Accepted Manuscript Title: Sodium hydroxide-mediated hydrogel of citrus pectin for preparation of fluorescent carbon dots for bioimaging Author: Xi Juan Zhao Wen Lin Zhang Zhi Qin Zhou PII: DOI: Reference:
S0927-7765(14)00519-0 http://dx.doi.org/doi:10.1016/j.colsurfb.2014.09.048 COLSUB 6649
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
29-5-2014 21-9-2014 23-9-2014
Please cite this article as: X.J. Zhao, W.L. Zhang, Z.Q. Zhou, Sodium hydroxide-mediated hydrogel of citrus pectin for preparation of fluorescent carbon dots for bioimaging, Colloids and Surfaces B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.09.048 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Sodium hydroxide-mediated hydrogel of citrus pectin for
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preparation of fluorescent carbon dots for bioimaging Xi Juan Zhaoa,b, Wen Lin Zhang a, Zhi Qin Zhoua, b, *
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a
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China, E-mail:
[email protected];
[email protected] 6
b
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Education, Chongqing 400715, China, Fax: 86-23-68251274; Tel: 86-23-68250229; E-
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mail:
[email protected] us
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College of Horticulture and Landscape Architecture, Southwest University, Chongqing 400716,
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Key Laboratory of Horticulture Science for Southern Mountainous Regions, Ministry of
*
Corresponding author, email:
[email protected] 1
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Highlights
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● NaOH-mediated hydrogel of citrus pectin can be used to synthesize fluorescent CDs.
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● The hydrogel can make the hydrothermal reaction of CDs down to 100 0C.
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● The hydrogel can avoid visually carbonized precipitates even up to 180 0C.
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● The fluorescent CDs with good biocompatibility make them successful in cell
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imaging.
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● The use of citrus pectin will promote pomace utilization of the genus Citrus L.
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Abstract
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The citrus process industry produces annually a huge amount of pomace, which is
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a rich source of citrus pectin. Here, we report the hydrogel of citrus pectin mediated by
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sodium hydroxide can be used to prepare fluorescent carbon dots (CDs). The
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introduction of hydrogel can not only make the temperature of the hydrothermal
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reaction down to 100 0C, but also avoid visually carbonized precipitates in the synthesis
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process even up to 180 0C. The as-synthesized CDs are well dispersed in water with an
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average size of 2.7 nm and show cyan fluorescence with high photostability, good
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biocompatibility. Furthermore, the CDs can act as a potential fluorescent probe for cell
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imaging. Citrus pectin as a non-toxic carbonaceous precursor for preparation of 2
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fluorescent CDs provides a new approach for the efficient utilization of citrus
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germplasm in future.
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Key Words: citrus pectin; hydrogel; fluorescent; carbon dots.
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1. Introduction
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Citrus is one of the most commonly consumed fruits in the world with an annual
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production of more than 100 million tons [1]. Citrus fruits can be consumed freshly or
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processed into dessert, juice and jam, and are good resources of vitamin C, folic acid,
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flavonoids, dietary fibre and many other health-promotion substances [2]. The citrus
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process industry produces annually a huge amount of pomace (usually 50% of the raw
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fruits). Besides the use as animal feed, the pomace can provide a rich source of pectin
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for food and other industries. Pectin is a naturally water-soluble polysaccharide with
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low production cost. Its backbone comprises linear chains of 1,4-linked α-D-
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galacturonic acid residues with some rhamnogalacturonic acid residue and α-L-
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rhamnopyranose by α-1-2 linkage, and contains many carboxyl groups and some methyl
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esters [3]. Owing to its biocompatibility, biodegradability and non-toxicity, pectin has
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been widely used in the pharmaceutical and food industries, e.g., acting as a carrier and
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coating material [4-6], as well as a gelling and thickening agent [7]. Nevertheless, the
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efficient use of citrus pomace has still been a problem unresolved. In this work, pectin is
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reported to be a non-toxic carbonaceous precursor for preparation of fluorescent carbon
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dots (CDs). The efficient use of citrus pectin will promote future germplasm utilization
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study of the genus Citrus L.
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CDs have attracting considerable attention since the discovery of fluorescent
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carbon nanoparticles in 2004 [8]. Different from semiconductor quantum dots with the 3
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known toxicity and potential ecosystem hazard [9-10], CDs have their fascinating
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properties such as low cytotoxicity and good biocompatibility for fluorescent labeling
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[11]. To date, various routes for preparing CDs have been reported [12-14], e.g., laser
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ablation of graphite [15], electrochemical oxidation of multiwalled carbon nanotubes
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[16], hydrothermal reaction of some carbonaceous precursors such as C60 [17], bombyx
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mori silk [18] and soy milk [19]. Among them, the hydrothermal route is popular owing
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to the simple reaction process and renewable carbonaceous precursors. And bio-
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precursors are particularly attractive considering the eco-friendly effects. Zhou et al. has
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synthesized fluorescent CDs from the hydrothermal carbonization of peach gum
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polysaccharide under the temperature of 180 0C for 12 h [20]. Such a high temperature
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can facilitate the carbonization of the precursors [21], but simultaneously lead to a lot of
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visually black particles in the products. While Sahu et al. reported that orange juice by
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the hydrothermal treatment of 120 0C can produce highly luminescent CDs [22].
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However, orange juice is a very nutritious drink, which is not suitable for mass
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production of CDs compared with the pectin that can be extracted from plenty of the
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residual pomace in the citrus processing. Here, we report a new kind of fluorescent CDs
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synthesized from the sodium hydroxide-mediated hydrogel of citrus pectin at relatively
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low temperatures down to 100 0C. The involvement of hydrogel plays an important role
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in lowering the reaction temperature and can avoid visually carbonized precipitates even
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up to 180 0C.
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2. Material and methods
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2.1. Reagents and chemicals All reagents were obtained from commercial sources and used as received if no
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additional statement. Pectin from citrus peel (Galacturonic acid ≥74.0 %, dried basis)
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was purchased from Sigma-Aldrich Co. LLC. (USA). Ultrapure water (18.2 MΩ)
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prepared with a Milli-Q system (Millipore, Bedford, MA, USA) was used throughout
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the experiments.
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2.2. Apparatus and characterization
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Transmission electron microscopy (TEM) and high-resolution transmission
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electron microscopy (HRTEM) were recorded with the Tecnai G2 F20 S-TWIN
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microscopy (FEI, USA). Elemental and functional groups analysis were made on an
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ESCALAB 250 X-ray photoelectron spectrometer and a FTIR-8400S Fourier transform
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infrared spectrometer (Shimadzu, Japan), respectively. Raman spectrum was taken on
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the Ag substrate using a LabRAM HR 800 Raman spectrometer (Horiba Jobin Yvon
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Inc., France). The zeta potential was recorded with Nano-ZS spectrometer (Malvern, UK)
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using the CDs after dialysis and in such case, the pH value of the CDs solution was
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determined to be about 7.0 with the pH test paper in order to make sure that residual NaOH
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and other small molecules have been detached. Fluorescence imaging was carried out
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with a DSU live-cell confocal microscope (Olympus, Japan) system. Absorption and
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fluorescence spectra were
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spectrophotometer (Hitachi, Japan) and an F-2500 fuorescence spectrophotometer
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(Hitachi, Japan), respectively.
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measured at
room temperature with a U-3010
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2.3. Preparation of CDs The CDs were synthesized using pectin by a hydrothermal method. In a typical
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synthesis, 20 mL Teflon-lined stainless steel autoclave was cleaned in a bath of fresh
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aqua regia and rinsed thoroughly in H2O before using. Then, 2 mL of 10 g/L pectin and
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2 mL of 1 mol/L NaOH were added to the autoclave. In such case, the hydrogels of
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pection formed. And the autoclave was maintained at 100 0C, 120 0C, 150 0C or 180 0C
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for 2 hour. After the autoclave cooled down naturally, a homogeneously light brown
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solution was obtained, indicating the formation of CDs. Next, the light brown solution
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was centrifuged at 15000 rpm for 10 min to get the upper solution. Through a dialysis
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membrane (1000 MWCO), residual NaOH and other small molecules will be detached.
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The obtained CDs solution was then freeze-dried under vacuum in order to get solid
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powder for further use.
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As for the contrast experiments, there is no NaOH-mediated hydrogel in the
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synthesis procedure. The details are shown as below. 2 mL of 10 g/L pectin and 2 mL of
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H2O were added to the 20 mL autoclave. After mixing thoroughly, the autoclave was
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maintained at 100 0C, 120 0C, 150 0C or 180 0C for 2 hour. When the autoclave cooled
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down naturally, we can obtain the corresponding products with different states.
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2.4. Biocompatibility testing
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The HEp-2 cells (2×105 cells/mL) in RPMI 1640 culture medium supplemented
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with 10% fetal bovine serum (FBS) were added to each well of a 96-well plate (100 L
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per well). The cells were first cultured for 24 h in an incubator (37 0C, 5% CO2), and for
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another 24 h, the culture medium was replaced with 100 L of RPMI 1640 (2% FBS)
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containing 10 L of the CDs prepared at 180 0C with different concentrations (0, 0.01, 6
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0.05, 0.1, 0.2, 0.5, 1 mg/mL). Cells cultured in the medium without adding CDs were
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taken as control. After that, the cells were washed with PBS buffer solution twice, and
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then, 90 L PBS buffer and 10 L of CCK-8 solution were added to each well and
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incubated for another 30 min. The optical density (OD) of the mixture was measured at
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450 nm with a Microplate Reader Model. The cell viability was estimated by the ratio
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of absorbance of the cells incubated with CDs to that of the cells incubated with the
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culture medium only.
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2.5. Intracellular uptake and fluorescent imaging
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The HEp-2 cells in RPMI 1640 culture medium containing 10% fetal bovine serum
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were added to each well of a 24-well plate (300 L per well). The cells were first
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cultured for 24 h in an incubator (37 0C, 5% CO2), and for another 24 h after the culture
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medium was replaced with 270 L of RPMI 1640 culture medium containing 30 L
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CDs obtained at 180 0C (1 mg/mL). After that, followed removing the culture medium,
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each well was washed with PBS buffer for three times. Then the cells were fixed with
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4% paraformaldehyde for 30 min, and mounted with glycerol on microscope slides for
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imaging.
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3. Results and discussion
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3.1. Synthesis of the fluorescent CDs
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(Fig. 1)
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The aqueous solution of pectin shows a distinct absorption band centered at 286
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nm with a shoulder peak at around 350 nm (Fig. 1A). Here, when an appropriate 7
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amount of the aqueous solution of NaOH is added to the pectin solution, hydrogel of
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pectin forms immediately accompanied with the color change to light-green. However,
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the absorption spectrum of pectin has no significant changes as shown in Fig. 1A. After
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the hydrogel was transferred to Teflon-lined stainless steel autoclave maintaining at 100
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0
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and the color of the product turns yellow or light brown to some extent (Fig. 1C),
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indicating the formation of CDs. Also, the absorption spectra have been changed. The
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featured absorption peak at 286 nm disappeared and a new one centered at 260 nm
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appeared gradually with increasing the temperature (Fig. 1B). Under 365 nm UV lamp
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light, the CDs can exhibit varying degrees of cyan fluorescence emission. And the
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higher the temperature, the stronger the fluorescence intensity will be (Fig. 1D). Since
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either pectin or the hydrogel has no fluorescence emission, the emission comes from the
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new generated CDs. Furthermore, the fluorescence spectra of the corresponding CDs
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are dependent of the excitation wavelength (Fig. 2 and Supporting Information, Fig. S1-
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S3). This excitation-dependent emission property is in accordance with that of CDs in
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previous reports. As shown in Fig. 2, the CDs obtained at 180 0C as an example have a
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broad UV absorption band with a featured peak at 260 nm and exhibit strong emission
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centered at around 460 nm when excitated at 360 nm. The emission peaks can shift
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from 460 nm to 554 nm with the increase of the excitation wavelength. And the
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quantum yield is calculated to be approximately 1.1% with quinine sulfate as the
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reference, which is comparable to that of the CDs obtained from natural gas soot (0.43%)
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as reported [13].
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C, 120 0C, 150 0C or 180 0C for two hours, respectively, the hydrogel will be destroyed
(Fig. 2)
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However, if no hydrogel is mediated in the synthesis procedure, no fluorescent
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CDs can be formed from pectin under the temperature of 100 0C and 120 0C for 2 hours
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(Supporting Information, Fig. S4). The color of the pectin solution has no changes, and
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under 365 nm UV lamp light, the aqueous solution shows very weak fluorescence
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emission and there is no distinct excitation-dependent emission property of CDs as
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shown in the fluorescence spectra (Supporting Information, Fig. S5). When the
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temperature reaches 150 0C, the product becomes brown with a lot of visible black
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precipitates, suggesting the pectin has been carbonized (Supporting Information, Fig.
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S4). After getting rid of the precipitates, the upper solution shows blue fluorescence
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(Supporting Information, Fig. S5). In fact, carbonization is more serious under 180 0C
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and the fluorescence of the upper solution is decreased (Supporting Information, Fig.
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S5). So, sodium hydroxide-mediated hydrogel can make the fluorescent CDs
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synthesized under the temperatures down to 100 0C and also can avoid visually
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carbonized precipitates even up to 180 0C.
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(Fig. 3)
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3.2. Characterization of the fluorescent CDs The TEM image reveals that the as-prepared CDs well separate from each other
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(Fig. 3A and Supporting Information, Fig. S6). The corresponding particle size
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distribution histogram shows that the CDs have a narrow size distribution with an
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average size of 2.7 nm. Composition and element analysis of the synthesized CDs were
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performed by XPS as shown in Fig. 3B and C. The wide scan XPS spectrum has three
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strong peaks ascribed to sodium, oxygen and carbon in turn. The sodium comes from
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NaOH added in the synthesis process of CDs. In addition, there is a very weak peak at
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about 400.0 eV, indicating the presence of nitrogen. Since pectin is a polysaccharide 9
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with chemical composition of C, H and O, the nitrogen might come from impurities. So,
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the peak at 285.7 eV in the C1s spectrum can be attributed to C–N band [18]. And the
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other three peaks at 284.6, 286.7 and 288.1 eV in Fig. 3C are attributed to the C–C, C–
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O, and C=O bands, respectively [18]. FTIR spectra show that the CDs obtained at 100
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0
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indicating that the four CDs have the same functional groups (Supporting Information,
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Fig. S7). The characteristic absorption bands of O–H stretching vibrations at 3422 cm-1,
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C=O stretching vibrations at 1643 cm-1, C=C stretching vibrations at 1600 cm-1, and C–
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H bending vibrations at 1447 cm-1 suggest that functional groups such as –COOH and –
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OH were attached on the surface of CDs (Supporting Information, Fig. S7). The zeta
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potential of the CDs was -41.3±1.1 mV, indicating the surface was negatively charged
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due to the carboxylic acid groups. Raman spectum taken on the Ag substrate was used
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to identify the state of carbon. A narrow G-line at around 1604 cm-1 and a broad D-line
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at around 1370 cm-1 indicated the presence of both sp2 carbon and sp3 hybridized carbon
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in the CDs (Fig. 3D).
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3.3. Mechanisms of sodium hydroxide-mediated hydrogel formation and CDs
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C, 120 0C, 150 0C and 180 0C have almost the same characteristic absorption bands,
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Usually, pectin is able to form hydrogel depending on several parameters such as
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saccharose, low pH values and polyvalent cations. Under alkaline conditions such as in
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the presence of sodium hydroxide, formation of the pectin hydrogel has hardly been
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reported before. It is known that under the alkaline conditions, polysaccharides will
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undergo various transformations such as alkaline hydrolysis, depolymerization, and
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oxidizing destruction [23]. And Zahran et al. indicated that these processes can take
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place for pectin to give galacturonic acid and some monosaccharides, e.g., galactose, 10
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arabinose, rhamnose, glucose, and mannose [24]. In this work, once the appropriate
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amount of NaOH was added to the pectin solution, hydrogel forms immediately. Since
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there are no divalent or trivalent metal ions, the formation of hydrogel is mainly
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attributed to the saccharides produced by pectin in the alkaline medium. Saccharides
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can serve as the dehydrating agent and promote the formation of hydrogen bonds
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between pectin molecules through competing with pectin to bind water. And further,
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micelles and three-dimensional network structures of pectin will form, finally leading to
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the formation of hydrogel. In fact, the sodium hydroxide-mediated hydrogel here is a
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multicomponent carbohydrate owing to the transformations of a part amount of pectin,
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which is different from the hydrogel of pectin formed in the other media.
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Compared to the pectin solution alone, the sodium hydroxide-mediated hydrogel of
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pectin is easier to produce fluorescent CDs under lower temperatures down to 100 0C. It
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is obvious that the hydrogel plays an important role. In our opinion, the hydrogel
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decreased some complex chemical reactions that pectin should undergo under
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hydrothermal conditions such as structure destruction, polymerization and carbonization
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[25]. In the hydrogel, there are already network structures of pectin mixed with the
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saccharides produced by pectin in the presence of sodium hydroxide. During the
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hydrothermal treatment, the hydrogel can be broken down into individual small
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nanoparticles yielding fluorescent CDs.
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3.4. Application of the fluorescent CDs
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(Fig. 4)
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The obtained CDs show excellent photostability, and no sign of photobleaching
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was observed even after 60 min of continuous excitation at 370 nm with the Xe lamp
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(Supporting Information, Fig. S8). Furthermore, the cytotoxicity of the CDs and their 11
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fluorescent imaging were evaluated by using HEp-2 cells as a sample. As shown in Fig.
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4A, the average cell viability was greater than 97% at all these tested doses of CDs
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ranging from 0 to 1.0 mg/mL. Even after the incubation time increased up to 24 hours,
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the cell proliferation was not affected, indicating the CDs are very biocompatible. Fig.
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4B and C display that the CDs possess good fluorescent signals for cell imaging, and
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are very likely to enter the cells through the cell membrane via endocytosis. The CDs
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can be located in the cell membrane and cytoplasm, lightening the cells. A control
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experiment of cells without incubation of CDs was also performed, showing that no
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obvious autofluorescence and negligible endogenous fluorescence can be observed
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under the same conditions (Supporting Information, Fig. S9). These results indicate that
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the CDs can serve as a potential fluorescent probe for cell imaging.
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4. Conclusions
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In this study, we report that sodium hydroxide can mediate the hydrogel formation
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of citrus pectin, which is different from commonly gel-forming conditions of pectin.
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And through the involvement of the hydrogel, we can successfully synthesize
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fluorescent CDs at low temperatures down to 100 0C by using the hydrothermal
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treatment. The CDs have high photostability, good biocompatibility and a potential use
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for fluorescent imaging in cells. Our study suggests a new approach for efficient use of
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citrus pectin in future, and has significance for the disposal of pomace produced in the
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citrus process industry.
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Acknowledgements
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This work was financially supported by China Postdoctoral Science Foundation (No.
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2014M552304), Chongqing Postdoctoral Science Foundation (Xm2014005), the
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Fundamental Research Funds for the Central Universities (XDJK2014A014), and the
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Program for Chongqing Innovation Team of University (KJTD201333).
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version,
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at http://www.sciencedirect.com.
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Captions for Figures Fig. 1. Optical properties of pectin and CDs. (A) UV-vis absorption spectra of pectin alone and pectin in the presence of NaOH. Inset: the optical images of pectin solution and the hydrogel of pectin formed in the presence of NaOH under daylight. (B) UV-vis absorption spectra of CDs
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prepared at 100 0C, 120 0C, 150 0C and 180 0C. (C) Optical images of CDs under daylight. (D) The
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corresponding images of CDs under 365 nm UV lamp light.
Fig. 2. Characteristic spectra of CDs obtained at 180 0C. (A) UV-vis absorption spectrum (left),
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excitation spectrum (middle) and emission spectrum (right) of the CDs. Inset: the optical images of CDs under daylight and UV light, respectively. (B) The fluorescence spectra of the CDs excitated
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with different excitation wavelengths from 360 nm to 500 nm. Inset: the normalized emission spectra of the CDs correspondingly.
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Fig. 3. Characterization of the CDs obtained at 180 0C. (A) TEM image of the CDs (scale bar: 5 nm), lower right inset: the size distribution of CDs based on statistics of the TEM image. (B) Wide XPS
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survey of the CDs. (C) High-resolution XPS C1s spectrum of the CDs. (D) Raman spectrum of the
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CDs. Laser wavelength: 532 nm; slit: 100 m; hole: 400 m; acquisition time: 1 s. Fig. 4. The practicality of CDs obtained at 180 0C for cell imaging. (A) Biocompatibility testing of
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the CDs. (B-C) Fluorescent images of HEp-2 cells incubation with CDs under bright field and excitation at GFP457, respectively.
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Fig. 1. Optical properties of pectin and CDs. (A) UV-vis absorption spectra of pectin alone and
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pectin in the presence of NaOH. Inset: the optical images of pectin solution and the hydrogel of pectin formed in the presence of NaOH under daylight. (B) UV-vis absorption spectra of CDs
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prepared at 100 0C, 120 0C, 150 0C and 180 0C. (C) Optical images of CDs under daylight. (D) The
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corresponding images of CDs under 365 nm UV lamp light.
Fig. 2. Characteristic spectra of CDs obtained at 180 0C. (A) UV-vis absorption spectrum (left), excitation spectrum (middle) and emission spectrum (right) of the CDs. Inset: the optical images of CDs under daylight and UV light, respectively. (B) The fluorescence spectra of the CDs excitated with different excitation wavelengths from 360 nm to 500 nm. Inset: the normalized emission spectra of the CDs correspondingly. 17
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Fig. 3. Characterization of the CDs obtained at 180 0C. (A) TEM image of the CDs (scale bar: 5 nm),
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lower right inset: the size distribution of CDs based on statistics of the TEM image. (B) Wide XPS survey of the CDs. (C) High-resolution XPS C1s spectrum of the CDs. (D) Raman spectrum of the
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CDs. Laser wavelength: 532 nm; slit: 100 m; hole: 400 m; acquisition time: 1 s.
Fig. 4. The practicality of CDs obtained at 180 0C for cell imaging. (A) Biocompatibility testing of the CDs. (B-C) Fluorescent images of HEp-2 cells incubation with CDs under bright field and excitation at GFP457, respectively.
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Graphical abstract
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