Peptide aptamer optical protein detection

Our bodies are made up of trillions of cells, which contain billions of proteins. Proteins are the workhorses of a cell, and until we understand how they work together, we will not be able to understand life at the level of each cell, nor will we be able to identify the changes that occur in triggering a disease. While the sequencing of the human genome has given us a catalogue of these proteins, we still lack the tools that allow us to study them both individually- is protein X expressed in this cell?- and collectively- do proteins X and Y, which are both expressed in a cell, actually interact with each other? There are two major hurdles to such studies. The first is that we need probes for each protein that allow us to detect a given protein even in the presence of the billions of other protein molecules in a cell extract. The second is that we need a way to sense when a target protein has bound to its specific probe- proteins are so small that any signal they generate is minute. One way around this has been to add labels to proteins that are capable of generating very large signals, but this solution has its own problems, as the label can modify the behaviour of the protein, for example to prevent a biologically-significant binding event, or to create a new, irrelevant interaction. We have solved both problems using a technique called protein engineering to create artificial proteins that specifically recognise target proteins, and that are sufficiently robust that they can be attached to sensing surfaces to generate label-free electrical or optical signals. Recent advances in our understanding of molecular fluorescence and the optical properties of nanometre-scale structures offer much in the generation of sensitive sensory interfaces. The goal of this project is to integrate receptive artificial proteins with these interfaces and identify the best means of applying our proof-of-concept work to the generation of detection methods both compatible with the needs of researchers but also enabling currently unanswerable questions to be answered. Such questions range from asking what is the smallest number of copies of protein that can be detected in a single cell, to what is the largest number of different proteins that can be detected simultaneously using a technology that is affordable to most biological/clinical labs.

A major bottleneck in the development of high density protein microarrays is the high throughput generation of robust capture agents of high affinity and specificity. Peptide aptamers can be selected from large libraries via phage display or yeast two hybrid screens, exhibit inherent (thermal) stability and can be tuned to the requirements of orientated non destructive surface immobilisation. Since the labelling of sample protein prior to any subsequent assay brings with unpredictable and variable effects, the utilisation of a label free approach is attractive. Optical assays are potentially cheap, highly sensitive and scaleable. Through the controlled immobilisation (or printing) of a fluorophore tagged peptide aptamer on glass and the subsequent incubation of this in a FRET partner tagged target protein a surface primed for highly responsive detection of unlabelled target protein is generated. Propagating surface plasmon resonances (utilized in SPR sensing) are evanescent electromagnetic waves with adsorption characteristics which are highly sensitive to changes in interfacial refractive index. In recent years, a nanoscale derivative of this concept, based on the extraordinary optical characteristics of noble metal nanoparticles or holes, has been introduced. The key advantage these 'localized' SPR (LSPR) assays have over standard SPR assays is the ease with which plasmons are excited (there is no requirement for a prism generated glancing angle of excitation incidence). This offers the possibility of cheap scale up, portability and multianalyte assaying. At sufficiently high dilution /spacing it is, additionally, possible to detect only a few tens of target molecules. This proposal seeks to utilize surface confined FRET and LSPR configurations (with metallic nanoparticles, nanoshells and perforated metallic film) with peptide aptamer technology in the establishment of multiprotein assays capable of highly selective protein detection in lysate.