Low-Cost Platform for Ultrasensitive Fluorescent Immunoblot

Abstract Western blot assays are fundamental tools used in biological and biomedical laboratories around the world. They have played a role in some of the most important discoveries of the last century with regards to diseases such as AIDS, Alzheimer’s, cancer, and drug-resistant infection to name just a few. As we “dig deeper” into the complex biomolecular networks that define living organisms, however, our requirements for our tools become increasingly demanding. For example, we now want to examine not simply the presence or absence of a particular protein in a sample, but would like to know the absolute concentration of that particular protein, the concentration of any post-translationally modified variants of that particular protein, and the concentration of that particular protein relative to other particular proteins. Many relevant proteins, and particularly their post- translationally modified variants are present at concentrations that are quite low. Western blot assays, and, in particular, fluorescent Western blot assays are an ideal tool for performing the measurements needed to “dig deeper” because they are capable of detecting and quantifying multiple specific proteins simultaneously, even if the proteins are of a similar size, such as is the case with post-translationally modified variants. Unfortunately, fluorescent Western blots suffer from feeble signal due to currently available fluorescent probes, restricting the absolute amount of protein which can be interrogated using fluorescent Western blots. Our product, which we call a Plasmonic Fluor (PF) and which is the subject of the present grant application, is an ultrabright fluorescent reagent designed to improve the detection sensitivity of fluorescent Western blots by orders of magnitude without requiring any changes to assay workflow or hardware. Since a major limitation of current fluorescence Westerns is the cost of the reader, we have also created a prototype reader which is incredibly inexpensive relative to current readers, but which can actually outperform them when combined with our PF technology. We will create plug-and-play PF reagents that are spectrally equivalent to the current state of the art LI-COR’s 680LT and 800CW. We will demonstrate that we can offer significantly enhanced performance at lower cost than existing techniques for this important methodology.