Sensitive chemoselectivity of cellulose nanocrystal films

Cellulose nanocrystals (CNCs), self-assembled into a chiral nematic structure film, create an advanced platform for the fabrication of remarkable sensing, photonic and chiral nematic materials. Despite extensive progress in the knowledge of functions of CNCs, their chemoselectivity has rarely been reported. Here, we report a brand-new perspective of CNCs in chemoselectivity, which shows sensitive selectivity even between isomers of monosaccharides and disaccharides by generating discernible crystal patterns. This sensitive selectivity of glucose homologs is attributed to the selective carbohydrate–carbohydrate interactions (CCIs) through generating hydrogen bonds between CNC units and the glucose homologs, endowing a remarkable color variation of the CNC films. Moreover, the CCIs are distinct for different immersion times and concentrations of glucose homologs, verified through Fourier Transform Infrared Spectroscopy (FTIR) spectra. Based on the sensitive CCIs, pristine CNC films are then used as templates to generate chiral mesoporous carbon films with tunable specific surface areas by assembly of CNC suspensions and the glucose homologs. We envision that the sensitive chemoselectivity of CNC films as well as precise structure modulation could provide insights into the recognition of carbohydrates and the preparation of mesoporous carbon in numerous practical applications. A pristine chiral nematic cellulose nanocrystal film with sensitive chemoselectivity to glucose homologues and even isomers of monosaccharides and disaccharide are prepared through self-assembly. For different kinds of glucose homologs, distinct crystal morphologies are formed due to the selective carbohydrate–carbohydrate interactions. Besides, the highly ordered left-helical layered structure of pristine cellulose nanocrystal film could be used as templates for the precise construction of delicate nanostructures.


Introduction
Cellulose nanocrystals (CNCs) are biodegradable and sustainable crystals of polymers that can be extracted from cotton (Atalla et al. 1984;Lu et al. 2010;Saito et al. 2006), tree pulp (Claro et al. 2018;Lima et al. 2004) and bacteria (Arserim-Ucar et al. 2021;Dugan et al. 2013). Through the sulfuric acid hydrolysis process (Xing et al. 2018), colloidal sulfate-functionalized CNC suspensions with the ability to be self-assembled to form chiral nematic phases possess remarkable optical properties and templating potentials. Moreover, due to their chiral nematic phase (Gray et al. 2020;Zhang et al. 2020), high surface area (Chen et al. 2021a, b;Elazzouzi-Hafraoui et al. 2008) and eco-friendly nature Sunasee et al. 2016), CNCs have been widely used in preparing chiral mesoporous nanomaterials for environmentally friendly sensors and energy storage devices (Wang et al. 2016;Lizundia et al. 2017).
Recent years have witnessed an increasing interest in developing cellulosic materials with different responses in multi-response systems by reacting the hydroxyl groups on the surface of CNCs with other functional groups through oxidation, esterification, etherification and graft copolymerization methods (Bardet et al. 2015;Eremeeva et al. 2020;Querejeta-Fernandez et al. 2014;Roy et al. 2009;Tang et al. 2017;Xiong et al. 2020;Zhang et al. 2020;Zhao et al. 2020). However, whether grafting polymers or adding nanoparticles to the CNC platform, the preparation process is generally complicated. Studies about the functions of pristine CNCs without grafting functional groups have aroused broad attention. Qing et al. (2019) reported the chemoselectivity of pristine CNC film to saccharides, along with the color variation through the CCIs. However, there is still scarce study of the chemoselectivity of cellulose in isomers of glucose homologs. It is known that there are slight differences in the chemical structures about the stereochemical orientation of one hydroxyl group between glucose homologs, also various expensive and complicated techniques have been developed for this distinction, such as colorimetric assays ) and smart polymers ). Therefore, it is highly desirable to develop pristine CNC films with sensitive selectivity for isomers of glucose homologs.
In this work, we fabricated pristine CNC films with chiral nematic phase via the self-assembly of rigid rodlike negatively-charged nanoscale particles (Parker et al. 2018;Eremeeva et al. 2020;Revol et al. 1998;Roman et al. 2005). The CNC film establishes sensitive selectivity between not only monosaccharides or disaccharide, but also their isomers, by forming distinct pattern morphologies in the crystalline process.
These distinct pattern morphologies are attributed to the CCIs between the pristine CNC film and glucose homologs through generating hydrogen bonds, as verified through the FTIR spectra. The CCIs are distinct for different immersion times and concentrations of glucose homologs, which further induced a remarkable color variation among the CNC films. Furthermore, the spatial CCIs were introduced to regulate the chiral nematic structure and fabricate mesoporous carbon films with a specific surface areas ranging from 700 to 1350 m 2 /g, after the hard template tetraethoxysilane is pyrolyzed and eliminated. We believe that the sensitive chemoselectivity as well as the tune of chiral mesoporous carbon films of CNC film can contribute to the development of sugar-based sensors (Egawa et al. 2011;Suresha et al. 2008), chiral recognition (Chen et al. 2021a, b;Li et al. 2021) and large-scale chiral structures (Lin et al. 2021;Tan et al. 2021).

Materials
Cotton was from a market (Dalian, China) without any further processing. The analytical grade chemical sulfuric acid (H 2 SO 4 , 98%), D-glucose, D-galactose, D-lactose, and D-sucrose were purchased through standard suppliers. Polystyrene Petri dishes with a diameter of 5 cm were supplied by Guangzhou Jet Bio-Filtration Co., Ltd.

Preparation and purification of CNC suspensions
The CNCs were prepared by acidic hydrolysis at a temperature ranging from 35, 45 to 65 ℃ and the time ranging from 60, 90, 120 to 150 min. The acid hydrolysis process was conducted at a ratio of 1:15 g/ mL (solid cotton to sulfuric acid). The sulfuric acid solution (64 wt%) was heated to a certain temperature in advance. After the temperature was constant, a certain proportion of the solid cotton was added to the solution and stirred vigorously. The hydrolysis reaction was terminated with a large amount of ultrapure water and the solution was allowed to stand for a night. The supernatant was then poured out, which was repeated several times until it was not layered.
After that, the suspension without stratification was centrifuged at 6000 rpm/min for 10 min. The dispersion and centrifugation processes were repeated at least three times. At last, it was dialyzed against water through membranes with a cutoff molecular weight of 12 kDa − 14 kDa (Membrane Filtration Products Inc., Seguin, TX, USA) until constant pH was reached (typically over 4-5 days). The process for cellulose hydrolysis with sulfuric acid is shown in Fig. S1 in Supplementary Material.

The sample sulfur content hydrolyzed by sulfuric acid
The sulfur content of the suspensions was often checked to evaluate the stability and charge content of the samples (Abitbol et al. 2013). After removal of excess acid, the suspension of CNC dispersion (2.0 wt%, 5 g) in presence of NaCl solution (1 mM, 75 g) was titrated with NaOH (50 mM). NaCl gave a certain ionic strength to limit repulsions in mixture due to the charged groups present at the surfaces of CNCs. The sulfur content in solution could be quantified ( Fig. S2 in Supplementary Material). Because of charge repulsion, CNCs were dispersed in water and organized into a chiral nematic lyotropic liquid crystalline phase that could be captured in a solid film when the dispersing medium was slowly evaporated.

Preparation of the mesoporous carbon films
Using the chiral phase of cellulose, the mesoporous silicon template (tetraethoxysilane, 200 μL) was introduced into the cellulose suspension (3 wt%, 5 mL). The same proportion of different types of glucose homologs (28 wt%) was added. The mixed solution was stirred at room temperature for 1 h, and then placed in a petri dish and dried for 24 h to obtain a mixed film.

Carbonization and de-templating of the nanocomposite cellulose film
The mixed film was put into a tube furnace filled with nitrogen with a heating rate of 2 ℃/min to 100 ℃ and kept for 2 h. Then the temperature was raised to 600 ℃ at the same heating rate, and maintained for 6 h. After that, the furnace was slowly cooled to room temperature, and a carbonized carbon/silica composite film was obtained. Finally, the black film was put in a 2 M NaOH solution (200 mL), and stored at 90 ℃ for 4 h to remove SiO 2 .

Carbonization and de-templating of the nanocomposite cellulose film
The TEM sample was prepared by putting dilute dispersion (~ 0.01 wt%) of cellulosic suspension onto carbon-coated grids. 10 μL CNC suspension was dispersed on a glow-discharged carbon-coated copper network (300-mesh copper, Ted Pella Inc) and filter paper was used to remove excess liquid and then characterized on a JEOL JEM-2100 (HR) with an accelerating voltage of 200 kV with a LaB6 filament.

Scanning electron microscopy (SEM) and polarized optical microscopy (POM)
The surface morphology was characterized using a Carl Zeiss electron microscope with ORION Nano-Fab. The sample was fractured after being frozen by liquid nitrogen, and the section was collected. POM was carried out on an Olympus BX53 optical microscope.

Atomic force microscopy (AFM)
An appropriate concentration of CNC dispersion (~ 0.005 wt%) was transferred to the mica plate and dried at room temperature. NanoWizard ULTRA Speed JPK in QITM mode was used to image the samples. To obtain a good characterization, a uniformly dispersed sample at a low concentration was prepared. Figure 1a shows the AFM images

Results and discussion
The hydrolysis time and temperature were first screened for extracting cellulosic suspension from cotton. Here, the hydrolysis time and temperature were changed from 60 to 150 min and 35 to 55 ℃, respectively. During the hydrolysis reaction, the amorphous regions of cellulose were selectively hydrolyzed, leaving the crystalline regions known as CNCs with spindle-shaped nanoparticles (Fig. S3 in Supplementary Material). Also, a protonated sulfate group (-OSO 3 ) was introduced into the surface of the CNC nanoparticles, which was detected by a conductivity titration method. The sulfur content and charge density under different reaction conditions are shown in Fig. S4 in Supplementary Material. With the reaction temperature increased, the sulfur content (wt%) increased first and then decreased due to the excessive hydrolysis of the CNC nanoparticles. Meanwhile, the sulfur content increased with the reaction time at the same temperature (Fig. S4a in Based on the charge repulsion induced by -OSO 3 , a chiral pristine CNC film was obtained via a simple and environmental evaporation-induced selfassembly method. The aspect ratio of obtained CNCs decreased as the hydrolysis time increased as shown in Fig. 1a, and the optimal hydrolysis temperature 45 ℃ was selected to obtain the largest CNC yield (~ 74.1 wt%) at this condition (Fig. 1b). Further optical photos showed that the structural color of pristine CNC films could be controlled from colorless to reddish with hydrolysis time varying from 60 to 150 min (Fig. 1c). Moreover, we found that a distinct fingerprint in macro-scale was generated on each CNC film, as shown in Fig. 1d. Figure 1e shows the SEM images of the micro-scale structures of CNC films, which manifested different pitches (P) ranging from 238 nm, 309 nm, 370 nm to 394 nm, respectively, with the increase of hydrolysis time. To explore the relationship between micro-structures and structural colors, we detected the reflection wavelength (λ) of the films by a spectrophotometer (Fig. 1f). It could be seen that the hydrolysis time resulted in a redshift of the reflection band. In addition, the reflected wavelength was positive to the pitch value P, which was consistent with De Vries equation (Devries et al. 1951): λ max = n avg P. Here, n avg was the average refractive index. We believe that the unique chiral nematic structures will provide a new insight in chemoselectivity.
To explore the chemoselectivity of the pristine CNC film, we examined the crystal morphologies of glucose homologs including monosaccharides, disaccharide and their isomers, whose molecular formulas are shown in Fig. 2a. During the experiment, we dripped the glucose homolog solutions (200 mmol/ mL, 200 μL) on the CNC films drop by drop. Then, the glucose homologs crystallized on the target surface, as shown in the schematic in Fig. 2b. The crystalline morphologies of various glucose homologs were visibly distinct among not only monosaccharides and disaccharide, but also their isomers, though there were only differences in the spatial isomerization of -OH in the molecular structure (Fig. 2a).
In the crystallization process, the glucose precipitated and spread gradually to generate glittering and translucent snowflake-like structures ( Fig. 2c and Fig.   S5 in Supplementary Material). However, the crystallization process of galactose (the isomer of glucose) was obviously different, forming a flower-like structure ( Fig. 2c and Fig. S6 in supplementary Material). Similarly, for lactose and its isomer, the crystal morphologies were also distinct, exhibiting structures of forked branches and patches of feathers, respectively (Fig. 2c). Moreover, we observed similar crystal morphologies of glucose homologs on glass plates, as shown in Fig. 2d. The above discrepancy shows that the pristine CNC films established sensitive chemoselectivity for not only monosaccharides and disaccharides, but also their isomers by forming distinct pattern morphologies in the crystallization process. Moreover, the XRD patterns of pristine CNC film, glucose homologs and the composite films are shown in Fig. S7. The CCIs between glucose homologs and the pristine CNC films may amplify the differences in crystals of glucose homolog.
FTIR spectroscopy measurement was adopted to confirm the existence of the CCIs between glucose homologs and the pristine CNC film. The broad absorption band at 3700-3100 cm −1 is attributable to the -OH (Kaushik et al. 2011), and the peak at 3520 cm −1 in pristine CNC film is due to -OH stretching vibration (Fig. 3a). Note that the stretching vibration of -OH is variational with different glucose homolog additives from 3520 to 3476, 3465, 3455 and 3444 cm −1 , corresponding to sucrose, glucose, galactose and lactose, respectively. Moreover, a redshift of -OH occurs, indicating the formation of hydrogen bonds between the CNC and glucose homolog additives. As the formation of the hydrogen bonds equalizes the electron cloud density, the frequency of the stretching vibration of hydroxyl groups decreased, and then the absorption peak of the proton donor shifted to a low wave number (Ni et al. 2019;SZEMIK et al. 1984, Zhao et al. 2020Bazrafshan 2020). Typically, the shifting to a lower wavelength from 3520 to 3477, 3474 and 3452 cm −1 originates from the strengthening of hydrogen bonds between pristine CNC films and glucose with different concentrations as 800 mM, 400 mM and 200 mM, respectively (Fig. 3b). As shown in Fig. 3c, a red-shift from 3550 to 3479, 3471, 3461 and 3456 cm −1 is observed as expected, corresponding to strengthened hydrogen bonds with the immersion time.
Further, UV reflectance spectra demonstrate that the interactions between glucose homologs and CNC nanoparticles affect the reflection wavelength λ. Figure 3d shows the red-shifts in reflection peaks with λ increased from 375 to 394, 513, 676 and 748 nm along with the increased P by adding glucose, galactose, lactose and sucrose, respectively, which is consistent with the aforementioned equation λ max = n avg P. Meanwhile, the reflection wavelength increases with the increased concentration of glucose and immersion time (Fig. 3e, f), which is due to the increasing P of the composite films. The POM images of the composite films are also collected as shown in Fig. 3g-j, in which the CNC/glucose films display a typical characteristics of the chiral nematic organization with the strong birefringence and fingerprint textures, accompanied by a shift in color from blue to red with different immersion time, which is consistent with the data Fig. 3f.
Note that, the CCIs between carbohydrates and sulfonated polysaccharide have been reported before (Bavireddi et al. 2013;Ji et al. 2019;Sletmoen et al. 2018), which can be detected by colloidal probe microscopy (Lorenz et al. 2012;Witt et al. 2016).

Fig. 3
The quantitative information about the carbohydratecarbohydrate interactions and dynamic fluctuation of interactions over immersion time and with different concentrations of glucose homologs. (a) FTIR spectra of the CNC film with glucose, galactose, lactose and sucrose, respectively; (b) FTIR spectra of the CNC film with different concentrations of glucose, e.g., 200 mM, 400 mM and 800 mM, respectively; (c) FTIR spectra of CNC/glucose with different immersion time; (d) Reflectance spectra of films with glucose, galactose, lactose and sucrose, respectively; (e) Reflectance spectra of films with different concentrations of glucose, e.g., 200 mM, 400 mM and 800 mM, respectively; (f) Reflectance spectra of CNC/glucose with different immersion time; (g-j) The POM images of CNC films with glucose at 1 day, 3 days, 6 days and 10 days, respectively Here, the schematic of CCIs between pristine CNC film and glucose homologs are shown in Fig. 4a. Different crystal morphologies are attributed to the hydrogen bonds with different conformations, formed by the isomerization of hydroxyl groups in isomers of glucose homologs. Moreover, the selective interaction between various glucose homologs and pristine CNC films can be used to regulate the porous structure of carbon (Gan et al. 2021;Gao et al. 2021;Wang et al. 2021). As CCIs between glucose homologs and pristine CNC film are weak, the influence of glucose homologs to the micro structure of CNC film would Nitrogen adsorption isotherms of mesoporous carbon film; (c) Photograph of a mesoporous carbon film with glossy black appearance obtained from pyrolysis and etching of the pristine CNCs/sucrose film and Barret − Joyner − Halenda pore size distribution of carbon film calculated from the adsorption branch of the nitrogen isotherms; (d) Surface areas of mesoporous carbon film with different glucose homologs based on Brunauer − Emmett − Teller (BET) theory be small. When glucose homologs are mixed into CNCs, the pitch value P could be disturbed due to the CCIs as shown in Fig. 4b. After further carbonization and de-templating process, a carbonized material with long range ordered mesopores is obtained assembled from CNC suspension and glucose homologs (CNC/ glucose homologs film), and a schematic is shown in Fig. 4c. Moreover, the pitch value P of the carbonized films is influenced by the types of glucose homologs, which are 327 nm, 384 nm, 346 nm and 365 nm, respectively, in contrast to the P of original CNC films (370 nm), as shown in Fig. 4d. This further verifies the distinction of selective interactions between various glucose homologs and CNCs.
Their applications are determined not only the chiral structure of carbonized films, but also the pore structure features of carbonized materials including specific surface area, pore volume and diameter. The following is an example of the mesopore carbonized material assembled from the CNCs and sucrose (CNCs/sucrose). The carbonized CNC/sucrose material has inherited the chiral structure as well as the functions of cellulose, as evidenced in the transmission electron microscopy (Fig. 5a). According to the nitrogen adsorption and desorption isotherm of carbonized CNC/sucrose material which exists a hysteresis loop (Fig. 5b), the adsorption isotherm could be labeled as Type V (Kazmierczak-Razna et al. 2017), which provides insight into the pore type of the material (Deng et al. 2020). Besides, we apply the Barret − Joyner − Halenda model (Fig. 5c) to calculate the peak pore diameter of CNC/sucrose material (~ 3.3 nm), which is an ideal carrier for catalyst.
Further, the influence of glucose homologs on specific surface area, pore volume and diameter was investigated. For CNC carbon films without glucose homologs, as shown in Fig. 5d and Table S1, the specific surface area, pore volume and diameter are 705 m 2 /g, 1.02 cm 3 /g and 3.7 nm, respectively. However, after monosaccharides or disaccharides are added into cellulose solution, the surface area of CNC carbon film is nearly doubled (Fig. 4d), while the pore volume and pore diameter are both slightly reduced (Table S1). This is attributed to the increased mesoporosity of CNC films with added glucose homologs. Therefore, we can fabricate mesoporous carbon films with tunable specific surface area assembled from CNC suspensions and various glucose homologs.

Conclusions
A pristine chiral nematic CNC film with sensitive chemoselectivity to glucose homologues and even isomers of monosaccharides and disaccharides are prepared through self-assembly methods. For different kinds of glucose homologs, distinct crystal morphologies are formed due to the selective carbohydrate-carbohydrate interactions. The CCIs are generated by forming hydrogen bonds between CNC units and glucose homologs that were verified by FTIR spectroscopy. Also, the CCIs are distinct for different immersion times and concentrations of glucose homologs, as reflected by red-shifts of the band in reflectance spectra and red-shift of hydroxyl group bands in the infrared spectrum. Besides, the highly ordered left-helical layered structure of pristine CNC film can be used as templates for the precise construction of delicate nanostructures by CCIs between glucose homologs and pristine CNC film. Through simple carbonization, a chiral porous carbon film is obtained. We envision that the sensitive chemoselectivity accompanied by the precise structure modulation of pristine CNC films can be applied in designing sugar-based sensors, electrode nanodevices, energy storage devices, etc.