Bioinspired design of a polymer gel sensor for the realization of extracellular Ca2+ imaging (original) (raw)

Although the role of extracellular Ca 2+ draws increasing attention as a messenger in intercellular communications, there is currently no tool available for imaging Ca 2+ dynamics in extracellular regions. Here we report the first solid-state fluorescent Ca 2+ sensor that fulfills the essential requirements for realizing extracellular Ca 2+ imaging. Inspired by natural extracellular Ca 2+-sensing receptors, we designed a particular type of chemically-crosslinked polyacrylic acid gel, which can undergo single-chain aggregation in the presence of Ca 2+. By attaching aggregation-induced emission luminogen to the polyacrylic acid as a pendant, the conformational state of the main chain at a given Ca 2+ concentration is successfully translated into fluorescence property. The Ca 2+ sensor has a millimolar-order apparent dissociation constant compatible with extracellular Ca 2+ concentrations, and exhibits sufficient dynamic range and excellent selectivity in the presence of physiological concentrations of biologically relevant ions, thus enabling monitoring of submillimolar fluctuations of Ca 2+ in flowing analytes containing millimolar Ca 2+ concentrations. Ca 2+ plays a crucial role in many important physiological and pathological processes in animals 1-17 and plants 9,18-23. Over the past several decades, many synthetic molecular and genetically encoded fluorescent Ca 2+ indicators have been developed, as represented by 1,2-bis(o-aminophenoxy)-ethane-N,N,N′ ,N′-tetraacetic acid (BAPTA) derivatives 24-27 and calmodulin-based proteins 28-32 , respectively. Ca 2+-imaging techniques that use such fluorescent indicators are indispensable in modern biology and medical science. In living organisms, Ca 2+ concentrations differ greatly depending on the compartment. Typically, the Ca 2+ concentration is ~100 nanomolar (nM) in intracellular cytosol, ~100 micromolar (μM) in the endoplasmic reticulum and mitochondria and ~1 millimolar (mM) in extracellular fluid and blood (Fig. 1a,b) 3. Plant vacuoles are also considered to contain mM-order Ca 2+ concentrations 20. Hence, Ca 2+ imaging in all of these compartments requires dedicated fluorescent indicators with specific dissociation constants (K d) that are appropriate for the respective background Ca 2+ concentrations. However, almost every Ca 2+ indicator known to date has a K d value ranging from nM to μM, and therefore allows for Ca 2+ imaging only in cytosol and organelles (Fig. 1a). Fluorescent Ca 2+ indicators with mM-order K d , compatible with extracellular Ca 2+ concentrations 27,32 , have scarcely been developed 9,10 , despite the fact that extracellular Ca 2+ , which is conventionally regarded as a diagnostic indicator for many diseases 3,7 , is now receiving considerable attention as a first messenger 3-17 in, for example, parathyroid gland 3,4 , neuron 12,13 , myocyte 14 , stem cell 15 and macrophages 16,17. In fact, there are major problems in the development of indicators for extracellular Ca 2+ imaging 9,10. First, such indicators should be designed to strike a balance between mM-order K d (i.e., a rather small affinity for Ca 2+) and high selectivity for Ca 2+ in the presence of excessive amounts of other physiological ions. Although simple Ca 2+ imaging against mM-order background concentration of Ca 2+ may be possible using existing indicators with μM-order K d , Ca 2+ indicators with one-order higher K d have a great advantage in monitoring Ca 2+ transients and oscillations in extracellular regions. Even more challenging in extracellular Ca 2+ imaging, one has to create a mechanism to avoid the outflow of indicators from an observation area through molecular diffusion. Obviously, this issue is intractable with existing molecular-based indicators.