Project Summary We propose to further develop, refine, and validate our emerging free-solution assay (FSA) technology and our relatively new compensated Interferometric reader (CIR).1,2 The development of FSA-CIR addresses a significant void, a blind spot in cancer research because it represents the only label-free, solution-phase, ultra- sensitive, enzyme-free, technology compatible with essentially any matrix. Unlike existing tools, FSA-CIR has been shown to be useful for; a) mechanism of Action (MOA) studies on unadulterated/unmodified targets and probes with no relative mass sensitivity, b) full-length membrane protein interaction studies in native matrix, c) defining allosteric modulation and weak protein-protein interactions, d) accelerating quantitative assay development, e) potentially addressing biomarker discovery/validation bottleneck, f) performing quantitative interactions across the matrix spectrum on a single platform and g) enabling ex-vivo measurements to guide first-in-human dose determinations (FIHD) (see Pfizer letter). FSA-CIR is a paradigm shifting technology based on a novel molecular interaction transduction method with fluorescence-level sensitivity, and capabilities for targeting, probing, and assessing molecular and cellular features of cancer biology, as well as improving early detection and screening, clinical diagnosis. FSA is mix-and-read, agnostic to the molecular interaction pair and compatible with complex matrices, making it uniquely applicable in both the basic and clinical cancer research arenas. CIR represents a major advancement in interferometric sensing, due to an unprecedented level of sample-reference compensation CIR is operated without external thermal control, a unique feature for a refractive index (RI) sensor with <10-7 RIU sensitivity. The optical engine in CIR is unique, patented and quite simple, consisting of a diode laser, capillary tube, mirror and detector. When combining the interferometer with a droplet generator for sample introduction, CIR facilitates quantification of molecular interactions without relative mass dependence, at picomolar sensitivity and allows good sample throughput (50 serum sample-reference pairs run in quintuplet, [5 replicate droplet pairs], plus calibrations in a day. Feasibility of our assay methodology is demonstrated for mechanism-of-action (MOA) studies, quantification of drug target engagement as needed in theranostics and ultrasensitive, volume constrained, biomarker assay development, and target quantification. Data indicate FSA-CIR has the potential for widespread applicability and adoption throughout the scientific community and is mature enough to be an R-33 project. At project completion we aim to provide the scientific and medical community with a user-friendly platform technology for biochemical mechanism of action studies, to aid in improving cancer prognostics, and the ability to measure properties such as molecular and/or cellular mechanisms important in cancer.

Public Narrative Under this project, we will further develop and validate an emerging platform technology that can be used to measure molecular interactions, in complex matrices, such as serum and urine, without altering the sample and at unprecedented sensitivity. Preliminary observations indicate that the Mix-and-Read, free-solution assay methodology can be used widely, with the potential to accelerate and/or enhance research in the areas of drug discovery, cancer biology, early detection and screening, and clinical diagnosis.