Ultrasensitive Detection of Attomolar Protein Concentrations by Dropcast Single Molecule Assays

Abstract

Measurements of very low levels of biomolecules, including proteins and nucleic acids, remain a critical challenge in many clinical diagnostic applications due to insufficient sensitivity. While digital measurement methods such as Single Molecule Arrays (Simoa), or digital ELISA, have made significant advances in sensitivity, there are still many potential disease biomarkers that exist in accessible biofluids at levels below the detection limits of these techniques. To overcome this barrier, we have developed a simple strategy for single molecule counting, dropcast single molecule assays (dSimoa), that enables more target molecules to be counted through increased sampling efficiency and with a simpler workflow. In this approach, beads are simply dropcast onto a microscope slide and dried into a monolayer film for digital signal readout. The dSimoa platform achieves attomolar limits of detection, with an up to 25-fold improvement in sensitivity over Simoa, the current state of the art for ultrasensitive protein detection. Furthermore, due to its simple readout process and improved cost-effectiveness compared to existing digital bioassays, dSimoa increases amenability to integration into point-of-care platforms. As an illustration of the potential utility of dSimoa, we demonstrate its ability to measure previously undetectable levels of Brachyury, a tissue biomarker for chordoma, in plasma samples. With its significantly enhanced sensitivity and simplicity, dSimoa can pave the way toward the discovery of new biomarkers for early disease diagnosis and improved health outcomes.

Framework for Rapid Comparison of Extracellular Vesicle Isolation Methods

“…We developed Simoa assays that can be used for ultrasensitive quantification of EVs in biological fluids and applied these assays to the analysis of EV isolation methods. Although several techniques have previously been applied to EV detection handling of the commercially-available Quanterix HD-X instrument, although our lab has developed other digital ELISA methods that do not rely on commercial instrumentation (13)(14)(15), which could be similarly applied to EVs.…”

“…Comparison of CSF EV recovery (top) and albumin contamination (bottom) across all tested methods ranked by EV recovery. Relative EV recovery was calculated by individually normalizing each tetraspanin to the sum of that tetraspanin in all fractions (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21) in the 10mL Sepharose CL-6B condition. The three tetraspanin percentages were then averaged to calculate the relative EV recovery.…”

Single-Particle Counting Based on Digital Plasmonic Nanobubble Detection for Rapid and Ultrasensitive Diagnostics

“…In the digital assays, increasing the total counting number benefits the sensitivity enhancement and reduces the measurement error (6) . In the DIAMOND platform, we can increase the data collection time, but this is a tedious way to acquire more data points ( Fig.…”

Trends of Bead Counting-Based Technologies Toward the Detection of Disease-Related Biomarkers

“…However, the sensitivity of Simoa is impeded by the low loading efficiency of beads, in which just about 5% of the total beads can be sampled into the microwells and analyzed (Cohen et al, 2020 ). To overcome this barrier, recently, the dropcast Simoa (dSimoa) system for the ultrasensitive protein detection by single molecule counting with a higher sampling efficiency is proposed (Wu et al, 2020 ). In this approach, instead of loading the beads into the microwells, the beads are simply dropcast onto a microscope slide for single molecule counting.…”