Multiplexed homogeneous bioassays provide an important alternative to microarrays for multiplexed analysis of biological samples, because they avoid some of assay development challenges and can greatly reduce assay turnaround time. For simultaneously detecting multiple targets in a homogeneous assay format, target-specific fluorescent labels (in various configurations, such as directly labeling and beacons) are an effective tool. Additional methods for multiplexed homogeneous bioassays, such as the Chelated Lanthanide Emission Fingerprinting (CLEF) method proposed here, would complement fluorescence methods and potentially offer advantages for some applications, such as those where filter wheels or multiple detectors are impractical due to the size or robustness requirements of the instrument. Multiplexed point of care diagnostic devices are improved by both the rapid turnaround time of homogeneous methods and the potential size and durability improvements achieved by avoiding filter wheels in the proposed multiplexing method. The CLEF assay scheme utilizes the long luminescent lifetimes of chelated lanthanides with molecular beacon technology to provide sensitive, multiplexed detection of nucleic acids. The assay takes place entirely in solution, speeding turnaround time. The CLEF assay uses luminescence-lifetime-based multiplexing;the different target-specific beacons each have different luminescent decay profiles, determined by the chelated lanthanide conjugated to the beacon. All the beacons are excited with a single LED, and all the beacons that are hybridized to their complementary target luminesce, while the energy of those that are not hybridized is quenched by a dark quencher. All emissions are read with a single emission filter and a single detector. The CLEF method is enabled by an instrument with a novel multiplexing algorithm and high-speed optics and data acquisition hardware. The CLEF algorithm separates the luminescent decay into the components contributed by each beacon, determines which beacons are signaling and which are quenched. In this 6-month R43 Phase I, work is organized around two specific aims designed to demonstrate the feasibility of this novel homogeneous multiplexing approach - Aim 1 establishing the reporter chemicals and conjugating them to probes, and Aim 2 enhancing the high-speed instrument and extending signal processing algorithms from duplex to greater degrees of multiplexing. 1
Homogeneous bioassays provide several advantages over assays that require the target to diffuse to probes on a planar solid phase, including the rapid turnaround time and simplicity in modeling the system kinetics. However, multiplexing is notoriously challenging in homogeneous bioassays, especially those with instruments suitable for use at the point of care. This proposal focuses on the development of a novel multiplexing method designed for use in homogeneous nucleic acid tests, made possible by novel probes, an advanced detector, and innovative signal processing algorithms. Nucleic acid tests using this multiplexing method can have a rapid turnaround time and can provide information about multiple infectious diseases or genotyping of a single disease, while reducing the cost per test over single target tests.