Figure 1. Principle of the NCCIM assay for evaluating neural progenitor cells.

(a) layout of the fully assembled NCCIM. The MIST array is fabricated on the surface of a sticky tape which is placed on a glass slide. A PDMS replica carrying microchambers is mated with a MIST array with a mechanical clamp (not shown). Cells cultured in the microchambers release cytokines which will be captured by the antibodies on the MIST array for ELISA detection. The biotinylated detection antibodies are labeled with streptavidin-horseradish (HRP) for tyramide-Cy3 assisted signal amplification. (b) Procedure to identify the type of secreted proteins detected on individual microbeads. After protein detection, the fluorescence intensity of each microbead is quantified as the cytokine expression level. 0.5M NaOH is added to the array to dissociate the hybridized DNAs and release the antibodies and the captured proteins. Only single-stranded oligo DNAs are left on the MIST array. In each of 4 cycles of the decoding process, a cocktail of fluorophore tagged oligo DNAs is applied, and the same array is imaged by a fluorescence microscope. Then the hybridized DNAs are dissociated before the next cycle. Once all the images are overlapped, the sequence of fluorescent colors on each microbead is obtained, which corresponds to a specific type of protein. Such reiterative hybridization and dissociation enable 10-plex detection of cytokines.