Dual-Signal-Encoded Barcodes with Low Background Signal for High-Sensitivity Analysis of Multiple Tumor Markers

The suspension array technology (SAT) is promising for high-sensitivity multiplexed analysis of tumor markers. Barcodes as the core elements of SAT, can generate encoding fluorescence signals (EFS) and detection fluorescence signals (DFS) in the corresponding flow cytometer channel. However, the bleed-through effect of EFS in the DFS channel and the reagent-driven non-specific binding (NSB) lead to background interference for ultrasensitive assay of multiple targets. Here, we report an ingenious method to eliminate background interference between barcode and reporter using low-background dual-signal-encoded barcodes (DSBs) based on microbeads (MBs) and quantum dots (QDs). The low-background DSBs were prepared via combination strategy of two signals containing scatter signals and fluorescence signals. Three types of MBs were distinguished by the scattering channel of flow cytometer (FSC vs. SSC) to obtain the scattered signals. Green quantum dots (GQDs) or red quantum dots (RQDs) were coupled to the surface of MBs by sandwich immune structure to obtain the distinguishable fluorescent signals. Furthermore, the amount of conjugated capture antibody on the MB's surface was optimized by comparing the change of detection sensitivity with the addition of capture antibody. The combination measurements of specificity and NSB in SAT platform were performed by incubating the capture antibody-conjugated MBs (cAb-MBs) with individual QD-conjugated detection antibody (QDs-dAb). Finally, an SAT platform based on DSBs was successfully established for highly sensitive multiplexed analysis of six tumor markers in one test, which suggests the promising tool for highly sensitive multiplexed bioassay applications.