Sequenece-specific detection of proteases using electronic p-Chips in multiplex format

DESCRIPTION provided by applicant The main goal of this project is to demonstrate the feasibility of combining novel cancer testing using F rster resonance energy transfer FRET with an emerging platform for clinical diagnostics The intent is to develop an assay capable of detecting and monitoring bladder cancer BlCa using a urine sample The assay targets a family of matrix metalloproteinases MMPs and hyaluronidase HA ase The presence and relative distribution of these enzyme markers is strongly correlated with progression of BlCa The principle of the assay involves the preparation of immobilized peptide substrates that are dual labeled with fluorescent dyes and the determination of the rate at which they are cleaved by MMPs or HA ase present in a patient sample This will be done by measuring the extent of FRET between the two fluorescent moieties linked through the target substrates for each respective MMPs or HA ase In order to differentiate between sequences each substrate will be immobilized on different p Chips which are ultra small integrated circuits with unique serial numbers Invented by PharmaSeq Inc each p Chipandapos s distinctive identification number ID is transmitted to an analyzer when the p Chip is illuminated with laser light The association of a specific substrate with an ID allows construction of a p Chip mounted database in which the identity of a target can be determined by the ID of the p Chip on which it resides Multiple p Chips carrying different substrates for different enzymes can be mixed together in a single assay andquot multiplexingandquot Results are derived by sequentially measuring the fluorescence and obtaining the ID of each p Chip in the assay This is accomplished by passing the p Chips through a flow based reader andquot Simuplexandquot also manufactured by PharmaSeq Sensitivity of the assay can be increased using modified p Chips onto which a silver nanoparticle film has been deposited prior to peptide dye conjugation The benefit of the proposed work is the creation of a highly sensitive multiplex enzymatic assay capable of simultaneously detecting low concentrations of several enzyme biomarkers that are indicative of cancer The research plan involves an evaluation of the suitability of the silver nanoparticle film prepared on the p Chip for maximum fluorescence enhancement and the application of the selected silver nanoparticle configuration toward enhanced detection of metastasis markers for bladder cancer We will use the capability of the PharmaSeq Simuplex flow reader to simultaneously excite and detect fluorescence at two wavelengths suited for two dyes Cy and Cy and evaluate the FRET ratio changes resulting from the release of a target fragment in the substrate cleaving process Our final goal is to take advantage of the metal fluorophore interactions and develop a highly sensitive assay on the p Chip platform to simultaneously detect the activity of MMP MMP as well as HA ase in the urine of bladder cancer patients being monitored for disease progression PUBLIC HEALTH RELEVANCE Tumor markers which can be found in the blood urine or tissues are produced by malignant cells and or by host tissues in response to invasion i e tumor associated inflammatory cells and then released into the bloodstream In the current project we propose to develop a non invasive ultra sensitive enzyme based test for three metastasis biomarkers in the urine of bladder cancer patients Procedures developed during this project have the potential to provide rapid and reliable quantitative diagnostic information that will allow diagnosis and monitoring of bladder cancer and will help guide the selection of proper medical therapies leading to an improvement of the health outcomes for affected patients