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In situ simultaneous monitoring of ATP and GTP using a graphene oxide nanosheet–based sensing platform in living cells

Nature Protocols volume 9, pages 1944–1955 (2014)Cite this article

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Abstract

Here we present a detailed protocol for in situ multiple fluorescence monitoring of adenosine-5′-triphosphate (ATP) and guanosine-5′-triphosphate (GTP) in MCF-7 breast cancer cells by using graphene oxide nanosheet (GO-nS) and DNA/RNA aptamers. FAM-labeled ATP aptamer and Cy5-modified GTP aptamer are used to construct the multiple aptamer/GO-nS sensing platform through 'π-π stacking' between aptamers and GO-nS. Binding of aptamers to GO-nS guarantees the fluorescence resonance energy transfer between fluorophores and GO-nS, resulting in 'fluorescence off'. When the aptamer/GO-nS are transported inside the cells via endocytosis, the conformation of the aptamers will change on interaction with cellular ATP and GTP. On the basis of the fluorescence 'off/on' switching, simultaneous sensing and imaging of ATP and GTP in vitro and in situ have been realized through fluorescence and confocal microscopy techniques. In this protocol, we describe the synthesis of GO and GO-nS, preparation of aptamer/GO-nS platform, in vitro detection of ATP and GTP, and how to use this platform to realize intracellular ATP and GTP imaging in cultured MCF-7 cells. The preparation of GO-nS is anticipated to take 7–14 d, and assays involving microscopy imaging and MCF-7 cells culturing can be performed in 2–3 d.

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Figure 1: Workflow of the process of in situ molecular probing in living cells by using the aptamer/GO-nS nanocomplex.
Figure 2: Schematic illustration of in vitro and in situ molecular probing in living cells by using the aptamer/GO-nS nanocomplex.
Figure 3: Fluorescence quenching ability test of GO-nS for single or multiple aptamer probes.
Figure 4: Selective response to ATP, GTP, CTP and TTP based on the aptamer/GO-nS sensing platform by recording the respective fluorescence channel.
Figure 5: Confocal images of in situ visualization for ATP and GTP.

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Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (no. 21235004, no. 21327806, no. 21305046), the National Basic Research Program of China (No. 2011CB935704) and the Tsinghua University Initiative Scientific Research Program. This work was also partially supported by a laboratory-directed research and development program at Pacific Northwest National Laboratory (PNNL). We are very grateful to T.J. Weber, D. Hu, C.-T. Lin and A.S. Lea (PNNL) for their help in experimental work and for helpful discussion.

Author information

Authors and Affiliations

  1. Department of Chemistry, Beijing Key Laboratory for Microanalytical Methods and Instrumentation, Tsinghua University, Beijing, China

    Ying Wang, Longhua Tang & Jinghong Li

  2. Pacific Northwest National Laboratory,, Richland, Washington, USA

    Zhaohui Li & Yuehe Lin

  3. School of Mechanical and Materials Engineering, Washington State University, Pullman, Washington, USA

    Yuehe Lin

Authors
  1. Ying Wang
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Contributions

Y.W., Z.L., J.L. and Y.L. designed the protocol and carried out the experiments. Y.W., L.T. and J.L. wrote the manuscript.

Corresponding author

Correspondence to Jinghong Li.

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Integrated supplementary information

Supplementary Figure 1 Characterization of GO-nS.

(a) AFM images of GO-nS. (b) Cross-sectional plot indicated with the blue line, lateral average over 400 nm. (c) TEM image of GO-nS dispersed on a carbon film. (d) Size distribution histogram of GO-nS. This figure is reproduced with permission from our previously published work38, copyright 2013 American Chemical Society.

Supplementary Figure 2 XRD pattern of GO-nS.

Powder X-ray diffraction (XRD) measurements were performed on Bruker D8-Advance X-ray powder diffract meter using a graphite monochrometer with Cu Ka radiation (k = 1.5406 Å). This figure is reproduced with permission from our previously published work38, copyright 2013 American Chemical Society.

Supplementary Figure 3 Raman spectra of GO-nS.

Raman spectra were obtained using a confocal microprobe Raman system (Renishaw, RM2000). This figure is reproduced with permission from our previously published work38, copyright 2013 American Chemical Society.

Supplementary Figure 4 FTIR spectrum of GO-nS.

The FTIR spectrum (800-4,000 cm-1) was measured using a Perkin Elmer Fourier transform infrared spectroscopy spectrometer with pure KBr as the background. This figure is reproduced with permission from our previously published work38, copyright 2013 American Chemical Society.

Supplementary Figure 5 Single nucleotide detection based on single-aptamer/GO-nS complex.

(a) Fluorescence emission spectra of 100 nM ATP aptamer-FAM quenched with 3 μg/mL GO-nS (black bottom line) and fluorescence recovery by addition of ATP with concentration ranging from 10 μM to 2.5 mM (from bottom to top). Inset: linear relationship between (F-F0)/F0 (where F0 and F are the fluorescence intensity without and with the presence of ATP, respectively) and ATP concentration. (b) Fluorescence emission spectra of 100 nM GTP aptamer-Cy5 quenched with 3μg/mL GO-nS (red bottom line) and fluorescence recovery by addition of GTP with concentration ranging from 0.01 to 2 mM (from bottom to top) in reaction buffer for 1 h at 25 °C. Inset: linear relationship between (F-F0)/F0 (relative fluorescence intensity, where F0 and F are the fluorescence intensity without and with the presence of GTP) and GTP concentration. Error bars were obtained from three parallel experiments. This figure is reproduced from published work37,38. Copyright 2010 and 2013, American Chemical Society.

Supplementary Figure 6 High-resolution cell image.

MCF-7 cells were cultured with 100 nM aptamer-FAM/GO-nS for 6 h with recommended media at 37 °C. Picture A and B were captured from two parallel experiments under the same operation conditions, respectively.

(Fluorescence imaging was carried out on an intensified charge-coupled device(ICCD)-based real-time fluorescence microscopy system, which was composed mainly of a TE2000 inverted microscope (Nikon, Japan) with a Nikon 100× N.A. 1.3 oil objective, an I-PentaMAX Gen IV ICCD camera (Roper Scientific, USA), and an xenon arc lamp in the Lambda DG-4 Wavelength Switcher (Sutter Instrument, USA). Selective excitation was produced through a 484 ± 7.5 nm band-pass filter. Fluorescence was monitored and quantitatively analyzed with the MetaMorph software (Universal Imaging Co., USA). Excitation light was adequately attenuated. Photo bleaching and cell damage were minimized by auto-synchronizing the exciting shutter and the exposure shutter, i.e. cells were exposed to light only during exposure but not at the interval. This ICCD fluorescence micro-imaging system possessed the advantages of high-performance thermoelectric cooling function to reduce noise, and a gain high enough to detect extremely low light.)

Supplementary Figure 7 Cleavage protection assay of GO-nS.

Agarose gel electrophoresis image for enzymatic cleavage protection assay: lane 1, DNA size ladder for 100 bp; lane 2, aptamer-FAM; lane 3, aptamer-FAM reacted with DNase I for 15 min; lane 4, aptamer-FAM reacted with DNase I for 40 min; lane 5, aptamer-FAM/GO-nS; lane 6, aptamer-FAM/GO-nS incubated with DNase I for 15 min; lane 7, aptamer-FAM/GO-nS incubated with DNase I for 40 min. Aptamer concentration is 100 nM, GO-nS is 3 μg/mL, and DNase I is 0.2 units/μL. Excitation wavelength for SYBR Green I: 494 nm.

Supplementary Figure 8 Cell viability assay.

Cell viability determined using a trypan blue assay after treatment of MCF-7 cells with 1 – 9 μg/ml GO-nS for 12 h (red), 24 h (green) or 72 h (blue). Values represent the mean ± se, n=3. *Significantly different from control, p < 0.05. This figure is adapted with permission from our previously published work38, copyright 2013 American Chemical Society.

Supplementary information

Supplementary Figure 1

Characterization of GO-nS. (PDF 144 kb)

Supplementary Figure 2

XRD pattern of GO-nS. (PDF 66 kb)

Supplementary Figure 3

Raman spectra of GO-nS. (PDF 81 kb)

Supplementary Figure 4

FTIR spectrum of GO-nS.. (PDF 93 kb)

Supplementary Figure 5

Single nucleotide detection based on single-aptamer/GO-nS complex. (PDF 115 kb)

Supplementary Figure 6

High-resolution cell image. (PDF 499 kb)

Supplementary Figure 7

Cleavage protection assay of GO-nS. (PDF 112 kb)

Supplementary Figure 8

Cell viability assay. (PDF 110 kb)

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Wang, Y., Tang, L., Li, Z. et al. In situ simultaneous monitoring of ATP and GTP using a graphene oxide nanosheet–based sensing platform in living cells. Nat Protoc 9, 1944–1955 (2014). https://doi.org/10.1038/nprot.2014.126

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