更多的科研Idea………
4、参数
磁镊主要包括磁路系统、显微成像系统以及数据采集和处理系统、温控系统等部分。作用力量程(pN):0.01 pN—100 pN
样品温度控制范围:14°C- 55°C
温度控制精度:1 / 100°C
图像漂移控制精度:20分钟内小于30 nm
高速实时测量速度:>60 Hz
CCD相机参数:使用768 ×576像素,60 Hz摄像机;或者使用1380 × 1040像素,31 Hz摄像机
样品固定装置磁铁与磁珠的最小距离:≥ 100um
图7 PicoTwist部分组件的参数
5、使用方法
样品(如DNA)的一端连接在直径1um的超顺磁性小球上,另一端连接在样品固定装置中的玻璃表面上。
两块钕铁硼磁铁置于样品上方,当移动磁铁接近样品时,通过超顺磁性小球给样品施加皮牛级的拉力
,同时旋转小磁铁可以向样品施加扭转应力.小球的位置通过一台倒置显微镜以大于60Hz的采样频率记录.
测量样品,如DNA 的力拉伸曲线、DNA长度与小磁铁旋转圈数关系曲线、计算得到DNA 超螺旋结构,
形成DNA超螺旋阈值处的结合和解离动力学等。
参考文献
A. Dawid, F. Guillemot, C. Breme, V. Croquette, F. Heslot, Mechanically controlled DNA extrusion from a palindromic sequence by single molecule micromanipulation. Phys. Rev. Lett. (2006) 96-18 p.
G. Lia, E. Praly, HA. Ferreira, C. Stockdale, YC. Tse-dinh, D. Dunlap, V. Croquette, D. Bensimon, T. Owen-hughes, Direct observation of DNA distortion by the RSC complex. Mol. Cell (2006) 21-3 p.417
R. Seidel, JGP. Bloom, J. Van Noort, CF. Dutta, NH. Dekker, K. Firman, MD. Szczelkun, C. Dekker, Dynamics of initiation, termination and reinitiation of DNA translocation by the motor protein EcoR124I. Embo J. (2005) 24-23 p.4188
KC. Neuman, OA. Saleh, T. Lionnet, G. Lia, JF. Allemand, D. Bensimon, V. Croquette,Statistical determination of the step size of molecular motors. J. Phys.-Condes. Matter (2005) 17-47 p.S3811
G. Charvin, TR. Strick, D. Bensimon, V. Croquette, Topoisomerase IV bends and overtwists DNA upon binding. Biophys. J. (2005) 89-1 p.384
H. Shroff, BM. Reinhard, M. Siu, H. Agarwal, A. Spakowitz, J. Liphardt, Biocompatible force sensor with optical readout and dimensions of 6 nm(3). Nano Lett. (2005) 5-7 p.1509
G. Charvin, A. Vologodskii, D. Bensimon, V. Croquette, Braiding DNA: Experiments, simulations, and models. Biophys. J. (2005) 88-6 p.4124
OA. Saleh, JF. Allemand, V. Croquette, D. Bensimon, Single-molecule manipulation measurements of DNA transport proteins. Chem. Phys. Chem. (2005) 6-5 p.813
DA. Koster, V. Croquette, C. Dekker, S. Shuman, NH. Dekker, Friction and torque govern he relaxation of DNA supercoils by eukaryotic topoisomerase IB. Nature (2005) 434-7033 p.671
A. Revyakin, RH. Ebright, TR. Strick, Single-molecule DNA nanomanipulation: Improved resolution through use of shorter DNA fragments. Nat. Methods (2005) 2-2 p.127
KC. Neuman, G. Charvin, D. Bensimon, V. Croquette,Single-molecule study of DNA unlinking by topoisomerase IV: Influence of the crossing angle. Biophys. J. (2005) 88-1 p.15A
J. Gore, MD. Stone, Z. Bryant, NJ. Crisona, S. Mitelheiser, A. Maxwell, NR. Cozzarelli, C. Bustamante, Single molecule investigations of the mechanochemical cycle of DNA gyrase. Biophys. J. (2005) 88-1 p.184A
D. Koster, S. Shuman, C. Dekker, NH. Dekker, Topoisomerase 1B: its mechanism and interactions with spinning DNA studied at the single molecule level. Biophys. J. (2005) 88-1 p.381A
JA. Abels, F. Moreno-herrero, T. Van Der Heijden, PTM. Veenhuizen, MM. Bruinink, C. Dekker, NH. Dekker, Single-molecule measurements of the persistence length of double-stranded RNA. Biophys. J. (2005) 88-1 p.570A
D. Ristic, M. Modesti, T. Van Der Heijden, J. Van Noort, C. Dekker, R. Kanaar, C. Wyman,Human Rad51 filaments on double- and single-stranded DNA: correlating regular and irregular forms with recombination function. Nucleic Acids Res. (2005) 33-10 p.3292
S. Neukirch, Extracting DNA twist rigidity from experimental supercoiling data. Phys. Rev. Lett. (2004) 93-19 p.
D. Skoko, B. Wong, RC. Johnson, JF. Marko, Micromechanical analysis of the binding of DNA-bending proteins HMGB1, NHP6A, and HU reveals their ability to form highly stable DNA-protein complexes. Biochemistry (2004) 43-43 p.13867
BD. Matthews, DA. Lavan, DR. Overby, J. Karavitis, DE. Ingber, Electromagnetic needles with submicron pole tip radii for nanomanipulation of biomolecules and living cells. Appl. Phys. Lett. (2004) 85-14 p.2968
Y. Harada, Studies on biomolecules using single molecule imaging and manipulation techniques. Sci. Technol. Adv. Mater. (2004) 5-5-6 p.709
附件:
现在已经有很多单分子的操纵技术:原子力悬臂、光镊、磁镊以及流场拖曳、生物膜力探针(BFP)等。
在这些实验中,生物分子的一端固定于一表面,另一端与力传感器相连。
表 1 各种生物单分子实验技术的比较
方法 | 力量程(pN) | 时间量程 | 实际运用 |
光镊 | 0.1—150 | >10 ms | 肌动蛋白、DNA、蛋白质、分子马达 |
磁镊 | 0.01—100 | >1 s | 拉伸、扭转 DNA |
微探针 | >0.1 | >100 ms | 拉伸、扭转、解旋DNA |
BFP | 0.5—1000 | >1 ms | 配体受体 |
AFM | >1 | >10 us | DNA、蛋白质 |
图1 力传感器的示意图
A. AFM 实验示意图,悬臂用来作分子间力作用的传感器,悬臂的位移由激光束获得;
B. 光学纤维作力传感器的例子;C. 光镊的示意图;D. 磁镊示意图。