Quantification of molecular interactions across the matrix spectrum enables cancer research.

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.

Grant Number: 5R33CA291185-02
NIH Institute/Center: NIH
Principal Investigator: DARRYL BORNHOP