Carbon Nanotube for Alkyne-Meditated

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Nanoconjugates of Ag/Au/Carbon Nanotube for Alkyne-Meditated Ratiometric SERS Imaging of Hypoxia in Hepatic Ischemia Xiaojie Qin, Yanmei Si, Dawei Wang, Zhaoyang Wu, Jishan Li, and Yadong Yin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05487 • Publication Date (Web): 04 Mar 2019 Downloaded from http://pubs.acs.org on March 7, 2019

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Analytical Chemistry

Nanoconjugates of Ag/Au/Carbon Nanotube for Alkyne-Meditated Ratiometric SERS Imaging of Hypoxia in Hepatic Ischemia Xiaojie Qin,† Yanmei Si,† Dawei Wang,‡ Zhaoyang Wu, *,† Jishan Li,*,† and Yadong Yin‡ †State

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

Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China ‡Department

of Chemistry, University of California-Riverside, California 92521,

United States

*To whom correspondence should be addressed: E-mail: [email protected], [email protected] Fax: +86-731-88821848

1

ACS Paragon Plus Environment

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

ABSTRACT We report a ratiometric surface-enhanced Raman scattering (SERS) nanoprobe for imaging hypoxic living cells or tissues, using azo-alkynes assembled on a single-walled carbon nanotube (SWCNT) surface-functionalized with Ag/Au alloy nanoparticles (SWCNT/Ag/AuNPs). Under a hypoxic condition, azobenzene derivatives pre-assembled on the surface of the nanostructures are reduced stepwise by various reductases and eventually removed from the surface of the SWCNT/Ag/AuNPs, resulting in the loss of characteristic alkyne Raman bands at 2,207 cm-1. Using 2D-band of SWCNTs at 2,578 cm-1 as the internal standard, we are able to determine the hypoxia level based on the ratio of two peak intensities (I2578/I2207) as demonstrated by the successful detection in different cell lines and rat liver tissue samples derived from hepatic ischemia surgery. By combining the outstanding anti-interference property of alkynes as SERS reporters and the distinct Raman responses of alkynes and SWCNTs in complex systems, this novel ratiometric SERS strategy holds a promise in becoming a very useful tool for in vitro and in vivo monitoring of hypoxia in research and clinical settings. KEYWORDS: Ag/Au alloy nanoparticle, single-walled carbon nanotube, azo-alkyne, surface-enhanced Raman scattering, hypoxia imaging

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Hypoxia, which is caused by inadequate oxygenation, is a feature of a variety of diseases, including cancers, ischemia and cardiovascular diseases.1-3 To date, detection of hypoxic regions have been achieved by utilizing immunostaining,4,5 positron emission tomography (PET) imaging,6,7 magnetic resonance imaging (MRI),8,9 and phosphorescence10,11 and fluorescence imaging.12−15 However, immunostaining and phosphorescence imaging have the drawbacks of being time-consuming and severe experimental conditions, while the PET and MRI face the challenges of radiation risk and/or poor diagnosis. Fluorescence imaging, while being highly sensitive and easy to use, can be influenced by environmental conditions, such as pH, polarity, and hydrophobicity. In addition, autofluorescence backgrounds of biological samples, photobleaching, and poor cell membrane-penetration remain challenges. Therefore, a new technology in hypoxic detection is still much needed. Surface-enhanced Raman scattering (SERS) is desirable in bioimaging in several ways, including low autofluorescence in the red to near-infrared range, resistance to photobleaching, lack of phototoxicity, and narrow emission peaks, which is useful for spectral multiplexing.16-20 Such properties might solve the aforementioned difficulties in fluorescence imaging and help SERS become a new choice to develop more reliable and versatile tools for hypoxia imaging. However, application of SERS techniques in in vitro/vivo imaging analysis still faces two significant challenges: 1) limitation of SERS substrates, such as lower plasmonic activity, poor chemical and structural stability; 2) biological background resulting from the overlap of Raman bands between reporter species and the intracellular 3



components. Thus, in this study, we first synthesized an Ag/Au alloy nanoparticles-coated single-walled carbon nanotube (SWCNT/Ag/AuNPs) as the SERS substrate, and then used for the construction of the ratiometric SERS nanosensor by combined usage of alkyne derivative which shows strong Raman signals in a cellular silent region (1,800−2,800 cm−1). The SWCNT provides a distinctive Raman scattering peak, and the Ag/Au alloy nanoparticles serve as a plasmonic-enhanced coating. The properties of SWCNTs include sharp scattering peaks, resistance to quenching and photobleaching, and non-overlap of their 2D-band (~ 2,578 cm-1) with the Raman scattering of the cellular molecules.21-23 The Ag/Au alloy nanoparticles produces SERS enhancements as high as that of pure Ag nanoparticles, but are significantly more stable chemically, and are of lower cytotoxicity, similar to Au nanoparticles.24-26 The other component, alkynes, has a Raman scattering peak in a cellular silent region, lacking background scattering.27-30 The hypoxia SERS sensor was prepared by assembling azo-alkynes (MPP, 4-((4-mercaptophenyl)diazenyl) phenyl 3-((4-(phenyethynyl) benzyl) thio) propanoate) on the SWCNT/Ag/AuNPs nanoconjugate's surface, and used for imaging in hypoxic living cells or tissues (Scheme 1). Initially, the two Raman scattering signal of MPP (2,207 cm-1) and SWCNTs (2,578 cm-1) will be shown and can be distinguished in the Raman spectra. Under hypoxic condition, azobenzene derivatives are stepwise reduced by various reductases, and the final reductive cleavage of the azo bond can remove the alkyne-groups from the surface of the 4


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SWCNT/Ag/AuNPs.15,31,32 As a result, the strong Raman signals at 2,207 cm-1, attributed to the alkyne-group of MPP molecules, decreases or disappears, whereas the band at 2,578 cm-1, attributed to the 2D-band of SWCNTs (internal standard), is not changed, thus allowing the detection of different levels of hypoxia based on the ratiometric peak intensity (I2578/I2207). EXPERIMENTAL SECTION Materials and Instruments. Single-walled carbon nanotubes (SWCNTs) were obtained from XF NANO, Inc. (Nanjing, China). Silver nitrate (AgNO3), sodium borohydride (NaBH4), hydrogen tetrachloroaurate (III) trihydrate (HAuCl4·3H2O), L-ascorbic acid (AA), poly(N-viny-2-pyrrolidone) (PVP, MW 10 000), sodium hydroxide (NaOH), sodium sulfite (Na2SO3), 4-iodobenzyl alcohol, phenylacetylene, liver microsomes and NADPH were purchased from Sigma-Aldrich Co. Ltd (Saint Louis, MO, USA). The human HIF1A ELISA KIT and rat Hif1a ELISA KIT were purchased from Shanghai MLBIO Biotechnology CO. Ltd (Shanghai, China). DNA oligonucleotides used in this study were obtained from Sangon Biotechnology (Shanghai, China), the sequence of the R-DNA is: 5’-ATC GTT ATC AGA CTG ATG GTA T-3’, that of the C-DNA is: 5’-CCC CCC CCC CCC CCC CCC CCC C-3’.

Synthesis

and

characterization

(Figures

S1-S14)

of

4-((4-mercaptophenyl)diazenyl)phenyl 3-((4-(phenyethynyl)benzyl)thio) propanoate (MPP) are shown in the Supporting Information (SI). Rat models, hepatic cancer cell line (HepG2), and human cervical carcinoma cancer cell lines (HeLa) were 5



provided by the Hunan Provincial Tumor Hospital (China). The study was approved by the Ethics Committee of Hunan Provincial Tumor Hospital. Hitachi U-4100 UV/Vis spectrophotometer (Hitachi, LTD., Tokyo, Japan). High-resolution transmission electron microscope (HRTEM, JEOL-EM-2100F; JEOL LTD., Tokyo, Japan). SynergyTM 2 Multi-Mode Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA). Raman microscope (In Via; Renishaw plc., Wotton-under-Edge, Gloucestershire, UK). Preparation of the SWCNT/Ag/AuNPs/MPP Nanoconjugate Probe. Synthesis of the SWCNT/AgNPs nanoconjugate and

the SWCNT/Ag/AuNPs nanoconjugate is

shown in the SI. To obtain the SWCNT/Ag/AuNPs/MPP nanoconjugate probe, MPP was added to the as prepared SWCNT/Ag/AuNPs nanoconjugate solution (with a final MPP concentration of 50 µM) and the reaction solution was incubated for 2 h. The mixture solution was then centrifuged to remove the free MPP, and ultimately dispersed in HEPES buffer. The SWCNT/Ag/AuNPs/MPP nanoconjugate probe was eventually obtained for use in the subsequent experiments. Raman Scattering Detection in Solution. For the Raman scattering spectrum analysis, a certain amount of the prepared SWCNTs-based nanoconjugate was dispersed in HEPES buffer solution (pH=7.4) containing various analytes and incubated for a certain time at 37 oC. For the determination of the stability of the SWCNT/Ag/AuNPs/MPP nanoconjugate probe, this nanoconjugate (0.0025 mg·mL-1) was dispersed in HEPES buffer at different pH value or different biologically relevant 6


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cellular component and then incubated for 6 h at 37 oC. To investigate whether the SWCNT/Ag/AuNPs/MPP nanoconjugate probe can be used as a ratiometric SERS nanosensor

for

the

detection

of

different

O2

levels

in

vitro,

the

SWCNT/Ag/AuNPs/MPP nanoconjugate probe (0.0025 mg·mL-1) was dispersed in a HEPES buffer (pH=7.4) solution containing NADPH (50 µM) and rat liver microsomes (75 µg·mL-1). Then, the mixture was incubated at different O2 concentrations (i.e., 21, 15, 10, 5 and 1%) for 6 h at 37 oC. Eventually, a certain volume of the prepared samples was transferred to a capillary tube to conduct SERS test. Raman Scattering Imaging in Living Cells With the SWCNT/Ag/AuNPs/MPP Nanoconjugate Probe. Cell culture is shown in the SI. To investigate the cell membrane penetrability of the nanoconjugates, HepG2 cells were co-incubated with the SWCNT/Ag/AuNPs/MPP nanoconjugate probe (0.0025 mg·mL-1) for 0.5, 1, 2, 3 and 4 h, respectively, and then the Raman scattering imaging was performed after three washes with PBS. For the hypoxia imaging assay, HepG2 or HeLa cells were co-incubated with SWCNT/Ag/AuNPs/MPP nanoconjugate probe (0.0025 mg·mL-1) for 2 h and washed several times with PBS (pH=7.4) to remove free unbound samples. Subsequently, 2 mL of DMEM was added into the cell culture dishes and the cells were further incubated under different O2 concentrations (i.e., 21, 15, 10, 5 and 1%) for another 6 h at 37 oC. The culture medium was removed and using PBS to wash the dishes three times. Cells were imaged using a Raman microscope with a 785-nm laser (50x long objective lens, 2 s for each acquisition). 7



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Raman Scattering Imaging in Tissue Samples With the SWCNT/Ag/AuNPs/MPP Nanoconjugate Probe. Preparation of the tissue samples is shown in the SI. Liver tissue slices (1-2 mm) were immersed in a HEPES buffer solution containing the SWCNT/Ag/AuNPs/MPP nanoconjugate probe (0.0025 mg·mL-1) and 10% goat serum at 37 oC for 3 h, and ultimately washed three times with PBS before Raman imaging. RESULTS AND DISCUSSION Preparation

and

Characterization

of

the

SWCNT/Ag/AuNPs/MPP

Nanoconjugate Probes. SWCNT-metal nanoconjugates were first synthesized using in situ DNA-templated silver nanoparticles synthesis and gold layer growth methods.33-36 The data presented in Figure S15 (SI) reveals that the Raman scattering signal of the C-rich DNA (C-DNA)-templated silver nanoparticles-coated SWCNTs is stronger than that of random DNA (R-DNA)-templated silver nanoparticles-coated SWCNTs. Moreover, the Raman scattering signal intensity is increased with the increase of the Ag+ concentration (up to C-DNA:Ag+ = 1:300, mol:mol), suggesting that the C-DNA sequence and its 1:300 molar ratio to Ag+ are the better choice for the synthesis of silver nanoparticles-coated SWCNT (SWCNT/AgNPs).33,37-39 After deposition of silver nanocrystals on the SWCNT's surface, gold layer growth was carried out to enhance the chemical stability and biocompatibility of the Ag nanostructure. The results shown in Figure S16 (SI) clearly indicate 8


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that the obtained SWCNT/Ag/AuNPs nanoconjugates underwent a change in color, and the Raman scattering signal intensity of the SWCNTs gradually decreased with the increased concentration of Au growth solution, which likely resulted from the deposition of the gold layer with the relatively weak plasmonic enhanced capacity on the surface of the AgNPs. Also, upon the etching treatment with H2O2, no apparent changes of the Raman scattering signal intensity can be observed when the molar ratio of Au to Ag is more than 3:1, indicating the formation of a perfect Au-coating layer. Accordingly, balancing the stability, biocompatibility, and the Raman enhancement of the SWCNT/Ag/AuNPs nanoconjugates, we chose the molar ratio of 1:300 for C-DNA/Ag+

and

3:1

for

Au/Ag

for

subsequent

experiments.

The

SWCNT/Ag/AuNPs nanoconjugates at the optimized conditions was also characterized by TEM, HAADF-STEM with EDS, UV/vis spectroscopy. The UV/vis spectra (Figure 1A) of SWCNT/C-DNA, SWCNT/AgNPs and SWCNT/Ag/AuNPs

displayed

an

absorption

at

around

410

nm

for

SWCNT/AgNPs and a continuous, broad absorption band (~600 nm) for SWCNT/Ag/AuNPs, both of which were greatly elevated compared to C-DNA-functionalized SWCNTs, indicating the successful formation of the target nanosensor. The TEM image (Figure 1B, Figure S17A in SI) of the synthesized SWCNT/Ag/AuNPs nanoconjugate revealed that the Ag/Au alloy nanoparticles were densely decorated on the surface of the SWCNTs after the DNA-templated silver nanocrystal growth and gold layer coating. In addition, 9



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the elemental composition determined by EDS analysis (Figure S17B in SI) and elemental mapping (Figure 1B) further evidenced the composition of the SWCNT-metal composites. Then, the hypoxic reporter element of MPP was immobilized on the surface of the SWCNT/Ag/AuNPs nanoconjugate via the strong S-Au bonding to form the ratiometric SERS nanoprobes of alkyne-functionalized SWCNT/Ag/AuNPs (SWCNT/Ag/AuNPs/MPP). The data presented in Figure 1C reveals a new strong Raman signals at 2,207 cm-1, assigned to the alkyne-group of MPP, upon addition of MPP molecules, indicating the immobilization of MPP molecules on the SWCNT/Ag/AuNPs surface

and

the

successful

formation

of

the

ratiometric

SERS

SWCNT/Ag/AuNPs/MPP nanoprobe. SERS Analysis of the SWCNT/Ag/AuNPs/MPP Nanoconjugates. We also studied the effect of different laser excitations on the Raman scattering enhancement capacity. The Raman spectra of the SWCNT/Ag/AuNPs/MPP nanoconjugates were recorded at three different excitation wavelengths (532, 633 and 785 nm). The data shown in Figure S18 (SI) reveals that, at a 785-nm excitation, the two peaks at 2,207 cm−1 and 2,578 cm−1 are significantly stronger than those obtained at either a 633-nm or 532-nm excitation, indicating that the SWCNT/Ag/AuNPs nanoconjugates exhibit stronger near infrared (NIR) plasmonic absorption and higher Raman scattering enhancement capacity at a 785-nm excitation. Additionally, at an excitation wavelength of 785-nm, both the excitation and scattering photons are in the NIR window (700−900 nm), making it ideal for optical imaging in biological samples.40,41 10


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We thus chose a 785-nm laser excitation for our subsequent biomedical studies. For the optical imaging assay, the laser power and spectral acquisition time also have very significant effects on the biological system itself and the spectroscopic imaging efficiency. Thus, we also carefully evaluated the SERS performance of the SWCNT/Ag/AuNPs nanoconjugate at various laser power (785 nm) and spectral acquisition time. From the results presented in Figure S19 (SI) clearly show that the Raman signals at 2,578 cm−1, corresponding to SWCNTs, cannot be observed even at the 100% laser power (300 mW) and a relative long spectral acquisition time of 8 s. In contrast, significant Raman scattering signals at both 2,207 cm−1 and 2,578 cm−1, corresponding to the SWCNT/Ag/AuNPs/MPP nanoconjugate, can be observed at only 1% laser power and a relative short spectral acquisition time of 0.1 s. These findings further indicate that the SWCNT/Ag/AuNPs/MPP nanoprobe has excellent SERS performance and is very suitable for optical imaging in biological systems. Stability

of

the

SWCNT/Ag/AuNPs/MPP

Sensing

Nanoconjugates

and

Ratiometric Measurement of Hypoxia In Vitro. To examine whether the SWCNT/Ag/AuNPs/MPP nanoconjugate can be used as a ratiometric SERS nanosensor for hypoxia detection in biological systems, we first performed an in vitro assay with rat liver microsomes (containing various reductases). As shown in Figure 2A, a significant decrease in the Raman signal at 2,207 cm−1, attributed to the alkyne-group of MPP, can be observed only under hypoxia. Moreover, the decrease of the O2 level dramatically increases the Raman scattering signal intensity ratio of I2578/I2207, and there is a positive correlation between I2578/I2207 and the decrease of the 11



O2 level, suggesting that the SWCNT/Ag/AuNPs/MPP nanoprobe was efficient at the specific signaling under hypoxic environment. Additionally, we also carefully investigated the influence of the pH value (Figure S20 in SI) and various intracellular components (Figure 2B), including metal ions (Na+, K+, Ca2+, Mg2+), glucose, ATP， amino acids (Cys, GSH), hydrogen sulfide, proteins (Cytochrome c, Caspase-3) and reactive oxygen species (H2O2, ClO-), on the stability of the SWCNT/Ag/AuNPs/MPP nanoprobe. The results presented in Figure S20 and Figure 2B reveals no obvious change in the Raman ratiometric signal of I2578/I2207 of the nanoprobes at various pH values or in the presence of these cellular components at their physiological concentrations, compared to the control. These findings indicate that the pH value and these cellular components at their physiological concentration exhibit negligible effects and further suggest that the SWCNT/Ag/AuNPs/MPP nanoprobe has high specificity for hypoxic measurement. Additionally, these results further confirm that the ratiometric nanosensor is practical and useful for hypoxia imaging in biological systems. Cell Uptake and Cytotoxicity of the SWCNT/Ag/AuNPs/MPP Nanoconjugate Probes. An intracellular nanoprobe should be able to enter cells efficiently and with high biocompatibility. Accordingly, we studied the cell uptake and toxicity of the SWCNT/Ag/AuNPs/MPP nanoconjugates before it was used in further biological experiments. The images displayed in Figure S21 (SI) show two intense Raman scattering signals detected at the 2,180-2,230 cm-1 channel and 2,520-2,640 cm-1 channel when the HepG2 (a hepatic cancer cell line used as a model here) cells were 12


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incubated with the sensing nanoconjugates for 2 h. MTT assay reveals no obvious cytotoxic effect of the SWCNT/Ag/AuNPs/MPP nanoconjugates on the HepG2 cells at the tested concentrations, compared with the SWCNT/AgNPs/MPP nanoconjugates (see SI; Figure S22). These results clearly indicate low cytotoxicity and biocompatibility of the SWCNT/Ag/AuNPs/MPP nanoconjugates, making them highly suitable for cell imaging applications. In Vitro Raman Imaging of the Cell Response to Hypoxia With the SWCNT/Ag/AuNPs/MPP Nanoconjugate Probe. After interrogating the various characteristics of the SWCNT/Ag/AuNPs/MPP sensing nanoconjugates, we next applied this nanosensor to live cells and investigated whether they can detect hypoxia. The SWCNT/Ag/AuNPs/MPP probes were incubated with HepG2 cells under normoxic conditions for 2 h. Free nanoconjugates were then removed and fresh cell-culture media was added. Next, the cells were incubated under decreased O2 concentrations (e.g., 21, 15, 10, 5 and 1%) for 6 h. This treatment resulted in a significantly increased expression of HIF1A (Figure S23 in SI), indicating the hypoxia in the cells.42-44 Cells were examined by confocal laser Raman microscopy (CLRM) imaging (Figure 3). The Raman signals in both the 2,520-2,640 cm-1 channel and the 2,180-2,230 cm-1 channel were strong when cells were incubated at 21% O2, indicating the normal oxygen levels. In contrast, in cells incubated at lower O2 concentrations (e.g. 15, 10, 5 and 1%), the signal intensity in the 2,180-2,230 cm-1 channel gradually decreased as a function of decreasing O2 concentration, but the signal in the 2,520-2,640 cm-1 channel remained unchanged, indicating development 13



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of hypoxia. Another cell line (HeLa) were also studied, and similar data were obtained (Figure S24 in SI). Therefore, the nanosensor is capable of visualization of the hypoxic status in a number of different cell lines using the ratiometric methodology. Raman

Imaging

Assay

of

Hypoxia

in

Hepatic

Ischemia

With

the

SWCNT/Ag/AuNPs/MPP Nanoconjugate Probe. The developed ratiometric SERS nanosensor was further employed to visualize the variations of hypoxic conditions in liver slices obtained from the normal rats (N), the rats with hepatic ischemia (HI) via HI surgery (HIS), and the octreotide (Oct)-pretreated hepatic ischemia rat group (Oct-HI) whose rats underwent HIS. HI is a temporary inflow and/or outflow blockage via vascular clamping to decrease intraoperative blood loss, which can cause apoptosis of liver cells and lead to injury of the liver.45 Oct is a synthetic somatostatin octapeptide whose primary role is to inhibit the secretion of growth

hormone,

but

has

been

found

to

significantly

reduce

hepatic

ischemia-reperfusion injury (HIRI)46-49 through a mechanism that remains unclear. Accordingly, herein, through the measurement of liver hypoxia by using the prepared ratiometric SERS nanosensor, we examine whether hepatic ischemia can generate a hypoxic environment. Additionally, we also investigate whether the anti-HIRI effect of Oct involves maintaining the liver cells under normoxic conditions to reduce injury to the liver. The results shown in Figure 4 reveal that, compared with the liver tissue from N group rats, the Raman signal in the 2,185-2,235 cm-1 channel significantly decreased and the peak intensity ratio of I2625/I2211 dramatically increased in the liver 14


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tissue from rats of the HI group, likely resulting from the development of a highly hypoxic environment due to hepatic ischemia. On the other hand, no apparent change of the signal in the 2,185-2,235 cm-1 channel and the peak intensity ratio of I2625/I2211 was observed in the liver tissue sample from the Oct-HI rat group. These findings suggest that the O2 level can be maintained at a relatively normal level when rats are pretreated with Oct before HIS, which keeps the liver cells with normal oxygen levels thus reduces the injury to the liver. Measurement of the Hif1a expression level, by ELISA analysis in homogenized liver tissue samples of the three evaluated rat groups, indicates that the hypoxic conditions have a similar trend (Figure S25 in SI). This finding suggest that Oct plays a role in the maintenance of the normoxic condition in the liver cells. In addition, the Z-scanning confocal Raman imaging results reveal that the hypoxic condition can still be detected up to a penetration depth of 600 μm (Figure S26 in SI). Additionally, a similar intensity ratio of I2625/I2211 can be observed at different sites in the normoxic and hypoxic liver tissue samples. Overall, the data presented here show that the ratiometric nanosensor is a high-contrast Raman imaging agent for hypoxia, capable of working in living cells and in tissue samples.

CONCLUSIONS In summary, we report a ratiometric SERS probe based on Ag/Au alloy nanoparticle-decorated SWCNTs and successfully demonstrated their application in the detection of hypoxia both in cells and animal tissues with high sensitivity and reproducibility. The Ag/Au alloy nanoparticle-decorated SWCNTs nanoconjugates 15



exhibited enhanced SERS performance, elevated chemical stability, and good biocompatibility. The ratiometric sensing method based on the highly distinguishable SERS signals of alkynes and SWCNTs promoted the reliability of the hypoxia detection in complex systems. More importantly, using this novel SERS nanoprobe, we found that hepatic ischemia can lead to the development of the liver hypoxic condition, and the anti-HIRI effect of Oct is partially mediated through maintaining the liver cells under normoxic condition. Considering the advantageous properties of the SERS sensing, this novel strategy combining the SWCNT/Ag/AuNPs with the cell-silent Raman reporters has the potential of becoming a useful approach for in vitro and in vivo monitoring of hypoxia in medical research and clinical diagnosis.

ASSOCIATED CONTENT Supporting Information. Experimental details for synthesis and characterization of MPP, cell culture, preparation of the tissue samples, MTT assay, ELISA analysis of HIF1A in cell lysates, ELISA analysis of Hif1a in homogenized rat liver tissues, and other additional information as noted in the text, including Figures S1-S26. This material is available free of charge via the internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author

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*[email protected] *[email protected] ORCID Jishan Li: 0000-0001-8144-361X Yadong Yin: 0000-0003-0218-3042 Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (21775035, 21475036, 21675045), and Hunan Provincial Natural Science Foundation (2016JJ1005).

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Page 24 of 31

Figure Captions Scheme

1.

Schematic

illustration

of

(A)

the

preparation

of

the

SWCNT/Ag/AuNPs/MPP conjugate-based SERS nanoprobe and (B) the sensing mechanism of hypoxia.

Figure 1. (A) UV-Vis absorption spectra of the SWCNT/C-DNA, SWCNT/AgNPs, and

SWCNT/Ag/AuNPs

nanoconjugate.

(B)

HADDF-STEM

imaging

and

EDS-mapping of the Ag and Au of the SWCNT/Ag/AuNPs nanoconjugate. (C) Raman scattering spectra of the SWCNT/Ag/AuNPs and SWCNT/Ag/AuNPs/MPP nanoconjugate in aqueous solution.

Figure 2. (A) Plots of the Raman scattering peak intensities ratio (I2578/I2207) versus O2 concentration. Insert: Raman scattering spectra of the SWCNT/Ag/AuNPs/MPP nanoprobe at different oxygen levels. (B) Effect of the biologically relevant cellular component (a to o: normoxia, hypoxia, 40 mM of Na+, 140 mM of K+, 2 mM of Ca2+, 2 mM of Mg2+, 1 mg·mL-1 of glucose, 1 mg·mL-1 of ATP, 10 mM of Cys, 10 mM of GSH, 100 μM of hydrogen sulfide, 100 μM of Cytochrome C, 350 ng·mL-1 of Caspase-3, 100 μM of H2O2, and 100 μM of ClO-, respectively) on the stability of the 24


Page 25 of 31 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


SWCNT/Ag/AuNPs/MPP nanoprobe. Each data point represents the average value calculated from three replicate Raman scattering spectra. The error bars indicate the standard deviation of the three repeated measurements.

Figure

3.

Confocal

Raman

images

of

HepG2

cells

treated

with

the

SWCNT/Ag/AuNPs/MPP nanoprobe (0.0025 mg·mL-1) and incubated at different oxygen levels. The signals were recorded in two channels (Ch1= 2,180-2,230 cm-1 and Ch2 = 2,520-2,640 cm-1) with a 785-nm laser, 30 mW of laser power, and 2 s of spectra acquisition time. Ch1/Ch2: Raman scattering ratiometric images displayed in pseudocolor. Raman spectra and the relative peak intensity ratio (I2578/I2207) were obtained from the linear regions across the HepG2 cells shown in "Ch1/Ch2". Scale bar: 5 μm.

Figure 4. Confocal Raman images of the rat liver tissue slice samples (obtained from N, Oct-HI and HI rat groups) treated with the SWCNT/Ag/AuNPs/MPP nanoprobe (0.0025 mg·mL-1). The signals were recorded in two channels (Ch1 = 2,185-2,235 cm-1 and Ch2 = 2,550-2,690 cm-1) with a 785-nm laser, 30 mW of laser power, and 1 s of spectra acquisition time. Ch1/Ch2: Raman scattering ratiometric images displayed in pseudocolor. Raman spectra and the relative peak intensity ratio (I2625/I2211) were obtained from the linear regions across the liver tissue slice samples

25



shown in "Ch1/Ch2". BF: Bright field images of the rat liver tissue slice samples. Scale bar: 5 μm.

Scheme 1 26


Page 26 of 31

0.9 0.6 0.3 0.0 300

B

SWCNT/C-DNA SWCNT/AgNPs SWCNT/Ag/AuNPs

BF

450 600 750 Wavelength (nm) DF

C

6

SWCNT/Ag/AuNPs SWCNT/Ag/AuNPs/MPP

3

A

Absorbance

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


Counts/x10

Page 27 of 31

4 2 0 2000

Ag

2200 2400 2600 -1 Raman Shift (cm ) Au

Figure 1

27


2800


6

Counts/x10

6 4

B

in buffer 21% 15% 10% 5% 2.5% 1%

3

8

4 2 0 2000

2200

2400

2600

I2578/I2207

A

I2578/I2207

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

2800

-1

Raman Shift (cm )

2 0

0

8 6 4 2 0

5 10 15 20 Oxygen level (%)

a b c d e f g h i j k l mn o Analytes

Figure 2

28


Page 28 of 31

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Figure 3

29


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 30


Page 30 of 31

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Figure 4

TOC

31


Carbon Nanotube for Alkyne-Meditated

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