Abstract
The authors describe the identification of an effective binding region of aptamers against glycated (HbA1c) and total haemoglobin (tHb) by using a fluorometric assay. Truncation of the originally selected aptamers from 60 to 46 and 34 nucleotides for HbA1c and tHb, respectively, enhances the affinity for their targets. Moreover, shortening the aptamer sequences leads to a better conformational change after target binding which enabled the integration of the aptamers in a graphene oxide (GO)-based fluorometric assay. First, fluorescein-labelled truncated aptamers were physically absorbed onto the surface of GO surface via pi-stacking interaction. This leads to quenching of fluorescence. Once the truncated aptamers bind the target protein, a conformational change is induced which results (a)) in the release of the aptamers from the surface of GO and (b) in the restoration of green fluorescence that is measured at 515 nm. The assay can be carried out in a microtiter plate format in homogeneous solution, this avoiding the steps of immobilization, incubation, and washing that are often necessary in immuno-assays. This also reduces the time and the costs of the overall assay and allows for high throughput screening for diabetes. HbA1c can be detected in the range from 5.4 to 10.6%. The assay is selective for HbA1c over other proteins that commonly exist in blood. The results obtained by using this method compare well with those of a turbidimetric immunoassay that is typically applied in clinical laboratories.