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Design, calibration and application of broad-range optical nanosensors for determining intracellular pH

Nature Protocols volume 9, pages 2841–2858 (2014)Cite this article

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Abstract

Particle-based nanosensors offer a tool for determining the pH in the endosomal-lysosomal system of living cells. Measurements providing absolute values of pH have so far been restricted by the limited sensitivity range of nanosensors, calibration challenges and the complexity of image analysis. This protocol describes the design and application of a polyacrylamide-based nanosensor (∼60 nm) that covalently incorporates two pH-sensitive fluorophores, fluorescein (FS) and Oregon Green (OG), to broaden the sensitivity range of the sensor (pH 3.1–7.0), and uses the pH-insensitive fluorophore rhodamine as a reference fluorophore. The nanosensors are spontaneously taken up via endocytosis and directed to the lysosomes where dynamic changes in pH can be measured with live-cell confocal microscopy. The most important focus areas of the protocol are the choice of pH-sensitive fluorophores, the design of calibration buffers, the determination of the effective range and especially the description of how to critically evaluate results. The entire procedure typically takes 2–3 weeks.

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Figure 1: Schematic illustration of the design, calibration and application of the nanosensor in this protocol.
Figure 2
Figure 3: Calibration curves of dual- and triple-labeled nanosensors.
Figure 4: Buffering capacity of the buffer system.
Figure 5: Determination of calibration buffer concentration.
Figure 6: Frequency distributions of intensity ratios of a triple-labeled nanosensor diluted in buffers of different pH.
Figure 7: Intracellular pH measurements with a triple-labeled nanosensor.
Figure 8: Intracellular pH measurements with a dual-labeled nanosensor.
Figure 9: Effect of PEI on the lysosomal pH.

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Acknowledgements

This work was financially supported by the Danish Cancer Society and the Danish Council for Independent Research (Technology and Production Sciences (FTP) grant no. 274-07-0172). We furthermore thank H. Sun and E.K.P. Kumar (Technical University of Denmark, Denmark) for synthesis of the nanosensors.

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Authors and Affiliations

  1. Department of Micro- and Nanotechnology, Technical University of Denmark (DTU) Nanotech, DTU, Lyngby, Denmark

    Rikke V Søndergaard & Thomas L Andresen

  2. Center for Nanomedicine and Theranostics, DTU, Lyngby, Denmark

    Rikke V Søndergaard, Jonas R Henriksen & Thomas L Andresen

  3. Department of Chemistry, DTU Chemistry, DTU, Lyngby, Denmark

    Jonas R Henriksen

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Contributions

R.V.S., J.R.H. and T.L.A. developed the protocol and analyzed the results. R.V.S. and T.L.A. designed the experiments and wrote the paper. R.V.S. performed all the experiments. J.R.H. wrote the code for image analysis.

Corresponding author

Correspondence to Thomas L Andresen.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Three different calibration types of the nanosensor.

Calibration in buffer was performed in pure buffer as described in the main protocol. Calibration in artificial cytoplasm and with nigericin was performed as described in BOX 3. Briefly; artificial cytoplasm was prepared by sonication of HeLa cells and mixed with buffers with controlled pH. In situ calibration with nigericin was performed by treatment of nanosensor-containing cells with nigericin in K+-rich buffers. Mean ± SD between three images are presented. Reproduced with permission from Benjaminsen et al. (Evaluating Nanoparticle Sensor Design for Intracellular pH Measurements. ACS Nano 5, 5864-5873 (2011)). Copyright 2011 American Chemical Society.

Supplementary information

Supplementary Figure 1

Three different calibration types of the nanosensor. (PDF 75 kb)

Supplementary Discussion

Potential expansion of the protocol to measure other metabolites. (PDF 105 kb)

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Søndergaard, R., Henriksen, J. & Andresen, T. Design, calibration and application of broad-range optical nanosensors for determining intracellular pH. Nat Protoc 9, 2841–2858 (2014). https://doi.org/10.1038/nprot.2014.196

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