Backfilling rolling cycle amplification with enzyme-DNA conjugates on antibody for portable electrochemical immunoassay with glucometer readout

A simple and feasible electrochemical immunosensing protocol with glucometer readout was designed for the detection of low-abundance disease-related biomarker (alpha-fetoprotein; AFP) on the basis of backfilling rolling cycle amplification (RCA) with invertase-DNA 2 conjugates on the detection antibody. The assay consisted of the immunoreaction, RCA reaction, DNA 2 -invertase hybridization and glucose measurement. Initially, a sandwiched immunoreaction was carried out between anti-AFP capture antibody-coated microplate between nanogold-labeled pAb 2 detection antibody conjugated with DNA 1 primer (DNA 1 -AuNP-pAb 2 ) in the presence of target ATP . Thereafter, the carried primers triggered the RCA reaction in the presence of circular DNA template, polymerase and dNTP, to produce numerous repeated oligonucleotide sequences for hybridization with many invertase-DNA 2 conjugates. The carried invertase molecules accompanying the hybridization reaction hydrolyzed sucrose into glucose, thereby resulting in the amplification of the detectable signal on a handheld personal glucometer (PGM). Under optimum conditions, the developed immunoassay exhibited high sensitivity for the quantitative screening of AFP within a dynamic range of 0.1–100 ng mL −1 at a low detection limit of 0.087 ng mL −1 . Other biomarkers and proteins did not interfere the signals of this system. In addition, this method was utilized to determine human serum samples containing target AFP, and received well-matched results with the referenced enzyme-linked immunosorbent assay (ELISA) method.