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Combined Signal Amplification Using a Propagating Cascade Reaction and a Redox Cycling Reaction for Sensitive Thyroid-Stimulating Hormone Detection.

Propagating cascade reactions based on two proteases are promising for obtaining high signal amplification. However, in many cases, biosensors that use cascade reactions do not have low detection limits because of the inherent slowness of proteolytic reactions. Here, we report a sensitive electrochemical immunosensor using a high-signal-amplification method that combines a propagating cascade reaction and a redox cycling reaction. The cascade reaction uses ecarin and prothrombin: the ecarin label proteolytically converts inactive prothrombin into active thrombin, which then proteolytically liberates electroactive p-aminophenol (AP) from an AP-conjugated peptide. The liberated AP is electrochemically oxidized to p-benzoquinone imine (QI), regenerated by the reduction of QI by NADH, and then electrochemically reoxidized. This electrochemical-chemical (EC) redox cycling reaction significantly increases the electrochemical signal. The developed immunosensor is also compared with an immunosensor that uses only a propagating cascade reaction and an immunosensor that uses a single proteolytic reaction and an EC redox cycling reaction. The detection limits for thyroid-stimulating hormone (TSH) obtained using the three immunosensors are 3 pg/mL, 2 ng/mL, and 4 ng/mL, respectively, indicating that the newly developed immunosensor is more sensitive than the other two. The measured concentrations of TSH in clinical serum are found to agree well with those determined using a commercial instrument.