Single Molecule Arrays (SiMoA)
Introduction
One platform being explored for the ReMeDx project utilizes a Single Molecule Array (SiMoA) microfluidic assay. This project is being developed in collaboration with Dr. David Walt’s group at Tufts University for the detection of various proteins of interest. Beads conjugated with antibodies can bind proteins from a blood sample and subsequently be reacted to form ELISA-based sandwich complexes. By loading the beads into discrete sub-femtoliter reaction wells, the signal from single proteins can be amplified in a low volume localized area to readily enable their detection. Determining the number of wells that display increased fluorescence after incubation with a fluorogenic enzyme substrate quantifies the protein concentration from the original sample. This microfluidic approach affords 1000-fold superior detection limits compared to immunoassays using conventional plate readers. Additionally, it is applicable to any protein with an available antibody pair which demonstrates the multiplexing potential of this technology.
SiMoA Strategy
• Incubate antibody-beads where number of beads >>> number of analyte molecules
• Only one or zero molecules bound per bead
• Introduce second antibody labeled with an enzyme
• Distribute beads in microwells with enzyme substrate and seal
• Measure fluorescence
• Concentration related to number of positive wells
Fluorescent images of beads in microwells
Beads in the array can be seen before (left) and after (right) sealing the wells with a fluorogenic enzyme substrate. Increases in fluorescence intensity in certain wells originate from ELISA complexes on those specific beads.