Self-assembled hydrogel fibers for sensing the multi-compartment intracellular milieu

Targeted delivery of drugs and sensors into cells is an attractive technology with both medical and scientific applications. Existing delivery vehicles are generally limited by the complexity of their design, dependence on active transport, and inability to function within cellular compartments. Here, we developed self-assembled nanofibrous hydrogel fibers using a biologically inert, low-molecular-weight amphiphile. Self-assembled nanofibrous hydrogels offer unique physical/mechanical properties and can easily be loaded with a diverse range of payloads. Unlike commercially available E. coli membrane particles covalently bound to the pH reporting dye pHrodo, pHrodo encapsulated in self-assembled hydrogel-fibers internalizes into macrophages at both physiologic (37°C) and sub-physiologic (4°C) temperatures through an energy-independent, passive process. Unlike dye alone or pHrodo complexed to E. coli, pHrodo-SAFs report pH in both the cytoplasm and phagosomes, as well the nucleus. This new class of materials should be useful for next-generation sensing of the intracellular milieu

In Vivo Fluorescence Imaging of Bacteriogenic Cyanide in the Lungs of Live Mice Infected with Cystic Fibrosis Pathogens

10.1371/journal.pone.0021387PLoS ONE67

Loss of Pluripotency in Human Embryonic Stem Cells Directly Correlates with an Increase in Nuclear Zinc

The pluripotency of human embryonic stem cells (hESCs) is important to investigations of early development and to cell replacement therapy, but the mechanism behind pluripotency is incompletely understood. Zinc has been shown to play a key role in differentiation of non-pluripotent cell types, but here its role in hESCs is directly examined. By mapping the distribution of metals in hESCs at high resolution by x-ray fluorescence microprobe (XFM) and by analyzing subcellular metal content, we have found evidence that loss of pluripotency is directly correlated with an increase in nuclear zinc. Zinc elevation not only redefines our understanding of the mechanisms that support pluripotency, but also may act as a biomarker and an intervention point for stem cell differentiation

Genetically encoded FRET sensors to monitor intracellular Zn2+ homeostasis

We developed genetically encoded fluorescence resonance energy transfer (FRET)-based sensors that display a large ratiometric change upon Zn2+ binding, have affinities that span the pico- to nanomolar range and can readily be targeted to subcellular organelles. Using this sensor toolbox we found that cytosolic Zn2+ was buffered at 0.4 nM in pancreatic ß cells, and we found substantially higher Zn2+ concentrations in insulin-containing secretory vesicles

Genetically encoded fluorescent probes for Intracellular Zn2+ imaging

In this chapter we provide an overview of the various genetically encoded fluorescent Zn2+ sensors that have been developed over the past 5 to 10 years. We focus on sensors based on Förster resonance energy transfer (FRET), as these have so far proven to be the most useful for detecting Zn2+ in biological samples. Our goal is to provide a balanced discussion of the pros and cons of the various sensors and their application in intracellular imaging. Following the description of the various sensors, several recent applications of these sensors are discussed. We end the chapter by identifying remaining challenges in this field and discussing future perspectives