式，測試距物鏡焦點0 ~ 15微米下系統影像比對的準確性，以評估系統解析度，結果顯示本系統於0 ~ 12 微米範圍中具有 ±79奈米的準確性。另調整磁鐵高度，在距離樣品室底部3.3、2.3、1.8、1.3、0.8、0.4及0.3 mm下測量磁力大小，並繪出磁力大小與磁鐵高度關係圖，顯示系統於磁鐵最低時可提供最高3.07±0.26皮牛頓的磁力。

關鍵字

磁鉗 ； 單分子操縱 ； 單分子錨定 ； 共價錨定

並列摘要

In recent years, single molecule manipulation systems have been widely used in research of biological systems. Magnetic tweezers can observe multiple biomolecules, including DNA and proteins, which could not be achieved by traditional systems. Magnetic beads were attached to the biomolecules and force was applied through magnets nearby. Location of the beads were measured through video microscopy. Orientation and height of magnetic beads was deduced through diffraction patterns formed by the beads. Magnetic force was calculated by equipartition theorem from Brownian motion.
In our research, magnetic tweezers system was built on top of an inverted microscope. For software development and system calibration, DNA molecules were anchored to the bottom of the chamber through incubation in buffer with specific pH. The x and y position of the beads were determined through iteration and boundary fill and the height through image recognition. Images were converted to radius vectors and compared with the Look Up Table of a calibration standard. Furthermore, we built a calibration program to measure magnetic force from Brownian motion of the bead. We also developed an assay to covalently attachment mutiple DNA molecules on PEG coated coverslips in one field of view through specific reaction between maleimide and thiol at neutral pH. This anchoring method is highly reliable and shows low nonspecific binding. The spatial resolution of system was tested up to 15 microns from the bottom of the coverslip, and we found that the accuracy of our image recognition method is 79 nm from 0 to 12 micron. Magnetic force was calculated when the magnets are 3.3, 2.3, 1.8, 1.3, 0.8, 0.4 and 0.3 mm from the bottom of the chamber and the maximum force this system can exert on a 1 micron diameter bead is 3.07±0.26 pN when the magnets is on top of the chamber.

並列關鍵字

Magnetic tweezers ； Single molecule manipulation ； Single molecule anchoring ； Covalently anchoring

參考文獻

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