Fig. 1
Immuno-digital ICA (idICA) for counting Aß-bound EVs. A, B Workflow of immune-digital ICA analysis of Aß-bound EVs. Ganglioside GM1-containing EVs are captured by cholera toxin B subunit (CTB)-coated magnetic beads (MB) and then reacted with the DNA oligo-conjugated detection antibody against an exosome marker protein CD9 or Aß (A). The resultant EV–bead–antibody complex in A and substrates for ICA are loaded into the digital device, enclosed into individual microwells by fluorinated oil, and analyzed by fluorescent imaging after the ICA reaction at 66 °C for 15 min (B). C Schematic illustration of ICA. (1) Invasive oligonucleotides and probe oligonucleotides hybridize to target DNA and generate 5′-flap structures in the probe oligonucleotides, which are cleaved by FEN-1. The target DNA is conjugated to detection antibodies. (2) The cleaved 5′-flaps bind to fluorescent probes and form 5′-flap structures between a quencher molecule [Q] and a fluorophore [F]. Cleavage of 5′-flaps by FEN-1 emits fluorescence signals. Unannealed 5′-flaps are shown in blue and yellow. Arrows indicate 5′-flap cleavage by FEN-1. D The digital device used in this study. There are 100 blocks of well arrays; 10,000 wells in each block correspond to the 106 microwells on a single fabricated device. Phase-contrast image and fluorescent image of a block of well array are shown. Scale bars, 1 mm