Label-free biosensor based on dsDNA-templated copper nanoparticles for highly sensitive and selective detection of NAD⁺

Abstract

We have developed a novel label-free fluorescence approach for nicotinamide adenine dinucleotide (NAD⁺) detection by coupling poly(AT-TA) dsDNA-templated fluorescent copper nanoparticles (CuNPs) as a fluorescence indicator with a dumbbell DNA probe designed for ligation. Herein, we designed a dumbbell probe which can form a self-complementary structure at both ends. In the absence of NAD⁺, the extension reaction can be performed from the exposed 3′-end, and instantly opened the hybridized structure at the 5′-end, producing a chimeric poly(AT-TA) double-stranded fragment which can be used as a highly-efficient template for the formation of CuNPs, showing high fluorescence. Whereas in the presence of NAD⁺, together with the E. coli DNA ligase, the 5′-phosphoryl end and the 3′-end of the DNA probe can be ligated to block the extension reaction, showing slight fluorescence. This novel NAD⁺ assay is label-free, fast and easy to operate, highly sensitive and selective, which holds wide applicability as a robust analytical tool in NAD⁺ related biological processes and clinical diagnosis.

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Introduction

Nicotinamide adenine dinucleotide (NAD⁺) is an important biological molecule, participating in many biological processes and plays a critical role in transcriptional regulation, DNA repair, calcium homeostasis, cell proliferation, and gene expression.^1–4 Known as coenzymes, NAD⁺ also serves as an essential substrate to many oxidoreductive enzymes.^5–7 Furthermore, recent studies showed that NAD⁺ could also dominate some significant life processes correlated with the metabolism in many organisms.^8,9 The intracellular NAD⁺ level is regarded as an important factor to correlate with many age-associated diseases, such as cancer, diabetes, and neurodegenerative diseases.^10–12 Therefore, due to its clinical and biological importance, sensitive and selective detection of NAD⁺ is of great significance for clinical diagnosis and therapy as well as understanding its basic roles in biological processes.

Traditionally, many strategies have been developed for NAD⁺ detection, such as high-performance liquid chromatography (HPLC),¹³ electrospray ionization mass spectrometry (ESI-MS),¹⁴ chemiluminescence,¹⁵ nuclear magnetic resonance (NMR) spectrometry,¹⁶ and capillary electrophoresis (CE).¹⁷ These methods, however, have some shortcomings of being time consuming, lacking sensitivity and requiring expensive equipment, or are unable to discriminate NAD⁺ from NADH or other analogues. Other alternative methods have been developed to solve these problems in recent years. For example, several methods based on fluorescence strategies have been reported.^18,19 Although these fluorescent methods have been proved more advantageous than traditional NAD⁺ assays, their shortcomings including the high cost of fluorescent modification and complicated design of molecular beacons are still unavoidable. Consequently, the development of simple, low-cost sensitive, and selective method for NAD⁺ assay is highly desirable.

Recently, nanomaterial-based probes have demonstrated unique advantages such as simplicity, sensitivity and low cost, as demonstrated in their wide applications in different fields.^20–23 Lately, emerging copper nanoparticles (CuNPs), which selectively form on double-stranded DNA (dsDNA) templates, offer excellent potential as novel fluorescent markers.^24–27 Poly(AT-TA) dsDNA was also found as the specific sequence which could act as a highly efficient template for the formation of the fluorescent CuNPs, and the fluorescence intensity was highly depend on the polymerization degree and the length of poly(AT-TA).²⁸ Meanwhile, poly(AT-TA) dsDNA-templated CuNPs formation exhibits a maximum λ_em at 600 nm with large MegaStokes shifting (up to 260 nm), which is suitabable for biological diagnostic techniques.²⁹ To the best of our knowledge, the exploration of dsDNA-templated CuNPs is still at a very early stage and has a great potential to be utilized in biochemical applications.

Herein, we proposed to develop a label-free, low-cost, sensitive, and selective approach for specific NAD⁺ detection based on dsDNA-templated fluorescent CuNPs and DNA ligation reaction. The 5′-end of dumbbell probe is modified with a phosphate group and the 3′-end is exposed. This protocol utilized NAD⁺ as an indispensable cofactor for E. coli DNA ligase form a ligase-adenylate intermediate. In the absence of NAD⁺, the extension reaction can be performed from the 3′-end, and instantly opened the hybridized structure at the 5′-end, producing a chimeric poly(AT-TA) dsDNA extended. The generated dsDNA product can be used as a template for the formation of CuNPs, showing high fluorescence signal. However, in the presence of NAD⁺, together with E. coli DNA ligase, the ligation between 3′-OH and 5′-PO₄ ends of dumbbell probe can be catalysed to form a closed circular DNA probe. The circular DNA probe with very short poly(AT-TA) dsDNA fragment does not support high fluorescent CuNPs' formation and so leads to a much lower fluorescence signal. Thus, the determination of NAD⁺ could be easily identified by CuNPs' fluorescence changes. Compared with traditional methods, the proposed strategy is convenient, exhibiting high analytical performance, and avoiding the need to design complicated fluorescence-labled DNA probes. This label-free method was used as an effective approach for NAD⁺ detection and successfully applied to quantify the NAD⁺ in the extract of A549 human lung adenocarcinoma cell, which showed great potential in sensitive quantification of NAD⁺ in NAD⁺ related biological process study and clinical diagnosis.

Experimental procedures

Chemicals and materials

The DNA sequences used in this work were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. The sequences of the DNA oligonucleotide were as follows:

Probe 1 (22AT): Phos-5′-CGA CATC ATA TAT ATA TAT ATA TAT ATA TGA TGT CGC ACA CAT ACA AAG TCT TAG CTG TG-3′

Probe 2 (P2): 5′-ATA TAT ATA TAT ATA TAT ATAT-3′

Probe 3 (14AT): Phos-5′-CGA CATC ATA TAT ATA TAT ATG ATG TCG CAC ACA TAC AAA GTC TTA GCT GTG-3′

Probe 4 (18AT): Phos-5′-CGA CATC ATA TAT ATA TAT ATA TAT GAT GTC GCA CAC ATA CAA AGT CTT AGC TGTG-3′

Probe 5 (28AT): Phos-5′-CGA CATC ATA TAT ATA TAT ATA TAT ATA TAT ATA TGA TGT CGC ACA CAT ACA AAG TCT TAG CTG TG-3′

Probe 6 (32AT): Phos-5′-CGA CATC ATA TAT ATA TAT ATA TAT ATA TAT ATA TAT ATG ATG TCG CAC ACA TAC AAA GTC TTA GCT GTG-3′

The adenosine diphosphate (ADP), adenosine monophosphate (AMP), and nicotinamide adenine dinucleotide phosphate (NADP) were obtained from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). E. coli DNA ligase, Klenow fragment polymerase (KF polymerase, without 3′ to 5′ exonuclease activity), and deoxyribonucleoside triphosphates (dNTPs) were obtained from New England Biolabs (Ipswich, MA, USA). Nicotinamide adenine dinucleotide (NAD⁺), the reduced form of nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate hydride (NADPH) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). 3-(N-Morpholino) propanesulfonic acid (MOPS), sodium ascorbate, copper sulfate and other salt reagents were commercially obtained from Dingguo Biotechnology Co., Ltd. (Beijing, China). The reaction buffer solution employed in this work was 10 mM Tris–HCl, 10 mM MgCl₂, and 50 mM NaCl (pH 7.9). The MOPS buffer (10 mM MOPS, 150 mM NaCl, pH 7.6) was used for the formation of fluorescent CuNPs. All reagents were used as received without any further purifications. All solutions were prepared by ultrapure water obtained through a Millipore Milli-Q water purification system (Billerica, MA, USA) with an electric resistance >18.2 MΩ.

The fluorescence measurements were carried out on an FL-4600 spectrometer (Hitachi, Japan). The optical path length of a quartz fluorescence cell was 1.0 cm. Excitation and emission slits were all set for a 5.0 nm band-pass and 700 V was chosen as the excitation voltage. The fluorescence emission spectra were collected from 500 nm to 680 nm at room temperature with a 340 nm excitation wavelength.

Fluorescence detection of NAD⁺

Briefly, a mixture containing 500 nM oligonucleotide, 0.09 U μL⁻¹ E. coli DNA ligase was prepared in a buffer (pH = 8.0) of 50 μL volume in the presence of varying concentrations of NAD⁺. The mixture was incubated at 37 °C for 30 min. Then, 1 μL dNTPs (10 mM μL⁻¹) solution and 0.5 μL Klenow Fragment (5 U μL⁻¹) polymerase solution were added into the mixture, and kept at 37 °C for 50 min. Then, 30 μL MOPS buffer (20 mM MOPS, 300 mM NaCl, pH 7.6), 10 μL of sodium ascorbate (50 mM), and 10 μL of CuSO₄ (2 mM) were added into the solution to give final volumes of 100 μL, the mixture was allowed to react for 15 min at room temperature (25 °C), followed by the fluorescence measurement with the excitation wavelength of 340 nm.

NAD⁺ detection in diluted cell extracts

A549 human lung adenocarcinoma cell lines were cultured in RPMI 1640 medium supplemented with 12% fetal calf serum, 100 μg mL⁻¹ streptomycin, and 100 units per mL penicillin. Cell extracts were prepared according to the previous reports.³⁰ The collected cells were resuspended in 20 μL of 10 mM Tris–HCl (pH 7.8) containing 150 mM NaCl. With the addition of 20 μL lysis buffer (20 mM Tris–HCl, 150 mM NaCl, 1% Triton X-100, 0.4 mM phenylmethylsulfonyl fluoride, pH 7.5), the mixture was incubated for 1.5 h at 4 °C with occasional shake. Cell debris was removed by centrifugation at 10 000 rpm for 10 min, and the supernatant was recovered. Diluted cell extracts were added to the assay solution (1%). The detection procedure was the same as those described in the aforementioned experiment for NAD⁺ detection in the clean reaction buffer.

Gel electrophoresis analysis

Electrophoresis analysis was carried out on 4% agarose gels by ethidium bromide staining, casting and running in 1×TBE buffer (45 mM Tris, 45 mM boric acid, 1.25 mM EDTA, pH 7.9) at room temperature. The electrophoresis procedure was performed at a constant potential of 100 V for 50 min with a loading of 10 μL of each sample into the lanes. Then the gel was imaged by a ChemiDoc XRD system (Bio-Rad).

Results and discussion

Strategy for NAD⁺ detection

By taking advantages of the fact that poly(AT-TA) dsDNA formation of fluorescent CuNPs is highly length dependent with an immense potential application to biochemical sensing, a novel label-free method for NAD⁺ detection has been proposed in this work. The principal design is illustrated in Scheme 1. The designed dumbbell probe contains three types of domains, a stem domain and two loop domains. The red part of stem domain and red loop domain can be hybridized to the complementary DNA forming poly(AT-TA) dsDNA as template for the formation of high fluorescence CuNPs through the reduction of Cu²⁺ by ascorbate. In the absence of NAD⁺, the extension reaction can be performed from the 3′-end, and instantly opened the hybridized structure at the 5′-end, producing a chimeric double-stranded poly(AT-TA) dsDNA fragment (red domain). The extended dsDNA product can be used as a highly-efficient template for the formation of CuNPs, and the formed dsDNA–CuNPs complexes show high fluorescence. Thus, once initiated, the polymerization and displacement reactions are continuously repeated to produce a large amount of fragments of dsDNA, which intensively increases the fluorescence signal as a result. In the presence of NAD⁺, together with E. coli DNA ligase, the ligation between 3′-OH and 5′-PO₄ ends of dumbbell probe can be catalysed to form a closed circular DNA probe with the action of ligase in an amount that is positively related to the concentration of the cofactor NAD⁺. Meanwhile, the circular DNA probe is an inactive substrate for DNA polymerase because of the polymerase elongation reaction is only initiated at the 3′-hydroxyl end of the primer. Thus, the circular DNA probe leads to a much lower fluorescence signal because of the CuNPs fluorescence intensity is highly dependend on the polymerization degree and the length of poly(AT-TA). Thus, through the fluorescence change of CuNPs, NAD⁺ might be successfully identified.

Scheme 1 Schematic illustration of fluorescence detection of NAD⁺ based on dsDNA-templated CuNPs and DNA ligation reaction.

Monitoring of the NAD⁺

Following the design, to demonstrate the feasibility of our assay for NAD⁺ detection, the fluorescence emission spectra under different conditions were investigated. First, the poly(AT-TA) dsDNA-templated CuNPs were confirmed by transmission electron microscopy (TEM) (Fig. 1). We can see that the size of the poly(AT-TA) dsDNA-templated fluorescent CuNPs is around 5 nm, which indicates that poly(AT-TA) dsDNA can serve as a template for fluorescent CuNPs formation. Then, the detection of NAD⁺ was realized by CuNPs' fluorescence monitoring. As shown in Fig. 2, the dumbbell Probe 1 can not be used as highly-efficient template for the formation of CuNPs, and so slight fluorescent signal can be observed (curve a). Probe 1 could be hybridized to the Probe 2 forming poly(AT-TA) dsDNA as template for the formation of CuNPs, and the formed dsDNA–CuNPs complexes show high fluorescence (curve c). Similarly, in the absence of NAD⁺, the fluorescence increased by addition of Klenow fragment DNA polymerase and DNA ligase owing to the extension reaction which produced a chimeric double-stranded poly(AT-TA) dsDNA fragment. The extended dsDNA product could be used as a template for the formation of CuNPs, which could also induce strong fluorescence at around 600 nm with the excitation of 340 nm (curve d). The formation of CuNPs was also evidenced by the TEM image which clearly showed that the CuNPs were spherical in shape, and the average size of them was about 5 nm (Fig. S1 in ESI†). However, upon the introduction of target NAD⁺, the polymerase elongation is not induced and as a result rather weak fluorescence signal was obtained (curve b), which may be ascribed to the ligation reaction of E. coli DNA ligase and incomplete extension reaction of Klenow fragment DNA polymerase. Thus, the excellent feasibility demonstrated that the ultrasensitive label-free fluorescent assay of NAD⁺ based on poly(AT-TA) dsDNA-templated CuNPs and DNA ligation reaction could be carried out.

Fig. 1 Typical TEM image of dsDNA-templated CuNPs.

Fig. 2 The fluorescence emission spectra of as obtained CuNPs under different conditions: (a) Probe 1 + Cu²⁺ + ascorbate (black line); (b) Probe 1 + E. coli ligase + NAD⁺ + KF polymerase + dNTPs + Cu²⁺ + ascorbate (green line); (c) Probe 1 + Probe 2 + Cu²⁺ + ascorbate (red line); (d) Probe 1 + E. coli ligase + KF polymerase + dNTPs + Cu²⁺ + ascorbate (blue line). (Probe 1, 500 nM; Probe 2, 500 nM; E. coli ligase, 25 U mL⁻¹; NAD⁺, 400 nM; KF polymerase, 10 U mL⁻¹; dNTPs, 100 μM; ascorbate, 5 mM; Cu²⁺, 200 μM).

To further verify the feasibility of the proposed strategy, an electrophoresis experiment was performed. As shown in Fig. S2,† in the absence of Probe 2, a DNA band of Probe 1 is observed in lane 1 (imaged under UV-light) after 50 min of gel electrophoresis. In the presence of Probe 2, it could be observed that there is only one significant bright band in lane 2. Additionally, the DNA band in lane 2 exhibites relatively low mobility in comparison with the DNA band in lane 1, demonstrating the impressive increase in the amount of double-helix DNA, the hybridization products. Similarly, in the presence of NAD⁺, together with DNA ligase, the dumbbell probe could be catalyzed to form a closed circular DNA probe, thereby a faint DNA band is observed in lane 4. In the absence of NAD⁺, the polymerization and displacement reactions are continuously repeated to produce a large amount of fragments of dsDNA, resulting in a highly significant bright band in lane 3, which indicates the successful growth of the double helix DNA. These electrophoresis results provided further evidence for the feasibility of the proposed strategy.

Optimization of assay conditions

The length of AT-TA plays an important role in the fluorescence of the CuNPs. In order to optimize the length of poly(AT-TA) DNA, five kinds of dumbbell probe, including poly(AT-TA)14, poly(AT-TA)18, poly(AT-TA)22, poly(AT-TA)32, and poly(AT-TA)36, were investigated for the formation of the fluorescent CuNPs. As shown in Fig. S3,† F₀/F reaches a maximum at poly(AT-TA)22 (F and F₀ are the fluorescence intensities of dsDNA-templated CuNPs platform in the presence and absence of NAD⁺, respectively), and decreased along with the length of poly(AT-TA) DNA up to 36. Thus, the Probe 1 (22AT) was used throughout subsequent experiments.

In order to optimize the experimental conditions, a series of measurements were researched in this work. It was found that the concentration of Cu²⁺ was an important factor influencing the fluorescence intensity of dsDNA-templated CuNPs. We first explored the effect of the concentration of Cu²⁺ on the fluorescence of dsDNA-templated CuNPs. We found that the fluorescent signal resulting from the metallization increased sharply upon increasing the concentration of Cu²⁺ ions and the best signal-to-background (S/N) ratio was obtained with 200 μM Cu²⁺ (Fig. S4 in ESI†). Thus, 200 μM of Cu²⁺ was used in all experiments.

To obtain more appropriate fluorescence intensity and accordingly better fluorescent response, we also optimized the sodium ascorbate concentration in the assay. Fig. S5† shows the effect of sodium ascorbate concentration on fluorescence intensity of CuNPs. The fluorescence intensity increases sharply between the sodium ascorbate concentrations of 0.5 mM and 2 mM and reaches equilibrium with that of 4 mM. Further increasing the sodium ascorbate concentration up to 5 mM does not significantly affect the fluorescence intensity. In order to obtain high reduction of Cu²⁺, we used a sodium ascorbate concentration of 5 mM in our work.

Moreover, to achieve the best sensing performance, the concentrations of dNTPs and KF polymerase were also optimized (Fig. S6 in ESI†). Experimental results showed that the following conditions provided the maximum S/N ratio for the sensing system: 10 U mL⁻¹ KF polymerase and 100 μM dNTPs. The effects of different concentrations of E. coli ligase ranging from 0 to 75 U mL⁻¹ were also investigated (Fig. S7 in ESI†). It was observed that the fluorescence response decreased rapidly with the increasing concentration of E. coli ligase, and reached a minimum value at 25 U mL⁻¹. Finally, 25 U mL⁻¹ E. coli ligase was chosen to ensure the efficient ligation reaction.

The effect of ligation reaction time was studied. It was observed that the change of the fluorescence response increased with increasing time within 30 min in the presence of the same concentration of NAD⁺ and reached a maximum value at 30 min (Fig. S8 in ESI†). Thus, 30 min was used as the optimal time of ligation reaction in the following experiments.

The extension reaction time is another important factor for excellent sensing systems. In order to obtain optimized assay time, the fluorescence intensity of different extension reaction time was measured. As presented in Fig. S9,† the fluorescence signal increases gradually with the increasing of the extension reaction time, and then reached equilibrium after 50 min. Thus, 50 min was chosen as the appropriate time in the following experiments.

Sensing performance of the proposed assay

The detection performance of the NAD⁺ assay is evaluated by exposing the sensor to a series of NAD⁺ concentrations under the optimized conditions. As shown in Fig. 3A, the fluorescence intensity decreases gradually with an increasing concentration of NAD⁺ from 0 to 400 nM, indicating that the formation of dsDNA-templated CuNPs is highly related with the concentration of target NAD⁺. Fig. 3B indicates that the fluorescence signal is linearly decreased with the NAD⁺ concentration ranging from 0.2 to 20 nM (regression coefficient R² = 0.994). The linear regression equation is y = −99.2565x + 4822.8262 (here, x is the concentration of NAD⁺ (nM), and y is the response peak fluorescence). The relative standard deviations of peak fluorescence readings are 3.6%, 2.8%, and 2.5% in three repetitive assays of 0.2 nM, 5 nM, and 10 nM NAD⁺, respectively, which shows that this proposed method has a good reproducibility. Meanwhile, the detection limit of 0.2 nM was obtained as calculated according to the rule of three times standard deviation over the background signal. This detection sensitivity is even superior to those determined using some previously reported methods.^18,31–35 The results indicate that the present label-free method could successfully detect the NAD⁺ with high sensitivity.

Fig. 3 (A) Fluorescence intensity–wavelength curves with different concentrations of NAD⁺ (top to bottom, 0, 0.2, 5, 10, 20, 40, 80, 200, 400 nM) in reaction buffer. (B) Fluorescence signal changes at 600 nm with the concentration of NAD⁺ ranging from 0 to 400 nM. Inset: linear responses in the range of 0.2–20 nM. The error bars represent for standard deviation (SD) across three repetitive experiments. (Probe 1, 500 nM; E. coli ligase, 25 U mL⁻¹; KF polymerase, 10 U mL⁻¹; dNTPs, 100 μM; ascorbate, 5 mM; Cu²⁺, 200 μM).

Assay selectivity

We also evaluate the selectivity of the sensing system towards NAD⁺. The fluorescence responses of several NAD⁺ analogs were tested under the same experimental conditions and the result was shown in Fig. 4. As can be seen from Fig. 4, upon addition of 0.5 μM of NADH or 5 μM of nicotinamide adenine dinucleotide phosphate (NADP), nicotinamide adenine dinucleotide phosphate hydride (NADPH), ATP, ADP and AMP, the results yield only negligible fluorescence changes compared with that to 0.5 μM of NAD⁺, indicating that this ligation reaction-based sensing system provides an excellent capability in differentiating NAD⁺ from its analogues, which makes it promising for practical applications.

Fig. 4 Selectivity of the ligation reaction-based sensing system for NAD⁺. The concentrations of NAD⁺ and NADH are 500 nM and the other compounds are 5 μM. Error bars are estimated from three replicate measurements. (Probe 1, 500 nM; E. coli ligase, 25 U mL⁻¹; KF polymerase, 10 U mL⁻¹; dNTPs, 100 μM; ascorbate, 5 mM; Cu²⁺, 200 μM).

Investigation of NAD⁺ detection in diluted cell extracts

The feasibility of the proposed strategy was also tested by the detection of NAD⁺ in complex biological samples. In order to examine the possibility of the as-proposed sensing platform for cellular NAD⁺ activity profiling, A549 human lung adenocarcinoma cell extracts were added into the buffer to simulate the intracellular environment during the test procedure. It was observed that the fluorescence signals decreased when the concentrations of NAD⁺ gradually increased from 0 to 400 nM (Fig. 5A). The fluorescence intensity and the concentration of NAD⁺ also exhibited a linear relationship in the range from 0.2 to 20 nM like that operated in Tris–HCl buffer (Fig. 5B). The above results demonstrate that this sensing platform works well in complex mixtures with other possible coexisting interfering species, suggesting that the label-free method could be further used for real sample analysis.

Fig. 5 (A) The fluorescence intensity with different concentrations of NAD⁺ (top to bottom, 0, 0.2, 5, 10, 20, 40, 80, 200, 400 nM) in reaction buffer containing 1% (v/v) cell extracts. (B) Fluorescence signal changes at 600 nm of the concentration of NAD⁺ from 0 to 400 nM. Inset: linear responses in the range of 0.2–20 nM. The error bars represent for standard deviation (SD) across three repetitive experiments. (Probe 1, 500 nM; E. coli ligase, 25 U mL⁻¹; KF polymerase, 10 U mL⁻¹; dNTPs, 100 μM; ascorbate, 5 mM; Cu²⁺, 200 μM).

Conclusions

In conclusion, based on dsDNA-templated CuNPs, a novel label-free fluorescent sensing platform for the assay of NAD⁺ has been developed. The assay relies on the principle that NAD⁺-dependent E. coli DNA ligation reaction and poly(AT-TA) dsDNA can act as a highly efficient template for the formation of the high fluorescence CuNPs. This method took advantage of the intrinsically extreme fidelity of E. coli DNA ligase to NAD⁺. The label-free design did not require any chemical modification for DNA or sophisticated equipment, thus exhibiting its simplicity and cost efficiency. The dsDNA-templated CuNPs and the label-free design improved the sensitivity with a low detection limit of 0.2 nM for the target NAD⁺ and the strategy was applicable to a real biological sample. In addition, this approach exhibited extreme specificity towards NAD⁺, compared with the excess of the NAD⁺ analogs. These advantages endow the NAD⁺ sensing strategy with a great potential for practical applications without sample purification, and may find wide applications in the environmental and biomedical fields.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (21505122, 21205108, J1210060), the Startup Research Fund of Zhengzhou University (1511316004), the Outstanding Young Talent Research Fund of Zhengzhou University (1521316003, 1421316038) and the Program for Innovative Research Team (in Science and Technology) in University of Henan Province (17IRTSTHN002). The authors are very grateful to Professor Yongjun Wu (Zhengzhou University) for providing A549 human lung adenocarcinoma cells.

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Footnote

† Electronic supplementary information (ESI) available: Additional figures. See DOI: 10.1039/c6ra17579a

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