Abstract
The aim of the ANALYSER project is the development of a flexible system for multiparametric in vitro monitoring of cellular cultures, with a focus on (but not limited to) electroactive cells like neurons and cardiac cells. The system will comprise an array of sensors based on an ultrasensitive organic device named organic charge modulated field effect transistor (OCMFET), specifically functionalized for the detection of a wide range of parameters and molecules, namely the electrophysiological activity (if any), ions like H+ and Ca2+, Dopamine, and Cortisol, with the idea of introducing a versatile and flexible tool that is able to conveniently investigate the multi-faceted extracellular environment by demonstrating the feasibility of detection of different paradigmatic species like ions, neurotransmitters, and hormones. The system that represents the outcome of the ANALYSER project will allow to acquire simultaneously the aforementioned parameters and analytes (or a subset of those, depending on the specific application), thus giving researchers in different areas the possibility of effectively probing different aspects of the state of a cell culture. This unprecedented capability makes this approach suitable for the comprehensive study of cells in vitro, allowing applications such as the study of neurodevelopment (with the aid of the new cellular engineering techniques that nowadays are able to provide several 3D structures and organoids), in vitro studies of neurodegenerative diseases (thanks to innovative approaches based on induced pluripotent stem cells), pharmacology and toxicology (thanks to the possibility of monitoring the effect of substances or drugs on cellular metabolism), and virology, just to mention a few of the most interesting ones. The functionalization techniques and materials will be specifically tailored in order to be compatible with the fabrication of a multisite sensors array, and adapted to the specific organic device that constitutes the core of the proposed system. The sensors will be characterized by an ultra-high sensitivity, thanks to the peculiar structure of the OCMFET structure, and will be mechanically flexible, low-cost and replicable in multiple copies on large areas, thanks to the low costs of the materials and fabrication techniques offered by organic electronics. In conclusion, the knowledge generated by the ANALYSER project will be of great interest in the in vitro field, and the technology that constitutes its outcome will represent the ideal candidate to address the new challenges that the new developments of cellular biology pose to researchers in several scientific disciplines, thus enabling the advancement and the expansion of both standard in vitro practice and novel approaches such as those based on pluripotent stem cells and in vitro tridimensional cellular aggregates and organoids.
Results achieved
The operative unit of Bologna, together with the IUSS unit, identified the architecture based on solution-gated extended gate OFET (ExG-OFET) as the most suitable choice for the development of chemical sensors. The UNIBO unit initially focused on identifying suitable materials and sensing strategies for the detection of pH, Ca2+, redox state and dopamine. In particular, electrochemically deposited membranes based on IrOx were selected for pH sensing. Several batches of IrOx surfaces were produced and characterized using standard electrochemical techniques, with the aim of subsequently integrating them with the transistors to test the sensing performance of the devices. For Ca2+ sensing, Au surfaces were electrochemically functionalized with a PEDOT film and subsequently modified by drop-casting the ion-selective membrane cocktail. These ion-selective sensors were then characterized via potentiometric techniques to assess all the relevant analytical parameters. The UNIBO unit also focused on the development of electrodes for measuring redox potential, a crucial parameter for monitoring cell cultures. The unit identified an optimal transducer structure that requires chemical modification of the printed measuring electrode with a PEDOT:ClO4 coating, in order to ensure adequate stability of the measurement system. Moreover, it was demonstrated that transistors based on the proposed technology are capable of detecting dopamine, a biologically relevant neurotransmitter that remains challenging to sense with this type of device. The transfer curves recorded from transistors equipped with platinum gate electrodes show a clear dependence of the threshold voltage on dopamine concentration, although the detailed sensing mechanism and the optimal operating conditions still require further investigation. UNIBO chemically functionalized the devices for the IUSS unit, which then characterized them, demonstrating very good performance in the determination of pH, Ca2+ and redox state, thereby underpinning the subsequent development stage towards a multi-sensor array. The design of a multi-sensor device started with the identification of a suitable protocol for integrating multiple sensors on the same substrate, along with the development of a functionalization procedure that accounts for compatibility among the different functionalization steps. The UNIBO operational unit implemented the chemical modifications and functionalization protocols required to create multisensing electrochemical transducers for the detection of Ca2+, pH and redox state on the arrays produced by the IUSS operational unit. Three different experimental protocols were used to assess the simultaneous response of the pH and calcium sensors, the pH and redox state sensors, and the calcium and redox state sensors. While systematically varying the relevant chemical variables—namely the pH, the calcium concentration, and/or the ratio between iron(II) and iron(III) concentrations in the test solutions in which the sensing surfaces were immersed—the responses of the sensors were recorded simultaneously, thus validating the feasibility of truly multiparametric measurements on the same platform. Overall, the achieved results demonstrate the feasibility of integrating multiple highly sensitive electrochemical transducers, based on ExG-OFET technology, into a coherent multisensor platform capable of simultaneously monitoring key physicochemical parameters relevant to in vitro cell culture studies. The successful development and characterization of pH, Ca2+ and redox, together with the implementation of compatible functionalization protocols for multi-site arrays, provide a solid technological and methodological foundation for the subsequent realization of the fully integrated ANALYSER system and its translation towards advanced applications in neurobiology, pharmacology and beyond.Project details
Unibo Team Leader: Isacco Gualandi
Unibo involved Department/s:
Dipartimento di Chimica Industriale "Toso Montanari"
Coordinator:
Istituto Universitario di Studi Superiori - Pavia(Italy)
Total Unibo Contribution: Euro (EUR) 122.500,00
Project Duration in months: 24
Start Date:
30/11/2023
End Date:
28/02/2026
