Advanced analytical methods for the study and characterisation of archaeological human and faunal remains

Accurate determination of biological sex is fundamental for reconstructing biological profiles of individuals in archaeological contexts. A robust approach to achieving this relies on detecting amelogenins (AMGs), a family of proteins that exhibit sexual dimorphism and account for approximately 90% of the total protein content of dental enamel. AMGs undergo partial degradation from the early stages of enamel formation, yet they persist in mature enamel for long periods, making them excellent biomarkers in ancient samples. Palaeoproteomics, the study of ancient proteins, has enabled significant advances in sex attribution by characterising AMG proteins via Mass Spectrometry (MS). Traditional immunochemical assays have recently been proposed as alternative techniques. However, there remains a lack of alternative AMG detection methods that are simple, cost-effective, environmentally sustainable, and suitable for in situ archaeological applications. In this context, we developed a protein extraction protocol for enamel, aiming to improve and simplify analytical workflows for both modern and archaeological samples. The protocol was successfully validated on modern samples using LC-MS/MS and assessed by FTIR and Raman spectroscopy before application to archaeological specimens. Due to its straightforward nature, involving only acid demineralisation, precipitation of the mineral phase, and neutralisation, the protocol was integrated into advanced biosensing workflows. Specifically, this research focuses on the application of Molecularly Imprinted Polynorepinephrine (MIPNE) to develop biomimetic receptors capable of detecting full-length AMGs from standard solutions. MIPNEs were synthesised using a template (a short synthetic peptide selected in silico on the AMGX amino acid sequence) to create specific recognition sites, mimicking the high specificity of an antibody-antigen interaction but offering advantages such as low cost, reusability, and environmental sustainability. Expanding upon the single epitope imprinting, a “Finger-Imprint” (FI) approach was developed by directly using the whole enamel protein extract as a template. This new approach enabled addressing the analytical challenges posed by complex matrices such as the intrinsically heterogeneous dental enamel. The FI biomimetic receptor was successfully coupled to Surface Plasmon Resonance (SPR), enabling the evaluation of imprinting efficiency and characterising the systems in terms of kinetic rates and binding affinity parameters towards the standard protein. Finally, following the need for analytical systems that combine operational simplicity and high sample throughput, the FI protocol was integrated with Bio-Layer Interferometry (BLI) and Localised Surface Plasmon Resonance (LSPR). Positive results from both standard solutions and real human dental extracts confirm the effectiveness of the biomimetic receptor and demonstrate the prospective applicability of the FI approach for personalised medicine and sex identification. In alignment with the principles of decolonisation and decentralisation in archaeological research, this work applied a MIPNE-based biosensing approach and laid the foundations for alternative, high-throughput, in situ analyses of archaeological enamel samples for sex attribution. The prospective goal is to significantly reduce analytical time, cost, and logistical constraints, promoting more sustainable and accessible methodologies.