Quantum Dot-Enriched Dendritic Mesoporous Silica Nanospheres for Chamber-Free Digital Detection: High Sensitivity, Easy Operation, and Clinical Accessibility

Digital highly sensitive biodetection is increasingly crucial for early disease diagnosis and treatment, particularly for accurate quantification of low-abundance biomarkers. Current digital biodetection methods represented as single-molecule assay (Simoa), enzyme- or DNA-assisted signal amplification strategy, the fluorescence nanosphere labeling-based digital biodetection, etc. often rely on microcompartments or time-consuming signal amplification steps, facing challenges in terms of cumbersome workflow, insufficient signal strength, and limited clinical accessibility. To address these limitations, we propose a high-sensitivity chamber-free digital immunoassay strategy based on a standard flow cytometer, which utilizes dendritic mesoporous silica nanoparticles (DMSNs) doped with quantum dots (QDs) forming ultrabright fluorescence nanospheres (DMSN@QDs@Silica, DQS) as signal label. Structure–function relationship (SFR) of DQS was systematically investigated to obtain DQS with optimal labeling kinetics and fluorescence brightness. The synthesized DQS are significantly brighter than commercial label fluorescent nanospheres, enabling direct digital signal detection via flow cytometry, which eliminates the need for isolation chambers and multistep signal amplification processes, simplifying the assay and reducing associated costs. We demonstrate that the high-sensitive DQS-based digital assay provides approximately 24-fold improvement in sensitivity over traditional flow cytometry fluorescence immunoassay, achieving a detection limit of 0.37 pg/mL for interleukin-6 (IL-6). This chamber-free digital detection approach holds significant promise for low-abundance biomarker detection, offering enhanced sensitivity, simplicity, and clinical applicability.