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Sensors and Actuators B: Chemical

journal homepage: www.elsevier.com/locate/snb

Fluorescent cellulose nanocrystals with responsiveness to solvent polarityand ionic strength

Weibing Wua,1, Ruyuan Songa,1, Zhaoyang Xub,⁎, Yi Jinga, Hongqi Daia,⁎, Guigan Fangc

a Jiangsu Co-Innovation Center for Efficient Processing and Utilization of Forest Resources, Nanjing Forestry University, Nanjing 210037, Chinab College of Materials Science and Engineering, Nanjing Forestry University, Nanjing 210037, Chinac Institute of Chemical Industry of Forestry Products, Chinese Academy of Forestry, Nanjing 210042, China

A R T I C L E I N F O

Keywords:Cellulose nanocrystalsFluorescenceIonic strengthSolvent permittivityDouble layer

A B S T R A C T

Fluorescent and stimuli-responsive cellulose nanocrystals (CNC) were prepared via covalent conjugation of a 1,8-naphthalimide dye onto TEMPO-oxided CNC. The structural and morphological integrity of CNC were es-sentially not affected by the functionalizaiton, in spite of their relatively high dye content of 0.1 ± 0.01 mmolg−1. The dye-labeled CNC show much higher fluorescence sensitivity to solvent polarity and ion concentrationcompared to pure dye. The fluorescence emission of dye-labeled CNC is greatly enhanced when reducing solventpermittivity or increasing ionic strength. The magnified fluorescence responsiveness is attributed to the effect ofaggregation-enhanced emission (AEE) based on the sensitive colloidal stability of CNC to medium conditions.Under high ionic strength and low solvent permittivity, the compressed double layers of CNC lead to spacerestriction and steric hindrance of dye groups, which limit the process of nonradiative transition. The ag-gregation of CNC triggered by the change of ion concentration was supported by dynamic light scattering (DLS)particle sizes and Zeta potential values. Owing to the special AEE effect, the fluorescent CNC are promisingsensing nanomaterials for wide applications.

1. Introduction

Nanomaterials with sensing capabilities have become researchhotspot in the last few years. They have been applied in various fieldsincluding bioimaging, drug deliver, sensors and environmental mon-itoring [1–3]. Cellulose nanocrystals (CNC), with a size of 50–300 nm inlength and 3–20 nm in width, are typically produced after acid hydro-lysis of the amorphous regions of native cellulose [4]. Besides theabundance, renewability, high strength, hydrophilicity, large specificsurface area, biodegradability and biocompatiblity, there are reactivegroups on the surface of CNC, such as hydroxyl, carboxyl, aldehyde orsulfate groups, facilitating further modification with chemical species[5–8]. CNC have emerged as promising candidate nanomaterial in theregard of sensors.

Surface modification strategies including oxidation [9], etherifica-tion [10], esterification [11], amidation [12], click chemistry [13] andgraft polymerization [14] have been generally adopted. Early researchwork mostly focused on the modification of hydroxyl groups on CNC.Fluorescein-5′-isothiocyanide (FTIC) and Rhodamine B isothiocyanate(RBITC) were labeled on CNC via a three-step reaction way [15].

Nielsen et al used another three-step approach including esterification,thiol-ene click reaction and amidation to label the hydroxyl groups ofCNC with isothiocyanate and succinimidyl ester fluorophores [16]. Tosimplify the dye-labeling procedure, a two-step method was applied toconjugate a pyrene-based succinimide ester dye and FTIC to aminosi-lane-functionalized CNC [17]. Navarro et al selectively labeled CNCwith a fluorescein derivative and coumarin via two click chemistryreactions: Diels-Alder cycloaddition and thiol-Michael reaction [13].Abitol et al modified cotton-source CNC with 5-(4, 6-dichlorotriazinyl)aminofluorescein (DTAF) in a facile, one-pot reaction under alkalineconditions [18]. Aldehyde groups have also been reported for the im-mobilization of dyes on CNC. Alex Fluor dyes with terminal amino-groups were successfully bound to CNC with dialdehyde structure viareductive amination [19]. Huang et al described one-step syntheticstrategy for fluorescent labeling the aldehyde groups of CNC with 7-hydrazino-4-methylcoumarin (HMC) and 7-amino-4-methyl-coumarin(AMC) [20]. It is noteworthy that all the reported labeling degrees ofdyes on CNC via the modification of hydroxyl and aldehyde groups arerelatively low, generally in the levels of μmol g−1 and even nmol g−1.By comparison, carboxyl groups can be a better choice to load

https://doi.org/10.1016/j.snb.2018.07.085Received 29 March 2018; Received in revised form 14 July 2018; Accepted 16 July 2018

⁎ Corresponding author.

1 These authors contributed equally to this paper.E-mail addresses: hughxzy@163.com (Z. Xu), hgdhq@njfu.edu.cn (H. Dai).

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fluorescent dyes with high contents. Rhodamine spiroamide-labeledCNC via carboxyl attachment was reported to possess dye content valueof 0.2 ± 0.01mmol g-1 [12]. Zhou et al synthesized multifunctionalCNC based on carboxylated CNC and the ratios of fluorescent dye tocarbohydrate were estimated to be 0.84:1 and 0.91:1 [21].

Fluorescent tagging of CNC has been reported for the study of cellviability [22], bioimaging [21,23], pH and temperature sensing[12,14,16], photoresponsive materials [24], and metal-ion detection[25,26], etc. 1, 8-Naphthalimide derivatives (NID), a popular fluor-ophore probe, have been widely used as reporters in colorimetric andfluorescent chemosensors owing to their superior brightness, photo-stability and tunable fluorescence emission [27]. In this work, we re-port a NID-labeled CNC probe that possesses sensitivity to solventpermittivity (solvent polarity) and ionic strength (electrolyte con-centration) in aqueous solution. Carboxylated CNC, obtained via 2, 2, 6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated oxidation [9], wereused as glyconanomaterial platform to fabricate the fluorescent nano-sensors. Their responsive fluorescence behavior and mechanism withrespect to solvent permittivity and ionic strength were investigated.

2. Materials and methods

2.1. Materials

2, 2, 6, 6-tetramethylpiperidine-1-oxyl (TEMPO) (98%) was pur-chased from Sigma-Aldrich. Sodium bromide (NaBr), sodium hypo-chlorite (NaClO), sodium hydroxide (NaOH), hydrochloric acid (HCl),sodium chloride (NaCl), acetic acid, sodium thiosulfate (Na2S2O3),disodium hydrogen phosphate (Na2HPO4), sodium dihydrogen phos-phate (NaH2PO4), 2-methoxyethanol, ethanol, chloroform were ac-quired from Nanjing Chemical Reagent Co., Ltd. Triethylamine, me-thylamine water solution (40%), N,N-dimethylformamide (DMF),methanol, dimethyl sulfoxide (DMSO) and ethylenediamine were pur-chased from Sinopharm Chemical Reagent Co., Ltd. 4-Bromo-1, 8-naphthalic anhydride (98.0%) was obtained from Zhengzhou AlphaChemical Co., Ltd. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC) and N-hydroxysuccinimide (NHS) were acquired from TCI(Shanghai) Co., Ltd. All chemicals were directly used without furtherpurification.

2.2. Preparation of carboxylated CNC via TEMPO-mediated oxidation(TOCNC)

First, carboxylated cellulose nanofibers were prepared from woodpulp through TEMPO-mediated oxidation according to reference [28].Bleached soft wood pulp (10 g) was suspended in 1000ml deionizedwater containing TEMPO (0.16 g), NaBr (1.6 g) and NaClO (110mL),and the pH was maintained at 10.5 by adding NaOH (0.2mol L−1) atroom temperature under mechanical agitation. The reaction wasstopped until no more decrease in pH was observed. The oxidized pulpwas purified through thoroughly washing with deionized water by fil-tration. Then the resultant products were ultrasonicated at consistencyof 1 wt% for 5min at 300W to obtain individualized nanofibers.

The cellulose nanofibers were transformed to CNC following aprotocol described by Salajková et al [29]. In a 500ml three-neckedflask equipped with condenser, solution of 3 g freeze-dried cellulosenanofibers dispersed in 150ml aqueous HCl solution (3mol L−1) wasrefluxed for 3 h. The hydrolysis reaction was subsequently stopped bydilution with deionized water. Then the result products were thor-oughly washed with deionized water by centrifugation (5000 rpm,10min) until a turbid supernatant appeared. Finally, the solution wasultrasonicated for 5min and dialyzed against distilled water untilneutral.

2.3. Synthesis of 4-((2-amnioethyl)amnio)-9-methyl-1,8-naphthalimide(AANI) [30,31]

4-Bromo-1, 8-naphthalene anhydride (14.0 g, 0.0505mol) andethanol (160mL) were added in a three-necked flask and heated to55 °C under stirring. 40% methylamine aqueous solution (4.5 g,0.051mol) was dissolved in 30ml ethanol, and slowly added to thethree-necked flask with a constant pressure dropping funnel. Then thetemperature was raised to 78 °C to make sure that the ethanol was re-fluxed for 4 h. After the reaction was completed, 150ml distilled waterwas added and the mixture was filtered to obtain the yellow solid of 4-bromo-9-methyl-1, 8-naphthalimide (12.9 g). Yield 92%, m. p. 180-182℃. 4-Bromo-9-methyl-1, 8-naphthalimide (2.9 g, 0.01mol), ethy-lenediamine (6.0 g, 0.1mol), triethylamine (1.0 g, 0.01mol), and 2-methoxyethanol (150mL) were added in a three-necked flask and re-fluxed for 4 h at 120 °C. After the solution was cooled to room tem-perature, 250ml deionized water was added. The precipitation wasfiltered and recrystallized in ethanol. The orange product of 4-((2-aminoethyl) amino)-9-methyl-1, 8-naphthalimide (AANI) was dried invacuum at room temperature (1.2 g). Yield 40.4%. 1H NMR (Methanol-D4, 400MHz) δ: 3.07 (t, 2 H), 3.44(s, 3 H), 3.57 (d, 2 H), 6.80 (d, 1 H),7.62 (t, 1 H), 8.29 (d, 1 H), 8.44 (d, 1 H), 8.50 (d, 1 H). ATR-FTIR ν:3468 cm−1 (-NH2); 3333, 3301 cm−1 (-NHR); 2971 cm−1 (−CH3);2864 cm−1 (−CH2-); 1678 cm−1 (C=O).

2.4. Preparation of AANI-labeled TOCNC (TOCNC-AANI)

The TOCNC solution (2mgmL−1, 500mL) was sonicated for 15minand the pH was adjusted to 5.2. EDC (1375mg, 7.173mmol) was addedinto the solution and maintained for 30min. Then NHS (162.5 mg,1.412mmol) was added and the pH was adjusted to 7.2 with PBSbuffer. AANI (20mg, 0.075mmol) in DMF (5mL) was added into abovemixture. The reaction was conducted under stirring for 16 h in the darkat room temperature. After the end of the reaction, the product waswashed by centrifugation (10, 000 rmp, 10min) for several times anddialyzed against distilled water until the dialysate was colorless andtransparent to obtain fluorescent CNC.

2.5. pH Buffer solutions

A series of different pH buffers were prepared for the study of pHdependence of TOCNC-AANI. The pH range of 4–9 was covered usingphosphate buffer solution with the concentration of 50mmol L−1.

2.6. Characterization and detection

Fourier transform infrared (FTIR) Spectra were recorded on a FTIR-650 spectrometer (Tianjin Gangdong Co., Ltd.) with single attenuatedtotal reflectance system. 32 scans were collected for each spectrumfrom the wavenumber of 4000–650 cm−1 at a resolution of 2 cm−1. Thenuclear magnetic resonance (NMR) data was obtained by a AVANCE IIIHD all-digital superconducting NMR spectrometer (400MHz, BrukerCo., Ltd.) using the solvent of methanol-D4.

The carboxyl content of TOCNC was measured by conductometrictitration. Freeze-dried nanocrystals (50mg) were dispersed in 50mlNaCl (1 mmol L−1) solution by sonication. The pH of the solution wasadjusted to 2 with HCl (0.1 mol L-1) to allow carboxyl groups to be intheir protonated form. The solutions were titrated against 0.2ml in-crement of 0.05mol L−1 NaOH. Titrations were duplicated three timesto obtain average values. The carboxyl content (CCOOH) of the samplewas calculated according to the following equation [32]:

=

−C C V Vm

( )COOH

2 1(1)

where C is the concentration of NaOH (0.05 mol L−1), V1 and V2 are theend point volumes of NaOH (L), and m is the weight of the TOCNC.

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UV-vis absorption was detected by UV–vis spectrophotometer (TU-1900, Beijing Purkinje General Instrument Co., Ltd.). The dye content ofTOCNC-AANI was assessed from UV absorbance with the calibrationcurves made from pure AANI standards.

X-ray diffraction (XRD) was conducted with multi-functional hor-izontal X-ray diffraction (Ultima IV, Rigaku Industrial Co., Ltd.). Theoperating conditions for the refractometer were Cu Kα radiation(l= 1.5418 Å), 2θ Bragg angle between 5° and 40°, step size of 0.05°,and a counting time of 300 s. Diffractograms from rotating specimenswere recorded on a position sensitive detector. The crystallinity index(CI) was calculated as follows [33,34]:

=

−I II

CI am200

200

where I200 is the intensity of the 200 lattice plane at 2θ of round22.4°and Iam represents the peak intensity at round 18°, which corre-sponds to the amorphous material in cellulose.

Transmission electron microscopy (TEM) images were acquired onTransmission Electron Microscope (JEM-1400TEM, JEOL Ltd.) with anaccelerating voltage of 80 kV. Aqueous CNC solution (0.005 wt%) wasfirstly sonicated for 3min and then dyed with uranyl acetate. A drop ofwell-dispersed solution was then placed on a carbon coated grid anddried under ambient conditions before analysis. Digital Micrographsoftware was used to measure the physical dimensions of CNC particles.

Atomic force microscopy (AFM) images were obtained by AtomicForce Microscope (Dimesion Edge,Bruker Co.). Samples were preparedby dropping the CNC solutions (0.001 wt%) onto the silicon wafer for afew seconds, followed by dried at 40 °C. The scan rate was 1.0 Hz, andthe scan range was 5 μm.

Thermogravimetric analysis (TGA) was carried out on a TAInstruments (TGA 209 F1, Netzsch). Samples were predried under va-cuum overnight. Data were collected after placing about 10mg ofsample in a clean platinum pan. All samples were subjected to heatingscan from 30 to 800 °C, with a heating rate of 10 °Cmin−1 and a ni-trogen flow rate of 40ml min−1.

The Zeta potentials were measured by NanoPlus zeta/nano particleanalyzer (NanoPlus-2, Micromeritics Instrument Co., Ltd.) at roomtemperature, using 0.7 wt% aqueous solutions of CNC with differentNaCl concentrations. A solid state laser with 664.30 nm wavelength wasused as light source. The final value is an average of five repeatedmeasurements for each sample.

Dynamic light scattering (DLS) for measuring the size of nanocrys-tals (0.35 wt%) was performed on dynamic light scattering nano laserparticle size analyzer(BT-90, Bettersize Instruments Co., Ltd.)at roomtemperature. A 20mW solid state laser light was 635 nm with mea-surement at scattering angle of 90°. Three independent measurementswere obtained for each sample.

Fluorescence spectra were recorded on Fibre-Optical Spectrometer(R4, Shanghai Ideaoptics Co., Ltd.) equipped with fp-405-T01-FL Laser.Fluorescence emissions were measured at excitation wavelength of405 nm with the slit width of 2 nm.

3. Results and discussion

3.1. Conjugation of AANIto carboxylated TOCNC.

It has been reported that acid hydrolysis of TEMPO-oxidized cellu-lose nanofibers achieved a higher degree of carboxylation than TEMPO-oxidation of cellulose nanocrystals [29]. This route was adopted here torealize high carboxyl content and facilitate further surface modifica-tion. Fluorescent AANI was covalently loaded on TOCNC via EDC/NHS-mediated reaction between the carboxyl and amino groups, as shown inScheme 1. The carboxyl content of TOCNC was determined via con-ductometric titration (Figure S2) and calculated to be 1.52mmol g−1

(Eq. (1)). The successful immobilization of AANI on the surface of CNCwas confirmed by FTIR (Fig. 1A). The characteristic absorption bands of

TOCNC at 3330, 2896, 1729 and 1067 cm-1 are associated to OeHstretching, C–H stretching, C]O stretching, and C–O stretching vibra-tion, respectively [35]. The C]O stretch absorption at 1729 cm-1 ofTOCNC-AANI decreases as a result of coupling reaction between theamino groups of AANI and carboxyl groups of TOCNC. The appearanceof absorption band at 1678 cm-1, which is ascribed to the stretchingvibration of amide, also proves the covalent conjugation of dye ontoTOCNC.

Fig. 1B compares the AFM images of the TOCNC before and afterdye conjugation. The size of TOCNC was measured to be in the range of6–15 nm in width and 80–250 nm in length via image software, re-spectively. After dye conjugation, TOCNC-AANI presented rod-likemorphologies similar to the unmodified TOCNC. The effect of the dyegrafting on the morphology of CNC was also investigated by TEMimaging, as shown in Figure S3. The observed average width dimensionwas determined to be 16.5 nm, which was a little higher than the rea-sonable size of dye-labeled CNC. Reason may be that the aggregation ofTOCNC-AANI occurred to some extent during the drying process. Thestructural and morphological integrity of the TOCNC-AANI did notappear to be affected by the conjugation of AANI. Fig. 1C presents theXRD patterns of TOCNC and TOCNC-AANI. TOCNC shows a clear cel-lulose Iβ polymorph with the 200 diffraction centered at 22.4°, a widepeak signal at 15.6° for 110 plane, and a weak signal at 34.4° for 004plane. The CI of TOCNC was estimated to be 70.2% according to Eq. (2).No discernable change in crystal structure after dye labeling wasmeasured by XRD. The characteristic signals and the calculated CI ofTOCNC-AANI generally remain as TOCNC, indicating that the crystalstructure was essentially not affected by the functionalization.

The thermal characteristics of nanocrystals were investigated byTGA, as seen in Fig. 1D. All the samples first show similar gradualweight loss due to the evaporation of water in the range of 20–200 °C.The onset temperatures of TOCNC and TOCNC-AANI occur at around232 and 247 °C, respectively. The degradation temperature for thelargest weight loss increases from 325° C of TOCNC to 340 °C ofTOCNC-AANI, indicating that the thermal stability of nanocrystals isimproved after covalent attachment of AANI (DTG data; Figure S4).

3.2. Absorption and fluorescent properties of TOCNC-AANI

The resulting TOCNC-AANI after the conjugation reaction werethoroughly washed and dialyzed to remove free AANI that might beadsorbed on the surface until no absorption of the supernatant wasdetected. The stability of the TOCNC-AANI solution remained the sameafter the conjugation reaction by visual comparison of the turbidity. Tofurther evaluate the stability of TOCNC-AANI solutions, their absorp-tion and fluorescence spectra were detected over time (Figure S5). Theabsorbance and fluorescent intensity of the TOCNC-AANI solutions re-mained essentially unchanged even after 24 h, suggesting that the la-beling did not have a negative impact upon the dispersity of CNC.Figure S6 shows the pictures of aqueous solutions of unmodified anddye-labeled TOCNC. The aqueous solution of TOCNC-AANI presents ayellow color under visible light, whereas TOCNC solution is colorless.Under UV illumination, the yellowish green fluorescence of TOCNC-AANI could be directly observed by naked eye.

Unmodified TOCNC show no absorption in the wavelength range ofvisible region. The absorption peak at 420 nm and the fluorescentemission peak at 550 nm of TOCNC-AANI are comparable to those ofpure AANI in aqueous solution (Fig. 2), respectively. Based on the si-milar UV–vis absorption spectra of TOCNC-AANI and AANI, reasonableassumption can be made that the basic chromophore of AANI did notchange either during the reaction or as a result of its bonding toTOCNC. This allowed us to use the absorption calibration curve (FigureS7) of pure AANI to determine the dye content bonded to TOCNC. Thedegree of substitution achieved in this work is 0.1 ± 0.01mmol g−1

which is higher than those of previously reported dye-labeled CNC. Forexample, Nielsen et al achieved FITC and RBITC contents of 2.8 and

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2.1 μmol g−1 cellulose [16], Abitol et al. obtained DTAF content ofround 11.4 nmol g−1 cellulose [18], and Grate et al reported Alexa dyecontent of about 5 μmol g−1 cellulose [19], respectively.

3.3. Effect of pH on the fluorescence behavior of TOCNC-AANI

Previously reported fluorescent CNC usually display remarkably pH-responsive fluorescence [12,15,16]. Their responsiveness was basicallyascribed to the sensitive dyes labeled on CNC. For example, FITC exists incationic, neutral, anionic, or dianionic form in aqueous solution with thechange of pH values, resulting in different emission wavelengths and

intensities. In this work, the fluorescence responsivenesses of AANI andTOCNC-AANI to pH were also investigated by measuring the fluores-cence spectra in PBS buffer solutions. As shown in Fig. 3, the fluorescentintensity of AANI at the emission peak of 550 nm is slightly reduced withan increase in pH from 4 to 9. Under normal circumstances, the effect ofpH on the fluorescence behavior of TOCNC-AANI should be similar tothat of AANI. However, the fluorescent intensity gradually increasesupon increasing pH value (Figure S8). The contrary change trend offluorescent intensity between TOCNC-AANI and AANI may be ascribedto the loading carrier of CNC, which is considerably sensitive to en-vironmental medium. The relatively small variation of fluorescent

Scheme 1. Synthesis of carboxylated TOCNC and dye-labeled TOCNC-AANI.

Fig. 1. FTIR spectra (A), AFM images (B), X-ray diffraction patterns (C) and TGA analysis (D) of TOCNC and TOCNC-AANI.

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intensity of TOCNC-AANI under different pH values indicates limitedfluorescence responsiveness to pH in aqueous solution. In the next part,the effects of solvent polarity and ion concentration on the fluorescencebehavior were further investigated to clarify the role of CNC carrier.

3.4. Effect of solvent polarity on the fluorescence behavior of TOCNC-AANI

Environment medium plays an important role in the fluorescencebehavior of fluorescent material. It is known that the fluorescencequantum efficiency of dye is dependent on the solvent polarity to agreat extent [36,37]. For TOCNC-AANI, solvent polarity affects not onlythe fluorescent intensity but also the dispersion ability of solution.Solvents with high polarities are required to ensure the dispersionstability and uniformity of CNC solutions, such as water, N, N-di-methylformamide, diemthyl sulfoxide, formamide, N-methyl for-mamide, etc [38–40]. Water possesses high dielectric permittivity (εr)of 80.20 and 3D hydrogen bond characteristics, whereas DMF has re-latively low εr of 38.25 and no solvent-solvent hydrogen bonds. Basedon this distinct difference, a series of component solvents of DMF andwater with different ratios, in which TOCNC-AANI solutions werecompletely isotropic and fluid at low concentration (0.01 wt%), wereused to investigate the solvent effect on the fluorescence behavior. Thefluorescence spectra of pure AANI were measured for comparasion(Fig. 4). In general, the polarity of the excited state of fluorescent

molecules is stronger than that of the ground state. Therefore, thefluorescent molecules in the excited state tend to interact with polarsolvents with high permittivities, leading to the change of electrondistribution and reorientation of the dipoles [41]. Since solvent effectpromotes nonradiative transition, the fluorescence quantum yield ofdye is reduced with an increase in solvent polarity. The fluorescentintensity of AANI at emission peak first gradually increases but thenslightly decreases as the volume ratio of DMF and H2O increases. Themaximum fluorescence emission occurs at the DMF proportion of 60%.The initial increasing trend, which is in accordance with the rule ofsolvent effect on fluorescence behavior, can be attributed to the re-duction of average permittivity. However, the final decreasing trend ofAANI emission is unexpected according to solvent effect. Similar ab-normal fluorescence behavior is also found in the component solventsof DMSO and H2O (Figure S9). The existence of H2O in the componentsolvent can form 3D hydrogen bonds [42], which may limit the colli-sion of dye molecules and reduce nonradiative transition. Since bothDMF and DMSO do not have solvent-solvent hydrogen bonds, theweakened fluorescence emissions of AANI at high proportions of DMFand DMSO are reasonable. When dye molecules are attached to thesurface of CNC, solvent polarity also affects the fluorescence behaviorof TOCNC-AANI. The difference is that the fluorescence responsivenessof TOCNC-AANI to solvent polarity is about 10 times higher than that ofAANI. Based on the structural characteristics of TOCNC-AANI, themagnification effect of fluorescence responsiveness could be attributedto the CNC supporter.

3.5. Effect of ion concentration on the fluorescence behavior of TOCNC-AANI

The effect of pH on the fluorescent intensity of TOCNC-AANI hasbeen discussed above. In the pH range of 4–9, pure AANI shows a slightdecrease in the fluorescent intensity with an increase in the pH value,whereas TOCNC-AANI presents a slightly increasing tendency (Fig. 3).Considering that there is no other influencing factor except the recipe ofPBS buffer solutions with different pH values, we speculate that thefluorescence emission of TOCNC-AANI is related to the effect of ionicstrength on the carrier of CNC. However, it is difficult to evaluate theionic strength because PBS buffer solutions are composed of weakelectrolytes of NaH2PO4 and Na2HPO4. Here, strong electrolyte of NaClwas used to further determine the effect of ionic strength on thefluorescence behavior of TOCNC-AANI (Figure S10). Fig. 5 shows thedetailed fluorescence behaviors of AANI and TOCNC-AANI under dif-ferent concentrations of NaCl. Both the fluorescence emission of AANIand TOCNC-AANI are responsive to the salt concentration in a largerange of 0–600mmol L−1. With an increase in NaCl concentration, the

Fig. 2. UV–vis absorption spectra (A) and fluorescence spectra (B) of TOCNC, AANI and TOCNC-AANI. AANI: 2.86×10−6 mol L-1, TOCNC-AANI: 0.01 wt%. (Forinterpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 3. Effect of pH on the fluorescence maxima of AANI (2.86×10−6 mol L-1)and TOCNC-AANI (0.01 wt%) in PBS buffers.

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fluorescent intensities successively go through first growth and laterdecline stages. The responsive sensitivity of TOCNC-AANI is greatlymagnified compared to that of AANI. Similar responsiveness and var-iation trends of fluorescent intensity were observed for other strongelectrolytes including LiCl, KCl, NH4Cl, MgCl2, CaCl2 and AlCl3, asshown in Fig. 6. It is noteworthy that TOCNC-AANI presents a muchsharper responsiveness to bivalent and trivalent ions than monovalentions, especially for Ca(II) and Al(III). However, the fluorescent intensity

gradually decreases for Al(III) and rapidly decreases for Ca(II) when theion concentration is higher than 15mmol L−1. There is precipitateformation that is visible to the naked eye for Ca(II). Obviously, themagnified fluorescence responsiveness to ionic strength is related to theCNC supporter. Considering that the permittivity of solution is reducedwith an increase in electrolyte concentration [43], we believe that themechanism of ionic strength affecting the fluorescence responsivenessis similar to that of solvent polarity.

Fig. 4. Photographs of AANI (A) and TOCNC-AANI (B) aqueous solutions under UV illumination. Fluorescence spectra of AANI (dash) and TOCNC-AANI (solid) inDMF/H2O component solvents with different DMF proportions (C). Plot of fluorescence maxima vs. proportion of DMF (D). AANI: 2.86× 10−6 mol L-1, TOCNC-AANI: 0.01 wt%.

Fig. 5. Photographs of AANI (A) and TOCNC-AANI solutions (B) with different NaCl concentrations. (C) Plot of fluorescence maxima vs. concentration of NaCl. AANI:2.86×10−6 molL-1, TOCNC-AANI: 0.01 wt%.

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3.6. Response mechanism of TOCNC-AANI

As discussed above, both the solvent polarity and ion concentrationslightly affect the fluorescence behaviors of AANI. The fluorescenceresponsiveness of AANI can be mainly explained by the variation ofmedium permittivity. However, it is not enough to elucidate the ex-tremely higher sensitivity of TOCNC-AANI compared to AANI. CNCcarrier must be another important factor that affects the fluorescencebehavior of TOCNC-AANI. Here, we put forward a model of aggrega-tion-enhanced emission (AEE) to explain the fluorescence behaviorbased on the colloidal stability of CNC carrier [40].

With a decrease in the average permittivity of DMF/H2O componentsolvent, the counterions are less solvated and more condense. As a re-sult, the efficiency of electrostatic stabilization of the TOCNC-AANI isreduced and partial aggregation of the TOCNC-AANI is formed. In ad-dition, the weak hydrogen bonding capability of DMF, which is solelyable to accept but not donate hydrogen bonds, may increase theprobability of forming hydrogen bond networks among nanocrystalsand further irreversible aggregation. The close approach of CNC createsa new compacted environment for dye groups, in which the rotationand vibration of dye groups are restricted, leading to less nonradiativetransition and enhanced fluorescence emission [14,44]. Besides thefluorescence characteristic of the attached AANI itself, the latter des-cending fluorescent intensity of TOCNC-AANI at high proportions ofDMF (Fig. 4D) may be ascribed to the excessive aggregation of CNCcarriers, which destroys the stability of solution and reduces thefluorescent signals that can be detected.

The remarkable magnification of fluorescent intensity with in-creasing ionic strength can also be explained by the colloidal stability ofTOCNC-AANI. Similar to the solvent effect, the shift toward a higherfluorescent intensity can mainly be ascribed to the aggregation of CNCunder high electrolyte concentrations [45]. To further support thefluorescence responsiveness of TOCNC-AANI based on AEE, Zeta po-tential and DLS measurements were conducted with different NaClconcentrations. Table 1 illustrates the Zeta potential and DLS size va-lues as a function of NaCl concentration. The Zeta potential of TOCNC-AANI is -30.81mV without any addition of NaCl, which is a little lowerthan the -33.53mV of unmodified TOCNC [46]. It can be explained bythe dye substitution of carboxyl group on the surface of TOCNC. In-creasing NaCl concentration actually lowers the absolute Zeta poten-tials of TOCNC-AANI [45,47]. Owing to counter-ions adsorption ontothe negatively charged surface of CNC, high ionic strength compressesthe electrostatic double layer surrounding the CNC and reduces the

electrostatic repulsion, resulting in the aggregation of nanocrystals.Although the DLS technique is designed for spherical particles, and notentirely suitable for rod-shaped CNC, the testing results can be used asreference to compare the relative dimensions of CNC. DLS measure-ments show that by adding NaCl to CNC aqueous solutions, theequivalent hydrodynamic size gradually increases in the concentrationrange of 0–600mmol L−1. The observed increase in the hydrodynamicsize of the nanocrystals at high NaCl concentrations is attributed to CNCaggregation. TOCNC-AANI presents a sharper responsiveness to Mg(II),Ca(II) and Al(III) than Na(I) (Fig. 6). It is reasonable since Mg(II), Ca(II)and Al(III) possess higher binding ability to the carboxyl groups, whichpromote the aggregation of CNC. The contrary decreases in the fluor-escent intensities of TOCNC-AANI when Ca(II) and Al(III) concentra-tions are higher than 15mmol L-1 are ascribed to the formation ofcarboxylate precipitates, especially for Ca(II).

The dispersion and aggregation models of TOCNC-AANI with re-sponsiveness to solvent permittivity and ionic strength are illustrated inFig. 7. TOCNC-AANI can be well dispersed in water. When addingelectrolyte into TOCNC-AANI solution, the double layers of CNC arecompressed via charge neutralization, leading to close approach andaggregation between nanocrystals. Similar aggregation of TOCNC-AANIoccurs when reducing the average permittivity of DMF/H2O componentsolvents via increasing the proportion of DMF. The magnified fluores-cence responsiveness of TOCNC-AANI is explained by AEE effect, whichis related to the sensitive colloidal stability of CNC carrier.

4. Conclusions

Responsive nanomaterials based on CNC were synthesized bycovalently grafting 1, 8-naphthalimide dye. High dye substitution de-gree of 0.1 ± 0.01mmolg−1 was obtained via EDC/NHS-mediatedreaction between the carboxyl groups of TOCNC and amino groups ofAANI. The crystal structure and water dispersibility of CNC were es-sentially not affected by the surface immobilization of AANI. Thefluorescent intensity of TOCNC-AANI is responsive to both the solventpermittivity and ionic strength. In particular, the responsive sensitivityof TOCNC-AANI is greatly magnified compared to that of the pure AANIowing to the aggregation of CNC carrier, which creates a favorableconfined environment for fluorescence emission of dye groups. The AEEtriggered by the change of ionic strength was supported by the DLSparticle size and Zeta potential. Based on the modifiability and ampli-fication effect of CNC, this type of fluorescent CNC probe can be po-tential candidate for nanosensor applications.

Fig. 6. Fluorescence maxima of TOCNC-AANI (0.01 wt%) in various electrolytesolutions with different concentrations.

Table 1Physico-chemical characteristics of TOCNC and TOCNC-AANI aqueous solu-tions with different NaCl concentrations.

Sample CNaCla

(mmol L−1)Dav

b

(nm)ζc

(mV)σd(mS cm−1)

TOCNC 0 47.9 −33.53 0.0610 60.3 −28.46 1.0120 79.1 −22.56 2.0150 97.6 −18.67 4.60100 101.9 −5.69 8.30200 131.1 −4.87 14.64

TOCNC-AANI 0 56.1 −30.81 0.0410 79.2 −20.06 1.0620 88.9 −17.70 2.0050 99.7 −13.64 4.51100 -d −12.25 8.13200 -d −5.47 14.75

a CNaCl:NaCl concentration.b Dav: Average particle size based on equivalent hydrodynamic volume de-

tected by NanoPlus particle analyzer.c ζ: Zeta potential.d The results are not repeatable due to extensive aggregation.

W. Wu et al. Sensors & Actuators: B. Chemical 275 (2018) 490–498

496

Acknowledgments

The support of this work by Natural Science Foundation of JiangsuProvince (BK20171450), the National Key Basic Research Program(2017YFD0601005), Priority Academic Program Development ofJiangsu Higher Education Institutions (PAPD), and National NaturalScience Foundation of China (31370583, 31770607) is gratefully ac-knowledged.

Appendix A. Supplementary data

Supplementary material related to this article can be found, in theonline version, at doi:https://doi.org/10.1016/j.snb.2018.07.085.

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Weibing Wu received a PhD (2008) in Material Physics and Chemistry from SoutheastUniversity, and then joined Nanjing Forestry University as an assistant professor. Hecarried out research work in Georgia Institute of Technology as visiting scholar(2013–2014). In 2018 he become a Professor in Pulp & Paper Enineering. His researchinterests are on nanocellulose materials, functional paper, biosensors and biomass fuelcell.

Ruyuan Song is currently a postgraduate student of Pulp & Paper Enineering at NanjingForestry University. Her research interest is fluorescent probe based on cellulose nano-crystals.

Zhaoyang Xu is a Professor of Materials Science at Nanjing Forestry University(NJFU)since 2018. He received a PhD in Wood Science and Technology at the NJFU in theCollege of Materials Science and Engineering in 2007. At the NJFU, His research interestsand fields is including wood-based panel manufacture, wood-plastic composites, nano-cellulose composites.

Yi Jing received a PhD in Pulp & Paper Enineering from Nanjing Forestry Universityin in2003. Dr Jing is presently a Professor in the College of Light Industry and FoodEngineering. His research interests are on wet end chemistry of paper making and coatingprocess technology.

Hongqi Dai is a Professor of Pulp & Paper Enineering having been at Nanjing ForestryUniversity since 2004. His research interests are on paper chemistry and engineering,clean paper making technology, fiber and paper based functional materials.

Guigan Fang received a PhD from Chinese Academy of Forestry in 2000. Dr. Fang ispresently a Professor at Institute of Chemical Industry of Forestry Products. His researchinterests are on clean pulping & papermaking technology, wastewater treatment andcellulose functional materials.

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