This work reports washing-free electrogenerated chemiluminescence (ECL) magnetic microbiosensors based on target assistant proximity hybridization (TAPH) for multiple protein biomarkers for the first time. As a principle-of-proof, alpha-fetoprotein (AFP) was chosen as a model analyte, and biotin-DNA1 bound streptavidin-coated magnetic microbeads (MMB@SA⋅biotin-DNA1) were designed as the universal capture MMB, while the corresponding two antibodies tagged with DNA2 or DNA3 were utilized as hybrid recognition probes, and ruthenium complex-tagged DNA4-10A was designed as a universal ECL signal probe. When the capture MMB was added into the mixture solution (containing the analyte, hybrid recognition probes, signal probe and tri-n-propylamine), biocomplexes were formed on the MMB. After the resulting MMB was efficiently brought to the surface of a magnetic glassy carbon electrode (MGCE), ECL measurement was performed without a washing step, resulting in an increase in the ECL intensity. A model for ECL measuring the second-order rate constants of hybridization reactions on MMB was derived. It was found that the rate constants for hybridization reactions on MMB in rotating mode are 1.6-fold higher than those in shaking mode, and a suitable DNA length of the signal probe can improve the signal-to-noise ratio. The washing-free ECL method was developed for the determination of AFP with a much lower detection limit (LOD) of 0.04 ng mL⁻¹. The developed flexible strategy has been extended to determine D-dimer with an LOD of 0.1 ng mL⁻¹ and myoglobinglobin with an LOD of 1.1 ng mL⁻¹. This work demonstrated that the proposed strategy of ECL TAPH on MMB at MGCE is a washing-free and flexible promising strategy, and can be extended to qualify other multiple protein biomarkers in real clinical assays.

这项工作首次报道了基于多种蛋白质生物标志物的目标辅助邻近杂交 (TAPH) 的免洗电化学发光 (ECL) 磁性微生物传感器。作为证明原则，甲胎蛋白 (AFP) 被选为模型分析物，生物素-DNA1 结合链霉亲和素包被的磁性微珠 (MMB@SA⋅biotin-DNA1) 被设计为通用捕获 MMB，而以相应的DNA2或DNA3标记的两种抗体为杂交识别探针，设计了钌络合物标记的DNA4-10A作为通用ECL信号探针。当将捕获 MMB 添加到混合溶液中时（包含分析物、杂交识别探针、信号探针和 tri- n-丙胺），生物复合物在 MMB 上形成。在将所得 MMB 有效地带到磁性玻璃碳电极 (MGCE) 的表面后，无需清洗步骤即可进行 ECL 测量，从而增加 ECL 强度。导出了用于测量 MMB 上杂交反应的二级速率常数的 ECL 模型。结果发现旋转模式下MMB杂交反应的速率常数比振荡模式高1.6倍，合适的信号探针DNA长度可以提高信噪比。免洗 ECL 方法是为测定 AFP 而开发的，检测限 (LOD) 低得多，为 0.04 ng mL ^-1。开发的灵活策略已扩展到确定 D-二聚体，LOD 为 0.1 ng mL^-1和 LOD 为 1.1 ng mL ^-1的肌红蛋白。这项工作表明，在 MGCE 上提出的 ECL TAPH 对 MMB 的策略是一种免洗且灵活的有前途的策略，并且可以扩展到在实际临床测定中鉴定其他多种蛋白质生物标志物。