Research Article pubs.acs.org/journal/ascecg
Facile Method for Fluorescent Labeling of Starch Nanocrystal Canxin Cai,†,‡ Benxi Wei,§ Zhengyu Jin,†,‡ and Yaoqi Tian*,†,‡ †
State Key Laboratory of Food Science and Technology and ‡School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China § School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, China ABSTRACT: Fluorescently labeled starch nanocrystal (FL-SNC) was synthesized using a simple, low-cost, and scalable two-step chemical modification process. Reactive amino groups were introduced onto the SNC surface through silanization with 3-aminopropyl triethoxysilane (APTES), and fluorescein isothiocyanate (FITC) groups were covalently attached through thiourea. Fourier transform infrared spectrometry, Xray photoelectron spectroscopy, solid-state cross-polarization magicangle spinning 13C NMR, UV−visible absorbance spectrophotometry, and fluorescence emission spectroscopy confirmed the successful introduction of the fluorescent groups. Transmission electron microscopy and X-ray diffraction data indicated that the dispersibility of FLSNC was significantly improved, and the original crystallinity and morphology were retained. Compared with a mixture of uncoupled FITC and SNC, covalently connected FL-SNC displayed a more obvious fluorescence intensity and higher photostability. Furthermore, FL-SNC was biocompatible with cells and could be easily internalized. In combination with the participation of active hydroxyls, this facile approach has potential use for synthesis of fluorescently polyhydroxyl nanoparticles and can be widely used for making biosensors and biomarker in food and biomedical industries. KEYWORDS: Starch nanocrystal, Fluorescent labeling, Surface modification, Triethoxysilane, Fluorescein isothiocyanate
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INTRODUCTION Polysaccharide nanocrystals derived from natural sources, such as cellulose nanocrystal (CNC),1 chitin nanocrystal,2 and starch nanocrystals (SNC)3 have uniform crystalline structures that confer rigidity and a high degree of crystallinity. They have acquired a reputation as having potential for biobased nanomaterials since they are easily processed, renewable, biocompatible, and nontoxic in comparison with inorganic nanoparticles.4,5 SNC are receiving increasing attention from the research community. They can be easily prepared by acid hydrolysis of starch and retain crystalline lamellas with a thickness of 5−7 nm, a length of 20−40 nm, and a width of 15−30 nm. SNC combine the advantages of organic materials with nanotechnology and are widely considered a good candidate for nanomaterials. Moreover, the starch surface can be easily modified with biocovalent groups, which can render SNC suitable for diverse applications thanks to their large specific surface area and reactive hydroxyl groups.6 By virtue of their unique nanoscale properties, as well as the active functional fluorophore groups via chemical modification, the fluorescent labeling methods for polysaccharide nanocrystal based on CNC,7,8 chitin nanocrystal,9,10 and other organic nanoparticles11,12 have been widely researched in recent years. For example, a mild procedure is presented to produce pHsensitive fluorescent CNC, with L-leucine amino acid as a spacer linker.13 Cellulose nanowhiskers modified through grafting of PG via click reaction and then QDs attached to © 2017 American Chemical Society
the functional group of CNW−PG by hydrogen bond formation enhanced suspension stability, highly fluorescent intensity, and biocompatibility.14 Wan et al. fabricated luminescent chitosan nanoparticles through a one-pot MCR and obtained amphiphilic WS-Chitosan@An-CHO copolymers.9 TEMPO-oxidized chitin nanocrystal was labeled with a fluorescent imidazoisoquinolinone dye and simultaneously conjugated with carbohydrate ligands, resulting in dually functionalized chitin nanocrystal with applications in bioanalysis and theranostic.15 Since specific spectral properties respond to environment changes, fluorescent dyes and their derivatives such as fluorescein isothiocyanate (FITC) and rhodamine B isothiocyanate are widely used in the preparation of biosensors.16−19 Amine functionality has been introduced to CNC by reacting with epichlorohydrin followed by ringopening in ammonium hydroxide solution.20 Meanwhile, Khaled et al. labeled CNC with rhodamine B isothiocyanate using a three-step pathway and observed no indication of cytotoxicity associated with the ability of the fluorescein-labeled nanoparticles to penetrate cells.21 Nielsen et al. have developed a three-step thiol−ene click reaction in order to improve the labeling method and produced a new type of fluoresceinlabeled CNC pH sensor.22 Huang et al. have labeled chitosan whiskers with FITC using an ionotropic gelation approach in Received: September 10, 2016 Revised: February 28, 2017 Published: March 30, 2017 3751
DOI: 10.1021/acssuschemeng.6b02157 ACS Sustainable Chem. Eng. 2017, 5, 3751−3761
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(Ultra Turrax T10, IKA, Staufen, Germany) at 6000 rpm for 3 min to avoid aggregates and stored at 4 °C following the addition of several drops of chloroform. The SNC concentration was determined by weighing freeze-dried powders of the homogeneous dispersions (10 mL) and was expressed as the weight percentage relative to the volume of the water phase (w/v). The final concentration of SNC suspensions was 3.45% (w/v). Synthesis of FL-SNC. Before the silane coupling modification, SNC were subjected to alkalization in order to improve interfacial adhesion with solvent.31 SNC suspension (1%, w/v) was soaked in sodium hydroxide solution (pH 10.0) and placed at ambient temperature for 5 h at a constant stirring speed of 200 rpm. Suspensions were then washed five times with ethanol−water (95/5, v/v) by centrifugation (3 min, 3200 × g, 25 °C) to replace the aqueous alkali. Amino groups were introduced to the surface of SNC by reaction between hydroxyl groups and the silane-coupling agent APTES. First, the silane-coupling agent (1%, w/v) was prehydrolyzed in ethanol− water (95/5, v/v) by adding benzoyl peroxide (0.5%, w/v) for 1 h at a constant stirring speed (200 rpm) at ambient temperature. Second, the alkaline treated SNC suspension (1%, w/v) was mixed with APTES hydrolysate for 3 h and treated as described above. The AM-SNC suspension was then dried in an air oven at 45 °C for 36 h. In order to remove residual unreacted APTES, the reaction mixture was washed by centrifugation (5 min, 3200 × g, 25 °C) in distilled water until the supernatant reached a constant pH. Next, the suspension was dried in an air oven at 45 °C for 6 h, and AM-SNC was ground and sieved (100 mesh). AM-SNC was then reacted with FITC in a mixture of methanol− water (90/10, v/v) for 36 h with stirring (200 rpm) at 20 °C in the dark. The final product was isolated by centrifugation (3 min, 3200 × g, 25 °C) with ethanol until UV/vis absorption spectroscopy detected no absorption peak at 400−500 nm. Reaction products were dried in a dark fume hood at ambient temperature, ground, and sieved (100 mesh). Meanwhile, an unlabeled SNC sample was treated by the same method to obtain fluorescein-absorbed SNC (FA-SNC). Characterization of FL-SNC Conjugates. FT-IR spectra of SNC, AM-SNC, and FL-SNC samples were collected between 4000 and 500 cm−1 on an FT-IR spectrophotometer (5DXC FT-IR, Nicolet Co. Madison, WI, USA) with 256 scans at a resolution of 4 cm−1. The samples (3 mg) were ground with spectroscopic-grade potassium bromide (200 mg) and pressed into 1 mm pellets. A blank disc was used as the background. XPS experiments were carried out using a multifunctional imaging electron spectrometer (Thermo ESCALAB 250XI, Waltham, Massachusetts, USA). The spectrometer was equipped with a monochromatic Al Kα (hν = 1486.6 eV) X-ray source running at 150 W and 15 kV. The kinetic energy of photoelectrons was determined by a hemispheric analyzer at 160 eV for wide-scan spectra and 20 eV for high-resolution spectra. During measurements, electrostatic charging of the samples was avoided by a low-energy electron source working in combination with a magnetic immersion lens. All recorded peaks were shifted by the same value to set the C 1s peak to 284.4 eV. Quantitative elemental compositions were determined from peak areas using sensitivity factors and the spectrometer transmission function. The spectrum background was subtracted according to the Shirley method.32 Atomic concentrations were calculated from the high-resolution photoelectron peak areas using Gaussian−Lorentzian deconvolution.33 Solid-state CP/MAS 13C NMR spectra were collected on a Brurker400 AVANCE III (Bruker, Switzerland) operating at 400 MHz for 13C. SNC, AM-SNC, and FL-SNC samples were spun at the magic angle (54.7°) with respect to the static magnetic field. Carbon chemical shifts relative tetramethylsilane were determined from the spectra, using solid glycine at ambient temperature as an external reference. Samples were packed into a rotor with a diameter of 5 mm in an identical state of humidity and spun at speeds of 5 kHz (magic angle spinning, MAS) in a probe of PABBO BB-1H/D Z-GRD. 13C NMR measurements were performed with acquisition parameters of 2 μs, a 90° proton pulse, and a 3 ms acquisition time. Variable delay times up
order to evaluate the effects of molecular weight and the degree of deacetylation on the cellular uptake and in vitro cytotoxicity of chitosan whiskers through chemical depolymerization and reacetylation.23 These studies demonstrate the potential of fluorescein-labeled polysaccharide nanocrystal to serve as novel drug nanocarriers, bioimaging probes, and biosensors. There is an abundance of hydroxyl groups on the surface of SNC that can react with active groups in modifying agents such as fluorescent dyes. However, the poor reactivity of the surface hydroxyl groups makes it difficult to form direct covalent links, and it is generally necessary to introduce modifications that activate the surface of SNC before reacting with fluorescent dyes.24 In previous work on CNC and chitin whiskers, two types of chemical modification have been performed:1 The first involved grafting a polymer onto the surface of SNC,25 while the second involved reacting a modifier that included an esterifying agent26,27 and a cross-linking agent27,28 with surface hydroxyl groups. The use of silane-coupling agents in ethanol/ water is a universal and easily performed method for improving the suitability of polysaccharides and introducing particular chemical groups.29 Unfortunately, these modification approaches are tedious and involve multiple (three or four) steps. Meanwhile, current one-pot treatments only achieve an intermediate degree of substitution. Thus, the development of a simpler and low-cost approach capable of achieving a high degree of fluorescein labeling is desirable. Using SNC and FITC as polyhydroxyl nanocrystal and fluorescein dye, respectively, the present study proposes a facile approach based on a simple and universally applicable method for fluorescein labeling via polar covalent binding. The reaction pathway consists of two steps: (a) introduction of reactive amino groups onto the surface of SNC via a silane coupling reaction, followed by (b) attack of the π-electrons of the isothiocyano groups by the lone electron pair of the introduced amino groups to form new thiourea covalent bonds via electrophilic addition with FITC. The product was characterized by Fourier transform infrared spectrometry (FT-IR), Xray photoelectron spectroscopy (XPS), and solid-state crosspolarization magic-angle spinning (CP/MAS) 13C NMR. The resultant fluorescently labeled SNC (FL-SNC; FITC/SNC) conjugates were verified, and their fluorescent properties and fluorescent quantum yield (QY) were investigated. The photostability was detected by fluorescence microscopy as the fluorescence loss during time intervals of irradiation. Moreover, the redispersibility, size distribution, and morphology of synthetic amino modified SNC (AM-SNC) and FL-SNC products were compared with unlabeled SNC to ensure that two-step modification was possible. Finally, MTT assay and cell uptake experiment of FL-SNC was further performed to ensure its bioimaging applications.
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MATERIALS AND METHODS
Materials. Waxy maize starch was donated by the Tianjin Tingfung Starch Development Co., Ltd. (Tianjin, China). All other chemicals and reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Suzhou, China) and were of analytical grade unless otherwise stated. Preparation of SNC. SNC was prepared by acid hydrolysis of waxy maize starch based on the description of Angellier et al. with minor modifications.30 Starch powder (80 g) was mixed with 800 mL of 3.16 M H2SO4 solution and placed at 40 °C for 7 days with stirring at 200 rpm. Suspensions were washed by successive centrifugation steps (5 min, 3200 × g, 25 °C) in distilled water until the supernatant reached a constant pH. The resultant suspensions were redispersed 3752
DOI: 10.1021/acssuschemeng.6b02157 ACS Sustainable Chem. Eng. 2017, 5, 3751−3761
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ACS Sustainable Chemistry & Engineering Scheme 1. Fluorescent Labelling of SNC with APTES and FITC by Two-Step Chemical Modification
Crystalline Integrity, Size Distribution, and Morphology. Prior to XRD analysis, SNC, AM-SNC, and FL-SNC samples were milled to a powder, passed through a 200 mesh sieve, and sealed in a vessel at 75% relative humidity using saturated sodium chloride. Samples (0.8 g) were pressed into a pellet (10 nm × 25 nm) with a hydraulic press. XRD patterns were obtained using a Bruker D8Advance XRD instrument (Bruker AXS inc., Karlsruhe, Germany). Diffractograms were collected at 40 kV and 30 mA with nickel-filtered Cu Kα radiation (wavelength, 1.5405 Å). Powdered samples were scanned from 3 to 45° (2θ) at a scanning rate of 4°/min. The relative crystallinity (RC) was calculated using Jade 6.0 software.35 The pH values of SNC, AM-SNC, and FL-SNC suspensions were adjusted to 10.0, and the size distribution of redispersed samples was measured using a commercial Zetasizer Nano ZS 90 instrument (Malvern, UK). The fluid and particle refractive indices were 1.33 and 1.53, respectively. Measurements were carried out in quintuplicate for error analysis. TEM observations were performed using a H-700 (Hitachi, Tokyo, Japan) at an acceleration voltage of 80 kV. Suspensions of SNC, AMSNC, and FL-SNC (0.01%, w/v) were sonicated at 4 °C for 15 min, and a single drop of suspension was spread onto a copper grid coated with carbon support film. After 1 min, excess liquid was blotted with filter paper, and the remaining film was allowed to dry before observation. Cell Culture and Cytotoxicity of FL-SNC. The human hepatoma cell line (HepG2) was purchased from the cell bank of the Type Culture Collection of Chinese Academy of Sciences. Cells were cultured in DMEM medium (Gibco, USA) with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, USA), at 37 °C and 5% CO2 for 24 h before the appropriate treatments. To explore the cytotoxicity of FL-SNC on HepG2 cells, 3(4,5-dimethylazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was performed. Cells were cultured in a 96-well plate overnight at an initial density of 1 × 104 cells per well. Subsequently, the culture medium was removed, and the cells in each well were incubated with different concentrations of FL-SNC (0, 25, 50, 100, 150, 200, and 300 μg/mL) at 37 °C and 5% CO2 for 24 h. Subsequently, the cells were rinsed by phosphate-buffered saline (PBS), and the medium was replaced with fresh medium. Then, 20 μL of MTT solution with a concentration of 5 mg/mL was added into each well and incubated for
to 20 ms were used for the proton experiments, and the crosspolarization time was set to 2 ms, with a recycle delay of 2 s and 4000 acquisitions. Fluorescent Properties of FL-SNC. The FITC content of FLSNC was determined from UV/vis adsorption spectra and fluorescence emission spectra. Due to the distinct effect of pH on absorbance, spectra were recorded in aqueous solution, and the pH was adjusted to >6.0 to ensure the dianionic state of FITC. Consequently, a maximum main absorption peak at 490 nm was present, and a direct correlation between absorption intensity and FITC content could be obtained. UV/vis absorption spectra were collected with a double beam UV/vis spectrophotometer (TU1900, Purkinje, Beijing, China). Fluorescence emission spectra were used to estimate the FITC content of covalently labeled FL-SNC. Fluorescence scans were obtained using a 2.5 nm slit width, a 495 nm excitation wavelength, and an emission scan ranging from 505− 800 nm. Fluorescence emission spectra were recorded on a Fluoro Max4 fluorescence spectrophotometer (Horiba JY, Edison, NJ, USA). In order to evaluate the photostability of FL-SNC, samples were continuously irradiated for different time intervals using a 100 W mercury lamp. The fluorescence loss was detected by fluorescence microscopy with the excitation of 460−490 nm and a barrier of 500− 600 nm at 40× magnification (BX41, Olympus, Tokyo, Japan). In order to guarantee the same observation conditions, equal qualities of the dry state samples were spread onto the slide with same area. The distance between lamp and lens was 15 cm, and the distance between lens and condenser was 6 cm. QY is an important factor for quantitatively evaluating fluorescent intensity of fluorescein. In this experiment, a well-established reference method was used for determining the QY values of FL-SNC as follows (Rhodamine B in anhydrous ethanol, literature QY: 98%).34 Φx = Φst(Ix /Ist)(ηx /ηst)2 (A st /A x ) where Φ is the QY, I is the integrated emission intensity, η is the refractive index of the solvent (ηethanol = 1.36, ηwater = 1.33), and A is the optical density. The subscripts “st” and “x” stand for standard with known QY and FL-SNC samples, respectively. To minimize reabsorption effects, the absorption value at the excitation wavelength is required to be smaller than or equal to 0.05. 3753
DOI: 10.1021/acssuschemeng.6b02157 ACS Sustainable Chem. Eng. 2017, 5, 3751−3761
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Figure 1. (a) FTIR spectra of SNC and AM-SNC and (b) of SNC, AM-SNC, and FL-SNC. the next 4 h. The culture medium was then removed, and DMSO (200 μL/well) was added to solubilize the formazan crystals. After dissolution of the formazan crystals, the optical density of the solution was measured using the absorbance readout through a microplate reader (BioTek Synergy2, BioTek, Vermont, USA) at a wavelength of 490 nm. The cells without FL-SNC addition (0 μg/mL) were considered as the control. Each concentration were prepared for six replicates. Cell viability was calculated by the percentage of absorbance in relative to the control. Cell Imaging with Confocal Microscopy. For confocal microscope magnification, HepG2 cells were seeded on a cover glass at a density of 1 × 104 cells per glass coverslip overnight, the medium was removed. Thereafter, cells were incubated with 200 μL of FL-SNC (100 μg/mL) which was dispersed in PBS for 2 and 4 h. The cells were washed continuously with 1 mL of PBS 3 times to remove unbound nanocrystal and analyzed by confocal laser scanning microscopy (CLSM, LSM710, Zeiss, Germany). The experiment was carried out with a Plan-Apochromat 40× 0.95 Korr M27 objective, and the excitation wavelength was set at 488 nm with a detected emission range of 493−634 nm. Statistical Analysis. Statistical analysis was performed using ORIGIN 9.0 (OriginLab Inc., Northampton, MA, USA). Strength data were analyzed with one-way analysis of variance. All comparisons were based on a 95% confidence interval.
the isothiocyano group, which made formation of the covalent bond between AM-SNC and FITC favorable and therefore facile. Characterization of AM-SNC and FL-SNC. FT-IR confirmed that the reactions described above resulted in the desired products (Figure 1a). SNC and AM-SNC clearly shared similar spectral properties at wavelengths of 1450 and 1370 cm−1 (C−H bending vibration), 1648 cm−1 (H−O−H bending vibration), 1157 cm−1 (C−O stretching vibration), and 1155, 1080, and 1020 cm−1 (C−O−O stretching vibration). However, the AM-SNC spectrum exhibited new peaks at 1491 and 1577 cm−1, indicative of an amino group and N−H bending vibration,36 respectively. Additionally, the intensity of the adsorption peak at 1197 cm−1 was increased, and the peak near 1020 cm−1 (C−O−C, Si−O−Si, and Si−O−C stretching vibration) was broadened.37 These results provided clear evidence that the amino group of APTES was chemically linked through a covalent bond to the SNC surface. In addition, the new peaks at 1419, 1512, and 1436 cm−1 (Figure 1b) were ascribed to the SN bond of the thiourea group and the aromatic ring of the fluorophore FITC.38 Together, these results confirmed the presence of a covalent link between FITC and SNC in the FL-SNC sample, rather than the intermolecular forces (such as hydrogen bonds) present in the AM-SNC sample. Furthermore, a weak adsorption peak appeared at 1560 cm−1, indicating an NO stretching vibration, presumably resulting from oxidation of the amino group during preparation, consistent with partial oxidation during the drying process. XPS data further confirmed that SNC was covalently linked to fluorescein. In AM-SNC spectra, silicon and nitrogen atoms were clearly present, presumably derived from the initial structure of APTES and indicating that silane had been successfully introduced onto SNC. In FL-SNC spectra, the presence of sulfur atoms characteristic of the isothiocyano group of FITC was apparent (Figure 2). Elemental surface
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RESULTS AND DISCUSSION Synthesis of FL-SNC. In order to enhance the reactivity of the hydroxyl groups of SNC, it was first treated with alkaline solution in order to promote the adsorption of hydrolyzed 3aminopropyl triethoxysilane (APTES) and subsequent silanization to introduce amino groups (Scheme 1a). The lone pair of electrons on the amino group of AM-SNC was then able to attack the π-electrons of the isothiocyano group of FITC via an electrophilic addition reaction (Scheme 1b), thereby forming new thiourea groups in the resultant covalently labeled FLSNC. The p−π conjugated system involving the amino group, thiourea group, and aromatic nucleus of the fluorophores was more stable than the original adjacent double bond structure of 3754
DOI: 10.1021/acssuschemeng.6b02157 ACS Sustainable Chem. Eng. 2017, 5, 3751−3761
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Figure 2. General spectra from XPS analysis. Samples are indicated in the figure.
with APTES. Similarly, Si 2p spectra showed one peak at 101.3 eV (Si−O−C bonds in AM-SNC), confirming the presence of a covalent C−O bond to silicon or siloxane networks of APTES. Compared with FL-SNC spectra, C 1s spectra of AM-SNC revealed an increase in the proportion of C−N bonds from 15.9 to 27.1% and a decrease in the hydrocarbon content from 28.0 to 18.7%. Furthermore, C 1s spectra displayed one peak at 291.4 eV (C−H bond in the benzene ring), and a peak characteristic of N−CS appeared at 400.7 eV in N 1s spectra. Both benzene ring and N−CS signals belong to the FITC fluorophore, which proved that the amino group of AM-SNC had been successfully linked to the isothiocyano group of FITC by a covalent bond. In addition, C 1s spectra showed an increase in CO and CS bonds from 2.6 to 10.5%, consistent with the incorporation of isothiocyano group CS bonds onto the surface of AM-SNC. Spectral assignment of C 1s, Si 2p, and N 1s regions therefore confirmed that the SNC had formed a covalent connection with fluorescein following treatment with APTES and FITC. Further evidence of C−N and N−CS bonds resulting from the covalent linkage between SNC and APTES, as between AM-SNC and FITC, was provided by solid state CP/ MAS 13C NMR. Spectra of SNC, AM-SNC, and FL-SNC alongside their molecular formulas (Figure 4) showed the characteristic carbon assignments and signal shifts described previously in the literature.39 For SNC sample, the peak at 60.8 ppm was assigned to carbon C6, and the region at 69.2−74.8 ppm was attributed to indistinguishable carbons C2, C3, and C5. The peak at 81.2 ppm was assigned to C4 carbons. Meanwhile, the region between 98.0 and 100.2 ppm was attributed to the C1 carbons of the waxy corn starch. After amino modification and fluorescein labeling, the samples exhibited quite different spectra. AM-SNC displayed three new peaks, corresponding to aliphatic aminopropyl carbons as follows: (1) The signal at 10.9 ppm corresponded to the C7 carbon of the methylene group linked to silicon. (2) The signal at 21.6 ppm corresponded to the C8 carbon of the methylene group connected to methylenes on both sides. (3) The peak at 41.5 ppm corresponded to the C9 carbon of the methylene group connected to the primary amine group. Moreover, FL-SNC spectra also exhibited unique peaks, with strong signals between 110.7 and 131.9 ppm corresponding to the Cx carbon atom of the protonated aromatic ring of FITC, and a small signal at 157.8 ppm corresponding to the Cy aromatic carbon connected to the substituted hydroxyl. The signal at 170.0 ppm was related to the C10 carbon of the isothiocyano group, and the chemical shift of this group increased due to the intensive p−π
concentration percentages are summarized in Table 1. Likewise, binding energies and the percentage of the surface containing functional groups are summarized in Table 2. Table 1. Elemental Surface Concentration Percentages for SNC, AM-SNC, and FL-SNC percentages (%) samples
O
C
S
N
Si
SNC AM-SNC FL-SNC
37.70 28.86 0.899
62.23 57.43 1.55
0.005
6.18 3.45
6.18 3.45
Table 2. Binding Energies of Surface Functional Groups Composition of SNC, AM-SNC, and FL-SNC Obtained from the Deconvolution of C 1s, Si 2p, and N 1s Signals percentages (%) SNC samples functional groups
BE (eV)a
C−Si C−C/C−H C−O CO/CS C−N C6H6 Si−O−C Si−O−Si N−C N−CS
284.4 285.0 286.5 288.0 286.0 291.4 101.3 102.4 399.9 400.7
AM-SNC
C 1s
C 1s
52.9 36.3 10.8
28.0 22.4 31.1 2.6
Si 2p
FL-SNC C 1s
N 1s
18.7 14.2 25.0 10.5 27.1 4.5 5.4 94.6 88.9 11.1
a
The binding energy for each signal was given with variation seen between different samples.
Figure 3 shows the deconvolution of the XPS patterns of C 1s, Si 2p, and N 1s regions. The C 1s signal revealed six peaks at 284.4, 285.0, 286.0, 286.5, 288.0, and 294.1 eV, arising from C1 (C−Si), C2 (C−C/C−H), C3 (C−N), C4 (C−O), C5 (CO/CS), and C6 (C6H6), respectively. The Si 2p signal exhibited two peaks at 101.3 and 102.4 eV, arising from Si1 (Si−O−C) and Si2 (Si−O−Si). Likewise, the N 1s signal showed two peaks at 399.9 and 400.7 eV, arising from N1 (N− C) and N2 (N−CS). Comparison with the SNC C 1s spectra indicated that the hydrocarbon content of AM-SNC decreased from 52.9 to 22.4%, whereas that of silicon increased from 0 to 28.0%. The result indicated that C−Si bonds had replaced a proportion of the C−H and C−C bonds through the amino modification 3755
DOI: 10.1021/acssuschemeng.6b02157 ACS Sustainable Chem. Eng. 2017, 5, 3751−3761
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Figure 3. High-resolution C 1s, Si 2p, and N 1s deconvoluted XPS spectra of (a) SNC, (b, c) AM-SNC, and (d−f) FL-SNC.
characteristic peak) and anionic (481 nm, the shoulder peak) forms of FITC, and these peaks were red-shifted and sharper due to the self-condensation of APTES during the amino modification. These results showed that the fluorescence properties of FL-SNC were distinct due to the amino functionality. As shown in Figure 5c, spectra of SNC and AM-SNC did not exhibit any absorption peak, which confirmed the absence of amino modification by APTES, whereas those of FITC, FLSNC, and FA-SNC all displayed an emission wavelength at 520 nm, indicating that the two-step modification did not affect the fluorescence emission wavelength of FITC. The fluorescence emission intensity of FL-SNC was higher than that of FA-SNC. This result suggests that regardless of the intermolecular forces, additional bonds were present between FITC and the modified SNC, suggesting that the grafting density of FITC was increased following covalent conjugation. In summary, the enhanced fluorescence properties of FL-SNC were verified by both UV/vis absorption and fluorescence emission spectra. Photostability measurements of the FL-SNC, FA-SNC, and FITC were demonstrated by fluorescence microscopy. Images were acquired at 120 min intervals. As FITC, FA-SNC, and FLSNC had the same excitation wavelength, their photostability could be compared under the same conditions. As shown in Figure 6, under illumination, the fluorescences of FITC and FA-SNC rapidly diminished by 84.3 and 80.8% after 30 min, respectively. On the contrary, the fluorescence of FL-SNC was remarkably stable, and its intensity was maintained at about 72.1% even after 120 min. Furthermore, a large number of small particles of FL-SNC (size within 1 μm) showed significant luminesce even after 90 min, while the small particles in FITC and FA-SNC samples no longer showed any luminescence. The comparison of the photostability results
conjugation resulting from linkage of the primary amine group of AM-SNC with the isothiocyano group of FITC. The final peak at 181.1 ppm was attributed to the C11 carbonyl carbon of the lactonic ring of FITC. These results confirmed that the modification of SNC by APTES and FITC was successful and that the amino group was incorporated and reacted with the isothiocyano group as intended. Fluorescence Properties of FL-SNC. The resulting FLSNC sample was thoroughly washed to remove unreacted dye, which might otherwise be physically absorbed on the surface by intermolecular forces. Covalent conjugation between FITC and SNC was confirmed by comparing the fluorescence properties of FL-SNC with FITC and those of FA-SNC with SNC from UV-lamp-induced apparent fluorescence, UV/vis absorption, and fluorescence emission spectra. Differences in apparent fluorescence of FA-SNC and FL-SNC samples were observed following exposure to a UV lamp (Figure 5a). The fluorescence intensity of FL-SNC was distinctly stronger than that of FASNC, suggesting the introduction of amino groups increased the grafting density of FITC significantly. In contrast, the weaker fluorescence intensity of control samples (SNC and FASNC) indicated that they did not react with FITC and suggested that removal of FITC by washing was successful. Therefore, no clear fluorescence was detected following exposure to UV light. UV/vis absorption spectra were obtained for SNC, FL-SNC, and FA-SNC in aqueous solution (Figure 5b). SNC and FA-SNC did not have an absorption peak within the 200−600 nm wavelength range, but FITC did exhibit significant absorption maxima for both the dianionic (483 nm, the characteristic peak) and anionic (463 nm, the shoulder peak) forms, in accordance with previous findings, when in suspension and at pH 6.0.40 In comparison, spectra of FL-SNC showed absorption maxima for both the dianionic (510 nm, the 3756
DOI: 10.1021/acssuschemeng.6b02157 ACS Sustainable Chem. Eng. 2017, 5, 3751−3761
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ACS Sustainable Chemistry & Engineering
Figure 4. Solid-state CP/MAS 13C NMR spectra and molecular structural formula. Samples are indicated in the figure.
Figure 5. Comparison of the fluorescent properties of FL-SNC and FA-SNC. (a) Suspension in UV light and visible light: (I) SNC, (II) FA-SNC, and (III) FL-SNC, (b) UV/vis absorbance spectra, and (c) fluorescence emission spectra (Excitation wavelength, λ excitation = 495 nm).
FITC,42 their brightness was sufficient for the application in bioimaging or biosensors. Effect of Two-Step Modification of SNC. Crystalline Integrity. Chemical modification of SNC was intended to modify the surface while preserving the integrity of the crystal lattice. The effects of amino modification and fluorescence labeling on the crystalline integrity were therefore investigated by X-ray diffraction (XRD) analysis. As shown in Figure 7a, SNC displayed a level of crystallinity that was typical of A-type
demonstrated that FL-SNC was photobleached at a lower rate than were FITC and FA-SNC. This may be result from the fact that covalently linked fluorophores concentrated on the surface of SNC which prevented the photobleaching of FL-SNC. Similar results were also obtained by Schulz et al.41 The QY value of FL-SNC was 0.57 in aqueous solution (pH 7.0) using Rhodamine B (QY = 0.89 in ethanol) as reference. Although the QY value of FL-SNC was approximately half that of 3757
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Figure 6. Time-course measurement of photostability. Images were taken at the same output light intensity and the same aperture size. (a) FITC, (b) FA-SNC, and (c) FL-SNC (scale bars = 20 μm).
Figure 7. Comparison of the properties of SNC, AM-SNC, and FL-SNC. (a) XRD spectra with the relative crystallinity; (b) water dispersibility of suspensions (0.05%, w/v): (I) SNC, (II) AM-SNC, and (III) FL-SNC; (c) particle size distribution of suspensions (0.01%, w/v); and (d) TEM images of samples.
cereal starch, with strong diffraction peaks at 2θ = 15 and 23° and a dual diffraction peak at 17 and 18°. After the two-step chemical modification by APTES and FITC, it was apparent that both crystalline features remained intact, since the basic Atype diffraction pattern was still clearly visible in the AM-SNC and FL-SNC data. However, in the AM-SNC and FL-SNC diffraction patterns, signals were present at 7.3°, indicating Vtype crystallinity, which presumably resulted from the addition of excess ethanol that complexed with SNC. The relative crystallinity was also calculated and found to be 45.6% (±2.5%) for SNC, 50.9% (±2.5%) for AM-SNC, and 57.1% (±2.5%) for FL-SNC. A significant increase in crystallization was therefore evident following addition of APTES and FITC, consistent with the results of Wang et al.,43 indicating that the cellulose crystal structure was affected by surface modification by the silanecoupling agent or alkali treatment. The presence of only small changes in crystallization further indicated that the two-step chemical modification of SNC did not significantly affect the crystal structure. Aqueous Redispersibility, Size Distribution, Morphology, and Dimensions. Figure 7b shows redispersed suspensions of SNC, AM-SNC, and FL-SNC samples. All samples formed stable colloidal suspensions in water that were slightly opaque, and the color was dependent on that of the powder added. Therefore, modification of SNC by APTES and FITC did not affect the redispersibility in water.
The SNC suspension exhibited a broad peak at 100−600 nm and a mean particle size of 265 nm, whereas the particle size ranged of AM-SNC and FL-SNC were 127−265 nm and 147− 307 nm, respectively (Figure 7c). Two-step chemical modification by APTES and FITC therefore shifted the size distribution to a smaller range, as well as improving the water dispersibility. TEM images of SNC, AM-SNC, and FL-SNC samples (Figure 7d) showed that after the two-step chemical modification particles were more individualized and monodisperse with an average size of ∼150 nm in the case of AMSNC and ∼200 nm in the case of FL-SNC consistent with the water dispersibility and particle size distribution results. Due to the introduction of amino groups, the pH of redispersed AMSNC (9.7−10.4) was higher than that of the SNC suspension (6.2−7.5), which improved agglomeration in alkali conditions.44 Gray objects were considered to be semicrystalline, while black objects were presumed to be more highly crystalline fragments. SNC nanoplatelets appeared to aggregate, presumably due to hydrogen bonding interactions with surface hydroxyl groups. Reaction with the silane-coupling agent and fluorescein improved the dispersibility of modified SNC significantly, even though their laminar shape remained similar to unmodified SNC, albeit with smoother edges and a more diffuse boundary. The edges of modified SNC were also lighter in color than the central regions of the particles, which suggests that the surface modification can alter the edge properties. 3758
DOI: 10.1021/acssuschemeng.6b02157 ACS Sustainable Chem. Eng. 2017, 5, 3751−3761
Research Article
ACS Sustainable Chemistry & Engineering Cellular Bioimaging Applications. FL-SNC may have great potential as biosensors or biomarkers due to their characteristics of superior photostability, good aqueous redispersibility, integrated crystallinity, and favorable size distribution. In order to explore the biocompatibility of FLSNC, the cell viability was quantitatively evaluated by the MTT assay. Figure 8 shows the effect of FL-SNC concentration on
The cell viability evaluation verified that the routine for fluorescent labeling of SNC did not produce additional cytotoxicity and confirmed that it could be used as biosensors or biomarkers. In order to evaluate the availability of FL-SNC used as a biomarker for live cell imaging, CLSM experiment was carried out. HepG2 cells treated with FL-SNC (100 μg/mL) for 2 h (Figure 9a−c) and 4 h (Figure 9d−f) were observed. After an incubation of 2 h, the majority of FL-SNC was incorporated by the cells with only a small fraction (
