Polydopamine Nanospheres Nanocomplex for in Situ

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Aptamer/Polydopamine Nanospheres Nanocomplex for in Situ Molecular Sensing in Living Cells Weibing Qiang, Hongting Hu, Liang Sun, Hui Li, and Danke Xu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03075 • Publication Date (Web): 10 Nov 2015 Downloaded from http://pubs.acs.org on November 16, 2015

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Analytical Chemistry

Aptamer/Polydopamine Nanospheres Nanocomplex for in Situ Molecular Sensing in Living Cells Weibing Qiang, Hongting Hu, Liang Sun, Hui Li, and Danke Xu* State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, 22 Hankou Road, Nanjing, Jiangsu 210093, China ABSTRACT: A nanocomplex was developed for molecular sensing in living cells, based on the fluorophore-labeled aptamer and the polydopamine nanospheres (PDANS). Due to the interaction between ssDNA and PDANS, the aptamer was adsorbed onto the surface of PDANS forming the aptamer/PDANS nanocomplex, and the fluorescence was quenched by PDANS through FRET. In vitro assay, the introduction of adenosine triphosphate (ATP) led to the dissociation of the aptamer from the PDANS and the recovery of the fluorescence. The retained fluorescence of the nanocomplex was found to be linear with the concentration of ATP in the range of 0.01-2 mM, and the nanocomplex was highly selective towards ATP. For the strong protecting capability to nucleic acids from enzymatic cleavage and the excellent biocompatibility of PDANS, the nanocomplex was transported into cells and successfully realized “signal on” sensing of ATP in living cells, moreover the nanocomplex could be employed for ATP semi-quantification. This design provides a strategy to develop biosensors based on the polydopamine nanomaterials for intracellular molecules analysis. For the advantages of polydopamine, it would be an excellent candidate for many biological applications, such as gene and drug delivery, intracellular imaging and in vivo monitoring.

Monitoring and visualization of physical events and molecules, the important method to understand cell biology, have a profound influence on advancement in biological science. Sensors with high spatiotemporal resolution, selective and quantitative signals for physical variables or biological molecules in living cells is a constant need in biological science.1,2 For the high sensitivity and non-invasiveness of fluorescent spectroscopic techniques, they offer sensing for intracellular analysis and intracellular signaling. From the morphological analysis of anatomical structures to sensitive measurements of intracellular molecules, fluorescent bioimaging is widely used in the applications of biomedical sciences.3 As fluorescent bioimaging is a non-destructive, selective, sensitive, real-time method without radioactivity4, it has some inherent advantages over those conventional imaging methods. Magnetic resonance imaging (MRI) is severely limited by non-specific biodistribution and expensive construction5, and micro singlephoton emission computed tomography/computed tomography (microSPECT/CT) is seriously limited by low spatial resolution6. The researchers have developed numbers of organic dyes and fluorescent proteins as powerful molecular probes for fluorescent biosensing. However, due to the poor photobleaching resistance, broad emission spectra and largely-shifted excitation bands of these molecular probes, there are some limitations for them to achieve reliable intracellular measurements.7 And what’s more, the possible chemical interactions or steric hindrance with biomolecules of these molecular probes would cause biotoxicity or perturbation to the investigated systems.8 Currently, the “always on” strategy was mostly used in the design of the detection probes, in which the probes are bound to the targets and then lead to an elevated signal with reference to surroundings.9-11 For the absence of signal change during the targeting of these probes, to eliminate interference from

excessive unbound probes, a time-consuming washing step is typically required in the in vitro utilization of “always on” probes. And during in vivo applications, the high background from constant signals in non-target tissues often affects target specificity and imaging contrast. As an alternative, some “signal on” activatable probes have been developed12-14, in which normally quenched signals are activated only after recognizing the targets, such as the molecular beacons (MBs)15,16. Some nanomaterials have be used as quenchers for developing “signal on” biosensors for the assay of biomolecules, such as nucleic acids and proteins. Typically, these nanomaterials are capable of quenching the fluorescence from different fluorophores with various emission frequencies through nonresonant energy transfer or electron transfer. For their advantage as universal quenchers, fluorescence-quenching nanomaterials eliminate the selection issue of a fluorophorequencher pair in conventional “signal on” biosensors. So far, gold nanoparticles (AuNPs)17 or nanorods18, carbon nanomaterials, such as carbon nanotubes (CNTs)19, carbon nanoparticles (CNPs)20 and graphene21, MoS2 nanosheets22, WS2 nanosheets23, carbon nitride nanosheets24, copper-oxide nanobelts25, metal-organic framework (MOF)26, and polyaniline nanofibers27 have been proved to be fluorescence-quenching nanomaterials. While, in the consideration of low cytotoxicity, only few of those fluorescence-quenching nanomaterials could be used for intracellular sensors, such as AuNPs, gold nanorods, and carbon nanotubes or graphene.28-31 In the intracellular biosensors, these nanomaterials act as not only a universal fluorescence quencher but also a nanocarrier for the recognition probes to improve the delivery. In the previous studies, the polydopamine nanospheres (PDANS) have been employed as a nanoquencher to develop the sensing platforms for the assay of biomolecules.32-34 And

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some reports have demonstrated that polydopamine have excellent biocompatibility and biodegradability35. Here, a “signal on” fluorescence biosensing nanocomplex was developed, based on the polydopamine nanospheres and the aptamers. Aptamers are single-strand DNA/RNA oligonucleotides with high specificity and affinity against many given targets, ranging from small inorganic and organic molecules to macromolecules and even cells.36-38 For the advantages of high affinity and specificity, low immunogenicity, as well as facile synthesis and modification,39,40 aptamers promise to be the nextgeneration recognition molecules in molecular diagnosis. In our study, we employed the carboxyfluorescein (FAM)labeled DNA aptamer and PDANS to create a biosensor for the adenosine triphosphate (ATP) sensing in living cells. ATP is the primary energy molecule in living cells, and it’s generally called as the “molecular unit of currency” for intracellular energy transfer.41,42 It is highly necessary for some biochemical reactions such as muscle contraction, membrane transportation, biomolecule synthesis and degradation, and signal transduction, etc.43,44 In the absence of the target, the aptamer/PDANS nanocomplex was initially in “off” state due to the efficient fluorescence quenching of FAM adjacent to the surface of PDANS. After the nanocomplex was incubated with sample containing the target or transported into cells, due to the binding of the aptamer by ATP, the nanocomplex would change into the “on” state as a result of the dissociation of the FAM from the surface of PDANS, thus providing greatly enhanced fluorescence emission intensity. The primary achievements indicated that this PDANS-based biosensor owned promising abilities for sensing of ATP in living cells, which will enable it to be applied in cellular imaging of the predicting biomarkers such as mRNAs and microRNAs.

MATERIALS AND METHODS Materials and Reagents. 96 microwell plates were obtained from Coring incorporated (NY, USA). Dopamine hydrochloride, oligomycin, adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and uridine triphosphate (UTP) were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). DNase I was obtained from Sigma-Aldrich (St, Louis, MO, USA). 1× PBS, complete Dulbecco’s Modified Eagle Medium (DMEM) (with 10 % fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin), Trypsin-EDTA, HeLa cells (human cervical carcinoma) and MTT cell proliferation and cytotoxicity detection kit were obtained from KeyGEN Biotech Co., Ltd. (Nanjing, China). MCF-7 cells (human breast cancer) was obtained from Hunan University. Glass bottom cell culture dishes (Φ15 mm) were purchased from Nest Biotechnology Co., Ltd. (Wuxi, China). Other reagents from commercial suppliers were analytical grade and used without further purification. Ultrapure water (electric resistance >18.25 MΩ) obtained through a Millipore Milli-Q water purification system (Billerica, MA, USA) was used throughout the experiments. 1× PBS (with 200 mM KCl, 5 mM MgCl2 and 0.1 mM EDTA) was used as the binding buffer for ATP assay. Carboxyfluorescein (FAM)-labeled ATP aptamer P1 (5’-FAMACCTGGGGGAGTATTGCGGAGGAAGGT-3’) and FAMlabeled random ssDNA P2 (5’-FAMAAAAAAGCTTGTGTTCGTTGGAAAAAA-3’) were synthesized and purified by Sangon. Apparatus. Scanning electron microscopy (SEM) images were recorded using an S-4800 scanning electron microscope

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(Hitachi, Japan). The absorption and fluorescence emission were measured on a Synergy H1 multi-mode reader (BioTek, USA). Confocal scanning laser microscope (CSLM) (TCS SP5, Leica, Germany) was used to take cell images. The laser excitation for FAM was 488 nm, the bright field image was recorded simultaneously by transmission PMT, and the fluorescence detection band was set to 505-575 nm for FAM. Preparation of the P1/PDANS Nanocomplex. PDANS was synthesized according to our previous report with some modifications.32 Briefly, 50 mg dopamine hydrochloride was added to a mixture of 100 mL Tris-buffer (10 mM) and 20 mL ethyl alcohol with stirring. After stirring for 72 h, the polydopamine nanospheres PDANS was obtained. The suspension was centrifuged and washed/resuspended with water several times. The precipitate was dried for the following experiments. After the synthesis of PDANS, enough PDANS was introduced into the binding buffer containing 100 nM P1, and the mixture was incubated at room temperature for 10 minutes to form the P1/PDANS nanocomplex. Then the obtained P1/PDANS nanocomplex was stored at 4 °C before further usage. The P2/PDANS nanocomplex was also prepared as the above descriptions. In Vitro Detection of ATP by the P1/PDANS Nanocomplex. In a typical assay, different concentration of ATP (ranging from 0 to 2 mM) or some other molecules was added into 100 nM P1/PDANS nanocomplex in the binding buffer and incubated at 37 °C for 1 h. After incubation, the fluorescence of the resulting solution was measured. The fluorescence emission spectra were recorded from 508 to 650 nm at an excitation wavelength of 480 nm, and the fluorescence intensity at 523 nm was used for quantitative analysis. Cytotoxicity Assays and Live Cell Imaging with the P1/PDANS Nanocomplex. HeLa cells and MCF-7 cells were grown in complete DMEM medium (with 10 % fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin) at 37 °C in a humidified atmosphere containing 5 % CO2. The MTT assay was used for the assessment of the cytotoxic effects from PDANS. HeLa cells were seeded into 96-well cellculture plate at 104 cell/well and then incubated for 24 h at 37 °C under 5 % CO2. After incubating the cells with various concentration of PDANS for 12 h, the standard MTT assay was carried out to determine the cell viabilities relative to the control untreated cells. In the cell imaging experiments, cells were seeded in glass bottom cell culture dishes and grown for 24 h. When the cells were about 90% confluent, 500 µL of fresh cell growth medium supplemented with the P1/PDANS nanocomplex, the P2/PDANS nanocomplex, the PDANS only, or P1 was added in the dishes, respectively. After an incubation of 12 h, 1× PBS was employed to wash the cells three times. The fluorescence imaging of the cells was observed with a confocal scanning laser microscope. And three dimensional images were taken by scanning the samples every 2 µm along the z-axis across a defined section.

RESULTS AND DISCUSSION The Principle of the P1/PDANS Nanocomplex for Molecular Sensing. As shown in Scheme 1, the aptamer and PDANS were employed to construct the aptamer/PDANS nanocomplex. As it has been proven that single-strand DNA sequences (ssDNA) could be assembled on the polydopamine surface with strong affinity45, the nanocomplex is constructed through the assembly of FAM-labeled ATP aptamer (P1) on


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PDANS. Large surface-to-volume ratio of PDANS makes it to be a suitable substrate for probes assembling. The assembly was caused by “π-π stacking” interactions between the nucleobases of the aptamer and the aromatic groups of PDANS.45 As shown in Scheme 1, the binding of the aptamer on PDANS guarantees the close proximity of fluorophores to the surface of PDANS. The following rapid and complete quench of fluorophores was led by the efficient energy transfer from fluorophores to PDANS. In direct contrast, after interaction with ATP, the conformation of the aptamer would change into a stable, hairpin structure.46 The weak binding ability of the hairpin-structured aptamer/target complex to PDANS makes fluorophores far away from the quencher surface, resulting in the fluorescence recovery of FAM. Referring to previous studies about the PDANS-based biosensors and the excellent biocompatibility of the polydopamine, we proposed that the P1/PDANS nanocomplex could be suitable for sensing of ATP based on the fluorescent “signal on” strategy. Hence, cell imaging studies were carried out on HeLa cells, which were incubated with the P1/PDANS nanocomplex. The fluorescent signal was observed by a confocal microscopy. As a result, bright fluorescent signals of FAM would been shown in the pictures corresponding to the aptamer releasing from the nanocomplex after interaction with cellular adenine derivatives (including ATP, ADP, AMP, and adenine) rather than only ATP, because of the lack of distinguishing ability of ATP-selective aptamer to adenine derivatives. Scheme 1. Schematic Illustration of Aptamer/PDANS Nanocomplex for the Molecular Sensing in Living Cells

Formation of the P1/PDANS Nanocomplex. Prior to the formation of the nanocomplex, the PDANS was prepared through a facile and low-cost method. We prepared monodisperse polydopamine nanospheres PDANS with a diameter of approximately 62.7 nm (Figure S1, Supporting Information). The SEM data indicates that PDANS falls within a size range that favors cellular uptake by mammalian cells.47 As PDANS shows broad band absorbance in the UV-vis spectrum (Figure S2, Supporting Information), it would quench the fluorescence of the fluorophores with different emission wavelengths due to FRET.32 The P1/PDANS nanocomplex was formed by mixing 100 nM P1 with PDANS for 10 min in the binding buffer. The quenching ability of PDANS toward P1 was evaluated (Figure 1). Fluorescence intensity of FAM decreased sharply during the increase of PDANS concentration due to energy transfer between the fluorophore and PDANS. When the concentration

of PDANS added reached 0.15 mg/mL, the fluorescence of P1 was nearly completely quenched, and the quenching efficiency was 95.05 %. Consequently, 0.15 mg/mL PDANS was considered optimal for the construction of the P1/PDANS nanocomplex with 100 nM P1.

Figure 1. Quenching efficiency of 100 nM P1 upon the introduction of different concentrations of PDANS. Inset: fluorescence emission spectra of 100 nM P1 with different concentrations of PDANS (0, 0.05, 0.10, 0.15, 0.20, and 0.25 mg/mL).

Figure 2. (A) Fluorescence emission spectra of 100 nM P1/PDANS nanocomplex in the presence of different concentrations of ATP (0, 0.01, 0.1, 0.5, 1, 1.5, and 2 mM). Inset: calibration curve for ATP detection. FI=2351.4c+630.1, (R2=0.9978). (B) Fluorescence intensity of 100 nM P1/PDANS nanocomplex with



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blank (a), 2 mM ATP (b), 2 mM CTP (c), 2 mM GTP (d), 2 mM UTP (e), and 20 U DNase I (h); and fluorescence intensity of 100 nM P2/PDANS nanocomplex without (f) and with 2 mM ATP (g).

In Vitro Detection of ATP. The P1/PDANS nanocomplex was used for the in vitro detection of ATP prior to sensing in living cells. Due to the energy transfer from FAM to PDANS, the fluorescence of FAM was quenched, and the nanocomplex was initially in “off” state. On being incubated with ATP for 1 h, the fluorescence of the nanocomplex was recovered (Figure 2A). Tracing the cause, the weak affinity of the hairpinstructured aptamer/target complex to PDANS makes fluorophore far away from the surface of PDANS and leads to fluorescence recovery of FAM. And the fluorescence intensity was found to be linear with the concentration of ATP in the range of 0.01-2 mM, and the LOD was calculated to be 0.01 mM (Figure 2A). The linear range of the nanocomplex met the need for the sensing of ATP in living cells, as the concentration of ATP in living cells is typically 1-10 mM48. Furthermore, the specificity of the nanocomplex for the detection of ATP was tested (Figure 2B). The target ATP led to the enhancement of the fluorescence of the P1/PDANS nanocomplex. While after the incubation of P1/PDANS with the three analogs CTP, GTP and UTP for 1 h, the fluorescence intensity of FAM was almost the same with the blank, and the nanocomplex was still in “off” state. This indicated that the P1/PDANS nanocomplex was selective for the assay of ATP. And this result successfully facilitates the following ATP sensing in living cells. Meanwhile, the nanocomplex constructed by the random DNA P2 and PDANS was also used for the specificity study. When the analyte was not presented, P2 was adsorbed on the surface of PDANS for the interaction between ssDNA and PDANS. As FAM was quenched through FRET, the P2/PDANS nanocomplex was in “off” state. While, the addition of 2 mM ATP didn’t lead to the recovery of the fluorescence, and the P2/PDANS nanocomplex was still in “off” state. The reason was that P2 could not react with ATP to change the conformation, the fluorescence kept quenched as P2 adsorbed on the surface of PDANS. The above results indicated that only ATP could lead to the fluorescence recovery of P1/PDANS, and none but P1/PDANS, the nanocomplex constructed by the aptamer and PDANS could be used for the sensing of ATP. Cleavage Protection and Cell Viability Assay of PDANS. To our knowledge, most nucleic acid probes, such as molecular beacons, are easily digested by cellular nucleases or degraded by cellular enzymes, which seriously limits their further applications in the studies of biomolecules and physical events in living cells. Therefore, the challenge in the biological application of aptamers in living cells is the delivery of aptamer probes into cells while protecting them from enzymatic cleavage. While, only a few nanomaterials (such as gold nanoparticles, carbon nanotubes, graphene, and silica nanoparticles)29,49 have proved with protection capabilities during molecular transport. Here, DNase I was employed to simulate enzymatic cleavage functions in living cells, which would nonspecifically cleave single- and double-stranded DNA. If PDANS could not protect the aptamer from enzymatic cleavage, P1 would be cleaved by DNase I that would cause the release of FAM from the surface of PDANS and the fluorescence recovery. While, the result shown that the introduction of DNase I didn’t lead to the fluorescence enhancement of P1/PDANS (Figure 2B), suggesting that PDNAS has the pro-

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tection capability to ssDNA against enzymatic cleavage, which has also been proved by a recent work34. Then, the aptamer P1 would target ATP after the delivery of the P1/PDANS nanocomplex into the cells. To realize the in situ target sensing in living cells, it’s expected that the P1/PDANS nanocomplex is with good biocompatibility and low toxicity. Consequently, the MTT assay, the standard cell viability assay, was employed for the evaluation of the cytotoxicities of PDANS on HeLa cells. After the HeLa cells were incubated with PDANS concentrations up to 0.45 mg/mL for 12 h, higher than 90 % cell survival rate was observed (Figure S3, Supporting Information). Throughout the present study for cell imaging, the concentration of PDANS used was less than 0.15 mg/mL, which would ensure high viability of all the tested cells.

Figure 3. Fluorescence images, bright-field images, and the merge of fluorescence and bright-field images of HeLa cells after incubation without (A) or with 100 nM P1 (B), 100 nM P1/PDANS nanocomplex (C), 100 nM P2/PDANS nanocomplex (D), and 0.15 mg/mL PDANS (E) for 12 h at 37 °C. Scale bar: 75 µm.

In Situ Live Cell Imaging of ATP. It has been demonstrated that the aptamer could be assembled on the surface of PDANS while retaining good specificity for ATP to dissociate from the aptamer/PDANS nanocomplex. To further test the


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employment of this nanocomplex for intracellular imaging study, HeLa cells were used to be incubated with P1/PDANS for 12 h. The nanocomplex consisting of random DNA P2 (P2/PDANS) was employed as a reference probe for the evaluation of the specificity of the P1/PDANS nanocomplex in living cells. And HeLa cells incubated with ATP aptamer P1 without PDANS were chosen as control to prove the transport ability of PDANS. As shown in Figure 3, a significant FAM fluorescence image was obtained from the cells cultured with P1/PDANS (Figure 3C), indicating successful intracellular aptamer delivery and ATP sensing in HeLa cells. While, almost no fluorescence signal was observed from HeLa cells incubated either with or without P1 as well as with P2/PDANS or PDANS only (Figure 3A, B, D, E). To confirm the internalization of P1/PDANS in HeLa cells, Z-scanning confocal imaging was further performed (Figure S4, Supporting Information). After the incubation of HeLa cells with P1/PDANS for 12 h, the images were taken by scanning the samples every 2 µm along the z-axis across a defined section. Bright FAM fluorescence was clearly present throughout the whole cells, suggesting the efficient delivery of this nanocomplex to the cytosol. Furthermore, HeLa cells were incubated with P1/PDANS at concentrations of 25, 50 and 100 nM, and after a 12 h incubation, images were captured by a confocal microscope (Figure S5, Supporting Information). Fluorescence intensity corresponding to cellular ATP increased with increasing nanocomplex concentration. This result demonstrated that this nanocomplex successfully realize the in situ sensing of ATP in HeLa cells. To further confirm the fluorescence signal resulting from the endogenously produced ATP of the HeLa cells, an assay for in situ ATP semi-quantification was designed, and the results were shown in Figure 4. Before culture with P1/PDANS, the HeLa cells were treated with 10 µM oligomycin (a wellknown inhibitor of ATP50) or with 5 mM Ca2+ (a commonly used ATP inducer51) for 30 min. As shown in Figure 4, the FAM fluorescence decreased dramatically upon treatment with oligomycin (Figure 4A), while a significant enhancement was observed when the cells were pre-incubated with Ca2+ (Figure 4C). These results demonstrate that the P1/PDANS nanocomplex could achieve reliable intracellular measurement of ATP in HeLa cells.

Figure 4. Fluorescence images, bright-field images, and the merge of fluorescence and bright-field images of HeLa cells treated with 10 µM oligomycin (A), medium (B) or 5 mM Ca2+ (C) followed by incubation with 100 nM P1/PDANS nanocomplex for 12 h at 37 °C. Scale bar: 75 µm.

Figure 5. Fluorescence images, bright-field images, and the merge of fluorescence and bright-field images of MCF-7 cells after incubation with 100 nM P1 (A), 100 nM P1/PDANS nanocomplex (B), and 100 nM P2/PDANS nanocomplex (C) for 12 h at 37 °C. Scale bar: 75 µm.

As the above results have demonstrated that the P1/PDANS nanocomplex could deliver the aptamer probe into HeLa cells and successfully achieve the in situ sensing of ATP. As ATP is the primary energy molecule in all living cells, we further test whether this nanocomplex could be used for intracellular sensing of ATP in other cells. MCF-7 cells were used to be incubated with P1, P1/PDANS and P2/PDANS for 12 h, respec-



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tively. As shown in Figure 5, fluorescence signal corresponding to FAM tagged on P1 in the nanocomplex was clearly observed (Figure 5B), while little/no fluorescence signal from P2/PDANS could be observed (Figure 5C). Meanwhile, fluorescence of FAM could not be observed from P1 without PDANS (Figure 5A). This indicated that the P1/PDANS nanocomplex could also be used for the intracellular sensing of ATP in MCF-7 cells. All these results demonstrate that the P1/PDANS nanocomplex can afford an excellent intracellular biosensor for high-contrast fluorescence imaging of biomolecules in living cells.

CONCLUSION In summary, a nanocomplex has been developed based on the fluorescence aptamer and the polydopamine nanospheres for in situ sensitive and selective assay of biomolecules. In vitro assays demonstrated that the P1/PDANS nanocomplex was a robust, sensitive, and selective biosensor for quantitative detection of ATP. And the confocal fluorescence microscopy experiments with HeLa cells and MCF-7 cells further suggested that the P1/PDANS nanocomplex was efficiently delivered into living cells and worked as an in situ “signal on” biosensor for specific, high-contrast imaging of target molecules. Excellent biocompatibility of PDANS along with noncovalent binding between oligonucleotides and PDANS have proved that PDANS was an efficient cargo and an excellent protector for cellular delivery of nucleic acids and possibly peptides or proteins in due course. What’s more, super quenching ability suggests PDANS as a universal sensing platform appropriate for various fluorescent probes. In conclusion, these advantages of polydopamine make it an excellent candidate for the employment in many biological fields, such as the assay of DNA and protein, the delivery of gene and drug, intracellular tracking as well as in vivo monitoring, etc.

ASSOCIATED CONTENT Supporting Information Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Tel/Fax: (+) 00862583595835. E-mail: [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We acknowledge the financial support of the National Basic Research Program of China (973 Program, 2011CB911003), National Natural Science Foundation of China (Grant no. 21227009, 21175066, 21328504, and 21475060), Fundamental Research Funds for the Central Universities (20620140439), and the National Science Funds for Creative Research Groups (21121091).

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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 ACS Paragon Plus Environment

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Figure 4 85x84mm (300 x 300 DPI)



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Figure 5 85x84mm (300 x 300 DPI)


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