Dual-color fluorescence and homogeneous immunoassay for the determination of human enterovirus 71

We have developed a new fluorescent immune ensemble probe comprised of a conjugated lower toxic water-soluble CdTe:Zn(2+) quantum dots (QDs) and Ru(bpy)(2)(mcbpy-O-Su-ester)(PF(6))(2)- antibody complex (Ru-Ab) for the dual-color determination of human Enterovirus 71 (EV71) in homogeneous solution. EV71 monoantibody was easily covalently conjugated with Ru(bpy)(2)(mcbpy-O-Su-ester)(PF(6))(2) to form a stable complex Ru-Ab, which acted both as an effective quencher of QDs fluorescence and the capture probe of virus antigen EV71. Herein, the target EV71 can break up the low fluorescent ionic ensemble by antigen-antibody combination to set free the fluorescent QDs and restore the fluorescence of QDs whereas the fluorescence intensity of Ru-Ab remains the same. Thus, the determination of EV71 by the complex Ru-Ab and QDs can be realized via the restoration of QDs fluorescence upon addition of EV71 and even can be directly evaluated by the ratio of green-colored QDs fluorescence intensity to Ru-Ab red-colored fluorescence intensity. The green-colored fluorescence of QDs was very sensitive to the change of EV71 concentration, and its fluorescence intensity increased with the increase of EV71 concentration between 1.8 ng/mL and 12 μg/mL. With this method, EV71 was detected at subnanogram per milliliter concentration in the presence of 160 μg/mL bovine serum albumin. More importantly, this strategy can be used as a universal method for any protein or virus by changing conjugated Antibodies in disease early diagnosis providing a fast and promising clinical approach for virus determination. In a word, a simple, fast, sensitive, and highly selective assay for EV71 has been described. It could be applied in real sample analysis with a satisfactory result. It was notable that the sensor could not only achieve rapid and precise quantitative determination of protein/virus by fluorescent intensity but also could be applied in semiquantitative protein/virus determination by digital visualization.