Development Of Digital ELISA Microfluidic Chip And Its Application On Protein Biomarkers Detection

Quantitative detection of small numbers of biomolecules at low concentrations is of great interest for health monitoring,biological applications,and the clinical diagnosis of disease.Enzyme-linked immunosorbent assay(ELISA)is widely used for the detection of protein biomarkers,as it amplifies the signals by using enzymes conjugated with antibodies.The conventional ELISA has a limit of detection of about 10-12 M concentration of the target,which compromises on detecting the samples with biomarkers at low concentrations of 10-16 to 10-12 M.Digital ELISA was developed for the detection of single molecules of proteins,in which each protein biomarker-enzyme complex was isolated into ultra-small reactor volumes(fL).Since the fluorescent product of the enzyme-substrate reaction is confined to a small volume rather than being free to diffuse into a large volume,it is possible to detect a single biomarker-enzyme complex using a conventional fluorescence microscope.A novel microfluidic chip employing power-free femtoliter-sized arrays was developed for the detection of low concentrations of protein biomakers by isolating individual paramagnetic beads into single wells.Arrays of femtoliter-sized wells were fabricated with polydimethylsiloxane(PDMS)using well-developed molding techniques.After capture the targets,beads was suspended 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.The use of power-free femtoliter-sized PDMS arrays greatly simplifies the loading of beads and the isolation of single beads in femtoliter volumes.This chip is low-cost in manufacturing and do not need complex surface modification.Then,to test the single-molecules detection ability of the chip,biotinylated ?-galactosidase(B?G)was detected by first capturing it with streptavidin-coated superparamagnetic beads.Since one million wells allowed us to simultaneously examine roughly 6 ×105 beads for the presence of biomolecules,the LOD of this chip achieved 930 zM for the detection of B?G.Moreover,digital ELISA of tumor necrosis factor-?(TNF-?)showed a LOD of?50.48 fg/mL(2.88 fM).One limitation of digital measurements is that the dynamic range is limited by the number of digital units in the assay.To improve the actual use value of the power-free PDMS femtoliter-sized arrays,we developed a digital-analog readout micro fluidic chip for highly sensitive detection with dynamic range.The chip is comprised of two region,digital readout region and analog readout region connected by an inverted triangular bell-mouth channel.The digital-analog readout chip achieved a dynamic range?8 orders of magnitude for the detection of B?G and the LOD down to 2 aM.And The digital-analog readout chip achieved a dynamic range?7 orders of magnitude for the detection of prostate-specific antigen(PSA).In summary,in this paper,we developed a novel power-free PDMS femtoliter-sized arrays and a digital-analog readout microfluidic chip for the detection of protein biomarker that combine high sensitivity of digital assays with large dynamic range with an end-point readout.Since the chip is cost-effective,simple operation,good performance on detection,we expect it can become a new chip-based digital immunoassay platform for diagnostic of biomarkers.