CHEMSUSCHEM FULL PAPERS DOI: 10.1002/cssc.201402432
A Facile and Green Method to Hydrophobize Films of Cellulose Nanofibrils and Silica by Laccase-Mediated Coupling of Nonpolar Colloidal Particles Oriol Cusola,*[a, b] M. Blanca Roncero,[a] Teresa Vidal,[a] and Orlando J. Rojas*[b, c] Hydrophobic particles based on dodecyl 3,4,5-trihydroxybenzoate (LG) were coupled onto the surface of cellulose nanofibrils (CNFs) and silica by treatment with a multicomponent colloidal system (MCS) derived from the laccase-mediated reaction of LG in the presence of a sulfonated lignin (SL). Surface modification upon treatment with MCS was monitored in situ and in real time by quartz crystal microgravimetry. The colloidal stability of MCS and its components in water was followed by measuring space- and time-resolved light transmission and back scattering. The sulfonated lignin increased dispersion stability and reduced the characteristic MCS particle size [from
4 to 80 nm, according to AFM and dynamic light scattering (DLS)]. It also facilitated the surface enzymatic reaction that led to adsorption and coupling of MCS onto CNFs and silica surfaces. The combined effect of reduced surface energy and surface roughness by MCS treatment produced an increase in water contact angle on CNFs and silica of about 90 and 808, respectively. Surface pretreatment with chitosan further increased the extent of MCS adsorption on the surfaces. This method represents a sustainable alternative to traditional approaches for cellulose hydrophobization and a step forward in implementing green routes for surface modification.
Introduction The utilization of renewable and biodegradable materials has increased tremendously in recent years. Their global market is expected to grow in the near future, following increasing societal awareness in relation to climate change and consumer perception.[1] Cellulose is the most abundant natural polymer in the biosphere and displays properties such as hydrophilicity, chirality, biodegradability, biocompatibility, and recyclability. In addition, it can be modified by a broad number of routes. Nanosized cellulose [cellulose nanofibrils (CNFs)] has attracted attention as a renewable resource that can be converted into high-value products and advanced functional materials; as such, it has been investigated for packaging materials, biomedical devices, adhesives, composites, electronics, and so forth.[2–7] CNFs differ significantly from common cellulosic fibers
[a] Dr. O. Cusola, Prof. M. B. Roncero, Prof. T. Vidal Textile and Paper Engineering Department Universitat Politcnica de Catalunya (UPC, BarcelonaTech) Colom 11, 08222 Terrassa (Spain) E-mail: [email protected] [b] Dr. O. Cusola, Prof. O. J. Rojas Department of Forest Products Technology School of Chemical Technology, Aalto University P.O. Box 16300, 00076 Aalto, Espoo (Finland) E-mail: [email protected] [c] Prof. O. J. Rojas Departments of Forest Biomaterials and Chemical and Biomolecular Engineering North Carolina State University Raleigh, NC 27695-8005(USA) Supporting Information for this article is available on the WWW under http://dx.doi.org/10.1002/cssc.201402432.
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
because they have a higher aspect ratio and specific surface area combined with higher intrinsic strength and flexibility.[8, 9] However, for a more comprehensive utilization of CNFs there is a need to develop hydrophobic CNF products. Waterproof coatings and films, packaging materials, and hydrophobic composites are but a few examples in which hydrophobized CNFs can be utilized. Hydrophobization of CNFs can improve industrial processing, including size pressing, printing, and conversion.[10, 11] More importantly, packaging applications demand ecologically friendly hydrophobizing agents that can facilitate product recyclability; at present such an issue is limited if conventional waxes and fluoropolymers are applied.[12] The wettability of a surface arises from the combined contribution of surface topography and low surface energy. Most methods aimed at obtaining high hydrophobicity tend to minimize contact between water droplets and the surface by hierarchical micro- and nanostructures.[13] There are a variety of hydrophobization methods that usually depend on the type of substrate. For example, wet chemical reactions and electrochemical deposition techniques are commonly applied to metals, whereas self-assembly and layer-by-layer deposition are used for glass. In contrast, textiles and other substrates are often modified through sol–gel and polymerization reactions.[14] Several studies have reported on a variety of approaches to hydrophobize different types of cellulosic surfaces. In some of the most relevant achievements, amphiphobic cellulose-based materials were fabricated by chemical etching and subsequent coating of functional thin films.[15] Highly hydrophobic cellulose materials possessing reversible wettability were fabricated by self-assembling monolayers onto an intermediate coating of titania.[16] Amphiphobic nanocellulose aeroChemSusChem 2014, 7, 2868 – 2878
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gel membranes were obtained by fluorination through chemical vapor deposition,[17] and microcrystalline cellulose was modified by using soybean oil.[18] A comprehensive review on the hydrophobization of cellulose expands on these and other methods.[19] To develop sustainable CNF-based products, green chemistry approaches based on enzyme reactions have gained interest in the scientific and industrial communities. Enzymatic functionalization of cellulosic fibers by using phenolic moieties to achieve antimicrobial or antioxidant effects have been reported.[20–22] The use of enzyme systems for surface hydrophobization is still in its infancy. A method based on the application of laccase (Lacc) enzymes combined Figure 1. Particle size of the MCS and its respective components (LG, SL, Lacc, or their with hydrophobic phenolic compounds has been combinations), as measured by DLS before and after filtration (0.45 mm pore size filtrasuccessful in endowing suspended cellulose fibers tion). and CNFs with hydrophobic properties.[23, 24] Interestingly, such treatments have been carried out in sults indicate the effect of the Lacc enzyme: the LG characterisaqueous dispersions. tic particle size was reduced (from > 3 mm to 300 nm) in the Herein, we present a new route to further utilize Lacc enpresence of Lacc (see also the Supporting Information). The zymes to hydrophobize cellulose fiber webs and CNF films by ability of SL to improve the characteristic size of the dispersion direct surface application of the enzyme together with co-adjuis apparent if one compares the particle size of the LG + Lacc vants. More specifically, we investigate the physicochemical insystem with that of MCS (= LG + Lacc + SL). teractions between CNFs or a model surface (silica) with a mulIt is thus hypothesized that the MCS can be effective in hyticomponent colloidal system (MCS) obtained after a Lacc-catadrophobization given the combined effect of the low surface lyzed reaction involving dodecyl 3,4,5-trihydroxybenzoate energy of LG[11, 27] and the surface topography upon adsorption [commonly known as lauryl gallate (LG)] in the presence of [25, 26] a sulfonated lignin (SL). (hierarchical micro- and nanostructures on the surfaces[28]). The application of the MCS is effective in hydrophobizing CNFs by simple immersion, spraying, The aqueous dispersions of the MCS and its components or size-pressing methods. The proposed methodology fulfills presented different turbidities and stabilities, as assessed by the requirements for facile application, renewability, and is light transmission (Tr) and backscattering (BS) measurements a green approach that meets recyclability criteria. (Turbiscan). Figure 2 shows the light transmission profiles for the MCS as well as its single or dual components. The LG system was unstable (Figure 2 a). On the contrary, the results Results and Discussion shown in Figure 2 b and d, which include the transmission data for the SL and SL + Lacc systems, respectively, confirm a high The colloidal stability and characteristic particle size of the MCS and its components in aqueous media is presented first, colloidal stability. The transmission values for the LG + Lacc followed by a discussion of the adsorption behavior and hysystem (Figure 2 c) also indicated good stability. This dispersion drophobization of silica and CNF films. The MCS system conwas dark, yielding low transmission values, in the range of 3 %. sisted of preformed particles obtained after reaction of LG, MCS presented a slight instability, but was more translucent Lacc, and SL, as indicated in the Experimental Section. (transmission values between 50 % and 60 %; Figure 2 e). The results in Figure 2 f correspond to the Lacc system, which was highly stable. Particle size and turbidity analyses Several effects of the Lacc enzyme on LG can be highlightHerein, treatments on silica and CNFs consisted of the deposied: Lacc reduced the LG particle size, increased the LG dispertion of MCS particles or respective components after a filtration sion stability, and increased the light absorption of the colloistep (0.45 mm pore size filters). MCS particle structures were dal dispersion, probably due to oxidation of LG. Additionally, coupled to the surfaces, whereby particle size and resultant the presence of the SL favored the stability of the dispersion roughness affected the hydrophobicity of the system. The parand reduced the LG particle size (see light transmission of the ticle size [dynamic light scattering (DLS)] of the MCS and its reaqueous dispersions; Figure 2). spective components before and after filtration are shown in To gain further understanding of the effect of Lacc and SL Figure 1. on LG, real-time Turbiscan online measurements were carried Filtration allowed us to better understand the effect of each out. Figure 3 includes the Tr and BS values measured during treatment and dispersion component, especially for cases in 27 h. which samples were highly polydisperse. The particle size re 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 2. Time- and space-resolved light transmission values [%] of aqueous dispersions of the MCS and its components (single or dual components). The different profiles are represented in such a way that the gray color intensity decreases with time, as a function of the height (horizontal axes) in the test tube at which the dispersion is contained. Key: Transmission of LG (a), SL (b), LG + Lacc (c), SL + Lacc (d), MCS (e), and Lacc (f). The scans were performed by using time intervals of 5 min over 1 h.
For the SL + Lacc system, Tr and BS remained unaltered during the enzymatic reaction. The tests showed a very slight change in both Tr and BS upon the introduction of the Lacc enzyme. However, SL oxidation catalyzed by Lacc occurred rapidly (instantaneously); no changes were observed during 30 h. For the LG + Lacc and MCS systems, Tr began to fall and reached 0 % at 4–5 h after the introduction of Lacc. Thereafter, Tr began to increase towards transmissions of about 50 % and 10 % in the case of MCS and LG + Lacc, respectively. This increase in Tr can be explained by the presence of the SL, which enhances the enzymatic effect, as discussed for the AFM and Turbiscan data. Notably, 50 % transmission achieved for the MCS is higher than that of 30 % recorded at the beginning of the reaction before the addition of the enzyme. The observed initial decrease in Tr is explained by the fact that Lacc oxidized the LG particles (which were several microns in size at the beginning of the reaction, see Figure 1), yielding a very dark color. After the reaction, the size was reduced and Tr increased. The BS measurements indicated similar effects to those noted for Tr. 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
The Turbiscan online measurements enabled calculation of the particle diameter (d) by using the Mie theory and the volume fraction (f) of the medium. The average particle size of the LG + Lacc system was about 17 mm before the enzymatic reaction, and it was reduced to around 180 nm after 27 h. For the MCS, the initial size was around 18 mm, and it was reduced to about 80 nm after 27 h of reaction time. This is a confirmation of the effects determined by Tr and BS; all indicate that SL enhanced dispersion and the enzyme activity. Adsorption of MCS and its components on silica First, silica was used as a model for cellulose. Silica carries free silanols and geminal silanol groups on the surface, which could represent the free hydroxyl groups of cellulose. By using silica, it was also possible to eliminate contributions from the otherwise soft and reactive nature of the cellulosic substrates: the silica surface, unlike cellulose, is flat and smooth with no porosity and negligible roughness and does not display any swelling, hydration, molecular diffusion, or so forth. ChemSusChem 2014, 7, 2868 – 2878
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the MCS given by changes in frequency never reached a plateau (Df linearly decreased with time). Figure 4 b includes results from similar experiments to those described before, but include adds a rinsing step with high ionic strength (0.1 m NaCl) electrolyte solution, which was applied before final rinsing with the background buffer. This was carried out to increase the ionic strength of the medium and determine the contribution of electrostatic affinity between silica and MCS. After rinsing with electrolyte solution, no desorption was observed; instead, a significant decrease in dissipation was noted. This can be explained by the compaction of a highly hydrated MCS adsorbed layer. Figure 4 c includes isotherms after preadsorbing a thin layer of chitosan (CHIT) onto the silica surface, prior to Figure 3. The percentage of light transmission, Tr (a), and back scattering, BS (b), upon enzyme reaction in MCS, LG + Lacc, and SL + Lacc systems measured during 24 h of observation. The introduction of the Lacc enzyme is inthe injection of MCS solution dicated by the vertical arrows. into the QCM chamber. Preadsorption of CHIT onto silica was carried out to determine the affinity of MCS for cationic surfaFigure 4 a includes QCM sensograms showing the evolution ces. The results indicate that, compared with bare silica, a very of the negative value of the frequency shift, related to mass thin layer of CHIT caused greater and faster adsorption of the uptake by the surface as a function of time. First, the buffer soMCS (see also Figure 4 a and b). In this case, adsorption lution (0.1 m sodium acetate) was introduced over 3 min or reached a Df plateau of about 160 Hz, which indicated until no changes in Df were observed. Thereafter, a filtered a much higher adsorption of the MCS components onto the MCS solution was injected, which resulted in a dramatic reducCHIT-coated silica surface. tion of frequency, and indicated adsorption or deposition of Treatments with MCS components (single or dual compoMCS onto the surface. To determine to what extent the reducnents; see Table 1) were also applied to silica to elucidate their tion in frequency was due to irreversible deposition of large contribution to adsorption. SL was effectively adsorbed onto aggregates, the sensor was rinsed with buffer after 70 min. A silica, reaching a Df value of about 14 Hz, which indicated slight reversal in frequency signal (Df increase) was observed the presence of a thin but firmly attached SL. LG adsorbed to due the removal of some MCS from the surface, but the frea very small extent (less than 2 Hz frequency shifts were requency value did not return to its initial value. This was taken as an indication of an irreversible adsorption process. The difference between the initial freTable 1. Composition of the aqueous MCS and single- and dual-component dispersions used for surface treatquency (Df signal in buffer solument of silica and CNF. tion) and the frequency after System LG SL Lacc Description rinsing is ascribed to firmly ad[g L1] [g L1] [U mL1] sorbed MCS particles on the MCS 1.2 1.2 1.2 product resulting from the reaction of LG + SL with Lacc silica surface (an effective Df of LG 1.2 – – LG sonicated at the enzymatic reaction concentration about 28 Hz was measured SL – 1.2 – SL dissolved at the enzymatic reaction concentration after 70 min of adsorption Lacc – – 1.2 Lacc at the enzymatic reaction concentration LG + Lacc 1.2 – 1.2 product resulting from reaction of LG with Lacc time). This fact indicated the SL + Lacc – 1.2 1.2 product resulting from the reaction of SL with Lacc physicochemical affinity beLG + SL 1.2 1.2 – LG sonicated + SL dissolved at the enzymatic reaction concentration tween silica and MCS. Notably, MCS 1.2 1.2 1.2 product resulting from the reaction of LG + SL with Lacc the adsorption isotherms for 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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www.chemsuschem.org It is very interesting to note that, after treatment with the enzyme, the aggregated particle size (in LG + Lacc dispersions) was reduced, which allowed the LG particles to pass through the 0.45 mm filter and become available for adsorption onto the silica surfaces. The adsorption data for SL + Lacc treatment indicated that SL also adsorbed onto the silica surface upon reaction with Lacc: a Df plateau value of about 14 Hz was measured, which was very similar to that observed in the experiment with the individual SL solution. The LG particle size is expected to play an important role in surface modification by MCS, resulting in different levels of hydrophobicity, depending on the conditions. The application of enzyme dispersions (Lacc dispersion) was also carried out and Df values of about 38 Hz were recorded after 60 min. Overall, there is evidence that the three-component MCS systems (LG, SL, and Lacc) can be adsorbed onto silica to different degrees.
Hydrophobization of CNF films
Figure 4. Quartz crystal microbalance (QCM) Df and DD isotherms upon injection of MCS dispersions on silica surfaces followed by rinsing with either background buffer (a) or electrolyte (0.1 m NaCl solution) and final rinsing with buffer solution (b). Results for similar experiments performed on CHITcoated silica surfaces are displayed in (c). The Df and dissipation, D, signals are indicated by * and ~, respectively.
corded after 60 min). This is an interesting result because quite different behavior was observed when LG was applied after the reaction with the enzyme system. 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
The adsorption of the MCS and respective components onto CNF substrates was investigated by using QCM measurements. The adsorption of MCS onto the CNF films is illustrated in Figure 5 a. After 85 min of adsorption time followed by rinsing (background buffer), Df reached a value of about 40 Hz, which indicated high affinity between the MCS and CNFs. By comparing this result with that obtained for silica, it can be concluded that MCS is adsorbed to a similar extent on both surfaces. Figure 5 b includes analogous experiments, but after application of preadsorbed cationic CHIT onto CNFs, prior to contact with the MCS. In this case, Df reached a value of about 110 Hz, which indicated that the thin layer of CHIT was effective in increasing MCS adsorption, similarly to that found in the case of silica. Figure 5 c and d includes data for adsorption from solutions of SL + Lacc and LG + Lacc on CNFs: Df values of about 14 and 564 Hz were obtained after the respective treatments. The SL + Lacc system adsorbed in very similar amounts on CNFs and silica, whereas the LG + Lacc system was adsorbed to a much larger extent onto CNFs. Upon adsorption of MCS components on silica and CNFs (without and with a CHIT preadsorbed layer; Figures 4 and 5), no frequency plateau was reached. This is taken as evidence of a slow and large buildup of MCS components on the surface, possibly in the form of multilayers. The Df values upon application of MCS on silica and CNFs reached values of 42 and 40 Hz (after 90 min contact), respectively, which reveals a similar extent of adsorption or surface modification by MCS. However, preadsorption of CHIT induced a larger change in frequency upon MCS treatment: 145 and 100 Hz (60 min contact time) for the silica and CNF surfaces, respectively. This indicates much greater adsorption on the cationic substrates. All components present in the MCS (SL, Lacc, SL + Lacc, or LG + Lacc) were built up to different extents on the silica and CNF surfaces. The results indicate that the LG + Lacc system adsorbed to the largest degree (on both silica and CNF films). ChemSusChem 2014, 7, 2868 – 2878
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Figure 5. The QCM Df and DD profiles upon adsorption of MCS on CNF surfaces (a) and on CHIT-coated CNFs (b). The profiles after treatment of the CNF substrates with SL + Lacc (c) and LG + Lacc (d) are also included. The Df and dissipation, D, signals are indicated by * and ~, respectively.
Attachment of enzyme-modified LG moieties onto cellulose The mechanistic pathway in the oxidation of gallic acid proposed by Tulyathan et al.[29] can be considered as the basis to understand the nature of the reaction between Lacc and LG. Lacc enzymes show specificity for phenols, o- and p-diphenols, methoxyphenols, aminophenols, benzenethiols, and hydroxyindoles; the formation of quinones by the oxidation of phenolic compounds by using Lacc enzymes has been widely reported.[30, 31] Such a mechanism involves the oxidation of the OH groups in the LG molecule to form the corresponding phenoxy radicals, which are further oxidized to quinones and its openring acidic product.[32] The formation of dimers through the regeneration of hydroquinones is expected to be difficult due steric hindrance by the long hydrocarbon chain in LG. The reaction produces a molecule carrying carboxylic acids anchored to the alkyl chain through the original ester. The formation of quinones can be identified by the dark coloration developed by the enzymatic products of LG. The QCM-D experiments indicated the attachment of high amounts of LG moieties onto CHIT-coated silica or CNF surfaces. This effect can be attributed to the affinity between the anionic MCS (possible presence of carboxylate groups in the enzyme-modified LG) and the cationic CHIT. Electrostatic effects can increase the buildup of adsorbates on the surface, which can then undergo esterification reactions. 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
We hypothesize that attachment of LG-containing colloidal particles onto the surfaces is produced by means of Fischer’s esterification reactions between the OH groups on the surface of silica or CNFs, and the COOH groups of LG in the MCS, upon enzymatic reaction.[32–34] In this reaction, the sodium acetate buffer used (and respective tartaric acid) may act as an acid catalyst to promote the protonation of the carboxylic groups. Strong bonding between LG and cellulose was reported in our previous work.[25] In addition, MCS solutions were used to hydrophobize paper, and the bonding strength was studied by subjecting the modified paper sheets to strong washing conditions (80 8C during 30 min) and Soxhlet extractions with acetone. No alteration of the hydrophobic character of the paper sheets was taken as an indication of strong LG attachment.
Water contact angle and surface hydrophobicity Water contact angle (WCA) measurements were performed for silica and CNF surfaces treated with the MCS and solutions of its components. This allowed further elucidation of the extent of adsorption and degree of hydrophobization of silica and CNF surfaces. Bare silica wafers and CNF films were treated by using the sequences described in previous sections (see Figures 4 and 5). ChemSusChem 2014, 7, 2868 – 2878
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Figure 7. Relationship between the WCA of the substrate and the shift in QCM frequency, Df (that correlates with the adsorbed mass) after treatment with MCS and LG + Lacc dispersions. Silica and CNF substrates are indicated by ^ and ^, respectively.
Silica and CNFs showed similar behavior, in terms of the resulting WCA: in both cases, the treatment that yielded the highest WCA was that of the solution of LG + Lacc; the same tendency to increase the WCA after adsorbing a thin CHIT layer was observed for MCS and CNF substrates. It is clear that there is a relationship between the amount of enzyme-modified LG adsorbed and the hydrophobicity achieved after the respective treatments: Figure 7 provides a summary of the QCM and WCA data upon enzyme-modified LG adsorption onto silica and CNFs (after treatment with MCS or LG + Lacc). The WCAs were measured from sensors after the QCM adsorption experiments introduced in the previous sections (prior to WCA measurements, the sensors were rinsed with Milli-Q water and dried under nitrogen). It is apparent that as the adsorbed mass increased (as indicated by the Df values) the WCA also increased. A plateau in the WCA is reached at Df 300 Hz. The Supporting Information contains a movie illustrating the increase in WCA after application of the MCS on cellulose. Figure 6. a) WCA measurements for silica surfaces treated with the MCS and its respective components after rinsing. b) Comparison of WCA values after treatment of bare silica and CNFs with the MCS or its respective component dispersions. BUFF = buffer rinsing.
As shown in Figure 6 a, the WCA of bare silica was about 158 and it increased slightly after exposure to buffer solution (by 58). MCS treatment produced an increase in the WCA of silica to about 358. MCS treatment on CHIT-coated silica increased the WCA to 508, which indicated increased adsorption and hydrophobization, in agreement with QCM adsorption isotherms. The increase in hydrophobicity is thus related to the extent of adsorption. Treatment with SL + Lacc and Lacc increased the WCA to about 35 and 558, respectively. The most effective treatment, as far as increased hydrophobicity of the surface was concerned, was that of LG + Lacc application, which yielded WCA values of 85 and 888 for silica and CHIT-coated silica, respectively. Figure 6 b provides a comparison of the final WCA after treatment of silica and CNFs (with and without adsorbed CHIT) with the MCS and with LG + Lacc and SL + Lacc systems. 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Silica surface analysis by AFM The surfaces of the QCM-D silica sensors treated with the MCS and its components were imaged by AFM (Figure 8). Figure 8 a shows the surface topography of the bare silica surface, which is featureless and smooth with a root-mean-square (rms) roughness of less than 1 nm. Figure 8 b corresponds to the silica sensor after exposure to a solution of Lacc: the enzymes adsorbed onto the silica sensor (roughness of the order of a few nm). Particles unevenly distributed on the surface were observed; these were likely to be enzyme aggregates. Figure 8 c shows the result after treatment of the silica surface with the SL + Lacc system (rms surface roughness of only few nanometers with a few unevenly distributed aggregates). These aggregates could be responsible for the relatively large particle sizes found for Lacc and SL + Lacc dispersions investigated in the particle size analyses. Figure 8 d shows the surface topography of the silica sensor after treatment with the LG + Lacc system, which shows evidence of large and uniformly disChemSusChem 2014, 7, 2868 – 2878
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Figure 8. AFM images of the silica surfaces after adsorption of the MCS and respective single- or dual-component systems: untreated bare silica surface (a) and silica surface after adsorption of Lacc (b); SL + Lacc (c), LG + Lacc (d), and MCS (e). An image for MCS adsorbed onto CHIT-coated silica is also included (f). All scan sizes were 10 10 mm2, except for image a), which was 5 5 mm2.
tributed particles (diameters and heights of 200–300 and 20–40 nm, respectively). The presence of such relatively large particles is only observed in enzyme-treated LG, since it was not observed in Lacc or LG systems. The particle diameters observed in Figure 8 d are of the same order of magnitude as those obtained from the DLS experiments (LG + Lacc system). The Supporting Information contains SEM images for the same system on CNF films. The silica surface treated with the MCS is shown in Figure 8 e and indicates the presence of uniformly distributed LG particles with a diameter and height of 100 and 10–20 nm, respectively. Again, the particle size of the adsorbed 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
MCS observed by AFM is of the same order of magnitude as that obtained from DLS analyses. Notice the effect of SL in the MCS system, whereby an enhancement of the enzyme role in reducing the size of the LG particles is observed (compare it with results from the LG + Lacc systems). The effect of a preadsorbed CHIT layer on silica was also accessed by AFM imaging (Figure 8 f). The images clearly indicated a significant increase in the number density of adsorbed LG particles from MCS; this effect was also observed in the QCM-D analyses discussed previously.
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A strong relationship between the WCA of the treated silica surfaces and their topography was observed. The treatment that provided the highest WCA was that after application of LG + Lacc on silica and CNFs, and this treatment was also the one that produced the highest roughness of the silica surface. The MCS system applied to silica (bare or carrying CHIT) also produced an important increase in WCA. AFM results confirmed the adsorption of LG particles onto silica. However, the most probable scenario for the hydrophobization of the substrates comprises both a physicochemical effect (adsorption) and esterification of the hydroxylated surface.
Sweden). CNF films were fixed on silica sensors that were first cleaned by immersion for 20 s in a 10 % solution of NaOH followed by rinsing with Millipore water and drying under nitrogen. The sensors were pretreated with polyethyleneimine (PEI) and dried, followed by spin coating with the dispersion of CNF (3000 rpm, 1 min). The CNF-coated sensors were rinsed with Millipore water, and dried in an oven at 80 8C for 15 min and stored until use.
Conclusions
The enzymatic treatments involved first the reaction of the enzyme with the hydrophobizing agent (LG). The selection of the preparation conditions was based on our previous reports.[23, 25]
We presented a sustainable biocatalytic method to obtain a multicomponent colloidal system (MCS) for the hydrophobization of silica and cellulose nanofibrils (CNFs). Adsorption of the MCS components [laccase (Lacc), sulfonated lignin (SL), and lauryl gallate (LG)] occurred on silica and CNF surfaces and such an effect was boosted by pretreatment of the substrates with chitosan (CHIT). Treatment with MCS increased the water contact angle (WCA) of CNFs and silica from 208 to about 908. A direct relationship between the amount of LG adsorbed onto surfaces and WCA was observed, and also between the roughness of the treated silica and CNF surfaces. Light scattering measurements revealed a reduction in the particle size from several microns down to 300 nm upon enzyme treatment in the presence of LG. The Lacc treatment in the presence of SL reduced the LG particle size even more to 80 nm through the dispersion effect of SL. Light transmission and backscattering data confirmed the effects of Lacc on LG: a reduction in the particle size and increased dispersion stability.
Experimental Section General The enzyme used herein was a laccase (Lacc) from Trametes villosa with an activity of 588 U mL1 and supplied by Novozymes. Dodecyl 3,4,5-trihydroxybenzoate (LG) and chitosan (CHIT, 50–190 kDa molecular weight, deacetylation degree of 75–85 %) were purchased from Sigma–Aldrich. Soluble sulfonated lignin (SL) with 5.9 kDa MW and 5 % total sulfur content was obtained from Borregaard (Sarpsborg, Norway) and was used as received. CNFs were obtained after mechanical deconstruction of bleached hardwood birch fibers by using a Masuko grinder (five passes) followed by a microfluidizer (20 passes). The individual CNFs were dispersed in water by using mechanical stirring and tip ultrasonication (10 min, 25 % amplitude). The resulting CNF suspension was centrifuged at 10 400 rpm for 45 min and nanofibrils were then collected from the supernatant by pipetting.[35] An AFM image of the CNF can be found in the Supporting Information.
Multicomponent colloidal system (MCS) and surface treatment The MCS refers to the product of the enzymatic reaction between T. villosa Lacc with the phenolic, low-surface-energy species (LG) in the presence of the SL.
The treatments were performed in 250 mL beakers containing LG (1.2 g L1), 1.2 U mL1 Lacc and the SL (1.2 g L1) dispersed in 0.1 m sodium acetate buffer (pH 4). LG was hydrophobic and insoluble in water; therefore, it was applied as a colloidal suspension after ultrasonication in water (Hielscher Ultrasonic Processor UP100 H, 30 min, 100 % amplitude). This reduced the LG effective aggregate size and ensured a homogeneous dispersion. LG and SL were kept under agitation for 10 min at 50 8C. Subsequently, the Lacc enzyme was added and the aqueous dispersion was stirred for 4 h of reaction. The reaction was stopped by quenching the system with cold water. Notably, after treatment with the enzyme, a colloidal stable dispersion of LG was produced. Several treatments were performed to elucidate the role of each of the compounds present in the MCS (Table 1). For QCM experiments, the MCS dispersion as well as single and dual components of the MCS were filtered by using 0.45 mm pore size syringe filters to eliminate any possible contribution from residual, large LG particles. In some experiments, aqueous dispersions of CHIT (0.5 g L1, sodium acetate buffer) were used to pretreat the surfaces.
QCM with dissipation monitoring (QCM-D) In situ QCM-D experiments were performed by using a Q-Sense E4 instrument (Vstra Frçlunda, Sweden) operated in continuous mode. The molecular interaction between MCS (and respective single or dual systems) with the substrate might produce a shift in the resonance frequency, (Df), which could be monitored and used to quantify mass gains on the surface (for example, through adsorption and coupling). A linear relationship between Df and the change in mass (including the contribution of coupled water) due to adsorption on the surface of the sensor can be determined through the Sauerbrey model, which assumes that adsorption is homogeneous and highly coupled [Eq. (1)]:[36] Dm ¼ CDf =n
ð1Þ
Substrate preparation
in which Dm, Df, C, and n are the variation of mass [ng cm2], the resonance frequency [Hz], a constant for the crystal [ng Hz1 cm2], and the overtone number (n = 1 for the fundamental frequency), respectively. For the present setup, C equals 17.7 ng cm2 s1 at f = 5 MHz. Herein, we used the third overtone (n = 3) to interpret the QCM data.
Substrates for adsorption tests consisted of bare silica wafers and QCM-D silica sensors obtained from Q-Sense (Vstra Frçlunda,
In addition to resonance frequencies, QCM-D measured the change in energy dissipation of the system, DD, which was moni-
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tored to determine frictional (viscous) losses on the surface. DD is related to the viscoelastic properties of the adsorbed layer(s) deposited on the sensor and can give an indication of its stiffness. For example, the presence of a soft layer on the sensor enhances damping of the vibration and results in a higher dissipation. The energy dissipation, D, is related to the frequency, Df, and decay time, t, through Equation (2): D ¼ 1=pDf t
ð2Þ
in which t values are obtained by periodically disconnecting the oscillating crystals from the main circuit through a computer-controlled relay. Time-resolved adsorption was followed on bare silica sensors and on silica sensors covered with CNFs. The QCM-D measurements were performed under a continuous flow rate of 100 mL min1 at 25 8C.
AFM A Nanoscope IIIa multimode scanning probe microscope (Digital Instruments, Inc., Santa Barbara, CA) was used to characterize the surface topography of the QCM-D silica sensors and the CNFs before and after surface treatment. The images were scanned in tapping mode by using a J scanner and silicon cantilevers (NSC15/ AIBS from Micromasch, Tallinn, Estonia). The radius of the curvature of the AFM tip, according the manufacturer, was less than 10 nm, and the typical resonance frequency of the cantilever was 325 kHz. At least two different areas were analyzed on each sample; scan sizes included 10 10, 5 5, and 1 1 mm2. AFM images were flattened by following first-order conversion. Image analysis was performed by using Nanoscope software (ver. V6.13 R1, Digital Instruments, Inc.) from which the rms roughness and Z sections in line profiles were determined.
Particle sizing The hydrodynamic diameter and size distributions of any colloidal particles present in the MCS, as well as control treatments, were determined by using a Malvern Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) light scattering device.
Water contact angle (WCA) The WCA was measured by means of a CAM-200 contact angle goniometer (KSV Instruments Ltd., Helsinki, Finland). Prior to measurements, silica and CNF-coated surfaces were rinsed with Milli-Q water and dried with nitrogen. A 4 mL water drop was gently deposited on the substrate from above and the profile of the drop placed on the silica and CNF surfaces was captured digitally by means of a high-resolution camera. The contact angle was determined upon contact, after stabilization of the water drop (time required to avoid initial transitory fluctuations, 2 s). The calculation of the contact angles was based on a numerical solution of the Young–Laplace equation by following a computational protocol built in the instrument.
ments, were placed in 80 mm long cylindrical glass cells. The detection head consisted of a pulsed near-infrared light source (l = 850 nm) and two synchronous backscattered (BS) and transmitted (Tr) flux detectors. The optical sensors collected the transmission and BS signals through vertical scanning of the sample with a step of 40 mm at given time intervals. At the beginning of the measurements, scans were performed every minute over 30 min. To evaluate the long-term stability, the time interval between data points was increased. The experimental results were represented as a set of curves that indicated the percentage of transmitted light as a function of the sample tube height. The optical analyzer in Turbiscan measurements detected the light flux backscattered at 1358 by a dispersed and low-transparency medium in a cylindrical cell. The backscattered flux (BS) is related to l* [as a first approximation, BS is inversely proportional to p (l*)] through Equations (3) and (4): p BS ¼ 1= ðl* Þ
ð3Þ
l* ¼ 2d=3fð1gÞQs
ð4Þ
in which l* (photon transport mean free path) represents the mean distance travelled by photons into the dispersion medium, f is the volume fraction of the particles, and d is the mean diameter. Qs and g are parameters given by the Mie theory. Transmission measurements were performed by means of the Turbiscan by sending a light beam through the cell and detecting photons that cross the dispersion without being diffused. The Lambert–Beer law gives an analytical expression of the transmitted flux (Tr) measured as a function of l [Eqs. (5) and (6)]: Tr ¼ Tr0 eð2ri =lÞ
ð5Þ
l ¼ 2d=3fQs
ð6Þ
in which l is the photon mean free path, ri is the internal radius of the measurement cell, and Tr0 denotes the transmittance of the continuous phase. The stability of MCS and single/dual systems were evaluated by analyzing the variation of the backscattered (BS) and transmitted (Tr) flux; both parameters are dependent on the particle volume fraction (f) and the particle mean diameter (d). Real-time Turbiscan online measurements were carried out by conducting the enzymatic reaction in a reaction vessel at constant temperature. Samples of the dispersions were flown from the reaction vessel to the measuring cell by means of the peristaltic pump. Data (Tr and BS) were collected at time intervals of 10 s.
Supporting Information It contains a movie illustrating the hydrophobizing effect of MCS after application on cellulose and also includes SEM images of the LG-containing particles deposited on CNF films as well as AFM and SEM images of CNF fibrils in films.
Acknowledgements Turbiscan and Turbiscan online tests The stability of the MCS and control treatments was evaluated by using Turbiscan MA 2000 (for space-resolved measurements) and Turbiscan online (Formulaction, France; for time-resolved measurements) instruments. The aqueous products, after the enzyme treat 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
We are grateful to the BIOSURFACEL-CTQ2012-34109 and BIOFIBRECELL projects (CTQ2010-20238-CO3-01) within the framework of the Spanish MICINN and the Academy of Finland’s Centres of Excellence Programme. Special thanks are also due to the conChemSusChem 2014, 7, 2868 – 2878
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CHEMSUSCHEM FULL PAPERS solidated research group AGAUR 2009SGR 00327 at Universitat de Barcelona (UB). Keywords: adsorption · biocatalysis · hydrophobization · enzymes · nanoparticles [1] C. Johansson, J. Bras, I. Mondragon, P. Nechita, D. Plackett, P. Simon, D. G. Svetec, S. Virtanen, M. G. Baschetti, C. Breen, F. Clegg, S. Aucejo, BioResources 2012, 7, 2506 – 2552. [2] O. Faruk, A. K. Bledzki, H. Fink, M. Sain, Prog. Polym. Sci. 2012, 37, 1552 – 1596. [3] W. Hamad, Can. J. Chem. Eng. 2006, 84, 513 – 519. [4] R. Kolakovic, L. Peltonen, A. Laukkanen, J. Hirvonen, T. Laaksonen, Eur. J. Pharm. Biopharm. 2012, 82, 308 – 315. [5] G. Siqueira, J. Bras, A. Dufresne, Polymer 2010, 2, 728 – 765. [6] Y. Zhang, T. Nypelç, C. Salas, J. Arboleda, I. C. Hoeger, O. J. Rojas, J. Renewable Mater. 2013, 1, 195 – 211. [7] T. T. T. Ho, T. Zimmermann, S. Ohr, W. R. Caseri, ACS Appl. Mater. Interfaces 2012, 4, 4832 – 4840. [8] T. Saito, R. Kuramae, J. Wohlert, L. A. Berglund, A. Isogai, Biomacromolecules 2013, 14, 248 – 253. [9] A. Ferrer, E. Quintana, I. Filpponen, I. Solala, T. Vidal, A. Rodrguez, J. Laine, O. J. Rojas, Cellulose 2012, 19, 2179 – 2193. [10] S. Li, S. Zhang, X. Wang, Langmuir 2008, 24, 5585 – 5590. [11] H. Ogihara, J. Xie, J. Okagaki, T. Saji, Langmuir 2012, 28, 4605 – 4608. [12] D. Stanssens, H. Van den Abbeele, L. Vonck, G. Schoukens, M. Deconinck, P. Samyn, Mater. Lett. 2011, 65, 1781 – 1784. [13] N. Aarne, J. Laine, T. Hnninen, V. Rantanen, J. Seitsonen, J. Ruokolainen, E. Kontturi, ChemSusChem 2013, 6, 1203 – 1208. [14] Z. Guo, W. Liu, B. Su, J. Colloid Interface Sci. 2011, 353, 335 – 355. [15] C. Jin, Y. Jiang, T. Niu, J. Huang, J. Mater. Chem. 2012, 22, 12562 – 12567. [16] C. Jin, R. Yan, J. Huang, J. Mater. Chem. 2011, 21, 17519 – 17525. [17] H. Jin, M. Kettunen, A. Laiho, H. Pynnçnen, J. Paltakari, A. Marmur, O. Ikkala, R. H. A. Ras, Langmuir 2011, 27, 1930 – 1934. [18] X. Dong, Y. Dong, M. Jiang, L. Wang, J. Tong, J. Zhou, Ind. Crops Prod. 2013, 46, 301 – 303.
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www.chemsuschem.org [19] J. Song, O. J. Rojas, Nordic Pulp Pap. Res. J. 2013, 28, 216 – 238. [20] A. Fillat, O. Gallardo, T. Vidal, F. I. J. Pastor, P. Daz, M. B. Roncero, Carbohydr. Polym. 2012, 87, 146 – 152. [21] E. Aracri, A. Fillat, J. F. Colom, A. Gutierrez, J. C. del Rio, A. T. Martinez, T. Vidal, Bioresour. Technol. 2010, 101, 8211 – 8216. [22] E. M. Cadena, X. Du, G. Gellerstedt, J. Li, A. Fillat, J. Garcia-Ubasart, T. Vidal, J. F. Colom, Bioresour. Technol. 2011, 102, 3911 – 3917. [23] J. Garcia-Ubasart, J. F. Colom, C. Vila, N. G. Hernndez, M. B. Roncero, T. Vidal, Bioresour. Technol. 2012, 112, 341 – 344. [24] P. Saastamoinen, M. L. Mattinen, U. Hippi, P. Nousiainen, J. Sipil, M. Lille, A. Suurnkki, J. Pere, BioResources 2012, 7, 5749 – 5770. [25] O. Cusola, C. Valls, T. Vidal, M. B. Roncero, Bioresour. Technol. 2013, 131, 521 – 526. [26] O. Cusola, C. Valls, T. Vidal, M. B. Roncero, Holzforschung 2014, 68, 631 – 639. [27] P. Roach, N. J. Shirtcliffe, M. I. Newton, Soft Matter 2008, 4, 224 – 240. [28] L. Mammen, X. Deng, M. Untch, D. Vijayshankar, P. Papadopoulos, R. Berger, E. Riccardi, F. Leroy, D. Vollmer, Langmuir 2012, 28, 15005 – 15014. [29] V. Tulyathan, R. B. Boulton, V. L. Singleton, J. Agric. Food Chem. 1989, 37, 844 – 849. [30] C. Navarra, C. Goodwin, S. Burton, B. Danieli, S. Riva, J. Mol. Catal. B 2010, 65, 52 – 57. [31] S. Witayakran, A. Zettili, A. J. Ragauskas, Tetrahedron Lett. 2007, 48, 2983 – 2987. [32] J. M. Gess, C. Dence, Tappi 1971, 54, 1114 – 1121. [33] M. J. Sobkowicz, B. Braun, J. R. Dorgan, Green Chem. 2009, 11, 680 – 682. [34] J. M. Gess in Paper Chemistry, 2nd Ed. (Ed.: J. C. Roberts), Springer, Dordrecht, 1995, pp. 120 – 139. [35] S. Ahola, P. Myllytie, M. sterberg, T. Teerinen, J. Laine, BioResources 2008, 3, 1315 – 1328. [36] G. Sauerbrey, Z. Phys. 1959, 155, 206 – 222.
Received: May 18, 2014 Published online on August 27, 2014
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A Facile and Green Method to Hydrophobize Films of Cellulose Nanofibrils and Silica by Laccase-Mediated Coupling of Nonpolar Colloidal Particles Oriol Cusola,*[a, b] M. Blanca Roncero,[a] Teresa Vidal,[a] and Orlando J. Rojas*[b, c] Hydrophobic particles based on dodecyl 3,4,5-trihydroxybenzoate (LG) were coupled onto the surface of cellulose nanofibrils (CNFs) and silica by treatment with a multicomponent colloidal system (MCS) derived from the laccase-mediated reaction of LG in the presence of a sulfonated lignin (SL). Surface modification upon treatment with MCS was monitored in situ and in real time by quartz crystal microgravimetry. The colloidal stability of MCS and its components in water was followed by measuring space- and time-resolved light transmission and back scattering. The sulfonated lignin increased dispersion stability and reduced the characteristic MCS particle size [from
4 to 80 nm, according to AFM and dynamic light scattering (DLS)]. It also facilitated the surface enzymatic reaction that led to adsorption and coupling of MCS onto CNFs and silica surfaces. The combined effect of reduced surface energy and surface roughness by MCS treatment produced an increase in water contact angle on CNFs and silica of about 90 and 808, respectively. Surface pretreatment with chitosan further increased the extent of MCS adsorption on the surfaces. This method represents a sustainable alternative to traditional approaches for cellulose hydrophobization and a step forward in implementing green routes for surface modification.
Introduction The utilization of renewable and biodegradable materials has increased tremendously in recent years. Their global market is expected to grow in the near future, following increasing societal awareness in relation to climate change and consumer perception.[1] Cellulose is the most abundant natural polymer in the biosphere and displays properties such as hydrophilicity, chirality, biodegradability, biocompatibility, and recyclability. In addition, it can be modified by a broad number of routes. Nanosized cellulose [cellulose nanofibrils (CNFs)] has attracted attention as a renewable resource that can be converted into high-value products and advanced functional materials; as such, it has been investigated for packaging materials, biomedical devices, adhesives, composites, electronics, and so forth.[2–7] CNFs differ significantly from common cellulosic fibers
[a] Dr. O. Cusola, Prof. M. B. Roncero, Prof. T. Vidal Textile and Paper Engineering Department Universitat Politcnica de Catalunya (UPC, BarcelonaTech) Colom 11, 08222 Terrassa (Spain) E-mail: [email protected] [b] Dr. O. Cusola, Prof. O. J. Rojas Department of Forest Products Technology School of Chemical Technology, Aalto University P.O. Box 16300, 00076 Aalto, Espoo (Finland) E-mail: [email protected] [c] Prof. O. J. Rojas Departments of Forest Biomaterials and Chemical and Biomolecular Engineering North Carolina State University Raleigh, NC 27695-8005(USA) Supporting Information for this article is available on the WWW under http://dx.doi.org/10.1002/cssc.201402432.
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because they have a higher aspect ratio and specific surface area combined with higher intrinsic strength and flexibility.[8, 9] However, for a more comprehensive utilization of CNFs there is a need to develop hydrophobic CNF products. Waterproof coatings and films, packaging materials, and hydrophobic composites are but a few examples in which hydrophobized CNFs can be utilized. Hydrophobization of CNFs can improve industrial processing, including size pressing, printing, and conversion.[10, 11] More importantly, packaging applications demand ecologically friendly hydrophobizing agents that can facilitate product recyclability; at present such an issue is limited if conventional waxes and fluoropolymers are applied.[12] The wettability of a surface arises from the combined contribution of surface topography and low surface energy. Most methods aimed at obtaining high hydrophobicity tend to minimize contact between water droplets and the surface by hierarchical micro- and nanostructures.[13] There are a variety of hydrophobization methods that usually depend on the type of substrate. For example, wet chemical reactions and electrochemical deposition techniques are commonly applied to metals, whereas self-assembly and layer-by-layer deposition are used for glass. In contrast, textiles and other substrates are often modified through sol–gel and polymerization reactions.[14] Several studies have reported on a variety of approaches to hydrophobize different types of cellulosic surfaces. In some of the most relevant achievements, amphiphobic cellulose-based materials were fabricated by chemical etching and subsequent coating of functional thin films.[15] Highly hydrophobic cellulose materials possessing reversible wettability were fabricated by self-assembling monolayers onto an intermediate coating of titania.[16] Amphiphobic nanocellulose aeroChemSusChem 2014, 7, 2868 – 2878
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gel membranes were obtained by fluorination through chemical vapor deposition,[17] and microcrystalline cellulose was modified by using soybean oil.[18] A comprehensive review on the hydrophobization of cellulose expands on these and other methods.[19] To develop sustainable CNF-based products, green chemistry approaches based on enzyme reactions have gained interest in the scientific and industrial communities. Enzymatic functionalization of cellulosic fibers by using phenolic moieties to achieve antimicrobial or antioxidant effects have been reported.[20–22] The use of enzyme systems for surface hydrophobization is still in its infancy. A method based on the application of laccase (Lacc) enzymes combined Figure 1. Particle size of the MCS and its respective components (LG, SL, Lacc, or their with hydrophobic phenolic compounds has been combinations), as measured by DLS before and after filtration (0.45 mm pore size filtrasuccessful in endowing suspended cellulose fibers tion). and CNFs with hydrophobic properties.[23, 24] Interestingly, such treatments have been carried out in sults indicate the effect of the Lacc enzyme: the LG characterisaqueous dispersions. tic particle size was reduced (from > 3 mm to 300 nm) in the Herein, we present a new route to further utilize Lacc enpresence of Lacc (see also the Supporting Information). The zymes to hydrophobize cellulose fiber webs and CNF films by ability of SL to improve the characteristic size of the dispersion direct surface application of the enzyme together with co-adjuis apparent if one compares the particle size of the LG + Lacc vants. More specifically, we investigate the physicochemical insystem with that of MCS (= LG + Lacc + SL). teractions between CNFs or a model surface (silica) with a mulIt is thus hypothesized that the MCS can be effective in hyticomponent colloidal system (MCS) obtained after a Lacc-catadrophobization given the combined effect of the low surface lyzed reaction involving dodecyl 3,4,5-trihydroxybenzoate energy of LG[11, 27] and the surface topography upon adsorption [commonly known as lauryl gallate (LG)] in the presence of [25, 26] a sulfonated lignin (SL). (hierarchical micro- and nanostructures on the surfaces[28]). The application of the MCS is effective in hydrophobizing CNFs by simple immersion, spraying, The aqueous dispersions of the MCS and its components or size-pressing methods. The proposed methodology fulfills presented different turbidities and stabilities, as assessed by the requirements for facile application, renewability, and is light transmission (Tr) and backscattering (BS) measurements a green approach that meets recyclability criteria. (Turbiscan). Figure 2 shows the light transmission profiles for the MCS as well as its single or dual components. The LG system was unstable (Figure 2 a). On the contrary, the results Results and Discussion shown in Figure 2 b and d, which include the transmission data for the SL and SL + Lacc systems, respectively, confirm a high The colloidal stability and characteristic particle size of the MCS and its components in aqueous media is presented first, colloidal stability. The transmission values for the LG + Lacc followed by a discussion of the adsorption behavior and hysystem (Figure 2 c) also indicated good stability. This dispersion drophobization of silica and CNF films. The MCS system conwas dark, yielding low transmission values, in the range of 3 %. sisted of preformed particles obtained after reaction of LG, MCS presented a slight instability, but was more translucent Lacc, and SL, as indicated in the Experimental Section. (transmission values between 50 % and 60 %; Figure 2 e). The results in Figure 2 f correspond to the Lacc system, which was highly stable. Particle size and turbidity analyses Several effects of the Lacc enzyme on LG can be highlightHerein, treatments on silica and CNFs consisted of the deposied: Lacc reduced the LG particle size, increased the LG dispertion of MCS particles or respective components after a filtration sion stability, and increased the light absorption of the colloistep (0.45 mm pore size filters). MCS particle structures were dal dispersion, probably due to oxidation of LG. Additionally, coupled to the surfaces, whereby particle size and resultant the presence of the SL favored the stability of the dispersion roughness affected the hydrophobicity of the system. The parand reduced the LG particle size (see light transmission of the ticle size [dynamic light scattering (DLS)] of the MCS and its reaqueous dispersions; Figure 2). spective components before and after filtration are shown in To gain further understanding of the effect of Lacc and SL Figure 1. on LG, real-time Turbiscan online measurements were carried Filtration allowed us to better understand the effect of each out. Figure 3 includes the Tr and BS values measured during treatment and dispersion component, especially for cases in 27 h. which samples were highly polydisperse. The particle size re 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 2. Time- and space-resolved light transmission values [%] of aqueous dispersions of the MCS and its components (single or dual components). The different profiles are represented in such a way that the gray color intensity decreases with time, as a function of the height (horizontal axes) in the test tube at which the dispersion is contained. Key: Transmission of LG (a), SL (b), LG + Lacc (c), SL + Lacc (d), MCS (e), and Lacc (f). The scans were performed by using time intervals of 5 min over 1 h.
For the SL + Lacc system, Tr and BS remained unaltered during the enzymatic reaction. The tests showed a very slight change in both Tr and BS upon the introduction of the Lacc enzyme. However, SL oxidation catalyzed by Lacc occurred rapidly (instantaneously); no changes were observed during 30 h. For the LG + Lacc and MCS systems, Tr began to fall and reached 0 % at 4–5 h after the introduction of Lacc. Thereafter, Tr began to increase towards transmissions of about 50 % and 10 % in the case of MCS and LG + Lacc, respectively. This increase in Tr can be explained by the presence of the SL, which enhances the enzymatic effect, as discussed for the AFM and Turbiscan data. Notably, 50 % transmission achieved for the MCS is higher than that of 30 % recorded at the beginning of the reaction before the addition of the enzyme. The observed initial decrease in Tr is explained by the fact that Lacc oxidized the LG particles (which were several microns in size at the beginning of the reaction, see Figure 1), yielding a very dark color. After the reaction, the size was reduced and Tr increased. The BS measurements indicated similar effects to those noted for Tr. 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
The Turbiscan online measurements enabled calculation of the particle diameter (d) by using the Mie theory and the volume fraction (f) of the medium. The average particle size of the LG + Lacc system was about 17 mm before the enzymatic reaction, and it was reduced to around 180 nm after 27 h. For the MCS, the initial size was around 18 mm, and it was reduced to about 80 nm after 27 h of reaction time. This is a confirmation of the effects determined by Tr and BS; all indicate that SL enhanced dispersion and the enzyme activity. Adsorption of MCS and its components on silica First, silica was used as a model for cellulose. Silica carries free silanols and geminal silanol groups on the surface, which could represent the free hydroxyl groups of cellulose. By using silica, it was also possible to eliminate contributions from the otherwise soft and reactive nature of the cellulosic substrates: the silica surface, unlike cellulose, is flat and smooth with no porosity and negligible roughness and does not display any swelling, hydration, molecular diffusion, or so forth. ChemSusChem 2014, 7, 2868 – 2878
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the MCS given by changes in frequency never reached a plateau (Df linearly decreased with time). Figure 4 b includes results from similar experiments to those described before, but include adds a rinsing step with high ionic strength (0.1 m NaCl) electrolyte solution, which was applied before final rinsing with the background buffer. This was carried out to increase the ionic strength of the medium and determine the contribution of electrostatic affinity between silica and MCS. After rinsing with electrolyte solution, no desorption was observed; instead, a significant decrease in dissipation was noted. This can be explained by the compaction of a highly hydrated MCS adsorbed layer. Figure 4 c includes isotherms after preadsorbing a thin layer of chitosan (CHIT) onto the silica surface, prior to Figure 3. The percentage of light transmission, Tr (a), and back scattering, BS (b), upon enzyme reaction in MCS, LG + Lacc, and SL + Lacc systems measured during 24 h of observation. The introduction of the Lacc enzyme is inthe injection of MCS solution dicated by the vertical arrows. into the QCM chamber. Preadsorption of CHIT onto silica was carried out to determine the affinity of MCS for cationic surfaFigure 4 a includes QCM sensograms showing the evolution ces. The results indicate that, compared with bare silica, a very of the negative value of the frequency shift, related to mass thin layer of CHIT caused greater and faster adsorption of the uptake by the surface as a function of time. First, the buffer soMCS (see also Figure 4 a and b). In this case, adsorption lution (0.1 m sodium acetate) was introduced over 3 min or reached a Df plateau of about 160 Hz, which indicated until no changes in Df were observed. Thereafter, a filtered a much higher adsorption of the MCS components onto the MCS solution was injected, which resulted in a dramatic reducCHIT-coated silica surface. tion of frequency, and indicated adsorption or deposition of Treatments with MCS components (single or dual compoMCS onto the surface. To determine to what extent the reducnents; see Table 1) were also applied to silica to elucidate their tion in frequency was due to irreversible deposition of large contribution to adsorption. SL was effectively adsorbed onto aggregates, the sensor was rinsed with buffer after 70 min. A silica, reaching a Df value of about 14 Hz, which indicated slight reversal in frequency signal (Df increase) was observed the presence of a thin but firmly attached SL. LG adsorbed to due the removal of some MCS from the surface, but the frea very small extent (less than 2 Hz frequency shifts were requency value did not return to its initial value. This was taken as an indication of an irreversible adsorption process. The difference between the initial freTable 1. Composition of the aqueous MCS and single- and dual-component dispersions used for surface treatquency (Df signal in buffer solument of silica and CNF. tion) and the frequency after System LG SL Lacc Description rinsing is ascribed to firmly ad[g L1] [g L1] [U mL1] sorbed MCS particles on the MCS 1.2 1.2 1.2 product resulting from the reaction of LG + SL with Lacc silica surface (an effective Df of LG 1.2 – – LG sonicated at the enzymatic reaction concentration about 28 Hz was measured SL – 1.2 – SL dissolved at the enzymatic reaction concentration after 70 min of adsorption Lacc – – 1.2 Lacc at the enzymatic reaction concentration LG + Lacc 1.2 – 1.2 product resulting from reaction of LG with Lacc time). This fact indicated the SL + Lacc – 1.2 1.2 product resulting from the reaction of SL with Lacc physicochemical affinity beLG + SL 1.2 1.2 – LG sonicated + SL dissolved at the enzymatic reaction concentration tween silica and MCS. Notably, MCS 1.2 1.2 1.2 product resulting from the reaction of LG + SL with Lacc the adsorption isotherms for 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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www.chemsuschem.org It is very interesting to note that, after treatment with the enzyme, the aggregated particle size (in LG + Lacc dispersions) was reduced, which allowed the LG particles to pass through the 0.45 mm filter and become available for adsorption onto the silica surfaces. The adsorption data for SL + Lacc treatment indicated that SL also adsorbed onto the silica surface upon reaction with Lacc: a Df plateau value of about 14 Hz was measured, which was very similar to that observed in the experiment with the individual SL solution. The LG particle size is expected to play an important role in surface modification by MCS, resulting in different levels of hydrophobicity, depending on the conditions. The application of enzyme dispersions (Lacc dispersion) was also carried out and Df values of about 38 Hz were recorded after 60 min. Overall, there is evidence that the three-component MCS systems (LG, SL, and Lacc) can be adsorbed onto silica to different degrees.
Hydrophobization of CNF films
Figure 4. Quartz crystal microbalance (QCM) Df and DD isotherms upon injection of MCS dispersions on silica surfaces followed by rinsing with either background buffer (a) or electrolyte (0.1 m NaCl solution) and final rinsing with buffer solution (b). Results for similar experiments performed on CHITcoated silica surfaces are displayed in (c). The Df and dissipation, D, signals are indicated by * and ~, respectively.
corded after 60 min). This is an interesting result because quite different behavior was observed when LG was applied after the reaction with the enzyme system. 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
The adsorption of the MCS and respective components onto CNF substrates was investigated by using QCM measurements. The adsorption of MCS onto the CNF films is illustrated in Figure 5 a. After 85 min of adsorption time followed by rinsing (background buffer), Df reached a value of about 40 Hz, which indicated high affinity between the MCS and CNFs. By comparing this result with that obtained for silica, it can be concluded that MCS is adsorbed to a similar extent on both surfaces. Figure 5 b includes analogous experiments, but after application of preadsorbed cationic CHIT onto CNFs, prior to contact with the MCS. In this case, Df reached a value of about 110 Hz, which indicated that the thin layer of CHIT was effective in increasing MCS adsorption, similarly to that found in the case of silica. Figure 5 c and d includes data for adsorption from solutions of SL + Lacc and LG + Lacc on CNFs: Df values of about 14 and 564 Hz were obtained after the respective treatments. The SL + Lacc system adsorbed in very similar amounts on CNFs and silica, whereas the LG + Lacc system was adsorbed to a much larger extent onto CNFs. Upon adsorption of MCS components on silica and CNFs (without and with a CHIT preadsorbed layer; Figures 4 and 5), no frequency plateau was reached. This is taken as evidence of a slow and large buildup of MCS components on the surface, possibly in the form of multilayers. The Df values upon application of MCS on silica and CNFs reached values of 42 and 40 Hz (after 90 min contact), respectively, which reveals a similar extent of adsorption or surface modification by MCS. However, preadsorption of CHIT induced a larger change in frequency upon MCS treatment: 145 and 100 Hz (60 min contact time) for the silica and CNF surfaces, respectively. This indicates much greater adsorption on the cationic substrates. All components present in the MCS (SL, Lacc, SL + Lacc, or LG + Lacc) were built up to different extents on the silica and CNF surfaces. The results indicate that the LG + Lacc system adsorbed to the largest degree (on both silica and CNF films). ChemSusChem 2014, 7, 2868 – 2878
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Figure 5. The QCM Df and DD profiles upon adsorption of MCS on CNF surfaces (a) and on CHIT-coated CNFs (b). The profiles after treatment of the CNF substrates with SL + Lacc (c) and LG + Lacc (d) are also included. The Df and dissipation, D, signals are indicated by * and ~, respectively.
Attachment of enzyme-modified LG moieties onto cellulose The mechanistic pathway in the oxidation of gallic acid proposed by Tulyathan et al.[29] can be considered as the basis to understand the nature of the reaction between Lacc and LG. Lacc enzymes show specificity for phenols, o- and p-diphenols, methoxyphenols, aminophenols, benzenethiols, and hydroxyindoles; the formation of quinones by the oxidation of phenolic compounds by using Lacc enzymes has been widely reported.[30, 31] Such a mechanism involves the oxidation of the OH groups in the LG molecule to form the corresponding phenoxy radicals, which are further oxidized to quinones and its openring acidic product.[32] The formation of dimers through the regeneration of hydroquinones is expected to be difficult due steric hindrance by the long hydrocarbon chain in LG. The reaction produces a molecule carrying carboxylic acids anchored to the alkyl chain through the original ester. The formation of quinones can be identified by the dark coloration developed by the enzymatic products of LG. The QCM-D experiments indicated the attachment of high amounts of LG moieties onto CHIT-coated silica or CNF surfaces. This effect can be attributed to the affinity between the anionic MCS (possible presence of carboxylate groups in the enzyme-modified LG) and the cationic CHIT. Electrostatic effects can increase the buildup of adsorbates on the surface, which can then undergo esterification reactions. 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
We hypothesize that attachment of LG-containing colloidal particles onto the surfaces is produced by means of Fischer’s esterification reactions between the OH groups on the surface of silica or CNFs, and the COOH groups of LG in the MCS, upon enzymatic reaction.[32–34] In this reaction, the sodium acetate buffer used (and respective tartaric acid) may act as an acid catalyst to promote the protonation of the carboxylic groups. Strong bonding between LG and cellulose was reported in our previous work.[25] In addition, MCS solutions were used to hydrophobize paper, and the bonding strength was studied by subjecting the modified paper sheets to strong washing conditions (80 8C during 30 min) and Soxhlet extractions with acetone. No alteration of the hydrophobic character of the paper sheets was taken as an indication of strong LG attachment.
Water contact angle and surface hydrophobicity Water contact angle (WCA) measurements were performed for silica and CNF surfaces treated with the MCS and solutions of its components. This allowed further elucidation of the extent of adsorption and degree of hydrophobization of silica and CNF surfaces. Bare silica wafers and CNF films were treated by using the sequences described in previous sections (see Figures 4 and 5). ChemSusChem 2014, 7, 2868 – 2878
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Figure 7. Relationship between the WCA of the substrate and the shift in QCM frequency, Df (that correlates with the adsorbed mass) after treatment with MCS and LG + Lacc dispersions. Silica and CNF substrates are indicated by ^ and ^, respectively.
Silica and CNFs showed similar behavior, in terms of the resulting WCA: in both cases, the treatment that yielded the highest WCA was that of the solution of LG + Lacc; the same tendency to increase the WCA after adsorbing a thin CHIT layer was observed for MCS and CNF substrates. It is clear that there is a relationship between the amount of enzyme-modified LG adsorbed and the hydrophobicity achieved after the respective treatments: Figure 7 provides a summary of the QCM and WCA data upon enzyme-modified LG adsorption onto silica and CNFs (after treatment with MCS or LG + Lacc). The WCAs were measured from sensors after the QCM adsorption experiments introduced in the previous sections (prior to WCA measurements, the sensors were rinsed with Milli-Q water and dried under nitrogen). It is apparent that as the adsorbed mass increased (as indicated by the Df values) the WCA also increased. A plateau in the WCA is reached at Df 300 Hz. The Supporting Information contains a movie illustrating the increase in WCA after application of the MCS on cellulose. Figure 6. a) WCA measurements for silica surfaces treated with the MCS and its respective components after rinsing. b) Comparison of WCA values after treatment of bare silica and CNFs with the MCS or its respective component dispersions. BUFF = buffer rinsing.
As shown in Figure 6 a, the WCA of bare silica was about 158 and it increased slightly after exposure to buffer solution (by 58). MCS treatment produced an increase in the WCA of silica to about 358. MCS treatment on CHIT-coated silica increased the WCA to 508, which indicated increased adsorption and hydrophobization, in agreement with QCM adsorption isotherms. The increase in hydrophobicity is thus related to the extent of adsorption. Treatment with SL + Lacc and Lacc increased the WCA to about 35 and 558, respectively. The most effective treatment, as far as increased hydrophobicity of the surface was concerned, was that of LG + Lacc application, which yielded WCA values of 85 and 888 for silica and CHIT-coated silica, respectively. Figure 6 b provides a comparison of the final WCA after treatment of silica and CNFs (with and without adsorbed CHIT) with the MCS and with LG + Lacc and SL + Lacc systems. 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Silica surface analysis by AFM The surfaces of the QCM-D silica sensors treated with the MCS and its components were imaged by AFM (Figure 8). Figure 8 a shows the surface topography of the bare silica surface, which is featureless and smooth with a root-mean-square (rms) roughness of less than 1 nm. Figure 8 b corresponds to the silica sensor after exposure to a solution of Lacc: the enzymes adsorbed onto the silica sensor (roughness of the order of a few nm). Particles unevenly distributed on the surface were observed; these were likely to be enzyme aggregates. Figure 8 c shows the result after treatment of the silica surface with the SL + Lacc system (rms surface roughness of only few nanometers with a few unevenly distributed aggregates). These aggregates could be responsible for the relatively large particle sizes found for Lacc and SL + Lacc dispersions investigated in the particle size analyses. Figure 8 d shows the surface topography of the silica sensor after treatment with the LG + Lacc system, which shows evidence of large and uniformly disChemSusChem 2014, 7, 2868 – 2878
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Figure 8. AFM images of the silica surfaces after adsorption of the MCS and respective single- or dual-component systems: untreated bare silica surface (a) and silica surface after adsorption of Lacc (b); SL + Lacc (c), LG + Lacc (d), and MCS (e). An image for MCS adsorbed onto CHIT-coated silica is also included (f). All scan sizes were 10 10 mm2, except for image a), which was 5 5 mm2.
tributed particles (diameters and heights of 200–300 and 20–40 nm, respectively). The presence of such relatively large particles is only observed in enzyme-treated LG, since it was not observed in Lacc or LG systems. The particle diameters observed in Figure 8 d are of the same order of magnitude as those obtained from the DLS experiments (LG + Lacc system). The Supporting Information contains SEM images for the same system on CNF films. The silica surface treated with the MCS is shown in Figure 8 e and indicates the presence of uniformly distributed LG particles with a diameter and height of 100 and 10–20 nm, respectively. Again, the particle size of the adsorbed 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
MCS observed by AFM is of the same order of magnitude as that obtained from DLS analyses. Notice the effect of SL in the MCS system, whereby an enhancement of the enzyme role in reducing the size of the LG particles is observed (compare it with results from the LG + Lacc systems). The effect of a preadsorbed CHIT layer on silica was also accessed by AFM imaging (Figure 8 f). The images clearly indicated a significant increase in the number density of adsorbed LG particles from MCS; this effect was also observed in the QCM-D analyses discussed previously.
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A strong relationship between the WCA of the treated silica surfaces and their topography was observed. The treatment that provided the highest WCA was that after application of LG + Lacc on silica and CNFs, and this treatment was also the one that produced the highest roughness of the silica surface. The MCS system applied to silica (bare or carrying CHIT) also produced an important increase in WCA. AFM results confirmed the adsorption of LG particles onto silica. However, the most probable scenario for the hydrophobization of the substrates comprises both a physicochemical effect (adsorption) and esterification of the hydroxylated surface.
Sweden). CNF films were fixed on silica sensors that were first cleaned by immersion for 20 s in a 10 % solution of NaOH followed by rinsing with Millipore water and drying under nitrogen. The sensors were pretreated with polyethyleneimine (PEI) and dried, followed by spin coating with the dispersion of CNF (3000 rpm, 1 min). The CNF-coated sensors were rinsed with Millipore water, and dried in an oven at 80 8C for 15 min and stored until use.
Conclusions
The enzymatic treatments involved first the reaction of the enzyme with the hydrophobizing agent (LG). The selection of the preparation conditions was based on our previous reports.[23, 25]
We presented a sustainable biocatalytic method to obtain a multicomponent colloidal system (MCS) for the hydrophobization of silica and cellulose nanofibrils (CNFs). Adsorption of the MCS components [laccase (Lacc), sulfonated lignin (SL), and lauryl gallate (LG)] occurred on silica and CNF surfaces and such an effect was boosted by pretreatment of the substrates with chitosan (CHIT). Treatment with MCS increased the water contact angle (WCA) of CNFs and silica from 208 to about 908. A direct relationship between the amount of LG adsorbed onto surfaces and WCA was observed, and also between the roughness of the treated silica and CNF surfaces. Light scattering measurements revealed a reduction in the particle size from several microns down to 300 nm upon enzyme treatment in the presence of LG. The Lacc treatment in the presence of SL reduced the LG particle size even more to 80 nm through the dispersion effect of SL. Light transmission and backscattering data confirmed the effects of Lacc on LG: a reduction in the particle size and increased dispersion stability.
Experimental Section General The enzyme used herein was a laccase (Lacc) from Trametes villosa with an activity of 588 U mL1 and supplied by Novozymes. Dodecyl 3,4,5-trihydroxybenzoate (LG) and chitosan (CHIT, 50–190 kDa molecular weight, deacetylation degree of 75–85 %) were purchased from Sigma–Aldrich. Soluble sulfonated lignin (SL) with 5.9 kDa MW and 5 % total sulfur content was obtained from Borregaard (Sarpsborg, Norway) and was used as received. CNFs were obtained after mechanical deconstruction of bleached hardwood birch fibers by using a Masuko grinder (five passes) followed by a microfluidizer (20 passes). The individual CNFs were dispersed in water by using mechanical stirring and tip ultrasonication (10 min, 25 % amplitude). The resulting CNF suspension was centrifuged at 10 400 rpm for 45 min and nanofibrils were then collected from the supernatant by pipetting.[35] An AFM image of the CNF can be found in the Supporting Information.
Multicomponent colloidal system (MCS) and surface treatment The MCS refers to the product of the enzymatic reaction between T. villosa Lacc with the phenolic, low-surface-energy species (LG) in the presence of the SL.
The treatments were performed in 250 mL beakers containing LG (1.2 g L1), 1.2 U mL1 Lacc and the SL (1.2 g L1) dispersed in 0.1 m sodium acetate buffer (pH 4). LG was hydrophobic and insoluble in water; therefore, it was applied as a colloidal suspension after ultrasonication in water (Hielscher Ultrasonic Processor UP100 H, 30 min, 100 % amplitude). This reduced the LG effective aggregate size and ensured a homogeneous dispersion. LG and SL were kept under agitation for 10 min at 50 8C. Subsequently, the Lacc enzyme was added and the aqueous dispersion was stirred for 4 h of reaction. The reaction was stopped by quenching the system with cold water. Notably, after treatment with the enzyme, a colloidal stable dispersion of LG was produced. Several treatments were performed to elucidate the role of each of the compounds present in the MCS (Table 1). For QCM experiments, the MCS dispersion as well as single and dual components of the MCS were filtered by using 0.45 mm pore size syringe filters to eliminate any possible contribution from residual, large LG particles. In some experiments, aqueous dispersions of CHIT (0.5 g L1, sodium acetate buffer) were used to pretreat the surfaces.
QCM with dissipation monitoring (QCM-D) In situ QCM-D experiments were performed by using a Q-Sense E4 instrument (Vstra Frçlunda, Sweden) operated in continuous mode. The molecular interaction between MCS (and respective single or dual systems) with the substrate might produce a shift in the resonance frequency, (Df), which could be monitored and used to quantify mass gains on the surface (for example, through adsorption and coupling). A linear relationship between Df and the change in mass (including the contribution of coupled water) due to adsorption on the surface of the sensor can be determined through the Sauerbrey model, which assumes that adsorption is homogeneous and highly coupled [Eq. (1)]:[36] Dm ¼ CDf =n
ð1Þ
Substrate preparation
in which Dm, Df, C, and n are the variation of mass [ng cm2], the resonance frequency [Hz], a constant for the crystal [ng Hz1 cm2], and the overtone number (n = 1 for the fundamental frequency), respectively. For the present setup, C equals 17.7 ng cm2 s1 at f = 5 MHz. Herein, we used the third overtone (n = 3) to interpret the QCM data.
Substrates for adsorption tests consisted of bare silica wafers and QCM-D silica sensors obtained from Q-Sense (Vstra Frçlunda,
In addition to resonance frequencies, QCM-D measured the change in energy dissipation of the system, DD, which was moni-
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tored to determine frictional (viscous) losses on the surface. DD is related to the viscoelastic properties of the adsorbed layer(s) deposited on the sensor and can give an indication of its stiffness. For example, the presence of a soft layer on the sensor enhances damping of the vibration and results in a higher dissipation. The energy dissipation, D, is related to the frequency, Df, and decay time, t, through Equation (2): D ¼ 1=pDf t
ð2Þ
in which t values are obtained by periodically disconnecting the oscillating crystals from the main circuit through a computer-controlled relay. Time-resolved adsorption was followed on bare silica sensors and on silica sensors covered with CNFs. The QCM-D measurements were performed under a continuous flow rate of 100 mL min1 at 25 8C.
AFM A Nanoscope IIIa multimode scanning probe microscope (Digital Instruments, Inc., Santa Barbara, CA) was used to characterize the surface topography of the QCM-D silica sensors and the CNFs before and after surface treatment. The images were scanned in tapping mode by using a J scanner and silicon cantilevers (NSC15/ AIBS from Micromasch, Tallinn, Estonia). The radius of the curvature of the AFM tip, according the manufacturer, was less than 10 nm, and the typical resonance frequency of the cantilever was 325 kHz. At least two different areas were analyzed on each sample; scan sizes included 10 10, 5 5, and 1 1 mm2. AFM images were flattened by following first-order conversion. Image analysis was performed by using Nanoscope software (ver. V6.13 R1, Digital Instruments, Inc.) from which the rms roughness and Z sections in line profiles were determined.
Particle sizing The hydrodynamic diameter and size distributions of any colloidal particles present in the MCS, as well as control treatments, were determined by using a Malvern Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) light scattering device.
Water contact angle (WCA) The WCA was measured by means of a CAM-200 contact angle goniometer (KSV Instruments Ltd., Helsinki, Finland). Prior to measurements, silica and CNF-coated surfaces were rinsed with Milli-Q water and dried with nitrogen. A 4 mL water drop was gently deposited on the substrate from above and the profile of the drop placed on the silica and CNF surfaces was captured digitally by means of a high-resolution camera. The contact angle was determined upon contact, after stabilization of the water drop (time required to avoid initial transitory fluctuations, 2 s). The calculation of the contact angles was based on a numerical solution of the Young–Laplace equation by following a computational protocol built in the instrument.
ments, were placed in 80 mm long cylindrical glass cells. The detection head consisted of a pulsed near-infrared light source (l = 850 nm) and two synchronous backscattered (BS) and transmitted (Tr) flux detectors. The optical sensors collected the transmission and BS signals through vertical scanning of the sample with a step of 40 mm at given time intervals. At the beginning of the measurements, scans were performed every minute over 30 min. To evaluate the long-term stability, the time interval between data points was increased. The experimental results were represented as a set of curves that indicated the percentage of transmitted light as a function of the sample tube height. The optical analyzer in Turbiscan measurements detected the light flux backscattered at 1358 by a dispersed and low-transparency medium in a cylindrical cell. The backscattered flux (BS) is related to l* [as a first approximation, BS is inversely proportional to p (l*)] through Equations (3) and (4): p BS ¼ 1= ðl* Þ
ð3Þ
l* ¼ 2d=3fð1gÞQs
ð4Þ
in which l* (photon transport mean free path) represents the mean distance travelled by photons into the dispersion medium, f is the volume fraction of the particles, and d is the mean diameter. Qs and g are parameters given by the Mie theory. Transmission measurements were performed by means of the Turbiscan by sending a light beam through the cell and detecting photons that cross the dispersion without being diffused. The Lambert–Beer law gives an analytical expression of the transmitted flux (Tr) measured as a function of l [Eqs. (5) and (6)]: Tr ¼ Tr0 eð2ri =lÞ
ð5Þ
l ¼ 2d=3fQs
ð6Þ
in which l is the photon mean free path, ri is the internal radius of the measurement cell, and Tr0 denotes the transmittance of the continuous phase. The stability of MCS and single/dual systems were evaluated by analyzing the variation of the backscattered (BS) and transmitted (Tr) flux; both parameters are dependent on the particle volume fraction (f) and the particle mean diameter (d). Real-time Turbiscan online measurements were carried out by conducting the enzymatic reaction in a reaction vessel at constant temperature. Samples of the dispersions were flown from the reaction vessel to the measuring cell by means of the peristaltic pump. Data (Tr and BS) were collected at time intervals of 10 s.
Supporting Information It contains a movie illustrating the hydrophobizing effect of MCS after application on cellulose and also includes SEM images of the LG-containing particles deposited on CNF films as well as AFM and SEM images of CNF fibrils in films.
Acknowledgements Turbiscan and Turbiscan online tests The stability of the MCS and control treatments was evaluated by using Turbiscan MA 2000 (for space-resolved measurements) and Turbiscan online (Formulaction, France; for time-resolved measurements) instruments. The aqueous products, after the enzyme treat 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
We are grateful to the BIOSURFACEL-CTQ2012-34109 and BIOFIBRECELL projects (CTQ2010-20238-CO3-01) within the framework of the Spanish MICINN and the Academy of Finland’s Centres of Excellence Programme. Special thanks are also due to the conChemSusChem 2014, 7, 2868 – 2878
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Received: May 18, 2014 Published online on August 27, 2014
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