Abstract
The ESILARANTE project aims to develop an innovative portable biosensing platform for the detection of cannabinoid in capillary blood samples, based on laccase biosensors fabricated via Electrospray Deposition (ESD), a green and cost-effective enzyme immobilization technique. The project combines enzyme purification, physico-chemical characterization and advanced electrochemical testing to identify the factors enabling high sensor stability and reusability. Particular attention is given to the analytical performance, with quantitative evaluation of enzymatic activity and validation against fully validated LC-MS/MS protocols. Analytical techniques also investigate enzyme integrity before and after deposition, helping to optimize the system. The expected result is a robust, selective, and miniaturized biosensor for rapid, on-site applications in forensic and roadside settings, representing a sustainable and scalable alternative to conventional laboratory-based cannabinoid testing.
Results achieved
Overall outcomes. The project developed a portable amperometric biosensor for the detection of cannabinoids, in particular Δ9-tetrahydrocannabinol (THC), in capillary blood, based on the enzyme laccase immobilized onto the electrode surface through a green, solvent-free, electrospray-based deposition. A first major result clarified the molecular basis of the biosensor's stability and reusability: the commercial Trametes versicolor laccase used as starting material was found to contain only a minor enzymatic fraction (about 0.4% of the powder), the remainder consisting largely of starch. This polysaccharide proved essential in anchoring the enzyme and preserving sensor performance over repeated use, whereas starch-free preparations lost their activity after a single washing cycle [M.C. Castrovilli, P. Gentili, A. Vitali, S. Cerra, F. Palmeri, I. Fratoddi, M. Polentarutti, G. Bais, L. Gullo, A. Cartoni, Electrospray deposition of starch-containing laccase: A green technique for low-cost and eco-friendly biosensors, Biosensors & Bioelectronics 267 (025) 116758]. The biosensor was subsequently applied to THC, yielding a concentration-dependent response and demonstrating the feasibility of the approach. Rapid on-site cannabinoid detection is of considerable interest in road-safety, forensic and clinical settings, where conventional laboratory analyses, although accurate, are time- and resource-intensive. A portable device operating on a finger-prick blood drop could therefore provide rapid preliminary results, complementing confirmatory laboratory testing. UNIBO unit results. The Alma Mater Studiorum - University of Bologna Unit (Research group of Pharmaco-Toxicological Analysis, PTA Lab) acted as the analytical reference of the project, with a twofold role: developing original mass spectrometry-based methods to benchmark and cross-validate the biosensor, and characterizing the enzyme preparations at the molecular level. The activities relied on high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) and to high-resolution quadrupole time-of-flight mass spectrometry (HRMS Q-TOF). An original HPLC-MS/MS method was developed and fully validated according to international bioanalytical guidelines, initially for THC and then extended to a seven-analyte panel comprising its main phase-I metabolites (11-hydroxy-THC and 11-carboxy-THC) and cannabidiol (CBD) together with its hydroxy- and carboxy-metabolites. The inclusion of the main phase-I metabolites alongside the parent compounds broadens the analytical information available and supports the interpretation of cannabis exposure. The method exhibited linearity in the low ng/mL range (with determination coefficients above 0.9940), limits of detection down to 0.2 ng/mL, intra- and inter-day precision below 5% and accuracy within ±15%, thus meeting the acceptance criteria for reliable quantification. The chemical stability of THC reference solutions was additionally verified by UV-Vis spectrophotometry over one month, ensuring the reliability of the calibration materials underpinning the quantitative work. Chromatographic separation was carried out under reversed-phase conditions with a rapid solvent gradient, while detection relied on selected precursor-to-product ion transitions specific to each analyte, ensuring high selectivity. Validation encompassed selectivity against endogenous interferences, matrix effect, extraction recovery and carry-over, all of which remained within acceptable limits when the method was transferred from standard solutions to biological matrices. A key deliverable was the orthogonal cross-validation of the biosensor: identical samples were analysed in parallel by HPLC-MS/MS and by the electrochemical device, both in buffered systems and in more complex, matrix-containing samples, yielding closely overlapping and consistently concentration-dependent results. This comparison between two independent measurement principles confirmed the analytical reliability of the biosensor. The biosensor itself provided a limit of detection in the low-micromolar range, adequate for the intended screening purposes, and its coupling with the fully validated HPLC-MS/MS reference method defines a screen-and-confirm workflow well suited to decentralised testing. The validated chromatographic method was then transferred to advanced microsampling formats, including whole-blood volumetric absorptive microsampling (VAMS), conventional and microfluidic-based dried blood spots (DBS), urine microsampling and sweat patches. This extended the project towards low-volume, minimally invasive and field-deployable strategies, consistent with sustainable microsampling and miniaturised sample preparation. Such dried, low-volume specimens also offer improved analyte stability during storage and transport and reduced biohazard. Part of this work was published [R. Mandrioli, R. Di Lecce, S. Noreen, M.C. Castrovilli, A. Kabir, M. Locatelli, Laura Mercolini, Michele Protti*, Novel microsampling approach using fabric-phase sorptive extraction (FPSE) for cannabinoid analysis in blood, Microchemical Journal 214 (2025) 113855)], introducing a fabric-phase sorptive extraction (FPSE) procedure for cannabinoid determination in blood. For each microsampling support the sample preparation and extraction steps were specifically optimised, and the influence of factors such as blood haematocrit and spot homogeneity on quantitation was assessed, in line with current recommendations for microsampling-based bioanalysis. The dried formats proved compatible with accurate cannabinoid determination, supporting their use for decentralised and remote sampling. At the molecular level, HRMS Q-TOF profiling of the laccase preparations revealed a dominant oligosaccharide pattern alongside a minor multiply charged protein envelope. Comparison with purified laccases from other fungal sources (Trametes villosa and Myceliophthora thermophila) confirmed the markedly excipient-rich nature of the commercial product, whose high starch content partially suppressed the ionization of the enzymatic fraction. The relative proportions of protein and polysaccharide across preparations were also compared, rationalising the batch-to-batch variability of the commercial material. These data provided independent, orthogonal confirmation of the results obtained by the partners through spectroscopic and electrophoretic techniques, and are also of practical relevance for the future quality control of biosensor manufacturing. Consistently with green analytical chemistry principles, the workflows were designed to minimise sample volumes, solvent and reagent consumption, and to be transferable across the different sampling formats, with only the sample-preparation step adapted to each support. Across the different matrices and supports, method performance was preserved, with recovery and matrix effect maintained within acceptance ranges, demonstrating the robustness and transferability of the analytical approach. From a translational standpoint, the combination of a green, reusable biosensor with validated microsampling-based reference methods provides a coherent analytical platform for rapid, sustainable and decentralised cannabinoid monitoring, readily extendable to other analytes of forensic and clinical interest. Overall, the Bologna unit delivered a reliable and transferable analytical framework that underpinned the validation of the biosensor and broadened the project towards sustainable, miniaturized cannabinoid testing of forensic, roadside and clinical relevance, providing a solid basis for future point-of-care developments.Project details
Unibo Team Leader: Michele Protti
Unibo involved Department/s:
Dipartimento di Farmacia e Biotecnologie
Coordinator:
CNR - Consiglio Nazionale delle Ricerche(Italy)
Total Unibo Contribution: Euro (EUR) 79.000,00
Project Duration in months: 24
Start Date:
28/09/2023
End Date:
28/02/2026
