Micromechanically Developed Magnetic Tweezers for Manipulating Single DNA Molecules

• Main Authors: Chi-Han Chiou, 邱祈翰
• Other Authors: Gwo-Bin Lee
• Format: Others
• Language: en_US
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/03682669143183296100

博士 === 國立成功大學 === 工程科學系碩博士班 === 93 === 　Directly studying the physical properties of biopolymers at the single-molecule level is often more informative than classical bulk experiments which average over several molecules. Bio-nanotechnology enables new methods of directly observing and manipulating individual biological molecules. The top-down approach can adopt MEMS (micro-electro-mechanical-systems) fabrication technologies to produce a micro-scale device, which can manipulate a single DNA molecule with a 2-nm diameter. The bottom-up process focuses on the molecular self-assembly, which operates using human-made synthesis to modify and address a single DNA molecule. This study develops a micro-magnetic platform using the synergy strategies of a top-down approach with a bottom-up process to investigate single DNA molecule properties. 　This study develops 2-D and 3-D micromachined magnetic tweezers for DNA manipulation using MEMS technologies. Essential platform technologies, including localized DNA immobilization, micro-magnetic device fabrication and microfluidics, can be integrated to form the micromachine-based DNA manipulation platform. For specific end anchoring, this study developed highly effective and strong binding methods, which are compatible with MEMS technologies, for constructing DNA molecules with two sticky ends. 　In the 2-D magnetic tweezers system, one end of a single DNA molecule was specifically bonded with a magnetic bead, and the other end was bonded with a gold surface. The molecule was then manipulated under a magnetic field generated by built-in hexagonally-aligned microcoils. This study successfully demonstrated the stretching and rotation of a single DNA molecule. To quantify the magnitude of magnetic forces acting on the DNA molecule, force calculation based on the gravity balance was performed and further verified by the Worm-Like chain (WLC) model. The measured DNA stretching forces were found to agree reasonably with the fitting values. The magnitude of forces acting on a DNA molecule is within the sub-pN range, enabling the study of DNA in an entropic region. 　To enhance magnetic forces, a new 3-D magnetic tweezers consisting of micro-electromagnets and a ling-trap structure was proposed. To improve the localized DAN immobilization efficiency, a novel ring-trapper structure was used to handle the vertical movement of magnetic beads which adhered to the DNA molecules. One extremity of the DNA molecule, which was bound to the thiol-modified magnetic bead, could be immobilized covalently on a gold surface. The other extremity, which was bound to another unmodified magnetic bead, could be manipulated under a magnetic field generated by micro-electromagnets. To measure the DNA stretching force, the magnetic force acting on the magnetic bead was calibrated by using the modified Stoke’s law. The force-extension curve was further fitted by the WLC model, Odijk’s model and Hooke’s law. A value of 453 pN for elastic modulus of DNA was obtained at an ionic strength of 10 mM Na+. This result reveals that DNA becomes more susceptible to elastic elongation at a low ionic strength due to electrostatic repulsion. In addition to a single DNA stretching, this study also successfully demonstrated the stretching of two DNA molecules using the 3-D magnetic tweezers. The experimental data reveals that DNA presents a highly nonlinear behavior. The apparatus can exert over 20-pN magnetic forces with less heating to extend the DNA molecule over the whole contour length to investigate its entropic and elastic regions. 　To connect the magnetic tweezers with the external large-scale fluid equipment efficiently, this study presented a simple and versatile micro-connector manufactured using PDMS casting. To eliminate the dead volume, a capillary was bridged to a micro-channel via a connection channel, which was formed by removing a metal wire after PDMS casting. The proposed method does not require any adhesive, precise drilling, delicate alignment procedure or micromachining processes. Additionally, the proposed method could prevent blocking of the capillaries, a phenomenon which was commonly observed when using adhesives. The proposed method can achieve detachable and reusable micro-connectors with a minimal dead volume. According to leakage tests, the micro-connector could withstand pressures up to 150 psi and a maximum flow rate of 50 μL/min. The pull-out tests show that the PDMS fitting could provide sufficient mechanical strength for practical applications. Not only does the novel micro-connector significantly eliminate the dead volume but it also raises the detection signal. Compared with the traditional Teflon tubing fitting, the micro-connector can reduce at least 50% the dilution effect for sample loading analysis because the dead volume is substantially eliminated. Most significantly, the proposed micro-connector couples capillaries to microfluidic chips more flexibly than other micro-connectors. 　With these methods, the size of the apparatus can be reduced markedly, allowing the magnetic tweezers platform to be mass-produced at low cost. Most significantly, the microfabricated system can be simplified without losing sensitivity and functionality, unlike in other methods such as the use of large-scale magnetic tweezers and optical tweezers.