Development of a Dual Fluorescence Signal-Enhancement Immunosensor Based on Substrate Modification for Simultaneous Detection of Interleukin-6 and Procalcitonin

High-sensitivity detection of biomarkers is of great significance to improve the accuracy of disease diagnosis and the rate of occult disease diagnosis. Using a substrate modification and two-color quantum dot (QD) nanobeads (QBs), we have developed a dual fluorescence signal-enhancement immunosensor for sensitive, simultaneous detection of interleukin 6 (IL-6) and procalcitonin (PCT) at low volumes (∼20 μL). First, the QBs compatible with QDs with different surface ligands were prepared by optimizing surfactants based on the microemulsion method. Through the use of a fluorescence-linked immunosorbent assay (FLISA), the feasibility of a dual signal-enhancement immunosensor was verified, and a 5-fold enhancement of fluorescence intensity was achieved after the directional coating of the antibodies on sulfhydryl functionalization (-SH) substrates and the preparation of QBs by using a polymer and silica double-protection method. Next, a simple polydimethylsiloxane (HS-PDMS) immunosensor with a low volume consumption was prepared. Under optimal conditions, we achieved the simultaneous detection of IL-6 and PCT with a linear range of 0.05-50 ng/mL, and the limit of detection (LOD) was 24 and 32 pg/mL, respectively. The result is comparable to two-color QBs-FLISA with a sulfhydryl microplate, even though only 20% of its volume was used. Thus, the dual fluorescence signal-enhancement HS-PDMS immunosensor offers the capability of early microvolume diagnosis of diseases, while the detection of inflammatory factors is clinically important for assisting disease diagnosis and determining disease progression.