|29th March 2015||
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Basic Technology Grant, RCUK
Biological and Biomedical Array Development
The new basic technology is targeted at biological and particularly biomedical research where strategies are undergoing paradigm-shifts in the manner of analysis and understanding but are somewhat limited by sensitivity of the available technology. Comprehensive high throughput genotyping and transcriptome analysis are fundamental to unlocking the complexities of multifactorial, multigene influenced disease conditions.
Within the biomedical sciences new post-genomic technologies underlie the major drive devoted towards mutation detection, tumour markers and metabolic profiling. Presently however screens for eg. mutation detection in genetic arrays sample ca. 6-10 parameters. Functional arrays with pixel densities of 256, 512 and 1024 squared will allow drug receptors or complementary DNA sequences to be placed on each array and binding monitored essentially in real time. Initially, oligonucleotide sequences will be implemented permitting validation with existing array technologies. Direct comparisons with commercially available arrays will be performed. Validation will take the form of monitoring binding and developing criteria for determination of 'false-positive' and 'false-negative' results. In the first instances, there is no requirement that every 'pixel' represents a different oligonucleotide sequence, simply that for any given pixel the 'nearest-neighbours' are different and with such large arrays the cost alone would make this approach prohibitive. Thus matrix-patterning can be implemented to provide realistic validation tests and 'addressability' of the technique. Similarly, as the feasiblility studies will require replicate assays on the same chip, statistical analysis can be performed with many replicates per measurement contributing to assessments of their significance. Once validation of the printing and detection technology is complete it will be feasible to print large arrays (512x512 or 1024x1024) with colossal oligonucleotide variability. The latter then opens up the possibility for mutational defect screening and tumour marker determination. In a similar manner drug screening may also be developed that will necessitate a drug target screen. The latter can be validated (as with the oligonucleotide screen) with a standard ligand-target pairing combinatorial library.
Printing the Array. The array will be printed by inkjet printing depositing 50 -100 μm spots on to the surface with a volume of order 500 pL and a periodicity of 60 μm. The total array dimensions for a 500 x 500 spot array will be 75 mm, scaling with size. The physical dimension places demands on the field of view of the camera, accurately focusing the array image onto the pixel array in the camera without aberration will be required. The array will initially be placed with the target substrate followed by a second printing on top of the first spot, and the response of each pixel monitored in real time to retrieve pharmakokinetic data. The array response will be measured by either e-CRDS or d-SPR with simultaneous lifetime f-EI. The fluorescence enhanced imaging f-EI will be developed with this part of the project to measure the response of the array during writing, simultaneously with the two SPR based detection technologies. The array fabrication development with biological functionalisation will be monitored using confocal microscopy, e-CRDS and fluorescence in the design cycle of the plasmon scaffold. The second half of the project will prepare the instruments with the 128x128 arrays using two cameras and all three detection technologies.
Our first array for medical diagnostics will be for COPD - Chronic Obstructive Pulmonary Disease. COPD is caused by the inhalation of toxins and pollutants that results in increasingly severe breathlessness. By 2020 it is estimated that COPD will be the 3rd commonest cause of death globally. In the UK there are 900,000 cases of COPD with a possible further 450,000 cases undiagnosed. This disease currently costs the UK £838 million pa.
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