Multiplex Sensing Platform for Predictive Biomarkers in Vascularized Composite Allografts Using Porous Laser-Engraved Graphene

Over two decades, vascularized composite allotransplantation (VCA) has been a promising solution for severe tissue deficits. However, the reliance on graft biopsies for monitoring and treatment guidance is hampered by invasiveness and sampling errors. To address this, we propose a novel, low-cost, portable, flexible, miniaturized, and site-of-care (SOC) platform for multiplex sensing of predictive biomarkers (e.g., interleukin 6 (IL6), lactate) and pH. This platform can detect instances of VCA rejection before apparent clinical signs of immunogenic reactions such as inflammation and tissue damage, potentially revolutionizing the way we monitor and manage VCA patients<i>.</i><br/>The sensor fabrication was performed on in-house fabricated porous laser-engraved graphene (PLEG)-electrode, utilizing the laser-induced carbonization (LIC) technique on a thin (12.5 µm) polyimide (PI) surface at optimized laser parameters (laser power-22%, speed-5.2%, and gas flow-50%). Compared to conventional screen-printed platforms, the PLEG electrode provides higher surface area, receptor loading, electrical conductivity, ease of surface modification, and improved sensitivity with an average resistance of 32 ± 3 Ω/cm. Subsequently, the connection pad and reference electrodes (RE) were coated with silver/silver chloride (Ag/AgCl) conductive paste for improved connection. The RE was stabilized by coating it with polyvinyl butyrate (PVB) and sodium chloride polymeric cocktail. Three working electrodes (WE) were designated as WE-1 (for IL6), WE-2 (for lactate), and WE-3 (for pH). The sensor construction was initiated with surface activation WEs in 0.50 M H₂SO₄ containing 0.10 M KCl as a supporting electrolyte by sweeping potential between 1.0 and -1.2 V vs. Ag/AgCl reference electrode. IL-6 and lactate sensors were fabricated by electrodeposition of prussian blue (PB) and gold nanoparticles (AuNPs) on preactivated WEs, crosslinked with 11-mercaptoundecanoic acid (11-MUA). Subsequently, IL-6 monoclonal antibodies or lactate oxidase were immobilized through EDC: NHS activation and passivated with bovine serum albumin. For pH sensing, a composite PVB/Ag/AgCl electrode was utilized as the RE to determine the open-circuit potential of a nafion/PANI/PLEG electrode. Functionalization of the PLEG electrode was characterized using Raman, FTIR, XRD, and SEM techniques. Gracilis myocutaneous (GMC) flap VCA were performed across fully SLA-mismatched swine donor-recipient pairs and followed to Banff Grade III-IV clinical AR or postoperative day (POD) 6.<br/>Post characterization, the sensor’s electrochemical measurements to detect IL6, lactate, and pH were performed in buffer, artificial interstitial fluid, porcine blood, and serum samples with a portable potentiostat for a practical demonstration. Under optimal experimental conditions, the devised sensor showed a sensitivity of 2.59 µA/(log10IL6), 0.021 µA/mM, and ~49.39 ± 2 mV in spiked serum for IL6, lactate, and pH. Subsequently, serum samples of porcine VCA recipients were collected on postoperative days (POD) 0, 2, and 6 and tested using the IL-6, lactate, and pH sensors, respectively. The sensors can successfully track the change in the levels of IL6 (increasing) and lactate (increasing) and the change in pH post-surgery and align with clinical observations of inflammation and ischemia post-allograft on POD6. The sensor results were also validated using commercial IL-6 ELISA and lactate kits using the same samples. They obtained a high correlation (r) of r = 0.9885 (n=3, for IL6) and r = 0.9896 (n=3, for lactate). These results validate the effectiveness of the developed miniaturized platform, instilling confidence in its potential for<i> real-time monitoring of biomarkers in VCA models.</i><br/> <br/><b>KEYWORDS: </b>Vascularized Composite Allotransplantation (VCA), Interleukin-6, Lactate, Graphene, Electrochemical sensor, Multiplex sensing