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Optical nanosensors and diagnostics have been designed to utilise the sensitivity of fluorescence to make quantitative measurements inside living cells. The nanosensor devices are less than 100 nm in diameter and small enough to be inserted into living cells with a minimum of physical perturbation. Nanosensors enable measurements of changes in small molecule concentrations inside single cells to be made in real-time. They exhibit advantages over widely used fluorescence dye based methods because the nanosensor matrix imparts two key benefits:
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Protection of the sensing component from interfering species within the intracellular environment.
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Protection of the intracellular environment from any toxic effects of the sensing component.
Nanosensors capable of measuring glucose, oxygen, calcium, zinc and pH are routinely been prepared in our laboratories and work is ongoing to widen the range of analytes that can be detected using this technology.
A further attractive feature of the nanosensors we are developing is that they are compatible with the standard imaging and quantification techniques widely used in life science research e.g., fluorescent plate readers, fluorescence and confocal microscopy.
The group is active in developing strategies for introducing nanosensors to cells. To date techniques including gene gun, picoinjection and liposomal delivery as well as cell-directed mechanisms such as phagocytosis and macrophage sequestration have been used. We have also developed a method using cell penetrating peptides (CPP) to internalise nanosensors within the cell (shown in the figure below). We are working towards having a range of generic ‘soft’ chemical methods available which will routinely deliver nanosensors to large numbers of living cells from various lineages in a controllable manner.
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Confocal fluorescence microscopy images of CPP-conjugated nanosensors delivered to (from left): CHO cells incubated with Ca-sensitive nanosensors (green) and DRAQ5 nuclear stain (blue); GH4 Pituitary lactotrope cells loaded with Ca-sensitive nanosensors; A172 human glioblastoma cell loaded with FITC nanosensors; Human mesenchymal stem cells (hMSC) loaded with Rhodamine B nanosensors (red) and counter stained with mouse anti human CD105:FITC antibody (green); hMSC cell loaded with Rhodamine B nanosensors (red) and co-stained with CD105:FITC membrane stain (green) and DRAQ5 nuclear stain (blue).
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Progress is underway to develop the next generation of instrumentation that will allow optical imaging of single nanosensors inside live cells. This remains a grand challenge as it requires Abbe’s diffraction limit of light to be defeated. We aim to achieve this using super-resolved optical techniques and patterned nanoarrays to generate nanometre resolution, leading to optical imaging at the nanoscale.
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