Washing-free electrogenerated chemiluminescence magnetic microbiosensors based on target assistant proximity hybridization for multiple protein biomarkers.

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. The developed flexible strategy has been extended to determine D-dimer with an LOD of 0.1 ng mL and myoglobinglobin with an LOD of 1.1 ng mL. 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.