Supplementary Figure 4: PDMS Surface Chemistry Functionalization Protocol and Differential Functionalization of Microwell Arrays
From: Seq-Well: portable, low-cost RNA sequencing of single cells at high throughput
(a) The surface of the PDMS device is initially treated with an air plasma under mild vacuum, terminating the surface in hydroxyls. This PDMS surface is aminated using (3-Aminopropyl)triethoxysilane (APTES). The amine surface is then activated with PDITC to create an isothiocyanate surface. The isothiocyanate on the top surface of the array (negative space) is covalently linked to chitosan polymers through their amine group. The hydrophobicity of the isothiocyanate surface prevents solvation of the microwells with the aqueous chitosan solution, preventing chitosan from reacting with the inner well surfaces (positive space). These surfaces are subsequently reacted with the free amine of poly(glutamic) acid polymers under vacuum to drive the solvation of the wells. (b) The top surface of a PDITC-activated array was coated with streptavidin-PE (red) and the inner well surfaces were coated with streptavidin-AF488 (green) using same method used to functionalize with chitosan and poly(glutamate). (c) Two chitosan/poly(glutamate) bifunctionalized arrays were submerged in MES buffer without (Panel 1) or with (Panel 2) 100 μg/mL EDC and 10 μg/mL NHS for 10 minutes. The arrays were washed and then submerged in PBS solution containing 1 μg/mL AF568-labeled antibody overnight. After washing, arrays were imaged for AF568 fluorescence.
