A Method for Designing Instrument-Free Quantitative Immunoassays.

Abstract

Colorimetric readouts are widely used in point-of-care diagnostic immunoassays to indicate either the presence or the absence of an analyte. For a variety of reasons, it is more difficult to quantify rather than simply detect an analyte using a colorimetric test. We report a method for designing, with minimal iteration, a quantitative immunoassay that can be interpreted objectively by a simple count of number of spots visible to the unaided eye. We combined a method called polymerization-based amplification (PBA) with a series of microscale features containing a decreasing surface density of capture molecules, and the central focus of the study is understanding how the choice of surface densities impacts performance. Using a model pair of antibodies, we have shown that our design approach does not depend on measurement of equilibrium and kinetic binding parameters and can provide a dynamic working range of 3 orders of magnitude (70 pM to 70 nM) for visual quantification.