Ascorbic acid-triggered electrochemical–chemical–chemical redox cycling for design of enzyme-amplified electrochemical biosensors on self-assembled monolayer-covered gold electrodes

l-Ascorbic acid 2-phosphate (AAP) is an optimal substrate for alkaline phosphatase (ALP) in electrochemical bioassays because of its low cost, good water solubility, less electrode passivation and high signal-to-background ratio. However, developing of electrochemical sensors with AAP as the enzyme substrate on self-assembled monolayer (SAM)-covered electrode is limited because the insulating SAM hinders the electron transfer between the electrode and ascorbic acid (AA, the enzymatic product of AAP). In this work, we first reported a strategy for developing AAP-based electrochemical biosensors on SAM-covered gold electrode. The method is based on AA-triggered “outer-sphere to inner-sphere” electrochemical–chemical–chemical (ECC) redox cycling with ferrocenecarboxylic acid (FcA) as the redox mediator. Specifically, AA produced from AAP facilitated the regeneration of FcA from its electrochemical-oxidation product (referred to as FcA+ in the text), leading to an increase in the anodic current of FcA. Electrochemically inert tris(2-carboxyethyl)phosphine (TCEP) was used as a chemical reducing reagent to regenerate AA from its oxidation product, thus amplifying the electrochemical signal. The applications and performances of the proposed method were demonstrated in the competitive assays of β-amyloid (Aβ) peptides. The theoretical simplicity and high sensitivity indicated that our work would be valuable for developing simple and sensitive electrochemical biosensors.