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Alkynes-DNA-Functionalized Alloyed Au/Ag Nanospheres for Ratiometric SERS Imaging Assay of Endonuclease Activity in Live Cells Yanmei Si, Yaocai Bai, Xiaojie Qin, Jun Li, Wenwan Zhong, Zhijun Xiao, Jishan Li, and Yadong Yin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04735 • Publication Date (Web): 05 Mar 2018 Downloaded from http://pubs.acs.org on March 6, 2018

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Analytical Chemistry

Alkynes-DNA-Functionalized Alloyed Au/Ag

Nanospheres for

Ratiometric SERS Imaging Assay of Endonuclease Activity in Live Cells Yanmei Si,† Yaocai Bai,‡ Xiaojie Qin,† Jun Li,† Wenwan Zhong,‡ Zhijun Xiao,† Jishan Li,*† Yadong Yin‡ †

State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and

Chemical Engineering, Institute of Chemical Biology and Nanomedicine, Hunan University, Changsha 410082, China. ‡

Department of Chemistry, University of California-Riverside, California 92521, United States.

*Corresponding author. E-mail: [email protected].

Fax: +86-731-8882 1848

ABSTRACT A novel ratiometric surface-enhanced Raman scattering (SERS) nanosensor has been developed to probe the activity of endonuclease under in vitro and in living cells conditions. The optimized alloyed Au/Ag nanoparticles (Au/AgNPs) were synthesized as the SERS substrate, which combined the superior properties of both the pure Au and Ag nanoparticles: they exhibit excellent plasmonic property with high chemical stability and low cytotoxicity. They were then employed for quantitative detection of endonuclease through functionalization with single-stranded DNA (ssDNA) carrying 3-(4-(phenylethynyl)benzylthio) propanoic acid (PEB) as endonuclease-responsive SERS signaling molecule, and 4-thiol phenylacetylene (TPA) as the internal standard SERS signaling molecule. In the presence of endonuclease, the ssDNA was cleaved, releasing the PEB molecules from the particle surface and decreasing the SERS signal at 2215 cm-1 from PEB. Since the SERS signal at 1983 cm-1 from alkynyl TPA remained the same, quantitative detection of endonuclease was achieved based on the ratiometric peak intensity of I1983/I2215, with a detection limit as low as 0.056 U/mL. A highly biocompatible and anti-jamming ratiometric SERS sensor was established by combining the alloyed Au/AgNPs with two unique alkynes molecules with Raman signal in the cellular silent region. The ratiometric sensor was successfully employed to detect intracellular endonuclease activity as well as endonuclease in living cells for the first time.

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

INTRODUCTION Endonucleases hydrolyze the phosphodiester linkages in the nucleic acid backbone. They play an essential role in many biological processes such as replication, repair, and recombination of nucleic acids, and are related directly to cell apoptosis.1-4 In addition, researchers have found that endonucleases may also be promising targets for the development of antimicrobial and antiviral drugs.5,6 Therefore, measurement of intracellular endonuclease activity is crucial for both biomedical research and drug screening. Conventionally, the enzyme activity monitoring has been carried out by gel electrophoresis,7 chromatographic separation,8 enzyme-linked immunosorbent assay (ELISA),9 colorimetric and fluorescence-based analysis,10-12 and so forth.13 Although impressive progress has been achieved, those techniques suffer from some shortcomings such as time-consuming, labor-intensive, requiring radioactive labeling of substrates, and especially the incapability to monitor the endonucleases in living cells. Therefore, it is highly desirable to develop a more effective technique for the measurement of the intracellular activity of endonucleases. Surface-enhanced Raman scattering (SERS)14 has been proved to be a powerful measurement technique and widely applied in biomedical fields, such as chemical and biomolecular sensing, imaging and diagnosis,15-20 due to its significant advantages over fluorescence methods, including resistance to photobleaching and phototoxicity, and narrow emission peaks for spectral multiplexing.21,22 However, the usage of SERS in biomedical sensing is still limited by the commonly used substrates made of spherical gold and silver colloids. Aggregation is typically required to generate the “hotspots” of the electromagnetic field on Au nanoparticles (AuNPs) to support the high signal enhancement. It is difficult to induce aggregation reproducibly.23,24 Although silver exhibits the highest plasmonic activity among various metals for supporting strong surface plasmon polarization,25-28 it has not yet been widely utilized in biomedical research because of its poor chemical and structural stability under the non-ideal chemical environments as well as high cytotoxicity.29 A powerful SERS-based detection technique typically requires a robust SERS substrate that can provide large extinction cross sections for improved SERS performance and possesses high chemical stability and biocompatibility for biomedical applications.30-32 Recently, we have successfully synthesized alloyed Au/Ag nanoparticles (Au/AgNPs) based on the strategy of interfacial atomic diffusion of Ag and Au at a high temperature of ~1000 °C.33 These alloyed NPs exhibit an excellent plasmonic property comparable to that of pure Ag nanoparticles (AgNPs), and significantly enhanced chemical stability even under extreme environmental conditions. Therefore, it is a fascinating prospect that such nanostructures would be ideal candidates for the fabrication of SERS nanosensors for applications in bioimaging and biosensing.


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In addition to the substrates, the reporter molecules, which are the other critical part that determines the effectiveness of a SERS-based sensing method, have not been studied as extensively as the SERS substrates. In biological systems, one major problem is that Raman bands of the typical reporters usually overlap with those originating from the interfering molecules in the complex biological matrices, resulting in significantly reduced detection accuracy. It is therefore of great importance to rationally select the reporter molecules for the complex detection system. Recently it has been shown that alkynes exhibit a distinct and strong Raman scattering peak in the cellular silent region of 1800−2800 cm−1, where most cellular components do not show Raman signals,34-37 avoiding the overlapping of Raman signals. Nevertheless, the broad application of alkyne-tag in biological sample analysis is still limited due to the extremely weak Raman scattering response of alkynyls. Fortunately, the conjugation of alkyne to an aromatic ring can significantly increase the Raman intensity.38 In addition, the Raman shifts of the alkynyl group can be greatly varied depending on the substitution pattern. In this work, we combined the highly stable and biocompatible alloyed Au/AgNPs with the modified alkynes to develop a novel SERS sensing method for measuring the activity of endonucleases both in vitro and in living cells. 3-(4-(phenylethynyl) benzylthio) propanoic acid (PEB) was prepared and labeled onto the thiol-modified DNA (HS-DNA-PEB), forming thiol/alkyne double-functionalized single-stranded DNA (ssDNA) as reporter molecules. 4-thiol phenylacetylene (TPA) was synthesized and used as an internal standard. An alkyne-DNA/alloyed Au/AgNPs spherical SERS nanoprobe was constructed via assembling the thiol/PEB double-functionalized ssDNA and TPA on the surface of the alloyed Au/AgNPs (Scheme 1). The Raman signals from both the PEB and TPA molecules on the SERS nanoprobe can be detected and identified. In the presence of an endonuclease, the PEB molecules coupled to the ssDNAs will be released from the particle surface due to the endonuclease-catalyzed DNA cleavage, resulting in a great decrease in their SERS signal. Since the Raman signal of the internal alkynyl compound (TPA) is not affected by the endonuclease, quantitative detection of endonuclease activity can be achieved by calculating the peak intensity ratio. Furthermore, this ratiometric strategy will overcome the problem of SERS signal irreproducibility which results from the inhomogeneous molecular adsorption and irregular distribution of the enhancement substrate. The hybrid utilization of the alloyed Au/AgNPs with Ag-like SERS enhancement capacity, Au-like stability and biocompatibility, and alkynes with distinct interfering-free SERS peak in the cellular silent region, results in an ideal SERS nanosensor in endonuclease detecting and imaging. To the best of our knowledge, this is the first report of an alkyne-DNA/alloyed Au/AgNPs spherical SERS nanosensor to be employed for intracellular measurement of the endonuclease.



EXPERIMENTAL SECTION Chemicals Endonuclease (DNase I, S1 nuclease) was purchased from New England Biotechnology (Beijing, China). The alkyne-tag DNA (5’ PEB-TTTTTTTTT-SH-3’) and the fluorophore-tag DNA (5’ TAMRA-TTTTTTTTT-SH-3’) were synthesized by Takara Biotechnology (Dalian) Co., Ltd. (Note: the alkynes molecules were prepared by ourselves). Synthesis of 4-thiol phenylacetylene (TPA) and 3-(4-(phenylethynyl)benzylthio) propanoic acid (PEB) was shown in supporting information (SI). Diethylamine, 16-mercaptohexadecanoic acid (MHA), 4-mercaptobenzoic acid (MBA) and tetraethyl orthosilicate (TEOS) was obtained from J&K Scientific (Beijing, China). gold (III) chloride hydrate (HAuCl4 ·3H2O), silver nitrate (AgNO3), sodium borohydride (NaBH4), ascorbic acid, poly(vinylpyrrolidone) (PVP) (Mw = 10000), potassium iodide and trisodium citrate salt (TSC) were purchased from Sigma-Aldrich Chemical Reagent Co. (China). Acetonitrile, ethanol and other chemical reagents were provided by Sinopharm Chemical Reagent Co. (China). All of the reagents used in this study were of analytical grade and were used without further purification. Deionized water (>18 MΩ) was supplied from a Millipore water purification system in all assays. Characterization. The transmission electron microscopy (TEM) images were taken with a high-resolution transmission electron microscope (HRTEM, JEOL JEM-2100F, Japan). The energy dispersive X-ray (EDX) spectra measurements were conducted using a TecnaiG2 F20 S-TWIN atomic resolution analytical microscope. The absorption spectra were measured by a Hitachi U-4100 spectrophotometer (Kyoto, Japan). MTT assay uses a Synergy™ 2 Multi-Mode Microplate Reader (Bio-Tek, Winooski, TV). SERS spectra were detected using a Raman microscope (InVia, Renishaw, Gloucestershire, UK) and spectra were acquired under a 633nm He-Ne excitation laser. Each spectrum was analyzed by least-squares analysis with Wire 3.4 Software (Renishaw). Synthesis of alloyed Au/AgNPs The synthesis of alloyed Au/AgNPs was conducted following a modified synthesis procedure reported previously, and the detailed procedures are also shown in SI.33,39 Briefly, the preparation of the alloyed Au/AgNPs was divided into six steps. The first, preparation of gold seeds (3 nm), and then AuNPs were obtained using a seeded-growth strategy. Next, a layer of silver shell was coated on the surface of AuNPs, formed Au@AgNPs, and then a layer of silica shell was coated on the Au@AgNPs. The so formed


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Au@Ag@SiO2 NPs were dried and annealed at about 900°C (3.5 h) in an argon atmosphere for effective alloying of gold and silver atoms. The sample after annealing was re-dispersed in an etching solution to remove the silica shell and obtained the alloyed Au/AgNPs. The synthetic route of different size alloyed Au/AgNPs is similar to the 30-nm NPs, the size of alloyed Au/AgNPs changed through changing the size of the AuNPs. The synthetic route of different Au/Ag mole ratio alloyed Au/AgNPs is similar to the 1/5 alloyed Au/Ag NPs, the Au/Ag ratio of alloyed Au/AgNPs changed through changing the adding amount of AgNO3. Cell viability assay The biocompatibility of alloyed Au/AgNPs was examined by the MTT (3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyltetrazolium bromide) assay, and the AuNPs was conducted as a control experiment. The HeLa cells were seeded in 96-well plates and incubated for 24 h at 37°C, then, 0.100 mL of NPs at different concentrations were added respectively, and incubated for 24 h. After that, 0.005 mL of MTT solution (5 mg/mL) was added to each well and was incubated at 37°C for another 2 h in the CO2 incubator. Then, 0.150 mL of DMSO was introduced into each well, and the absorbance of the wells was measured on a microplate reader (Bio-Rad model 680) with a test wavelength of 570 nm. Cells incubated in the absence of nanoparticles were used as controls. Preparation of alkyne-tag Au/AgNPs SERS nanoprobe. The alloyed Au/AgNPs solution was cleaned by centrifugation to remove PVP, which hinders the formation of Au−S bond between DNA-PEB or TPA and alloyed Au/AgNPs, and then re-dispersed in deionized water. The solution of 0.200 mL HS-DNA-PEB (100 uM) was mixed with 1.6 mL NPs and the mixture was incubated for 12 h. After that, 0.200 mL TPA (100 uM) was injected and incubated for another 2 h. The sample was then centrifuged at room temperature for twice (each for 10 min) to remove free DNA-PEB and TPA and finally dispersed in 2.0 mL water solution for the subsequent experiments. For quantifying the number of DNA-PEB on each alloyed Au/AgNP, a thiol labeled DNAs strand with a fluorophore of 3'-TAMRA tag was used to modify alloyed Au/AgNPs according to the preparation above of the alkyne-tag Au/AgNPs SERS nanoprobe. Considering the same modification process, theoretically speaking, the surface coverage of the 3'-TAMRA-tagged DNAs strand was the same as the DNA-alkynes functionalized alloyed Au/AgNPs.40,41 For determining the amount of 3'-TAMRA-tagged strand in the as-prepared alloyed Au/AgNPs’ surface, the 3'-TAMRA-modified DNAs in supernatant after centrifugation in modification process was quantified using standard solutions of the 3'-TAMRA-modified DNA, and then the binding amount of DNA



strands were calculated based on the known total added amount. The surface concentration of oligonucleotide was estimated to be ca 300 strands per alloyed Au/AgNP. SERS detection of endonuclease in solution. A solution of endonuclease (DNase I, S1 nuclease) was freshly prepared by the dilution of endonuclease stock solution with specific buffer solution. In 0.500 mL tubes, a 0.005 mL endonuclease solution was mixed with 0.015 mL DNA-alkyne functionalized alloyed Au/AgNP nanoprobe and allowed for incubation for 30 min at 37°C in a water bath. Finally, a portion of the reaction solution was transferred to capillary tubes to conduct SERS detection. SERS imaging of endonuclease in living cells. The HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin and were maintained in an incubator containing 95% air and 5% CO2 at 37°C. For preparing live cells for spectroscopic detection and imaging, the cells were seeded in 6 cm dishes and allowed to adhere overnight, and then incubated for 2 h with culture medium containing nanoprobes (1.0060 x 10^-10 M/L). After that, the culture medium was removed, and the cell culture dishes were washed three times with PBS to remove free unbound samples. The culture dish was then mounted to a small incubator on the microscope stage for cell imaging and spectroscopic.

RESULTS AND DISCUSSION Preparation and characterization of the alloyed Au/AgNPs. Figure 1 shows the typical characterizations of the alloyed Au/AgNPs and their intermediates during the synthesis. After the overgrowth of Ag shell over AuNPs, core/shell Au@AgNPs were obtained in high yield, as indicated in the TEM image (Figure 1A) and unambiguously confirmed by energy spectrum (Figure 1B). A resonance peak similar to AgNPs appeared at ~410 nm after a layer of Ag coating on AuNPs (Figure 1D), indicating that the optical response of noble-metal nanoparticles is very sensitive to the outer-shell composition. After silica coating, high-temperature annealing, and silica removal, the resultant alloyed Au/AgNPs were nearly perfect nanospheres (Figure 1C), in contrast to the irregular shapes of the original core/shell NPs. They showed a characteristic resonance peak that was nearly symmetric and much narrower than the peak of the original core/shell NPs (Figure 1D), thanks to their excellent monodispersity and uniform spherical shapes. Furthermore, the elemental analysis (Figure 1E) of the alloyed Au/AgNPs (30 nm) evidenced that the high-temperature annealing significantly increased the diffusion rates of Au and Ag atoms and enabled ACS Paragon Plus Environment

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complete atomic mixing of the two metals, leading to the formation of alloyed NPs. As we all know, stability is an essential consideration in the selection of SERS substrate. For example, despite the strong SERS enhancement effect, AgNPs can be oxidized and etched easily under non-ideal conditions, which limited their application in SERS detection. The stability of AuNPs, Au@AgNPs and alloyed Au/AgNPs were first tested in an H2O2 solution. As shown in Figure S1 (SI), the UV-vis spectra of alloyed Au/AgNPs remained at their original intensity even after 36 h of etching. On the contrary, the resonance peak of core/shell Au@AgNPs disappeared after the same treatment, indicating that alloy nanoparticles possess Au-like stability. The excellent chemical stability of the alloy particles may greatly expand their use in many plasmon-based applications, especially when corrosive reagents are involved. In addition to the reactive oxygen species (e.g., H2O2) that are problematic in the application of nanomaterials as SERS substrate, the low pH in endosomes, as well as the high salt content, could also be damaging to the particles. Thus, the stability of the alloyed Au/AgNPs toward the potential interfering factors including pH and high salt content were evaluated (Figure S2). One can see from Figure S2 that the plasmon resonance band of the alloyed Au/AgNPs remained at their original intensity both under the pH from 4 to 9 (Figure S2 A) and under different salts at their physiological concentration (Figure S2 B). The high stability of the alloyed Au/AgNPs under potential disturbance factors in the cell further guarantees the reliability of the material as a SERS substrate in biological detection. Effect of composition of the alloyed Au/AgNPs on SERS. Considering the composition of the particles usually has a significant impact on the SERS performance, the alloyed Au/AgNPs with different Au/Ag molar ratios (from 1/1 to 1/7) were synthesized and investigated. The UV−vis spectra of the core/shell Au@AgNPs with different Au/Ag molar ratios (Figure S3) show that coating of a thicker Ag layer produces a more Ag-like plasmon resonance and that the intensity of the extinction peak increases with the increase of the mole fraction of Ag. The highly symmetric absorption spectra of alloyed Au/AgNPs with different Au/Ag molar ratios further indicate the effective interdiffusion of gold and silver atoms even with high silver content (Figure 2A). To investigate the influence of the composition of alloy nanoparticles on SERS enhancement, we have obtained the SERS spectra of the 4-mercaptobenzoic acid (MBA)-absorbed alloyed Au/AgNPs (~30 nm) with different Au/Ag molar ratios. As shown in Figure 2B, the intensity of SERS signals increases with the increasing of Ag amount. However, the stability of alloy NPs is also dependent on the Au/Ag ratio, and it decreases obviously as the amount of Ag increases.33 After full consideration of the stability, biocompatibility and the SERS enhancement effect of the alloyed Au/AgNPs, the



molar ratio of 1/5 was selected as the best choice, and subsequent experiments all use the proportion. Effect of size of the alloyed Au/AgNPs on SERS. The magnitude of SERS enhancement is crucially dependent on the plasmonic properties of metal nanostructures, which are determined by not only the composition but also the size of the nanoparticles. Therefore, the effect of the size of the alloyed Au/AgNPs (e.g., 30 nm, 50 nm and 90 nm) on the enhancement of SERS signals was explored. The synthetic route for 50-nm and 90-nm NPs was similar to that of the 30-nm NPs, where the size of alloyed Au/AgNPs could be tuned through changing the size of the initial AuNPs.39 As shown in Figure S4 A-C, all the alloyed Au/AgNPs exhibited good monodispersity and uniform spherical shape. UV−vis spectra (Figure S4 D) showed that the 50-nm alloyed Au/AgNPs displayed a similar highly symmetric absorption peak with slight red-shift compared to the 30-nm NPs, while the resonance peak of the 90-nm alloyed Au/AgNPs broadened with obvious red-shift. Stability experiments showed that both the 50-nm and 90-nm alloyed Au/AgNPs maintained their plasmon band intensity of the original value after H2O2 etching (Figure S5), further indicating their excellent stability against etching agents. The influence of particle size on the Raman enhancement was then evaluated. As shown in Figure 2C, the SERS signals for the 50-nm alloyed Au/AgNPs were enhanced more significantly than those for the 30-nm alloyed Au/AgNPs under the same concentrations of both alloyed NPs and Raman reporter molecules, while the signal intensity decreased clearly for the 90-nm alloyed Au/AgNPs under the same conditions. The enhancement factor of the alloyed Au/AgNPs of different sizes were also calculated theoretically based on the experimental data.42 The calculations clearly showed that the alloyed NPs of 50 nm exhibited the highest SERS enhancement factor, which is consistent with the SERS spectra, further indicating that the 50-nm alloyed NPs possess a relatively better enhancement effect. Thus, the alloyed NPs with the size of 50 nm and Au/Ag molar ratio of 1/5 are the best choice for this work. SERS investigation of AuNPs, core/shell Au@AgNPs and alloyed Au/AgNPs with the same size. The enhancement capacity of AuNPs, core/shell Au@AgNPs and alloyed Au/AgNPs with the same size of 50 nm (Figure S6) was also investigated. As shown in Figure 2E, compared with AuNPs and core/shell Au@AgNPs, the alloyed Au/AgNPs showed the strongest SERS signals. It is worth noting that the SERS enhancement of the core/shell Au@AgNPs was weaker than that of the alloyed Au/AgNPs, which may be attributed to the oxidative etching of the Ag shell layer that leads to the reduced SERS capacity of the core/shell nanoparticles as well as a lower binding density of reporter molecules (MBA) on the particle surface. Besides, the enhancement factor of these NPs (Figure 2F) was calculated as 7.4168x10^4 for 50-nm AuNPs,


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2.1025x10^5 for 50-nm core/shell Au@AgNPs and 1.7810x10^6 for 50-nm alloyed Au/AgNPs, respectively, which is also consistent with the SERS spectra of these NPs. These data further suggest that the NPs with excellent plasmonic property comparable to that of pure Ag nanoparticles and significantly enhanced chemical stability could be achieved in alloyed Au/AgNPs. Cytotoxicity of the alloyed Au/AgNPs. The cytotoxicity of the alloyed Au/AgNPs was evaluated via the standard MTT assay by using HeLa cells as the models. As shown in Figure 3A, the cell viability decreased slightly with the increasing dose of both AuNPs and alloyed Au/AgNPs, suggesting that the alloyed Au/AgNPs presented similar biocompatibility as AuNPs. In addition, no apparent cytotoxicity observed even with the concentration of NPs as high as 5.0300x10^-10 M. Therefore, it can be concluded that the alloyed Au/AgNPs exhibit not only excellent SERS enhancement but also good biocompatibility, which is promising as SERS substrate in biodetection. Preparation and characterization of the alkynes-DNA functionalized alloyed Au/AgNPs nanoconjugates. For investigating the establishment of the DNA-alkynes functionalized alloyed Au/AgNPs nanoconjugates, the establishment and characterization of the preparation process were conducted comparably using Raman spectroscopy. One can see from Figure 3B that strong Raman signals of 2215 cm-1 and 1983 cm-1 vibrations were observed for DNA-PEB/TPA modified alloyed Au/AgNPs, which can be attributed to the alkynyl of PEB and alkynyl of TPA, respectively. No signals were detected for either alloyed Au/AgNPs in the absence of DNA-PEB/TPA or DNA-PEB/TPA short of alloyed Au/AgNPs. Thus, the endonuclease-responsive element of DNA-PEB is believed to be successfully conjugated to the SERS substrate of alloyed Au/AgNPs, forming DNA-alkynes double-functionalized alloyed Au/AgNPs nanoconjugates. To ensure the effectiveness of the nanoconjugates in cell detection, we evaluated the stability of the nanoconjugates in the devitalized cell lysates. Here, the nanoconjugates were mixed with the devitalized cell lysate with different concentrations for SERS tests. As shown in Figure 3C, no obvious changes in the peak intensity ratio (I1983/I2215) were observed under different concentrations of cell lysates, suggesting that the nanoconjugates are stable in the presence of cell interfering components. It can be thus speculated that the proposed SERS nanoprobe will be stable enough for endonuclease detection in live cells. Sensing performance of the SERS nanoprobes. The capability of the nanoconjugates as an endonuclease SERS nanosensor was first evaluated in an aqueous solution. As can be seen from the SERS spectra (Figure 4A), the peak intensity at 2215 cm-1 attributed to



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alkynyls of PEB decreased accordingly with increasing the concentration of DNase I, while the peak intensity at 1983 cm-1 attributed to alkynyls of TPA remained unchanged, thus resulting in an increase of the ratiometric peak intensity of I1983/I2215 and achieving a ratiometric SERS detection of endonuclease. Figure 4B also suggests that there is a positive correlation between the peak intensity ratio of I1983/I2215 and the concentration of DNase I over the range of 0–80 U/mL, and moreover a detection limit of ca 0.056 U/mL can be estimated according to the 3 σ rule. The dynamic detection range and limit of the developed method were also compared with the current assays for endonuclease reported elsewhere (Table S1), showing the better or comparable detection sensitivity. Furthermore, we also investigated the sensing capability of the SERS nanosensor for S1 nuclease in solution. Figure 4C showed the SERS spectra of the DNA-alkynes functionalized alloyed Au/AgNPs nanoconjugates before and after the addition of S1 nuclease. One can see from the results that S1 nuclease can also lead to the intensity decrease of 2215 cm-1 band, likely a result of the cleavage reaction by S1 nuclease, indicating that our nanosensor is also capable of sensing S1 nuclease, thus realizing the measurement of the total amount of endonuclease in living cells. Live-cell imaging assay of endonuclease using the prepared SERS nanosensor. After confirming the effectiveness of the ratiometric SERS strategy in the aqueous solution, we further explore the detection of endonuclease in live cells. The cell permeability of the SERS nanosensor toward HeLa cells was first examined. Figure S7 shows that the sensing nanoconjugates can enter cells efficiently after only 1 h of incubation as indicated by the intense Raman signal at 1983 cm-1 from TPA. As a result, the DNA-alkynes functionalized alloyed Au/AgNPs exhibit rapid cell membrane penetration capacity, which may be ascribed to the extraordinary cellular uptake property of spherical nucleic acids (SNA).43 To further verify the internalization of the nanoconjugates in HeLa cells, Z-scanning confocal SERS imaging of Hela cells incubated for 2 h was carried out. As shown in Figure S7, strong Raman signal at 1983 cm-1 of TPA was present throughout the whole cells, suggesting the efficient delivery of the nanosensing conjugates to the cytosol. Raman imaging was finally performed for the SERS nanoprobes-incubated HeLa cells before and after the addition of an apoptotic agent (2-phenethyl isothiocyanate (PEITC), an organic molecule which can cause cell apoptosis)44. As shown in Figure 5A, the SERS signals at 1983 cm-1 and 2215 cm-1 exhibit strong peak intensities for the HeLa cells without treatment of PEITC, indicating a lower level of endonucleases in normal cells. In contrast, for the HeLa cells treated with PEITC, the SERS band at 1983 cm-1 remained unchanged, while the peak intensity at 2215 cm-1 decreased significantly (Figure 5B), which indicates the over-expression of endonucleases in the process of cell apoptosis. Besides, the significant differences in the peak intensity ratio


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of I1983/I2215 between normal cells (a, b, c of Figure 5A) and apoptosis cells (d, e, f of Figure 5B), further indicates that the prepared ratiometric SERS nanosensor via the assembly of DNA-alkynes on the alloyed Au/AgNPs has the excellent performance to screen the endonucleases in live cells.

CONCLUSIONS In summary, we have successfully developed a novel ratiometric SERS nanosensor based on the alkyne-DNA-functionalized alloyed Au/AgNPs for the detection of endonuclease in living cells with high sensitivity and excellent signal reproducibility. Outstanding SERS performance for the alloyed Au/AgNPs has been demonstrated, which is attributed to the excellent plasmonic property comparable to pure AgNPs, significantly enhanced chemical stability, and low cytotoxicity similar to AuNPs. The successful conjugation of alkynes as SERS reporters to the SERS substrate of alloyed Au/AgNPs has further guaranteed strong Raman signals in cell silent area without interference. Besides, the ratiometric SERS sensing approach based on the differentiable SERS signals of alkynes has enhanced the reliability of the endonuclease detection in complex systems. Considering the unique properties of the SERS technique, this novel ratiometric SERS strategy by combining the alloyed Au/AgNPs with the cell-silent Raman reporters may hold great potential for in vitro and in living cells applications in medical research and clinical diagnostics.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Detailed experimental process and additional spectroscopy data 1HNMR,

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CNMR of the products; Chemical

stabilities of alloyed Au/AgNPs (Figure S1, Figure S2); UV−vis spectra of the core/shell Au@AgNPs (Figure S3); TEM images and of UV−vis spectra of the alloyed Au/AgNPs with different size (Figure S4); Chemical stabilities of alloyed Au/AgNPs with different size (Figure S5); TEM images of AuNPs, core/shell Au@AgNPs, alloyed Au/AgNPs (Figure S6); Comparison of detection performances among different assays for endonuclease detections (Table S1). SERS imaging of the nanoprobe-loaded HeLa cells for different incubation time (Figure S7).

AUTHOR INFORMATION Corresponding Author *Corresponding author, E-mail: [email protected].

Fax: +86-731-8882 1848



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Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT This work was supported by the National Natural Science Foundation of China (No. 21475036, No. 21775035, No. 21527810), and Hunan Provincial Natural Science Foundation (No. 2016JJ1005). The authors also thank Professor Chuanbo Gao at Xi'An Jiaotong University for his helpful suggestions in the synthesis of alloyed Au/AgNPs.


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Scheme 1 Schematic illustration of the DNA-alkynes functionalized alloyed Au/AgNPs-based SERS nanosensor for ratiometric detection of endonuclease.



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Figure 1 TEM images of core/shell Au@AgNPs (A) and alloyed Au/AgNPs (C) obtained from a typical synthesis of alloyed Au/AgNPs (Au/Ag = 1/5); (B) Energy spectrum of core/shell Au@AgNPs; (D) Optical evolution from AuNPs to alloyed Au/AgNPs: (a) AuNPs, (b) core/shell Au@AgNPs, (c) core/shell Au@Ag@SiO2NPs and (d) alloyed Au/AgNPs. The spectra have been normalized relative to the spectral maximum of the core/shell Au@Ag nanoparticles. (E) Elemental analysis of the alloyed Au/AgNPs (Au/Ag = 1/5, ~30 nm): (a) DF-STEM image; (b, c) EDX elemental maps of Au and Ag, respectively.


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Figure 2 (A) UV−vis spectra of alloyed Au/AgNPs with different Au/Ag molar ratios (a-g: 1/1-1/7); (B) SERS spectra of MBA (50 μM) in the presence of alloyed Au/AgNPs with different Au/Ag molar ratios (a-g: 1/1-1/7). (C) SERS spectra of MBA (50 μM) in the presence of alloyed Au/AgNPs (Au/Ag = 1/5) with different size: (a) ~30 nm, (b) ~50 nm, (c) ~90 nm; (D) SERS enhancement factors of the alloyed Au/AgNPs (Au/Ag = 1/5) with different size: (a) ~30 nm, (b) ~50 nm, (c) ~90 nm. (E) SERS spectra of MBA (50 μM) in the presence of (a) 50-nm AuNPs, (b)50-nm core/shell Au@AgNPs (Au/Ag = 1/5), and (c)50-nm alloyed Au/AgNPs (Au/Ag = 1/5); (F) SERS enhancement factors of (a)50-nm AuNPs, (b)50-nm core/shell Au@AgNPs (Au/Ag = 1/5) and (c) 50-nm alloyed Au/AgNPs (Au/Ag = 1/5).



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Figure 3 (A) Cell relative viability of HeLa cells after treated with different concentrations of AuNPs (red bars) and alloyed Au/AgNPs (blue bars). (B) SERS spectra of (a) 50-nm alloyed Au/AgNPs (Au/Ag = 1/5), (b) HS-DNA-PEB and TPA solution, (c) 50-nm alloyed Au/AgNPs and HS-DNA-PEB/TPA solution. (C) Stability of the SERS nanoprobe in the presence of devitalized cell lysate with different concentration.


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Figure 4 (A) SERS spectra of the DNA-alkynes functionalized alloyed Au/AgNPs nanosensor upon addition of DNase I with different concentrations; (B) Correlation of the peak intensity ratios of I1983/I2215 and DNase I concentrations; (C) SERS spectra of the DNA-alkynes functionalized alloyed Au/AgNPs nanosensor before (a) and after (b) treatment with S1 nuclease.



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Figure 5 SERS imaging of the nanoprobe-loaded HeLa cells before (A) and after (B) treatment with PEITC. SERS spectra and ratiometric peak I1983/I2215 obtained from points (a-c, d-f) denoted in the Raman ratiometric images in the same row. (I1983/I2215: Raman ratiometric images displayed in pseudocolor) Scale bar: 10 μm.


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