Field effect sensing for protein microarrays

We are made up of trillions of cells, and each cell is made of billions of proteins. To understand how our bodies work, we need to understand how the proteins in a cell interact. The first step in this understanding was the sequencing of the human genome, the set of genes whose DNA encodes the proteins we wish to understand. This has allowed us to design probes- in some cases using human DNA itself- that can be printed in very high densities on microscope slides. We can print a probe that is specific for every gene onto a single microscope slide, and so ask which genes are being turned on, or off to make RNA or not, at every stage in the life of a cell. This ability has already created a new understanding of biology, and led to a new way of thinking about how cells work. Though impressive, this still paints but a very incomplete picture of cellular machineries at work, because although they tell us a lot about the expression of normal genes as RNA, they do not tell us anything about whether or not protein is made from that RNA (it isn¿t always), nor do they tell us about the expression of proteins whose genes carry mutations that will lead to disease. To rectify this, we would need to print probes that recognise all of the different forms of expressed proteins. This has been attempted, using either antibodies or human proteins themselves as the probes. While this makes a lot of sense, because both antibodies and proteins have evolved to recognise specific isoforms of each protein, it has proved difficult, for two reasons. First, because antibodies and proteins are very fragile, printing them onto glass slides causes them to lose their ability to recognise their target proteins in cell extracts. Second, proteins are minute, and detecting the interaction between two is correspondingly hard. So far, the best way of doing this is to attach a dye to the proteins in cell extracts, so that the printed protein lights up if it recognises its target protein in the extract. This is problematic: attaching the dye can change the protein, and we run the risk that it may no longer be recognised by our probes or may even be recognised by the wrong ones. We plan to address each of these difficulties. First, we have access to libraries of artificial antibodies that have the ability to recognise cellular proteins, but are very stable and can be printed onto a slide without losing this ability. We know that this is true for the first ones we have tried, and part of this proposal will allow us to ask why this works, and whether we can learn from the behaviour of these proteins ideas that will help us with other, less stable proteins. Second, we plan to expand upon our finding that when proteins are printed on the surface of an electrode, they change the electrical properties of the electrode in a measurable way. Then, if a second protein (from a cell extract) binds to the first protein, we get another change- so if we have many electrodes, each carrying one receptor protein, we will be able to measure the binding of each cell protein in turn, and thus the behaviour of and interactions between, every protein in a cell at any given time. This will allow us to gain intimate insights into the molecular basis of life in a cell.

The ultimate goal of this inter-disciplinary proposal is to allow the creation of very high density arrays of detector proteins that can be used for label free detection of protein-protein interactions in solution. A unique requirement for success in this venture is for chemists, electrical engineers, microfabrication specialists and biologists to be able to work together. The common goal is to solve a major problem in the post-genome era: obtaining and integrating information regarding the whole proteome of a cell, ie. The expression, quantity and activation state of all of its proteins, simultaneously. While this is a very ambitious goal, the technology described here will go a long way to making it possible. The proposal has its base in recent work from the Cambridge Engineering group showing that the interaction between single stranded DNA molecules can be specifically detected using filed effect sensing, and that this can be extended to peptide aptamer/protein interactions. We seek to identify the optimal chemistry to minimise interference from solvent and from non-specific interaction of proteins with the sensor, and to allow the immobilisation of oriented peptide aptamers on gold electrodes. Peptide aptamers are small artificial proteins that can be readily engineered, which will both allow us to explore the interactions of proteins with a gold surface in molecular detail and allow us to create functional detectors for intermolecular interactions. We will also explore the advantages of different adlayers: mixtures of polymers that will be used both to protect the electrode form the aqueous solution that comprises the cellular lysates we will interrogate and to minimise the non-specific adsorption of proteins in the lysate to the electrode. The Oxford chemistry group have extensive experience both in coupling proteins to such surfaces, in creating and characterising adlayers and in characterising the spacing, packing and orientation of proteins on gold. Finally, we will need to explore the microfabrication of field effect sensing devices that are compatible with the requirements of the immobilisation and detection mechanisms, while also being compatible with biological applications. This clearly represents a major challenge. The Central Microstructure Facility at RAL have access to existing structures that fulfil some of these requirements, and a world-class track record in solving intricate problems in fabrication and electronic processing. The various groups have a record of being able to work together in various combinations (RAL and Oxford; Cambridge Engineering and the Cambridge partner, RAL, Oxford and the Cambridge partner), maximising the potential yield from this proposal.