The National Cancer Institute has called for new tools for cancer research and diagnosis that are accurate, practical and affordable. Alderon Biosciences, Inc. proposes to fill this need by developing an open biosensor platform technology for quantitative studies of cancer gene expression, with the goal of replacing slower and more costly methods. The working hypothesis of this SBIR proposal on """"""""Innovative Tools for the Molecular Diagnosis of Cancer'' is that the disposable sensor arrays and electrochemical readers of Alderon's innovative enzyme-enhanced electrochemical system (eSystem) for cancer gene expression analysis will greatly facilitate studies of cancer biomarkers. Feasibility of using Alderon's biosensor platform technology for quantitative cancer gene expression analysis has been demonstrated in Phase I work and differential gene expression was quantified in induced and non-induced cancer cells. The major milestone achieved in Phase I work was demonstration of target detection sensitivity sufficient for enzyme-enhanced probe-based detection of even rarely expressed messages in small tissue samples without need for target amplification (quantification of mRNA targets down to 1-3 copies per cell in samples as small as 104 cells). In Phase II, new eSystem readers and ePlates with small sensor-area electrodes that require very small volumes of samples and reagents will be further developed and validated. Phase II Project Aims are: 1) make and test new eSystem components (improved assays, small-sensor ePlates and ePlate readers) for simultaneous quantification of levels of expression of multiple cancer gene targets without use of target amplification steps; 2) produce manufacturing prototypes of new eSystem readers and 96-sensor microplates; and 3) refine performance until equivalent or better (speed, sample size, cost, reliability) assays of gene expression are obtained with the eSystem compared to quantitative real-time RT-PCR assays. Highly sensitive gene expression measurements without the complexity or cost of existing methods have been proven possible with new small sensor-area microplates that require very small volumes of samples and reagents. This advanced electrochemical technology will be further developed for cancer research and diagnostics, and then extended to other human health products. When disease-related gene expression products reach clinical use in an estimated 5-7 years, markets will likely grow to exceed a billion dollars/yr for each major disease. We intend to reach a strategic share of the market in research and testing with the aid of proven technology and strategic partnerships. ? ? ?