Aptamer-based sensing of ß-casomorphin-7.

ABSTRACT

ß-Casomorphin-7 (BCM-7), a seven amino acid peptide, is released during digestion of ß-casein A1 variant of milk which is speculated to be associated with certain diseases. Fifteen ssDNA aptamers having high affinity toward BCM-7 were identified from a 72 nt long random library after ten rounds of systematic evolution of ligands by exponential enrichment. Dissociation constant values of selected aptamers were in the range of 7.7-156.7 nM. Seq6 aptamer exhibited the lowest Kd value. Nine aptamers were evaluated for their binding toward BCM-7, BCM-9A1, and BCM-9A2 peptides, and binding was variable. SeqU5 exhibited the lowest binding with BCM-9A1 and BCM-9A2. Aptamer-coated gold nanoparticles (GNPs) resulted in color change of GNPs in the presence of BCM-7, thereby establishing recognition of BCM-7 by aptamers. The enzyme-linked aptamer-sorbent assay (ELASA) was evaluated as an assay of BCM-7 in biological fluids. BCM-7-peroxidase competed with BCM-7 in ELASA, performed with BCM-7 solution and BCM-7 spiked urine pretreated with urease, plasma, and ß-casein digest samples.