Classifications

• • H—ELECTRICITY
  • H01—ELECTRIC ELEMENTS
  • H01B—CABLES; CONDUCTORS; INSULATORS; SELECTION OF MATERIALS FOR THEIR CONDUCTIVE, INSULATING OR DIELECTRIC PROPERTIES
  • H01B1/00—Conductors or conductive bodies characterised by the conductive materials; Selection of materials as conductors
  • H01B1/06—Conductors or conductive bodies characterised by the conductive materials; Selection of materials as conductors mainly consisting of other non-metallic substances
  • H01B1/12—Conductors or conductive bodies characterised by the conductive materials; Selection of materials as conductors mainly consisting of other non-metallic substances organic substances
  • H01B1/122—Ionic conductors

Definitions

• the present disclosure relates to unprotected tripeptide ionogel compositions, respective production processes and characterization with application in a variety of technological fields.
• Hydrogels are materials typically composed of more than 90% (w/w) of water. Peptides can self-assemble into hydrogels, representing a promising class of soft biomaterials that has received increasing attention during the last decades.
• the process of peptide self organization occurs at a molecular level in a 'bottom-up' approach where the simplest biomolecules, such as peptides or amino acids, interact with each other in aqueous solutions in a coordinated manner. In this process, one-dimensional peptide aggregates evolve to form fibrils that coil into larger fibres, crosslinking to form a gel.
• hydrogels weakness is the low stability in non-aqueous environments. When exposed to air, for example at ambient room conditions, hydrogels rapidly lose moisture, shrink and turn brittle due to water evaporation. On the other hand, to turn hydrogels into conductive materials there is usually the need to incorporate dopants in the hydrogel formulation [10].
• Ionic liquids are molten salts composed by at least one organic salt of composition X + and Y , wherein X + represents the cation of the salt and Y represents the anion of the salt.
• the melting point of ionic liquids is below 100°C, and they show very low volatility.
• Ionic liquids are designer solvents in the sense that the anion and cation can be varied and tuned to different purposes.
• Ionic liquids are considered green solvents with very interesting properties, namely high ionic conductivity, large electrochemical window, low vapour pressure, excellent thermal, electrochemical and chemical stability, non flammability, non-toxicity, biocompatibility and biodegradability.
• ionogels retain the interesting features of the ionic liquids they are composed of, but the immobilization of ionic liquids to yield advanced functional materials is still challenging [11] The entrapment of ionic liquids within biological matrices following a designed "bottom-up" approach is challenging and largely unexplored.
• Low molecular weight gelators are small molecules with molecular weight less than 2000 Da.
• the physical gelation of low-molecular- weight compounds results from non-covalent bonds, such as hydrogen bonds.
• gelator molecules are first self-assembled, producing fibrous assemblies. Then, these fibrous assemblies form a three-dimensional network structure, and gelation occurs by trapping solvent in the networks.
• Short peptide sequences including unprotected tripeptides, fall within this category of LMWGs.
• Dutta and co-workers reported LMW amino acid-based molecules as ionogelators [13] These were prepared from a precursor scaffold of hydrogelators and organogelators, an amino acid/dipeptide moiety at the head of an amphiphilic molecule with a free amine group at the N-terminus and a long hydrophobic chain at C-terminus.
• ionogelators prepared from a precursor scaffold of hydrogelators and organogelators, an amino acid/dipeptide moiety at the head of an amphiphilic molecule with a free amine group at the N-terminus and a long hydrophobic chain at C-terminus.
• ionogelation process systematic variations of amino acids and protective groups were performed.
• the developed ionogels were exploited as adsorbing agents in environmental clean-up and as templates in the synthesis of TiC nanoparticles.
• amino acid or peptide-based gelators are chemically modified with other chemical groups that promote the self-assembly process, which reduces the easiness of production of the low molecular weight gelator, and also reduces the sustainability, biodegradability and biocompatibility of the final gel.
• the present disclosure relates to a peptide ionogel composition (or formulation), wherein unprotected tripeptides are used as gelators.
• the tripeptide ionogels result from the gelation of ionic liquids with unprotected tripeptides that present self-assembly propensity.
• protected peptides with self-assembly propensity are those chemically modified with functional moieties at one or more different sites, namely the C-terminus, the N-terminus or at the amino acid side chains.
• the protecting groups used for the modification have self-assembling properties usually due to the hydrophobicity and aromaticity, and include the 9-fluorenylmethyloxycarbonyl (Fmoc) group, or any groups containing aromatic tails, naphtyls, lipids, fatty acids, cyclic compounds, sugars, carbamide, or aliphatic tail-moieties [19].
• Unprotected peptides are those that are not modified at the C-terminus, N-terminus or side chains with protecting groups presenting self-assembling propensity, as listed above.
• the tripeptide ionogels here described are supramolecular peptide assemblies in ionic liquids. They are stable at ambient conditions, have thermal, electrochemical and chemical stability in a wide range of temperature and humidity conditions, are conducting, soft, self- healing, thermoreversible, have a large electrochemical window, are non-flammable, non toxic, biocompatible and biodegradable materials. These materials can further encapsulate other stimuli-responsive elements, as for example optical probes made of liquid crystals, including liquid crystals presenting nematic, smectic or chiral phases.
• the major advantages and unique properties of the formulations and production methods of tripeptide ionogels described in the present disclosure are: i) easy production of tripeptides, without the need to protect the tripeptide or to chemically modify the tripeptide with functional groups yielding self-assembling properties, as observed in all previous examples of small-peptide based low- molecular weight ionogelators;
• tripeptide ionogels chemical diversity and functionality of tripeptide ionogels is greater than in tripeptide hydrogels.
• hydrogels chemical functionality arises solely from the tripeptide sequence, whereas in ionogels both components (tripeptide and ionic liquid) confer chemical and functional richness to the system;
• tripeptide ionogels present faster gelation times when compared to equivalent tripeptide hydrogels, making them suitable for additive manufacturing; v) when compared to equivalent tripeptide hydrogels, the resulting tripeptide ionogels remain chemically and physically unaltered when stored at ambient conditions of temperature (18-27°C), pressure (atmospheric pressure, 1013.25hPa) and relative humidity (30-80%), due to the low volatility of the ionic liquid employed;
• the resulting tripeptide ionogels present ionic conductivity over time in the same range as the ionic liquid used to produce the ionogel, which is not possible to observe in hydrogels where conductivity is typically given by dopants;
• tripeptide ionogels are thermally stable and thermoreversible
• tripeptide ionogels are self-healing materials
• tripeptide ionogels retain the physico-chemical and functional features of the tripeptides and of the ionic liquids they are composed of;
• tripeptide ionogels represent a way of immobilizing ionic liquids in stable formulations and gel matrices using very mild processing conditions
• the tripeptide ionogel formulations and resultant materials withstand several processing and fabrication techniques including spin coating, flow-coating (doctor blade), electro- and blow-spinning, 3D-printing, patterning, metal depositions, to give rise to a variety of structures;
• tripeptide ionogel formulations and resultant materials can be processed into different formats including thin films - spread on flexible or rigid substrates with or without patterning and with or without chemical functionalization - or other structures with different ID, 2D and 3D geometries (e.g. fibres, particles) to yield stable materials with distinct geometries;
• tripeptide ionogels can incorporate other elements, as for example liquid crystals which self-assemble within the ionogel in a stimuli-responsive manner and which could further contribute as opto-electrical transducers to chemical and physical changes;
• xv the intrinsic properties of tripeptide ionogels and their potential to provide electrical and optical signals in the presence of chemical analytes in gas or liquid phases, or as a response to physical changes (e.g. temperature, pressure, electromagnetic field), allows its utilization as sensitive films, able to detect analytes and physical changes through different transduction principles (optical, electrical, opto-electric, piezoelectric).
• sensitive films can be arranged due to the wide range of components available for the films, allowing the optimization of response patterns for a variety of applications;
• tripeptide ionogels the production method of tripeptide ionogels is scalable and compatible with mass production.
• An aspect of the present disclosure relates to an ionogel composition
• an ionogel composition comprising: at least one ionic liquid selected from imidazolium ionic liquids, ammonium-based ionic liquids, or mixtures thereof; and
• At least one unprotected tripeptide as a gelator, preferably ionogelator, wherein the tripeptide has self-assembly propensity and is selected from the following list: Lysine- Tyrosine-Phenylalanine, Lysine-Phenylalanine-Phenylalanine, Arginine-Phenylalanine- Phenylalanine, Aspartic acid-Phenylalanine-Tyrosine, and Tyrosine-Phenylalanine-Aspartic acid; or
• a gelator is a substance capable of forming a gel.
• an ionogelator is defined as a molecule responsible for the gelation of ionic liquids. Gelation is defined as a process in which the immobilization of a given solvent leads to the formation of soft materials.
• the tripeptide concentration ranges from 10 - 700 mM.
• the tripeptide concentration ranges from 20 - 600 mM, preferably 100 - 400 mM, more preferably 50 - 400 mM.
• the tripeptide concentration ranges from 20 -200 mM, preferably 20 - 100 mM.
• the unprotected tripeptides have an amide at the C-terminal, preferably Aspartic acid-Phenylalanine-Tyrosine, and Tyrosine-Phenylalanine-Aspartic acid peptides.
• the unprotected tripeptides comprise a molecular weight equal or inferior to 2000 g/mol, preferably between 270-540 g/mol.
• the ionic liquid concentrations range from 20-98 % (w/w).
• the ionic liquid is selected from phosphate-containing ionic liquids, chloride-containing ionic liquids, dicyanamide-containing ionic liquids, quaternary ammonium salts, or mixtures thereof.
• the imidazolium-based ionic liquids are selected from a list comprising: l-butyl-3-methylimidazolium dicyanamide, l-ethyl-3-methylimidazolium dicyanamide, l-butyl-3-methylimidazolium chloride, l-ethyl-3-methylimidazolium chloride, 1 or mixtures thereof.
• the ammonium-based ionic liquids are selected from a list comprising: choline chloride, choline hydroxide, choline phosphate buffer, 2- hydroxyethylammonium formate, choline dihydrogenphoshate, dicholine monohydrogenphosphate, or mixtures thereof.
• the choline phosphate ionic liquid buffer system (choline phosphate buffer) is produced by the addition in equimolar amounts of the ionic liquids choline dihydrogenphosphate and dicholine monohydrogenphosphate.
• the water content of choline phosphate buffer ranges from 16-22% wt.
• the tripeptides ionogelation is triggered by changes on the pH, increasing the pH to 7-9, preferentially 7.5-8.5.
• the pH is increased by addition of sodium hydroxide, choline hydroxide or l-butyl-3-methylimidazolium hydroxide.
• the tripeptides ionogelation is triggered by changes on the temperature or a buffer composition.
• the ionogels' water concentration is below 90% (w/w), preferably between 2-75% (w/w).
• the molecular self-assembly of the tripeptide occurs by non- covalent interactions such as hydrogen bonding, hydrophobic, electrostatic, van der Walls, and p-stacking, p-cation, p-sulphur, dipole-dipole, ionic, or metal-chelating interactions.
• the ionogel composition may further comprise an additive selected from the following list: citric acid, tartaric acid, ascorbic acid, glucose, sucrose, xylose, arginine, histidine, proline, urea, sorbitol, xylitol, glycerol, monoethylene glycol, menthol, lactic acid, betain, or mixtures thereof.
• an additive selected from the following list: citric acid, tartaric acid, ascorbic acid, glucose, sucrose, xylose, arginine, histidine, proline, urea, sorbitol, xylitol, glycerol, monoethylene glycol, menthol, lactic acid, betain, or mixtures thereof.
• ionogel composition might comprise choline dihydrogenphosphatexitric acid, choline dihydrogenphosphate:tartaric acid, choline dihydrogenphosphate:ascorbic acid, choline dihydrogenphosphate:glucose, choline dihydrogenphosphate:sucrose, choline dihydrogenphosphate:xylose; choline dihydrogenphosphate:arginine; choline dihydrogenphosphate:histidine; choline dihydrogenphosphate:proline; choline dihydrogenphosphate:sorbitol; choline dihydrogenphosphate:xylitol; choline dihydrogenphosphate:monoethylene glycol; choline dihydrogenphosphate:lactic acid; dicholine monohydrogenphosphate: citric acid; dicholine monohydrogenphosphate:tartaric acid; dicholine monohydrogenphosphate:ascorbic acid, dicholine monohydrogenphosphate:glucose, dicholine monohydrogenphosphat
• the ionogel is stable at ambient conditions (temperature - 18-27°C, pressure - atmospheric, 1013.25hPa, relative humidity - 30-80%).
• the ionogel is soft, self-healable, thermoreversible, stimuli- responsive, non-flammable, non-toxic, biocompatible and biodegradable.
• the ionogel composition may further comprise a stimuli-responsive component.
• the stimuli-responsive component is a liquid crystal.
• the ionogel composition may be obtainable by moulding, casting, spin-coating, film coating, spinning, electrospinning, microfluidic, 3D-printing, ink-jet printing, patterning or metal deposition method.
• Another aspect of the present disclosure relates to a disc, film, fiber, sphere, cube, mesh, channel or patterned structure comprising the ionogel composition of the present disclosure.
• Another aspect of the present disclosure relates to an article comprising the ionogel composition of the present disclosure.
• the article is a sensing device, electronic device, opto-electronic device, flexible device, wearable device, conducting device, coating material, electrochemical device, electrolyte, semi-solid lubricant, adsorbent agent; pharmaceutical formulation, food product, or cosmetic product.
• Another aspect of the present disclosure relates to use of the ionogel composition described in the present disclosure for additive manufacturing, chemocatalysis or biocatalysis, encapsulation, drug delivery and/or cell assays.
• Another aspect of the present disclosure further relates to a kit or a reagent comprising the ionogel composition of the present disclosure.
• Another aspect of the present disclosure further relates to a method for preparing the ionogel composition of the present disclosure, comprising the following steps: obtaining at least one unprotected tripeptide, selected from the following list: Lysine- Tyrosine-Phenylalanine, Lysine-Phenylalanine-Phenylalanine, Arginine-Phenylalanine- Phenylalanine, Aspartic acid-Phenylalanine-Tyrosine with the C-terminus capped with an amide, and Tyrosine-Phenylalanine-Aspartic acid with the C-terminus capped with an amide.;
• the gelation is promoted by heating the solution up to 40-110°C, preferably 40-80°C.
• Figure 1 depicts a schematic and simplified representation of the formulation and production process of tripeptide ionogels.
• FIG. 2 illustrates a schematic representation of the tripeptide structures used to produce the ionogels.
• the tripeptides are grouped in System I and System II, according to the gelation trigger identified in tripeptide hydrogels.
• Tripeptides belonging to System I self- assemble into hydrogels when the gelation trigger is pH-dependent.
• Tripeptides belonging to System II self-assemble into hydrogels when the gelation trigger combines buffer composition (typically phosphate dependent) and temperature.
• Figure 3 shows a schematic representation of the experimental procedure and overall results of KYF ionogels in [Bmim][DCA] and [Emim][DCA]. It also compares the results obtained with the hydrogel, 40mM KYF in deionized water. Additionally, being produced in different laboratories, with different tripeptide's batch provided by different commercial houses, it also shows the reproducibility of this procedure.
• Figure 4 illustrates the importance of water content (% w/w) in the formulations of KYF gels: when the water content in the formulation is 98.7% (w/w), the tripeptide concentration is 27mM (hydrogel photo); when the water content in the formulation is 71.3% (w/w), the tripeptide concentration is 50mM (ionogel photo); when the water content in the formulation is 26.9% (w/w), the tripeptide concentration is 86mM (ionogel photo); when the water content in the formulation is 11.31% (w/w), the tripeptide concentration is 257mM (ionogel photo). Photos depict inverted glass vials, to show the formation of self-supporting gels (inverted glass vial test).
• Figure 5 shows an embodiment that illustrates the stability of the tripeptide ionogels at temperature, humidity and pressure of ambient conditions, 2 days after production: photographic images of 560 mM KYF ionogels prepared in [Bmim][DCA] and 40 mM KYF hydrogels prepared in deionized water.
• Figure 6 schematically shows an embodiment of the experimental procedure, and overall results of KFF ionogels in [Bmim][DCA] and [Emim][DCA]. It also compares the results obtained with the hydrogel, 40mM KYF in deionized water. Additionally, being produced in different laboratories, with different tripeptide's batch provided by different commercial houses, it also shows the reproducibility of this procedure.
• Figure 7 illustrates an embodiment of optical microscopy images of produced KFF (146mM) ionogels in the ionic liquid [Bmim][DCA], final water content 12.0 %.
• KFF 146mM
• DCA ionic liquid
• Figure 8 shows photographic representations of gels made of 352 mM KFF in [Emim][DCA] (final water content: 15.25% (w/w)) and 191 mM KFF in the ionic liquid [Bmim][DCA], (final water content: 11.26% (w/w)) between day 1 and month 5.5 after ionogel production. Photos depict inverted glass vials, to show the formation of self-supporting gels (inverted glass vial test).
• the insets illustrate (A and B) bright field and (C and D) polarised optical microscopy images of KFF self-assembly in the ionic liquid [Bmim][DCA] when the ionogel formulation comprises 191 mM KFF in the ionic liquid [Bmim][DCA], (final water content: 11.26 %) at day 1 and month 4 after production.
• Figure 9 illustrates the ATR-FTIR transmission spectra of (a) KYF ionogels in (i) [Bmim][DCA] and (ii) [Emim][DCA] and (b) KFF ionogels in (i) [Bmim][DCA] and (ii.) [Emim][DCA].
• ATR-FTIR spectra of the ionic liquids [Bmim][DCA] and [Emim][DCA] are also shown.
• FIG. 10 illustrates an embodiment of the conductivity of tripeptide ionogels, giving as an example the conductivity of KYF ionogel in [Bmim][DCA] (320 mM KYF in [Bmim][DCA], final water content: 13.43% (w/w)).
• A) it is shown a schematic of the apparatus to demonstrate the ion conduction of the tripeptide ionogel by lightening up a light-emitting diode (LED); in B) a photograph of the real set-up is shown. Similar results are observed for KYF in [EMIM][DCA], KFF in [BMIM][DCA] and KFF in [EMIM][DCA]
• Figure 11 illustrates the chemical structure of the ionic liquids choline dihydrogenphosphate (ChDFIP) and dicholine monohydrogenphosphate (DChFIP) used to produce the choline phosphate buffer and the additive citric acid (CA) used on the non- conventional solvent DChFIP:CA.
• ChDFIP choline dihydrogenphosphate
• DChFIP dicholine monohydrogenphosphate
• CA additive citric acid
• Figure 12 illustrates an embodiment of gels obtained with distinct formulations using System II tripeptides.
• the hydrogel presented for RFF has 30 mM tripeptide in sodium phosphate buffer 0.1 M pH 8.0
• the hydrogel presented for DFY-NFI2 has 20 mM tripeptide in sodium phosphate buffer 0.1 M pH 8.0
• the hydrogel presented for YFD-NFI2 has 20 mM tripeptide in sodium phosphate buffer 0.1 M pH 8.0.
• compositions are as follows: 30mM RFF ionogel in choline phosphate buffer, 16% (w/w) water and 20 mM DFY-NFI2 in choline phosphate buffer, 16% (w/w) water.
• self-supported tripeptide ionogel composed of RFF:dicholine monohydrogenphosphatexitric acid with the following molar ratios: 0.008:1:1. Photos depict inverted glass vials, to show the formation of self-supporting gels (inverted glass vial test).
• Figure 13 schematically shows an embodiment of the experimental procedure and overall results of RFF in [Bmim][DCA] and its successful gelation in choline phosphate buffer.
• Figure 14 schematically shows an embodiment of the experimental procedure and overall results of DFY-NFI2 in [Bmim][DCA] and in choline phosphate buffer.
• Figure 15 illustrates the ATR-FTIR transmission spectra of the 20 mM hydrogels (in 0.1 M D2O sodium phosphate buffer pH 8.0) and 20 mM ionogels (in D2O choline phosphate buffer, apparent pH 7.1) using the tripeptides DFY-NFI2 and YFD-NFI2.
• Figure 16 illustrates A) an embodiment of the mechanical properties of DFY gels, hydrogel and ionogel. Storage (G') and loss modulus (G") as a function of the angular frequency of the gels formed by DFY (30 mM in choline phosphate buffer at an apparent pH 7.1 and sodium phosphate buffer at pH 8.0). B) Photographic image showing that despite being a gel, it is still possible to be pipetted.
• Figure 17 illustrates A) a transmission electron microscopy image (TEM) for an embodiment of 30 mM DFY-NH2 ionogel prepared in choline phosphate buffer, apparent pH
• Figure 18 shows an embodiment of the ionic conductivity of 30 mM DFY-NH2 ionogel at day 1 and day 50 after production, at 20°C. At 1 kHz, ionic conductivity of DFY-NH2 ionogel was l.OxlO 3 S.cm 1 . Choline phosphate buffer is also presented. The inset presents ionic conductivity of 30 mM DFY-NH2 ionogel 1 hour after production.
• Figure 19 illustrates A) an embodiment of the stability over time of DFY gel (30 mM in choline phosphate buffer at an apparent pH 7.1 and sodium phosphate buffer at pH 8.0), as compared to DFY hydrogel when exposed to environmental conditions.; and B) the variation of ionogel weight over time.
• Figure 20 illustrates an embodiment of the thermoreversibility of 30 mM DFY-NH2 ionogels in choline phosphate buffer, upon heating the ionogels from room temperature to 75°C, and then cooling down back to room temperature.
• Figure 21 illustrates optical microscopy images of thin films (produced by film coating method) of 30 mM and 50 mM DFY-NH2 ionogel formulations in choline phosphate buffer. At day 3, a scratch was inflicted in the gel and at day 6 the scratch is no longer visible, indicating the self-healing properties of the ionogel materials.
• Figure 22 illustrates the conductivity of tripeptide ionogels, giving as an example conductivity of 20 mM DFY-NH2 ionogel prepared in choline phosphate buffer, apparent pH
• A) a schematic representation of an embodiment of the apparatus used to demonstrate the conduction of the tripeptide ionogel by lightening up a light-emitting diode (LED) is shown; in B) a photograph of the real set-up is shown. In C) it is shown an embodiment of a thin film of choline dihydrogenphosphatexitric acid (1:1) conducting material mediating electron transfer into a LED.
• LED light-emitting diode
• Figure 23 shows an embodiment of the humidity effect on the electrical properties of 50mM DFY-NH2 ionogel thin films.
• Figure 23A presents a schematic illustration of an in-house built electronic nose used to perform the experiment.
• B) the electrical signals of DFY-NH2 ionogel thin films when exposed to cycles of humidification and drying between 0% and different relative humidity (RH) levels (i.) 58%, ii.) 50%, iii.) 44% and iv.) 25%) are observed.
• RH relative humidity
• Figure 24 shows an embodiment of polarised optical microscopy images of 517 mM KYF ionogels in [Bmim][DCA], 23.70% (w/w) water content and 560 mM KFF ionogels in [Bmim][DCA], 17.58% (w/w) water content.
• Both ionogels incorporate the liquid crystal 5CB (4- Cyano-4'-pentylbiphenyl), as stimuli-responsive optical probes, preferentially droplets within radial configuration.
• the present disclosure relates to a new soft gel material, prepared through the gelation of ionic liquids using unprotected tripeptides.
• the rationale to design the tripeptide ionogel formulations and respective production methods started by understanding the gelation trigger and gelation protocols of previously described unprotected tripeptide hydrogelators.
• the gelation trigger includes, for example, changes in pH, ionic strength or temperature [7-9]
• the ionic liquids were designed to mimic this trigger.
• the ionogel production protocols were designed accordingly. With this approach, the aqueous solvent of the hydrogels was totally or partially substituted by rationally designed ionic liquids.
• KYF has also shown the capability to form hydrogels when dissolved in other aqueous solvents such as sodium chloride (5 mM), addition of sodium hydroxide (1 M) for the final pH value, or sodium phosphate buffer solution 0.1 M pH 8 (Table 1), but only when the pH value is in the range of 7.5 ⁇ 1.0. KFF did not self-assembly in sodium phosphate buffer (0.1 M pH 8.0).
• Table 1 System I hydrogels, tripeptide hydrogelation triggered by pH.
• DFY-NH2 was also able to form a transparent gel when dissolved by heat in 0.2 M Citrate-0.1 M Phosphate buffer pH 7.0. However, this tripeptide precipitates when the solvent is 0.1 M Citrate buffer pH 6.2. In System II, the gelation trigger seems to be mainly phosphate dependent.
• Table 2 System II hydrogels, tripeptide hydrogelation triggered by buffer composition and temperature.
• the imidazolium-based ionic liquids can be selected from a list comprising l-butyl-3- methylimidazolium dicyanamide ([Bmim][DCA]), l-ethyl-3-methylimidazolium dicyanamide ([Emim][DCA]), l-butyl-3-methylimidazolium chloride ([Bmim][CI]), l-ethyl-3- methylimidazolium chloride ([Emim][CI]), or mixtures thereof.
• the ammonium-based ionic liquids considered were choline chloride, choline hydroxide, choline phosphate buffer, and 2- hydroxyethylammonium formate ([HEtNHsHCC ]).
• the choline phosphate buffer is an ionic liquid buffer system formed by the mixture of two ionic liquids (i) choline dihydrogenphosphate and (ii) dicholine monohydrogen phosphate in equimolar amounts, to which 16-17% (w/w) deionized water are added.
• the pH of the choline phosphate buffer is 7.1 ⁇ 1.0, and is called the apparent pH ( Figure 11).
• Imidazolium-based ionic liquids are defined as those where the cation is a charged heterocycle organic molecule, an imidazole ring where one of the nitrogen atoms is protonated such as dialkylimidazolium.
• ammonium-based ionic liquids are composed by an ammonium cation, a positively charged or protonated amine or a quaternary ammonium cation of the basic structure NI , where one or more hydrogen atoms can be replaced by alkyl groups and/or alkyl groups with hydroxy group attached (R).
• an alkylammonium As example an alkylammonium.
• tripeptides KYF and KFF were dissolved in ionic liquids at a given concentration in a glass vial at room temperature (18-27°C), by means of vortex and sonication, followed by pH increasing to 7.5 ⁇ 1, by dropwise addition of alkaline solutions, such as, sodium hydroxide (1 M or 2 M), potassium hydroxide, [Bmim][OH], or choline hydroxide.
• alkaline solutions such as, sodium hydroxide (1 M or 2 M), potassium hydroxide, [Bmim][OH], or choline hydroxide.
• KYF and KFF were ionogelators.
• KYF and KFF tripeptides when dissolved in ionic liquids of imidazolium-family, [Bmim][DCA], [Bmim][CI], [Emim][DCA] and [Emim][CI], and after rising the pH to 7.5 ⁇ 1.0 (by the addition of NaOH solutions (1 M, 2 M), [Bmim][OH] or choline hydroxide), formed self-supporting gels ( Figures 3 and 6).
• the gelator KYF formed a transparent and clear gel when dissolved in [Bmim][DCA] and in [Emim][DCA] ( Figure 3).
• the molecular self-assembly occurred at 412 mM, a high gelation concentration when compared with the one needed for a hydrogel formation (20 mM) (Table 1).
• the tripeptide KFF formed opaque gels with self-assembled structures with birefringence properties when dissolved in [Bmim][DCA] and [Emim][DCA] and after rising the pH to 7.5 ⁇ 1.0 (by the addition of NaOFI solutions (1 M, 2 M), [Bmim][OFI] or choline hydroxide).
• the formation of self-assembled structures with birefringence properties was observed in real-time by optical microscopy ( Figures 7 and 8) in a similar process to the one described by Wang et al. [20]. With time, the opaque gels, containing the birefringent structures, changed to transparent clear gels, suggesting the 'evolution' to fibrillary structures ( Figure 8).
• Table 3 Examples of gelation formulations for the System I tripeptides KYF and KFF.
• RFF and DFY-NFI2 tripeptides formed self-supported gels when dissolved in choline phosphate buffer at concentrations of 30mM and 45mM, respectively.
• the tripeptide RFF formed an opaque gel with self-assembly microstructures, while the gelator DFY-NFI2 was capable of forming a transparent clear gel ( Figure 12). It is noteworthy that, depending on the batch used, the gelation time for DFY-NFI2 in choline phosphate buffer was much faster than for the corresponding hydrogel (observed for the tripeptide batch within counter ion chloride), occurring instantaneously.
• Choline phosphate buffer is composed by choline dihydrogenphosphate and dicholine monohydrogenphosphate (equimolar amounts) and 16- 17% (w/w) deionized water. Figures in parenthesis are gelation concentrations.
• Tripeptide ionogels of both systems, I and II were characterized by several techniques.
• self-supporting hydrogels and ionogels can be checked visually by the glass vial inversion test.
• the macroscopic manifestation of successful gelation is the absence of observable material flow in the vials wall upon inversion of the glass vial. Examples are shown in Figures 3, 4, 6 and 8 for system I, and in Figure 12-14 and 20 for system II.
• Gel-like properties of biomaterials can also but determined through rheological measurements.
• the rheological properties of DFY-NFI2 gels (system II) were measured with an Anton Paar MCR 302 rheometer with the temperature controlled at 25°C using a parallel sandblasted 10 mm plate. Amplitude sweep was performed to determine the critical strain. Frequency sweep was performed to measure G' and G" at a constant strain (0.5 and/or 0.1), in the angular frequency range 0.1-100 rad/s. The experiments were carried out approximately 24h after ionogel sample cooling.
• ATR-FTIR total reflection Fourier transform infrared
• ATR-FTIR spectra obtained for KYF in [Bmim][DCA], KYF in [Emim][DCA], KFF in [Bmim][DCA] and KFF in [Emim][DCA] showed peak formation in amide I region for tripeptide ionogels ( Figure 9).
• the results suggested different interactions, and consequently self-assembly for KYF and KFF in ionic liquids as macroscopic and microscopically observed.
• the peak observed at 1672 cm 1 is due to the presence of trifluoroacetic acid in the tripeptide sample.
• DFY-NF ionogel, DFY-NFh hydrogel and YFD-NFh hydrogel have shown peaks in the amide I region which may suggest periodical organization either through helical or b-sheet structures ( Figure 15).
• the morphology of the gels was characterized by microscopy.
• the gels were characterized by different microscopy analysis, namely optical and polarised microscopy and by transmission electron microscopy (TEM).
• TEM transmission electron microscopy
• KFF ionogel (system I) samples were observed by optical microscopy using a microscope Zeiss Axio Observer.Zl/7 coupled with an Axiocam 503 color camera equipped with crossed polarizers.
• Microscope images showed the formation of birefringent microstructures associated to the self-assembly of KFF in [Bmim][DCA] or [Emim][DCA]. These microstructures are only observed for opaque gel and not for translucid KFF ionogels. Examples are shown in figures 7 and 8.
• DFY-NFh ionogel (system II) nanostructures were characterized by TEM.
• carbon-coated grids were purchased from Electron Microscopy Sciences.
• a drop (5mI) of the sample solution was applied to the carbon-coated grid and incubated for one minute.
• the sample was then washed with water to remove ionic liquids and then stained with 5mI of 2% (w/v) uranyl acetate solution for 30 seconds. After blotting the excess stain solution, the grid was left to air dry.
• the negatively stained sample was imaged in a FEI TITAN Halo TEM operating at 300kV. Images were recorded in the low-dose mode (20e A 2 ) on a FEI CETA 16M camera (4096 x 4096 pixels). DFY-NH2 ionogels showed a dense three-dimensional nanofibrous network as can be seen of Figure 17. The average diameter of the individual fibers was about 8-12 nm.
• the ionic conductivity of ionogels was measured by electrochemical impedance spectroscopy at environmental conditions (temperature 18-27°C, atmospheric pressure (1013.25hPa), and RH 30-80%), covering a frequency range from 100 kHz to 0.1 Hz.
• ionic conductivity of DFY-NFI2 ionogel (System II) was l.OxlO 3 S.cm 1 , very similar to the one exhibited by its solvent choline phosphate buffer ( Figure 18).
• ionogel formation does not affect the ionic conductivity of the ionic liquids and the same value ranges were observed in the corresponding ionogels (for example, at 1 kHz for [Emim][DCA] 18x10 1 S.cm 1 , for [Bmim] [DCA] 10x10 1 S.cm 1 and for dicholine monohydrogenphosphate_citric acid (1:1) 4.7xl0 5 S.cm 1 ).
• Conductive properties of the tripeptide ionogels (Systems I and II) were shown by lightening up light-emitting diodes (LED) as shown in Figures 10 (System I) and 22 (System II). It was observed that the LED lights up when using the tripeptide ionogels, independently of its water content.
• thermoreversibility of tripeptide ionogels is illustrated in Figure 20.
• obtained ionogels were heated to 75 ⁇ 35°C for 10 minutes. Afterwards, they were left to cool down to room temperature (18-25°C). The gels were observed by the glass vial inversion test before heating, after heating and after cooling down. This is shown to System II tripeptides but the same is observed for System I.
• Figure 21 illustrates an embodiment of the potential of the ionogel formulations to be spread as thin films on a substrate (here for example spread on an unmodified glass slide). This Figure 21 also shows the self-healing properties of tripeptide ionogels.
• Figures 5 and 19 it is possible to see the processing of the tripeptide ionogel macroscopic structures, moulded by casting the formulation in a 3D printed mould.
• the addition of the tripeptide and ionic liquid is made in defined orders and concentrations, at specific conditions of pH, temperature, sonication and agitation, and with added additives when required.
• the pH of the tripeptide dissolved in the ionic liquid is increased by the addition of NaOFI, KOFI, [Bmim][OFI], or choline hydroxide.
• an additive capable of hydrogen bonding can be citric acid, tartaric acid, ascorbic acid, glucose, sucrose, xylose, arginine, histidine, proline, urea, sorbitol, xylitol, glycerol, monothylene glycol, menthol, lactic acid, betain, or mixtures thereof.
• the final tripeptide ionogels contain variable amounts of water (always below the 90% (w/w) threshold value found in hydrogels; typically, in tripeptide ionogels the water content is found between 2-75%).
• the formulations and respective tripeptide ionogels can be processed by diverse techniques and moulded into different formats, yielding advanced functional materials.
• the formed supramolecular structures can be processed in different geometries selected from discs, films, fibers, spheres, cubes, mesh, channels or patterned structures, using different methods as moulding, casting, spin coating, film coating, spinning, electrospinning, microfluidics, 3D-printing, ink-jet printing, patterning, metal depositions and fabrication methods. They can be casted into several substrates such as glass slides, silicon wafers, interdigitated electrodes, with or without chemical or physical modification.
• the tripeptide ionogels are stable at ambient conditions, have thermal, electrochemical and chemical stability in a wide range of temperature and humidity conditions, are conducting, soft, self-healing, thermoreversible, have a large electrochemical window, are non-flammable, non-toxic, biocompatible and biodegradable materials.
• the ionogels are stimuli-responsive. They possess electric, ionic and/or protonic conductivity. They possess optical and piezoelectric properties. The conductivity, optical and piezoelectricity changes when in presence of chemical (e.g. analytes in gas or aqueous phases, cells) or physical stimuli (e.g. temperature, pressure) can be used as transducing signals to develop sensors.
• chemical e.g. analytes in gas or aqueous phases, cells
• physical stimuli e.g. temperature, pressure
• the tripeptide ionogels may find application in a variety of technological fields, such as sensing devices; electronic devices; opto-electronic devices; flexible and wearable devices; conducting devices; materials for additive manufacturing; chemocatalysis and biocatalysis; coating materials; metal recovery; encapsulation, drug delivery and/or cell assays; pharmaceutical formulations; electrochemical devices such batteries, capacitors and solar cells; electrolytes; semi-solid lubricants; magnetorheological fluids and electrochemical devices; adsorbents; purification matrices; food; and cosmetics products.
• Unprotected tripeptides KYF, KFF, RFF, DFY-NFI2 and YFD-NFI2 were produced in-house by conventional peptide synthesis methods or obtained from commercial suppliers.
• the ionic liquids used were purchased to loLiTec (Germany).
• the water content of all ionic liquids used was determined by Karl-Fischer titration using an 831 KF Coulometer with a generator electrode without diaphragm with Flydranal Coulomat AG as the analyte. The final value noted was the average of a minimum of three experiments.
• the tripeptide KYF was dissolved in l-butyl-3-methyl imidazolium dicyanamide (water content estimated by Karl Fisher as 0.5 ⁇ 0.5% (w/w)) by vortex and sonication, followed by increasing the pH to 7.5 ⁇ 1 by dropwise addition of sodium hydroxide solution (NaOFI) 2 M.
• NaOFI sodium hydroxide solution
• the final peptide concentration range in the ionogel was ideally between 50 - 550 mM, with 2- 72% (w/w) water.
• KYF ionogel can also be prepared in l-ethyl-3-methylimidazolium dicyanamide (water content estimated by Karl Fisher as 0.5 ⁇ 0.5%(w/w)) following the experimental protocol described above, yielding a final peptide concentration ideally between 50-500 mM, with 2-72% (w/w) water. All characterization of the gels took place at least 24 hours after ionogel preparation. Samples for ATR-FTIR analysis were prepared with NaOD solution instead of NaOFI. As an alternative, l-butyl-3-methyl imidazolium chloride or 1-ethyl- 3-methylimidazolium chloride can be used to prepare the ionogels. Examples of KYF ionogels or related characterization can be seen in Figures 3 - 5, 9, 10 and 24.
• KFF was dissolved in l-butyl-3-methyl imidazolium dicyanamide (water content estimated by Karl Fisher as 0.5 ⁇ 0.5% (w/w)) by vortex and sonication, followed by increasing the pH to 7.5 ⁇ 1 by dropwise addition of sodium hydroxide solution (NaOFI) 2M.
• NaOFI sodium hydroxide solution
• the final peptide concentration in the ionogel was ideally between 50-200 mM, with 2-75% (w/w) water.
• KFF ionogel can also be prepared in l-ethyl-3-methylimidazolium dicyanamide (water content estimated by Karl Fisher as 0.5 ⁇ 0.5% (w/w)) following the experimental protocol described above, yielding a final peptide concentration ideally between 50-500 mM, with 2- 75% (w/w) water. Characterization of the gels took place at least 24 hours after ionogel preparation. Samples for ATR-FTIR analysis were prepared using NaOD solution instead of NaOFI. As an alternative, l-butyl-3-methyl imidazolium chloride or l-ethyl-3- methylimidazolium chloride can be used to prepare the ionogels. Examples of KFF ionogels or related characterization can be seen in Figures 6 - 9, 24.
• the choline phosphate ionic liquid buffer system is an ionic liquid hydrated buffer formed by the mixture of two ionic liquids (i) choline dihydrogenphosphate and (ii) dicholine monohydrogenphosphate in equimolar amounts, to which 16-17% (w/w) deionized water are added.
• the buffer is homogenized by means of vortex and sonication.
• Ionic liquid buffer water content was determined using a Karl Fischer; apparent pH was verified to be at 7.1 ⁇ 1. Chemical structures of choline dihydrogenphosphate and dicholine monohydrogenphosphate are presented in Figure 11.
• DFY-NH2 was dissolved in choline phosphate buffer (apparent pH 7.1 ⁇ 1, 16%-17% (w/w) water content) heating at 75 ⁇ 35 °C following by vortex and cooling down to room temperature, or just cooling down to room temperature.
• the final peptide concentration ideally is between 20-100 mM, with 16-20 % (w/w) water. Characterization of the gels took place at least 24 hours after ionogel preparation. Samples for ATR-FTIR analysis, choline phosphate buffer was prepared using D2O instead of H2O. Examples of DFY-NH2 ionogels or related characterization can be seen in Figures 12 and 14-23.
• RFF was dissolved in choline phosphate buffer (apparent pH 7.1 ⁇ 1, 16%-17% (w/w) water content) heating at 75 ⁇ 35 Q C following by cooling to room temperature.
• the final peptide concentration in the ionogel ideally is between 20-100 mM, with 16-20 % (w/w) water. Characterization of the gels took place at least 24 hours after ionogel preparation. Samples for ATR-FTIR analysis, choline phosphate buffer was prepared using D2O instead of H2O. Examples of RFF ionogels can be seen in Figures 12, 13.
• RFF ionogel was prepared by heating and stirring method. This method consist in mixing the components of the gel: (i) ammonium quartenarium salt: choline dihydrogenphosphate, dicholine monohydrogenphosphate or choline chloride (ii) an additive which has the capability of H-bond donor or receptor, such as citric acid, and (iii) an unprotected tripeptide, RFF according with the amounts calculated considering different molar ratios preferentially 1:1:0.5 - 1:1:0.005, ideally 1:1:0.1 - 1:1:0.005. The system was heated to 80 ⁇ 20 Q C and stirred at 300 rpm, until a clear transparent viscous solution or gel is obtained. Examples of RFF ionogels with additive can be seen in Figure 12.
• Liquid/viscous samples were prepared by pipetting IOOmI of the solution into an 0.5 ml Eppendorf previously fill with IOOmI PDMS in order to decrease the amount of biomaterial need for the characterization.
• the gold coated electrodes (connection pines) were inserted in an in-house 3D printed adapter and position in the Eppendorf being partially submersed in the biomaterial.
• Gel samples were prepared using an in-house 3D printed o-ring with 6mm inner diameter and 0.6mm height. The system, ring - hydrogel, was sandwiched between two gold contacts.
• Ionic conductivity of the gels and viscous/liquid samples was determined at environmental conditions by Electrochemical Impedance Spectroscopy using a potentiostat Gamry Instruments - Reference 3000 (measurement conditions: frequency range from 100 kHz - 0.1 Hz and Vpp lOOmV (AC)). The conductivity was calculated using the following equation where s is the conductivity, R is the resistance; and I and A are the thickness and area of the samples, respectively.
• the results of ionic conductivity studies for 30 mM DFY-NFI2 ionogel in choline phosphate buffer are shown in Figure 18.
• Figures 10 and 22 demonstrate the ion conduction of the tripeptide ionogel by lightening up a light-emitting diode (LED).
• LED light-emitting diode
• a humidity sensor was placed in the outlet of the detection chamber, in order to measure the variations of RH applied to the ionogel thin films when they are exposed alternately to humid and dry nitrogen. The results are shown in Figure 23. Similar results were obtained when exposing the thin films to volatile organic compounds.
• the project leading to this invention has received funding from the European Research Council through the grant reference SCENT-ERC-2014-STG-639123 and from Fundagao para a Ciencia e Tecnologia through the PhD scholarship SFRH/BD/113112/2015.

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