Discovering melamine-specific bioreceptors via phage display, constructing its validation method based on the quenching on nanocomplex, and applying screened bioreceptor to the electrochemical assay of melamine

Blending melamine into foodstuffs is prohibited, but despite its harmfulness, melamine is still added to dairy products for falsifying nutritional composition. In this study, to discover the bioreceptor recognizing melamine, phage display was introduced. For the technique, two types of haptens and two kinds of phage display methods were used to improve its specificity through cross-checking each acquired result. The melamine hapten was synthesized by reacting 6-aminohexanoic acid with 2-chloro-4,6-diamino-1,3,5-triazine, which function as a spacer and epitope site, respectively, and hapten was decorated on the bovine serum albumin (BSA) as a carrier material to manufacture BSA-hapten. Based on these haptens, the phage-displayed peptides are identified by sequencing, and five peptide bioreceptors were selected according to the frequency and similarity. To construct the simple verification procedure for the discovered peptides, graphene oxide quantum dot (GOQD)-Hg²⁺ quenching system was adopted, and their selectivity and sensitivity were determined. Briefly, the melamine-Hg²⁺ complex could easily be attached on the surface of GOQD through π-π stacking, thus, the fluorescence of GOQD is quenched via charge transfer. However, if the screened peptides possess an affinity to melamine, the diminished fluorescence intensity will be recovered after adding the bioreceptor to the system resulting from hindering the formation of the melamine-Hg²⁺ complex. We successfully figured out the valid peptide sequences among the candidates through this approach, and the best hit was applied to the electrochemical impedance spectroscopic system. As a result, it shows a high sensitivity to the melamine with a low detection limit.