Cellulose nanocrystals (CNCs) are rising star nanomaterials due to their merits, including abundant natural resources, excellent hydrophilicity, high specific surface area, and so forth [1,2]. Moreover, the unique properties such as non-trivial biocompatibility and cell membrane penetration capability enable the biomedical applications of CNCs [3]. It has been witnessed that CNCs were used for devising tissue reparation scaffolds [4], drug delivery systems [5], bio-sensing/imaging systems [[6], [7], [8], [9]], etc. For the CNC-based sensors and probes, signal transduction units are needed to produce detectable sensing/imaging signals. One decade ago, researchers assembled colorimetric moieties such as polypyrrole [10], Schiff-base [11], and Methyl Red [12] with CNCs to construct sensors responsive to humidity, metal ions, and pH, respectively. The sensors exhibited noticeable color changes upon the changes in target concentration/strength. Wang et al. [13] and Bi et al. [14] devised strain and amino acid sensors that outputted electrical signals by assembling CNCs with conductive nanomaterial or Au electrode.

Fluorescent sensing techniques are characterized by higher sensitivity and accuracy, signal-read easiness, and fast detection [15]. Inspired by this, Mohmoud et al. reported the construction of fluorescently labeled CNCs (F-CNCs) by attaching fluorescein isothiocyanate and rhodamine B isothiocyanate for probing the cellular uptake and cytotoxicity of CNCs [6]. Thereafter, a diverse range of F-CNCs was developed by linking/adsorbing/synthesizing fluorophores on the surface of CNCs. The covalent modification usually utilized the reactions between hydroxyl or carboxyl groups of CNCs with the active groups (amino group, thiol group, halogenated groups) of fluorophores [7,[16], [17], [18]]. Therefore, the reactions were selective and dependent upon the chemical structures of both CNCs and fluorophores, which might limit the successful modification. Moreover, additional crosslinking reagents were required for the construction of F-CNCs. The physical adsorption approaches involved the electrostatic/hydrophobic interactions between CNCs and fluorophores, featuring simplicity and cost−/labor-effectiveness. Nevertheless, F-CNCs stabilized by physical interaction were vulnerable to the interferences of complex biological fluidics, leading to structural collapse and diffusions of fluorophores. In-situ synthesis of fluorescent nanomaterials is a promising strategy for constructing F-CNCs. On one hand, the impacts on morphology and mono-dispersity of CNCs were negligible. On the other hand, the bonding between CNCs and fluorescent nanomaterials is stable and insensitive to changes in ionic strength or pH. Aiming to explore a CNC-based fluorescent probe for cell imaging, Chen et al. [19] reported a one-pot synthesis of CdS@ZnS quantum dots on the surface of CNCs. The constructed CNC/CdS@ZnS featured long-term colloidal stability and excellent photobleaching resistance. Similarly, Tam's [20] and Gong's groups [21] synthesized CdS and ZnS quantum dots, respectively, on the surface of CNCs. From the perspective of biomedical application, F-CNCs prepared via the in-situ synthesis approach are mainly limited by the unsatisfying quantum yield (QY) of quantum dots synthesized under low temperatures and the cytotoxicity issue brought by heavy metals in quantum dots.

Carbon dots (CDs) are the most appealing fluorescent nanomaterials presently due to their high QYs, low toxicity, ease of synthesis, etc., making them ideal alternatives to conventional organic dyes or semi-conductor quantum dots [22,23]. Moreover, the emission wavelength of CDs can be facilely modulated by controlling the particle diameter [24], introducing molecular emission centers [25], or doping with heteroatoms [26]. The advanced optical, as well as chemical properties of CDs encouraged researchers to conjugate CNCs with CDs. For instance, Guo et al. constructed F-CNCs for living cell imaging by anchoring CDs onto CNCs via carbodiimide coupling [27]. Imea's group constructed a CNC/APTES/folic acid/CDs system for imaging and photodynamic/photothermal treatments of cancer cells [28]. The assembling of CDs was achieved via electrostatic interaction. Noticeably, the reported CNC-CDs nanohybrids were still constructed by post-treatment strategies, either chemically or physically [29,30]. Exploring an approach that can directly synthesize CDs on the surface of CNCs is anticipated to address the limitations of chemical modification or physical adsorption and significantly prompt the biomedical applications of F-CNCs.

Organic acids were extensively used for extracting CNCs from bioresources [31,32]. Simultaneously, organic acids, citric acid (CA) in particular, were the most classic precursors in synthesizing CDs [33,34]. By harnessing the dual function of CA, a successive hydrolysis‑carbonization approach for in-situ synthesis of CDs on CNCs was proposed in our previous work [35]. By hydrolyzing wood pulp with CA, the amorphous region of cellulose was removed, remaining CNCs decorated with dense CA (CNC-CA) via esterification. Subsequently, the CA moieties were partially carbonized to form CDs. The prepared CNC-CDs nanohybrid was challenged to detect metal ions. However, the single-emissive CNC-CDs nanohybrid was not compelling for multi-purpose applications, such as multiplexed sensing/imaging. In this work, by realizing the urgent need to expand the application scope of F-CNCs, we go one step further to tune the emission wavelength of CDs on the surface of CNCs. As shown in Scheme 1, the CNCs are extracted from bulk cellulosic filter paper, followed by in-situ carbonization of CA to synthesize CDs with varying emission colors. The emission wavelength tuning is achieved by precursor engineering. Ethylenediamine (EDA) and urea are served as N sources to produce blue-emissive CDs (bCDs) and green-emissive CDs (gCDs) on the surface of CNCs, respectively. While thiourea is adopted to synthesize N, S-codoped red-emissive CDs (rCDs). The chemical and morphological properties of F-CNCs with varying emission colors are well-characterized. Moreover, the mechanism of emission wavelength tuning of CDs is elaborated. The biomedical application is exemplified by a multiplexed cytoplasm imaging experiment.