Figure 1
Integrated microfluidic immunosensing chip for rapid, automated and parallel cytokine detection from biological solutions.
(a) Schematic of AlphaLISA bead conjugation. Initially, analytes and AlphaLISA assay components were separated through isolation valves. Programmed micropumps promoted the first mixing step between analytes, anti-analyte AlphaLISA acceptor beads and biotinylated antibodies, before the second mixing with streptavidin-coated AlphaLISA donor beads. (b) Principle of AlphaLISA immunosensing. (c) Chip layout. The two-layered PDMS microfluidic chip was integrated with microvalves and micropumps for flow isolation, metering and pumping. The chip consisted of two main regions: a circulation array for homogeneous mixing and conjugation of cytokines (injected from eight sample ports in blue) with AlphaLISA components (streptavidin-coated AlphaLISA donor beads in red and anti-analyte AlphaLISA acceptor beads and biotinylated antibodies in yellow) and an imaging chamber array that centralized and amplified AlphaLISA fluorescent signals. Both the circulation and imaging chamber arrays comprised eight identical replicates of circulation loop and imaging chamber, respectively, allowing for eight different samples (as illustrated by different blue colors) processed and detected simultaneously. Furthermore, a programmable cleaning scheme which flushed through chip compartments with air and blocking solution (in green) via a common outlet (in purple) was integrated into the chip to help recover the chip for repeated AlphaLISA immunoassays. (d–i) Chip operation. Flow visualization and operation optimization were conducted using food dyes. Initially, compartments of the microfluidic chip were all empty (d). The circulation array was first filled with analyte solutions (in blue), AlphaLISA acceptor beads / antibodies (in yellow) and AlphaLISA donor beads (in red) (e). The first mixing step was then carried out to mix analytes and AlphaLISA acceptor beads / antibodies (f). After 20 sec of the first mixing process, solution color appeared very even, suggesting that mixing could be achieved rapidly. The second mixing was performed to mix analytes, AlphaLISA acceptor beads / antibodies and AlphaLISA donor beads (g). AlphaLISA conjugation mixtures were then transported to the imaging chamber array for detection (h). Finally, a cleaning process was carried out to remove all solutions and recover the chip to the initial clean state before next cycles were initiated (i).
