A Multidisciplinary Approach to Protein Nanoarrays

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.