Novel Single Bio-Molecule Measurement using Micro/Nano Machining Technology

F1-ATP酶是已知的最小的旋转马达，它在水解ATP时沿逆时针方向旋转。单分子实验指向三个催化事件每转，在协议的复杂的分子结构。F1的生理功能是合成ATP。在普遍存在的F0 F1复合物中，这种能量上的上坡反应由F0驱动，F0是F1的伴侣马达，它迫使F1向后（顺时针）旋转，导致ATP合成。在这里，我们设计了一个实验，结合单分子操作和微加工技术来测量这种机械化学转化的产量。单个F1分子被封闭在飞升大小的密封室中，并使用磁性镊子顺时针旋转。当磁场关闭时，F1分子以与合成ATP的量成比例的速度进行反时针旋转。我们在这里提供的直接证据表明，F1的设计， 关于我们 将其催化反应与机械旋转相结合。我们的研究结果表明，1-亚基在ATP合成过程中具有重要的功能。此外，我们提出了一种新的技术，用于图案化活性蛋白质在玻璃基板上使用穿孔的聚二甲基硅氧烷（PDMS）片筛。筛，其中有锥形孔，是通过旋涂聚二甲基硅氧烷在一个陶瓷形状的模具制造。通过该筛，FITC（荧光异硫氰酸酯，牛）-白蛋白被成功地点样在阵列中。图案化的斑点被完美地分离，这消除了蛋白质与不期望的区域非特异性结合的问题。为了表明蛋白质在图案化后保持其活性，我们使用了F1-ATP酶生物分子马达;它们的活性可以通过观察图案化后的旋转运动来容易地验证。用三种荧光微珠的选择性图案化表明了通过使用荫罩在同一基底上图案化不同蛋白质的可能性。少

F1-ATPase is the smallest known rotary motor, and it rotates in an anticlockwise direction as it hydrolyses ATP. Single-molecule experiments point towards three catalytic events per turn, in agreement with the molecular structure of the complex. The physiological function of F1 is ATP synthesis. In the ubiquitous F0F1 complex, this energetically uphill reaction is driven by F0, the partner motor of F1, which forces the backward (clockwise) rotation of F1, leading to ATP synthesis. Here, we have devised an experiment combining single-molecule manipulation and microfabrication techniques to measure the yield of this mechanochemical transformation. Single F1 molecules were enclosed in femtolitre-sized hermetic chambers and rotated in a clockwise direction using magnetic tweezers. When the magnetic field was switched off, the F1 molecule underwent anticlockwise rotation at a speed proportional to the amount of synthesized ATP. We provide here direct evidence that F1 is designed to tightly … More couple its catalytic reactions with the mechanical rotation. Our results suggest that the 1-subunit has an essential function during ATP synthesis. Moreover, we propose a novel technique for patterning active proteins on a glass substrate using a perforated polydimethylsiloxane (PDMS) sheet-sieve. The sieve, which has tapering holes, is fabricated by spin-coating PDMS on a pyramidal-shaped mold. By means of this sieve, FITC (fluorescent isothiocyanate, bovine)-albumin was successfully spotted in an array. The patterned spots were perfectly isolated, which eliminates the problem of non-specific binding of proteins to undesired areas. To show that proteins maintained their activity after the patterning, we used F1-ATPase biomolecular motors ; their activity can easily be verified by observing their rotary motion after patterning. Selective patterning with three kinds of fluorescent micro beads indicated the possibility of patterning of different proteins on the same substrate by using the shadow mask. Less