Asymmetric cellulose nanocrystals: thiolation of reducing end groups via NHS EDC coupling

Transcription

1 Cellulose (2014) 21: DOI /s ORIGINAL PAPER Asymmetric cellulose nanocrystals: thiolation of reducing end groups via NHS EDC coupling Lokanathan R. Arcot Meri Lundahl Orlando J. Rojas Janne Laine Received: 8 July 2014 / Accepted: 30 August 2014 / Published online: 10 September 2014 Ó Springer Science+Business Media Dordrecht 2014 Abstract Cellulose nanocrystals (CNC) were functionalized in aqueous media at the reducing, aldehyde ends of cellulose. CNC oxidation to produce carboxyl groups was followed by carbodiimide-mediated reaction to install thiol groups. The selectivity and extent of thiolation at the reducing ends was qualitatively confirmed by imaging (transmission electron microscopy) silver nanoparticles that tagged the CNC termini and by X-ray photoelectron spectroscopy, respectively. The adsorption of thiolated CNC onto gold surfaces as well as the viscoelastic property of the formed adlayer was investigated by using quartz crystal microgravimetry. The thiolated CNC chemisorbed on the surfaces were further analyzed for surface density and distribution by using atomic force microscopy. Overall we introduce a facile, mild Lokanathan R. Arcot and Meri Lundahl have contributed equally to this work. L. R. Arcot (&) M. Lundahl O. J. Rojas J. Laine Department of Forest Products Technology, School of Science and Technology, Aalto University School of Chemical Technology, Vuorimiehentie 1, P.O. Box 16100, Espoo, Finland lokanathan.arcot@aalto.fi O. J. Rojas (&) Departments of Forest Biomaterials and Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, USA ojrojas@ncsu.edu asymmetric thiolation procedure as an efficient alternative to conventional reductive amination. Keywords Cellulose nanocrystals Reducing end Thiolation Chemisorption Introduction Plant biomass is the most abundant renewable resource available to humans (Klemm et al. 2005); it consists mainly of cellulose and heteropolysaccharides (Habibi et al. 2010), which are candidates to drastically reduce mankind s dependence on nonrenewable and ecotoxic materials (Klemm et al. 2005). The potential applicability of cellulosic materials in fabricating high-tech devices is evidenced from the flurry of reports on piezoelectric materials (Lee et al. 2009), haptic sensors (Yun et al. 2010), wireless communication devices (Kim et al. 2008), damage detection sensors (Wandowski et al. 2011), electro-active materials (Kim et al. 2010), self-cleaning aerogels or gas permeable dirtresistant coatings (Jin et al. 2011). Long range order in hybrid (organic inorganic) cellulosic materials can enable control over the nano-architecture and facilitate improved electrical, mechanical, thermal, optical and magnetic properties (Habibi et al. 2010; Klemm et al. 2005; Moon et al. 2011). Among the constituents of plant biomass, CNC carry versatile chemical groups such as hydroxyls and aldehydes, which can be

2 4210 Cellulose (2014) 21: modified using numerous reactions to enable different nanotechnologies (Habibi et al. 2010; Moon et al. 2011; Lin et al. 2012; Klemm et al. 1998, 2005). Anisotropy in the location of the reducing groups in CNC can allow precise manipulation of the way cellulose crystals associate with each other and with other components in, for example, composite and hybrid materials. Understanding the relationship between the nano-architecture of the CNC and their properties will ensure control over their self-and directed assembly. In turn, related discovery and design of novel functions can further expand the applicability of cellulosic nanomaterials (Klemm et al. 2005). Several studies have successfully demonstrated various chemical routes to isotropically modify CNC in order to make them suitable for a variety of materials such as ferrocene-based electron conductors (Eyley et al. 2012), fluorescent tagged bioimaging agents (Dong and Roman 2007), surfactant-modified drug delivery carriers (Jackson et al. 2011), stimuli-responsive including temperature sensitive (Zoppe et al. 2010), magnetically responsive (Nypelö et al. 2014) and imidazolium-functionalized ion exchangers (Eyley and Thielemans 2011). In contrast to non-sitespecific modification of CNC, which has been extensively investigated, fundamental physicochemical studies related to topochemically modified CNC have hardly been reported. Reducing end-modified CNCs could allow precise control over their self-assembly behavior on surfaces or in composite materials. Establishing efficient chemical routes to introduce robust functional groups is a prerequisite to broadening the applicability of CNC in advanced high-end technologies. Here we report for the first time a simple chemical route to introduce chemically versatile thiol groups at the reducing ends of CNC. Thiol groups are chemically very robust due to the fact that they take part in a number of reactions including Click coupling, chemisorption to noble metal surface and disulfide linkage formation. Most of previously reported CNC end functionalization has been used as a tool to elucidate mechanistic and structural aspects related to the parallel configuration of cellulose I (Hieta et al. 1984), parallel-up packing in cellulose I a and I b (Koyama et al. 1997) and the mode of action of cellulolytic enzymes (Velleste et al. 2010). However, unlike most reported efforts, our proposed route based on versatile thiol chemistries allows versatile control over CNC interactions (Hermanson 2008; Karaaslan et al. 2013), manipulation of self-assembly processes and is expected to facilitate mechanistic studies taking advantage of reducing end modification (Hieta et al. 1984; Koyama et al. 1997; Velleste et al. 2010). The proposed method involves reaction of an active ester intermediate on the carboxylic ends of CNC followed by nucleophilic reaction with amine containing molecules carrying thiol termini (Fig. 1). Confirmation of the topochemical nature of thiolation to produce asymmetric nanoparticles was assessed by transmission electron microscopy (TEM) and the extent of reaction was estimated by using X-ray photoelectron spectroscopy (XPS). The topochemically thiolated CNC were examined with regards to behaviors similar to those displayed by alkane thiol chains that self-assemble as adsorbed monolayers on the surface of noble metals (Love et al. 2005). Thus, the chemisorption of thiolated CNC on Au was Fig. 1 Schematic illustration of the procedure used to thiolate the reducing end of CNC. Left conventional reductive amination procedure included here as a reference. Right proposed chemical route involving an initial oxidation step followed by thiolation mediated by NHS EDC chemistries

3 Cellulose (2014) 21: investigated by using quartz crystal microgravimetry with dissipation monitoring (QCM-D), which also shed light on the viscoelasticity of the respective adlayers. The proposed method aims to further previous reducing end thiolation procedure (Arcot et al. 2013). Materials and methods Chemicals 6-amino-1-hexanethiol hydrochloride (NH 2 R SH), N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), polyethylene imine (PEI), sodium chlorite (NaClO 2 ) and potassium chloride (KCl) were purchased from Sigma-Aldrich Finland Oy. Hardened ash-less filter paper was acquired from Whatman GmbH, Dassel, Germany. Spectra/por dialysis membrane (MWCO ) was purchased from Spectrum Laboratories Inc., Rancho Dominguez, California. Milli-Q water (MQ, resistivity 18.2 MX) dispensed by Millipore Synergy UV system was used in the preparation of all aqueous solutions. Reducing end thiolation of CNCs CNC produced by sulfuric acid hydrolysis of ash-less Whatman filter paper was used in the thiolation procedure. The powdered filter paper (10 g) was added to sulfuric acid (175 ml, 64 % w/w) and the hydrolysis maintained at 45 C for 45 min followed by quenching with MQ water (1,800 ml). The resulting dispersion was centrifuged and the washed sediment was dialyzed against MQ water, followed by 24 h incubation with 1 g of mixed bed resin. The resulting dispersion was filtered to remove the resin particles, hence leaving a filtrate of CNC. The aldehyde groups on the reducing end of CNC were oxidized to carboxyl groups following the procedure reported earlier (Hieta et al. 1984), using 10 mg ml -1 CNC suspension to which appropriate amount of NaClO 2 was added for a final NaClO 2 concentration of 250 mm. Once the NaClO 2 dissolved completely, the ph of the suspension was adjusted to 3.5 using acetic acid, followed by stirring for 20 h at room temperature. The reaction mixture was subsequently dialyzed against MQ water to remove excess reactants and side products. The resulting CNC carrying carboxyl functionalized ends (CNC COOH) were used for further thiolation, as described below. A 2 mg ml -1 suspension of CNC COOH was degassed by bubbling nitrogen for 30 min and the ph was adjusted to 7.0, followed by addition of NHS (5 lm). Following, EDC (50 lm) was added to the reaction mixture and the ph adjusted to 6.5. Appropriate amounts of KCl were added such that the final suspension was 1 M in KCl. Once the ionic strength of the reaction mixture was increased, a solution of NH 2 R SH (ph 9.2) was added so that the resulting reaction mixture was 1 lm in NH 2 R SH. Following the addition of NH 2 R SH, the ph of the reaction mixture was adjusted to 9.2 and incubated at room temperature for 2 h. It should be noted that the addition of KCl causes aggregation of CNC COOH, hence the procedure included sonication for the first 30 min of the 2 h incubation time. After the 2 h incubation, the resulting reaction mixture was dialyzed against degassed MQ water. The resulting CNC carrying end-thiols (CNC SH) were stored at 4 C until use. Chemisorption studies Chemisorption of CNC SH on gold surfaces was monitored by using a QCM-D (E4 instrument, Q-Sense, Sweden). Gold coated quartz crystals were cleaned by UV ozone treatment for 30 min followed by sonication in acetone for 30 min. Experiments were performed with 0.1 mg ml -1 CNC COOH or CNC SH dispersions. All measurements were performed by flowing CNC dispersions on gold (100 ll min -1 )ata constant temperature of 23 C. Firstly, MQ water was passed through the chambers to get a stable base line for 6 min followed by flowing CNC COOH or CNC SH dispersion for 30 min. Later the chambers were rinsed with MQ water. Once the QCM measurement was finished, the crystals were dried using a jet of nitrogen, followed by topographical analysis using AFM. All QCM-D sensograms presented correspond to the normalized frequency and dissipation changes measured from the 7th overtone signal. AFM imaging CNC COOH and CNC SH chemisorbed on QCM gold crystals were imaged using an AFM (Nanoscope IIIa Multimode scanning probe microscope from

4 4212 Cellulose (2014) 21: Digital Instruments Inc., Santa Barbara, CA). All images (scan size lm 2 ) were obtained by using tapping mode in air with silicon cantilevers. The recorded images were flattened and the height scale was adjusted using NanoScope analysis 1.2 software. Surface chemical analyses XPS high resolution spectra were recorded using a Kratos Axis Ultra DLD instrument (Kratos Ltd, Telford, UK) equipped with a monochromated aluminum anode (Al K a 1,486 ev) operating at 100 W power (12.5 kv and 8 ma) with 20 ev pass energy. A hybrid lens mode was employed during analysis (electrostatic and magnetic). The photoelectron take-off angle with respect to the surface s normal in all measurements was 0. The measured binding energies were charge corrected with reference to ev, corresponding to the C C/C H species. Sample preparation involved drop casting a suspension of CNC, CNC COOH or CNC SH on a cleaned silicon wafer followed by evaporation. Drop casting was done multiple times so as to allow the formation of a thick layer of the CNC (thicker than the analysis depth of XPS technique *10 nm). In the high resolution spectra of S, the doublet centered at 169 ev binding energy was assigned to S 2p of the sulfate half ester groups (Littlejohn and Chang 1995) and the peak centered at ev binding energy was assigned to S 2p of thiol groups (Castner et al. 1996). The concentration of sulfate half ester groups in CNC SH is known (Li and Ragauskas 2011), hence it is possible to estimate the concentration of thiol groups based on the area ratio of XPS peaks in S 2p spectra corresponding to sulfate half ester and thiol groups. In the N high resolution spectrum, the peak centered in the region ev was assigned to amide N (400.0 ev binding energy; Siow et al. 2006), while the peak centered at 402 ev binding energy was assigned to protonated amine N, carrying a positive charge (Strother et al. 2000). CNC morphology and evidence of asymmetrical modification Bright field TEM images were recorded using a FEI Tecnai T12 microscope (Eindhoven, Netherlands) operating at 120 kv. To 2 ml of 1 mg ml -1 CNC (CNC SH or CNC COOH) aqueous dispersions, 200 ll of aqueous silver nitrate solution (10 mm) was added, followed by addition of 2 ml aqueous sodium borohydride (5 mm) and this final dispersion (aged for 24 h) was used in the sample preparation procedure for TEM. Sample preparation was carried out by pipetting 10 ll suspension containing silver nanoparticles and CNC (CNC SH or CNC COOH) onto carbon grids, followed by removal of excess liquid using the edge of a Whatman filter paper after 1 min equilibration. The CNC SH particles had higher affinity for the metallic part of the grid, thus just 1 min equilibration was sufficient to obtain a significant number of particles on the TEM grid. On the other hand in case of neat CNC or CNC COOH, the carbon grid was first treated with 1 wt% PEI solution, to introduce cationic charges onto the surface of the carbon grid, followed by placing 10 ll suspension containing CNC or CNC COOH and silver nanoparticles, and final removal of excess liquid using a Whatman filter paper. Once the excess liquid was removed, the carbon grids were allowed to dry at ambient conditions. Results and discussion Asymmetric thiolation of CNC It is well known that thiol groups have very high affinity for noble metal surfaces such as Au and Ag. Thus, silver nanoparticles exposed to CNC SH would bind to just one end if the reducing end groups of the CNC are in the parallel configuration (Arcot et al. 2013). TEM images of silver nanoparticles synthesized in presence of CNC, CNC COOH and CNC SH are presented in Fig. 2. Three images for each sample have been included so as to emphasize that TEM evidence applies generally for the asymmetric functionalization, i.e., it is not a fortuitous occurrence affected by the preparation procedure, etc. Upon comparison it is clear that a significant number of CNC and CNC COOH particles (Fig. 2a f) bind nonselectively the silver nanoparticles and here is hardly any evidence for end-functionalization. In contrast, the TEM images corresponding to CNC SH (Fig. 2g, h) show evidence of silver nanoparticles binding to single end of the nanoparticles. In case of thiolated CNC, the silver nanoparticles are always found at the end of the CNC whereas in case of non-thiolated ones the nanoparticles are found to be nonspecifically

5 Cellulose (2014) 21: Fig. 2 TEM images of silver nanoparticles (darker particles) synthesized in the presence of neat CNC (a, b, c), CNC COOH (d, e, f) and CNC SH (g, h, i). Three images are provided in each case for a more complete assessment of topochemical functionalization. Scale bar in all images 200 nm bound along the length of the CNC. Additionally, it should be noted that in case of non-thiolated CNC there is a significant number of nanocrystals without any silver nanoparticle attached. The thiolated CNC images show nanocrystals that are almost always attached to silver nanoparticles, thus indicating that the thiolation process significantly enhanced the interaction between CNC and the silver nanoparticles. This end specific presence of silver nanoparticles can be taken as a qualitative confirmation of the thiolation protocol catalyzed by NHS EDC which topochemically functionalized the reducing end of the CNC with thiol groups. Covalent versus electrostatic thiolation of CNCs The efficiency of thiolation was quantitatively ascertained using XPS. High resolution S 2p and N 1s spectra are presented in Fig. 3a, b, respectively. By comparing the S 2p spectra of CNC, CNC SH and CNC COOH it becomes clear that all samples have a prominent peak centered at around 169 ev binding energy, which corresponds to sulfate half ester groups present on the cellulose nanoparticle after sulfuric acid hydrolysis. More importantly the CNC SH sample appears to have a low intensity peak in the ev binding energy range, which is absent in the other two samples (CNC and CNC COOH). This peak in the S 2p region can be attributed to S species of lower oxidation state such as thiol or disulfide. The NH 2 R SH is the sole source of lower oxidation state S species that was added during the functionalization reaction. The presence of thiol related species raises an interesting question related to the nature of bonding between NH 2 R SH molecules and the cellulose nanocrystals (CNC). There are two possible routes of interaction: firstly, electrostatic interactions owing to the fact that CNC have anionic sulfate half ester groups while the protonated amine of NH 2 R SH is positively charged; secondly, a covalent amide bond formed by reaction between amine of NH 2 R SH and the carboxyl groups of CNC COOH

6 4214 Cellulose (2014) 21: Fig. 3 High resolution XPS spectra, S 2p (a) and N 1s (b) of various cellulose nanocrystal samples: CNC SH (top), CNC COOH (middle) and CNC (bottom). All spectra were charge corrected. The binding energy (B.E., ev) and intensity (counts per second) are plotted in the x- and y-axes, respectively after NHS EDC treatment. The answer to this question becomes clear upon a closer inspection of the N 1s spectra shown Fig. 3b. In the N 1s spectrum corresponding to CNC SH there are two prominent peaks centered at 402 and 400 ev binding energies, which correspond to N from protonated cationic amine and from amine bonds, respectively. The significantly larger peak area of amide nitrogen relative to protonated amine clearly indicates that majority of NH 2 R SH molecules in CNC SH sample are bound to the nanocrystals covalently at the reducing end through amide bond rather than along the entire length of the nanoparticle, through electrostatic interaction. Based on the ratio of peak areas of sulfate half ester and thiol in S 2p high resolution spectrum, the concentration of thiol in CNC SH was estimated to be 25 lmol g -1, thus the efficiency of reducing end aldehyde conversion is calculated to be approximately 60 % if one assumes the reducing end concentration to be between 35 and 45 lmol g -1. Chemisorption of CNC SH on gold Chemisorption of CNC SH, CNC COOH and CNC on gold was quantitatively studied by using QCM-D and the results are presented in Fig. 4a, b. The reduction in oscillation frequency upon adsorption of the CNCs is directly proportional to the amount of mass adsorbed on the surface of the gold-coated QCM crystal. Upon comparison it becomes very clear that the frequency decreases by a factor of 10 upon adsorption of CNC SH relative to the values observed in the case of CNC COOH and CNC. This enhanced ability of CNC SH to bind onto the gold surface can be attributed to the Au-thiol interaction similar to what is observed in self-assembly of alkane thiols on the same metal (Love et al. 2005). The dissipation plots presented in Fig. 4b also indicate trends that support the evidence provided by the frequency sensograms. The dissipation values plotted against the shift in frequency (or the DD DF plot) gives a profile whose slope is inversely related to the rigidity of the adsorbed layer (Höök et al. 1998). Thus in addition to the extent of adsorption, QCM-D technique also provides information about the viscoelastic properties of the adlayer (Dixon 2008). As such, the slopes in the DD DF profiles for CNC, CNC COOH and CNC SH were found to be -0.03, and -0.1, respectively. The higher slope for CNC SH is similar to that reported previously for CNC SH that was synthesized through a reductive amination route that also indicated chemisorption in an up-right orientation (Arcot et al.

7 Cellulose (2014) 21: EDC mediated and reductive amination methods are carried out in basic ph conditions ([9) that may partially remove sulfate half ester groups from the surface of CNC that were prepared by sulfuric acid hydrolysis (Hasani et al. 2008). Since the colloidal stability of CNC is dependent on the electrostatic stabilization due to the presence of the surface sulfate half ester groups, a milder chemical reaction would result in thiolated CNC with higher dispersibility or less aggregation in aqueous media. Figure 5 includes AFM topography images of thiolated CNC synthesized by the NHS EDC mediated route (Fig. 5a) and by reductive amination (Fig. 5b). Upon closer inspection it is very clear that after NHS EDC reaction the resultant CNC are well dispersed while in case of reductive amination a significantly larger CNC aggregation is apparent (compared the number of individualized or single particles and the aggregated domains of CNC). Importance of KCl addition before NH 2 R SH addition or ph control Fig. 4 QCM-D sensograms for CNC adsorption on gold surfaces at 0.1 mg ml -1 concentration (23 C, flow rate of 100 ll min -1 ). Profiles corresponding to the frequency (a) and dissipation (b) shifts after adsorption are included. Three types of CNC were used: CNC, CNC COOH and CNC SH. The aqueous suspensions of CNC were introduced at 6 min time, followed by MQ rinsing at 36 min, as indicated by the respective arrows in the plots 2013). Thus, there is indirect evidence that the thiolated CNC synthesized through NHS EDC chemistries are chemically similar to those synthesized via reductive amination and possibly chemisorb onto Au in up-right orientation. Compared to reductive amination, the NHS EDC mediated route for CNC functionalization is milder in terms of temperature and reaction time, which is important for the maximum retention of the sulfate half ester groups on the nanoparticles. Both NHS The thiolated CNCs (or CNC SH particles), denote the nanocrystals produced in the presence of 1 M KCl before addition of NH 2 R SH or a ph rise which followed the activation of carboxyl with NHS EDC. EDC activation of carboxyl occurs most efficiently in the ph range below 7 while the stability of active ester intermediate drastically diminishes in basic ph ([8); additionally, the reaction between the active ester intermediate and amine requires that the amine is in deprotonated form, in other words the ph of the medium should be higher than isoelectric ph of the amine (*9 in case of NH 2 R SH). Thus this reaction should be ideally carried out by EDC activation at acidic ph followed by addition of amine and then increased ph to a value higher than the isoelectric ph of the amine. The amine molecules added to a reaction mixture at acidic ph would be protonated and thus interact electrostatically with any anionic groups present (sulfate half ester groups in case of CNC), which might significantly diminish the availability of amine groups for amide bond formation. NHS EDC mediated carboxyl-amine coupling through amide bond formation is a well-known procedure but in our thiolation procedure we added 1 M KCl before the

8 4216 Cellulose (2014) 21: Fig. 5 AFM topography images of QCM gold crystals carrying adsorbed CNC SH: CNC SH synthesized by NHS EDC chemistry (a) and CNC synthesized through reductive amination as reported by Arcot et al. (b). Image size 2 lm 9 2 lm addition of NH 2 R SH. This salt addition before reaction with NH 2 R SH ensures that the electrostatic interactions are screened. In order to demonstrate the importance of the order in which KCl is added, NH 2 R SH addition and ph rises were performed and the chemisorption of the produced CNC was studied by interchanging the order of NH 2 R SH addition and ph raise, without KCl addition during the 2 h incubation. The chemisorption studies performed using QCM-D is presented in Fig. 6. Three different samples are considered: first, ph raise followed by NH 2 R SH addition; second, NH 2 R SH addition followed by ph raise; third, 1 M KCl addition before NH 2 R SH reaction and ph raise. It must be noted that the first and second procedures involved a final 1 M KCl equilibration step (after 2 h reaction time) to remove any electrostatically bound NH 2 R SH molecules. In case of the third procedure, the KCl addition was carried out before the 2 h reaction period. As observed in Fig. 6a, the frequency decrease is approximately 4 Hz for both first and second procedure, which excluded KCl addition before the addition of NH 2 R SH. In contrast, the third procedure, which included the KCl addition, exhibited a frequency decrease of approximately 40 Hz, a clear indication that the increase in ionic strength significantly increased the efficiency of amide bond formation. The shift in dissipation (Fig. 6b) that took place during the adsorption of the CNC following the three procedures followed by thiolation corroborates the observations made from the QCM frequency data: successful thiolation occurred under increased ionic strength conditions. Thus, compared to the reductive amination procedure (Arcot et al. 2013), the proposed NHS EDC mediated thiolation method offers a superior synthesis route in terms of lower reaction time and higher colloidal stability of the functionalized CNC. Conclusions Cellulose nanocrystals were topochemically thiolated at their reducing ends by using a simple procedure involving initial carboxylation of the reducing end aldehyde groups followed by NHS EDC mediated activation and reaction with nucleophilic amine molecules carrying thiol termini. The successful specific thiolation of CNC was confirmed by imaging of tagged silver nanoparticles. XPS was used to quantify the extent of thiolation and to demonstrate that the amine in NH 2 R SH molecules associated with the CNC COOH through amide bond formation while a minor proportion of molecules interacted physically by electrostatic interactions. Adsorption studies indicated that the thiolated CNC chemisorbed on gold in the up-right orientation. In contrast to CNC SH produced by reductive amination, the nanoparticles produced by this new method displayed limited or no aggregation. Additionally, KCl addition before reaction of NH 2 R SH was found to be critical for successful thiolation of CNC. Overall, we introduce a new method based on mild NHS EDC mediated thiolation that can be carried out in aqueous medium and at room temperature for the synthesis of asymmetric CNC.

9 Cellulose (2014) 21: Fig. 6 QCM-D data for CNC adsorption on gold surfaces from aqueous dispersions of 0.1 mg ml -1 concentration (23 C, flow rate 100 ll min -1 ). The shift in frequency (a) and dissipation (b) upon adsorption are included. Three different procedures were used in the thiolation of the CNCs: first, ph raise followed by NH 2 R SH addition; second, NH 2 R SH addition followed by ph raise; third, 1 M KCl addition before NH 2 R SH reaction and ph raise. The respective CNC suspension was introduced at 6 min, followed by MQ rinsing at 36 min, as indicated by the arrows in the plots Acknowledgments The TEM images presented in this work were recorded at the Aalto University Nanomicroscopy Center (Aalto-NMC) premises. This work was partly supported by the Academy of Finland through its Centres of Excellence Programme ( ) and under Project HYBER. References Arcot LR, Nykänen A, Seitsonen J, Johansson LS, Campbell J, Rojas OJ, Ikkala O, Laine J (2013) Cilia-mimetic hairy surfaces based on end-immobilized nanocellulose colloidal rods. Biomacromolecules 14(8): Castner DG, Hinds K, Grainger DW (1996) X-ray photoelectron spectroscopy sulfur 2p study of organic thiol and disulfide binding interactions with gold surfaces. Langmuir 12(21): Dixon MC (2008) Quartz crystal microbalance with dissipation monitoring: enabling real-time characterization of biological materials and their interactions. J Biomol Tech 19(3): Dong S, Roman M (2007) Fluorescently labeled cellulose nanocrystals for bioimaging applications. JACS 129(45): Eyley S, Thielemans W (2011) Imidazolium grafted cellulose nanocrystals for ion exchange applications. Chem Commun 47(14): Eyley S, Shariki S, Dale SEC, Bending S, Marken F, Thielemans W (2012) Ferrocene-decorated nanocrystalline cellulose with charge carrier mobility. Langmuir 28(16): Habibi Y, Lucia LA, Rojas OJ (2010) Cellulose nanocrystals: chemistry, self-assembly, and applications. Chem Rev 110: Hasani M, Cranston ED, Westman G, Gray DG (2008) Cationic surface functionalization of cellulose nanocrystals. Soft Matter 4(11): Hermanson GT (2008) Bioconjugate Techniques, 2nd edn. Academic press, Waltham Hieta K, Kuga S, Usuda M (1984) Electron staining of reducing ends evidences a parallel-chain structure in Valonia cellulose. Biopolymers 23(10): Höök F, Rodahl M, Brzezinski P, Kasemo B (1998) Energy dissipation kinetics for protein and antibody-antigen adsorption under shear oscillation on a quartz crystal microbalance. Langmuir 14(4): Jackson J, Letchford K, Wasserman B, Ye L, Hamad W, Burt H (2011) The use of nanocrystalline cellulose for the binding and controlled release of drugs. Int J Nanomed 6: Jin H, Kettunen M, Laiho A, Pynnolnen H, Paltakari J, Marmur A, Ikkala O, Ras RHA (2011) Superhydrophobic and superoleophobic nanocellulose aerogel membranes as bioinspired cargo carriers on water and oil. Langmuir 27(5): Karaaslan MA, Gao G, Kadla JF (2013) Nanocrystalline cellulose/b-casein conjugated nanoparticles prepared by click chemistry. Cellulose 20(6): Kim J-H, Kang K, Yun S, Yang S, Lee M-H, Kim J-H, Kim J (2008) Cellulose electroactive paper (EAPap): the potential for a novel electronic material. MRS Proc. doi: / PROC-1129-V05-02 Kim J, Lee H, Kim HS (2010) Beam vibration control using cellulose-based electro-active paper sensor. Int J Precis Eng Manuf 11(6): Klemm D, Philipp B, Heinze T, Heinze U, Wagenknecht W (1998) Systematics of cellulose functionalization: comprehensive cellulose chemistry Wiley. KGaA: 1 31 Klemm D, Heublein B, Fink H, Bohn A (2005) Cellulose: fascinating biopolymer and sustainable raw material. Angew Chem Int Ed 44: Koyama M, Helbert W, Imai T, Sugiyama J, Henrissat B (1997) Parallel-up structure evidences the molecular directionality during biosynthesis of bacterial cellulose. PNAS 94(17):

10 4218 Cellulose (2014) 21: Lee SW, Kim JH, Kim J, Kim HS (2009) Characterization and sensor application of cellulose electro-active paper (EA- Pap). Chin Sci Bull 54: Li Y, Ragauskas AJ (2011) Cellulose nano whiskers as reinforcing filler in polyurethanes. Advances in diverse industrial application of nanocomposites. In: Boreddy R (ed) InTech:17 36 Lin N, Huang J, Dufresne A (2012) Preparation, properties and applications of polysaccharide nanocrystals in advanced functional nanomaterials: a review. Nanoscale 4(11): Littlejohn D, Chang S (1995) An XPS study of nitrogen sulfur compounds. J Electron Spectrosc Relat Phenom 71(1):47 50 Love CJ, Estroff LA, Kriebel JK, Nuzzo RG, Whitesides GM (2005) Self-assembled monolayers of thiolates on metals as a form of nanotechnology. Chem Rev 105(4): Moon RJ, Ashlie M, Nairn J, Simonsen J, Youngblood J (2011) Cellulose nanomaterials review: structure, properties and nanocomposites. Chem Soc Rev 40(7): Nypelö T, Rodriguez Abreu C, Rivas J, Dickey MD, Rojas OJ (2014) Magneto-responsive hybrid materials based on cellulose nanocrystals. Cellulose. doi: /s Siow KS, Britcher L, Kumar S, Griesser HJ (2006) Plasma methods for the generation of chemically reactive surfaces for biomolecule immobilization and cell colonization: a review. Plasma Process Polym 3(6 7): Strother T, Hamers RJ, Smith LM (2000) Covalent attachment of oligodeoxyribonucleotides to amine-modified Si (001) surfaces. Nucleic Acids Res 28(18):3535 Velleste R, Teugjas H, Väljamäe P (2010) Reducing end-specific fluorescence labeled celluloses for cellulase mode of action. Cellulose 17(1): Wandowski T, Malinowski P, Ostachowicz WM (2011) Damage detection with concentrated configurations of piezoelectric transducers. Smart Mater Struct 20(2): Yun G, Kim J, Kim J, Kim S (2010) Fabrication and testing of cellulose EAPap actuators for haptic application. Sens Actuators A Phys 164:68 73 Zoppe JO, Habibi Y, Rojas OJ, Venditti RA, Johansson L, E- fimenko K, Österberg M, Laine J (2010) Poly(N-isopropylacrylamide) brushes grafted from cellulose nanocrystals via surface-initiated single-electron transfer living radical polymerization. Biomacromolecules 11(10):