Constructing 2D Nanosheet-assembled MnCo2O4 Nanotubes for

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Constructing 2D Nanosheet-assembled MnCo2O4 Nanotubes for Pressure and Colorimetric Dual-signal Readout Detection of Cancer Cells in Serum Samples Erli Ding, Jun Hai, Fengjuan Chen, and Baodui Wang ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00895 • Publication Date (Web): 31 Jul 2018 Downloaded from http://pubs.acs.org on August 1, 2018

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ACS Applied Nano Materials

Constructing 2D Nanosheet-assembled MnCo2O4 Nanotubes for Pressure and Colorimetric Dual-signal Readout Detection of Cancer Cells in Serum Samples Erli Ding, Jun Hai, Fengjuan Chen*, Baodui Wang,*

State Key Laboratory of Applied Organic Chemistry and Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, Lanzhou University, Gansu, Lanzhou, 730000, China.

ABSTRACT Developing a facile and reliable strategy for detecting cancer cells in early stages in aqueous systems remains a challenge, although strategy is crucial in biomedicine. Here, a green approach is proposed for synthesis of MnCo2O4 nanotubes. The resulting nanotubes have been shown to have peroxidase activity that catalyzes the oxidation of 3, 3’, 5, 5’- tertamethylbenzidine (TMB) by O2, resulting in a typical color reaction from colorless to blue. Moreover, such nanotubes exhibit excellent catalytic properties for the decomposition of H2O2 into O2, resulting in a significant increase in pressure in the bottle. Thus, a new sensor system using MnCo2O4 nanotubes as an artificial peroxidase, generating O2 as pressure signal, and TMB as a color change reporter molecule for dual-mode pressure-based (pressuremeter) and colorimetric (naked eye) detection of cancer cells was established. By using folic acid

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(FA) as a recognition element, a total of 50 folate receptor (FR) positive cancerous cells can be distinguished by naked-eye observation and pressure meter. The clinical applicability of such dual-mode strategy has been tested in detecting cancer cells in serum samples. We envisaged that such dual-signal readout dual-mode strategy based on MnCo2O4 nanotubes offers a promising biosensing platform for early clinical diagnosis.

KEYWORDS: Dual-Signal; MnCo2O4 nanotubes; cancer cells; pressure-based; colorimetric

1. INTRODUCTION Early diagnosis and treatment of cancer are the keys to improve patient survival rate. In order to select effective and accurate treatment approaches and improve the treatment effect, sensitive and simultaneous diagnosis of cancer based on multimodal strategies is particularly critical in early-stage cancer diagnosis.1-4 Many kinds of technologies,

including

cytometric

methods,

cell-enrichment

methods,

and

polymerase chain reaction (PCR)-based methods, have been applied for detecting cancer cells.5-10 However, the above analysis methods have defects in real sample analysis due to the time-consuming, expensive, and the need for advanced instruments. Therefore, developing a multimode detection strategy with convenience, low-cost, high sensitivity and selectivity is highly needed for point of early care diagnosis.11 In recent years, point-of-care testing (POCT) has been paid more and more attention in healthcare diagnostics, especially in resource-limited settings, as they


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offer the advantages of speed, simplicity, and low cost as well as miniaturization of the devices.11-17 To achieve reliable and facile assay, the signal transduction and signal readout strategies play an important role in the development of POCT. Among the most common techniques, colorimetric methods, especially based on the oxidation of 3, 3’, 5, 5’-tertamethylbenzidine (TMB), have the advantages of fast response, simple, and visual detection. They have been used to discriminate large quantities of chemicals and biological analytes.3,9,18-21 However, when these methods are used to detect the early stage cancer, it is easy to get false-positive/negative results because of the low and limited number of both biomarkers and occult cancer cells. In order to solve these problems, great efforts have been made to the development of multimode detection techniques and thus to provide more comprehensive information relative to a single detection method in early cancer diagnosis and prevention. Currently, pressure-based bioassays, based on measurement of gas pressure, as signaling techniques, have been used as powerful biosensing platforms owing to their cost-effective, portable monitoring, speedy and simple method.12,22,23 In such bioassays, the biomolecular recognition signal of analytes could be readout through the pressuremeter, many reports use such method for detecting biomolecules

22

In

addition, our group reported to use the pressure based detection method for detecting cancer sensitively.23 However there are still some challenges in the quantitative detection of biomolecules based on pressure. We envisaged if multiple techniques based on the combination of colorimetric methods and pressure based strategies are applied for detection of cancer cells, the accuracy of detection will be improved.



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However, such strategies are rarely reported and are therefore urgently needed. Recent studies suggest that the synthesis of multifunctional nanomaterials is crucial to the construction of multi-mode detection platform. Moreover, nanomaterials with high activity and biocompatible can improve the sensitivity of colorimetric and pressure-based detection. Nowadays, two-dimensional (2D) nanomaterials24-26 have attracted great concern in many fields such as catalysis and absorption of pollution due to its large surface area and high active site.27,28 However, the low stability and circulation of 2D materials have always been a problem.29 Recent studies suggested that intergating 2D and one dimensional (1D) materials in one nanostructure can greatly improve the stability of the materials.30-33 On the basis of the above strategy, we prepared the 2D nanosheet-assembled hollow MnCo2O4 nanotubes by the spinneret electrospinning and thermal treatment method. Here, we choose MnCo2O4 nanotubes, because this material has a high catalytic activity, especially in the oxidation reaction.33 We demonstrated that the resulting

hollow

MnCo2O4

nanotubes

exhibit

excellent

catalytic

activity

for decomposition of H2O2, which can result in the measureable pressure change in the reaction bottle. Also, they also exhibit high catalytic activity for oxidation of TMB, which can produce naked eye recognizable color changes. Based on the above features, we designed folic acid (FA)-conjugated MnCo2O4 nanotubes based POC strategy for dual-mode colorimetric (naked eye) and pressure (pressuremeter)-based detection of cancer cells (Scheme 1). Meanwhile, the feasibility of such colorimetric and pressure readout dual-mode strategy based on MnCo2O4 nanotubes for specific


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detecting cancer cells in serum samples was also demonstrated.

Scheme 1 Preparation of Folic Acid (FA)-Conjugated MnCo2O4 Nanotubes and Schematic Diagram of the Construction of the Colorimetric and Pressure-Based Detection Platform.

2. EXPERIMENT SECTIONS 2.1. Synthesis of MnCo2O4 Nanotubes. 0.05 g PAN nanofibers

23

were added into

the 50 mL of ethanol containing 2 mmol of cobalt (II) acetate and 1 mmol of manganese acetate (II). The obtained mixture reacted for 4 h under sonication at 25 0C. The formed composite was thoroughly washed by ethanol for three times and then dispersed into 10 mL of ethanol containing 5 mg of NaBH4 to react for 30 mins. The obtained composite nanofibers were thoroughly washed by water for three times and dried at 40 0C in an oven. Finally, the composite nanofibers were calcinated at 400 0C for 2 h at a heating rate of 1 0C min-1.



2.2. Catalytic Decomposition of Hydrogen Peroxide. The catalytic activity of MnCo2O4 nanotubes was evaluated by decomposition of H2O2 to generate O2, in which the generation O2 can result in the presser change of the sealed glass bottle that can be easily detected using a pressuremeter. In a typical experiment, 5, 10, 15 or 20 µL of H2O2 (30%), and different amounts of MnCo2O4 nanotubes (0, 0.05, 0.1, 0.2, and 0.25 mg) were transferred to a 2 mL bottle. A pressuremeter was inserted into the bottle to test the change of pressure at 25 oC. 2.3. Catalytic Oxygenation of TMB. In a typical experiment, 50 µL of TMB (16 mM) dissolved in ethanol was transferred to 2 mL of HAc-NaAc buffer (pH = 5.0), and different quality of MnCo2O4 nanotubes (0, 1.25, 2.5, 5, 10 and 14 µg) were added. The absorbance at 652 nm was monitored by a 721E visible spectrophotometer per a certain time. 2.4. Colorimetric and Pressure Based Detection of Cancer Cells. NIH3T3 cells, A549 cells, and Hela cells seeded in 6-well plate at density of 1 × 105 cells per well were incubated with different concentration of FA-MnCo2O4 nanotubes (50 to 150 µg/mL) and washed three times. Later, TMB were mixed. The final experiments results were read by enzyme reader. Except for replacing TMB with H2O2, the pressure assay of cancer cells is the same as the above steps. The final experiments results were read by a portable home-made pressuremeter. 2.5. Detection of Cancer Cells in Serum Sample. NIH3T3 cells, A549, and Hela cells (∼6 × 105) were first spiked into 10% bovine serum albumin. Then, different


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concentrations of FA-MnCo2O4 nanotubes was mixed with the above serum samples. After the mixtures were incubated for 1h, the serum samples were washed using PBS buffer and mixed with TMB. The final experiments results were read by enzyme reader. Except for replacing TMB with H2O2, the pressure assay of cancer cells in samples is the same as the above steps. The final experiments results were read by a portable home-made pressuremeter. 3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of the Nanosheet-Based Hollow MnCo2O4 Nanotubes. The formed intermediate products of PAN/Mn-Co acetate hydroxide and ultrathin nanosheet constructed PAN/Mn-cobalt hydroxide nanofibers were confirmed by X-ray diffraction (XRD), shown in Figure S1 and S2, respectively.34 The evolution of morphology of nanostructures in the process formation of nanosheet-based MnCo2O4 nanotubes was tracked by SEM (Figure 1A, 1B, S3 and S4). Compared with PAN nanofibers (Figure 1A), after formation of PAN/Mn-cobalt hydroxide nanofibers, the PAN nanofibers were fully decorated with uniform Mn-Co hydroxide nanosheets and the nanosheets evenly distributed across and connected to each other (Figure S4). Figure 1B showed that the final nanostructures formed nanosheet-based MnCo2O4 nanotubes when the PAN/Mn-cobalt hydroxide nanofibers calcinated at 400 0

C.



Figure 1. SEM images of PAN nanofibers (A) and nanosheet constructed hollow MnCo2O4 nanotubes (B); (C) TEM images of nanosheet constructed hollow MnCo2O4 nanotubes; (D) HRTEM image of nanosheet constructed hollow MnCo2O4 nanotubes; E) The corresponding scanning transmission electron microscopy (STEM) and X-ray energy-dispersive spectroscopy (EDS) elemental mapping images of Mn (F), Co (G), and O (H) in hollow MnCo2O4 nanotubes.

Figure 1C and D show the transmission electron microscopy (TEM) and high resolution transmission electron microscopy (HRTEM) of MnCo2O4 nanotubes. As shown in Figure 1C, the diameter of nanotubes is about 600-800 nm and the diameter of the pore is about 550 nm.The nanosheet assembled nanotubes are porous and composed of 5 nm MnCo2O4 nanoparticles. The HRTEM image (Figure 1D) illustrates that the MnCo2O4 nanoparticles shows an interplanar spacing of 0.24 nm, corresponding to the (311) planes of cubic spinel MnCo2O4.35 EDS mapping of


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MnCo2O4 nanotubes shows that Mn, Co, and O elements coexisted and distributed uniformly in the whole nanotube (Figure 1E-H), which was further confirmed by EDX spectrum (Figure S5). The inductively coupled plasma-atomic emission spectrometry (ICP-AES) further confirmed that the atomic ratio of Co/Mn is 2.20/1.

Figure 2. A) Mn 2p XPS spectrum of the catalyst; B) Co 2p XPS spectrum of the catalyst; C) XRD patterns of MnCo2O4 nanotubes; D) N2 adsorption/desorption isothermals of hollow MnCo2O4 nanotubes.

Typical XPS spectra (Figure S6) revealed that the O, Mn, Co, and adventitious C coexisted in the MnCo2O4 nanotubes. As shown in Figure 2A, the Mn 2p spectrum shows Mn 2p3/2 peak at ∼641.6 eV and the Mn 2p1/2 peak at ∼653.6 eV, corresponding to the Mn2+.36 Also, the Co 2p spectrum indicates Co 2p3/2 peak at ~780 eV and Co 2p1/2 peak at ~795.4 eV (Figure 2B), together with a spin-energy separation of around 15.4 eV, proving the presence of Co3+.37 The XRD patterns of



MnCo2O4 nanotubes was shown in Figure 2C. Tne set of diffraction peaks at 30.8, 36.2, 58.4, 64.2 of nanotubes were ascribed to the planes of MnCo2O4 (220), (311), (511) and (440), respectively. Nitrogen adsorption measurements indicate that the MnCo2O4 nanotubes existed the mesopores structures with a pore size in the ranges of 2-100 nm and the pores volume of 0.47 cm3 g-1 (Figure 2D).

Figure 3. A) The pressure change rusult by oxygen evolution in aqueous solution (1 mL) containing different volume of H2O2 (30%) in the presence of 0.2 mg of MnCo2O4 nanotubes at 298 K; B) The pressure change rusult by oxygen evolution evolution in aqueous solution (1 mL) containing 15 µL of H2O2 (30%) in the presence of different quality of MnCo2O4 nanotubes at 298 K.

3.2. Catalytic Decomposition of Hydrogen Peroxide. The as-synthesis MnCo2O4 nanotubes were first tested for the catalytic decomposition of 15 µL of H2O2 (30%) at room temperature in the bottle (used for gas chromatography analysis). In our experiment, the volume of the bottle is 2 mL, and we kept the liquid volume up to 1 mL by adding H2O. Notably: the gas leakage will not happened when the pressuremeter inserted into reaction bottle, because there is a rubber mat at the top of the bottle. The generation O2 could be easily detected using a pressure meter. As shown in Figure 3A, only in two mins, the pressure in 2 mL bottle can reach up to


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150 kPa. This pressure can be easily detected by a pressure meter. The influence of the amount of catalyst for H2O2 degradation is also studied. Figure 3B shows that with the amount increase of catalyst the change of pressure is much faster. The pressure of 0.2 mg of catalyst is 150 kPa, which is higher than 0.05 mg catalyst (75 kPa). What is more, such catalyst can also catalyze the decomposition of H2O2 to produce significant pressure in the presence of serum as shown in Figure S7. But the change of pressure in serum samples is smaller than that in aqueous solution, which may be result from the bubbles in bottle when serum samples present in reaction system, shown in Figure S8, Figure S9 and S10 show that the MnCo2O4 nanotubes exhibit good stability.

Figure 4. (A) Images and (B) corresponding UV/vis spectra of the oxidation color reaction of TMB by MnCo2O4 in 2 mL acetic acid−acetate buffer (pH=5.0) with or without 10% serum samples at 35 °C for 5 min. (a, 50 µL of 16 mM TMB alone; b, 40 µg MnCo2O4 alone; c, both; d, both in 10% serum samples); (C) Structure illustration and the catalytic oxidation of TMB.



3.3. Catalytic Oxygenation of TMB. The as-synthesis MnCo2O4 nanotubes were used as peroxidase to test the activity of catalytic oxidation of TMB. As shown in Figure 4A, it was found that MnCo2O4 nanotubes can catalyze the oxidation of TMB in the absence of H2O2 in HAc-NaAc buffer (pH=5.0) and quickly produce a typical blue color change. Also, it can be seen a typical blue color change in 10% serum samples, and the MnCo2O4 nanotubes were stable in 10% serum samples (Figure S9 and S10). Meanwhile, two strong absorption peaks occurred at 370 and 652 nm (Figure 4B) with the progress of oxidation reaction, indicating that the catalyst exhibited strong peroxidase like activity in the air. Figure 4C shows the catalytic oxidation of colorless TMB to blue ox-TMB. In order to verify the oxidant comes from oxygen, the reaction was carried out in nitrogen atmosphere. From the Figure S11, compared with the O2, the solution appears lighter blue under the protection of nitrogen.

Figure 5. (A) Michaelis−Menten curve of MnCo2O4 nanotubes with TMB. (B) Double-reciprocal plots for MnCo2O4 nanotubes with the varied concentration of TMB.

3.4. Specificity and Sensitivity of the Dual-Mode Assay. The as-synthesis MnCo2O4 nanotubes exhibit high peroxidase-like activity over a wider range of pH of


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2.6-6 (Figure S12A and S13). Moreover, MnCo2O4 nanotubes show excellent peroxidase-like activity over a range of temperature between 20 and 45 oC (Figure S12B). Compared to the most reported catalysts,38-42 the reaction conditions of catalytic oxidation of TMB by MnCo2O4 catalyst in this work were not limited, which is essential for practical applications.43 In this work, temperature and pH value of MnCo2O4 catalyst that we choose are 35 oC and 5.0, respectively. Figure S14 shows that the absorbance at 652 nm increases with the increasing of the amount of catalyst. What’s more, there was a good linear relationship between the concentration of catalyst and the absorbance at 652 nm. In addition, the oxidation reaction catalyzed by MnCo2O4 nanotubes also follows the typical Michaelis-Menten behavior toward TMB (Figure 5A). According to Line Weaver-Burk plot (Figure 5B), the calculated values of Km and Vmax for the MnCo2O4 nanotubes with TMB were 0.063 mM and 2.17 × 10−5 M s−1, respectively, which is better than the reported catalysts.43,44

Figure 6. A) Pressure-change profiles of cells after incubation with FA-MnCo2O4 nanotubes for 1 h and then addition of H2O2. B) Quantitative detection of HeLa cells using FA-MnCo2O4 nanotubes and H2O2 based on pressure-change. Values are expressed as mean ±SD.

Since the MnCo2O4 nanotubes exhibit amazing peroxidase activity toward the decomposition of H2O2 and high catalytic activity for the oxidation of TMB, they



were used to design as dual mode sensing platform for colorimetric and pressure-based detection of cancer cells. Scheme 1 shows a folic acid (FA)-functionalized MnCo2O4 nanotubes-based nanoplatform for dual mode detection of cancer cells. FA binding on nanotubes was further characterized by FT-IR and UV absorption spectra. The resultant FA-MnCo2O4 nanotubes were characterized by UV-vis and FT-IR spectra (Figure S15 and S16). Cytotoxic experiments suggested that the FA-MnCo2O4 nanotubes have low cytotoxicity (Figure S17). In order to verify the application performance of the FA-MnCo2O4 nanotubes based pressure detection platform, different amounts of FA-MnCo2O4 nanotubes incubated with HeLa cells with high-expressed folate receptor(FA),45 A549 cells with low-expressed folate receptor (FR)45 and NIH3T3 cells without FR46 for 1h, respectively, followed by the addition of a certain volume of hydrogen peroxide (30%). Here, polymerase chain reaction (PCR) analysis and agarose gel electrophoresis was used to confirm the expression amount of FR in cancer cells.45 The pressuremeter is inserted into the bottle to detect the change of the pressure produced by the gas during the reaction. As shown in Figure 6A, three kinds of cells with different FR expression levels can easily be distinguished by pressuremeter. Next, we also tested the sensitivity of this method for the cancer cells detection. Varying HeLa cell numbers ranging from 0~104 cell mL-1 were incubated with a certain concentration (150 ug/mL) of FA-MnCo2O4 nanotubes and H2O2 (30%). As shown in Figure 6B, a linear relationship between the pressure and cell numbers was observed. Based on this linear relationship, we directly read the LOD, which reach to 50 cells


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mL-1. The results indicated that the FA-MnCo2O4 nanotubes based pressure strategy could sensitively detect specific cancer cells. Moreover, the confocal images show that FA-MnCo2O4 nanotubes only adhere to the surface of Hela cells, while FAMnCo2O4 nanotubes are not found on the surface of NIH3T3 cells (Figure S18)

Figure 7. A) Absorbance-change in 652 nm profiles of cells after incubation with FA-MnCo2O4 nanotubes for 1 h and then additon of 20 µL of 16 mM TMB in 200µL HAc-NaAc (pH=5.0); Photographs of (B1) HeLa cells incubated with TMB and HAc-NaAc in absence of FA-MnCo2O4 nanotubes, (B2) HeLa cells, (B3) A549 cells and (B4) NIH3T3 cells incubated with 100 µg/mL FA-MnCo2O4 nanotubes for 1 h and then additon of 20 µL of 16 mM TMB in 200µL HAc-NaAc (pH=5.0).



Figure 8. Quantitative detection of HeLa cells using FA-MnCo2O4 nanotubes and TMB based on a UV spectrophotometer. Values are expressed as mean ±SD.

In order to further verify the accuracy of pressure-based detection, colorimetric bioassay was also used. NIH3T3 cells, A549 cells, and HeLa cells were incubated with different amounts of FA-MnCo2O4 nanotubes for 1h, followed by the addition of 20 µL of 16 mM TMB. Figure 7A shows the absorption intensities of HeLa cells and A549 cells at 652 nm are much higher than NIH3T3 cells. The color changes produced by different cells could easily be recognized by naked eyes (Figure 7B1-B4 and Figure S19), in which the solution color of HeLa cells and A549 cells is blue, while the solution color of NIH3T3 cells is almost colorless. These results suggested that colorimetric bioassay is easy to distinguish between folate receptor overexpressed cancer cells and normal cells. Next, we also tested the sensitivity of this method for the cancer cells detection. Varying HeLa cell numbers ranging from 0~103 cell mL-1 were incubated with a certain concentration (150 ug/mL) of FA-MnCo2O4 nanotubes


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and TMB. As shown in Figure 8, the absorption intensities at 652 nm is increased with the increase of the number of HeLa cells. Based on the linear relationship between the absorbance and cell numbers, we directly read the LOD, which also reach to 50 cells mL-1. The LOD is higher than that previously reported colorimetric method in the presence of H2O2.47,48 The results indicated that the FA-MnCo2O4 nanotubes based colorimetric strategy by a UV spectrophotometer could sensitively detect specific cancer cells.

Figure 9. A) Pressure-change profiles of cells after incubation with FA-MnCo2O4 nanotubes for 1 h and then addition of H2O2 in 10% serum samples; B) Absorbance-change in 652 nm profiles of cells (6*105/mL) after incubation with FA-MnCo2O4 for 1 h and then addition of 20 µL of 16 mM TMB in 200 µL HAc–NaAc (pH = 5.0) which containing 10% serum samples. Values are expressed as mean ±SD.

To further test feasibility of this colorimetric and pressure-based strategy for detecting cancer cells in biological fluids, cell samples were prepared from bovine serum albumin with a volume fraction of 10%. Pressure and colorimetric signals induced by the group of NIH3T3 cells incubated with FA-MnCo2O4 nanotubes were much lower than A549 cells and Hela cells groups (Figure 9). It suggests that the



integrating colorimetric and pressure-based cancer cell detection method is simple, low-cost, intuitive, specific, and sensitive and could be used in complicated environments.

4. CONCLUSION In conclusion, we integrated colorimetric and pressure-based dual-mode strategy for convenient, low-cost, intuitive, selective and highly sensitive detecing cancer cells. This novel dual-signal readout assay is based on the nanosheet assembled hollow MnCo2O4 nanotubes as an artificial enzyme. MnCo2O4 nanotubes were prepared by the spinneret electrospinning and thermal treatment method. The resultant MnCo2O4 nanotubes exhibit excellent catalytic properties for the TMB oxidation and the decomposition of H2O2 into O2. When FA, as recognition units, modified to the MnCo2O4 nanotubes, the resultant nanoplatform can integrate colorimetric and pressure-based strategies for reliable, facile, intuitive, and ultrasensitive detection of specific cancer cells. Moreover, such colorimetric and pressure-based dual-mode strategy that was capable of specifically detecting cancer cells in serum was also realized. To the best of our knowledge, this is the first FA-MnCo2O4 nanotubes based dual-mode strategy for detecting cancer cells that combined pressure meter and colorimetric method. We expect that such dual-signal readout strategy based on MnCo2O4 nanotubes could be used for early stage cancer diagnosis. ASSOCIATED CONTENT Supporting Information


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Characterizations, Supporting Figures are available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. The authors declare no competing financial interest. ACKNOWLEDGMENTS The work was supported by the National Natural Science Foundation of China (21671088, 21431002, and 21501080), and Fundamental Research Funds for the Central Universities (lzujbky-2017-105 and 2018-it03). We wish to thank the Electron Microscopy Centre of Lanzhou University for the microscopy and microanalysis of our specimens. REFERENCES (1) Nam, T.; Park, S.; Lee, S.; Park, K.; Choi, K.; Song, I.; Han, M.; Leary, J.; Yuk, S.; Kwon, I.; Kim, K.; Jeong, S. Tumor Targeting Chitosan Nanoparticles for Dual-modality Optical/MR Cancer Imaging. Bioconjugate Chem. 2010, 21, 578-582. (2) Dong, J.; Song, L.; Yin, J.; He, W.; Wu, Y.; Gu, N.; Zhang, Y. Co3O4 Nanoparticles with Multi-enzyme Activities and Their Application in Immunohistochemical Assay. ACS Appl. Mater. Interfaces 2014, 6, 1959-1970. (3) Kulkarni, B.; Jayakannan, M. Fluorescent-tagged Biodegradable Polycaprolactone Block Copolymer FRET Probe for Intracellular Bioimaging in Cancer Cells. ACS Biomater. Sci. Eng.



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Constructing 2D Nanosheet-assembled MnCo2O4 Nanotubes for

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