Fingerprints from the past: A new biosensing approach for the analysis of archaeological enamel samples

Amelogenins (AMG) are crucial proteins for sexing archaeological remains due to their resilience and relative abundance in dental enamel. Herein, we introduce an innovative approach to imprint mimetic receptors based on molecularly imprinted polynorepinephrine to streamline AMG detection in modern and archaeological teeth. This easy, rapid, and cost-effective biosensing tool can detect and bind full-length AMG from standard solutions and AMG residues from enamel extracts. A digestion-free protein extraction protocol was established from 1.0 – 2.0 mg of enamel powder and the whole extract was used to directly imprint polynorepinephrine. The biomimetic receptor was coupled with Surface Plasmon Resonance and Bio-Layer Interferometry to assess the imprinting efficiency and characterise the systems in terms of kinetic rates and binding affinity parameters towards the target protein. Expanding upon the single epitope imprinting, this original “Finger-Imprint” (FI) approach addresses the analytical challenges posed by complex matrices such as the intrinsically heterogeneous dental enamel. High-resolution nanoLC-MS/MS analysis served the dual purpose of validating the effectiveness of the extraction protocol and enabling the precise identification of the extract components. The characterisation and semi-quantification of 69 modern and 21 archaeological human enamel extracts revealed non-tryptic peptides primarily associated with AMGX across all samples and AMGY exclusively in male extracts. Further exploration of AMG isoforms and splice variants revealed complete coverage of the hydrophilic parts of the proteins, namely the tyrosine-rich amelogenin peptide and the C-terminal region, supporting the design and development of the mimetic receptors. These successful results prove the efficacy of the biosensing approach and the effectiveness of the FI-based biomimetic receptor, which was specifically tailored to the extract and requires a quick and straightforward preparation process involving biocompatible and biodegradable reagents.