Cross-talk-free simultaneous fluoroimmunoassay of two biomarkers based on dual-color quantum dots

In this article, we demonstrate the fabrication and simultaneous fluorescent detection of two biomarkers related to lung cancer. Polystyrene microspheres (PSM) were introduced as biomolecular immobilizing carriers and a 96-well filter plate was used as the separation platform. The whole experiment could be effectively carried out in a homogeneous system, as exemplified by the detection of carcinoembryonic antigen (CEA) and neuron specific enolase (NSE). First, two capture antibodies for CEA and NSE were immobilized on the PSM surface. Next, they reacted successively with two antigens and two modified detection antibodies. Finally, these two biomarkers could be recognized by streptavidin-conjugated quantum dots (QD) and goat-anti-FITC conjugated QD with a detection limit of 0.625 ng mL−1, which was lower than the clinical cut-off level. The protocol showed good precision within 6.36% and good recovery in the range of 90.86–105.02%. Compared with several other assay formats reported previously, our new technique is competitive or even better. Furthermore, the immunosensor was successfully illustrated in 20 serum samples. Overall, this new immunoassay offers a promising alternative for the detection of biomarkers related to cancer diseases, taking advantage of simplicity, specificity, sensitivity and cost-efficiency.

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► A new cross-talk-free duplex fluoroimmunoassay for cancer related biomarkers was developed using multiple QD as detection elements with the LOD of 0.625 ng/mL.
► A fast homogeneous immunoreaction as well as a simple heterogeneous separation process was achieved by the coupling of the submicrometer-sized polystyrene microspheres as the carrier and the 96-well filter plate as the reaction and separation container.
► This new approach could also be extended to detect other biomarkers relating to other cancers, such as alpha fetoprotein and prostate specific antigen associated with liver cancer and prostrate cancer, etc.