A Multidisciplinary Approach to Protein Nanoarrays

蛋白质是所有生物体的分子机器，并执行生命所必需的所有功能。在每一个生物体中，大量的蛋白质以一种高度协调的方式发挥作用，从营养物质的处理到生物体的繁殖。因此，蛋白质的功能对健康有重大影响，因为许多疾病是由各种蛋白质的活性改变、缺乏或过量产生引起的。蛋白质的活动也是依赖于生命系统的任何人类经济活动的基础，例如利用发酵的工业和农业部门。因此，为了充分了解生命系统，有必要鉴定蛋白质，测量存在的数量，发现它们的功能并阐明它们相互作用的机制。这些都包含在被称为蛋白质组学的科学领域中。研究蛋白质的一种方法是将蛋白质固定在二维表面上，如玻片或芯片，每个蛋白质被放置在表面的指定位置。这提供了一种处理大量蛋白质和同时测试它们的方便方法。通过这种方式，整个芯片及其蛋白质集合可以进行各种测试，如果在载玻片上的特定位置检测到感兴趣的生物活性，则可以识别引起该活性的蛋白质。目前的技术允许在单个芯片上生产大约10,000个蛋白质点的阵列，斑点大小约为百分之一毫米。然而，自然界中可以检测的蛋白质数量是巨大的。仅在人类中，人类基因组计划已经鉴定了大约5万种蛋白质。此外，各种蛋白质的类型和活性在不同的细胞之间和细胞生命周期的不同时间是可变的。许多有趣的蛋白质也以非常少的量存在。为了能够检查来自如此广泛不同来源的如此大量的蛋白质，需要进一步增加可以放置在芯片上用于分析的蛋白质数量的生产方法。带有更小蛋白质斑点的芯片也意味着测试只需要少量的蛋白质，这些蛋白质可能是罕见的。进一步缩小这些点可以通过利用纳米技术来实现，纳米技术是在纳米尺度（一米的十亿分之一）上构建物体的科学，原则上甚至可以缩小到单个分子。因此，本提案旨在使用两种纳米技术在硅氧烷表面（一种类似玻璃的材料）上构建这些蛋白质阵列。这两种技术分别是浸入式纳米光刻技术，将一个非常细（纳米宽）的尖端浸入化学墨水中，用于在表面上书写图案，以及扫描近场光刻技术，它使用一个非常细的孔来引导激光在表面上书写图案。然而，这个提议也包括了一些其他的科学领域，这些领域将需要构建蛋白质纳米阵列。分子生物学技术将被用于生产蛋白质，这些蛋白质可以特异性地附着在表面上已经形成图案的区域上，这样蛋白质附着的方式就很好地定义了。为了将表面纳米技术和生物学结合起来，合成化学将被用于制备新型墨水，这种墨水与生物系统兼容，适合书写，并且可以在激光下反应产生斑点（或其他图案），这些斑点随后可以以特定的方式附着在蛋白质上。虽然已经有使用纳米技术制造这种规模的一种或两种蛋白质阵列的例子，但这里提出的关键突破是多种蛋白质的阵列，这将与蛋白质组学直接相关。

Proteins are the molecular machinery of all living organisms and perform all the functions necessary for life. In every organism, large numbers of proteins act in a highly orchestrated manner to perform tasks from the processing of nutrients to the reproduction of the organism. The function of proteins therefore has a major bearing on health as many diseases are caused by the altered activity, deficiency or overproduction of various proteins. The activity of proteins also underlies any human economic activity which is reliant on living systems such as industries which utilise fermentation and the agricultural sector. Thus, in order to fully understand living systems, there is a need for the identification of proteins, measurement of the amounts present, discovery of their function and elucidation of the mechanisms by which they interact with each other. These are encompassed in the scientific field known as proteomics . One method of enabling the study of proteins is to anchor them to a two-dimensional surface, such as a glass slide or chip , where each protein is placed at a defined location on the surface. This offers a convenient means of handling large numbers of proteins and a means to test them simultaneously. In this way, an entire chip and it's collection of proteins can be subjected to various tests and if a biological activity of interest is detected at a particular location on that slide, the protein which caused that activity can be identified. Current technology allows the production of arrays of approximately 10,000 protein spots on a single chip with spot sizes of about a hundredth of a millimetre.However, the number of proteins in nature that could be examined is vast. In humans alone, the Human Genome Project has identified approximately 50,000 proteins. Moreover, the types and activity of various proteins are variable between different cells and at different times in a cell's life cycle. Many interesting proteins are also present in very small amounts. To be able to examine such a large number of proteins from such widely varied sources, production methods are needed which further increase the number of proteins that can be placed on a chip for analysis. Chips with smaller protein spots would also mean that only tiny amounts of proteins which may be rare are needed for testing. Further miniaturisation these spots could be achieved by harnessing the techniques developed in nanotechnology, the science of constructing objects at nanometre scales (a billionth of a metre) and in principle, down to even a single molecule. Accordingly, this proposal aims to use two nanotechnological techniques to construct these protein arrays on siloxane surfaces, a glass-like material. These techniques are dip-pen nanolithography, where a very fine (nanometre wide) tip is dipped in a chemical ink and used to write patterns on surfaces, and scanning near-field photolithography which uses a very fine hole to direct laser light to write patterns on the surface. However, this proposal also includes a number of other scientific areas which will be needed to build a protein nanoarray . Molecular biology techniques will be employed to produce proteins which can be specifically attached to areas on the surface that have been patterned such that the way in which the protein is attached is well defined. To bring the surface nanotechnology and biology together, synthetic chemistry will be employed to prepare novel inks which are compatible with biological systems, suitable for writing and can react under laser light to produce spots (or other patterns) which can subsequently attach proteins in a specific manner. While there have already been examples where nanotechnology has been used to make arrays of this scale with one or two proteins, the key breakthrough that is being proposed here is an array of multiple proteins which would be directly relevant in proteomics.