Highly Stable and Multi-Emissive Silver Nanoclusters Synthesized in

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Biological and Medical Applications of Materials and Interfaces

Highly Stable and Multi-Emissive Silver Nanoclusters Synthesized in situ in DNA Hydrogel and Their Application for Hydroxyl Radical Sensing Jing Li, Jiantao Yu, Yishun Huang, Haoran Zhao, and Leilei Tian ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b09152 • Publication Date (Web): 12 Jul 2018 Downloaded from http://pubs.acs.org on July 13, 2018

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ACS Applied Materials & Interfaces

Highly Stable and Multi-Emissive Silver Nanoclusters Synthesized in situ in DNA Hydrogel and Their Application for Hydroxyl Radical Sensing Jing Li, Jiantao Yu, Yishun Huang, Haoran Zhao, and Leilei Tian* Department of Materials Science and Engineering, Southern University of Science and Technology, 1088 Xueyuan Blvd., Nanshan District, Shenzhen, Guangdong 518055, P. R. China. KEYWORDS DNA hydrogel, silver clusters, multi-color cell imaging, reactive oxygen/nitrogen species, ratiometric fluorescence probe

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ABSTRACT

Oligonucleotide-stabilized silver nanoclusters (AgNCs) show promising applications in bioimaging and bio/chemo-sensing. However, unsatisfactory photostability limits their practical applications. In this work, fluorescent AgNCs were synthesized in situ in a DNA hydrogel, consisting of cross-linked enzymatically amplified polymeric DNAs with cytosine-rich sequences in the presence of Ag+. The fluorescence property of the resultant AgNCs was optimized by a rational design of the DNA sequences to cover a broad spectrum with comparable green and red emissions. Under the protection of the DNA hydrogel, the AgNCs showed significantly improved photostability in an ambient oxygen environment, as well as low cytotoxicity even at a high concentration. Therefore, these properties show the rolling-circleamplification (RCA)-stabilized AgNCs to be a promising possible fluorescence probe for the detection of reactive oxygen/nitrogen species (ROS/RNS) in live cells because red-emitting species are susceptible to oxidation and consequently convert to green-emitting species. Finally, the as-prepared AgNCs were demonstrated to be a sensitive and specific probe for cellular imaging and the monitoring of ROS/RNS levels, which broadens the applications of AgNCs and provides a new tool for related biological investigations.


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INTRODUCTION Oligonucleotide-stabilized silver nanoclusters (AgNCs) have attracted much research attention due to their facile preparation, water solubility, and high fluorescence, comparable to that of organic dyes and quantum dots.1-2 The emission of AgNCs is highly dependent on the sequences of the oligonucleotide ligands and can be continuously tuned to cover the entire visible and nearinfrared regions,3-4 thus accommodating various bioimaging applications. Moreover, the emission of AgNCs is sensitive to changes in the oligonucleotide conformation and the surrounding environment, making them popular bio-sensing materials for the detection of pH, temperature, metal ions, biomolecules, etc.5-8 For example, Zhu and co-workers utilized AS1411 aptamer-stabilized AgNCs as specific fluorescent probes to image and target human breast cancer cells.5 Werner and co-workers discovered the emission enhancement effect when AgNCs were near guanine-enriched DNA sequences; consequently, they developed related biosensors to detect influenza viruses.9 Despite their rapid development, there are still shortcomings for oligonucleotide-stabilized AgNCs. For example, the photostability of most oligonucleotidestabilized AgNCs is still not satisfactory.10 Liu and coworkers reported that the red-emitting AgNCs showed poor stability, starting to decay from the first hour and turning to green-emitting 24 hours later.11-12 The investigations found that, the decrease in red fluorescence upon UV-light exposure is accompanied by an increase in green fluorescence, demonstrating that this conversion is an oxidation process because UV light stimulates the generation of reactive oxygen species.11,13-18 According to further structual characterization, upon oxidation, the sizes of redemitting AgNCs are reduced to generate smaller clusters (which might be the green- or yellowemitting species) and Ag+ ions, accompanied by electron loss. Moreover, the oxidation process can be reversed by the addition of a reducing agent, such as NaBH4, resulting in the conversion


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of green-emitting AgNCs to the red-emitting species. Therefore, the interconversions between the two species are essentially a reversible redox process driven by the intra- or inter-strand transfer of Ag+ ions and accompanied by electron loss and gain. The redox processes are reversible because that the AgNCs are always associated with DNAs during the structural interconversion processes. In addition, there are some other issues that would limit the further development of AgNCs, such as their potential biotoxicity, the high cost of fabricating a large quantity of oligonucleotides and the under exploration of their properties. Nanoparticle-hydrogel composites have attracted much research interest,19 because the incorporation into hydrogels decreases the toxicity of the nanoparticles; meanwhile, the combination of these two types of materials has been thought to generate not only structural diversity but also property enhancement. Among all the hydrogel materials, DNA hydrogels have become a popular research topic due to the uniqueness of DNAs,20-22 which has been utilized as a matrix for controlling the growth of metal nanoparticles.19, 23-25 On one hand, the strong affinity between DNA and transition metals allows an efficient concentration of metal precursors inside the hydrogel.26-30 On the other hand, with respect to oligonucleotides, condensed DNA in a hydrogel may provide better protection for functional species.31 Herein, we used an RCA method to develope a new functional DNA hydrogel that is cross-linked at the cytosine-rich sequences through cytosine-Ag+-cytosine interactions (Scheme 1). In the hydrogel, AgNCs were synthesized through the reduction of the encapsulated Ag+, which would be in situ stabilized by the surrounding aggregated DNAs. Therefore, these RCA-stabilized AgNCs (hereafter abbreviated RCA-AgNCs) exhibited greatly improved photo- and thermostability and much lower toxicity in the ambient environment. As the red-emission AgNCs are susceptible to oxidation, recently, some well-designed AgNCs samples have been applied to intracellular


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imaging and monitoring of reactive oxygen or nitrogen species (ROS/RNSs), especially hydroxyl radical (•OH).32-33 The superior photostability of our RCA-AgNCs will benefit selectivity in the detection of ROS/RNS, because only strong oxidation agents can quench the red fluorescence. Moreover, the AgNCs are protected by very long and condensed RCA products, and thus their reversible response to redox switches might be improved to allow for a ratiometric response with improved detection accuracy. Therefore, multicolor RCA-AgNCs were synthesized through a rational design of the DNA sequences, whose red emission is weakened and whose green emission is simultaneously enhanced under the strong oxidation condition. We finally demonstrated that this kind of RCA-AgNCs is a sensitive and specific ratiometric fluorescence probe for the detection of ROS/RNS in live cells and shows the best selectivity for •OH. It is well-known that the intercellular level of ROS/RNS is important for a wide variety of physiological processes; in particular, •OH is closely related to cellular damage, mutagenesis, cancer, and degenerative diseases.34-35 Therefore, this work not only broadens the application horizon of DNA-stabilized AgNCs but also provides a potential tool for antioxidant therapy and related biological research.

EXPERIMENTAL SECTION Reagents. Silver nitrate (AgNO3), sodium borohydride (NaBH4), acetic acid (HAc), phenol /chloroform /isoamyl alcohol (25:24:1), sodium acetate (NaAc), ethylene diamine tetraacetic acid disodium salt (EDTA-Na2), dimethyl sulfoxide (DMSO), hydrogen peroxide (H2O2, 30%), sodium nitrite (NaNO2), sodium hypochlorite (NaClO, 5% available chlorine), 2,2′-azobis (2methylpropionamidine)-dihydrochloride (AAPH), magnesium chloride (MgCl2), sodium chloride (NaCl), potassium chloride (KCl), zinc acetate Zn(OAc)2, ferric chloride (FeCl3), ferrous sulfate


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(FeSO4), copper sulfate (CuSO4), calcium chloride (CaCl2), lead dichloride (PbCl2), mercuric perchlorate {Hg(ClO4)2}, D-cysteine (D-Cys), L-cysteine (L-Cys), L-glutamine (L-Glu), Lphenylalanine (L-Phe), glycine (GLy), bovine serum albumin (BSA), ascorbic acid (Vc), and glutathione (GSH) were purchased from Aladdin, China. Absolute alcohol, lipopolysaccharides from Escherichia coli (LPS, L2880), and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich Chemistry Co. Ltd, China. The DNA sequences in Table S1 were purchased from Sangon Biotech, China. The 15,000 bp DNA marker was purchased from Artibody & Life service, China. GelRed (×10,000) was obtained from Biotium, USA. The ultrapure water was purified by a Milli-Q water purification system (Millipore Corp., MA, USA). The modified phosphate-buffered saline (M-PBS) was made up of 60 mM disodium phosphate, 50 mM monosodium phosphate, 2.7 mM monopotassium phosphate and 2 mM MgCl2 (pH 7.2). T4 ligase and deoxyribonucleotide triphosphates (dNTPs) were purchased from Takara, China. Phi 29 polymerase was purchased from Thermo Fisher, USA. Fetal bovine serum (FBS), trypsin−EDTA solution, penicillin/streptomycin and Dulbecco’s Modified Eagle’s Medium (DMEM) were obtained from Gibco BRL Co., Ltd., USA. Human cervical carcinoma cells (HeLa), human breast cancer cells (MCF-7), and human alveolar epithelial cells (A549) were kindly provided by Professor Ying Sun at the Department of Biology of Southern University of Science and Technology (SUSTech). The Ramos cell line was purchased from Fu Cheung Biotechnology, China. Instruments. The RCA reactions were conducted by an Eppendorf ThermoMixer®C. The rheological behavior of Ag+ crosslinked DNA hydrogel was characterized by a HAAKE MARS III rheometer. The UV-Vis absorbance spectra were measured on a BioTek Cytation 3 and the fluorescence spectra were measured on a HORIBA FluoroMax-4. The samples of RCA-AgNCs


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were diluted, dropped on a fresh mica substrate, and completely dried at room temperature; thereafter, the samples were characterized by a Dimension Edge atomic force microscope (AFM) from Bruker, Germany. The dynamic light scattering (DLS) test was conducted in M-PBS buffer by a NanoBrook instrument (Brookhaven Instruments Corporation, USA). The X-ray photoelectron spectroscopy (XPS) measurements were performed on a Thermo Fisher Scientific Escalab 250xi spectrometer with a monochromatic Al K-alpha source (1486.6 eV), for which the tube current was 12 mA with a tube voltage of 15 kV and the diameter of the beam spot was 500 µm. The RCA-AgNC samples were observed by transmission electron microscopy (TEM) and high-resolution TEM on a Hitachi HT7700 and a Tecnai F30, respectively. The mass spectra of OS6 stabilized AgNCs (Table S1) were determined by a Q-Exactive electrospray ionization (ESI) mass spectrometer (MS) in negative ion mode from Thermo Fisher Scientific™, USA. The cellular imaging was observed by a laser scanning confocal microscope (TCS SP8, Leica, Germany). RCA reaction and product purification. The protocol forthe standard RCA reaction is as follows: (1) Annealing: The RCA template sequence (0.3 µM) was mixed with the primer sequence (0.6 µM) in a T4 ligase buffer [50 mM Tris-HCl, 10 mM MgCl2, 10 mM dithiothreitol (DTT), 1 mM ATP, pH=7.5]. After being heated at 90°C for 10 min, the mixture was slowly cooled to room temperature. (2) Ligation: The template-primer hybrid was mixed with T4 DNA ligase (10.4 U µL-1), reacted at 16°C for 16 h, and subsequently heated at 65°C for 10 min to inactivate the ligase. (3) Amplification: The ligation product was mixed with dNTPs (2 mM each) and phi 29 DNA polymerase (0.2 U µL-1) in an RCA reaction buffer [50 mM Tris-HCl, 10 mM MgCl2, 10 mM (NH4)2SO4, 4 mM DTT]. The amplification reaction was processed at 30°C for 24 h, and then heated at 85°C for 10 min to inactivate the DNA polymerase. After the RCA


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products (200 µL) were dissolved with EDTA (0.5 M, 20 µL), phenol extraction and ethanol precipitation were conducted according to the protocol we reported previously.

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Finally, the

RCA pellet was resuspended in ultrapure water and further quantified according to the absorption at 260 nm using a BioTek microplate reader. Preparation of OSx- or RFx-stabilized AgNCs. For a typical process, the oligonucleotides (OS1 ~ OS6; their sequences are listed in Table S1) and RF serial sequences (RF1 ~ RF9; their sequences are listed in Table S1) were first separately mixed with the AgNO3 solution and incubated at room temperature for 10 min. Thereafter, NaBH4 was added to the previous mixture under vigorous shaking (lasting for 1 min). The molar ratio of fresh NaBH4 to AgNO3 to DNA base was fixed at 1:1:3. The as-prepared AgNCs were stored at room temperature and kept protected from light. Preparation of RCA-AgNCs. A solution of AgNO3 (different concentrations for different reaction ratios) was slowly added to the purified RCA product (1.36 mg mL-1), and the mixture was incubated at room temperature until a homogeneous DNA hydrogel was formed. This Ag+crosslinked DNA hydrogel was suspended in NaBH4 solution (0.2 mM) under occasional agitation. Agarose gel electrophoresis characterization. Gel electrophoresis using 0.5 wt.% agarose was conducted to characterize the purified RCA products and RCA-AgNCs, and the gel was stained by 3×GelRed and imaged by Tanon 3500 (Tanon, China). Rheological analysis. For the characterization of rheological behavior, first the amplitudesweep oscillatory tests were conducted with 200-µL samples on a 20-mm parallel plate at the frequency of 1 Hz. The oscillatory strain ranged from 0.01% to 100% for the determination of


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the linear viscoelastic region. The frequency-sweep oscillatory tests were carried out at the strain of 1%. Testing the photo- and thermostability of AgNCs stabilized by OS6, RF8, and RCA products. The photostabilities of AgNCs stabilized by different DNAs were determined by monitoring their fluorescence changes under certain conditions. For the photostability under the ambient environment at room temperature (avoiding light), the red emissions (all were excited at 560 nm) of AgNCs stabilized by OS6 (emission at 633 nm), RF8 (emission at 626 nm), and RCA products (emission at 633 nm) were monitored during 30 days of storage. In addition, excited at 560nm, the red emissions of AgNCs stabilized by OS6, RF8, and RCA products were monitored when the samples were exposed to sunlight for 2, 5, and 10 hours. For thermostability, the red emissions of AgNCs stabilized by OS6 and RCA products were monitored every 10 min when incubated at 37°C and at 50°C, respectively. The sensitivity and selectivity for detecting ROS/RNS. The RCA-AgNCs (the final concentration of 0.01 mg mL-1 was calculated according to the concentration of DNA in equivalent; similar hereinafter) were used to detect different concentrations of •OH (0.2-80 µM) after a 30-min incubation, and •OH was produced by a Fenton reaction. In the selectivity experiment, 80 µM •OH (produced by a Fenton reaction of 80 µM Fe2+ and 160 µM H2O2) was applied. Hypochlorite anion (ClO-) was supplied by NaClO (80 µM). Alkyl peroxyl radical (ROO•) was produced by AAPH (80 µM) through an incubation at 310 K. H2O2 and NaNO2 were applied to produce peroxynitrite (ONOO-, 80 µM). H2O2 and NaClO were applied to produce single oxygen (1O2) (80 µM). These ROS/RNS species were obtained after a 30-min incubation with the reactive agents at room temperature, and then they were mixed with RCA-AgNCs. K+, Na+, Mg2+, Ca2+, Fe3+, Fe2+, Cu2+, Zn2+, Mn2+, Hg2+, Pb2+, D-Cys, L-Cys, L-


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Glu, L-Phe, Gly, BSA, Vc, and GSH were prepared at a final concentration of 80 µM. The fluorescence ratiometric responses of RCA-AgNCs in the presence of various interferents were monitored. Redox titration. The RCA stabilized AgNCs (the final concentration of the experiment system was

0.01

mg

mL-1

in

100

µL)

were

used

to

conduct

the

redox

titration.

The oxidizing environment was created by the addition of the mixture of Fe2+ and H2O2 (finally generating 10 µM of •OH), which was neutralized by the addition of NaBH4 (10 µM). For every titration, 1 µL oxidizing or reducing agent was added to the experiment system, followed by vigorous shaking for 10 s. After 30 min, the fluorescence spectrum of the experiment system was detected under excitation at 440 and 560 nm. Intracellular detection. The HeLa, MCF-7, and A549 cells were cultured to the exponential growth phase in DMEM medium supplemented with 10% (v/v) fetal bovine serum, 4 mM Lglutamine and 1% penicillin/streptomycin. All cells were cultured in a humid incubator at 37°C and 5% CO2. Then, they were washed with M-PBS buffer and digested with 0.25% trypsinEDTA. Finally, the cells were centrifuged and resuspended in complete medium. For the determination of cytotoxicity, the A549 cells were seeded in a 96-well plate (8000 cell/well) for a 24-h preincubation. Then, the cells were washed twice and incubated with OS1, OS2, OS4, OS6, RF8, and RCA stabilized AgNCs at the same concentration and with RCAAgNCs at different concentrations. After a 24-h incubation at 37°C and 5% CO2, the cells were washed twice and MTT was added (5 mg mL-1) for another 4-h incubation in the humid incubator. Next, the MTT solution was removed carefully and 150 µL DMSO was added. Finally, the plate was placed on a shaking table at 100 rpm for 15 min and the absorbance at 490 nm for each well was recorded.


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For the cellular imaging, the HeLa, A549, and MCF-7 cells were seeded in a 96-well plate (3000 cell/ well), for a 36-h preincubation. In addition, the cells were washed twice with PBS, and incubated with 27 µg mL-1 RCA stabilized AgNCs at 37°C and 5% CO2 for 2 h. Finally, the cells were washed twice with PBS, and the cell images were taken under the CLSM. The excitation wavelengths used to detect the multicolor AgNCs were 448 and 552 nm, and the fluorescence signals were collected at the wavelength ranges of 500-560 nm and 610-700 nm, respectively. In addition, the photostabilities of OS6- and RCA-AgNCs were determined under the scanning of a 552 nm laser of the CLSM. Initially, we collected the fluorescence signals of the A549 cells at wavelengths ranging from 610 nm to 700 nm, and then continuously scanned for 15 min, the fluorescence signals were collected again. For the detection of •OH in live cells, 1 µg mL-1 LPS was used to induce oxidative stress for different incubation times, and then the CLSM was applied to obtain the fluorescence images. The fluorescence signals were collected at the wavelength ranges of 500-560 nm and 610-700 nm.

RESULTS AND DISCUSSION


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Scheme 1. Illustration of the preparation of RCA-AgNCs. A DNA sequence, which was to be amplified hundreds of times through an RCA reaction, was carefully screened according to the properties of the resultant AgNCs. We attempted to synthesize AgNCs exhibiting comparable green and red emission. In addition to being applied in multi-color cell imaging, the AgNCs were used as a ratiometric fluorescence probe for the detection of ROS/RNS in live cells, which is based on the fact that the red- and green-emitting AgNCs show differentiated responses to the oxidative environment. For this purpose, six short oligonucleotides OS1 ~ OS6 (the sequences are listed in Table S1),3, 14, 36 which can stabilize AgNCs (OSx-AgNCs, x=1-6) to exhibit particular emissions (Figure S1), were used as references to design the DNA sequence. Among the nine candidate sequences, RF1 ~ RF9 (the sequences are listed in Table S1), RF8 was selected because the AgNCs stabilized by this sequence exhibited a homogenous distribution of different emissive species (Figure S2). Finally, the complementary sequence of RF8 was used as a template to synthesize a high quantity of RCA products comprising of RF8 as repeat fragments, realizing a high-throughput method to synthesize AgNCs. The solution of RCA products (1.36 mg mL-1) in the presence of Ag+ (1.52


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mM) gradually lost flowability and could be taken out by a pipette tip as a whole (Figure 1a).The rheological analysis exhibited a storage modulus (G’) one order of magnitude higher than that of the loss modulus (G’’), evidencing the formation of a DNA hydrogel (Figure 1b). As the strong cytosine-Ag+-cytosine interactions can better immobilize Ag+ to prevent agglomeration during reduction, most of the oligonucleoside ligands for AgNCs are cytosine-enriched sequences. Therefore, it is not difficult to understand why the very long cytosine-rich RCA products could convert to a DNA hydrogel in the presence of Ag+ ions. Actually, according to previous reports, nanoparticles synthesized in situ in a hydrogel network show improvements in quantity, size and dispersion uniformity23 and better stability and lower toxicity.19 Thereafter, the Ag+cross-linked DNA hydrogel was soaked in a NaBH4 solution to synthesize fluorescent AgNCs. The reaction conditions, such as the concentration of reactants, reaction temperature, and reducing time, have been carefully optimized according to the optical properties of the resultant AgNCs (Figure S3). Finally, according to the amount of DNA bases (1.36 mg mL-1 RCA products), 1/3 equivalent Ag+ was used for gelation and 1/3 equivalent NaBH4 was applied for reducion at room temperature. The as-prepared RCA-AgNCs were characterized by TEM, as shown in Figure 1c. Distinct silver nanocrystals with uniform diameters of ~2 nm (according to the statistical chart of size distributions in Figure 1d) were observed, which showed a fringe spacing of 0.2 nm (the insert in Figure 1c), well matching the {200} crystal face of Ag.37 Further, the RCA-AgNCs were characterized by XPS. As shown in Figure S4, the Ag 3d XPS spectrum showed two groups of binding energies at 368.33 eV (Ag 3d5/2) and 374.36 eV (Ag 3d3/2), as well as at 369.61 eV (Ag 3d5/2) and 375.62 eV (Ag 3d3/2), which are characteristics of Ag0 and Ag+, respectively. According to previous reports,38-39 Ag0 is the main component of the cores of the AgNCs and Ag+ is located at the surfaces of the AgNCs. These results demonstrated that AgNCs were


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successfully synthesized in the DNA hydrogel. As most of the Ag+ ions were converted to AgNCs after reduction, the strength of the Ag+-RCA hydrogel was greatly reduced. After dilution with water, a homogeneous solution of RCA-AgNCs was obtained. Therefore, we further characterized the morphology of the RCA-AgNCs. As shown in Figure S5, the asprepared RCA-AgNCs and their precursors, the RCA products, were characterized by agarose gel electrophoresis, which exhibited similar bands near the sample-loading zones. The result indicates that the long RCA products remained intact without visible degradation after the synthesis of the AgNCs. Thereafter, DLS and AFM were conducted to determine the average particle size and morphology of the RCA-AgNCs. The DLS results revealed that the hydrodynamic diameters of the RCA-AgNCs were ~230 nm (as shown in Figure 1e), which matched well with the AFM results. The AFM image (Figure 1f) indicated that the RCA-AgNCs were well-defined spherical structures. As the diameters of the AgNCs are ~2 nm according to the TEM result, we can prove that the RCA-AgNCs are nanocomposites consisting of the RCA sequences and the encapsulated AgNCs.


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Figure 1. (a) A digital photograph of the Ag+ cross-linked DNA hydrogel taken out of a tube by a pipette tip. (b) Rheological analysis of the Ag+-cross-linked DNA hydrogel. (c) TEM image of the RCA-AgNCs, with the insert showing a high-resolution image. (d) The size distribution histogram of the RCA-AgNCs summarized according to the TEM image. (e) DLS analysis of the RCA-AgNCs nanocomposites. (f) AFM characterization of the RCA-AgNCs nanocomposites. Similar to the fluorescence property of the RF8-AgNCs, the RCA-AgNCs displayed excitationdependent emissions covering a wide range from 538 to 653 nm (Figure 2a). It has been reported that the red-emitting AgNCs are more susceptible to photobleaching, so we compared the photostability of different AgNCs stabilized by OS6, RF8, and the RCA product. In the ambient environment, protected from light, the red emission from the OS6-AgNCs was only 20% of the initial fluorescence after 5 days (Figure 2b); on the other hand, during 30 days of storage, the red emission of the RCA-AgNCs remained almost constant and that of the RF8-AgNCs decayed to 70% of its maximum (Figure 2c). The photostability of these three kinds of AgNCs upon


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exposure to sunlight are also compared in Figure 2d. After a 10-h exposure to sunlight, the intensity of red emissions of the OS6-AgNCs, RF8-AgNCs, and RCA-AgNCs decreased by 70%, 40%, and 15%, respectively. Additionally, the thermostability of the RCA-AgNCs was studied and compared with that of the OS6-AgNCs. As shown in Figure 2e, when incubated at 37 °C for 60 min, the RCA-AgNC sample remained ~ 90% of its original fluorescence, while the OS6AgNCs lost ~80% of its fluorescence. In a further test, we demonstrated that the RCA-AgNCs showed good thermostability even at 50 °C (Figure S6). According to a previous report, longer oligonucleotides can promote the stability of the resultant AgNCs,2 as was proven by the comparison between the OS6-AgNCs and RF8-AgNCs. For RCA-AgNCs, in addition to the further elongated DNA sequences, the condensed hydrogel environment might also contribute to the superior stability of the clusters. Such good stability in the ambient environment makes RCA-AgNCs a promising sensing probe in further applications. Next, the cytotoxicity of the RCA-AgNCs at different concentrations to the A549 cell line was determined by an MTT assay, and the data are summarized in Figure 3a. The cell viability of A549 cells showed no significant decrease when the concentration of the RCA-AgNCs increased up to 90 µg mL-1 (the concentration of AgNCs was quantified according to the DNA concentration; similar hereinafter). Compared with the AgNCs stabilized by OSx and RF8, the RCA-AgNCs showed a relatively lower toxicity (Figure S7), indicating that the hydrogel encapsulation can endow AgNCs with a more biocompatible property in addition to the stabilization function. Thereafter, the RCA-AgNCs (27 µg mL-1) were incubated with the different cells, and the fluorescence images of the cells were taken by the CLSM, as shown in Figure 3b. Strong green and red emissions were collected from all the A549, HeLa, and MCF-7 cells (adherent cell lines), and the Ramos cells (a suspension cell line) under laser excitations at


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448 nm and 552 nm, respectively. The RCA-AgNCs showed a high uptake efficiency in a variety of cell lines, demonstrating that it is a widely applicable reagent for multicolor cell imaging. More importantly, after a 15-min exposure under laser excitation at 552 nm, the emission from the RCA-AgNCs showed no obvious fading, while the fluorescence of the OS6-AgNCs was completely quenched (Figure 3c). Combined with the previous results, we can conclude that the red emission of the RCA-AgNCs shows a superior stability not only in an ambient environment but also under laser exposure, which ensures the reliability of their further application in ROS/RNS probing in live cells.


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Figure 2. (a) The normalized fluorescence spectra of the RCA-AgNCs under 440-580 nm excitation with a 20 nm interval. (b) The photostability of OS6 stabilized AgNCs in an ambient oxygen environment. (c) The photostabilities of the RF8- (black) and RCA product- (red) stabilized AgNCs in ambient oxygen environment, avoiding from light; (d) The photostabilities of OS6- (blue), RF8- (black), and RCA product- (red) stabilized AgNCs under sunlight exposure. (e) The thermostabilities of the OS6- (blue) and RCA product- (red) stabilized AgNCs under 37 °C incubation.


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Figure 3. (a) The cytotoxicity of the RCA-AgNCs to A549 cells. (b) Fluorescence images of various cells illuminated by the RCA-AgNCs; the green channel shows excitation at 448 nm and emission at 500-560 nm, and the red channel shows excitation 552 nm and emission at 610-700 nm (scale bar equals 50 µm). (c) The photostabilities of OS6- and RCA-stabilized AgNCs before and after a 15-min exposure under laser excitation at 552 nm (scale bar equals 100 µm). Although it has been demonstrated that the RCA-AgNCs are well stabilized by DNA hydrogels in an ambient oxygen environment, the green- and red-emitting species may show distinct responses to the presence of a strong oxidative substance because of their nature. Therefore, we further explored the possibility of utilizing RCA-AgNCs as a ratiometric fluorescence probe to detect ROS/RNS in live cells. For sensing applications in live cells, it is necessary to investigate the potential interferents that may interact with Ag and induce false results. Therefore, in addition to a few common ROS/RNS species, several amino acids, metal


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ions and antioxidants and a protein were further investigated, and their influences on the fluorescence of the RCA-AgNCs were analyzed. As shown in Figure 4a, the ratios of emission integrations (R = Igreen / Ired) between the ranges of 500-560 nm (from the green-emitting AgNCs) and 610-700 nm (from the red-emitting AgNCs) in the absence (R0) and presence (RF) of various analytes, were summarized to evaluate the selectivity of the RCA-AgNCs. The ratio {(RFR0)/R0} significantly increased over 10 times in the presence of •OH, and slightly increased by 2~3 times for 1O2, Hg2+, and D-Cys, whereas, the ratio negligibly increased for the other ROS/RNS species (such as ROO•, ClO−, ONOO−, and H2O2), amino acids and protein (L-Cys, L-Glu, L-Phe, GLy, and BSA), and antioxidants (Vc and GSH). It is worth noting that some reagents, such as BSA, Vc, and GSH, enhanced the red emissions of the RCA-AgNCs and resulted in negative ratios. The reason is presumably that these reagents act as protecting agents or reducing agents to further stabilize the red-emitting species.38,40 Additionally, we demonstrated that the common intracellular metal ions showed negligible interference with the emissions of the RCA-AgNCs. All the results certified that the RCA-AgNCs showed the best selectivity for the detection of •OH, the most reactive form of oxygen. In addition, as •OH was produced by a Fenton reaction in the in vitro experiment, which induces reagents other than •OH, the effect of the Fenton reagents (including H2O2, Fe2+, and Fe3+) on the ratiometric fluorescence response was investigated (Figure 4b). As a whole, the Fenton reagents resulted in a significant increase in the green emission and a decrease in the red emission of the as-prepared AgNCs. When only H2O2 or Fe2+ was present, the effects on the emissions of the AgNCs are negligible. Notably, Fe3+ showed some influence, quenching both the green and red emissions of the AgNCs to certain extent. To further clarify the roles of Fe3+ and •OH, we used NaF and DMSO to mask Fe3+ and •OH in the Fenton reaction, respectively (Figure 4c); however, NaF and DMSO by


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themselves showed negligible effects to the fluorescence of the RCA-AgNCs (Figure 4d). When Fe3+ was masked from the Fenton reaction by F-, both the green and red emissions showed increases; the intensities of both emissions were slightly higher than those with the presence of the Fenton reagents on the whole. On the other hand, when •OH was masked from the Fenton reaction by DMSO, the green emission greatly decreased and the red emission increased; however, the intensities of both emissions were slightly lower than those of the original emissions of the as-prepared AgNCs, which clearly arose from the effect of Fe3+. Therefore, we conclude that Fe3+ shows an undifferentiated quenching effect on both emissions of the RCAAgNCs, probably because Fe3+ would competitively bind to DNA and result in the structural deconstruction or agglomeration of the AgNCs, finally causing the quenching of the emissions.41 Accordingly, these results proved that •OH is the only effective component in a Fenton reaction that can cause the ratiometric fluorescence variations of RCA-AgNCs. Figure 5a shows the titration curves of the RCA-AgNCs with increasing •OH concentration; the results illustrated that the green emissions excited at 440 nm showed an increasing trend in intensity, and the intensity of the red emissions excited at 560 nm decreased simultaneously. The ratio of Igreen/Ired showed a linear relation with the concentration of •OH ranging from 0.2 to 80 µM {Igreen/Ired = 0.9713 + 0.1220×[•OH] (R2 = 0.991)}, and the detection limit was determined to be 58 nM based on the 3 σ rule (Figure 5b), which is comparable to the sensitivity of other fluorescence probes recently reported.32-35, 42-44


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Figure 4. (a) The selectivity of the RCA-AgNCs for the detection of ROS/RNS and the reliability characterization in the presence of different interferents (the concentration for all chemicals is 80 µM). (b) The fluorescence spectra (at excitation wavelengths of 440 nm and 560 nm) of the RCA-AgNCs in the absence (solid black) and presence of various Fenton reagents: 40 µM Fe2+ and 80 µM H2O2 (solid red line); 40 µM Fe2+ (dashed cyan line); 40 µM Fe3+ (dashed blue line); and 80 µM H2O2 (dashed purple line). (c) The fluorescence spectra of RCA-AgNCs in the absence (solid black line) and presence (solid red line) of Fenton reagents, and the spectral changes when Fe3+ was masked by 1.5 mM NaF (dashed blue line) and •OH was masked by 1.5 mM DMSO (dashed cyan line). (d) The influences of NaF (dashed blue line) and DMSO (dashed cyan line) to the emissions of the RCA-AgNCs (solid black line).


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Regarding the sensing mechanism of the RCA-AgNCs for the selective detection of •OH based on the ratiometric responses, we assume it involves the conversion from the red-emitting species to the green-emitting species under the oxidation condition.13, 15, 17 For our system, the very long RCA sequences can effectively stabilize the resultant AgNCs, creating a greater barrier to the conversion of the red-emitting to the green-emitting species; therefore, the conversion of the RCA-AgNC samples can only be induced by the strong oxidation agents. The improved stability of the RCA-AgNCs will benefit its good selectivity for •OH and reversible response to redox switches. According to Figure 5c, when •OH was first added to the as-prepared RCA-AgNCs, the red emission decreased, and the green emission increased. Further, by adding a strong reducing agent, NaBH4, the fluorescence changes were quickly reversed, i.e., the red emission increased and the green emission decreased back to their as-prepared states. The interconversions between the red and green species in response to the redox switches can be repeated for several cycles, which agrees with literature reports.11 As the RCA sequences are long (generally >10,000 nucleotides), it is difficult to directly characterize the molecular structures of the RCA-AgNCs by mass spectrometry. Therefore, we selected the OS6-AgNCs as the red-emitting counterpart model, whose structural changes under oxidation were characterized by ESI mass spectrometry. As shown in Figure S8, species, such as Ag10 and Ag13, were abundant in the as-prepared OS6AgNC sample and were found stabilized by the two OS6 strands.27 After oxidation, in the oxidized sample, Ag11 and other smaller clusters became more abundant, compared with Ag13. When the oxidized sample was reduced again by NaBH4, the smaller clusters and Ag11 converted back to Ag13. The results well coincide with those of previous reports,11, 13-17 which demonstrated that the interconversion between red-emitting and green-emitting species is a redox process driven by intra- or inter-strand transfer of silver ions, accompanied by electron loss and gain. The


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capability to reversibly respond to redox switches provides the RCA-AgNCs the good reliability for the real-time monitoring of •OH levels in live cells.

Figure 5. (a) The titration curves of the RCA-AgNCs with an increase in •OH concentration; the green emission excited at 440 nm and the red emission excited at 560 nm were recorded. (b) The relationship between Igreen/Ired ratios and concentrations of •OH. (c) The reversible changes of the two emissions, Igreen (green line) and Ired (red line), in response to redox changes.

For live-cell application, it is very important to monitor the dynamic level of ROS/RNS in various cellular states to study the related bioprocesses. LPS, which can induce oxidative stress mediated by ROS/RNS at the cellular level, were applied to the cultures of A549 cells (which were stained by the RCA-AgNCs in advance) with different incubation times. As a function of treatment time from 0 (control) to 60 min, the fluorescence images changed in a time-dependent manner (Figure 6). The red emission gradually weakened until it completely disappeared; at the same time, the green emission increased, resulting in the changing of the overlaid fluorescence


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from orange to green with time. The success in monitoring the dynamic levels of ROS/RNS in live cells demonstrated that the RCA-AgNCs are a potential tool for future applications in antioxidant therapies and related biological studies.

Figure 6. Using the RCA-AgNCs as a ratiometric fluorescence probe to monitor the dynamic levels of ROS/RNS in LPS-treated A549 cells at various incubation times (scale bar equals 50µm). CONCLUSIONS In summary, taking advantage of the RCA method, we achieved the cost-efficient fabrication of a large quantity of cytosine-rich DNA sequences, which were cross-linked to form a DNA hydrogel in the presence of Ag+. After reduction, multicolor AgNCs were quantitatively synthesized. Surrounded by the DNA hydrogel, the RCA-AgNCs exhibited improved photo/thermostability and better biocompatibility. In addition, the resultant RCA-AgNCs showed a broad emission covering the whole visible spectrum in an excitation-dependent manner. As the


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red-emitting species could be converted to the green-emitting species under a strong oxidation condition, the RCA-AgNCs were demonstrated to be a promising ratiometric fluorescence probe for the intracellular detection of •OH, showing high sensitivity and selectivity against other ROS/RNS species and many common biological interferents. Overall, the RCA-AgNC with outstanding fluorescence properties and application-oriented advantages is a promising nanomaterial for the biomedical applications of multicolor cell imaging and intracellular sensing.

ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. A list of the DNA sequences used in this work; sequence screening for an RCA template; the optimization of synthesis conditions of RCA-AgNCs; XPS and agarose gel electrophoresis characterization of RCA-AgNCs; the thermostability of RCA-AgNCs; the cytotoxicity of AgNCs stabilized by different DNAs; the ESI-mass spectrometric characterization of OS6-AgNCs upon redox switch. (PDF)

AUTHOR INFORMATION Corresponding Author *[email protected] ACKNOWLEDGMENT


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The work is supported by grants from the National Natural Science Foundation of China (Nos. 51503096

and

21705074),

Shenzhen

Fundamental

Research

Programs

(No.

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