Ultrasensitive Automatic Detection of Small Molecules by Membrane Imaging of Single Molecule Assays

Determination of trace amounts of targets or even a single molecule target has always been a challenge in the detection field. Digital measurement methods established for single molecule counting of proteins, such as single molecule arrays (Simoa) or dropcast single molecule assays (dSimoa), are not suitable for detecting small molecule, because of the limited category of small molecule antibodies and the weak signal that can be captured. To address this issue, we have developed a strategy for single molecule detection of small molecules, called small molecule detection with single molecule assays (smSimoa). In this strategy, an aptamer is used as a recognition element, and an addressable DNA Nanoflower (DNF) attached on the magnetic beads surface, which exhibit fluorescence imaging, is employed as the output signal. Accompanied by digital imaging and automated counting analysis, E2 at the attomolar level can be measured. The smSimoa breaks the barrier of small molecule detection concentration and provides a basis for high throughput detection of multiple substances with fluorescence encoded magnetic beads.