Instantaneous detection of αₛ-casein in cow's milk using fluorogenic peptide aptamers

The most abundant protein in cow's milk is αₛ-casein, which is an allergen responsible for causing allergic reactions, including the life-threatening, hyper-sensitive immune reaction anaphylaxis. Casein sensors are used widely to avoid its ingestion or food contamination; however, these sensors use expensive antibodies and require several tens of minutes to process the multiple steps required for analysis. To overcome these drawbacks, we used fluorogenic peptide aptamers that instantaneously enhance their fluorescence upon binding to a target molecule. Two fluorogenic peptide aptamers, Cas1 and Cas2, were selected using ribosome display. Both aptamers instantaneously enhanced their fluorescence in the presence of αₛ-casein. Although both aptamers slightly enhance their fluorescence in the presence of a negative control protein, β-lactoglobulin, we found that modification at the N-terminus of Cas1 with polyethylene glycol (PEG-cas1) can suppress fluorescence enhancement for β-lactoglobulin without compromising the fluorescence enhancement and affinity for αₛ-casein, presumably because the modification suppresses unwanted interactions between PEG-cas1 and β-lactoglobulin. The detection limit of our fluorogenic aptamer system was found to be about 0.04 μM (∼1 ppm), which is comparable with that of commercially available immunochromatography kits for αₛ-casein. Notably, our system can detect αₛ-casein in an exceptionally short time (i.e., 20–25 s) when compared with the 15 min required by immunochromatography kits. We also demonstrated that the prompt fluorescence enhancement of PEG-cas1 in the presence of αₛ-casein can be observed even by naked eyes. The results of this study demonstrate the remarkable ability of fluorogenic aptamers and their potential application for other target molecules.