Subscriber access provided by AUT Library
Article
Ratiometric Upconversion Luminescence Nanoprobe with Near-infrared Ag2S Nanodots as the Energy Acceptor for Sensing and Imaging of pH in vivo Caiping Ding, Shasha Cheng, Cuiling Zhang, Youran Xiong, Mingqiang Ye, and Yuezhong Xian Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00404 • Publication Date (Web): 09 May 2019 Downloaded from http://pubs.acs.org on May 9, 2019
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
Ratiometric upconversion luminescence nanoprobe with near-infrared Ag2S nanodots as the energy acceptor for sensing and imaging of pH in vivo Caiping Ding, Shasha Cheng, Cuiling Zhang,* Youran Xiong, Mingqiang Ye, Yuezhong Xian,* Shanghai Key Laboratory of Green Chemistry and Chemical Processes, Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241, China. *E-mail:
[email protected];
[email protected]. ABSTRACT: A luminescence resonance energy transfer (LRET) system was successfully developed using near-infrared (NIR) Ag2S nanodots (NDs) as the energy acceptors and upconversion nanoparticles (UCNPs) as the energy donors. The system possessing the properties of NIR excitation (980 nm) and NIR emission (795 nm), was used for the ratiometric detection and bioimaging of pH in tumor cells and zebrafish. Glutathione and mercaptopropionic acid (MPA) co-modified Ag2S NDs (GM-Ag2S NDs) were prepared by ligand exchange with an excellent pH-responsive property over a pH range of 4.0 to 9.0. The NIR GMAg2S NDs were covalently grafted with silica coated UCNPs, and an efficient LRET platform was developed via modulation of the thickness of the silica coating. Due to the LRET process between UCNPs and GM-Ag2S NDs, a ratiometric luminescence nanoprobe with the properties of NIR excitation-NIR emission was constructed for pH biosensing and bioimaging. Based on high contrast bioimaging, the nanoplatform can distinguish between tumorous tumor and normal tissue in the zebrafish model.
Lanthanide-doped upconversion nanoparticles (UCNPs) which are able to convert near infrared (NIR) light into ultra-violet, visible and NIR light have attracted considerable interest due to their unique properties, such as high photo-stability, low photodamage, and long luminescence lifetimes.1, 2 Tailored UCNPs are promising candidates for various bioapplications,3 including bio-fluorescence imaging,4 photodynamic tumor therapy,5 siRNA delivery6 and biosensing.7 The developments in these fields are generally dependent on the luminescence resonance energy transfer (LRET) process, which can be constructed by efficient meditation of the energy transfer from the energy donor to the acceptor. UCNPs are commonly used as the energy donor in LRET biosystems.8-13 In addition, the properties of the energy acceptor show a strong impact on the LRET efficiency and analytical performance. Nowadays, small organic dyes,14-19 two dimensional materials (such as graphene20 and MnO221, 22), and metal nanomaterials (such as gold nanoparticles23 and silver nanoclusters24) have been employed as energy acceptors to develop versatile LRET nanoplatforms with superior performance. However, low energy-transfer efficiency, poor visible emission, low photostability and complex preparation are the most significant restrictions encountered when using organic dyes as energy acceptors. In addition, in most UCNPs-inorganic nanomaterials-based LRET system, acceptors usually quench the luminescence of UCNPs, and no new emission can be obtained. Thus, the probability of observing a false positive signal might be greatly increased. Given the aforementioned problems, there is an urgent need to develop new acceptors for LERT-based platforms for use in comprehensive biological applications. NIR fluorescence possesses noninvasive properties and is beneficial for the detection and imaging of biomolecules.25 For
example, NIR fluorescent Ag2S nanodots (NDs) have been widely applied in bioimaging and chem-/bio-detection.26-29 Ag2S NDs exhibit low toxicity or can even be nontoxic due to being free of toxic metals (such as Cd2+, Pb2+, Hg2+) and ultralow solubility product constant (Ksp = 6.3× 10-50).30 Owing to their excellent biocompatibility, NIR Ag2S NDs have been employed as nanoprobes for bioassays31 and bioimaging in living cells32, 33 and tissues.34, 35 The above applications of Ag2S NDs were commonly used as labeled fluorescence probes to achieve long term and dynamic in vivo cell tracking. In Ag2S NDs-based LRET bioassays, the NDs usually act as the energy donor and their fluorescence is easily quenched by various quenching agents. For example, Kuang et al. reported the ultrasensitive detection and bioimaging of microRNAs in living cells based on the energy transfer from Ag2S NDs to AuCu9S5 nanoparticles.36 Au nanorod-Pt@Ag2S core-satellite nanostructures were fabricated through complementary DNA assembly, which were used for fluorescent detection and imaging of intracellular microRNA-21 based on the disassembly of the core-satellite structures and observing the fluorescence induced by DNA hybridization.37 In addition to the aforementioned features of Ag2S NDs, there is still great demand for searching Ag2S NDs with new properties so that can be used for complex biological applications. In this work, we report a novel LRET nanoplatform for in vivo ratiometric pH sensing, in which NIR Ag2S NDs modified with 3-mercaptopropionic acid (3-MPA) and glutathione (GSH) (GM-Ag2S NDs) were used as the energy acceptor and UCNPs were employed as the energy donor. As shown in Scheme 1, GM-Ag2S NDs can be assembled with silica coated UCNPs by a coupling reaction. GM-Ag2S NDs were for the first time used as the energy acceptor, which could be excited via nonradiative energy transfer and reabsorption from NaGdF4:Yb/Er UCNPs
ACS Paragon Plus Environment
Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
and emit its own NIR luminescence. The LRET nanoplatform exhibits superior performance with regards to bioassays due to NIR excitation (980 nm) and NIR emission (795 nm), which is suitable for efficient elimination of background luminescence interference from biological systems. Interestingly, the GMAg2S NDs obtained through ligand exchange between GSH and 3-MPA modified NDs show a broad dynamic response toward pH in the range of 4.0-9.0. Ratiometric pH detection was realized by monitoring the pH-dependent red luminescence from the GM-Ag2S NDs at approximately 795 nm and the green luminescence of the NaGdF4:Yb/Er at approximately 540 nm as the reference signal. The nanoplatform has been successfully applied in the biological system for the measurement of intracellular pH and differentiation of tumor and normal tissue in zebrafish.
Scheme 1. Schematic illustration of the developed LRET nanoplatform as a ratiometric nanoprobe using pH-responsive GM -Ag2S NDs as the energy acceptor and UNCPs as the energy donor in tumor cells and tumor zebrafish. EXPERIMENTAL SECTION Materials and Reagent. AgNO3, NaHPO4‧12H2O, KH2PO4, NaH2PO4, NaCl, KCl, CaCl2, MgSO4 and ethylene glycol (EG, ≥99%) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 3-MPA (99%) was supplied from J&K Chemical Technology Co., Ltd. (Shanghai, China). N-(3dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) was supplied from Alfa Aesar Co., Ltd. GSH, NH4F (AR, 98%), 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) were obtained from Aladdin Industrial Co. Ltd. (Shanghai, China). Ln(NO3)3·xH2O (Ln = Gd, Yb, and Er, 99.99% metals basis), PEI (average MW ~25 000) and nigericin were purchased from Sigma-Aldrich (Beijing, China). N-hydroxysuccinimide (NHS) was supplied from Macklin Co., Ltd. (Shanghai, China). Cell counting Kit-8 (CCK-8) and trypsin were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Dulbecco's modified eagle medium (DEME) and Dulbeccos phosphate-buffered (D-PBS) was supplied from Sangon Biotech Co., Ltd. (Shanghai, China). Fetal bovine serum (FBS) and penicillin-streptomycin (P/S) solution were obtained from Dingguo Biotech Co., Ltd. (Shanghai, China). The living Hela cells were obtained from the China Infrastructure of Cell Line Resources (Shanghai, China). The ultrapure water was used throughout the experiment. Preparation of GM-Ag2S NDs. According to our previous method, 29Ag2S NDs with 3-MPA as the ligand were prepared, and then GM-Ag2S NDs were synthesized by further modification. In brief, 200 µM GSH was added into the colloid
of 100 μg/mL of MPA-Ag2S NDs and reacted for 12 h at room temperature. The obtained GM-Ag2S NDs were then washed with deionized water using Amicon-Ultra centrifugal filters (3000 Da cut off) and stored in the dark at 4 °C. In order to disclose the composition on the surface of Ag2S NDs, elemental analysis was performed. For ligand exchange, 5.0×10-5 mol GSH was added into the 10 mL unpurified MPAAg2S NDs containing 2.5×10-5 mol MPA for 12 h at room temperature. After that, the mixture was purified using Amicon-Ultra centrifugal filters (3000 Da cut off) and freezedried for analysis. As for the controlled experiment, the ligand exchange reaction was conducted as above-mentioned procedure, and then, the mixture was used for elemental analysis after freeze-drying. Preparation of PEI-Capped NaGdF4:Yb/Er UCNPs. PEIcapped NaGdF4:Yb/Er UCNPs were synthesized by a facile one-step solvothermal method. In brief, 0.4 g of PEI were dispersed in 18 mL of EG with vigorous stirring and then 2.4 mmol of NaCl were added. Then, 1.2 mmol of lanthanide dopants Gd(NO3)3, Yb(NO3)3, and Er (NO3)3 with a molar ratio of 78:20:2 were added to the above-mentioned solution to form a transparent solution. After that, 0.2413 g NH4F in 12 mL of EG were added to the above mixture and stirred for 10 min, and then the solution was transferred into a 50 mL Teflon-lined stainless steel autoclave and heated at 200 °C for 2 h. Subsequently, the as-synthesized UCNPs were separated from the reaction mixture by centrifugation and washed several times with ethanol and water. The UCNPs were then dried in a vacuum at 60 °C for 12 h. Preparation of UCNPs@SiO2-NH2. Firstly, 1mg/mL UCNPs were dissolved in 4 mL deionized water, and then 0.01 g CTAB and 0.08 mL NH3·H2O were added to the above solution and sonicated for 20 min. Then, different amounts of TEOS (10, 20, 30 and 50 μL) were added and the mixtures were rotated for 6 h with speed 800 rmp. The silica shellcoated UCNPs were washed with water three times. In order to realize the amination, the nanoparticles were redispersed in 4 mL of water, and then, 15.5 μL of APTES was added. After reaction for 8 h at room temperature, the obtained products were washed three times with water and dried in a vacuum for further use. Assembly of UCNPs@SiO2-NH2 with GM-Ag2S NDs. Carbodiimide chemistry was used to realize the covalent coupling between UCNPs@SiO2-NH2 and GM-Ag2S NDs. Approximately 5 mg of GM-Ag2S NDs was activated in 50 mM EDC and 50 mM NHS in 1.5 mL PBS (0.01 M, pH 6.8) at room temperature with gentle shaking for 30 min. Subsequently, the activated GM-Ag2S NDs were separated by Amicon-Ultra centrifugal filters (3000 Da cut off) and washed with 0.01 M PBS (pH 7.2) three times. Then, the NDs were resuspended in 1 mL PBS (0.01 M, pH 7.2) and reacted with 200 μg of UCNPs@SiO2-NH2 for approximately 12 h under continuous shaking at room temperature. The resulting UCNPs@SiO2-Ag2S NDs were washed with PBS and then stored at 4 °C for use. UCNPs@SiO2-Ag2S nanoprobe for pH sensing. A series of standard pH solutions were prepared with 0.1 M HCl and 0.1 M NaOH. 200 μL of 1 mg/mL stock solution of UCNPs@SiO2-Ag2S NDs nanoprobe was added into 1800 μL of standard pH solution and the fluorescence signal over the range of 400-900 nm was collected by excitation at 980 nm.
ACS Paragon Plus Environment
Page 2 of 9
Page 3 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry Cytotoxicity of UCNPs and UCNPs@SiO2-Ag2S nanoprobe. Cytotoxicity of UCNPs and UCNPs@SiO2-Ag2S NDs was determined by CCK-8 assays. Before the experiment, 5.0×104 Hela cells were seeded in a 96-well plate for 12 h and were incubated with various concentrations of UCNPs (10, 20, 40, 60, 80, 100 and 200 μg/mL) and UCNPs@SiO2-Ag2S NDs (10, 20, 40, 60, 80, 100, 200 and 400 μg/mL) in 100 µL of DMEM medium supplemented with 10% FBS in a humidified incubator at 37 °C containing 5% CO2. After 24 h incubation, the medium was removed and replaced with a mixture containing 100 µL of fresh DMEM and 10 µL of CCK-8 reagent solution. After 4 h incubation, the absorbance of the solution at 450 nm was measured using an AC100-120 microplate reader. UCNPs@SiO2-Ag2S nanoprobe for pH sensing in tumor cells. Hela cell-spiked samples were prepared according to the following procedure. Hela cells were stained with 400 μg/mL UCNPs@SiO2-Ag2S NDs for 4 h. Before to use, adherent cells were washed three times with PBS to remove excess UCNPs@SiO2-Ag2S nanoprobe. The UCNPs@SiO2-Ag2S labeled Hela cells were incubated with high K+ ion buffer solution (30.0 mM NaCl, 120.0 mM KCl, 1.0 mM CaCl2, 0.5 mM MgSO4, 1.0 mM NaH2PO4, 5.0 mM glucose, 20.0 mM HEPES) containing 10.0 μM negericin at different pHs at 37 ℃ for 30 min. Upconversion luminescent bioimaging in Hela cells at different pH conditions was observed using a Nikon confocal microscope. The excitation wavelength was 980 nm and the collection emission wavelengths were 520±25 nm and 795±25 nm. UCNPs@ SiO2-Ag2S nanoprobe for pH sensing in tumorbearing zebrafish. 72 hpf of zebrafishes were microinjected with 2.0 μg of GM-Ag2S NDs or 4.0 μg of UCNPs@SiO2-Ag2S and then cultured at 28 °C in 5 mL of embryo media at different pH (6.0-9.0) for 1.0 h. After that, the zebrafishes were observed by fluorescence imaging. To build a tumor model, 3 day old zebrafish embryos were microinjected with Hela cells and cultured for 24 h, and then 4.0 μg of UCNPs@SiO2-Ag2S nanoprobe were microinjected and cultured over different periods of time. The upconversion luminescence bioimaging in zebrafish was observed using a Nikon confocal microscope. RESULTS AND DISCUSSION Characterization of GM-Ag2S NDs. GM-Ag2S NDs were prepared via a ligand exchange reaction between GSH and MPA on the surface of the Ag2S NDs. The thiol groups of GSH are able to form stable Ag-S bonds on the surface of the Ag2S NDs by the competition with MPA.38 To investigate the composition on the surface of the Ag2S NDs after ligand exchange, we measured the carbon (C) and nitrogen (N) content in GM-Ag2S NDs by elemental analysis. The N source (relative atomic mass of 14.007) originates from the GSH, and C (relative atomic mass of 12.01) is from MPA and GSH. As for the controlled experiment, the theoretical mass ratio of C and N should be 3.287 based on the molar ratio of GSH/MPA of 2:1 in the unpurified sample. As for the purified Ag2S NDs, the mass ratio of C and N should be 2.858 if only GSH is present on the surface of Ag2S NDs. The results for elemental analysis are shown in Table S1. The C/N mass ratio for the controlled experiment (unpurified sample) is approximately 3.283, which is very close to the theoretical value of ~3.287. Whereas the C/N mass ratio for purified Ag2S NDs is approximately 3.045 (2.858
