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Hypothermic machine perfusion (HMP) provides preservation superior to cold storage and may allow for organ assessment prior to transplantation. Since flavin mononucleotide (FMN) in perfusate has been proposed as a biomarker of organ quality during HMP of donor livers, the aim of this study was to validate FMN as a biomarker for organ quality in the context of HMP preserved kidneys. Perfusate samples (n = 422) from the paired randomised controlled COPE-COMPARE-trial, comparing HMP with oxygenation (HMPO2) versus standard HMP in kidneys, were used. Fluorescence intensity (FI) was assessed using fluorescence spectroscopy (excitation 450nm; emission 500-600nm) and validated by fluorospectrophotometer and targeted liquid chromatography mass spectrometry (LC-MS/MS). Fluorescence intensity (FI)(ex450;em500-600) increased over time during machine perfusion in both groups (p<0.0001). This increase was similar for both groups (p = 0.83). No correlation, however, was found between FI(ex450;em500-600) and post-transplant outcomes, including day 5 or 7 serum creatinine (p = 0.11; p = 0.16), immediate graft function (p = 0.91), creatinine clearance and biopsy-proven rejection at one year (p = 0.14; p = 0.59). LC-MS/MS validation experiments of samples detected FMN in only one perfusate sample, whilst the majority of samples with the highest fluorescence (n = 37/38, 97.4%) remained negative. In the context of clinical kidney HMP, fluorescence spectroscopy unfortunately appears to be not specific and probably unsuitable for FMN. This study shows that FMN does not classify as a clinically relevant predictive biomarker of kidney graft function after transplantation. [ABSTRACT FROM AUTHOR]

Antonio Alessio Leonardi,1– 3 Rosalia Battaglia,4 Dario Morganti,1,2 Maria Josè Lo Faro,1,3 Barbara Fazio,2 Chiara De Pascali,5 Luca Francioso,5 Gerardo Palazzo,6,7 Antonia Mallardi,8 Michele Purrello,4 Francesco Priolo,1 Paolo Musumeci,1,* Cinzia Di Pietro,4,* Alessia Irrera2,* 1Dipartimento di Fisica e Astronomia, Università di Catania, Catania, 95123, Italy; 2CNR-IPCF, Istituto per i Processi Chimico-Fisici, Messina, 98158, Italy; 3CNR-IMM UoS Catania, Istituto per la Microelettronica e Microsistemi, Catania, 95123, Italy; 4Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy; 5CNR-IMM, Institute for Microelectronics and Microsystems, Via Monteroni, University Campus, Lecce, 73100, Italy; 6Chemistry Department, University of Bari ‘Aldo Moro’, Bari, 70125, Italy; 7CSGI, Center for Colloid and Surface Science c/o Chemistry Department, Bari, 70125, Italy; 8CNR-IPCF, Institute for Chemical-Physical Processes, c/o Chemistry Department, Bari, 70125, Italy*These authors contributed equally to this workCorrespondence: Paolo Musumeci; Cinzia Di Pietro Email paolo.musumeci@ct.infn.it; dipietro@unict.itIntroduction: Small extracellular vesicles (sEVs), thanks to their cargo, are involved in cellular communication and play important roles in cell proliferation, growth, differentiation, apoptosis, stemness and embryo development. Their contribution to human pathology has been widely demonstrated and they are emerging as strategic biomarkers of cancer, neurodegenerative and cardiovascular diseases, and as potential targets for therapeutic intervention. However, the use of sEVs for medical applications is still limited due to the selectivity and sensitivity limits of the commonly applied approaches.Methods: Novel sensing solutions based on nanomaterials are arising as strategic tools able to surpass traditional sensor limits. Among these, Si nanowires (Si NWs), realized with cost-effective industrially compatible metal-assisted chemical etching, are perfect candidates for sEV detection.Results: In this paper, the realization of a selective sensor able to isolate, concentrate and quantify specific vesicle populations, from minimal volumes of biofluid, is presented. In particular, this Si NW platform has a detection limit of about 2× 105 sEVs/mL and was tested with follicular fluid and blastocoel samples. Moreover, the possibility to detach the selectively isolated sEVs allowing further analyses with other approaches was demonstrated by SEM analysis and several PCRs performed on the RNA content of the detached sEVs.Discussion: This platform overcomes the limit of detection of traditional methods and, most importantly, preserves the biological content of sEVs, opening the route toward a reliable liquid biopsy analysis.Keywords: silicon nanowires, biosensor, small extracellular vesicles, label-free, luminescence