Preparation of iron oxide-based calcium sensors for MRI

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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