Stereo-assembled DNA–dye molecules on magnetic microspheres with ultralarge coding capacity for suspension arrays

As the central element of suspension arrays, fluorescent-encoded microspheres have become a prevalent technology for multi-index organism detection. The agglomeration and self-quenching effect of fluorescent materials at high concentration is one of the main reasons the coding capacity of fluorescent coding microsphere fails to rise. To overcome these shortcomings, this study proposed a preparation method for fluorescent-encoded microspheres by stereo-assembling two dye molecules on magnetic microspheres. The coding capacity of fluorescent microspheres prepared by this developed method in the same particle size with two colors is 104 based on the variation of type and concentration of DNA–dye molecules, which is the largest coding capacity compared with that reported previously. These encoding microspheres exhibit excellent stability for long-term storage. Two kinds of fluorescent microspheres (i.e., S4R5 and S4R7) were chosen to fix specific captured antibodies for simultaneous detection of classical tumor markers, including alpha-fetoprotein and carcinoembryonic antigen; a “sandwich” type immunoassay via flow cytometry was used to demonstrate the ability of the developed suspension array to detect multiple targets simultaneously. StayGold, a kind of green fluorescent protein, was expressed as probe labeled onto detecting antibodies. For the detection of alpha-fetoprotein and carcinoembryonic antigen, we achieved a wide linear range of 1×10¹–1×10⁵ pg/mL (R² = 0.9915) and 5×10¹–2.5×10⁵ pg/mL (R² = 0.9903) while meeting the detection limits of 3.7 and 7.8 pg/mL, respectively. This developed suspension array detection system shows great alternative prospects in multiple analysis across various fields, including clinical diagnostics, genomics, proteomics, environmental monitoring, and drug screening.