A novel microfluidic chip employing power-free polydimethylsiloxane (PDMS) femtoliter-sized arrays was developed for the detection of low concentrations of protein biomakers by isolating individual paramagnetic beads in single wells. Arrays of femtoliter-sized wells were fabricated with PDMS using well-developed molding techniques. Paramagnetic beads were functionalized with specific antibodies to capture the antigens. These antigens were labeled with enzymes via conventional multistep immunosandwich approach. After suspending in aqueous solutions of enzyme substrate, the solutions were delivered to the arrays using a conventional micropipette. The aqueous solutions were introduced into the microwells by capillarity and the beads were loaded into microwells by gravity. A fluorocarbon oil was then flowed into the chip to remove excess beads from the surface of the array and meanwhile isolated the femtoliter-sized wells. All processes were achieved by conventional micropipette, without external pumping systems and valves. Finally, the arrays were imaged using standard fluorescence imaging after incubation 30 min for digital counting enzyme molecules. It was demonstrated that the chip platform possessed the performance of digital counting with a linear dynamic range from 1 aM to 1 fM for the detection of biotinylated β-galactosidase (BβG), achieving a limit of detection (LOD) of 930 zM. Using this chip, a digital immunoassay to detect Tumor Necrosis Factor α (TNF- α) was developed. Since the chip fabrication is low-cost and circumvents the surface modification, we expect it can become a new chip-based digital immunoassay platform for ultrasensitive diagnostic of biomarkers.
Keywords: Digital counting; Digital immunoassays; PDMS; Paramagnetic beads.
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