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Fluorescent carbon dots derived from Maillard reaction products: Their properties, biodistribution, cytotoxicity and antioxidant activity Dongmei Li, Xiaokang Na, Haitao Wang, Yisha Xie, Shuang Cong, Yukun Song, Bei-Wei Zhu, and Mingqian Tan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05643 • Publication Date (Web): 23 Jan 2018 Downloaded from http://pubs.acs.org on January 24, 2018
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Journal of Agricultural and Food Chemistry
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Fluorescent carbon dots derived from Maillard reaction products: Their
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properties, biodistribution, cytotoxicity and antioxidant activity
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4
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Dongmei Li1,2, Xiaokang Na1,2, Haitao Wang1,2, Yisha Xie1,2, Shuang Cong1,2, Yukun
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Song1,2, Xianbing Xu1,2, Bei-Wei Zhu1,2 and Mingqian Tan1,2*
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8
1
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Seafood, Dalian Polytechnic University, Qinggongyuan 1, Ganjingzi District, Dalian
School of Food Science and Technology, National Engineering Research Center of
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116034, Liaoning, People’s Republic of China
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2
12
116034, Liaoning, People’s Republic of China
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*Corresponding
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[email protected], ORCID: 0000000275350035)
Engineering Research Center of Seafood of Ministry of Education of China, Dalian
author
(Tel
&
Fax:
+86-411-86318657,
ACS Paragon Plus Environment
E-mail:
Journal of Agricultural and Food Chemistry
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Abstract
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Food-borne nanoparticles have received great attention owing to their unique
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physico-chemical properties and potential health risk. In this study, carbon dots (CDs)
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formed during one of the most important chemical reactions in food processing field,
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the Maillard reaction from the model system including glucose and lysine, was
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investigated. The CDs purified from Maillard reaction products emitted a strong blue
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fluorescence under ultraviolet light with a fluorescent quantum yield of 16.30%. In
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addition, they were roughly spherical with sizes of around 4.3 nm and mainly
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composed of carbon, oxygen, hydrogen, and nitrogen. Their surface groups such as
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hydroxyl, amino and carboxyl groups were found to possibly enable CDs to scavenge
25
DPPH and hydroxyl radicals. Furthermore, the cytotoxicity assessment of CDs
26
showed that they could readily enter HepG2 cells while caused negligible cell death at
27
low concentration. However, high CDs concentrations were highly cytotoxic and led
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to cell death via interference of glycolytic pathway.
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Keywords: carbon dots, Maillard reaction, anti-oxidation, cell metabolism,
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cytotoxicity
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Introduction
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The bio-effect of engineered nanomaterials in food items is an emerging research
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topic in both academic and industrial communities1-3. Nanoscale materials (sizes: 1 -
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100 nm) exhibit unique physico-chemical properties, such as high surface to volume
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ratio, enhanced reactivity and distinct photocatalytic ability compared with their
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respective bulk materials.4 Food supplemented with nanomaterials has a variety of
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benefits, including extended shelf life and enhanced flavors.5 However, nanoscale
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materials can penetrate cell and may cause adverse health effects due to their distinct
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physico-chemical properties.6,7 Most current studies of nanomaterials in food mainly
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focus on the assessments of properties and potential toxicity of engineered
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nanomaterials that are added to food as food additives.8,9 These studies, however,
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disregard nanomaterials that can be generated during food processing. It is important
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to understand the origin of such foodborne nanomaterials, especially its potential
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toxicity and biological impacts.
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Carbon dots (CDs), which were first discovered in 2004,10 generally refer to the
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spherical fluorescent nanoparticles with sizes of below 10 nm and are mainly
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composed of carbon, oxygen, hydrogen, and/or nitrogen.11-14 The associations of CDs
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with food have been reported. In 2012, Sk et. al.,15 reported the presence of CDs in
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bread and suger; their formations were ascribed to the heating of carbohydrate. The
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observation was further validated by Al-Hadi, who used human mesenchymal stem
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cells as a model to prove the toxic effects of CDs found in bakery products.16 CDs
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generated during food processing are the same as those engineered CDs produced by ACS Paragon Plus Environment
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hydrothermal synthesis. However, the formation mechanisms, properties, and
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potential health risks of CDs in food are not well understood.
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Maillard reaction, also known as ‘non-enzymatic browning reaction’, is a
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heat-induced browning reaction widely used in the food industry.17 Generally,
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condensation reaction between reducing sugars and amino groups is the first step in
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Maillard reaction. The reaction is then followed by the advanced stage, in which a
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series of complex reactions, including dehydration, rearrangement, isomerization and
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condensation reactions, take place to ultimately lead to the formation of brown
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nitrogenous polymers, known as ‘melanoidins’.17-19 It is important to note that the
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reactions that occur during the advanced stage of Maillard reaction, especially the
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condensation reaction, are analogous to the hydrothermal synthesis process of CDs
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prepared from molecular precursors, such as citric acid and ammonia solution.20,21
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The products from Maillard reaction can emit fluorescence when irradiated with UV
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light. Fluorescent products derived from Maillard reaction of glutathione and ascorbic
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acid have been used as probes for detection of Hg2+ irons or small molecule.22 CDs,
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which were thought to link with Maillard reaction, have recently been used in the
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fabrication of tunable multicolor display.23 Nevertheless, because the exploration of
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CDs formation mechanism is still needed, we are motivated to explore CDs derived
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from the products of Maillard reaction in thermal food processing.
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We have previously investigated the presence of CDs and their properties in
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food items, such as instant coffee,24 beer25 and commercial beverages.26 In addition,
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the formation mechanism of CDs in protein-based food items, such as pike eel27 and ACS Paragon Plus Environment
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beef,28 has recently been demonstrated. These studies have shown that the CDs are
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nitrogen-containing materials while their properties are closely linked to heating
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temperature and raw materials. Moreover, the products generated from Maillard
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reaction are found to highly correlate with types of food and heating temperature.29
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Therefore, it is possible that Maillard reaction may be involved in the formation of
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CDs in food.
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In this study, CDs formed during Maillard reaction of a model system: glucose
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and lysine were investigated. The physic-chemical properties of CDs, such as particle
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size, optical properties, surface group and elemental composition were characterized.
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The reduction activity of CDs was assessed though DPPH- and hydroxyl-radicals
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scavenging activities. Fe2+-chelating ability of CDs (through inhibition of Fenton
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reaction) was studied to assess their antioxidant mechanism. The biodistribution of
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CDs was investigated in onion epidermal and HepG2 cells. Toxicological risks of
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CDs were studied in HepG2 cells. Finally, real-time analysis of cellular respiration
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was conducted to evaluate the effects of CDs on mitochondrial and glycolytic
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functions in HepG2 cells.
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MATERIALS AND METHODS
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Materials
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Glucose, lysine, hydrogen peroxide, quinine sulfate (98%) and (ethylenedinitrilo)
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tetraacetic acid (EDTA) disodium salt were purchased from Aladdin Technology
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(Shanghai, China). Onion was purchased from a local grocery store in Dalian China.
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2,2-Pipheugl-1-picrylhydrazyl
(DPPH)
and
3-(4,5-dimethyl-2-thiazoyl)-2,5-
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diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis,
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USA). Fetal bovine serum and Dulbecco’s minimum essential medium were
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purchased from Hyclone (Logan, UT, USA). All chemicals were of analytical grade
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and were used without further purification.
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Separation of CDs from Maillard reaction products
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Glucose (0.5 g) and lysine (0.5 g) were first dissolved in 5 mL of deionized water. The
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solution was transferred to a 25 mL poly(tetrafluoroethylene) and then autoclaved at
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180 oC for 10 h. The obtained crude product was purified using a D101 macroporous
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adsorption resin column using water as an eluent. The fluorescent fractions were
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collected, concentrated by a vacuum rotary evaporator to remove solvent, and
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lyophilized to yield brown powder (0.25 g, 25%).
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Instrumentation and characterization
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The morphology of the CDs was examined by a JEM-2100 transmission electron
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microscope (TEM) (JEOL, Tokyo, Japan) operation at 200 KV. Fluorescent spectra
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were obtained by a F-2700 spectrophotometer (Hitachi, Tokyo, Japan) at ambient
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conditions. Ultraviolet visible spectra were obtained by a Lambda 35 UV-vis
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spectrophotometer (Perkin Elmer, Norwalk, USA). Lifetime was measured by Fluoro
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Max-spectrofluorometer (Horiba Scientific Co, French) with a 450 nm laser as the
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excitation source. The following equation was used to calculate the fluorescence
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lifetime (τ) of CDs:
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τ = ( A1τ1 + A2 τ 2 + A3 τ3 ) /( A1 + A2 + A3 )
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Where Ai is the fractional contribution of time-resolved decay lifetime of τi.
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The chemical groups on the surface of CDs were analyzed using a Fourier transform
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infrared (FT-IR) spectrometer (Perkin Elmer, USA) with the KBr pellet technique
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ranging from 500 to 4000 cm-1. X-ray photoelectron spectroscopy (XPS) spectra were
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recorded by an Escalab 250Xi X-ray photoelectron spectrometer (ESCALAB250,
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Thermo VG, Waltham, USA). X-ray diffraction (XRD) was measured on a
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diffractometer (XRD-6100, Shimadzu, Kyoto, Japan).
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Measurement of fluorescence quantum yields
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The quantum yield (QY) of CDs was determined with quinine sulfate (dissolved in
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0.1 M H2SO4, QY=55%) as a reference and calculated using the following equation:
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A´ I n2 Ф=Ф´· I´ ·A · 2 n´
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Where Φ is the QY of the CDs, I is the integrated emission intensity of CDs, n is the
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refraction index, and A is the optical density. Ф´, A´, n´ and I´ are QY, optical density,
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refraction index and integrated emission intensity of quinine sulfate, respectively. A
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series of solutions of CDs and quinine sulfate were prepared with optical absorbance
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values less than 0.1. The fluorescent spectra were recorded and their intensities were
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integrated. QY was determined by comparison of the integrated fluorescent intensity
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vs optical density. All the solutions were measured under the same instrumental
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conditions.
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Scavenging of DPPH free radicals
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Scavenging capability of CDs for DPPH free radicals was estimated in this study.
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Two milliliter of fresh prepared DPPH ethanol solution (50 µg mL-1) and 1 mL CDs
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solution with different concentrations was mixed evenly, the absorbance at 519 nm
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was recorded after incubation in dark for 60 min. Ethanol was used as the blank
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control. DPPH radical scavenging capacity was calculated as follows:
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DPPH radical scavenging activity %=(1-Asample/Acontrol)×100%
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Scavenging of hydroxyl free radicals
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Electron spin resonance (ESR) spectroscopy technology was used to determine the
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hydroxyl radical scavenging activity according to the previous method.30 Briefly, 10
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µL of CDs with different concentrations (1.56, 3.12, 6.25, 12.50, 25.00 and 50.00
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mg/mL) were dispersed in deionized water (52 µL), 6 mM EDTA-Na2 (10 µL), 6%
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H2O2 (8 µL), and 1 M 5,5-dimethyl-1-pyrroline N-oxide (DMPO, 10 µL). Ten
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microliters of 6 mM FeSO4 was added to initiate the reaction. After incubated at
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40 °C for 30 min, 40 µL of the mixture was transferred into a glass capillary tube
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(Blaubrand intraMARK, Brand, Germany) for spectral acquisition with an ESR
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spectrometer A200 (Bruker, Karisruhe, Germany). The amplitude of the second peak
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of the spectrum represents the amount of DMPO-OH adducts, which is related to the
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amount of hydroxyl radicals scavenged. The instrumental parameters were used as
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receiver gain: 1.0 × 105, modulation amplitude: 1.0 G, microwave power: 74.8 mw,
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microwave frequency: 9.44 GHz, time constant: 163.84 ms, conversion time: 480 ms
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and modulation frequency: 100.00 kHz. Deionized water was used as a blank control.
Hydroxyl free radicals scavenging activity %=(1- Asample/Acontrol)×100%
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Fe2+-chelating ability
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The Fe2+-chelating capacity was investigated by a modified method as described by
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previous work.31 500 µL of CDs solutions with varying concentrations (1, 2, 4, 6, 8,
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and 10 mg/mL) was mixed with 25 µL FeCl2 (2 mM) and 500 µL distilled water. After
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5 min incubation, 50 µL of ferrozine (5 mM) was added and incubated at room
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temperature for 5 min. After centrifuged at 2000 rpm for 10 min, the absorbance of
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supernatant at 562 nm was measured. The capability of the sample to chelate Fe2+ was
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calculated by the following equation:
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Fe2+ Chelating ability(%) = [1 - Asample/Acontrol] × 100%.
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Bio-distribution of CDs in onion epidermal cells
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A small piece of the epidermal membrane of a fresh onion was treated with 1 mg/mL
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CDs aqueous solution for 10 min. The membrane sample was laid flat on the surface
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of a slide, and then covered with a thin glass coverslip ensuring that there was no air
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bubble. The onion epidermal cells were then imaged by a fluorescence microscope
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(Nikon, Tokyo, Japan) at the excitation wavelength of 405 nm.
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Bio-distribution of CDs in HepG2 cells
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HepG2 cells purchased from the Cell Bank of Type Culture Collection of the Chinese
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Academy of Sciences, Shanghai, China were treated with 1.5 mg/mL CDs aqueous
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solution at 37 °C for 24h.
Then the cells were thoroughly washed with phosphate
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buffer saline (PBS) (500 µL each time) for three times and kept in PBS for optical
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imaging. An inverted laser scanning confocal microscopy (SP8, Leica, Wetzlar,
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Germany) with excitation wavelength of 408, 458 and 488 nm were used for blue,
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green and red region images collection, respectively.
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Cytotoxicity
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The HepG2 cells were seeded in a 96-well plate with the density of 7000 cells/well
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and incubated for 24 h. After incubation with different concentrations of CDs for 12 h,
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20 µL of MTT (5 mg/mL) solution was added and incubated for another 4 h.32 The
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HepG2 cells was further washed with PBS thrice and the resulted formazan crystal
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was dissolved in DMSO. The absorbance of DMSO solution was recorded at 570 nm
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using an Infinite 200 multi-mode microplate reader (Tecan, Hombrechtikon,
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Switzerland). The cell viability was calculated as the following formula:
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Cell viability (%) = Asample/ Ablank×100%
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where Asample and Ablank are the optical density of the CDs and blank control,
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respectively .
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Real-time analysis of cellular respiration
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The effects of CDs on mitochondrial and glycolytic function were studied using an
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XFp Extracellular Flux Analyzer (Seahorse Bioscience, MA, USA), in which oxygen
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consumption rate (OCR) and extracellular acidification rate (ECAR) of HepG2 cells
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were measured in real-time. Briefly, sensor probes were incubated with seahorse XF
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calibration solution at 37 °C for 12 h. HepG2 cells were seeded (in 50 µL medium) in
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an 8-well cell culture plate with an initial cell density of 1x104 cells/well for 4 h. CDs
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derived from Maillard reaction were diluted to 1, 2, 5, 10, and 20 mg/mL with the
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medium supplemented with glucose (10 mM), sodium pyruvate (1 mM) and
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glutamine (2 mM), pH 7.3 - 7.4. The cells were first incubated for 17 min and then
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CD solutions were added. OCR and ECAR were then measured in real-time every
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seven minutes, for a total of 48 min. Blank medium was used as a control.
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Results and discussion
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The hydrothermal reaction of glucose and lysine in a typical carbonyl ammonia
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condensation reaction that takes place between glucose and lysine as a result of
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Maillard reaction.33 Glucose possesses reactive carbonyl group, which reacts with the
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nucleophilic amino group of the amino acid lysine, and forms a complex mixture of
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Maillard reaction products. When irradiated with a UV light, strong blue fluorescence
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was observed from Maillard reaction products prepared from glucose and lysine
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through hydrothermal method, whereas that was not observed from glucose and lysine
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solution prior to Maillard reaction (Fig. 1A, inset). The UV-vis absorption spectrum of
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the fluorescent products (Fig. 1A) showed that two peaks associated with the π-π*
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electronic transition in C=C and the n-π∗ transition in C=O were observed at 275 and
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325 nm, respectively.34 The fluorescence spectra (Fig. 1B) exhibited the classical
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bathochromic emission phenomenon with a maximum emission wavelength of 440
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nm under an excitation wavelength of 360 nm. The result was similar to that of CDs ACS Paragon Plus Environment
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that were previously reported to have excitation-dependent fluorescence behavior
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(one of its characteristics),35 attributing to their size differences and multiple surface
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emissive trap statuses.36,37 The CDs derived from Maillard reaction of glucose and
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lysine had a fluorescence quantum yield of 16.30% in relative to the reference,
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quinine sulfate. The TEM image displayed in Fig. 1C showed that the products were
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composed of spherical nanoparticles with sizes ranging from 2.3 to 6.8 nm (Fig. S1).
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In addition, it had an inherent characteristic with a fluorescence lifetime (time an
228
electron spends to return to its ground state) of 9.5 ns (Fig. 1D). These results strongly
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suggest that Maillard reaction of glucose and lysine leads to the formation of
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fluorescent nanoparticle products.
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The elemental analysis based on the XPS spectrum of CDs showed that three
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predominant peaks at 285, 400 and 532 eV (Fig. 2A) were associated with elements C,
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N and O, respectively; and with relative contents (calculated based on the integral
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area) of ca. 69.5%, 8.6% and 21.6%, respectively. This result further signifies that the
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nanoparticles from Maillard reaction products are carbon-rich fluorescent nanodots
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that are highly analogous to the artificially synthesized CDs.38 The N/C atomic ratio
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of CDs was calculated to be 12.3%, indicating high concentration of nitrogen.
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High-resolution C1s XPS spectrum exhibited the characteristic peaks of C=C/C-C,
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C-N, C-O and C=O at 284.6, 285.5, 286.4 and 287.4 eV, respectively (Fig. 2B). In
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addition, high-resolution N1s peaks observed at around 399.2 and 400.8 eV can be
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assigned to the pyridinic-like N and N-H groups, respectively (Fig. 2C). O1s XPS
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spectrum was decomposed into peaks at 530.7, 531.4, 532.3 and 533.2 eV
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corresponding to O-H, O=C-O, C-O and O=C-O, respectively (Fig. 2D). A broad peak
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at around 21.82° (Fig. 2E) was due to highly disordered carbon atoms.
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The surface groups of CDs were determined using FT-IR spectroscopy. As
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shown in Fig. 2F, a broad peak centered at 3408 cm-1 was assigned to the stretching
247
vibrations of –OH and N-H bounds. A peak centered at 2936 cm-1 was due to the
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vibration of -CH. A strong peak with high intensity at 1060 cm-1 indicated the
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presence of aromatic alkoxy bonds. In addition, peaks at around 1600 cm-1 may be
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due to the symmetric stretching vibration of C=O. These results indicate that there are
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high contents of hydroxyl, amino and carbonyl groups on the surface of CDs.
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As demonstrated in Fig. 3A, the effects of pH on the fluorescence intensity of
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CDs can be neglected. While the fluorescence intensity was not influenced by ionic
254
strength of up to 10 mg/L NaCl (Fig. 3B), it decreased by about 10% under extremely
255
high ionic strength conditions (i.e. at higher than 8 mg/L NaCl). The effect of
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different metal ions on the fluorescence intensity of CDs was also investigated.
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Common metal ions, except for Cu2+ and Fe3+, had some influence on the
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fluorescence intensity of CDs (Fig. 3C), incoherent with the previously reported
259
data.39 In addition, CDs exhibited excellent light stability when irradiated with UV
260
light (Fig 3D), as indicated by the fluorescence intensity that remained constant for up
261
to 1800 s.
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Maillard reaction products have reportedly possess the characteristic reduction
263
activity against reactive oxygen species (ROS).40 In this study, two typical free
264
radicals, DPPH and hydroxyl radicals were used in experiments investigating the ACS Paragon Plus Environment
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scavenging activity of ROS for the Maillard reaction-derived CDs. DPPH radicals are
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stable free radicals because a lone pair electron on N atom is surrounded by three
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benzene rings; however, a reaction with an antioxidant can result in the formation of a
268
stable DPPH-H complex together with the color change, from purple to light yellow.
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As shown in Fig. 4A, the scavenging ability of CDs against DPPH increased with
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increasing concentration of CDs, and a linear relation (with an equation: Y = 7.018 +
271
0.755X) between inhibition rate and CDs concentrations was observed. While CDs
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had the half-maximal effective concentration (EC50) of 570 µg/mL, compared with
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9.86 µg/mL for vitamin C (Vc), and the DPPH scavenging activity is however about
274
1/57th of Vc. The result demonstrated that although CDs had scavenging activity
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against DPPH, the removal rate is relatively low compared with Vc.
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Furthermore, the scavenging activity of CDs against hydroxyl radical was
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investigated. Hydroxyl radicals are one of the most active chemical substances that
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are harmful to human health due to their ability to attack primary biological
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macromolecules, such as proteins, sugars, nucleic acids, lipids and other essential
280
molecules.41 Therefore, rate of hydroxyl radicals removal is a major indicator
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indicating the capacity of an antioxidant. As shown in Fig. 4B, the signal intensity for
282
hydroxyl radicals decreased with increasing CDs concentration. The CDs from
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Maillard reaction products had an EC50 towards hydroxyl radicals of 12.23 mg/mL.
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This result further confirms the hydroxyl radicals’ scavenging ability of CDs derived
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from Maillard reaction products. Nonetheless, the scavenging capability of CDs
286
against either DPPH or hydroxyl radicals is lower than those of N- and S-doped CDs
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prepared from garlic.42
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Antioxidants scavenge hydroxyl radicals in two ways: they can either directly
289
react with hydroxyl radicals (as discussed above) or indirectly inhibit the formation of
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hydroxyl radicals by chelating metal ions involving in such formation. The data in Fig.
291
4C showed that the chelating ability increased with the increase of CDs concentration.
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The EC50 of CDs towards hydroxyl radicals scavenging was determined to be 4.595
293
mg/mL, which is much higher than that of EDTA-Na2 (0.017 mg/mL). This suggests
294
that the metal ions involved in the formation of hydroxyl radicals are chelated by the
295
CDs, resulting in improved hydroxyl radicals scavenging capacity. These results
296
indicate that CDs derived from Maillard reaction products exhibit certain hydroxyl
297
radicals scavenging capacity through both the direct and the indirect mechanisms.
298
Bio-distribution of CDs in representative cells, onion epidermal cells and liver
299
cancer cells (HepG2), were examined. The inner skin of an onion was immersed in
300
CDs solution and then placed on glass slides. The distribution of CDs in onion cells
301
was visualized using a fluorescence microscope under an excitation wavelength of
302
405 nm. The bright-field images in Fig. 5 showed that the cells before (Fig. 5A) and
303
after (Fig. 5D) treatment with CDs were not different. However, a blue fluorescence
304
was observed under the excitation wavelength of 405 nm in the epidermal cells
305
treated with CDs (Fig. 5E) in contrast with that in the control cells (Fig. 5B). The
306
fluorescence signal also indicated that CDs were distributed in the cell wall, but not in
307
the cytoplasm. The result is different from that previously reported for CDs obtained
308
from kvass.26 ACS Paragon Plus Environment
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The bright-field and fluorescent images in Fig. 6 show that HepG2 cells
310
treated with CDs exhibit blue (Fig. 6B), green (Fig. 6C), and red (Fig. 6D)
311
fluorescence with excitation wavelengths of 408, 458 and 488 nm, respectively. The
312
fluorescence was found distributed mainly in the cytoplasm (not in nucleus), similarly
313
to the previous observation.26 This observation has also been seen for carbon quantum
314
dot prepared from cabbage, used as imaging probe in biomedical applications.43 The
315
results show that CDs from Maillard reaction products can easily enter both plant and
316
animal cells, and are distributed either inside the cell wall or the cytoplasm.
317
In vitro cytotoxicity of CDs was evaluated in HepG2 cells, which were used as
318
model cells. The result in Fig. 7A shows that cell survival rate was 90% after 24 h
319
exposure to CDs of concentrations below 1 mg/mL. Further increasing CDs
320
concentration significantly decreased cell viability. Approximately 80% of HepG2
321
cells were dead when the concentration of CDs reached 10 mg/mL. These observation
322
demonstrates that the cytotoxicity of CDs is dose-dependent, which is consistent with
323
that previously reported.38 To further understand the cytotoxicity mechanism of CDs,
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the effect of CDs on energy metabolism of HepG2 cells was examined.44 Mammalian
325
cells produce adenosine triphosphate via two major mechanisms: glycolysis and
326
oxidative phosphorylation (OXPHOS).45 Nevertheless, most cancer cells with intact
327
mitochondria convert OXPHOS to the less energetically favorable glycolysis.45
328
Specifically, the ECAR and OCR, indications of the glycolytic activity and
329
mitochondrial function, respectively, can be determined simultaneously within the
330
same small population of HepG2 cells. The result in Fig. 7B shows that OCR value
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(including those at high concentration 20 mg/mL) remained unchanged compared
332
with control, and the mitochondrial respiration is almost unaffected by CDs derived
333
from Maillard reaction products. Unlike OCR, the value of ECAR dramatically
334
decreased when the concentration of CDs was higher than 1 mg/mL, in consistent
335
with the result from MTT assay. Moreover, such decrease suggests that CDs may
336
inhibit the glycolysis in HepG2 cells (Fig. 7C). The real-time data of OCR and ECAR
337
(Fig. 7D) further showed that while the OCR value was not substantially changed
338
compared with control prior to the addition of CDs, the ECAR value remarkably
339
decreased to lower than the base value. The ECAR value decreased to almost zero
340
when the CDs concentration was increased to 20 mg/mL. We can conjecture that CDs
341
may bind to certain key enzymes involving in glycolysis and inhibit their
342
complexes,46 nonetheless, further investigations are needed in order to better
343
understand such mechanism of actions.
344
In summary, we demonstrated that fluorescent CDs were the nanoparticles
345
derived from the products of Maillard reaction of glucose and lysine (a model system).
346
The fluorescent nanostructures were also found in real food like grilled fish. The
347
overall production yield of the nanostructures was about 1.67% as calculated based on
348
the weight of turbot flesh.47 Herein, the CDs derived from Maillard reaction are
349
mono-dispersed and exhibit the bathochromic emission phenomenon with a
350
fluorescent quantum yield of 16.30%. They are composed of four elements, including
351
C, H, O and N, with high contents of hydroxyl, amino and carbonyl/ carboxylate
352
groups on their surface. The CDs had unique antioxidant capacity as demonstrated by
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free-radical scavenging activity. Importantly, these CDs were found to be mainly
354
distributed inside the cell wall of onion epidermal cells and the cytoplasm (not
355
nucleus) of HepG2 cells. Moreover, the CDs may inhibit the glycolysis of HepG2
356
cells, thus causing cytotoxicity. Further investigations are however needed to
357
elucidate such inhibition mechanism.
358
ASSOCIATED CONTENT
359
Supporting Information
360
The Supporting Information is available free of charge on the ACS Publications
361
website at DOI: XXXXXXX.
362
Size distribution histogram of the CDs formed in the Maillard reaction
363
(Fig.S1).
364
Acknowledgement:
365
This work was supported by the National Key Research and Development Program of
366
China (2017YFD0400103, 2016YFD0400404). We also thank Tao Liu from Liaoning
367
Bai Hao Biological Technology Co. Ltd. (Benxi, Liaoning, China) for his technical
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assistant.
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FIGURE CAPTIONS.
524
Figure 1. (A) UV-visible absorption spectrum of CDs formed in the Maillard reaction
525
using glucose and lysine as a model system. Insets show the photographs of glucose
526
and lysine solution (1, 1’), and CDs (2, 2’) formed in the Maillard reaction under the
527
illumination of visible (1, 2) and UV light (1’, 2’), (B) fluorescence spectra, (C) TEM
528
image (Inset shows the high resolution TEM image) and (D) fluorescence decay curve
529
of the CDs formed in the Maillard reaction.
530
Figure 2. (A) XPS survey, high resolution spectra of (B) C1s, (C) N1s, (D) O1s for the
531
CDs formed in the Maillard reaction. (E)XRD pattern and (F) FTIR spectra the CDs
532
formed in the Maillard reaction.
533
Figure 3. Effect of (A) pH, (B) ionic strength, (C) metal ions, and (D) continuous UV
534
radiation on fluorescence intensity of the CDs formed in the Maillard reaction. F.L.
535
represents fluorescence.
536
Figure 4. (A) Scavenging ability of the CDs formed in the Maillard reaction and
537
vitamin C (inset) against DPPH. (B) ESR spectra of DMPO/•OH adducts at different
538
concentrations of the CDs formed in the Maillard reaction. (C) Chelating ability of the
539
CDs formed in the Maillard reaction and EDTA-Na2 (inset) to Fe2+.
540
Figure 5. Bright field and fluorescence microscope images of onion epidermal cells at
541
the illustration of (A) visible light and (B) excitation wavelength of 405 nm, (C)
542
overlay of (A) and (B). Bright field and fluorescence microscope images of onion
543
epidermal cells incubated with CDs at the illustration of (D) visible light and (E)
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excitation wavelength of 405 nm. (F) overlay of (D) and (E). Scale bar = 100 µm.
545
Figure 6. (A) Bright-field and fluorescent images of HepG2 cells incubated with CDs
546
at the excitation wavelengths of (B) 408 nm, (C) 458 nm, and (D) 488 nm,
547
respectively. Scale bar=30 µm.
548
Figure 7. (A) Cell viability of HepG2 cells by MTT assay after incubation with CDs
549
formed in the Maillard reaction. (B) Relative OCR (oxygen consumption rate) and (C)
550
ECAR (extracellular acidification rate) of HepG2 cells in the addition of CDs. (D)
551
Real-time analysis of OCR and ECAR in HepG2 cells upon adding CDs (20 mg/mL)
552
at 17 min for a total of 48 min. (Mean ± SD, n = 3).
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554
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Fig.1
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Intensity (a.u.)
N1s
200 400 600 800 Binding Energy (eV)
D
N-H 400.8
pyridinic N 399.3
398 400 402 404 Binding Energy (eV)
E
F
100 50 0 10
20
30
40
50
*O=C-O 531.4
60
2θ(Degree)
O-H 530.7
O=C-O* 533.2
Glu
CDs
Lys
200 C=N N-H 150 100
C=C N-H
50 C-O-C 0
O-H
C-H
C=O
3500 3000 2500 2000 1500 1000 500
-1 Wavenumber (cm )
561
562
532.3 C-O
526 528 530 532 534 536 538 Binding Energy (eV)
21.82o
150
C-N 285.4 C-O 286.3 C=O 287.6
280 282 284 286 288 290 292 Binding Energy (eV)
Transmittance (%)
Intensity (a.u.)
C
396
C-C/C=C 284.5
Intensity (a.u.)
0
Intensity (a.u.)
B
O1s
C1s
Intensity (a.u.)
A
Fig. 2.
563
564
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0.5
0.0
F.L. Intensity (a.u.)
3
4
5
6
7
8
9
** ** ** ** ** ** **
0.5
0.0 0
1
2
3
4
5
6
7
8
9 10
Concentration (mg/mL)
D
1.0
1200
** ** **
0.5
**
0.0
*
10 11
pH
C
567
** **
2
566
B 1.0
**
F.L. Intensity (a.u.)
** ** **
**
F.L. Intensity (a.u.)
F.L. Intensity (a.u.)
A 1.0
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900 600 300 0
+ 2+ 2+ 2+ 2+ 2+ 2+ 3+ 2+ trol 2 Con Mg Mn Zn Ca Co Fe Ni Cu Fe
0
300
600
900
1200
Time (s)
Ion species Fig. 3.
568
569
570
571
572
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1500
1800
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Fig. 4.
576
577
578 579
Fig. 5.
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581 582
Fig. 6.
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0.8
*
**
0.6 0.4 ** 0.2 0.0 0
0.25 0.5
1
2
5
90 60 30 0 0
10
1
C
20
120 **
100
100
OCR (pmol/min)
Normalized ECAR (%)
10
OCR
80
60 40
** **
20
2
5
10
**
100 **
80
60
20
20 **
0
20
6
**
**
**
**
12 18 24 30 36 42 48
Time (min)
CDs (mg/mL)
60 40
40
0
1
**
Injection:CDs (20 mg/mL)
** 0
**
ECAR
80
0
587
5
D 120
120
586
2
CDs (mg/mL)
CDs (mg/mL)
Fig. 7.
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589
590
591
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ECAR (mpH/min)
Cell viability
Normalized OCR (%)
B 150
A 1.0
Journal of Agricultural and Food Chemistry
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TOC
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