Simultaneous quantitative detection of multiple tumor markers in microfluidic nanoliter-volume droplets

We developed a simultaneous detection method for multiple tumor markers (TMs) in microfluidic droplets based on a multiple fluorescence resonance energy transfer (FRET) system. In this system, graphene oxide (GO) was used as the single quencher, while the multi-color quantum dots (QDs) labeled on different aptamers were employed as energy donors. When the aptamers were adsorbed onto GO due to the pi-pi stacking interaction, QDs were drawn to the surface of GO and quenched by it. Once the TMs were introduced, the corresponding fluorescence of QDs was recovered obviously owing to the preferential interaction of aptamers with the TMs. Here, the multi-FRET system was encapsulated into nanoliter-volume droplets by a simple T-junction micro-fluidic chip. The targets could be detected rapidly as the generated droplets flew through the integrated on-line detection zone. Three tumor markers, carcinoembryonic antigen (CEA), prostate-specific antigen (PSA) and vascular endothelial growth factor (VEGF(165)) could be detected simultaneously in 33 nL-volume droplets, which is only 1/3000 of the volume of the sample consumed in the conventional fluorescence spectrophotometer. In addition, the signals corresponding to different TM targets in one nanoliter-volume droplet could be read out at the same time, and the signals could be output continuously owing to the uninterruptible generation of droplets. Even with a signal acquisition frequency of 55 droplets per minute, the multi-FRET biosensing system has linear ranges of 0.50-70 ng mL(-1)for CEA, 0.25-70 ng mL(-1) for PSA and 0.50-70 ng mL(-1) for VEGF(165). The detection limits of CEA, PSA and VEGF 165 were calculated to be 0.15 ng mL(-1), 0.035 ng mL(-1) and 0.11 ng mL(-1), respectively. The method was also validated by analyzing human serum sample dilutions. The proposed multi-FRET-based system has potential to become a powerful tool for rapid, low-cost and simultaneous detection of multiple tumor markers.