Detection of cancer markers in a fast and sensitive fashion is important in determining the type and stage of the disease and hence can facilitate early detection of cancer. Today, most methods used to detect biomolecules utilize fluorescent and radiolabels, which increases the time, cost and expertise required for assays. Micro- and nanotechnology has enabled researchers to develop label-free detectors that offer scalability (down in size and up in number of sensors) for detecting multiple analytes. However, the majority of micro/nanosensors have not yet achieved the sensitivity levels of label-based detection schemes. This proposal explores a novel set of methods to enhance the sensitivity of fluorescent/radio-label-free platforms to enable detection of cancer markers in clinically significant conditions and concentrations.
The specific aims of the proposal focus on adapting allosteric nucleic acid catalysts (aptazymes) to 1) a nanomechanical cantilever biosensor, which is a model label-free platform which has attracted an immense amount of academic attention in recent years but has not received much clinical acceptance due to insufficient sensitivity, and 2) a bead-based diffraction biosensor, which is a much newer detection fluorescence-free platform. Aptazymes that can covalently bind to immobilized substrates on a solid surface (upon activation by target proteins) will be developed based on their functionality on a surface (Aim 1) and adapted for operation with the cantilever and the bead-based diffraction biosensor (Aim 2). For the cantilever sensor, large aptazymes (constructed either by introducing additional nucleotides or by conjugating aptazymes to micron-sized particles) activated by target molecules will ligate to the functionalized cantilever surface and yield an amplified signal. For the diffraction biosensor, magnetic beads functionalized with aptazymes will be used to selectively capture target molecules from complex mixtures. The capture will activate the aptazymes and allow their ligation (and hence the ligation of the beads) to a solid surface that is functionalized in an alternating pattern. The binding of the beads will result in a solid diffraction grating that generates very sensitive signals. Further signal enhancement in both platforms will be achieved by performing rolling circle amplification. It is expected that detection of proteins via aptazymes will generate far greater signals in comparison with those generated by direct detection of non-covalent protein-receptor binding. Two different cancer-relevant proteins will be used as model targets against which aptazymes will be developed: basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). It is expected that adapting these new aptazymes to the nanomechanical cantilever sensor and the new bead-based diffraction biosensor will enable 50 fM and 500 fM detection limits for bFGF and VEGF. The developed strategy will be tested in biologically significant media such as serum and cell lysate (Aim 3). The aptazyme development will be performed at The University of Texas at Austin while all experimental work will be undertaken by Purdue University. Many cancers can be treated if detected early. Detecting cancer markers in body fluids is a promising way to achieve early detection. This project will constitute a milestone in development of sensitive but relatively simple platforms for detection of cancer markers. ? ? ?
| Lee, Joonhyung; Icoz, Kutay; Roberts, Ana et al. (2010) Diffractometric detection of proteins using microbead-based rolling circle amplification. Anal Chem 82:197-202 |
