Highly-sensitive electrochemical immunosensing method based on dual amplification systems.

In this work, a novel immunosensing method has been developed on the basis of the sensitive determination of a product generated by an enzyme reaction with dual amplification system combining an electrochemical-redox cycling and coulometric signal transduction using a galvanic cell. Analytes were captured on microparticles to form sandwich-type immunocomplexes and then labeled with β-galactosidase (β-gal). 4-Aminophenol (PAP) produced by enzyme reaction of β-gal was introduced into the anode compartment consisting of a comb type of an interdigitated array (IDA) electrode. PAP was oxidized at the IDA electrode by the coupled reduction of silver ions at the glassy carbon (GC) electrode of the cathode, resulting in the deposition of silver metal on the GC electrode. The other comb of the IDA electrode was used to reduce quinoneimine generated by the oxidation of PAP, regenerating PAP. The deposited silver was collectively converted to a signal by anodic stripping voltammetry. The amount of silver deposited corresponded to the degree of PAP oxidation by redox cycling, which leads to an enhancement of the stripping signal due to the conversion of the product (PAP) and accumulation of the insoluble silver metal. Using carcinoembryonic antigen as a model analyte, the present immunosensing method showed linear behavior over two orders of magnitude with detection limits down to 0.01 ng/mL. Dual signal amplification with redox cycling and coulometric signal transduction provides a promising, sensitive, and simple method for the determination of marker proteins.