A type of novel Fluorescent magnetic carbon quantum dots for cells imaging and detection Su Xi1, Xu Yi1 2 3∗∗, Che Yulan1, Liao Xin1, Jiang Yan1 1
Chemistry and Chemical Engineering College, Chongqing University, Chongqing,
400044, China; 2
Defense Key Disciplines Lab of Novel Micro-nano Devices and System Technology,
Chongqing, 400044, China; 3
International R & D center of Micro-nano Systems and New Materials Technology,
400044, China
∗
Correspondence to: Xu Yi;
e-mail address: [email protected].
Contract grant sponsor: Nature and Science Fund of Chinese National Educational Committee; contract grant number: 21375156) Contract grant sponsor: The national excellent doctoral dissertation of PR China; contract grant number: FSNEDD-200941. Contract grant sponsor: Technologic Research Foundation Project of Chongqing Science and Technology Committee; contract grant number: 2010AC5050.
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/jbm.a.35468 This article is protected by copyright. All rights reserved.
Journal of Biomedical Materials Research: Part A
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Abstract: A new type of multifunctional fluorescent magnetic carbon quantum dots SPIO@CQDs(n) ([superparamagnetic iron oxide nanoparticles (SPIO), carbon quantum dots, (CQDs)]) with magnetic and fluorescence properties was designed and prepared through layer by layer self-assembly method. The as-synthesized SPIO@CQDs
(n)
exhibited different emission colors including blue, green and red,
when they were excited at different excitation wavelengths, and its fluorescent intensity increased as the increase of CQDs layer (n). SPIO@CQDs(n) with quite low toxicity could mark cytoplasm with fluorescence by means of non-immune markers. The mixture sample of liver cells L02 and hepatoma carcinoma cells HepG2 was taken as an example, and HepG2 cells were successfully separated and detected effectively by SPIO@CQDs(n), with a separation rate of 90.31%. Importantly, the designed and prepared SPIO@CQDs(n) are certified to be wonderful biological imaging and magnetic separation regents.
Key Words: carbon quantum dots, Layer by layer self-assembly, cell imaging and detection, cell magnetic sorting INTRODUCTION
The research on fluorescence and magnetic properties are important for biochemical targets labeling and detection. The multi-functional fluorescent and magnetic composites were of various available functions, such as labeling, separation and detection, which could greatly improve their application value in the biochemical tests fields. Fluorescent magnetic composites were usually prepared by assembling organic 2 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
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dyes, quantum dots and precious metals together with magnetic iron oxide nanoparticles, 1-4 which were of good properties and application prospect in the field of separation and test of cells and heavy metal ions. 5, 6 Quantum dots, one of the widely used fluorescent markers, were also used for preparing fluorescent magnetic composites, which could be applied in many biomedical researching fields for their good fluorescent property. 7 However, classic quantum dots were usually obtained by a mixture of lead, cadmium, silicon and others, which suffered from toxicity and poor biodegrade ability. As a new type of quantum dots, carbon quantum dots (CQDs) with similar optical properties as semiconductor quantum dots, was discovered by American scientists Clemson University in 2004. Due to its favorable properties, such as low cost, environmental friendly preparation, low toxicity, good biocompatibility, easy synthesis and functionalization, 8-10 CQDs was expected to somehow replace semiconductor quantum dots in the biomedical fields. The studies on optical properties and biochemistry for CQDs were still at a primary stage, while there were many works to be done for their application. This is because the modifying groups -NH2, -OH, -COOH and so on, made it easy to link and package with biological materials. 11 The researches on biological imaging, analysis testing and photo-catalyst were reported, 12 while the magnetic carbon quantum dots composites prepared by assembling CQDs onto SPIO were rarely reported so far.
Due to the fluorescence quenching effect of magnetic nanoparticles on quantum dots (QDs), chemists paid much more attention to prepare multi-functional fluorescent magnetic composites by assembling QDs with magnetic nanoparticles. 3 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
Journal of Biomedical Materials Research: Part A
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Subramanian et al. prepared Fe2O3-CdSe composites using doping method, of which high temperature is required and the process was complicated and difficult to control. 2
By means of polymers embedding method, Prasad et al. fabricated Fe3O4-QDs
composites, of which the QD surface was modified to avoid fluorescence quenching, but the amount of QD and Fe3O4 was difficult to control during the process.
13
Sreenivasan et al. proposed the synthesis process of Fe3O4-QDs composites, by which the amide bond between Fe3O4-COOH and QD-NH2 was formed with condensation reaction using EDC/NHS (EDC, 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; NHS, N-Hydroxysuccinimide).14 However, quenching phenomenon occurred on their surface when the Fe3O4-QDs prepared by EDC/NHS linking with other reagents.15 Fe3O4-QDs composites were also prepared by electrostatic adsorption using layer by layer self-assembly method with the starting materials of Fe3O4 and QDs, and the whole process was simple, easy to control and no complicated equipment requirements. The particle size could be easily controlled by adjusting the number of QDs layers.6, 15-17 However, there was hidden toxicity of the connected QDs, and the QDs had to be further modified by amino, hydroxyl, and carboxyl groups.
In this work, fluorescent magnetic carbon quantum dots (SPIO@CQDs(n)) with multi-layer structure were prepared through layer by layer self-assembly mode by alternating adsorption of the opposite electric charged polymer electrolyte and CQDs. Based on the characterization of its structure and fluorescence properties, and as well as its biocompatibility and cellular toxicity, the feasibility of the new type of 4 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
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fluorescence labeling regent used for cells was investigated. In turn, SPIO@CQDs
(n)
was used to separate the target cells HepG2 from the liver cells L02. The results showed that HepG2 cells could be marked by SPIO@CQDs(n), and the target HepG2 cells were efficiently separated and quantitatively detected. MATERIALS AND METHODS
Materials Gelatin was purchased from Shanghai chemical reagent factory (China). Ethylene glycol was supplied by Chuandong chemical Co., Ltd (Chongqing, China). SPIO was purchased from Beijing DK Nano technology Co., Ltd (China). Ethyl silicate, Poly dimethyl diallyl ammonium chloride, 2-Amino-2-(hydroxymethyl)-1, 3-propanediol, NaCl and HCl were purchased from Sinopharm Chemical Regent Co., Ltd (China). DMEM medium, fetal bovine serum, penicillin streptomycin and PBS powder were purchased from HyClone Company (USA).
CQDs preparation 0.2 g gelatin was heated to dissolve in the glycol. The mixture was poured into a TFR100-Teflon reaction kettle and react in the microwave for 40 min. Finally, the reactor was cooled to room temperature. The ultrapure water was added to the reactor, and the yellow brown solution was filtered to yield the aqueous solution of CQDs, which should be preserved in the dark.
SPIO@CQDs(n) preparation 40 mL SPIO (1 mg·mL-1) was added to 200 mL isopropyl alcohol. Then, the mixture was dispersed in an ultrasonic bath. In turn, 7 mL NH3·H2O and 0.6 mL TEOS were 5 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
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added into the mixture drop by drop. SPIO@SiO2 was prepared for 9 h at 30 ℃. The SPIO@SiO2 were separated via a strong magnet and washed with ultrapure water and absolute ethyl alcohol for several times and dried by vacuum freezing. 0.3 mg·mL-1 of SPIO@SiO2 was added to 20 mL 0.2% of PDDA solution containing 20 mM Tris and NaCl. The mixture was deal with ultrasonic. The PDDA was allowed to be absorbed for 30 min under shaking. PDDA coated SPIO@SiO2 was collected by applied magnetic fields, and washed several times with 20 mM Tris aqueous containing 20 mM NaCl. Finally, they were dispersed again in the solution of CQDs with the above washing liquid. Negatively charged CQDs was then deposited onto the coated SPIO@SiO2 for 3 h by shaking. The SPIO@CQDs
(n)
was collected
by magnetic separation and obtained by washing several times. The above mentioned process was repeated until the desired SPIO@CQDs(n) (n=3, 6, 9, 12, 15) were obtained.
Characterization CQDs, SPIO, SPIO@SiO2 and SPIO@CQDs(n) were measured by IRPrestige-21 infrared spectrophotometer (Shimadzu) using KBr pellets as the sample matrix. Fluorescence spectra of SPIO@CQDs(n) were obtained by RF-5301PC Fluorescence spectrometer (Shimadzu). Zeta potential of CQDs, SPIO@SiO2, SPIO@SiO2@PDDA and SPIO@CQDs(n) nanoparticles were measured by Zetasizer Nano ZS90 zeta potential analyzer (Malvern). The prepared nanoparticles were also morphologically characterized by a HITACHI H-600 transmission electron microscope (Japan).
Toxicity assay of SPIO@CQDs(n) 6 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
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SPIO@CQDs(n) were soaked in 75% alcohol to sterilize. A series of SPIO@CQDs(n) solution were prepared in the fresh cell culture fluid at concentrations of 0, 20, 40, 60, 80,100 µg·mL-1, respectively. Hepatoma carcinoma cell HepG2, liver cells L02 and fibroblast cells 3T3-LE were cultured in DMEM Medium at 37°C under 5% CO2 atmosphere after overnight, supplemented with penicillin/streptomycin (100 units·mL-1) and 7% fetal bovine serum (FBS). In the 96-well plates, 103 cells were added into each well. In the second day, the medium was replaced with 200 µL fresh medium containing different concentrations of SPIO@CQDs(n). The cells were cultured for another 24h. Then, 20 µL of MTT regent was added. After 4h, the medium was removed, and 150 µL DMSO was added. The optical density (OD) value of each well was measured at 490 nm using the Varioskan Flash multifunctional enzyme label reading (Thermo Fisher Scientific, USA). The cell toxicity was evaluated by the MTT assay.
Liver cell L02 imaging experiments 103cells·mL-1 of liver cells L02 were seeded on 24-well plates and cultured using established procedures after overnight. Afterwards, the medium was replaced with 600 µL fresh medium containing different concentrations of SPIO@CQDs(n), and the cells were cultured for another 6 h. The liver cells L02 were washed with PBS for three times. Finally, the liver cells L02 were imaged using a LSM780 confocal laser scanning microscopy (CLSM, Zeiss, Germany) to optimize the layer number (n) and concentration of SPIO@CQDs(n). 7 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
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Similarly, the medium was replaced with 600 µL fresh medium containing the optimum concentration of SPIO@CQDs(n) (n = 12), the cells were respectively cultured for 1, 3 and 6h. Finally, the liver cells L02 were imaged using CLSM to optimize the time that liver cells L02 were labeled by SPIO@CQDs(12). Target HepG2 cells labeling and separating experiments HepG2 cells were labeled with SPIO@CQDs(12) at the same way as L02 cells, and they were imaged using CLSM. HepG2 cells (105 cells·mL-1) were seeded in culture bottle and then cultured using established procedures after overnight. After the HepG2 cells were washed with PBS, 1 mL fresh medium containing 0.2 mg SPIO@CQDs(12) was added and then it was cultured for another 14h. After removing the unincorporated SPIO@CQDs(12) and washing with PBS, the labeled HepG2 cells were digested with pancreatic enzyme. The cells were collected through a TD45 low speed centrifuge at 800 rpm for 5 min, and then the labeled HepG2 cells were dissolved in the suspensions with PBS 102, 103, 104, 105 cells·mL-1
at 10,
respectively, and then their fluorescence intensity
values were detected by fluorescence spectrophotometer. At last, a standard curve of the logarithm of concentration and fluorescence intensity values was plotted. The samples were obtained by mixing labeled HepG2 cells and liver cells L02 with equal amount. The labeled HepG2 cells were collected by magnetic separation and then washed with PBS. At last, the target HepG2 cells were detected by fluorescence
8 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
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spectrophotometer. The quantity of captured cells was gained according to the standard curve, and capture rate of the target HepG2 cells were calculated. RESULTS
Design and preparation of SPIO@CQDs(n) The schematic illustration of the procedure for fabrication of SPIO@CQDs(n) was shown in Figure 1. As shown in Figure 2A, the TEM imaging suggested that the diameter of the prepared SPIO@SiO2 was adjusted to 40 nm, with silica shell thickness of 30 nm. As depicted in Figure 2B-C, the average diameter of SPIO@CQDs(n) was 200 nm, and the composites obviously showed core-shell structure. As illustrated in Figure 2D, the zeta potential of nanoparticles changes between ±40 mV attributed to their different surface material. The Fourier infrared (FT-IR) spectra of SPIO, SPIO@SiO2, CQDs and SPIO@CQDs(n) were shown in Figure 2E. The characteristic absorption bands of SiO-H and SiO2 in SPIO@SiO2 were at 3480, 1094 cm-1 respectively. In FT-IR spectra of CQDs and SPIO@CQDs(n), the characteristic absorption bands of –NH2 and -OH appeared were at 3400-3500 cm-1; the stretching vibration bands of –NH2 were at 2850-3000 and 2400-2500 cm-1; the characteristic absorption band of –COOH was at 1600 cm-1. Characterization of fluorescent properties
9 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
Journal of Biomedical Materials Research: Part A
Fluorescent spectra of SPIO@CQDs(n) were shown in Figure 2F. When n≥9, SPIO@CQDs(n) had a significant fluorescence characteristic, and then the fluorescence intensity was cumulative with the increase of n.
In addition to the down-converted PL properties, SPIO@CQDs(n) exhibited an up-converted characteristic and the emission wavelength of SPIO@CQDs(n) was increased with the increase of excitation wavelength, as illustrated in Figure 3(A-B). It was shown in Figure 3 (C-D) that a typical down/up-converted PL spectrum of SPIO@CQDs(n) with maximum peak at 430 nm was excited by 345 nm and 680 nm light respectively. The down-converted PL emission and excitation spectra were also depicted in Figure 3E. Interestingly, for the same irradiation time, the fluorescent intensity of CQDs decreased by 63% when that of SPIO@CQDs(n) decreased by 14% (Fig. 3F).
Cytotoxicity assay of SPIO@CQDs(n) MTT assay results were shown in Figure 4. Liver cells L02, hepatoma carcinoma cells HepG2 and fibroblast cells 3T3-LE were respectively incubated with gradient concentrations of SPIO@CQDs (n) for 24 h, and all of them showed high viabilities. Liver cells L02 labeling with SPIO@CQDs(n) Liver cells L02 were cultured with 60 µg·mL-1 of SPIO@CQDs(n) (n=3, 6, 9, 12, 15) at 37℃ for 6h, and after removing the unincorporated SPIO@CQDs(n) by PBS washing, the confocal images were recorded as shown in Figure 5A. With the increase of CQDs layers (n), the fluorescent intensity of the liver cells L02 swallowed 10 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
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SPIO@CQDs(n) were enhanced, and the CLSM images were much clearer. When the CQDs layers n≥9, cells L02 were labeled effectively by SPIO@CQDs(n) and showed strong fluorescent. SPIO@CQDs(n) with concentrations gradient (20, 40, 60, 80 µg·mL-1; n=12) were incubated with liver cells L02, respectively. After the same treatment, the CLSM images were measured and shown in Figure 5B. The CLSM images became clear, and the fluorescent of SPIO@CQDs(12) labeled liver cells was enhanced as the concentration increase of SPIO@CQDs(12). When the concentration was over 60 µg·mL-1, the fluorescent intensity of liver cells L02 didn’t change any more.
SPIO@CQDs(12) was incubated with L02 cells for 1, 3, 6 h, respectively. After the same treatment, the CLSM images were detected and shown in Figure 6. The luminescent spots widely appeared in the membrane and cytoplasm of the L02 cells, while the fluorescence at the central region involved with nucleus was very weak (Fig. 6.D1-D3). The fluorescent variation with incubation time of L02 cells with SPIO@CQDs(12) was shown in Figure 6A0-C3. When SPIO@CQDs(12) was incubated with L02 cells for only 1h, the L02 cells showed fluorescent, and the mark effect increased with the increase of interaction time between SPIO@CQDs(12) and L02 cells.
HepG2 cells marking and sorting with SPIO@CQDs(n) HepG2 cells were labeled nonspecifically by SPIO@CQDs(12), and the CLSM images were shown in Figure 7A0-A3. After labeling, fluorescence spectrophotometer was 11 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
Journal of Biomedical Materials Research: Part A
used to detect fluorescence response signals of different concentrations of HepG2@ SPIO@CQDs(12), and standard curve of the logarithm of concentration and fluorescence intensity value was plotted (Fig. 7) in the range of 10-105 cells·mL-1 using the equation y = 0.984 x + 6.574 with R2= 0.9258.
HepG2@SPIO@CQDs(12) and liver cells L02 incubated without SPIO@CQDs(12) at the same condition were mixed with 1: 1 ratio. The HepG2@ SPIO@CQDs(12) were collected by the magnet for 30s, and the remaining liver cells L02 were removed by PBS washing. The enriched HepG2@SPIO@CQDs(12) were dispersed to the initial volume of the mixed cells by PBS and detected with fluorescence spectrometer. According to the standard curve (Fig. 7C), the amount of the enriched HepG2@ SPIO@CQDs(12) was calculated. The capture rate (formula Ⅰ) s obtained and it was 90.31%.
In the
equation,
c0 and c1 were
the initial concentration of
HepG2@SPIO@CQDs(12), and the concentration of HepG2@SPIO@CQDs(12) isolated from the hybrid cells, respectively. Capt ur e r at e=
c1 × 100% c0
Ⅰ
DISCUSSION
There was growing focus on magnetic and fluorescent quantum dot nanoparticles due to their outstanding dual function. However, the SPIO cores have a fluorescent quenching effect on QDs due to the electronic coupling and energy transfer. In addition, the broad absorbance spectrum of SPIO also attenuates both the excitation light and emitted fluorescence. In our study, shell structure of SiO2 was adopted as 12 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
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outside protective layer of SPIO to increase the distance and interval between SPIO and CQDs, and this can reduce the fluorescent quenching effect on the QDs.
In order to obtain fluorescent and magnetic multifunctional fluorescent magnetic composites, a new kind of nano fluorescent reagent was prepared, by which the carbon quantum dots with low biological toxicity was layer-by-layer self-assembled to the SiO2 coated superparamagnetism iron oxide nanoparticles (SPIO) (Fig. 1). SPIO@SiO2 core-shell magnetic nanoparticles were obtained from the silica source, SPIO and ethyl silicate (TEOS). The hydrolysis of TEOS in alkaline condition can form gel with three-dimensional structure, which let it coat on SPIO. By controlling the concentration of SPIO, the speed and time of stirring, SPIO@SiO2 nanoparticles with uniform particle size and good dispersive capacity were generated. This was confirmed by the TEM results.
CQDs were quickly prepared by Microwave-assisted hydrothermal method using gelatin as carbon source, ultrapure water and Ethylene glycol as solvent. The experimental CQDs had the characteristics of high fluorescent yield, stable fluorescent performance, good solubility in water and negative charge.
Fluorescent magnetic carbon quantum dots composites (SPIO@CQDs
(n))
were
prepared by repeating the above steps (Fig. 1). Firstly, the strong cationic polyelectrolyte PDDA was absorbed onto SPIO@SiO2 with abundant negative charges by the electrostatic force, and then the compound SPIO@SiO2@PDDA became positively charged, and it could absorb negatively charged CQDs. In the 13 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
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assembly process, in order to increase the effective adsorption of CQDs by SPIO@SiO2, the Tris aqueous of pH 9.0 was employed to enhance the negative charge of the surface and improve the stability of the nanoparticles 11. The changes of zeta potentials during the preparation were also used to monitor the assembly process, which suggested that driven by the electrostatic force, positively charged strong cationic polyelectrolyte PDDA and negatively charged CQDs were alternately absorbed onto SPIO@SiO2. On comparison the Fourier infrared (FT-IR) spectra of SPIO, SPIO@SiO2 and CQDs with that of SPIO@CQDs
(n),
the characteristic absorption bands of SiO-H,
SiO2 and CQDs were all in Fourier infrared (FT-IR) spectra of SPIO@CQDs(n) suggested that SPIO@CQDs
(n)
was successfully obtained by layer by layer
self-assembly.
The fluorescent spectra of SPIO@CQDs(n) excited at the same wavelength (λ = 350 nm) indicated that, with the isolation effects of the silica layer, the innermost layer of CQDs still suffered from the quenching effect of the iron oxides when n
Chemistry and Chemical Engineering College, Chongqing University, Chongqing,
400044, China; 2
Defense Key Disciplines Lab of Novel Micro-nano Devices and System Technology,
Chongqing, 400044, China; 3
International R & D center of Micro-nano Systems and New Materials Technology,
400044, China
∗
Correspondence to: Xu Yi;
e-mail address: [email protected].
Contract grant sponsor: Nature and Science Fund of Chinese National Educational Committee; contract grant number: 21375156) Contract grant sponsor: The national excellent doctoral dissertation of PR China; contract grant number: FSNEDD-200941. Contract grant sponsor: Technologic Research Foundation Project of Chongqing Science and Technology Committee; contract grant number: 2010AC5050.
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/jbm.a.35468 This article is protected by copyright. All rights reserved.
Journal of Biomedical Materials Research: Part A
Page 2 of 28
Abstract: A new type of multifunctional fluorescent magnetic carbon quantum dots SPIO@CQDs(n) ([superparamagnetic iron oxide nanoparticles (SPIO), carbon quantum dots, (CQDs)]) with magnetic and fluorescence properties was designed and prepared through layer by layer self-assembly method. The as-synthesized SPIO@CQDs
(n)
exhibited different emission colors including blue, green and red,
when they were excited at different excitation wavelengths, and its fluorescent intensity increased as the increase of CQDs layer (n). SPIO@CQDs(n) with quite low toxicity could mark cytoplasm with fluorescence by means of non-immune markers. The mixture sample of liver cells L02 and hepatoma carcinoma cells HepG2 was taken as an example, and HepG2 cells were successfully separated and detected effectively by SPIO@CQDs(n), with a separation rate of 90.31%. Importantly, the designed and prepared SPIO@CQDs(n) are certified to be wonderful biological imaging and magnetic separation regents.
Key Words: carbon quantum dots, Layer by layer self-assembly, cell imaging and detection, cell magnetic sorting INTRODUCTION
The research on fluorescence and magnetic properties are important for biochemical targets labeling and detection. The multi-functional fluorescent and magnetic composites were of various available functions, such as labeling, separation and detection, which could greatly improve their application value in the biochemical tests fields. Fluorescent magnetic composites were usually prepared by assembling organic 2 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
Page 3 of 28
Journal of Biomedical Materials Research: Part A
dyes, quantum dots and precious metals together with magnetic iron oxide nanoparticles, 1-4 which were of good properties and application prospect in the field of separation and test of cells and heavy metal ions. 5, 6 Quantum dots, one of the widely used fluorescent markers, were also used for preparing fluorescent magnetic composites, which could be applied in many biomedical researching fields for their good fluorescent property. 7 However, classic quantum dots were usually obtained by a mixture of lead, cadmium, silicon and others, which suffered from toxicity and poor biodegrade ability. As a new type of quantum dots, carbon quantum dots (CQDs) with similar optical properties as semiconductor quantum dots, was discovered by American scientists Clemson University in 2004. Due to its favorable properties, such as low cost, environmental friendly preparation, low toxicity, good biocompatibility, easy synthesis and functionalization, 8-10 CQDs was expected to somehow replace semiconductor quantum dots in the biomedical fields. The studies on optical properties and biochemistry for CQDs were still at a primary stage, while there were many works to be done for their application. This is because the modifying groups -NH2, -OH, -COOH and so on, made it easy to link and package with biological materials. 11 The researches on biological imaging, analysis testing and photo-catalyst were reported, 12 while the magnetic carbon quantum dots composites prepared by assembling CQDs onto SPIO were rarely reported so far.
Due to the fluorescence quenching effect of magnetic nanoparticles on quantum dots (QDs), chemists paid much more attention to prepare multi-functional fluorescent magnetic composites by assembling QDs with magnetic nanoparticles. 3 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
Journal of Biomedical Materials Research: Part A
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Subramanian et al. prepared Fe2O3-CdSe composites using doping method, of which high temperature is required and the process was complicated and difficult to control. 2
By means of polymers embedding method, Prasad et al. fabricated Fe3O4-QDs
composites, of which the QD surface was modified to avoid fluorescence quenching, but the amount of QD and Fe3O4 was difficult to control during the process.
13
Sreenivasan et al. proposed the synthesis process of Fe3O4-QDs composites, by which the amide bond between Fe3O4-COOH and QD-NH2 was formed with condensation reaction using EDC/NHS (EDC, 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; NHS, N-Hydroxysuccinimide).14 However, quenching phenomenon occurred on their surface when the Fe3O4-QDs prepared by EDC/NHS linking with other reagents.15 Fe3O4-QDs composites were also prepared by electrostatic adsorption using layer by layer self-assembly method with the starting materials of Fe3O4 and QDs, and the whole process was simple, easy to control and no complicated equipment requirements. The particle size could be easily controlled by adjusting the number of QDs layers.6, 15-17 However, there was hidden toxicity of the connected QDs, and the QDs had to be further modified by amino, hydroxyl, and carboxyl groups.
In this work, fluorescent magnetic carbon quantum dots (SPIO@CQDs(n)) with multi-layer structure were prepared through layer by layer self-assembly mode by alternating adsorption of the opposite electric charged polymer electrolyte and CQDs. Based on the characterization of its structure and fluorescence properties, and as well as its biocompatibility and cellular toxicity, the feasibility of the new type of 4 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
Page 5 of 28
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fluorescence labeling regent used for cells was investigated. In turn, SPIO@CQDs
(n)
was used to separate the target cells HepG2 from the liver cells L02. The results showed that HepG2 cells could be marked by SPIO@CQDs(n), and the target HepG2 cells were efficiently separated and quantitatively detected. MATERIALS AND METHODS
Materials Gelatin was purchased from Shanghai chemical reagent factory (China). Ethylene glycol was supplied by Chuandong chemical Co., Ltd (Chongqing, China). SPIO was purchased from Beijing DK Nano technology Co., Ltd (China). Ethyl silicate, Poly dimethyl diallyl ammonium chloride, 2-Amino-2-(hydroxymethyl)-1, 3-propanediol, NaCl and HCl were purchased from Sinopharm Chemical Regent Co., Ltd (China). DMEM medium, fetal bovine serum, penicillin streptomycin and PBS powder were purchased from HyClone Company (USA).
CQDs preparation 0.2 g gelatin was heated to dissolve in the glycol. The mixture was poured into a TFR100-Teflon reaction kettle and react in the microwave for 40 min. Finally, the reactor was cooled to room temperature. The ultrapure water was added to the reactor, and the yellow brown solution was filtered to yield the aqueous solution of CQDs, which should be preserved in the dark.
SPIO@CQDs(n) preparation 40 mL SPIO (1 mg·mL-1) was added to 200 mL isopropyl alcohol. Then, the mixture was dispersed in an ultrasonic bath. In turn, 7 mL NH3·H2O and 0.6 mL TEOS were 5 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
Journal of Biomedical Materials Research: Part A
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added into the mixture drop by drop. SPIO@SiO2 was prepared for 9 h at 30 ℃. The SPIO@SiO2 were separated via a strong magnet and washed with ultrapure water and absolute ethyl alcohol for several times and dried by vacuum freezing. 0.3 mg·mL-1 of SPIO@SiO2 was added to 20 mL 0.2% of PDDA solution containing 20 mM Tris and NaCl. The mixture was deal with ultrasonic. The PDDA was allowed to be absorbed for 30 min under shaking. PDDA coated SPIO@SiO2 was collected by applied magnetic fields, and washed several times with 20 mM Tris aqueous containing 20 mM NaCl. Finally, they were dispersed again in the solution of CQDs with the above washing liquid. Negatively charged CQDs was then deposited onto the coated SPIO@SiO2 for 3 h by shaking. The SPIO@CQDs
(n)
was collected
by magnetic separation and obtained by washing several times. The above mentioned process was repeated until the desired SPIO@CQDs(n) (n=3, 6, 9, 12, 15) were obtained.
Characterization CQDs, SPIO, SPIO@SiO2 and SPIO@CQDs(n) were measured by IRPrestige-21 infrared spectrophotometer (Shimadzu) using KBr pellets as the sample matrix. Fluorescence spectra of SPIO@CQDs(n) were obtained by RF-5301PC Fluorescence spectrometer (Shimadzu). Zeta potential of CQDs, SPIO@SiO2, SPIO@SiO2@PDDA and SPIO@CQDs(n) nanoparticles were measured by Zetasizer Nano ZS90 zeta potential analyzer (Malvern). The prepared nanoparticles were also morphologically characterized by a HITACHI H-600 transmission electron microscope (Japan).
Toxicity assay of SPIO@CQDs(n) 6 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
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Journal of Biomedical Materials Research: Part A
SPIO@CQDs(n) were soaked in 75% alcohol to sterilize. A series of SPIO@CQDs(n) solution were prepared in the fresh cell culture fluid at concentrations of 0, 20, 40, 60, 80,100 µg·mL-1, respectively. Hepatoma carcinoma cell HepG2, liver cells L02 and fibroblast cells 3T3-LE were cultured in DMEM Medium at 37°C under 5% CO2 atmosphere after overnight, supplemented with penicillin/streptomycin (100 units·mL-1) and 7% fetal bovine serum (FBS). In the 96-well plates, 103 cells were added into each well. In the second day, the medium was replaced with 200 µL fresh medium containing different concentrations of SPIO@CQDs(n). The cells were cultured for another 24h. Then, 20 µL of MTT regent was added. After 4h, the medium was removed, and 150 µL DMSO was added. The optical density (OD) value of each well was measured at 490 nm using the Varioskan Flash multifunctional enzyme label reading (Thermo Fisher Scientific, USA). The cell toxicity was evaluated by the MTT assay.
Liver cell L02 imaging experiments 103cells·mL-1 of liver cells L02 were seeded on 24-well plates and cultured using established procedures after overnight. Afterwards, the medium was replaced with 600 µL fresh medium containing different concentrations of SPIO@CQDs(n), and the cells were cultured for another 6 h. The liver cells L02 were washed with PBS for three times. Finally, the liver cells L02 were imaged using a LSM780 confocal laser scanning microscopy (CLSM, Zeiss, Germany) to optimize the layer number (n) and concentration of SPIO@CQDs(n). 7 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
Journal of Biomedical Materials Research: Part A
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Similarly, the medium was replaced with 600 µL fresh medium containing the optimum concentration of SPIO@CQDs(n) (n = 12), the cells were respectively cultured for 1, 3 and 6h. Finally, the liver cells L02 were imaged using CLSM to optimize the time that liver cells L02 were labeled by SPIO@CQDs(12). Target HepG2 cells labeling and separating experiments HepG2 cells were labeled with SPIO@CQDs(12) at the same way as L02 cells, and they were imaged using CLSM. HepG2 cells (105 cells·mL-1) were seeded in culture bottle and then cultured using established procedures after overnight. After the HepG2 cells were washed with PBS, 1 mL fresh medium containing 0.2 mg SPIO@CQDs(12) was added and then it was cultured for another 14h. After removing the unincorporated SPIO@CQDs(12) and washing with PBS, the labeled HepG2 cells were digested with pancreatic enzyme. The cells were collected through a TD45 low speed centrifuge at 800 rpm for 5 min, and then the labeled HepG2 cells were dissolved in the suspensions with PBS 102, 103, 104, 105 cells·mL-1
at 10,
respectively, and then their fluorescence intensity
values were detected by fluorescence spectrophotometer. At last, a standard curve of the logarithm of concentration and fluorescence intensity values was plotted. The samples were obtained by mixing labeled HepG2 cells and liver cells L02 with equal amount. The labeled HepG2 cells were collected by magnetic separation and then washed with PBS. At last, the target HepG2 cells were detected by fluorescence
8 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
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Journal of Biomedical Materials Research: Part A
spectrophotometer. The quantity of captured cells was gained according to the standard curve, and capture rate of the target HepG2 cells were calculated. RESULTS
Design and preparation of SPIO@CQDs(n) The schematic illustration of the procedure for fabrication of SPIO@CQDs(n) was shown in Figure 1. As shown in Figure 2A, the TEM imaging suggested that the diameter of the prepared SPIO@SiO2 was adjusted to 40 nm, with silica shell thickness of 30 nm. As depicted in Figure 2B-C, the average diameter of SPIO@CQDs(n) was 200 nm, and the composites obviously showed core-shell structure. As illustrated in Figure 2D, the zeta potential of nanoparticles changes between ±40 mV attributed to their different surface material. The Fourier infrared (FT-IR) spectra of SPIO, SPIO@SiO2, CQDs and SPIO@CQDs(n) were shown in Figure 2E. The characteristic absorption bands of SiO-H and SiO2 in SPIO@SiO2 were at 3480, 1094 cm-1 respectively. In FT-IR spectra of CQDs and SPIO@CQDs(n), the characteristic absorption bands of –NH2 and -OH appeared were at 3400-3500 cm-1; the stretching vibration bands of –NH2 were at 2850-3000 and 2400-2500 cm-1; the characteristic absorption band of –COOH was at 1600 cm-1. Characterization of fluorescent properties
9 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
Journal of Biomedical Materials Research: Part A
Fluorescent spectra of SPIO@CQDs(n) were shown in Figure 2F. When n≥9, SPIO@CQDs(n) had a significant fluorescence characteristic, and then the fluorescence intensity was cumulative with the increase of n.
In addition to the down-converted PL properties, SPIO@CQDs(n) exhibited an up-converted characteristic and the emission wavelength of SPIO@CQDs(n) was increased with the increase of excitation wavelength, as illustrated in Figure 3(A-B). It was shown in Figure 3 (C-D) that a typical down/up-converted PL spectrum of SPIO@CQDs(n) with maximum peak at 430 nm was excited by 345 nm and 680 nm light respectively. The down-converted PL emission and excitation spectra were also depicted in Figure 3E. Interestingly, for the same irradiation time, the fluorescent intensity of CQDs decreased by 63% when that of SPIO@CQDs(n) decreased by 14% (Fig. 3F).
Cytotoxicity assay of SPIO@CQDs(n) MTT assay results were shown in Figure 4. Liver cells L02, hepatoma carcinoma cells HepG2 and fibroblast cells 3T3-LE were respectively incubated with gradient concentrations of SPIO@CQDs (n) for 24 h, and all of them showed high viabilities. Liver cells L02 labeling with SPIO@CQDs(n) Liver cells L02 were cultured with 60 µg·mL-1 of SPIO@CQDs(n) (n=3, 6, 9, 12, 15) at 37℃ for 6h, and after removing the unincorporated SPIO@CQDs(n) by PBS washing, the confocal images were recorded as shown in Figure 5A. With the increase of CQDs layers (n), the fluorescent intensity of the liver cells L02 swallowed 10 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
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Journal of Biomedical Materials Research: Part A
SPIO@CQDs(n) were enhanced, and the CLSM images were much clearer. When the CQDs layers n≥9, cells L02 were labeled effectively by SPIO@CQDs(n) and showed strong fluorescent. SPIO@CQDs(n) with concentrations gradient (20, 40, 60, 80 µg·mL-1; n=12) were incubated with liver cells L02, respectively. After the same treatment, the CLSM images were measured and shown in Figure 5B. The CLSM images became clear, and the fluorescent of SPIO@CQDs(12) labeled liver cells was enhanced as the concentration increase of SPIO@CQDs(12). When the concentration was over 60 µg·mL-1, the fluorescent intensity of liver cells L02 didn’t change any more.
SPIO@CQDs(12) was incubated with L02 cells for 1, 3, 6 h, respectively. After the same treatment, the CLSM images were detected and shown in Figure 6. The luminescent spots widely appeared in the membrane and cytoplasm of the L02 cells, while the fluorescence at the central region involved with nucleus was very weak (Fig. 6.D1-D3). The fluorescent variation with incubation time of L02 cells with SPIO@CQDs(12) was shown in Figure 6A0-C3. When SPIO@CQDs(12) was incubated with L02 cells for only 1h, the L02 cells showed fluorescent, and the mark effect increased with the increase of interaction time between SPIO@CQDs(12) and L02 cells.
HepG2 cells marking and sorting with SPIO@CQDs(n) HepG2 cells were labeled nonspecifically by SPIO@CQDs(12), and the CLSM images were shown in Figure 7A0-A3. After labeling, fluorescence spectrophotometer was 11 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
Journal of Biomedical Materials Research: Part A
used to detect fluorescence response signals of different concentrations of HepG2@ SPIO@CQDs(12), and standard curve of the logarithm of concentration and fluorescence intensity value was plotted (Fig. 7) in the range of 10-105 cells·mL-1 using the equation y = 0.984 x + 6.574 with R2= 0.9258.
HepG2@SPIO@CQDs(12) and liver cells L02 incubated without SPIO@CQDs(12) at the same condition were mixed with 1: 1 ratio. The HepG2@ SPIO@CQDs(12) were collected by the magnet for 30s, and the remaining liver cells L02 were removed by PBS washing. The enriched HepG2@SPIO@CQDs(12) were dispersed to the initial volume of the mixed cells by PBS and detected with fluorescence spectrometer. According to the standard curve (Fig. 7C), the amount of the enriched HepG2@ SPIO@CQDs(12) was calculated. The capture rate (formula Ⅰ) s obtained and it was 90.31%.
In the
equation,
c0 and c1 were
the initial concentration of
HepG2@SPIO@CQDs(12), and the concentration of HepG2@SPIO@CQDs(12) isolated from the hybrid cells, respectively. Capt ur e r at e=
c1 × 100% c0
Ⅰ
DISCUSSION
There was growing focus on magnetic and fluorescent quantum dot nanoparticles due to their outstanding dual function. However, the SPIO cores have a fluorescent quenching effect on QDs due to the electronic coupling and energy transfer. In addition, the broad absorbance spectrum of SPIO also attenuates both the excitation light and emitted fluorescence. In our study, shell structure of SiO2 was adopted as 12 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
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outside protective layer of SPIO to increase the distance and interval between SPIO and CQDs, and this can reduce the fluorescent quenching effect on the QDs.
In order to obtain fluorescent and magnetic multifunctional fluorescent magnetic composites, a new kind of nano fluorescent reagent was prepared, by which the carbon quantum dots with low biological toxicity was layer-by-layer self-assembled to the SiO2 coated superparamagnetism iron oxide nanoparticles (SPIO) (Fig. 1). SPIO@SiO2 core-shell magnetic nanoparticles were obtained from the silica source, SPIO and ethyl silicate (TEOS). The hydrolysis of TEOS in alkaline condition can form gel with three-dimensional structure, which let it coat on SPIO. By controlling the concentration of SPIO, the speed and time of stirring, SPIO@SiO2 nanoparticles with uniform particle size and good dispersive capacity were generated. This was confirmed by the TEM results.
CQDs were quickly prepared by Microwave-assisted hydrothermal method using gelatin as carbon source, ultrapure water and Ethylene glycol as solvent. The experimental CQDs had the characteristics of high fluorescent yield, stable fluorescent performance, good solubility in water and negative charge.
Fluorescent magnetic carbon quantum dots composites (SPIO@CQDs
(n))
were
prepared by repeating the above steps (Fig. 1). Firstly, the strong cationic polyelectrolyte PDDA was absorbed onto SPIO@SiO2 with abundant negative charges by the electrostatic force, and then the compound SPIO@SiO2@PDDA became positively charged, and it could absorb negatively charged CQDs. In the 13 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
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assembly process, in order to increase the effective adsorption of CQDs by SPIO@SiO2, the Tris aqueous of pH 9.0 was employed to enhance the negative charge of the surface and improve the stability of the nanoparticles 11. The changes of zeta potentials during the preparation were also used to monitor the assembly process, which suggested that driven by the electrostatic force, positively charged strong cationic polyelectrolyte PDDA and negatively charged CQDs were alternately absorbed onto SPIO@SiO2. On comparison the Fourier infrared (FT-IR) spectra of SPIO, SPIO@SiO2 and CQDs with that of SPIO@CQDs
(n),
the characteristic absorption bands of SiO-H,
SiO2 and CQDs were all in Fourier infrared (FT-IR) spectra of SPIO@CQDs(n) suggested that SPIO@CQDs
(n)
was successfully obtained by layer by layer
self-assembly.
The fluorescent spectra of SPIO@CQDs(n) excited at the same wavelength (λ = 350 nm) indicated that, with the isolation effects of the silica layer, the innermost layer of CQDs still suffered from the quenching effect of the iron oxides when n
