Assay’s methodology and experimental setup. (A) The methodology followed in our automated ELISA on-chip assay included the steps listed below: (1) blood collection and serum extraction; (2) on-chip immobilization of viral antigens (SARS-CoV-2 spike protein); (3) pumping serum samples through the microfluidic device (if present, anti-SARS-CoV-2 antibodies interact with the immobilized SARS-CoV-2 spike proteins); (4) pumping anti-IgG-HRP through the microfluidic device to detect IgG antibodies; and (5) analysis of the colorimetric reaction, either by recovering the resulting reaction and performing the analysis with a microplate reader or by carrying out a color intensity analysis using ImageJ software. (B) Diagram of the experimental setup assembled for our automated ELISA on-chip. The components are: (1) software that controls all the peripheral devices of the setup; (2) flow control unit; (3) ELISA reagents (wash buffer, blocking buffer, and anti-IgG-HRP conjugate antibody); (4) 12/1 bidirectional microfluidic rotary valve; (5) serum samples; (6) microfluidic valve controller; (7) pinch valves; (8) 3/2-way switching valves; (9) microfluidic device; and (10) connections between components and reservoirs (color code: blue is for pneumatic connections, green is for fluid connections, and black is for electrical connections).