The Harbron research lab focuses on molecular and macromolecular systems that modulate their fluorescence properties in response to a specific stimulus. Stimulus-induced changes in fluorescence intensity and/or color can be exploited in a variety of sensing and imaging applications including analyte detection in biological systems and probes for super-resolution microscopy. Our lab is currently focused on the use of light, metal ions, and pH as the stimuli for fluorescence modulation.
Fluorescence systems that respond to a light signal can be used to create photoactivated (or deactivated) fluorophores for microscopy and optical data storage, among other applications. One strategy for achieving fluorescence intensity photomodulation is to combine a fluorophore with a photochrome, a molecule that has two different molecular forms that interconvert in response to specific wavelengths of light. The photochrome is selected such that it is an efficient fluorescence quencher in only one of its two forms. Thus, switching the form of the photochrome with light activates or deactivates fluorescence quenching.
Ideally, the fluorophore in this type of fluorophore-photochrome construct should possess desirable photophysical properties including a large extinction coefficient, high fluorescence quantum yield, and good photostability. We currently use conjugated polymer nanoparticles (CPNs or Pdots) as fluorophores because they possess these features as well as the ability to act as light harvesting units, transferring the excitation energy of many chromophores to photochromic quenchers via fluorescence resonance energy transfer (FRET). An additional key advantage of CPNs as fluorophores is that they are stably suspended in water, enabling study of biological or environmental samples. We have demonstrated on-off fluorescence intensity modulation in CPNs doped and covalently functionalized with photochromic dyes using ultraviolet light as "off" switch. We are currently investigating off-on fluorescence intensity modulation using a visible light stimulus.
We can adapt the dye-functionalized CPN strategy outlined above to develop highly sensitive probes for heavy metal analytes and have demonstrated this approach for the detection of mercury in water. CPNs are doped with a masked rhodamine dye that is colorless and non-fluorescent until it encounters mercury, which induces transformation of the dye to its fully colored and highly fluorescent rhodamine form. By using CPNs that can act as a FRET donors for the rhodamine dye, we have a fluorescent system with two emission colors and can use the ratio of those two colors as our signal. This type of ratiometric sensing eliminates spurious readings due to instrumental or environmental fluctuations. Our CPNs emit green fluorescence until encountering mercury, which unmasks the rhodamine dyes that then act as FRET acceptors and emit red fluorescence. The significant changes in the red-to-green fluorescence ratio shows that we can detect sub-ppb levels of mercury in water.
Rhodamine spirolactam (RSL) dyes possess a spirocyclic structure that renders them colorless and non-fluorescent until a stimulus induces a ring-opening reaction that converts them to their fully conjugated, highly colored, fluorescent form. RSLs can be opened by acid, but the rate and pH at which this reaction occurs vary significantly among different derivatives. We seek to understand the structural factors that control the ring-opening reaction. Our goal is to achieve tunability to provide RSL pH probes that become fluorescent at a specific rate and pH for fluorescent pH probe applications.