A Multidisciplinary Approach to Protein Nanoarrays

蛋白质是所有生物体的分子机器，并执行生命所需的所有功能。在每一种生物体中，大量的蛋白质以高度协调的方式发挥作用，执行从营养素加工到生物体繁殖的任务。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、缺乏或过度产生引起的。蛋白质的活性也是任何依赖于生命系统的人类经济活动的基础，例如利用发酵和农业部门的工业。因此，为了充分了解生命系统，需要鉴定蛋白质，测量存在的量，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。一种能够研究蛋白质的方法是将它们锚在二维表面上，例如载玻片或芯片，其中每个蛋白质被放置在表面上的限定位置。这为处理大量蛋白质提供了一种方便的方法，并提供了一种同时测试它们的方法。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，并且如果在该载玻片上的特定位置检测到感兴趣的生物活性，则可以鉴定引起该活性的蛋白质。目前的技术允许在一个芯片上产生大约10 000个蛋白质点的阵列，点的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确定了大约50，000种蛋白质。此外，各种蛋白质的类型和活性在不同细胞之间以及在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量的形式存在。为了能够检测来自如此广泛的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。具有较小蛋白质点的芯片也意味着只需要少量的蛋白质（可能是罕见的）进行测试。通过利用纳米技术发展起来的技术，可以实现这些斑点的进一步简化，纳米技术是在纳米尺度（十亿分之一米）上构建物体的科学，原则上，甚至是单个分子。因此，该提案旨在使用两种纳米技术在硅氧烷表面（一种玻璃状材料）上构建这些蛋白质阵列。这些技术是蘸笔纳米光刻，其中一个非常精细（纳米宽）的尖端浸入化学墨水中并用于在表面上写入图案，以及扫描近场光刻，其使用非常精细的孔来引导激光在表面上写入图案。然而，该提案还包括许多其他科学领域，这些领域将需要建立蛋白质纳米阵列。将采用分子生物学技术来产生蛋白质，所述蛋白质可以特异性地附着到表面上的区域，所述表面上的区域已经被图案化，使得蛋白质附着的方式被明确限定。为了将表面纳米技术和生物学结合在一起，将采用合成化学来制备与生物系统相容的新型墨水，适合于书写，并且可以在激光下反应以产生斑点（或其他图案），随后可以以特定的方式附着蛋白质。虽然已经有纳米技术用于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一种与蛋白质组学直接相关的多种蛋白质阵列。

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.