SBIR-STTR Award

Proximity technology motivated by an array of potential applications offers a robust foundation for the development of cell type specific quantitative intramolecular epitopes of analytes in tissue sections. In this proposal, we highlight how our CamelTM (dual peaks) quantum dots (QDs) are greatly suitable fluorophores, with spectroscopic properties that can be controlled via fluorescence resonance energy transfer (FRET) interactions. We expect that a ratio of activated signal transducers as phosphorylated proteins to their total protein levels in cancer cells can be determined by FRET signals. In addition, those signals can be widely and simply captured and visualized to cell types as well as quantitated via conventional microscopy without a fluorescence lifetime imaging microscopy (FLIM) technique. We will further evaluate this QD-FRET assay in human head and neck cancer xenograft tissues for the quantification of phosphorylated proteins to their total protein levels in each cell. We believe this QD-antibody based proximity assays will represent a significant advancement in a rapid detection of targets with high sensitivity and specificity.

NIH Spending Category:
Bioengineering; Biotechnology; Cancer; Nanotechnology; Rare Diseases

Project Terms:
Antibodies; base; Biological Assay; Camels; cancer cell; cell type; Cells; Development; Epitopes; Fluorescence; Fluorescence Resonance Energy Transfer; fluorophore; Foundations; Head and Neck Cancer; Human; Immunoassay; Liquid substance; Methods; microscopic imaging; Microscopy; phase 2 study; Preparation; Property; Proteins; Quantum Dots; rapid detection; Sensitivity and Specificity; Signal Transduction; Slide; Techniques; Technology; Tissues; Transducers; Validation; Xenograft procedure

This proposal is to develop a novel slide based proximity assay platform to quantify protein phosphorylation on tissues. The novelty of this assay platform is derived from the use of the quantum dot with two emission peaks for fluorescence resonance energy transfer (FRET): one emission as internal standard and the other as FRET donor. Change of ratio of the two emission intensity during the FRET can be used to quantify the ratio of two epitopes on a target protein. Phase I project demonstrated the feasibility of quantifying AKT and ERK phosphorylation on both cell lines and tissues. Phase II project will 1) complete validation of the quantification methods with patient derived animal model; 2) study c-Met protein phosphorylation; 3) validate the kits with PDX animal model, 4) establish quality control procedures and parameters and study the reproducibility and stability of the assays; and 5) establish GLP documentation of the assays. The final deliverable is four kits, AKT kit, ERK kit, c-Met kit and an universal kit.