*2.1. Device Fabrication*

The reported microfluidic device (Figure 1a) consists of three layers of microstructures made of polydimethylsiloxane (PDMS; Dow Corning, Midland, MI, USA): a control layer (height: 20 μm), a flow layer (gas channel height: 20 μm) and a micro−grating layer, with the design layout as illustrated in Figure 1b. The device was fabricated by multilayer soft lithography [25] and the required replica molds were fabricated by photolithography, as described in Figure S1 in the Supplementary Material.

**Figure 1.** (**a**) Representative microscopic image of a microfluidic immunoassay device integrated with an ECM protein-coated topologic environment. Inset: microscopic image of the parallel grating array in cell culture chamber (grating width: 18 μm, the depth: 18 μm). Scale bar: 100 μm. (**b**) The integrated microfluidic immunoassay device microchannels in the control layer consist of two functional units: valves (*cyan*) for micromixers and valves (*red*) for flow control. The flow channels are composed of an array of microchambers (*green*) for microbead trapping, connecting channels (*black*) between the cell culture region and the downstream cytokine detection arrays. Specially, the connecting microchannels surrounding cell culture chamber contained multiple micro-gaps (height 4 μm; width 50 μm) to allow liquid flow while simultaneously confining the cells in the culture chamber. Scale bar: 4 mm. (**c**) Schematic of the integrated microfluidic immunoassay device operation. Immune cells (macrophages) and nasopharyngeal cells (NPC) were co-cultured in the grating array-embedded cell culture chamber, and the media with secreted cytokines from the cell culture chamber were extracted and incubated with pre-loaded antibody-conjugated microbeads for capturing cytokines in one of the cytokine detection-microchamber. Fluorescent-conjugated detection antibodies were used to label the captured cytokine for microscopic imaging-based multiple cytokine quantification.

The layout of the microfluidic device was designed with Adobe Illustrator CS6 software. Plastic photomasks for each layer were printed by Newway Photomask, Inc. for fabricating the molds. The mold of parallel micro-gratings (width: 18 μm; depth: 18 μm) was fabricated by deep reactive ion etching with an AZ50XT positive photoresist (AZ Electronic Materials, Somerville, NJ, USA) as the sacrificial layer. On the other hand, molds of the control layer and the flow layer were also fabricated by photolithography of SU−8 photoresist (SU−8 2010, Microchem, Westborough, MA, USA) on planar silicon wafers. Microchannel patterns of the flow layer mold were fabricated with AZ50XT with a height of 20 μm, reflowed at 120 ◦C for 1 min, while the microchambers and the filtering microstructure around the cell culture region (height: 30 μm) of the mold were aligned with the microchannel patterns and fabricated with an SU-8 2010 photoresist (Microchem, Westborough, MA, USA). All the molds were then silanized by trichloro (1H, 1H, 2H, 2H-perfluoro-octyl) silane (Sigma-Aldrich, St. Louis, MO, USA) for 12 h to facilitate the release of the molded PDMS layers in the later procedures.

Afterwards, PDMS pre-polymer was prepared by mixing the base and the curing agent with a weight ratio of 10:1. The microfluidic device with multiple PDMS layers were fabricated by replica molding of PDMS from the molds using the multiplayer soft lithography as described in Figure S1, with the PDMS applied on the molds with different thickness: 5 mm by pouring on the control layer mold, 35 μm by spin-coating over the flow layer mold, and 1 mm by pouring on the micro-grating mold. All the following bonding processes between PDMS layers were then achieved by air plasma (energy 10 kJ; Harrick plasma cleaner PDC-002, Ithaca, NY, USA). According to the layout of the microfluidic device, as shown in Figure 1a, the PDMS control layer was aligned and bonded onto the flow layer, with holes punched at the gas/liquid inlets and outlets (diameter: 1 mm; WHAWB100073, Sigma-Aldrich, St. Louis, MO, USA), followed by punching a hole (diameter: 6 mm; WHAWB100082, Sigma-Aldrich, St. Louis, MO, USA) at the cell culture region. The PDMS substrate was then bonded on the micro-grating layer, with the micro-grating structures (Figure 1a and Figure S1 in the Supplementary Material) facing the culture chamber. The culturing region was further covered by bonding with another PDMS layer (thickness: 5 mm), with two punched holes as the sample inlet and outlet. Finally, the entire multilayer PDMS substrate was bonded onto a glass slide (Cytoglass, Nanjing, China) for physical support.
