The purpose of the present project is to demonstrate a new type of portable analyzer and the feasibility of performing multiplex immunoassays utilizing this platform technology. The multiplex capability is made possible by using small, electronic p-Chips. The initial proof-of-concept application will be an immunoassay determining the concentration of antibodies to dengue virus (DENV) in serum or plasma samples. The assay will be designed to rapidly determine whether a patient has been previously infected with one of the four viral serotypes. This information is crucial as pre-existing immunity to DENV can drastically worsen disease progression and ultimately disease outcome upon re-exposure to heterologous viral serotypes. The long-term goal is to provide a portable assay platform for multiplex immunoassays that analyze key biomarkers for a variety of diseases. The advantages of this system are rapid assay speed (turnaround time from drawing a patient's blood sample to the reporting of the results), minimal sample handling and low cost for both the analyzer and consumables. The electronic chips are laser light-activated microtransponders (p-Chips) that are small (600 m x 600 m x 100 m), chemically inert and highly stable. p-Chips, each of which contains a unique serial number, are derivatized with probes, in this case antibodies to specific protein biomarkers, and then incubated with a sample and binding detected with fluorescent reagent(s). A key feature of p-Chips is that they are passive devices that become active only when directly illuminated with pulsed light, thus enabling them to be individually read while spinning in a closed, rotating tube exposed to a focused excitation laser beam. The readout relies on the simultaneous determination of the intensity of fluorescence and the chip serial number (ID), which identifies the binding probe. Multiplex assays are constructed by placing different probes on p-Chips within the same test tube in a manner that lends itself readily to reading dozens to hundreds of p-Chips and reporting results in a very short period of time. The main goals of this project are to (1) build a prototype reader and develop software that will both control the device and analyze the data; and (2) demonstrate and characterize a one-tube-one-step-no-wash immunoassay for detecting dengue virus. The results for the proposed approach will be a novel system for testing the exposure of a patient, either recently or in the past, to the dengue virus. The system will be composed of the portable analyzer (Cyclone) and the assay configured on p-Chips quantifying four serotypes of the anti-dengue Abs in serum. The system can be expanded easily to include additional biomarkers of interest.
Current methods for performing multiplex assays require complex and expensive instrumentation. This project introduces a new type of portable analyzer and reagent scheme that will significantly reduce the complexity, time and cost to perform clinical diagnostic tests in many areas, including infectious diseases; blood, heart and lung diseases; and cancer. The first application of this new system will be an immunoassay to detect pre- existing immunity to dengue virus infection.
