DNA Barcode Chip Patterning and Validation.

DNA barcode patterns are prepared as described elsewhere (7, 8) with slight modifications. After bonding of PDMS device to the PLL slide, 0.1% PLL solution (Sigma-Aldrich) flowed through the channels followed by air blow dry. Then a library of 5′-amine modified ssDNA was dissolved in a mixture of DMSO and deionized water (vol/vol = 2:3) with a final concentration of 300 μM and mixed with a 2 mM PBS solution of BS³ linker (Thermo Fisher) at 1:1 ratio (vol/vol). A library of freshly prepared DNA solutions were flowed into different channels, and the assembly was incubated at room temperature for 2 h. The glass slide was then separated from the PDMS slab and washed with 0.02% SDS solution and water. Each DNA barcode slide was validated before bonding to the single-cell assay chip. A small area close to the edge was validated to check the DNA loading and uniformity. To do this, fluorescent Cy3-labeled cDNA mixture in 1% BSA (Sigma-Aldrich) in 1× PBS (Irvine Scientific) were hybridized for 1 h. After washing three times with 1% BSA/PBS and PBS, the slide was dried by nitrogen gun and scanned by Axon GenePix 4400A. Under laser power of 15% and gain of 450, fluorescence intensity above 30,000 was acceptable for cytoplasmic protein detection at the single-cell level.