(a) Exploded view of the proposed microfluidic chip. Four layers of PDMS were used to construct the negative-pressure-driven microfluidic chip. The bottom layer was an air chamber layer for the actuation of a pneumatic micromixer, the third layer was a fluidic channel layer, the second layer was another air chamber layer for the actuation of the normally closed valves, and the top layer was a flat PDMS layer that sealed the air chamber. (b) The chip was equipped with a micromixer incorporated into the reaction chamber at the center of the chip and with five reservoirs (four for sample loading and one for a wash buffer). There were six normally closed valves located between the reservoirs and the reaction chamber. A syringe filter was integrated into the antigen-loading chamber to filter out any debris in the urine samples. The driving force for fluid movement relied on a suction force provided from the waste outlet. All of the liquid handling can be performed with the aid of the integrated microfluidic components. (c) Photograph of the assembled microfluidic chip with dimensions of 40 mm × 40 mm.
Illustration of the working principles behind the bead-based ELISA using the microfluidic chip. (a) A urine sample containing the target protein and antibody-coated magnetic beads was introduced into the chip. (b) After the incubation process, the target protein was captured, and the unwanted protein was washed out. (c) and (d) The secondary antibody was bound to the antigen, and the excess antibody was washed out. (e) and (f) The enzyme was linked to the secondary antibody, and after the excess enzyme was washed out, a substrate was used to quantitatively measure the target protein.
Design and characterization of the normally closed valve. (a) Working principle of the valve. When the air in the chamber was suctioned out, the PDMS membrane was deflected, and the fluid could pass through the valve. (b) Photograph of the valve in the open and closed positions. (c) The DI water started flowing through the normally closed valve when the actuation pressure surpassed −4.2 kPa. The normally closed valve was almost fully open when the applied pressure was greater than −45 kPa.
Design and characterization of the suction force-driven micromixer. (a) Cross-sectional view of the micromixer. The mixer has two air chambers connected by a narrow air channel to delay the movement of air from one chamber to the other. When pulses of suction force were applied, the fluid above the PDMS membrane vibrated, thus enhancing the mixing efficiency. (b) Series of photographs of the mixer actuated for a period of 30 s. The ink and DI water were mixed together gradually. (c) Characterization of the mixing index of the micromixer with different negative air pressures and driving frequencies.
The detection ability of the proposed chip. (a) The detection range was up to 2000 ng ml−1 with 20 μl of magnetic beads (1 × 109 beads ml−1), and the detection linearity was 0.996 in this range. (b) The detection limit was approximately 10 ng ml−1 for APOA1 for the chip.
Comparison of the results for five patient samples obtained using the proposed chip and using traditional 96-well ELISA. The differences in the results were 0.9%, 6.8%, 9.4%, 1.8%, and 5.8% for samples 1, 2, 3, 4, and 5, respectively.
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