SEMI-SIMOA: A Chip-scale Flow-based Single-Molecule Assay at the Point-of-Care using Semiconductor Technology

Project Summary Over the past two decades, single-molecule assays (SIMOA) have transformed the detection landscape for low- abundance protein biomarkers, such as cytokines and neurological markers, across a vast dynamic range. Sourced from a variety of samples, these assays provide exceptional capabilities that enhance our knowledge of diseases and fine-tune diagnostic methodologies. While various platforms have been developed and commercialized, they invariably involve intricate workflows and depend on highly specialized, bulky equipment. Such complexities confine the use of this technology to centralized labs, necessitating controlled environments and skilled operators. To meet these challenges, we introduce a compact SIMOA system that seamlessly integrates electronics and sub-µm sized microfluidics within a single integrated circuit (IC) chip, produced by semiconductor foundries. The creation of the microfluidics leverages a unique single-step wet-etching process, which facilitates the efficient integration of thousands of fluidic and electronic channels onto a compact, millimeter-sized CMOS chip. Drawing from the principles of flow-cytometry-based and nanopore-centric SIMOA platforms, our system electronically counts individual sandwiched immunocomplexes as they pass through the on-chip 500-nm pores. Notably, the IC itself stands as the main instrument, enabling results to be directly viewed on a personal device, eliminating the need for a reader. The project objective will be achieved by four specific aims: (1) Aim I will focus on implementing the CMOS- embedded microfluidics, the sensing on-chip pores, integrated on-chip electrodes, and the magnetic separation module. (2) Aim II will focus on developing resistive-pulse-sensing low-noise readout circuits and signal processing units for digitization and identification of the binding events. The integrated microfluidics/electronics system will be packaged, tested, and validated. (3) Aim III will focus on the functionalization of nanoparticles using target-specific antibodies. We will target three biomarkers, ɑ-synuclein, NfL, and GFAP, that are related to neurodegenerative diseases. (4) Aim IV will optimize the workflow and validate the assay by benchmarking with the established commercial SIMOAs. This project holds significant promise; its successful completion could transform future diagnostics for many diseases that require detecting low-abundant biomarkers, particularly benefiting settings with limited resources.