Dye-Based Fluorescence Quantification
Quickly quantify nucleic acids and measure encapsulation efficiency
Get lit with dye-based fluorescence
Fluorescence is a powerful technique for quantification due to the selectivity and sensitivity of dye-based fluorophores. Fluorophores that selectively bind to nucleic acids are a great way to quantify DNA or RNA, even in the presence of other biomolecules. Fluorescence is also more sensitive than other techniques, allowing reliable quantification of samples below 1 ng/µL of nucleic acid. Taking advantage of the need for dye-ligand interactions is the basis of many highly selective and sensitive assays.
Time to get excited
When a fluorophore is excited by photons of light, it absorbs that light which puts its electrons into an excited state. This excited state is much less stable, so these electrons are looking to return to their stable ground state as soon as possible. To return to the ground state, the excited fluorophore needs to shed some energy and it does so in the form of emitting light at a lower energy, which will be a higher wavelength. Instruments with fluorescence capabilities have light sources to excite fluorophores and detectors behind emission filters which collect emitted light over a range of wavelengths. The amount of collected emission light is proportional to the amount of fluorophore present, so fluorescence is a quantitative method to determine concentration.
Get glowing results with the right fluorophores
For a molecule to have fluorescent properties, electrons within its structure need some breathing room. This is made through conjugated pi bonds, usually in ring structures, which allow pi electrons to freely move between atoms within the conjugated structure. The shape of the molecule affects its specific fluorescent properties including the wavelength of light that gives maximum excitation and the highest intensity of emitted light (λmax ). Fluorophores do not have a single wavelength for excitation and emission, but rather a spectrum or range of wavelengths that will excite or emit. To optimize fluorescence emission from a fluorophore, it’s best to select an excitation source near the absorbance maximum. Additionally, an emission filter that collects the most light (area under the emission spectrum) will lead to the best sensitivity.
Set the standard
In order to take the final step and convert fluorescence data from relative intensities to actual concentrations, all measurements should include a standard curve that correlates a fluorescence intensity value to a known amount of analyte being measured. Checking the shape of the standard curve also ensures that all measurements are within the appropriate dynamic range of the assay.
Shine a light on quality of your LNPs
Fluorescence really shines when measuring encapsulation efficiency (EE%) in LNP preps. Assays to measure EE% rely on detecting the increase in visible fluorescence that happens when a fluorescent dye (e.g. RiboGreen®) binds to free RNA in solution. Encapsulated RNA is protected from RiboGreen® and won’t be quantified in a prep of intact nanoparticles. Combining the fluorescence quant of free RNA with UV/Vis for total RNA gives all the values necessary to calculate EE%.
Stunner
Stunner is the only system that pulls together UV/Vis concentration with rotating angle dynamic light scattering (RADLS) on the same 2 μL sample. Add on dye-based fluorescence with Stunner AF (Add Fluorescence) and see even more in the same run. Nail down your lipid nanoparticle quality by knocking size, encapsulation efficiency (EE%), particle concentration and detection of aggregates off your list in one hit. Without skipping a beat, you’ll know if your nanoparticle is good to go.
Ready for more?
Nanoparticle researchers can now use the right tool for LNP characterization studies with an instrument that combines UV/Vis, rotating dynamic light scattering (RADLS), and dye-based fluorescence. Have a question or ready to find out more?