An Ultrasensitive Single-Particle Collision Electrochemical Immunoassay at Subfemtogram-per-Milliliter Levels via Cascaded Hybridization Chain Reaction Amplification

Herein, we developed an ultrasensitive single-particle collision-based electrochemical immunosensing platform for detecting a total prostate-specific antigen (tPSA) as a model target. It relies on the collision events resulting from the enzyme-catalyzed, in situ generated silver shells on gold nanoparticles. To boost the sensitivity of the method, a branched hybridization chain reaction (bHCR) was employed to introduce abundant alkaline phosphatase (ALP) for signal amplification. Initially, target tPSA and an aptamer-linker DNA conjugate were captured on the antibody-coated plate. The terminal ends of the DNA probe subsequently served as initiators to trigger the bHCR process. This cascade reaction created multiple binding sites for ALP and thereby accelerated the production of ascorbic acid (AA). The generated AA subsequently mediates the reduction of silver ions, facilitating the formation of silver shells on the Au NPs and yielding detectable collision signals from Au@Ag NPs. As a result, the concentration of tPSA can be directly reflected and quantified by the recorded collision signals. Benefiting from the dual signal amplification of bHCR and Au NP metallization, the collision immunosensor achieved a highly sensitive tPSA detection with a detection limit as low as 0.07 fg/mL. Besides the excellent selectivity, reproducibility, and anti-interference ability, the practical applicability of the method was validated using serum samples. This work provides a robust and ultrasensitive platform for immunological analysis, holding promising potential for clinical diagnostics.