Ultrasensitive Detection of Urine LAM for Point-of-Care Diagnosis of All Forms of Tuberculosis

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Project Summary Tuberculosis (TB) remains one of the most challenging diseases with ten million new cases and two million deaths yearly, and severe problems due to underdiagnosis and inadequate testing. A point-of-care (PoC) test for all kinds of TB that is sensitive, quick, simple, and not sputum-based is urgently needed. The most promising avenue to accomplish this is an immunoassay based on the TB biomarker Lipoarabinomannan (LAM). Fluxus Inc.’s long-term goal is to develop a novel suite of ultrasensitive, target-agnostic, low-complexity diagnostic instruments based on optofluidic single-molecule technology that combines integrated optics and microfluidics on a single chip-based system. The objective of this project is to develop the first PoC compatible system for detection of LAM in urine for TB diagnosis in collaboration with FIND (Foundation for Innovative New Diagnostics), the world’s leading non-profit organization for diagnostics for poverty-related diseases. The test will use single protein detection to achieve the sensitivity of PCR with the relative simplicity and speed of an immunoassay. Phase I will introduce the innovative elements at the core of the project and result in the demonstration of the critical performance parameters for a PoC test. Our objectives will be accomplished by completing the following specific aims that introduce new innovative concepts: (1) Demonstration of an ultrasensitive LAM assay; (2) Introduction of grating coupled waveguides on the standard optofluidic architecture; and (3) Integration of an optimized sample-preparation process. Under the first Aim, we will show that LAM can be identified and quantitated with single-molecule analytical sensitivity. Specifically, we will develop a bead-based solid-phase extraction assay using best-in-class antibodies (provided by FIND) and bright fluorescent nanoparticle reporters. An analytical limit of detection of 1 pg/mL and a dynamic range of 5 logs will be demonstrated – 500x more sensitive than the only available LAM assay (Alere LF-LAM). The second Aim introduces a new approach using a grating couple to deliver light into the optofluidic chip. This method is optimized for mass fabrication and PoC instrument complexity and cost. We will adapt the current chip fabrication process to incorporate the grating coupler and demonstrate 20% light coupling efficiency. Under the third Aim, we will introduce a disposable pre-prep cartridge for single-step urine LAM extraction/separation across a fluid-fluid interface with at least 90% capture efficiency. The proposed work is significant because it will lead to the first ultrasensitive LAM assay for TB diagnosis from urine at the PoC. The test will have the specificity and sensitivity of PCR detection, but at the low complexity and cost of an immunoassay, and for diagnosis of all kinds of TB. This concept will then serve as the blueprint for a broader class of single-molecule diagnostic instruments for rapid pathogen and biomarker detection. 1