Droplet-free and enzyme-free digital immunoassay based on fluorescent microspheres for protein detection

Abstract

Digital immunoassays with high sensitivity offer the early disease diagnosis and therapeutic management of various diseases. By using fluorescent microspheres and magnetic microparticles (MMPs), a droplet-free and enzyme-free digital immunoassay method was proposed for protein detection. The utilization of fluorescent microspheres offers several advantages. Firstly, they are anchored onto the MMPs in digital detection, thereby eliminating the need for droplets. This innovative feature streamlines the assay process and enhances its efficiency. Secondly, the fluorescent microspheres were embedded with a lot of fluorescent molecules, allowing the detection of single fluorescent nanospheres. Signal amplification was achieved by the embedding of fluorescent molecules within the microspheres, thus eliminating the need for enzyme-based amplification methods of the signal. This approach not only simplifies the assay but also reduces costs associated with enzyme procurement and utilization. To validate the efficacy of this digital immunoassay method, interleukin 6 (IL-6) was chosen as the target protein for detection. The results showed a significant linear relationship between IL-6 concentrations of 0.01–50 pg/mL (R2 = 0.9982), highlighting the robustness of the assay. Furthermore, the assay showed exceptional sensitivity, with a limit of detection (LOD) of 0.006 pg/mL. Thus, this digital immunoassay method using fluorescent microspheres is a simple and low-cost technique, holding great potential for the industrialization of digital immunoassays.