Elements and systems for ultrasensitive protein analysis

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The single most significant factor determining the outcome of many diseases, and notably cancer, is disease progression at diagnosis. Unfortunately, diseases are typically only detected at an advanced stage, after symptoms appear. A hallmark of diseases is the dysregulation of proteins in cells, and which are released into the blood, and can be used as biomarkers for disease diagnosis. It follows that it might be possible to screen and diagnose diseases early if protein indicators of disease can be discovered. Current blood tests used in the clinic detect a protein constituting as little as one part in 10 billion of blood proteins. However, to discover biomarkers for early diagnosis, technologies that can measure multiple proteins simultaneously and are a thousand to a million times more sensitive are needed. Ultrasensitive protein analysis technologies have emerged recently, but they are complicated, can only operate in the lab, and multiplex protein measurement is either not feasible, or only possible at the expense of sensitivity and reliability because the assay design did not take into consideration cross-reactivity among multiple reagents. Here, we propose to develop new technologies for ultrasensitive, multiplex analysis of proteins for biomarker discovery and for point-of-care testing. The work will be organized along three axes: (i) A digital nanodot array that comprise millions of nanoscale-dots that each can capture proteins will be made using a low-cost patterning method we developed. Captured proteins will be detected with single-molecule sensitivity, and counted. We introduce a novel noise suppression algorithm based on spatial discromination that can enhance assay sensitivity and robustness. (ii) A novel microbead-based assay with colocalized immobilization of reagents will be developed. Beads will be added to the sample, and mixed, thus “scooping” up all the proteins, which will then be detected. Both (i) & (ii) are designed with multiplexing in mind and are designed to suppress cross-reactivity. (iii) We will develop 3D printed capillary microfluidic circuits that are designed from a library of elements, akin to electronic circuits, and can be made within a few hours by rapid prototyping. These capillary microfluidics are self-powered and programmable, and will be developed into low cost, ultrasensitive test for laboratory and point-of-care analysis. This program is poised to significantly impact assay science and technology, and benefit human health. This grant will also provide annual support for training of 5 undergraduate and graduate students within a transdisciplinary environment encompassing nanotechnology, healthcare and commercialization, and train tomorrow’s scientists, entrepreneurs, and leaders.