The Euroanalysis biannual international conference of the Division of Analytical Chemistry of the European Chemical Societytook place in Geneva, Switzerland, 27 – 31 August, 2023. The 21st edition was jointly organized by the Division of Analytical Sciences of the Swiss Chemical Society (DAS) and the University of Geneva.
RELIANCE partners from Fribourg School of Engineering and Architecture (HEIA-FR) participated in the event and presented the project with a poster entitled “Bio-based antimicrobial peptides for smart response self-disinfected surfaces”. They shared some preliminary results regarding the extraction of antimicrobial peptides (AMPs) from keratin from chicken feathers through the application of various hydrolysis methods. These peptides undergo detailed characterization of their bioactive properties in order to be incorporated in the unique nanoparticles RELIANCE develops. The process will be further optimized for broader porduction and acquisition of fully active AMPs, to be integrated into materials, thus driving advancement in self-disinfecting applications.
Euroanalysis provides a forum for the European Analytical World to discuss the latest developments in analytical sciences. The theme of the 2023 meeting was: Analytical Probing of Complex Systems. It reflects the urgent need to observe chemical changes in complex systems, from materials surfaces, to living cells and ecosystems, in real time and with adequate spatial resolution. The conference is problem-oriented and has an interdisciplinary spirit. It brings together researchers that work in fundamental and applied sciences, but also decision makers and people intimately familiar with the systems of interest. It actively involves people from academia, federal research institutes, industry and regulatory bodies.
As part of work package 2 activities, RELIANCE proposes the development of mesoporous inorganic nanoparticles as potential nanocontainers of bioactives, due to their high stability, biocompatibility, large specific surface area, tunable pore diameter, and easy surface functionalization. They can be obtained by sol-gel technology which offers the possibility to scale up the synthesis process due to its cost effectiveness since it does not require expensive equipment and can be applied at low processing temperatures. Additionally, the mesoporous structure of the particle allows for the possibility of adding other elements during the same manufacturing process, such as copper, which can be easily incorporated thus enabling the contact killing action of the mesoporous silica nanoparticles. The contact killing action allowed by Cu-SMIN will be combined synergistically with non-toxic biobased actives such as essential oils and antimicrobial peptides.
These particles, which have dimensions in the nanometer range, exhibit unique antimicrobial properties and once their synthesis is finalized, they can be applied in a wide range of fields, and can also be incorporated in coatings to prevent the proliferation of harmful microorganism in contact surfaces and high traffic areas.
During the first year of the project, in addition to having optimized the synthesis procedures for obtaining smart-release nanoparticles made of silica, at lab scale, we are working on the incorporation of copper and its functionalization. We are excited with these promising first results which will help our continuing fight with infections caused by bacteria, virus and fungi in the future.
Microbial colonization of surfaces burdens today’s societies by causing significant cost to human lives and the economy. Its prevention remains a global challenge for humanity and we are constantly seeking ways to reduce the transmission of microorganisms. A solution RELIANCE project works on is smart response self-disinfectant antimicrobial surfaces achieved through the design and development of a new range of antimicrobial nanocoating with a contact-killing action. The nanocoating consists of copper-functionalized mesoporous silica nanoparticles modified with Antimicrobial Compounds based on either essential oils or amino peptides isolated from protein-containing waste streams.
Our Swiss partner Haute école d’ingénierie et d’architecture Fribourg (HEIA-FR) is responsible for identifying and isolating Antimicrobial Peptides (AMP) from chicken feathers. Millions of tons of feathers are generated annually as byproducts from the poultry industry causing serious environmental issues and impacting human health safety. Even though feathers are of interest due to their composition of 90% wt. of Keratin protein, they are still underexploited because of their high stability and resistance to common proteolysis protocols.
Therefore, within this project, the Institute of Chemical Technology at HEIA-FR aims to develop effective, profitable, and sustainable processes to use this resource and transform waste feathers into AMPs. Given the difficulty and the challenges behind this mission, three approaches are currently being investigated for the isolation of bioactive peptides with optimal bioactive properties. A screening of the best hydrolysis conditions of each approach is being evaluated and an initial bioactive peptide fraction has been isolated and successfully tested against E. Coli and S. Aureus. The antimicrobial assays are assessed by our partner UNITOV.
HEIA-FR will further develop and work out the scale-up of AMP production. Moreover, the Institute of Chemical Technology will cooperate with the Plastics Innovation Competence Center (PICC) on the anchoring of the produced AMPs to the surface of Cu-dopped mesoporous silica nanoparticles developed by TEKNIKER and further coating while utilizing the cold atmospheric plasma technology to produce smart antimicrobial coated surfaces for application in the automobile industry.
On June 22, RELIANCE partner Centexbel, leader of the work activities on the synthesis and formulation of biobased polyurethane nanocoatings and their applicaition through digital printing, presented the project at the INFOhappening Textile Coating, Finishing, Dyeing & Printing workshop. The annual event took place in Zwijnaarde, Belgium and was attended by 40 industry representatives of the coatings industry, some of who joined online.
The main objective of the workshop was to showcase to the participants innovative approaches in the textiles coating industry, new projects and associated novel technologies, interesting results and regulatory matters.
The RELIANCE project was presented with preliminary results on the activities of digital finishing. Digital finishing is a very new approach to the textiles coating and finishing industry. It allows for a decrease of the use of antimicrobials up to 90%! Digital printers apply an invisible antimicrobial finish locally, where they are needed, which is done in lower amounts, precisely controlled by digital printers. The visitors of the workshop were able to see some samples at the RELIANCE booth, assisted by the scientific experts involved in the project who properly addressed all their questions.
The fourth consortium meeting of RELIANCE, marking 12 months since the start of the project, was hosted by our partner MILLIDYNE in Finland, nestled amid lush green landscapes and crystal-clear lakes.
The event took place in a charming wooden cabin with large windows offering breathtaking views of a lake and the surrounding forest. This proved to be the perfect setting for sharing results, achieving milestones and overcoming challenges, discussing further strategies and exchanging ideas on what approaches to apply in the next stages of work activities.
Upon presentations’ conclusion, everyone was ready to enjoy some relaxing time and traditional Finnish delicacies. As the sun began to descent, the RELIANCE team took a boat trip on the lake, back to Tampere’s city center, and recharged to continue their intense work on the development of the antimicrobial nanoparticles and the incorporation of the essential oils and keratin based antimicrobial peptides.
RELIANCE partners from Fribourg School of Engineering and Architecture (HEIA-FR) took part in the 16. Freiburger Symposium organized by the Swiss Chemical Society, Division of Industrial and Applied Chemistry (DIAC) in Fribourg, Switzerland, 27 – 28 April 2023. They presented the progress within Work Package 2 “Synthesis of mesoporous nanoparticles with improved antimicrobial effect” with a poster entitled “Bio-based antimicrobial peptides for smart response self-disinfected surfaces”.
HEIA-FR team leads the activities related to optimizing keratin extraction from poultry feathers, which also involve a study of enzymatic methods for pure peptide fractions isolations, their characterization, and proper modification for achieving optimum boactive properties when attached to the copper doped mesopourous silica nanoparticles (Cu-SMIN).
The Freiburger Symposium is a well established platfrom for exchanging research results through posters and we are pleased that its 16th edition held under the topic “From Lab to Industrial Application – Trends/Innovation in Process Chemistry & Technology” of co-chaired by one of RELIANCE project’s key consrotium members Dr. Roger Marti, HEIA-FR. The objective of this biennial conference is to provide DIAC’s more than 300 members, chemists and chemical engineers interested in industrial chemistry, chemical production and chemical process development with opportunities for professional training and networking.
RELIANCE project aims to design and develop smart response self-disinfectant antimicrobial nanocoatings based on a new range of smart antimicrobial nanoparticles. They will consist of mesoporous silica nanoparticles with metallic copper in their structure, modified with biobased bioactive compounds.
To ensure that the antimicrobial nanocoatings developed within the RELIANCE project are sustainable for humans and the environment, to increase recycling and use of resources in a more efficient way, efforts are required from an early stage of design and manufacturing.
Fundamental to the whole effort is the topical concept of ‘Safe-and-Sustainable-by-Design‘, SSbD, which implies the design of safe chemicals and materials, minimising their emission into the environment and the use of natural resources, with the aim to reduce the negative impacts to human health and environment. It integrates circularity, climate neutrality, functionality and safety of materials, products and processes throughout the life cycle.
The European Commission has recently established a set of recommendations[1] that proposes a European framework for SSbD chemicals and materials be established for Research and Innovation activities. The EC Joint Research Center has proposed a first SSbD framework[2] that sets a common baseline for evaluating safety and sustainability – an important step to increase the protection of human health and the environment against hazardous substances. The Sustainability criteria should consider environmental, social, and economic dimensions while safety should be included in all of the dimensions.
On this framework developed by JRC for SSbD criteria implementation, a two-phase approach is recommended:
first a design phase in which a number of guiding principles are proposed to support the design of chemicals and materials,
SSbD1
Material efficiency
SSbD2
Minimize the use of hazardous chemicals/materials
SSbD3
Design for energy efficiency
SSbD4
Use renewable sources
SSbD5
Prevent and avoid hazardous emissions
SSbD6
Reduce exposure to hazardous substances
SSbD7
Design for end of life
SSbD8
Consider the whole Life Cycle
and then a stepwise hierarchical approach is proposed to address the comprehensive safety and sustainability assessment of the new designs by carrying out different analysis on chemical safety, direct toxicological or ecotoxicological impact, aspects of environmental sustainability as well as social and economic conditions.
RELIANCE is aligned with this SSbD philosophy and in the first steps of the project the SSbD guiding principles are being considered during the antimicrobial nanocoatings design phase. In this preliminary sustainability analysis special attention was paid to verify that the initial selection of materials will allow the environmental and societal acceptance of the final antimicrobial nanocoatings developed within the project.
As a second step within the RELIANCE project, a Safety and Sustainability assessment will be conducted as shown in Figure 1, from a Life Cycle Engineering perspective that sequentially applies safety (material hazard and human health), environmental, economic and social comprehensive studies:
Figure 1. Sustainability assessment by RELIANCE project (based on the JRC framework)
[2] (2022, European Commission and JRC), Caldeira, C., Farcal, R., Garmendia Aguirre, I., Mancini, L., Tosches, D., Amelio, A., Rasmussen, K., Rauscher, H., Riego Sintes, J. and Sala, S., Safe and sustainable by design chemicals and materials – Framework for the definition of criteria and evaluation procedure for chemicals and materials, EUR 31100 EN, Publications Office of the European Union, Luxembourg, 2022, ISBN 978-92-76-53280-4, DOI 10.2760/487955 (online)
RELIANCE was presented at LOPEC 2023 Conference that was recently held in Munich, Germany, attended by experts from 22 countries who shared their expertise in around 170 presentations. The paper-based electrochemical (bio)sensors that will be used as an analytical method in the project were presented in the session dedicated on “Challenges and use cases of large area, printed or organic electronics in biomedical applications which included all applications in biomedical and healthcare such as sensors, diagnostic devices and wearable health patches that contain significant printed and flexible elements”.
“As the world’s most important communication platform for the industry, the LOPEC Conference presented the latest state of the technology and provided valuable insights for both new users and industry insiders,” said Wolfgang Mildner, General Chair of LOPEC.
In various conference formats, companies such as BMW, Microsoft, Pirelli, Samsung, Sun Chemical and Varta presented trends and new applications in printed electronics.
“The LOPEC Conference is a great yearly event for me to bring myself up to date,” stated Dr. Edzer Huitema, Chief Technology Officer at E Ink.
Focus topics of the exhibition and conference were Smart Living and Mobility, as in the words of Mildner, “sustainability has come into even sharper focus, because printed electronics are produced in a resource-efficient manner and enable green solutions”.
Antimicrobial characterization and nanotoxicity study
Characterizing the antimicrobial activity of bioactive compounds, mesoporous additives, and obtained nanocoatings, selected and developed within Reliance project, is the overriding goal of work package 7 (WP7). These materials will be tested, considering a wide range of bacteria, fungi, and viruses, to include SARS-CoV-2. Furthermore, nanotoxicological study of the nanocoating developed will be addressed, including environment and in-vivo test (inhalation). The testing activities will ensure the effectiveness of the bioactive compounds, mesoporous additives, and obtained nanocoatings as well as their safety.
The activities in WP 7 are carried out by Reliance project partners Tor Vergata University, Policlinico Militare (Italian Ministry of Defence), Centexbel, Ineris, and Tekniker. During the first six-month period, they started to design the set-up for virus and bacteria analyses using the reference methods while also adding testing of some essential oils such as Carvacrol and Eugenol.
Carvacrol and Eugenol were initially tested to evaluate their antibacterial activities towards Escherichia coli (G-) and Bacillus clausii (G+). The Minimal Inhibitory Concentration (MIC) tests were performed by the broth microdilution method. The MIC is defined as the lowest concentration of the essential oil at which the bacteria does not demonstrate visible growth. The essential oils were firstly dissolved in an equal volume of Dimethyl sulfoxide (DMSO), then serial two-fold dilutions from 5.000%–0.002% (v/v) of the essential oils were prepared, and 200 μL of each dilution dispensed into a 48-well micro-titer plate. Each well was then inoculated with 200 μL of the bacterial suspension containing 105 CFU, and the micro-titer plate was incubated at 37 °C for 24 h. The preliminary experiments reported in Figure 1, highlighted Carvacrol MIC=0.08 % for B. clausii (Gram+) while a MIC=0.15 % is reported for Eugenol. In the case of E. coli (Gram-), Carvacrol MIC=0.04 %, and Eugenol MIC=0.08 % were observed, suggesting a higher inhibitory activity of Carvacrol vs. Eugenol for the tested bacteria.
Fig.1
Antimicrobial activity evaluation by MIC for Carvacrol (CAR) and Eugenol (EUG) on B. clausii and E. coli. Nc, negative control (no bacteria), Pc, positive control (absence of EO).
To determine the mechanism of action of the selected essential oils on different viruses, the first experiments were carried out using chikungunya virus (CHIKV). CHIKV was isolated from a clinical sample by the Scientific Department of Policlinico Militare (Italian Ministry of Defence), and propagated in Vero cells. The viral titer was determined by plaque assay. In order to determine the virucidal activity of Carvacrol and Eugenol, equal volumes (0.5 ml) of CHIKV suspension, containing 10^5 PFU/mL, and essential oils were mixed with a final concentration of 0.5%, that resulted not toxic to the cells (Figure 2).
Figure 2. Study of cytotoxic effect and DMSO effect.
The effect of the essential oil addition at different time points (1, 3, 5, 15, 30, 45 and 60 min) was evaluated. As shown in Figure 3, viral inhibition by 50% was observed starting from 1- and 3-minutes treatment with Carvacrol and Eugenol, respectively. Interestingly, the antiviral activity of the compounds increased over time, reaching a percentage of inhibition of about 100 % upon 15 min incubation.
Figure 3. CHIKV survival after treatment over time
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Electrochemical paper-based devices to be used in RELIANCE presented at Materials for Biosensing & Bioimaging 2023 Workshop in Madrid
The workshop “Materials for Biosensing & Bioimaging 2023” was organised in the Universidad Autónoma de Madrid through the Institute for Advanced Research in Chemical Sciences – Universidad Autónoma de Madrid (IADCHEM).
The audience included an international body of students, faculty and staff from Universidad Autónoma de Madrid and other universities in Spain.
Fabiana Arduini, partner in RELIANCE project and leader of the antimicrobial characterization work activities, was invited as a speaker to share on her research activity in the electrochemical paper-based devices. The principle will be applied in RELIANCE project for evaluating the antimicrobial efficiency of the developed nanocoatings by using paper-based devices combined with a smarphone-assisted potentiostat. This method presents a high interest due to the findings that paper-based sensors can overcome the limitation of the classical miniaturized electrochemical sensors, with published articles in several high-impact factor journals, including Biosensors and Bioelectronics (IF 12.5) and invited reviews (e.g. Nanomaterials and paper-based electrochemical devices: merging strategies for fostering sustainable detection of biomarkers Journal of Materials Chemistry B 10 (44), 9021-9039, selected for cover page).
Reportedly, paper-based electrochemical (bio)sensors demonstrate an ability to overcome the limitations of the other printed electrochemical sensors in the measurement of entirely liquid samples by detecting the target analyte in the aerosol phase or solid sample, without the additional sampling system. More information on that could be found in the invited review ”Electrochemical paper-based devices: When the simple replacement of the support to print ecodesigned electrodes radically improves the features of the electrochemical devices” published in Current Opinion in Electrochemistry SI: Emerging Opinions (2022) (Arduini, F., 2022. Electrochemical paper-based devices: When the simple replacement of the support to print ecodesigned electrodes radically improves the features of the electrochemical devices. Current Opinion in Electrochemistry, p.101090).
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