Preparation of iron oxide-based calcium sensors for MRI

Abstract

We describe a protocol for the preparation of a family of calcium indicators for MRI. The indicators consist of superparamagnetic iron oxide nanoparticles functionalized with the calcium-sensing protein, calmodulin, and its target peptides. Calcium-dependent protein–protein interactions drive particle clustering and produce large changes in T2 relaxivity. To prepare these indicators, interacting polypeptides are purified, biotinylated and then conjugated to streptavidin-coated nanoparticles. Conjugates are isolated, mixed to obtain functional sensors and incubated in the presence or absence of calcium to test response properties. Calcium-dependent responses are assayed by dynamic light scattering and MRI. The whole series of procedures can be carried out in 12 h. The iron oxide-based calcium sensors discussed here are suitable for the detection of calcium concentration changes of 1 μM in vitro.

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Figure 1: MRI calcium sensor mechanism.
Figure 2: Anticipated results using superparamagnetic iron oxide-based calcium sensors.
Figure 3: Calcium sensitivity range.

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Acknowledgements

This work was generously funded by the Raymond and Beverley Sackler Foundation, the McKnight Endowment Fund for Neuroscience and the National Institutes of Health (R21 EB005723 to A.J.). A.J. is a Raymond and Beverley Sackler Foundation Scholar. The authors are grateful to Atsushi Miyawaki for the Xenopus CaM D64Y E104Q template sequence.

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Correspondence to Alan Jasanoff.

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Atanasijevic, T., Jasanoff, A. Preparation of iron oxide-based calcium sensors for MRI. Nat Protoc 2, 2582–2589 (2007). https://doi.org/10.1038/nprot.2007.377

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