1. (aii) (ai) (bi) (bii) (ci) (cii) SensorART BioMEMs Specifications and Development: Biosensors for early detection of VAD-related complications INTRODUCTION SensorART aims to provide a set of technologies for heart assistance, supporting patients with chronic heart failure, treated at home without renouncing to access high medical expertise, and healthcare professionals, keeping under control the performance of cardiovascular implanted Ventricular Assist Devices (VAD) by tele-control services. Recent advances in cardiac surgery have decreased cardiac morality, but a high number of postoperative deaths can be attributed to inappropriate myocardial protective techniques applied during the operative phase of extracorporeal circulation or to the a cytokine storm that is a potentially fatal immune reaction consisting of a positive feedback loop between cytokines and immune cells, with highly elevated levels of various cytokines. Therefore, the development of a rapid procedure for assessing concentrations of inflammatory biomarkers would be a valuable tool for diagnosis and therapy evaluation in comparison to ELISA-based techniques. One of the new approaches in the SensorART project was to improve the response by impedancemetric measurements, whereby the immobilization of the specific antibodies on gold microelectrodes were analyzed against different types of antigens associated with both pro & anti-inflammatory biomarkers (Tumor Necrosis Factor-α, Interleukin-1α, Interleukin-6, Interleukin 10, and Interleukin 1 receptor antagonist). The fabrication of gold microelectrode on PI: (a) casting of PDMS on the silicon mold, (b, c) peeling of the PDMS stamp from the silicon mold with microelectrode geometry, (d) inking the PDMS stamp with octadecylthiol (ODT), (e) microcontact printing (µCP) of the inked stamp with the gold substrate, (f) self-assembled monolayer (SAMs) formation onto the gold layer, (g) wet etching of gold not protected with SAMs, and (h) optical image of the chip based on a PI substrate. Biosensor Production on Polyimide Fabrication of Silicon Master Molds (a) (b) (c) (d) Silicon molds were developed by standard micro-optical lithography masks for the fabrication of the (a) microelectrodes and (b) microfluidic system. Eight working electrodes (WE’s) were developed for multiplex detection. This included a counter electrode (CE) and two reference electrodes (RE’s). (c) The alignment of the microelectrodes and microfluidic body. (d) The final concept for microelectrodes on polyimide (PI) and the microfluidic system in polydimethylsiloxane (PDMS). (a) Schematic biorecognition on the gold WE, (b) IL-10 detection on the WE’s, (c) modified diazonium-mAb’s on the gold WE’s with an integrated Ag/AgCl RE’s, and (d) TNF-α detection with non- fluorescence attributable to the negative test. Biorecognition and Lab-on-Chip The completed SensorART Lab-on-a- Chip. PDMS microfluidic system chemically bonded to the PI substrate. Electrochemical Impedance Spectroscopy (EIS) for the Detection of Cytokines Starting date: 01/03/2010 Duration in months: 48 Call (part) identifier: FP7-ICT-2009-4 Budget: 8,500.000.00 € Coordinator: IFC-CNR GENERAL INFORMATION Nyquist plot of EIS on modified gold microelectrodes for human cytokine detection ranging from 1 pg/mL to 15 pg/mL with: (Top) (a) IL-6, (b) IL-1β, and (c) IL-10, and (Bottom) Normalization of the curves with interferences attributable to non-specific binding of inactive proteins. ELISA versus EIS measurements for LVAD patient plasma IL-10 cytokine levels Prof. Abdelhamid Errachid: abelhamid.errachid@univ-lyon1.fr abelhamid.errachid@univ-lyon1.fr Dr Nicole Jafreeiz-Renault, Dr Nadia Zine, Dr Carole Chaix-Bauvais, Dr Monique Siguad, Dr Abdellatif Baraket, Dr Michael Lee, and Alvaro Gracia-Cruz The UCBL Team