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。单分子实验指向每回合三个催化事件，与配合物的分子结构一致。F1的生理功能是ATP的合成。在无处不在的F0F1复合体中，这种能量上坡反应是由F0驱动的，F0是F1的搭档马达，它迫使F1向后（顺时针）旋转，从而导致ATP合成。在这里，我们设计了一个结合单分子操作和微加工技术的实验来测量这种机械化学转化的产率。单个F1分子被封闭在飞升大小的密封腔中，并用磁镊子顺时针方向旋转。当磁场关闭时，F1分子以与合成ATP量成比例的速度进行逆时针旋转。我们在这里提供了直接的证据，证明F1被设计成紧密耦合其催化反应和机械旋转。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，我们提出了一种利用多孔聚二甲基硅氧烷（PDMS）筛板在玻璃衬底上对活性蛋白进行图图化的新技术。该筛具有锥形孔，通过在锥体型模具上旋转涂覆PDMS来制造。通过这种筛，FITC（荧光异硫氰酸盐，牛）白蛋白成功地在阵列中被发现。这些有图案的斑点是完全分离的，这就消除了蛋白质与不需要的区域非特异性结合的问题。为了证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；它们的活动可以很容易地通过观察图案后的旋转运动来验证。三种荧光微珠的选择性图图化表明，利用阴影掩膜在同一底物上对不同蛋白质进行图图化的可能性。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