Abstract
Bloodstream infections caused by drug-resistant bacteria is a global health problem. The total number of deaths per year related to antimicrobial resistance (AMR) is predicted to overcome in 2050 those caused by cancer today, creating economic costs of about 100 billion dollar. Within the EU, Italy is most affected with 30% of the 33'000 reported deaths in the EU per year. AMR continues to increase, for instance by the constant over-use of antibiotics to treat patients. Unnecessary, empirical and broad spectrum prescriptions are due to uncertainty rather than a diagnosed pathogenic infection. A major problem is that the time-to-result periods of state-of-the-art methods for bacteria and antibiotic resistance detections exceed the time-to-treatment window up to days, as the concentration of bacteria in blood is below the limit of detection. This requires bacteria culture for hours to days pre-analysis. In this project, the further development of an electroanalytical Point-Of-Care (POC) platform for the detection of model living bacterial cells and antibiotic resistance was pursued. The electrochemical detection is based on the bacterial metabolism, which has the potential to reduce the detection time compared to optical measurements. The electrochemical component of the POC platform was aimed to be fabricated by using inkjet printing. Bacteria were aimed to be immobilized by a specific bacteria capturing unit near the sensor surface. Microfluidic modules and heating modules were to be developed for advancing sensing automation at optimized bacterial conditions.
Results achieved
The analytical, geometrical, microfluidic, and technological requirements necessary for the development of the point-of-care (POC) platform were established. The analytical specifications required for living bacterial cell capture and detection were defined. A representative and complementary panel of bacterial models was selected as target bacterial strain and negative control. The most suitable recognition elements, i.e., antibodies and aptamers, were selected based on robustness, commercial availability, and compatibility with all materials of the POC platform as well as sample solutions. An electroanalytical module and glass-based Lab-on-Chip (LoC), integrating thermal control and optical back-up functionalities, were successfully designed and fabricated, constituting in combination the technological core of the POC platform. A multiplexed sensing configuration requiring only microliter sample volumes was realized, providing an optimal balance between high sensing performance and low material as well as sample consumption. The LoC integrated temperature sensors, a thin-film heater for precise temperature control at 37 °C, and photosensors. Numerical simulations validated the heater design, confirming uniform temperature distribution and stable operation under the required experimental conditions for bacteria capture and detection. The electroanalytical module was successfully realized through the combination of inkjet printing of graphene-based electrochemical sensors and 3D printing of the electrochemical cell components. A key achievement was the development of an innovative inkjet printing process for fabricating graphene electrodes coated with UV-photopolymerized hydrogels. Optimized printing strategies enabled reliable hydrogel deposition with tunable composition and functional group density. Carboxylic acid functional groups were introduced into the hydrogel matrix to enable the covalent immobilization of bacterial recognition elements required for selective bacterial capture. Extensive characterization using fluorescence microscopy validated the successful immobilization of the recognition elements and living bacterial cell capture, leading to the identification of the hydrogel composition, which provided the best compromise between antibody immobilization efficiency, hydrogel printability, and sensor stability. An important complementary result was the development of evanescent-wave-assisted photopolymerization of the hydrogel on optical fibers, demonstrating the successful transfer of the sensing chemistry to optical sensors and further expanding the technological versatility of the bacteria detection concept. Living bacterial cells were successfully captured by the hydrogel-based capturing layers and detected both electrochemically by measuring the metabolic conversion of suitable redox indicators and by surface plasmon resonance. The development of the electrochemical antimicrobial susceptibility test (AST) encountered challenges due to hydrogel fabrication reproducibility issues. Nevertheless, the underlying detection principles were successfully validated, and an alternative optical-fiber-based strategy was successfully established. The scientific outcomes of the project were widely disseminated through international and national conferences, including oral presentations, several of which were invited as well as keynote lectures, and poster communications at the 76th Annual Meeting of the International Society of Electrochemistry (ISE), Giornate dell'Elettrochimica Italiana (GEI), the International Workshop on Surface Modification for Chemical and Biochemical Sensing (SMCBS), AISEM Conferences, the European Biosensor Symposium, and international online conferences on sensors and applied sciences. Project results have also been disseminated through high-quality peer-reviewed publications in international journals, including Electrochimica Acta and Optical Materials, with all published works made available in open access.Project details
Unibo Team Leader: Andreas Stephan Lesch
Unibo involved Department/s:
Dipartimento di Chimica Industriale "Toso Montanari"
Coordinator:
ALMA MATER STUDIORUM - Università di Bologna(Italy)
Total Eu Contribution: Euro (EUR) 199.944,00
Total Unibo Contribution: Euro (EUR) 82.393,00
Project Duration in months: 24
Start Date:
28/09/2023
End Date:
28/02/2026
