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 approximately 12 h. The iron oxide-based calcium sensors discussed here are suitable for the detection of calcium concentration changes of approximately 1 microM in vitro.
Atanasijevic
Tatjana
T
Department of Nuclear Science and Engineering, Massachusetts Institute of Technology, 150 Albany Street, NW14-2213, Cambridge, Massachusetts 02139, USA.
Jasanoff
Alan
A
D000818:Animals; D019921:Biotinylation; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry; D002417:Cattle; D003287:Contrast Media / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005290:Ferric Compounds / Q000737:chemistry*; D008279:Magnetic Resonance Imaging / Q000379:methods*; D053768:Metal Nanoparticles / Q000737:chemistry*
D002147:Calmodulin; D003287:Contrast Media; D005290:Ferric Compounds; C000499:ferric oxide; D002118:Calcium