Category: Blog

Essential oils have been employed for centuries in various cultures for their therapeutic properties. Beyond their delightful fragrances, essential oils are gaining increasing recognition for their potent antimicrobial activity. These volatile and concentrated extracts derived from plants possess a remarkable ability to combat a wide array of harmful microorganisms, including bacteria, viruses, fungi, and even some parasites. The exploration of essential oils as natural alternatives to synthetic antimicrobial agents has sparked significant interest in recent years, fuelled by the growing awareness of the limitations and potential risks associated with conventional antibiotics and disinfectants. Among the wide variety of essential oils available, thymol, menthol, eugenol, and carvacrol stand out for their remarkable potency in terms of antimicrobial properties. In this comprehensive exploration of essential oils for antimicrobial activity, RELIANCE is currently in progress, merging the potential of nanomaterials with essential oils to develop a highly promising antimicrobial surface. Given that one method depends on the gradual release of essential oils from loaded nanoparticles, monitoring the amount of essential oils present on the surface in real-time can serve as a valuable, fast analytical approach. In this context, it has recently been demonstrated that the paper can be used as support for the development of electrochemical devices useful for this purpose.

Paper-based sensors

Paper-based electrochemical (bio)sensors have emerged as highly attractive analytical devices for their superior sustainable features, such as avoiding the use of polyester as support and the reduction of waste, being incinerated after use. However, paper-based electrochemical (bio)sensors have recently demonstrated further advantages, including the simple combination with vertical microfluidics and their use as a reservoir to deliver smart electrochemical (bio)sensors that are able to i) contain the reagents, ii) preconcentrate the target analyte, and iii) synthesize the nanomaterials inside the paper network. Furthermore, these devices have shown 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. Herein, we developed in RELIANCE a paper-based device for the detection of essential oils on the surface, as a smart tool to evaluate the availability of these compounds on the functionalized surface.

Preparation of paper-based sensor

Filter paper-based screen-printed electrodes were home-produced. Filter paper sheets were firstly modified with an ad hoc designed wax pattern in order to delimitate the hydrophilic area in which liquid samples were dropped, avoiding them to reach the electrical contacts through capillary permeation. The wax pattern was printed onto filter paper by means of a special printer and treated at 100 °C for 1 min in order to allow the wax to homogeneously permeate through the paper network. Then, conductive inks were used to print a three-electrode system onto wax-modified filter paper sheets. The working and counter-electrodes were obtained using a graphite-based ink and the pseudo-reference electrode was printed using Ag/AgCl-based ink (Fig. 1).

Electrochemical detection of essential oils

Different pulse voltammetry was selected for essential oils detection by connecting the paper-based sensor to a portable potentiostat connected to a laptop to easily manage the data. The modification of the working electrode surface with nanomaterial dispersion in order to improve the analytical performance of the sensor was demonstrated to be critical for the sensitive detection of essential oils. The electrochemical measurements were performed by sampling the biological compounds putting the paper strip in contact with the site wetted by oils. The calibration curves were carried out by analyzing different solutions of thymol, eugenol, and carvacrol in the concentration range comprised between 2-16 ppm.

Conclusion

The results obtained have demonstrated the ability to detect essential oils such as thymol, eugenol, and carvacrol on the surface using a paper-based device. This method offers the advantage of simplicity, sustainability and provides broad and promising applications for the detection of these substances.

Contributor: University of Rome Tor Vergata

The goal of RELIANCE project is to design and develop smart response self-disinfectant antimicrobial nanocoatings based on a new range of antimicrobial nanoparticles. The framework, on which the actual functionalities that bring out the antimicrobial effects are being built, comprises of mesoporous silica nanoparticles (SMIN). The silica particles are obtained via modification of the well-established Stöber process, using a soft-template method. Although the process to prepare these particles is relatively simple per se, the upscale of chemical processes or reactions using larger quantities of reactants rarely goes smoothly giving comparable results to laboratory scale. The upscale of mesoporous silica nanoparticles is no exception. Furthermore, with reference to the myriad of scientific articles dealing with the silica nanoparticle synthesis, it is evident that even minute changes in some parameter may cause a significant effect on the result. Therefore, to obtain conditions that are robust enough to enable the synthesis in a reproducible manner, would be of paramount importance from the upscaling point of view.

In RELIANCE, SMIN are prepared in batch processes. Unfortunately, there is no real practical way to monitor the progress of the reaction during the synthesis itself and to do adjustments as necessary to steer the reaction into the wanted direction. Therefore, the evaluation of the outcome will be done only after the process is completed and the product isolated and purified. The method of “trial and error” is both laborious and time consuming.

One of the key targets in RELIANCE is to produce particles with defined particle size distribution. When going to ever smaller particle sizes (i.e. nanoparticles), the task becomes more challenging. Multiple experiments have been carried out do date, to close the gap between the results obtained at lab-scale and the initial upscaled trials. The figure below shows a scanning electron microscope image of the product obtained in very recent upscaled experiment (50x reactant amounts compared to labscale). The particle size distribution measured from the image, and further supported by an independent laser diffraction analysis, are in agreement of a result very close to the targeted values. In the near future, the quest will focus on fine tuning of the current experimental parameters and set-up to still improve the particle size distribution.

When speaking about true colloidal solutions where the particles (with their size characterised in the nanometer scale, ≈ 100 nm and below) are separate and dispersed throughout the media, their isolation from the reaction mixture is not possible by simple filtration techniques and more advanced methods need to be utilised, like for example centrifugation. Preparing SMIN in quantities as set up by RELIANCE would require upscaling to volumes of tens of liters and that would no longer be viable to be processed by centrifugation. By changing the synthesis parameters, it has been observed that the formed particles start to assemble into larger aggregated structures. Formation of aggregates has a huge effect on the following step: when forming larger sized aggregates, simple vacuum or pressure filtration of the particles is again possible, therefore simplifying the work-up remarkably.

Although the aggregation is beneficial to ease the product isolation process, there is a downside. For the final coatings, particles need to be separated from each other to give well-dispersed and homogenous coating solutions. The experiments done so far demonstrate that using suitable techniques together with possible processing aids can lead to obtaining good dispersions of the particles.

Contributor: MILLIDYNE

Home appliances are commonly used in our daily life, with their surfaces being exposed various sources of virus and microbes, and human bodies. The research work in RELIANCE project addresses the above by bringing to the European consumers a set of innovative anti-microbial and antiviral coatings for everyday life products and applications.

While there are high expectations for some surfaces, such as the glass shelves of refrigerators, to be capable of suppressing bacteria or virus proliferation, the same applies to the technologies behind these surfaces’ functionalities. The novel biobased and self-disinfectant compounds RELIANCE develops will be added to the new nanocoatings, triggering a contact-killing action against bacteria and viruses. However, the matrix of the coating in which these newly designed active ingredients will be embedded represents an exciting challenge in itself. Through chemical design and surface nano- structuration, RELIANCE aims at intrinsically offering virus and microbe-repelling coatings, with a global antimicrobial action.

In the recent years, a large amount of omniphobic and repellent coatings, both inorganic and organic, were developed that were based on fluorine compounds. At the same time, European citizens are becoming increasingly concerned with health and environmental issues due to persistent chemicals waste and regulations expected to impose further restrictions on the use of controversial chemicals, and even mandate their overall replacement. Some fluorine-based chemicals are on the list.

All of the above calls for a new, technically ambitious approach, reflected in RELIANCE work package three, led by partner POLYRISE SAS. Their team focuses on the design and development of hybrid nanocoatings. The work activities so far have led to the obtaining of the first sol-gel-based hybrid inorganic-organic coatings exhibiting both pronounced hydrophobic features and dynamic oleophobicity. In other words, oily liquid or aqueous-based dirt-stain solutions will roll off on such coated surfaces without wetting and soiling them, meaning that the new hybrid coatings will achieve these characteristics without using fluorine-based compounds.

To impart a kind of omniphobicity, hybrid coatings based on functionalized nanosilica and low surface energy sol-gel and siloxane networks were developed, to prepare the coatings of desired transparency for application on substrates such as glass or stainless steel, commonly used in home appliances. The transparency and good aesthetics are assessed by Haze Measurement for clarity of the coated substrate, the mechanical resistance is assessed by hardness, while hydro- and oleophobicity are assessed by both Water or Hexadecane Contact Angle. Along with liquid repellency, these are the specifications for such coatings and formulations, enabling the achievement of the following characteristics when applied on glass substrates:

Property	Characteristics	Values
Transparency	Transparency	0,3 %
Mechanical resistance	Hardness ISO
Easy-to-clean	Water Contact Angle	98°
	Hexadecane Contact Angle	38°

Oleophobicity can also be demonstrated through the behaviour of an olive oil droplet on the coated surface that effortlessly rolls off glass, without wetting it.

The picture below shows glass-coated substrates obtained by a dip-coating process followed by a thermal curing process at a moderate temperature of 160°C.

As an objective of the project, the nano structuration of the coated surface could also be observed and determined with Atomic Force Microscopy (AFM), displayed in the image below:

To conclude, the development of the coatings must also achieve sustainable development targets. In addition to no fluorine compound use, the new hybrid coatings will be mostly based on aqueous composition while addressing the need for being compatible with spray application deposition.  These are the next challenges we are going to tackle in RELIANCE.

Stay tuned!

RELIANCE project develops an entirely new class of biocidal additive. The antimicrobial action of the Cu-SMIN nanoparticles is enhanced due to the contact-killing antimicrobial properties provided by non-ionic copper on their structure, combined with the antimicrobial activity of encapsulated essential oils (EOs). Essential oils are natural, eco-friendly, safe and easily biodegradable agents that have been reported to be effective against bacteria, fungi and viruses in certain concentrations. Due to their multicomponent nature, their antimicrobial activity is not attributed to a specific mechanism but is instead, the result of the action on multiple targets in the cells. For this reason, their application does not lead to bacterial resistance and they appear to be suitable to fight multi-drug resistant bacteria.

To establish the antimicrobial activity of the selected essential oils in RELIANCE, namely thymol, eugenol, carvacrol, and menthol, the initial research phase of the project involves rigorous testing using laboratory-based techniques and procedures. This phase is important for understanding the effectiveness of the selected essential oils on the inhibition of the microorganisms’ growth.

ISO Method for antibacterial activity on the surface (ISO 22196):

1. Preparation of test specimens: Non-porous materials are cut into standardized pieces.
2. Inoculation of bacteria: A known quantity of the target bacteria is applied to the test specimens.
3. Incubation: The specimens are incubated at a specified temperature and humidity to allow bacterial growth.
4. Measurement of bacterial activity: After a specified time, the bacterial activity on the test specimens is assessed by measuring the bacterial population.
5. Calculation of antimicrobial activity: The reduction in bacterial population on the test material compared to a control material is used to determine the antimicrobial activity, typically expressed as a percentage reduction.

ISO Method for Viruses:

Antimicrobial susceptibility testing for viruses, especially when it comes to antiviral drugs, is a more specialized area and may not follow ISO standards in the same way as bacteria. The methods for assessing antiviral susceptibility often vary depending on the virus, the drug being tested, and the laboratory’s capabilities. However, some general principles are followed:

1. Virus Isolation and Propagation: Enveloped viruses tested in these experiments are isolated from clinical specimens and then propagated in cell cultures until 80% of cellular lysis is observed. The clarified supernatant containing the virus is kept at 80°C until use. Non-enveloped virus MS2 Phage, was purchased by ATCC and propagated in its host cells: E. coli bacteria. After overnight incubation, the supernatant containing the virus was filtered to remove the host-cells and stored at 4 °C until use. These processes are necessary to ensure a sufficient quantity of the virus for testing.
2. Determination of Viral Titers: Viral titer is determined by plaque assay. Briefly, virus is ten-fold diluted and inoculated for 1 hour in a confluent monolayer of cells and then cultured in a semisolid overlay medium containing 1,5% tragacanth and 2%FBS (final concentration). After several days, depending on the virus, cells are washed and stained with crystal violet and units forming plaques (PFU) are counted. For MS2 Phage, after propagation, it is ten-fold diluted and plated in a soft agar overlay containing E. coli cells. After overnight incubation, PFU are visible and countable.
3. Virucidal activity: A viral suspension was incubated with a specific concentration of the drugs at different time points. Then, the mixture was used to infect confluent cell monolayer and after 1 h incubation, plaque assay was performed to evaluate the possible virucidal effect of the compounds against the viruses studied.
4. Measurement of Viral Replication: Viral Replication was evaluated through plaque assay. The inhibitory activity of the oils was estimated by comparing the number of plaques obtained in treated-virus with that obtained in untreated control virus.
5. Interpretation: The results are analyzed to determine the susceptibility or resistance of the virus to the tested antiviral drugs.

It’s essential to note that specific methodologies for virus susceptibility testing can vary widely, depending on the virus in question and the available laboratory resources. While ISO standards may provide some guidance for quality control and best practices, the exact protocols often depend on the unique characteristics of each virus and drug combination.

Results

Dilution tests are central in assessing the efficacy of antimicrobial agents against microorganisms. In these tests, microorganisms are exposed to a range of dilutions of the antimicrobial agent in a broth. The lowest concentration of the antimicrobial agent that, under specific laboratory conditions, prevents visible growth of the microorganisms within a defined timeframe is referred to as the Minimum Inhibitory Concentration (MIC). The MIC serves as a quantitative measure, assisting clinicians in determining the susceptibility of the microorganism to the antimicrobial agent and guiding treatment decisions. It is crucial to maintain strict control and standardization to ensure reproducibility of results within and between laboratories, as variations can significantly affect outcomes.

With these assumptions, the antimicrobial capacity of essential oils is tested and preliminary tests indicate that Carvacrol exhibits higher inhibitory activity compared to Eugenol. Additionally, it is observed that E. coli is more sensitive to the tested essential oils than B. clausii.

The Minimum Bactericidal Concentration (MBC) is determined by subculturing the broths used for MIC determination onto fresh agar plates. MBC represents the lowest concentration of the antimicrobial agent that results in the death of 99.9% of the bacteria being tested. For both Eugenol and Carvacrol, the MBC is found to be 1-2 dilutions higher than the MIC, indicating that a slightly higher concentration is needed to achieve bacterial killing.

Notably, after just 5 minutes of incubation, the results show an impressive 98% inhibition of bacterial growth. At a concentration of 0.6%, both essential oils successfully eliminate 100% of E. coli, S. aureus, and B. clausii within just one minute. Interestingly, Thymol demonstrates that S. aureus is more susceptible to its inhibitory effects compared to E. coli. This rapid and effective inhibition demonstrates the potency of the tested essential oils against the microorganisms.

The virucidal activity of the oils was assessed by plaque assay. The virus, incubated with an established concentration of the oils = 0,5%, was ten-fold diluted and inoculated in a cell monolayer for 1 hour. Then, cells were cultured with a semisolid overlay for several days and finally washed and stained using Crystal violet to visualize and count plaques forming units.

Different results have been obtained, depending both on the oil and the virus tested. Moreover, it has been observed that the number of infectious particles incubated with the oils decreases in a time-dependent manner. Preliminary tests showed that Carvacrol and Eugenol diluted to 0.5%, were able to inhibit CHIKV in a few minutes of contact: virus survival was 58.9% after 1 minute of treatment with Carvacrol and 53.1% after 3 minutes of treatment with Eugenol. Similar percentages have been observed also against SARS-CoV-2. Eugenol has been tested also against the pandemic influenza virus H₁N₁ /09 showing a two-log reduction (98% inhibition), after 5 minutes of incubation.

Bacteriophage MS2, a model for non-enveloped viruses, has been incubated at different times with Eugenol and Carvacrol. The oils have been diluted to a final 0,5% concentration as for all the experiments described above, but some virucidal effect has been observed even at 60 minutes of incubation. On the contrary, Eugenol to a 1% final concentration, showed a two-log reduction after 15-30 minutes exposure.

In conclusion, the obtained results confirm those available in the literature: essential oils are more efficient against enveloped viruses than non-enveloped ones.

These findings provide valuable insights into the potential use of essential oils as antimicrobial agents and highlight their effectiveness in inhibiting bacterial growth.

Looking to the Future

The next work activities will extend to deliver smartphone-assisted electrochemical paper-based devices for on-site detection of antimicrobial surface effectiveness.

Contributor: University of Rome Tor Vergata & ISBD

Brecht Demedts (Centexbel), Yasmine Van Thuyne (Alsico High Tech)

Fabrics are fibrous substrates with a very larger surface area in result of which they absorb liquids very well. The large absorption of fluids can happen both by capillary effects and through molecular swelling (e.g., water uptake by interacting through hydrogen bonds with cellulose). While this is a big advantage for comfort, it is an unwanted feature when adding coatings to textiles because the coating pastes soak into the fabric, rendering uncomfortable feel and touch. In order to circumvent this, typically fluorinated pretreatments are given to the textiles in order for coatings to remain on the surface of the fabric. The figure below presents a simple method based on hydrogels that RELIANCE partner Centexbel uses to prevent protrusion of coating pastes in a fabric.

An urgent need for alternatives to fluorinated chemicals

Part of the RELIANCE project focuses on the development of sustainable water repellency using fluor-free chemistries. This is important because fluorochemicals are notoriously persistent in nature. Nonetheless, fluorochemicals like PFOA and PFOS have been used extensively due to their excellent performance. In this context, fluorochemicals were increasingly popular in use until it became clear they were very persistent and barely degrade in nature. The use of PFOA/PFOS substances have been restricted in Europe by REACH under Annex XVII, and has recently been replaced by the Stockholm convention on persistent organic pollutants (POP) that has been in place since July 4th 2020. This novel legislation still foresees exceptions for the use of PFOA in textile applications where oil- and water repellent textiles are needed to protect employees against dangerous fluids, which include possibly pathogenic blood spat or mucosal aerosols. These applications could only make use of PFOA until July 4th 2023, indicating even the phasing out of PFOA in Europe is difficult for certain demanding applications. In most of the textile products PFOA is being replaced by perfluorohexane sulfonic acid (PFHxS) or other less harmful fluorochemicals. Under novel regulatory developments led by the German excellence BAuA, ECHA is currently investigating a further restriction also of PFHxS, as these components are also bio-accumulative. Even though exceptions for medical textiles are foreseen (25 ppb of PFHxS salts), it illustrates the difficulties in providing powerful alternatives for oil- and water repellency needed in protective clothing. The RELIANCE project is taking a radically different approach by using pretreatments with hydrogels.

Hydrogels efficiently prevent impregnation of coating pastes into the fabric

RELIANCE made use of hydrogel formation of certain polysaccharides when they are combined with salts. The main examples are alginate and pectin, that make hydrogels when combined with calcium ions and gellan gum that makes hydrogels when combined with natrium, potassium or calcium. We tried different approaches in which coatings can be applied on top of pretreated fabrics where a hydrogel formation occurs rapidly preventing the paste from further protruding in the fabric.

Next steps and optimization

In order to ensure that the coatings adhere well to the fabric, different setups have been made comparing pectin, gellan gum and alginate. Pectin proved to be unsuccessful in preventing the protrusion, but both gellan gum and alginate were very effective to achieve protective topcoatings. In a next step we tested two methods where either the hydrogel is added to the textile before coating the biobased polyurethane or whether the hydrogel forming polysaccharide could be added to the biobased polyurethane and coated on a calcium pretreated fabric. Both approaches were successful in achieving nice topcoatings, but when wash tests were performed, the first method rendered delamination of the coatings, while the second method resisted washing well.

Sustainability in protective clothing & the reliance project

Centexbel is an R&D centre for the textiles and plastics industry that has a large focus on developing sustainable methods & chemistries for textiles. For this project Centexbel works together with Alsico High tech to treat textiles with next generation antimicrobials and water-repellent chemistries suited for protective clothing. Protective clothing has an important role as it protects employees from dangerous situations (e.g. blood spat, virus particles), or it can protect production environments from human contaminations (e.g. prevent skin flakes from entering cleanrooms in electronics or production of pharmaceutical components). Alsico High Tech specializes in cleanroom clothing and has high standards in developing sustainable clothing. Alsico High Tech aims for a holistic approach for sustainable materials covering environmental, economic and social aspects of the protective clothing it develops (see Alsico’s sustainability report). But sometimes some specifications and requirements are particularly challenging. This is the case for certain classes of workwear where antimicrobial or water repellency is required. We are delighted that a consortium of companies led by Tekniker is tackling this multidisciplinary issue in the Horizon Europe project RELIANCE, where we can use the expertise of Europe’s top researchers to deal with some of the most challenging issues that our industry has to deal with.