Sulfuric acid hydrolysis is the earliest and most effective method for preparing chiral nematic liquid crystals of CNCs, and it is also one of the most studied methods^[25]. While hydrolyzing cellulose, sulfuric acid also introduces sulfonic acid negative charge groups on its surface, thereby allowing CNC particles to be stably dispersed in water due to electrostatic repulsion from the negative charges on their surfaces^[13]. As the suspension concentration increases, rod-like molecules undergo self-assembly under a combined effect of electrostatic repulsion and intermolecular forces to form chiral nematic phase liquid crystals.

During the preparation of CNCs chiral liquid crystals by sulfuric acid hydrolysis method, the hydrolysis conditions significantly affect the yield and morphology of CNCs. Numerous studies have shown that the optimal sulfuric acid concentration is approximately 64 wt%, with a hydrolysis temperature ranging from 45 to 60 ℃. The hydrolysis time generally varies between 30 and 120 minutes depending on the type of cellulose raw materials^[31]. When the sulfuric acid concentration is lower than 60 wt%, the acid hydrolysis reaction is incomplete, making it impossible to obtain a stable CNCs suspension; while when the concentration exceeds 70 wt%, the crystalline regions tend to swell or even hydrolyze into glucose, thereby reducing the yield of CNCs^[32]. Qi et al.^[33] investigated the relationship between sulfuric acid hydrolysis conditions and the morphology of CNCs (Fig. 4). Experiments showed that using 64 wt% sulfuric acid under different hydrolysis conditions could produce nanocellulose with varying morphologies. When the hydrolysis temperature was 45 ℃ and the time was 45 min, the resulting rod-like CNCs exhibited a type I crystal structure. Fingerprint textures were successfully observed at a CNCs concentration of 3 wt%. However, under hydrolysis conditions of 35 ℃ and 75 min, only cellulose nanospheres (CNS) could be obtained. Furthermore, Dai Linlin et al.^[34] used bleached conifer pulp as the raw material and conducted single-factor experiments to study the influence of reaction conditions during sulfuric acid hydrolysis on the characteristics of CNCs. The results indicated that excessively high hydrolysis temperatures or prolonged hydrolysis times would lead to varying degrees of cellulose carbonization. When the sulfuric acid concentration was 64 wt%, the temperature was 45 ℃, and the hydrolysis time was 30 min, the prepared CNCs had lengths of approximately 200–400 nm and diameters around 5–15 nm. Self-assembly behavior began to occur when the CNCs suspension concentration reached 2.2 wt%, and clear fingerprint textures were observable when the concentration increased further to 2.83 wt%.

The sulfuric acid hydrolysis method is currently the most commonly used approach for industrial CNCs production due to its mature and stable process. However, this method suffers from high production costs, significant equipment corrosion, and difficulties in sulfuric acid recovery. Moreover, the CNCs obtained via sulfuric acid hydrolysis exhibit poor thermal stability. Therefore, from the perspective of current industrial practice, the large-scale preparation of CNC cholesteric liquid crystals using concentrated sulfuric acid hydrolysis remains unsustainable in terms of environmental impact, economic feasibility, or product performance.

The TEMPO oxidation method employs the TEMPO oxidation reagent (2,2,6,6-tetramethylpiperidin-1-yl)oxyl to selectively oxidize the primary hydroxyl groups at the C6 position in the cellulose molecular structure into carboxyl groups under relatively mild conditions, thereby achieving carboxylation of cellulose. This method can also disrupt the hydrogen bond network in the amorphous and paracrystalline regions of cellulose, softening its rigid structure, which makes cellulose fibers easier to process without altering their original crystalline structure. The oxidized cellulose can be readily dispersed into CNCs and CNFs through mechanical treatment. Compared with traditional sulfuric acid hydrolysis, the TEMPO oxidation method offers milder processing conditions, lower equipment requirements, and higher operability. The resulting nanocellulose contains a large number of carboxyl groups, exhibits excellent dispersion properties, and possesses a larger aspect ratio and higher specific surface area; therefore, it has attracted considerable attention from researchers.

TEMPO reagent was first used to oxidize the primary alcohol hydroxyl groups at the C6 position of dextran into carboxylic acid groups[38]. Subsequently, this system has gradually been applied to cellulose oxidation and has become a common preparation method for oxidizing cellulose. Combining this method with mechanical or acid hydrolysis methods can yield nanocellulose with surface carboxylation and strong negative charge. Due to the high stability of carboxylated CNCs suspensions, they can also self-assemble into cholesteric liquid crystals. He et al.[39] used cotton cellulose as raw material, first hydrolyzing it with hydrochloric acid at 60 °C for 7 h, followed by TEMPO-mediated oxidation under pH 10-11 for 10 h, obtaining a well-dispersed CNCs suspension. The study found that when the CNCs suspension concentration reached 4.1 wt%, an anisotropic phase began to form along with obvious optical birefringence (Fig. 5a); as the concentration increased to 9.0 wt%, a fingerprint texture could be clearly observed (Fig. 5b), indicating a cholesteric liquid crystal pitch of approximately 6 μm (Fig. 5c). On this basis, Xu et al.[40] successfully prepared carboxylated CNCs by performing TEMPO oxidation after hydrochloric acid hydrolysis on bleached cotton pulp, obtaining films with left-handed chiral nematic structures through EISA, with POM and cross-sectional SEM results confirming the chiral structure (Fig. 5d-f).

TEMPO oxidation is the most commonly used method for preparing carboxylated CNCs. This method offers mild preparation conditions and simple operation, and the resulting carboxylated CNCs possess advantages such as easy modification and good thermal stability. However, TEMPO reagent is relatively expensive and has certain toxicity. The preparation process requires strict control of the reaction system's pH, and the reaction time is long, which is not conducive to industrial production.

Compared with CNCs prepared by the sulfuric acid method, carboxylated CNCs exhibit higher surface activity and suspension stability, as well as excellent self-assembly capability, making them more widely applicable in the field of chiral nematic liquid crystals. However, due to the limitations of the TEMPO oxidation method, other oxidation systems such as potassium permanganate^[41] and ammonium persulfate^[42] have been gradually developed and applied in the preparation of carboxylated CNC-based chiral nematic liquid crystals.

Zhou et al.^[43] prepared carboxylated CNCs by hydrolyzing pulp under relatively mild conditions (50 ·C) using potassium permanganate and oxalic acid in an H₂SO₄ (1.0 wt%) medium, obtaining rod-like CNCs with average diameters of 10-22 nm and lengths of approximately 150-300 nm. The reaction process and mechanism are shown in Fig. 6a and 6b. Experiments revealed that when the concentration of the carboxylated CNC suspension reached 7.0 wt%, a chiral nematic liquid crystal self-assembly behavior similar to that of CNCs prepared via traditional H₂SO₄ hydrolysis could also be observed (Fig. 6c). This report provides an effective and low-cost method for preparing carboxylated CNCs; however, due to the difficulty in removing Mn²⁺ introduced into the reaction solution, which demands high operational requirements, its practical application is somewhat limited.

Ammonium persulfate (APS) tends to decompose into sulfate radicals with stronger oxidizing properties when heated, thereby increasing the acidity of the system. As a result, APS can disrupt the amorphous regions of cellulose while oxidizing it, enabling the one-step preparation of carboxylated CNCs and significantly shortening the preparation cycle, which offers unique advantages over traditional TEMPO oxidation and other oxidation methods^[44]. This method was first discovered by Leung et al.^[28], followed by Gray et al.^[45], who reported that carboxylated CNC suspensions prepared using this method could form cholesteric liquid crystals at a concentration of 5 wt%. However, this report did not investigate parameters such as critical concentration or pitch variation. On this basis, He et al.^[18] used tunicate cellulose as raw material and synthesized carboxylated CNCs with high surface charge density through APS oxidation and ultrasonication (Fig. 7a). Their study found that phase separation occurred in the CNC suspension at a concentration of 3.5 wt%, and a distinct fingerprint texture became observable when the concentration increased to 4.1 wt% (Fig. 7b), confirming the formation of chiral nematic liquid crystals from carboxylated CNCs. Although APS is inexpensive, its consumption during preparation is extremely high (228.01 g of ammonium persulfate is required for every 10 g of pulp), leading to excessively high preparation costs and limiting the large-scale application and promotion of this method.

In recent years, researchers have found that organic acids (such as p-toluenesulfonic acid, oxalic acid, and maleic acid) can be used to hydrolyze cellulosic materials for the preparation of CNCs^[46-48]. Compared with other methods, the organic acid hydrolysis method involves milder reaction conditions, causes less corrosion to equipment, and allows the hydrolyzed organic acids to be recovered through simple recrystallization at low temperature or room temperature. This effectively addresses the issue of difficulty in recovering acids in traditional acid hydrolysis methods, opening up a green, economical, and sustainable pathway for the preparation of CNCs^[49].

Existing studies have shown that when certain dicarboxylic acids are used, the hydroxyl groups on cellulose molecules can undergo a Fischer-Speier esterification reaction with organic acids to obtain carboxylated CNCs^[50], making possible the self-assembly of chiral nematic liquid crystals from their suspensions. Researchers have reported a simple and efficient method for preparing functionalized cellulose nanocrystals with high yield, in which hardwood dissolving pulp is subjected to one-step esterification and hydrolysis with molten oxalic acid dihydrate at 110°C, yielding carboxylated CNC suspensions exhibiting obvious birefringence under polarizers^[51] (Figure 8a). To gain a clearer understanding of the liquid crystalline behavior of CNCs prepared using the oxalic acid method, Jia et al.^[52] systematically compared the properties of carboxylated CNCs prepared by the oxalic acid method and sulfuric acid method using filter paper as the raw material. The study revealed that unlike the liquid crystalline behavior of sulfuric acid-prepared CNCs, the carboxylated CNCs obtained via the oxalic acid method could not self-assemble into highly ordered structures independently. However, when cationic solutions were externally added for regulation, the hydrogel exhibited significant birefringence (Figure 8b). Nevertheless, the report did not further evaluate its self-assembly behavior, leaving it still unclear whether carboxylated CNCs prepared using organic acid methods possess the ability to self-assemble into chiral nematic liquid crystal phases. In response, our research group previously explored the self-assembly behavior of carboxylated CNCs prepared by organic acid hydrolysis of defatted cotton^[53]. It was found that rod-like carboxylated CNCs obtained via maleic acid hydrolysis of defatted cotton displayed birefringence at a mass concentration of 4.6%, but no fingerprint texture was observed upon further concentration. When catalysts and esterifying agents were introduced during the hydrolysis process, the resulting CNC suspension exhibited an obvious fingerprint texture under polarized light microscopy at a mass concentration of 9%, confirming the formation of a chiral nematic liquid crystalline phase. Furthermore, cross-sectional SEM images of the solidified films confirmed that the CNCs self-assembled into highly ordered layered structures (Figure 8c). This demonstrates that through property modulation, carboxylated CNCs prepared via organic acid hydrolysis can also undergo self-assembly to form chiral nematic liquid crystals. However, the addition of catalysts and esterifying agents during hydrolysis increases production costs and complicates the composition of recoverable organic acid components in subsequent processing.

To address this issue, our research group subsequently used defatted cotton as a raw material to prepare carboxylated cellulose nanocrystals (OA-CNCs) via hydrolysis and esterification with recyclable oxalic acid under conditions without any catalysts or esterifying agents. By optimizing the hydrolysis process and conditions, rod-like nanocrystals with aspect ratios of approximately 1.9–5.1 were obtained, with surface COOH contents of about 0.11–0.17 mmol/g. The results showed that OA-CNC suspensions begin to undergo phase separation at a concentration of about 1.0 wt% and completely transition from isotropic to anisotropic phases at around 3.0 wt% (Fig. 9a, b). The clear fingerprint texture observed under polarized light microscopy is shown in Fig. 9c. The used oxalic acid can still be utilized for the preparation of CNC chiral liquid crystals after five cycles (Fig. 9d).

In summary, it can be seen that through process control during preparation, recyclable organic acid hydrolysis can also yield carboxylated CNCs capable of self-assembling into chiral liquid crystals, providing a sustainable route for the large-scale production of CNC-based chiral liquid crystals.

The formation of chiral nematic liquid crystals from CNCs highly depends on the characteristics of the CNC particles, including their particle size, aspect ratio, and surface properties^[56]. By adjusting hydrolysis conditions such as acid concentration, oxidant dosage, reaction time, and reaction temperature, the degree of reaction can be controlled, thereby modifying the properties of CNC particles to achieve regulation of the chiral liquid crystal structure.

Abidi et al.^[57] systematically investigated the influence of CNCs size on their self-assembly behavior. The study found that compared with larger-sized CNCs, smaller-sized CNCs tend to self-assemble into cholesteric nematic phases with larger pitches (Figure 11a). Subsequently, Duan Min^[58] experimentally demonstrated that during the preparation of CNCs cholesteric liquid crystals via sulfuric acid hydrolysis, increasing the hydrolysis time or reaction temperature can reduce the particle size of CNCs, thereby increasing the pitch of the CNCs chiral films. In addition to hydrolysis time and temperature, the structure of cholesteric liquid crystals can also be controlled by adjusting the acid-to-material ratio (i.e., the ratio of acid amount to raw material mass). When the acid-to-material ratio increases, the CNCs size decreases, and the critical concentration for phase separation of the cholesteric nematic liquid crystal increases, consequently reducing the pitch of the CNCs chiral films^[59]. Moreover, the structure of CNCs cholesteric liquid crystals can be regulated through selection of different cellulose raw materials. Zhang et al.^[60] employed sulfuric acid hydrolysis to isolate CCNCs, ACNCs, and TCNCs from cotton pulp, algae, and tunicate cellulose respectively. Experimental results showed that the obtained CNCs exhibited aspect ratios of 15±10, 18±12, and 75±60 respectively, while the critical concentrations for cholesteric liquid crystal phase separation decreased successively, and the pitches of the chiral films increased accordingly (Figure 11b).

Based on the excellent mechanical properties and unique optical characteristics of CNCs, their films can be designed as security marks, labels, and optical components that sense external environmental changes through structural color variation. Moreover, CNCs chiral films exhibit non-fading structural colors and angle-dependent color variation, showing great potential in the field of anti-counterfeiting functional materials^[82]. Many scholars both domestically and internationally have already developed CNCs anti-counterfeiting materials with humidity responsiveness, fluorescence responsiveness, and other properties.

Zhao et al.^[83] designed and synthesized a CNC/PAA iridescent coating by utilizing the electrostatic interaction between CNCs and polyacrylic acid (PAA). This coating not only retains the chiral structure of CNCs and exhibits rapid humidity-responsive properties, but also shows angle-dependent color changes under crossed polarization conditions (Fig. 16a, b); based on these characteristics, a "RH-RA-color" ternary anti-counterfeiting code system was established. By adjusting the relative humidity and rotation angle, the appearance and disappearance of patterns on the film can be controlled, thereby achieving a dual anti-counterfeiting pattern effect.

Constructing CNCs hybrid materials with multiple optical states as anti-counterfeiting labels and information encryption materials can significantly enhance their anti-counterfeiting capabilities to prevent information theft or counterfeiting. Xing et al.^[84] selected a Eu³⁺ complex exhibiting red fluorescence emission and a green fluorescence-emitting TPE derivative, tetrakis[4-(4'-carboxyphenyl)phenyl]ethylene (TPEC), as fluorescent guests to fabricate dual-emissive CPL materials with photonic switching properties composed of CNCs and emissive guests via the EISA process. The material can be customized into films of various shapes, and the resulting films exhibit bright red and cyan fluorescence colors under excitation at 254 nm and 365 nm, respectively (Figure 16c top); when both 254 nm and 365 nm UV lamps are turned on simultaneously, the pattern displays a light pink fluorescence (Figure 16c top). Additionally, when used as ink, a smiley printed via stamping becomes invisible under a 365 nm UV lamp but is clearly observable only under 254 nm UV irradiation (Figure 16c middle); "CNC" written as fluorescent ink remains colorless under natural light but exhibits bright colors under UV irradiation, making it easily identifiable and showing great potential for application in the field of anti-counterfeiting (Figure 16c bottom).

In order to obtain intelligent photonic materials capable of real-time switching of circularly polarized signals, Duan et al. ^[85] initially incorporated fluorescent carbon quantum dots (F-CQDs) into the chiral structure of CNCs to obtain multicolored circularly polarized fluorescence (CF) films. They further constructed a novel dual photonic bandgap structure composed of CNC and CF (CNC/CF) films, which not only enables flexible switching of the intensity, wavelength, and direction of circularly polarized fluorescence (CF), but also excites dual circularly polarized reflections for advanced multi-level encryption (Figure 16d). For example, the encrypted information within the composite film can be recognized under right-handed circular polarizers or fluorescent illumination, whereas the composite QR code pattern cannot be identified under sunlight or left-handed circular polarizers (Figure 16e).

Chen et al.^[86] reported a rewritable photonic chiral paper composed of CNCs and polycations. The designed "sandwich" structure (Fig. 16f): the top layer can only be decoded under right-handed polarized light, while the bottom layer can be decoded by wetting with water. Due to the depolarization effect of the sandwich structure, an invisible QR code pattern inkjet-printed on the bottom film can only be recognized under right-handed circularly polarized light when the bottom film is wet and the top film is dry, thereby achieving multidimensional encryption (Fig. 16g).

The research on using CNCs as a chiral source for preparing templated functional materials can be traced back to 1993, when Revol et al.^[9] found that the chiral structure was preserved in solid films obtained from CNCs through the EISA process, which laid a solid theoretical foundation for using CNCs as templates for preparing chiral materials. In recent years, with the rapid development of CNC-based chiral research, significant progress has been made in the co-assembly of CNCs as chiral templates with polymers or inorganic materials to prepare advanced materials, which show broad application prospects in fields such as sensors, optoelectronic devices, and smart displays.

Shopsowitz et al.^[87] synthesized mesoporous silica films using CNCs as templates. After removal of the CNC templates, the chiral structures and high specific surface areas of the CNCs were accurately replicated in the inorganic solids, demonstrating for the first time that template methods can produce free-standing mesoporous silica films with long-range ordered structures. The chiral helical structure of the mesoporous film imparts birefringent properties, with a reflection peak wavelength that can be tuned across the entire visible spectrum and near-infrared range (Figure 17a). Based on the above research, Shopsowitz^[88] et al. further utilized the chiral mesoporous silica film obtained through the aforementioned method as a template to prepare mesoporous anatase titanium dioxide materials, wherein the chiral structures were again replicated and preserved within the titanium dioxide material (Figure 17b). This chiral mesoporous titanium dioxide film material can be applied in photocatalysis and dye-sensitized solar cells. Sun et al.^[89] prepared photonic crystal composite films (GCP) of different colors by combining cellulose nanocrystals (CNCs) with two different surface charge densities and particle sizes, phenol-glutaraldehyde resin, and graphene oxide (GO) at varying ratios. Flexible photonic film actuators (GP) were obtained after alkaline treatment to remove the CNCs. Due to uneven distribution of GO throughout the film, the upper and lower surfaces of GP exhibited differing swelling behaviors. This characteristic enables GP to simultaneously display structural color changes and solvent-responsive actuation, allowing rapid water-induced color change and bending when exposed to water (Figure 17c). These features indicate promising applications for detecting the presence of water in various solvent mixtures, as well as in optical actuators and solvent sensors.

Leng et al.^[90] proposed a strategy for preparing flexible photonic latex films using CNCs chiral liquid crystals as templates. This strategy fabricated composite films of organosilane-modified acrylate emulsion and CNCs through the EISA method (Fig. 17d), where the structural color of the composite film originates from the chiral structure of the CNCs, and its color can be adjusted by varying the emulsion content. After selective removal of the CNCs via alkali treatment, two-dimensional porous latex films retaining the chiral structure were obtained. These films exhibit reversible humidity-responsive structural color, which can be tuned through water absorption and desorption (Fig. 17e, f), showing significant potential for application in humidity indicators.

The chiral nematic structure formed by CNC self-assembly endows materials with responsiveness to external environments (such as pressure, temperature, humidity, pH, etc.)^[91], enabling them to sense environmental changes and provide detectable visual or color signals. This chiral structure is tunable, allowing adjustment of the material's morphology and an increase in specific surface area. Therefore, utilizing CNCs to fabricate functional materials with environmental responsiveness and adjustable morphology can be applied in fields such as mechanically stretchable materials, indicators, electromagnetic interference (EMI) shielding materials, and pollutant degradation. However, due to their highly crystalline structure and ordered molecular arrangement, CNCs inherently exhibit poor mechanical properties, limiting their practical applications. The mechanical strength of CNC-based films can be enhanced through blending with other reagents.

Kose et al.^[92] developed a stretchable CNC/elastomer composite material with a chiral structure (Fig. 18a), which exhibits reversible stretch-induced chromatic stimulus-responsive properties due to the incorporation of CNCs. When stretched, noticeable color changes can be observed under crossed polarizers; upon removal of the tensile force, the material rapidly recovers its original shape and color (Fig. 18b). This material enables visualization of mechanical stress and can be applied in the development of multifunctional sensors for automatic crack detection in bridges, buildings, and other structures.

Jin et al.^[93] intercalated CNCs between reduced graphene oxide (RGO) sheets as reinforcements and dispersants to form highly ordered nacre-like structured CNC/RGO films through a self-assembly process (Fig. 18c). The highly ordered layered structure improved the mechanical strength and electromagnetic interference (EMI) shielding effectiveness of the film (Fig. 18d). Furthermore, the morphology and aspect ratio of the CNCs influenced the thickness, mechanical properties, electrical conductivity, and EMI shielding effectiveness of the film. Higher aspect ratios and smaller diameters resulted in lower film porosity, denser structures, and more uniform distributions, thereby achieving higher electrical conductivity and shielding effectiveness, demonstrating great application potential as EMI shielding materials in aerospace and flexible electronics fields.

Feng et al.^[94] prepared a CNCs composite film with high strength and excellent water resistance through co-assembly of CNCs with oxidized starch and tannic acid. The three-dimensional cross-linked network formed by hydrogen bonding interactions among CNCs, oxidized starch, and tannic acid endows the composite film with broad solvent resistance and outstanding mechanical properties, showing excellent stability in water, common organic solvents, strong acid (pH=0.5), and strong base (pH=14) environments (Fig. 18f). Additionally, due to the high swelling capacity of cellulose in methanol and its easy permeability into the amorphous regions of starch, the film exhibited a red shift in color before and after immersion in methanol (Fig. 18g), making it an effective test paper for distinguishing methanol from water and ethanol, and enabling its use as a colorimetric sensor for specific detection of methanol in methanol-ethanol mixtures.

Wang et al.^[95] developed a novel method to control the size of Au nanoparticles by utilizing the chiral structure of CNCs. This approach regulates the chiral liquid crystal structure formed by CNC self-assembly by adjusting the concentration of CNCs in solution, thereby controlling the size of Au nanoparticles at the nanoscale (Figure 18h). The synthesized CNC@Au exhibited excellent catalytic performance in the reduction reaction of the 4-nitrophenol model substrate, achieving high catalytic efficiency (conversion rate of 98.0%) within 30 minutes. This study demonstrated that the chiral nematic structure of CNCs has great potential in regulating the size of functional nanomaterials.

CNCs' excellent biocompatibility and degradability make them ideal candidates for constructing biomedical fields such as drug carriers and medical detection[96]. Ganguly et al.[97] constructed a magnetically controlled three-dimensional wound repair scaffold by utilizing the responsiveness of CNCs under magnetic field stimulation. This scaffold, prepared from alginate-silk fibroin and CNCs, exhibited high anisotropy and could control the alignment direction of the scaffold through induced ordering of CNCs under an external magnetic field, thereby inducing three-dimensional ordered orientation growth of cells (Fig. 19a). Physiological responses of cells cultured on the scaffold confirmed enhanced cell activity, and the wound healing capability of the scaffold was also evaluated in a rat model, further proving its bioactivity (Fig. 19b). This research provides a theoretical basis for using CNCs in tissue engineering materials. Liu et al.[98] constructed a ratiometric upconversion fluorescent nanosensor for pH monitoring and imaging by using CNCs as bridges to chemically bond upconversion nanoparticles donors with fluorescein isothiocyanate and rhodamine receptors. The rod-like structure of CNCs improved the stability of the probe, solving issues such as dye aggregation, long distance between donor and acceptor, and poor water dispersibility of the probe. The probe showed good reversibility, selectivity, and biocompatibility, and demonstrated excellent pH monitoring and imaging capabilities in live cells and in vivo, making it applicable for early diagnosis and treatment of certain diseases (Fig. 19c).