Intumescent features of nucleic acids and proteins

Thermochimica Acta 591 (2014) 31–39

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Intumescent features of nucleic acids and proteins Jenny Alongi *, Fabio Cuttica, Alessandro Di Blasio, Federico Carosio, Giulio Malucelli Dipartimento di Scienza Applicata e Tecnologia, Politecnico di Torino, Viale Teresa Michel 5, 15121 Alessandria, Italy

A R T I C L E I N F O

A B S T R A C T

Article history: Received 30 May 2014 Received in revised form 10 June 2014 Accepted 11 June 2014 Available online 21 July 2014

Are nucleic acids and proteins intumescent molecules? In order to get an answer, in the present manuscript, powders of deoxyribose nucleic acids (DNA) and caseins have been exposed to different heat fluxes under a cone calorimeter source and to the direct application of a propane flame. Under these conditions, DNA and caseins exhibited a typical intumescent behaviour, generating a coherent expanded cellular carbonaceous residue (char), extremely resistant to heat exposure. The resulting volumetric expansion as well as the resistance of the formed char turned out to be dependent on (i) the chemical structure of the chosen biomacromolecule, (ii) the evolution of ammonia and (iii) the adopted heat flux in cone calorimetry tests (namely, 25, 35, 50 and 75 kW/m2). The presence of ribose units within the DNA backbone determined the formation of highly expanded and coherent residues as compared to those obtained from caseins. Indeed, under a heat flux of 35 kW/m2, when a carbon source (i.e. common cane sugar) was added to caseins, the resulting char was similar to that formed by DNA. Furthermore, the char expansion was ascribed to the evolution of ammonia released by these biomacromolecules upon heating, as detected by thermogravimetry coupled to infrared spectroscopy, and confirmed by scanning electron microscopy experiments performed on the bubbles present in the residues of flammability tests. ã 2014 Elsevier B.V. All rights reserved.

Keywords: DNA Caseins Intumescence Combustion Cone calorimeter Carbon sources Volatile products evolution

1. Introduction

 a carbon source (i.e. pentaerythritol, saccharides, polysaccharides,

The term “intumescence” is generally used to describe the behaviour of some materials having flame retardant (FR) features, in particular for plastics or textiles [1,2]; when it was used for the first time, this noun focused on defining substances able “to grow and to increase in volume against the heat” and “to show an expanding effect by bubbling”. Upon heating, intumescent materials begin to swell and then to expand: as a consequence, a foamed char, that protects the underlying material from the heat flux or the flame, is formed on the surface [3–6]. In doing so, the char layer acts as a physical barrier that slows down heat, oxygen and mass transfer between gas and condensed phases. By this way, intumescent systems are able to interrupting the self-sustained combustion of the polymer during its thermal degradation that involves the formation of gaseous fuel; hence, the intumescence process results from a combination of charring and foaming of the surface reactions. Furthermore, the density of the char decreases with increasing the temperature. Usually, an intumescent material consists of three main components:

 a blowing agent (i.e. melamine, etc.), which, upon heating,

etc.),

 an acid source (i.e. ammonium phosphates or polyphosphates,

etc.),

* Corresponding author. Tel.: +390131229337; fax: +390131229399. E-mail address: [email protected] (J. Alongi). http://dx.doi.org/10.1016/j.tca.2014.06.020 0040-6031/ ã 2014 Elsevier B.V. All rights reserved.

releases great amounts of expandable or non-combustible gases (ammonia or carbon dioxide). The foamed char is the result of a sequence of reactions, occurring in between the three components of the intumescent formulation. In order to achieve the formation of a consistent and coherent char, and thus the maximum effect of the intumescent formulation as flame retardant, the three components should react in proper time/temperature conditions. Indeed, nowadays, intumescent systems can be considered as the most performing solution available to withstand the fire threat. However, the design of environmentally-friendly intumescent materials could represent a significant step-forward in flame retardancy; therefore, the use of biomacromolecules containing all three components of an intumescent formulation could represent a key point, worthy to investigate. These green intumescent systems are already present in nature: indeed, deoxyribonucleic acid (DNA) possesses this feature, because of its chemical structure, which contains nitrogen bases, deoxyribose units and phosphate groups. As recently demonstrated, this biomacromolecule can be considered an all-in-one intumescent compound able to promote the char formation either when deposited on cotton fabrics through simple impregnation [7–9], layer-by-layer deposition [10], or when coated on thick films of ethylene vinyl acetate (EVA) copolymers [11]. Caseins have also

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J. Alongi et al. / Thermochimica Acta 591 (2014) 31–39

and to the direct application of a propane flame. The morphology of the expanded char formed during combustion has been investigated by using Scanning Electron Microscopy (SEM). Finally, the evolution of the volatile species generated upon heating has been studied by coupling high resolution thermogravimetry to infrared spectroscopy, although the rates of combustion and thermal oxidation are significantly different. 2. Experimental 2.1. Materials

Fig. 1. HRR curves of DNA as a function of temperature under different heat fluxes.

shown a similar behaviour when applied to cotton [12] or polyester and polyester-cotton blends [13]. It is worthy to note that these products can be derived from industrial wastes: indeed, DNA can be extracted from salmon and herring sperm as well as caseins from bovine milk [14]. The results collected up to now have raised the following questions: are nucleic acids and proteins intumescent additives? How do they behave upon heating? In order to answer to these questions, in the present manuscript, we have investigated the combustion resistance of DNA and caseins to different heat fluxes (namely, 25, 35, 50 and 75 kW/m2) generated by a cone calorimeter

Powders of DNA from herring sperm and caseins from bovine milk (12–15 a-s1, 3–4 a-s2, 9–11 b and 2–4 k) were purchased as grade reagents by Sigma–Aldrich, Inc., and used as received. A common phosphorus flame retardant, ammonium polyphosphate (APP, PHOS-CHEK1 P30) was purchased from ICL Performance Products. Caster sugar (food grade) was also employed. 2.2. Preparation of samples for cone calorimetry tests 3 g of powder was deposited on an aluminium foil and subsequently compressed at room temperature, using a compression moulding press (applied pressure: 5 MPa). 2.3. Characterization techniques Cone calorimeter tests (Fire Testing Technology, FTT) were performed according to the ISO 5660 standard [15]. The samples (50  50  3 and 50  50  1 mm3 for DNA and caseins, respectively)

Fig. 2. DNA residues after cone calorimetry tests under different heat fluxes.

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Table 1 Residues and expanded volume percentage of biomacromolecules after cone calorimetry tests. Sample

TTI (s)

PHRR (kW/m2)

time at PHRR (s)

FO (s)

Notes

Residue (wt.%)

Volumetric expansion (%)

DNA_25 kW/m2 DNA_35 kW/m2 DNA_50 kW/m2 DNA_75 kW/m2

– 1 1 1/34

– 37 63 129/99

– 8 9 2/44

– 12 25 60

No ignition Char formation and expansion continues after FO Char formation and expansion continues after FO 2 ignitions: after 20 s, FO is reached, but at 34 s a ignition confined on the specimen bottom occurs

66 56 43 30

1600 1700 1200 750

Caseins_25 kW/m2 Caseins_35 kW/m2

8 2/118

150 199/160

34 20/136

70 250

68 20

3500 4900

Caseins_50 kW/m2

1

224/145

14/104

205

18

2900

Caseins_75 kW/m2

1

311/181

10/78

140

14

2400

Caseins–sugar_35 kW/m2

3

144

16

206

– 2 ignitions. After 62 s, FO is reached, but at 118 s a further ignition of the specimen occurs 2 PHRRs as the char formed during the first step of combustion cracks 2 PHRRs as the char formed during the first step of combustion cracks Coherent and consistent final residue

24

1600

Theexperimentalerrorwas10%.

were placed on a sample holder and irradiated at different heat fluxes (namely, 25, 35, 50 or 75 kW/m2) in horizontal configuration. These samples were prepared using the same amount of DNA and caseins (namely, 3 g). Because of the different density of the two biomacromolecules, the specimen thicknesses after compression were 3 and 1 mm for DNA and caseins, respectively. For each formulation, the tests were repeated three times at 23  C and 50% R.H. and an experimental error of 10% has been calculated as standard deviation for all the measured parameters, i.e. time to ignition (TTI, s), heat release rate peak (PHRR, kW/m2), time at PHRR (s) and flame out (FO, s). Flammability tests were carried out applying a propane flame (25 mm length) directly to the powder of DNA and caseins placed on a ceramic backing pad; three flame applications of 5 s were performed. The cross-section of the residues after combustion tests was observed by using a LEO-1450VP scanning electron microscope (beam voltage: 5 kV). To this aim, cross-section was cut from the residue, pinned up to conductive adhesive tapes and goldmetallized. An X-ray probe (INCA Energy Oxford, Cu-Ka X-ray source, k = 1.540562 Å) was used to perform elemental analysis. The evolution of gases was monitored by high-resolution thermogravimetry (Q500 TA Instrument) and thermogravimetry coupled to infrared spectroscopy (Spectrum GX FTIR system, PerkinElmer). To this aim, 10 mg samples were heated in platinum pans in air (30 ml/min) from 50 to 800  C at 10  C/min (transfer line temperature = 250  C; cell temperature = 220  C). 3. Results and discussion 3.1. Investigation on DNA intumescence DNA resistance to an irradiative heat flux has been assessed by cone calorimetry: Fig. 1 plots the HRR curves as a function of temperature under 25, 35, 50 and 75 kW/m2; the final residues after these tests are presented in Fig. 2. In detail, under a low heat flux (namely, 25 kW/m2), DNA does not ignite: it undergoes to pyrolysis until the end of the test (after 100 s) and forms an expanded cellular carbon structure, resistant to the heat exposure; in doing so, a final residue of 66 wt.% with a volumetric expansion of about 1600% has been observed (Table 1 and Fig. 2). When the heat flux increases up to 35 kW/m2, DNA immediately ignites (TTI = 1 s) reaching a PHRR of 37 kW/m2 in 8 s and the FO in 12 s. Also in this case, although the formation of an expanded char has been observed during combustion, its expansion continued after

Fig. 3. Residues of neat (A) and burnt DNA (B and C) after flame application.

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Fig. 4. SEM micrographs of DNA exposed to 25 (A), 35 (B), 50 (C) and 75 kW/m2 (D) – 500X.

Fig. 5. SEM micrographs of DNA exposed to 25 (A), 35 (B), 50 (C) and 75 kW/m2 (D) – 5000X.

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Fig. 6. TG and dTG curves of DNA in air (A) and IR spectrum at 250  C (B).

FO, reaching 1700%. Under 50 kW/m2, DNA combustion proceeds analogously to what observed at 35 kW/m2: the only differences can be referred to the measured volumetric expansion. Indeed, in this case, the expansion is lower as compared with that of DNA under 35 kW/m2. As the expansion is due to the generation of bubbles, under 50 kW/m2, the heating rate is too high and all the bubbles generated during heating quickly explode, thus limiting the expansion of the residue. On the other hand, when DNA is subjected to the highest heat flux (i.e. 75 kW/m2), it immediately burns, reaching a PHRR of 129 kW/m2 in 2 s, and forming a thermally stable char that resists up to 34 s. After that, DNA ignites again (reaching a second PHRR of 99 kW/m2 at 44 s) as some cracks located at the base of the expanded structure appear. In this case, the volumetric expansion observed during combustion is much lower (i.e. 750%, Table 1) as compared to that achieved under the other heat fluxes: more specifically, also in this case, the expanded structure grows up in a remarkable way during combustion, but at a certain moment collapses as it lacks of compactness. DNA behaves in a similar way when it is put in direct contact with a propane flame. Fig. 3 shows neat DNA powder (A) in comparison with the residue (B) obtained after three ignitions (5 s each); once again, an expanded cellular

carbon structure able to protect the surrounding material has been formed, as shown in Fig. 3C where unburned powder is still visible, after the removal of the protective char. The morphological characterization of the residues after cone calorimetry has been carried out by using SEM coupled with elemental analysis. Figs. 4 and 5 depict the typical DNA residues at different magnifications and formed under 25 (Figs. 4 and 5A), 35 (Figs. 4 and 5B), 50 (Figs. 4 and 5C) and 75 kW/m2 (Figs. 4 and 5D). From an overall consideration, the intumescent feature of DNA is revealed even at low magnification; indeed, bubbles generated upon heating can be found in all the residues, regardless of the adopted heat flux. Such structures are rather similar and mainly consist in phosphorus-rich phases (as evidenced by elemental analysis in the inlets). Furthermore, the effect of the different heat fluxes can be appreciated if the integrity of bubbles is considered: indeed, the residue left under a heat flux of 75 kW/m2 (Fig. 4D) appears heavily damaged and fragmented accordingly to the lowest volumetric expansion already observed at a macroscopic scale (Fig. 2). In addition, at higher magnification further information can be collected concerning the walls of bubbles, the formation of which is ascribed to the aggregation of smaller ones still present in their fragments (Fig. 5).

Fig. 7. HRR curves of caseins as a function of temperature at different heat fluxes and corresponding residues.

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Fig. 8. SEM micrographs of caseins exposed to 25 (A), 35 (B), 50 (C) and 75 kW/m2 (D).

From a chemical point of view, it is reasonable to suppose that the formed char consists of a carbon and phosphorus-rich structure deriving from the reactions taking place between phosphate groups and ribose units of DNA, and that bubbles are generated from blowing species like nitrogen bases. In order to qualitatively understand the type of gases released during DNA combustion, high resolution thermogravimetry coupled to infrared spectroscopy has been employed, although the rates of combustion and thermo-oxidation are significantly different. Fig. 6A plots TG and dTG curves of DNA in air in between 50 and 400  C. It is noteworthy that DNA has a significant weight loss between 190 and 275  C (namely, 20 wt.%): this can be ascribed to the production of ammonia, as confirmed by FTIR spectroscopy performed on the degradation products. As an example, the typical FTIR spectrum referring to this temperature range is plotted Fig. 6B: the two characteristic adsorptions of ammonia at 930 and 965 cm 1 are well visible [16]. In conclusion, upon exposure to a heat source and in particular under a heat flux of at least 35 kW/m2 or after a flame application, DNA burns for a short time, producing an expanded cellular carbon structure derived from the dehydration of its ribose units (probably induced by reacting with phosphate groups) and swollen by the ammonia release from the nitrogen bases. Furthermore, the final residues found at the end of these tests have proven to depend on the adopted heat flux: the higher the heat flux, the lower is the final residue and its expansion (see Table 1). The behaviour already observed for DNA makes it a suitable flame retardant for bulk polymers or copolymers as well as a possible FR additive for coatings or paints. This hypothesis has been recently proven in the case of ethylene vinyl acetate copolymers (containing 18 wt.% of vinyl acetate) when DNA was added in bulk or used as a coating [11]. In particular, the collected results have shown that the DNA coating is more effective as compared to its bulk addition in EVA, since the former is able to block the ignition of the copolymer under a heat flux of 35 kW/m2

(PHHR, [CO] and [CO2] reductions of 80%), as well as to greatly postpone and reduce the combustion kinetics under a heat flux of 50 kW/m2 ([CO] and [CO2] reductions of 80 and 70%, respectively). These findings can be ascribed to the exceptional thermal shielding properties exerted by DNA on EVA surface: this biomacromolecule was capable of protecting the underlying material from a butane/propane torch during three consecutive flame applications for 5 s. However, although DNA was present on EVA surface, the volumetric expansion already observed for pure DNA has not been observed. This is probably due to the intimate interactions occurring between DNA and EVA. A similar behaviour has been observed when DNA has been used as coating for cotton [7–9], eventually coupled with chitosan in layer-by-layer assembly [10]. Indeed, these coatings were able to extinguish flame in horizontal flammability tests and strongly reduced the heat released during combustion. In addition, the deposition a neat DNA coating on cotton fabrics (19% final dry addon), did not allow igniting the fabric under a heat flux of 35 kW/m2 generated by a cone calorimeter, meanwhile increasing LOI from 18 (neat cotton) to 28% (DNA-treated cotton) [8]. On the other hand, with 20 bi-layers of DNA/chitosan, a significant decrease of cotton THR and PHRR ( 32 and 41%, respectively) and increase of its LOI (from 18 to 24%) was observed. In this case, the obtained char turned out to be thermally stable and thus able to provide good thermal shielding features to cotton [10]. Once again, the macroscopic swelling typical of DNA powder did not occur, but, probably for the first time, the intumescence phenomenon was revealed by the formation of a microbubble-rich residue, as assessed during horizontal flame spread tests. 3.2. Investigation on casein intumescence Following an experimental procedure similar to DNA, caseins have been also exposed to the chosen different heat fluxes: the HRR curves as a function of time, as well as the residues at the end of

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Fig. 9. HRR curves of caseins and caseins-sugar mixture as a function of temperature (A) and corresponding residue (B) (heat flux = 35 kW/m2) and residues of neat and burnt caseins (C and D) and caseins-sugar mixture (E and F) after flame application.

tests are presented in Fig. 7A and B, respectively. Table 1 lists the collected data. As compared to DNA, caseins ignition occurs also when these proteins are subjected to a heat flux of 25 kW/m2, reaching a PHRR of 150 kW/m2 and leaving an expanded residue. Increasing the heat flux up to 35 kW/m2, similarly to the DNA exposed to 75 kW/m2, caseins ignite very early (TTI = 2 s) and reach the FO in 62 s (PHRR1 = 199 kW/m2); however, upon continuous heating, caseins ignite again at 118 s, giving a second combustion step (PHRR2 = 160 kW/m2). This latter is due to char generated during the first combustion step that is not so coherent and consistent: some cracks appear and thus caseins ignite again as the cracks allow the release of volatile combustible gases entrapped within the foamed char. Further increasing the heat flux to 50 and

75 kW/m2, caseins behaviour changes as they ignite only once. During the first exposure step, caseins burn reaching PHRR1 (224 and 311 kW/m2 for 50 and 75 kW/m2 of heat flux) and generating an expanded char that is not thermally stable: indeed, at a certain time, some cracks are formed, through which gases and combustible species are released, and thus a second PHRR is reached (145 and 181 kW/m2 for 50 and 75 kW/m2 heat flux). As in the case of DNA, the residues found at the end of these tests depend on the adopted heat flux: the higher the heat flux, the lower is the final residue and its expansion (see Table 1). In addition, it is important to highlight that the volumetric expansions measured for caseins are higher than those assessed for DNA due to the different thickness of the specimens at the beginning of the test

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Fig. 10. TG and dTG curves of caseins and APP in air (A and B) and IR spectra at 255 and 270  C of caseins (C) and APP (D).

(namely, 1 and 3 mm for caseins and DNA). Once again, SEM analyses did not indicate significant differences among the obtained residues: indeed, regardless of the employed heat flux, all the residues show an expanded cellular carbon structures rich in bubbles, as evidenced by the SEM micrographs reported in Fig. 8 It is noteworthy that during combustion the char formed by DNA is stronger and more coherent than that generated from caseins: this finding can be ascribed to the compositional differences between the two biomacromolecules. Indeed, although they both contain some nitrogen-based species able to release ammonia as well as phosphate groups, in the case of caseins, the carbon source is not present, unlike DNA that bears ribose units. For this reason, a 1:1 mixture of caseins and common cane sugar was prepared and tested under 35 kW/m2 in the cone calorimeter. Fig. 9A reports the HRR curve of this mixture in comparison with that of neat caseins: the former ignites in 3 s, similarly to caseins, but produces a carbonaceous structure that is more thermally stable than that of caseins. Indeed, the mixture burns reaching a PHRR of 144 kW/m2 and then goes on until the end of test, without the formation of cracks, leaving a higher final residue with respect to neat caseins (24 vs. 20 wt.% for caseins-sugar and caseins, respectively), which is more coherent and consistent, but less expanded (Fig. 9B). Independently of the sugar presence, caseins react also to a propane flame application, generating an expanded structure able to protect the surrounding material, as depicted in Fig. 9C–F. The presence of bubbles in casein residues can be ascribed to the ammonia release, as already observed for DNA. Fig. 10A and B report TG and dTG curves of caseins: they show a significant 25% weight loss in between 200 and 300  C. Monitoring the volatile species evolving in this temperature range, the typical bands of ammonia at 965 and 930 cm 1 have been found (Fig. 10C). As previously discussed [12],

caseins can be considered as polyaminoacids having numerous phosphate groups in their micellar structure that decompose similarly to APP. Indeed, the TG and dTG curves of caseins and APP show very similar thermal degradation profiles (Fig. 10B and C). Both the molecules significantly lose weight in between 200 and 300  C, releasing ammonia and generating (poly) phosphoric acid, able to favour the formation of a residue. This is in agreement with the data reported in the literature [17]. Obviously, the decomposition temperatures of APP are higher than those of caseins, since in APP the phosphate groups are linked by covalent bonds in a long chain, while in the caseins the same groups are present only on the micelle shell. The evolution of ammonia has been confirmed by infrared spectroscopy: as typical examples, the IR spectra of caseins and APP at 255 and 270  C are compared in Fig. 10C and D. Two examples of caseins FR efficiency have been recently reported by our group for cotton [12], polyester and polyestercotton blends [13]. Similarly to DNA, caseins enhanced the resistance of cotton, polyester and polyester-cotton blends to a methane flame, reaching the self-extinguishment in the case of cotton and strongly reducing the burning rate in the case of polyester and polyestercotton blends. Unlike the blend, for which no substantial changes were found, LOI values significantly increased for cotton (from 18 to 24%) and polyester (from 21 to 26%) [13]. Once again, the evidence of the intumescence was assessed by scanning electron microscopy. 4. Conclusions In the present work, the intumescent character of DNA and caseins has been thoroughly investigated; more specifically, if intumescence means “to grow and to increase in volume against the heat” and “to show an expanding effect by bubbling”, DNA and

J. Alongi et al. / Thermochimica Acta 591 (2014) 31–39

caseins can be considered as intumescent biomacromolecules when subjected to a certain irradiative heat flux (under a cone calorimeter) or to the application of a propane flame, since they are both able to generate an expanded cellular carbon structure rich in bubbles. In detail, DNA burns under 35, 50 or 75 kW/m2 and pyrolyses under 25 kW/m2 only. However, in all cases, it produces a coherent and consistent final residue rich in carbon and phosphorus that is swollen due to the evolution of ammonia during combustion. The same behaviour has been observed when the biomacromolecules have been subjected to three applications of a propane flame (5 s each). On the other hand, caseins burn and expand upon any adopted heat flux or when ignited by a propane flame. The most significant difference between these two biomacromolecules is the amount of final residue found at the end of these tests as well as its consistence. The absence of a carbon source in caseins, which in turn is present in DNA as ribose units, induces the formation of a weak and non-thermally stable char during combustion. Acknowledgement The European COST Action “Sustainable flame retardancy for textiles and related materials based on nanoparticles substituting conventional chemicals”, FLARETEX (MP1105) is gratefully acknowledged. References [1] S. Bourbigot, M. Le Bras, S. Duquesne, M. Rochery, Recent advances for intumescent polymers, Macromol. Mater. Eng. 289 (2004) 499–511. [2] H.L. Vandersall, Intumescent coating systems their development and chemistry, J. Fire Flammabil. 2 (1971) 97–140. [3] S. Bourbigot, S. Duquesne, Intumescent-based fire retardants, in: C.A. Wilkie, A.B. Morgan (Eds.), Fire Retardancy of Polymeric Materials, CRC, Press, Boca Raton, 2010, pp. 163–206.

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