Extracellular vesicles contain a wide range of different types of molecular cargo, such as lipids, carbohydrates, nucleic acids, and proteins. However, due to their small size (~150 nm for exomes), the number of molecules they contain is limited. For example, there can be ~20,000 protein molecules per EV vesicle. Because the proteome is estimated to consist of >100,000 different proteins, there is significantly less than a single copy of a particular protein per EV. As such, specialized techniques have been developed to search for these rare proteins, such as OLink, which uses the proximity ligation assay and quantitative PCR to detect rare events. Challenges include the complicated and long workflow and the use of expensive antibodies. To address this issue, we are developing a nanotechnology to quantify and identify single protein molecules based on peptide fingerprinting and exopeptidase digestion of single peptides.

Resistive Pulse Sensing (RPS) is a label-free and single-molecule detection approach that requires simple instrumentation to implement and as such, can be mobilized to be integrated into in vitro diagnostic assays for not only detecting but identifying key disease-associated biomarkers with high analytical sensitivity, such as proteins. Thus, RPS is a logical choice for coupling with liquid biopsy markers for the precision management of a variety of diseases due to the mass limits imposed on any assay in which a liquid biopsy marker is used. We have developed a unique measurement modality and sensor technology (dual in-plane nanopore sensor) that couples RPS to nanoscale electrophoresis. Not only does the sensor generate the typical RPS measurement parameters, but also the molecular-dependent electrophoretic mobility, which we call the time-of-flight (ToF). The RPS parameters coupled with the ToF and machine learning lead to high detection efficiency and classification accuracy of single molecules harvested from liquid biopsy markers, including proteins. Our devices, which are made from plastics via high-scale production modalities (nano-injection molding), consist of channels with dimensions ranging from 1 to 100 nm (effective diameter) that are 10’s of microns in length.

In this talk, I will discuss the operational parameters and unique applications of our dual in-plane nanopore sensor for peptide fingerprinting of single protein molecules and subsequent exopeptidase digestion of single proteins for determining their amino acid composition. The technology I will discuss involves the use of an immobilized nanoscale enzymatic reactor (INER) containing a proteolytic or exopeptidase enzyme to digest input molecules. Operational characteristics of this sensor will be shared.