(150a) Power and Lithography Free Highly Sensitive Digital Protein Detection Platform Using an Inexpensive Track-Etched Polycarbonate (PCTE) Membrane

Absolute and accurate quantification of several protein biomarkers present in a small volume of blood, cerebrospinal fluid, saliva, or urine can enable early disease diagnostics, prognosis and monitoring of further treatment in patients. Current state of the art technique for protein detection is Enzyme-Linked Immunosorbent Assay (ELISA) which suffers from low limit of detection (~nM-pM) beyond which several clinically relevant protein biomarkers (~pM-fM) of cancer, neurodegenerative, cardiovascular, inflammatory and autoimmune diseases remain undetected. In order to solve this issue, single-molecule arrays (Simoa) have been developed in which a protein molecule is sandwiched between an antibody coated magnetic beads and an enzyme conjugated detection antibody to form an immunocomplex. Subsequently, the immunocomplex is sampled into thousands of fL reaction chambers keeping the total protein concentration within the Poisson limit such that each bead contains either one or zero molecule. The fluorescence signal generated from the enzymatic reaction gives a digital readout providing an absolute quantification of the protein biomarker. However, Simoa are mostly performed in specially designed microwells which requires complex and expensive cleanroom based microfabrication techniques, costly and bulky experimental setup, fluidics control setups such as syringe pump, centrifuge, vacuum, or other specialized equipment’s. Also, the total number of microwells present in the device which determines the sensors dynamic range is limited by the master mold which is cumbersome to tune. Moreover, these complex workflows require good amount of expertise and present a bottleneck for using them for point of care applications in resource limited areas where a use and throw sensor is preferable. In order to solve some of the abovementioned issues, we report a novel user friendly, power and clean room lithography free digital protein detection platform using an inexpensive, use and throw track-etched polycarbonate (PCTE) membrane.

Commercially available PCTE membranes are manufactured by ion irradiation in a nuclear reactor and have a high density (~10⁶ pores/cm²) of uniformly sized cylindrical through holes at various desired hole diameters with low protein binding properties. The through pores of a wetted membrane completely filled by capillary action can be easily converted into microwells when sealed on a sticky surface without generating any air bubbles which was confirmed from confocal microscopy; thus avoiding the use of vacuum pumps. Subsequently, when 2.8 µm sized magnetic beads are settled down by gravity on the 5 µm diameter membrane, less than 10% of the microwells are filled with magnetic beads. Single magnetic beads filling efficiency was further increased to more than 80% by keeping a magnet at the optimized distance at the bottom and mechanically shaking it. The microwells can then be easily isolated from each other by sealing with an oil phase making sure each microwell acts as an individual reaction chamber. A single enzymatic reaction produces a fluorescent product which is detected from standard fluorescent microscopy within 45 minutes as the reaction product is concentrated in a ~300 fL microwell reactor. Magnetic bead containing microwell that lights up will denote it’s ‘on’ state and similarly, the ones that do not show any signal will denote ‘off’ state. The ratio of the enumerated ‘on’ and ‘off’ microwells using a custom written image analysis code correlated linearly with the protein concentration. Using this digital sensor, we achieve a dynamic range of pM-fM with a limit of detection of sub fM for biotin conjugated enzyme on streptavidin functionalized magnetic beads. This digital ‘on’ and ‘off’ readout format circumvents any bias that usually occurs in other analog sensors based on absolute intensity, current and voltage measurements.

The same device design was further developed to digitally detect specific extracellular vesicles (EVs) which are heterogeneous membrane-bound vesicles and are actively recognized as one of the most promising as well as stable biomarkers for liquid biopsy. Two color coded magnetic beads were functionalized with two different monoclonal antibodies respectively to capture EVs based on their surface protein from a 100ul sample. Then, the fluorescence signal generated from the enzymatic reaction is used to enumerate specific tumor-derived EVs based on the color of magnetic beads. Collectively, the inexpensive (< $0.1 per membrane) and user friendly PCTE membrane based digital ELISA can provide great platform for digital quantification of single protein biomarkers in a lithography and power free manner thus enabling point of care applications in laboratory resource constraints areas.