- Nano Express
- Open Access
Plant Cell Imaging Based on Nanodiamonds with Excitation-Dependent Fluorescence
© The Author(s). 2016
- Received: 5 June 2016
- Accepted: 19 September 2016
- Published: 23 September 2016
Despite extensive work on fluorescence behavior stemming from color centers of diamond, reports on the excitation-dependent fluorescence of nanodiamonds (NDs) with a large-scale redshift from 400 to 620 nm under different excitation wavelengths are so far much fewer, especially in biological applications. The fluorescence can be attributed to the combined effects of the fraction of sp2-hybridized carbon atoms among the surface of the fine diamond nanoparticles and the defect energy trapping states on the surface of the diamond. The excitation-dependent fluorescent NDs have been applied in plant cell imaging for the first time. The results reported in this paper may provide a promising route to multiple-color bioimaging using NDs.
- Cell imaging
- Plant cell
Fluorescent nanodiamonds (NDs) have been investigated extensively for bioimaging, single-photon source, and drug delivery due to their remarkable optical properties, such as high photostability, facile surface functionalizability, aqueous dispersibility, good biocompatibility, and inertness [1–12]. To date, most of the bioimaging studies using fluorescent NDs are based on their color centers, especially nitrogen vacancy (N-V) centers [13–15]. However, the fluorescence efficiency of the color centers is usually low. Although some methods like ion implications and ion irradiating have been adopted to improve the fluorescence efficiency of color centers in NDs, cost-intensive techniques are always needed, which impede the applications of the fluorescent NDs in a large scale. In addition, the N-V centers in NDs are usually prepared by irradiation using a high-energy electron beam, proton beam, or helium ions, in which sophisticated equipment and complex experimental procedure are required [2, 16–18]. Therefore, finding a simple and cost-effective method for preparing fluorescent NDs is urgently needed. Recently, by taking advantage of the surface functionalization, researchers realized surface defect-related emission by modified NDs with various functional groups. For example, Mochalin et al. reported blue fluorescent NDs through wet chemistry route by conjugating carboxylated NDs with octadecylamine . The fluorescence from functional groups can be excited by a common light source. However, the water solubility of such NDs is poor, which hinders the applications of the NDs in biotechnology. Xiao et al. have reported NDs with excitation-dependent fluorescence properties, and the fluorescence was attributed to the diverse functional groups residing on the NDs . Nevertheless, the report on bioimaging using the excitation-dependent NDs has not been reported to date, which cast a shadow on whether such NDs can be employed in bioimaging. Moreover, theoretical calculations demonstrated that the fluorescence of carbon nanoparticles can also be adjusted by controlling the content of the sp2-hybridized carbons isolated by sp3 carbons and the size of the conjugated sp2 domains in isolated carbon nanoparticles [21, 22]. Nevertheless, experimental data are still absent currently. At present, most bioimaging research based on NDs is centered on animal cells, but the work associating plant cells and NDs is very rare. Therefore, it is meaningful to expand the application field based on NDs in plant word.
Herein, we reported an easy way to prepare NDs with excitation-dependent fluorescence, and the fluorescence can be attributed to the combined effects of the defect states on the surface of the NDs and sp2-hybridized domains. Synthetic type Ib diamond nanoparticles with a mean particle diameter of 50 nm were obtained by annealing at 420 °C for 30 min. The excitation-dependent fluorescent NDs have been applied in plant cell imaging for the first time. The results reported in this paper may provide a promising route for multiple-color bioimaging using NDs.
Synthesis of Fluorescent NDs
Synthetic type Ib diamond powders were purchased from Zhongnan Jete Superabrasives Co., Ltd (Zhengzhou, China). The NDs were sintered at 420 °C in air for 30 min to regulate the content of the sp2-hybridized carbon on the ND surface. The annealed NDs were dissolved in deionized water and separated by 8500 rpm centrifugation to remove the possible large agglomerates or impurities. The centrifuged NDs were then dispersed in deionized water (1 mg ml−1) for further research.
Mung Bean Sprout Cultivation
Mung bean seeds having uniform size were placed in a Petri dish. Fifty milliliters of fluorescent NDs solution (1 mg ml−1) has been employed as the culture medium. The mung bean seeds were cultivated in the fluorescent NDs aqueous solution for 48 h at 25 °C in the dark until cotyledons emerged; after that, the spouts were rinsed by deionized water for five times to remove the possibly adsorbed NDs and contaminations. The control experiment was carried out in the same conditions except the culture medium was replaced by the deionized water.
The mung bean hypocotyl was sliced with freezing microtome and the thickness of the thin slice was about 40 mm. Confocal microscope images were taken to evaluate the cell imaging applications of the fluorescent NDs. Cell images were observed with a confocal laser scanning microscope (CLSM) Zesis 710 3-channels (Zesis, Germany) with the excitation wavelength at 405 nm, 488 nm and 480-550 nm, 600-680 nm of the emission filter.
The morphology and microstructure of the fluorescent NDs were characterized by a field emission scanning electron microscope (FESEM, JSM 6700F) and a high-resolution transmission electron microscope (HRTEM, FEI Technai G2 F20). The structural properties of the fluorescent NDs were characterized using a micro-Raman spectroscope (Renishaw RM 2000) and an X-ray diffractometer (XRD, PA National X’ Pert Pro). The Fourier transform infrared (FTIR) spectra of the NDs were recorded on a Bio-Rad Excalibur spectrometer (Bruker vector 22). A small amount of NDs was mixed with potassium bromide (KBr), grinded adequately, and then made into a pallet for a test. The absorption spectrum was measured by a Shimadzu UV-2401 instrument.
The fluorescence spectra of the NDs were measured by a double-grating spectrophotometer (Horiba FL-322). The transient fluorescence of the NDs was also measured by the Horiba FL-322 spectrometer using the following instrumental settings: 280 nm NanoLED; time range of 200 ns; peak preset at 5000 counts; repletion rate at 1 MHz; and synchronous delay of 50 ns.
To clarify the composition of the functional groups on the surface of the NDs, FTIR spectrum of the NDs has been measured, as shown in Fig. 2c. The vibration modes at around 800–1000 cm−1 can be attributed to the C–O stretching mode, while the peak at around 3450 cm−1 to the stretching vibration mode of the O–H bond . The functional groups on the surface of the NDs may play an important role in determining the fluorescent properties of the NDs. The absorption spectrum of the NDs in aqueous solution is indicated in Fig. 2d. The spectrum shows a broad absorption extending to the visible region, and a well-defined peak appears at 229 nm (5.42 eV), which may be originated from the band edge absorption of diamond.
Fitted decay times of the fluorescence of the NDs at different wavelengths under a 280-nm excitation
Based on the above results, the possible mechanism for the excitation-dependent fluorescence from the NDs has been proposed. There exist various sizes of conjugated sp2 domains around the isolated sp3 ND cores. By increasing the sp2 bonding fraction and the size of the conjugated sp2 domains, the fluorescence induced by radiative recombination redshifts gradually. Meanwhile, the defect energy trapping states increase correspondingly, which may facilitate the occurrence of the nonradiative recombination. Therefore, the fluorescence peak of the NDs redshifts and the corresponding intensity diminishes gradually as the excitation wavelength increases.
In conclusion, water dispersible fluorescent NDs have been prepared, and excitation-dependent fluorescence has been observed in the NDs. The excitation-dependent fluorescent NDs have been employed in mung bean cell imaging for the first time. Note that the excitation-dependent fluorescence may help to avoid the fluorescence interference of organism in plant and animal cells; thus, potential applications in high-quality cell imaging based on NDs may be achieved in the future.
This work is financially supported by the National Science Foundation for Distinguished Young Scholars of China (61425021), the Natural Science foundation of China (11374296 and 51272238), the China Postdoctoral Science Foundation (2015M582192, 2016T90671), and Startup Research Fund of Zhengzhou University (1512317006).
CXS conceived the idea and supervised the project. LXS and QL designed and conducted the experiments. LXS, QL, CXS, and ZJ performed the data analysis. LXS, QL, CXS, and ZJ wrote the manuscript. All of the authors discussed the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
- Aharonovich I, Greentree AD, Prawer S (2011) Diamond photonics. Nat Photonics 5(7):397–405View ArticleGoogle Scholar
- Chang YR, Lee HY, Chen K, Chang CC, Tsai DS, Fu CC, Lim TS, Tzeng YK, Fang CY, Han CC, Chang HC, Fann W (2008) Mass production and dynamic imaging of fluorescent nanodiamonds. Nat Nanotechnol 3(5):284–288View ArticleGoogle Scholar
- Song PA, Yu YM, Wu Q, Fu SY (2012) Facile fabrication of HDPE-g-MA/nanodiamond nanocomposites via one-step reactive blending. Nanoscale Res Lett 7(1):355Google Scholar
- Wort CJH, Balmer R (2008) Diamond as an electronic material. Mater Today 11(1–2):22–28View ArticleGoogle Scholar
- Zhang XY, Yin JL, Kang C, Li J, Zhu Y, Li WX, Huang Q, Zhu ZY (2010) Biodistribution and toxicity of nanodiamonds in mice after intratracheal instillation. Toxicol Lett 198(2):237–243View ArticleGoogle Scholar
- Zhang XY, Wang SQ, Fu CK, Feng L, Ji Y, Tao L, Li SX, Wei Y (2012) PolyPEGylated nanodiamond for intracellular delivery of a chemotherapeutic drug†. Polym Chem 3(10):2716–2719View ArticleGoogle Scholar
- Zhang XY, Wang SQ, Liu MY, Hui JF, Yang B, Tao L, Wei Y (2013) Surfactant-dispersed nanodiamond: biocompatibility evaluation and drug delivery applications. Toxicol Res 2(5):335–342View ArticleGoogle Scholar
- Gismondi A, Reina G, Orlanducci S, Mizzoni F, Gay S, Terranova ML, Canini A (2015) Nanodiamonds coupled with plant bioactive metabolites: a nanotech approach for cancer therapy. Biomaterials 38(38):22–35View ArticleGoogle Scholar
- Gismondi A, Nanni V, Reina G, Orlanducci S, Terranova ML, Canini A (2016) Nanodiamonds coupled with 5,7-dimethoxycoumarin, a plant bioactive metabolite, interfere with the mitotic process in B16F10 cells altering the actin organization. Int J Nanomedicine 11(1):557–574View ArticleGoogle Scholar
- Perevedentseva E, Hong SF, Huang KJ, Chiang IT, Lee CY, Tseng YT, Cheng CL (2013) Nanodiamond internalization in cells and the cell uptake mechanism. J Nanopart Res 15(8):1834View ArticleGoogle Scholar
- Mochalin VN, Shenderova O, Ho D, Gogotsi Y (2011) The properties and applications of nanodiamonds. Nat Nanotechnol 7(1):11–23View ArticleGoogle Scholar
- Kurantowicz N, Strojny B, Sawosz E, Jaworski S, Kutwin M, Grodzik M, Wierzbicki M, Lipińska L, Mitura K, Chwalibog A (2015) Biodistribution of a high dose of diamond, graphite, and graphene oxide nanoparticles after multiple intraperitoneal injections in rats. Nanoscale Res Lett 10(1):398View ArticleGoogle Scholar
- Liao WH, Wei DH, Lin CR (2012) Synthesis of highly transparentultrananocrystalline diamond films from a low-pressure, low-temperature focused microwave plasma jet. Nanoscale Res Lett 7(1):82Google Scholar
- Treussart F, Jacques V, Wu E, Gacoin T, Grangier P, Roch JF (2006) Photoluminescence of single colour defects in 50 nm diamond nanocrystals. Physica B Condens Matter 376:926–929View ArticleGoogle Scholar
- Krüger A, Liang Y, Jarre G, Stegk J (2006) Surface functionalisation of detonation diamond suitable for biological applications. J Mater Chem 16(24):2322–2328View ArticleGoogle Scholar
- Neugart F, Zappe A, Jelezko F, Tietz C, Boudou JP, Krueger A, Wrachtrup J (2007) Dynamics of diamond nanoparticles in solution and cells. Nano Lett 7(12):3588–3591View ArticleGoogle Scholar
- Lawson SC, Fisher D, Hunt DC, Newton ME (1998) On the existence of positively charged single-substitutional nitrogen in diamond. J Phys Condens Matter 10(27):6171–6180View ArticleGoogle Scholar
- Davies G, Hamer MF (1976) Optical studies of the 1.945 eV vibronic band in diamond. Proceedings of the Royal Society: A Mathematical Physical and Engineering Sciences 348(1653):285–298View ArticleGoogle Scholar
- Mochalin VN, Gogotsi Y (2009) Wet chemistry route to hydrophobic blue fluorescent nanodiamond. J Am Chem Soc 131(13):4594–4595View ArticleGoogle Scholar
- Xiao J, Liu P, Li L, Yang G (2015) Fluorescence origin of nanodiamonds. J Phys Chem C 119(4):2239–2248View ArticleGoogle Scholar
- Qu S, Zhou D, Li D, Ji W, Jing P, Han D, Liu L, Zeng H, Shen D (2016) Toward efficient orange emissive carbon nanodots through conjugated sp(2)-domain controlling and surface charges engineering. Adv Mater 28(18):3516–3521View ArticleGoogle Scholar
- Sk MA, Ananthanarayanan A, Huang L, Lim KH, Chen P (2014) Revealing the tunable photoluminescence properties of graphene quantum dots. J Materials Chem C 2(34):6954–6960View ArticleGoogle Scholar
- Li W, Irle S, Witek HA (2010) Convergence in the evolution of nanodiamond Raman spectra with particle size: a theoretical investigation. ACS Nano 4(8):4475–4486View ArticleGoogle Scholar
- Wang X, Low XC, Hou W, Abdullah LN, Toh TB, Rashid MMA, Ho D, Chow EK (2014) Epirubicin-adsorbed nanodiamonds kill chemoresistant hepatic cancer stem cells. ACS Nano 8(12):12151–12166View ArticleGoogle Scholar
- Hsu MH, Chuang H, Cheng FY, Huang YP, Han CC, Chen JY, Huang SC, Chen JK, Wu DS, Chu HL (2014) Directly thiolated modification onto the surface of detonation nanodiamonds. ACS Appl Mater Interfaces 6(10):7198–7203View ArticleGoogle Scholar
- Zhang XY, Wang SQ, Zhu CY, Liu MY, Ji Y, Feng L, Tao L, Wei Y (2013) Carbon-dots derived from nanodiamond: photoluminescence tunable nanoparticles for cell imaging. J Colloid Interface Sci 397:39–44View ArticleGoogle Scholar
- Li XM, Zhang SL, Kulinich SA, Liu YL, Zeng HB (2014) Engineering surface states of carbon dots to achieve controllable luminescence for solid-luminescent composites and sensitive Be2+ detection. Sci Rep 4(2):4976Google Scholar
- Tan DZ, Zhou SF, Xu BB, Chen PP, Shimotsuma Y, Miura K, Qiu JR (2013) Simple synthesis of ultra-small nanodiamonds with tunable size and photoluminescence. Carbon 62:374–381View ArticleGoogle Scholar
- Collins AT, Thomaz MF, Jorge MIB (1983) Luminescence decay time of the 1.945 eV centre in type Ib diamond. J Phys C 16(11):2177–2181View ArticleGoogle Scholar