Performance Comparison of Fluorescent Sodium Ion Indicators

Detecting, analyzing, and understanding the Sodium ion (Na+) in biological systems is an important aspect of scientific research. Because sodium is an extremely important metal in living organisms, and because it is prevalent in all systems, it is vital to have a concrete means in which to measure its activity to maintain a normal level of physiological function.

Sodium levels attribute to a host of biological activities: increases and decreases in in extracellular volume, plasma volume and blood volume in and around cells, to the generation of action potentials in nervous and cardiac tissue, to maintaining normal cellular homeostasis, to the regulation of one's blood pressure and immune responses, as well as fluid and electrolyte balance within the body. Sodium ions also help modulate neuronal activity, nutrient transporting and molecule-signaling. Fluctuations in sodium levels account for many diseases and disorders that can be better understood with additional research on measuring both intracellular and extracellular activity levels. With further understanding of sodium transporters and sodium-permeable channels, scientists and researchers are able to combat cardiovascular and physiological diseases, as well as better equip us in treating hypertension, liver disorders and kidney diseases. Fluctuations can also occur during intense exercise and during some surgical procedures, furthering the need to better understand how best to regulate it.

One way to measure sodium levels is through fluorescent indicators, though this is not without its challenges. Designing specific sensors particularly for sodium is an excellent analytical method of detecting quantitative data on the activity of sodium channels and transporters, as they will exhibit fluorescence upon binding with Na+. The use of fluorescent sensors is a highly specialized technique, because not only does the fluorescent sensor need to attract and detect Na+ ions at an appropriate range, but also must detect and discriminate against other ions such as potassium and other widespread physiological cations.

Until more recently, sodium concentrations were quantified using potentially less-reliable or expensive methods such as flame tests, chromatography, gravimetry, and atomic emission spectroscopy. The introduction of fluorescent indicators provides scientists and researchers with the ability to isolate sodium ions in mitochondria, and measure both intracellular and extracellular efflux.

Summary of Common Sodium Indicators

Indicator	Excitation (nm)	Emission (nm)	Fluorescence	Membrane permeable
SBFI	337	527	Green	N*
Sodium Green Na+	480	540	Green	N*
SoNa™ 520	491	511	Green	N*
CoroNa Green	492	516	Green	Y
CoroNa Red	540	590	Red	Y
ING-1	525	545	Yellow-green	Y
ING-2	525	545	Yellow-green	Y

1. Cell-permeable versions of these probes are commercially available

When determining the best approach in use of these sodium-specific sensors, it is necessary to consider multiple factors: brightness, the ability to absorb photons, and the probability in which the photons are emitted for each one absorbed (quantum yield), sensitivity, affinity, specificity, photostability, pH sensitivity, kinetics and targetability. Small molecule-based synthetic indicators and protein-based indicators that are genetically encodable are the two primary classifications. Synthetic indicators tend to be more photostable and have more brightness to them, while genetically coded indicators can generally be observed at a specific location within the cell via cellular machinery.

As the use of fluorescent sensors for sodium advances, there are now a small number of prominent probes being used today. Sodium binding benzofuran isophthalate (SBFI) is one of the first developed and is a UV-excitable, ratiometric green indicator for intracellular sodium quantities. Ratiometry is an ideal method in comparing intensities of more than one wavelength to detect input changes of Na+ ions. Though proven useful, SBFI tends to have low brightness, directly related to a low quantum yield. Its advantage is that it reduces cell morphology, dye loading and photobleaching. SBFI is also membrane impermeable, with excitation levels at 340-380nm and emission levels of 505nm. This wide range reflects signal wavelength change of bound vs. free indicator as well as the variation in fluorescence response over the entire spectral profile.

FAQs:	Spectrum Viewer:
What are the alternatives to SBFI for measuring sodium levels in cells? How is SBFI introduced into cells or samples for measurement? How does SBFI work as a sodium indicator?	Sodium-bound and Sodium-free SBFI

Sodium Green Na+ indicator, a non-ratiometric dye excited at 488nm, has proven to be a significant alternate indicator to SBFI with a longer wavelength absorption and a much higher fluorescence quantum yield. CoroNa Red is a fluorescent indicator that only fluoresces in the absence of Na+, whereas CoroNa Green will fluoresce upon the binding of the Na+ ion, with excitation levels at 492nm and peak emission at 516nm. Both CoroNa Red and CoroNa Green are membrane-permeable and linked to a crown ether to best exhibit an increase in fluorescence emission intensity. Their smaller size also contributes to more successful loading.

These four previously mentioned methods are quickly being replaced with more highly sensitive indicators such as Ion Natrium Green-1 (ING-1) and Ion Natrium Green-2 (ING-2), which was formerly Asante Natrium Green (ANG-2). ING-1 is a lower-affinity indicator with a yellow-green fluorescence that is membrane-permeable. ING-2 is also yellow-green, but with a higher affinity for sodium and used for high throughput screening of Na+ channels and transporters. With both ING-1 and ING-2 exhibiting excitation levels at 525mm and emission levels at 545 nm, these relatively small synthetic fluorochromes are proving to respond to changes in Na+ levels with great compatibility with a wide variety of detectors, and under conditions in which the Na+ binds or doesn't bind.

Even more recent than these, and with an emission profile in the common green channel, SoNa™ 520 is a new sodium-sensitive fluorescent indicator dye used to detect sodium levels in biological samples. It perhaps has the highest detection sensitivity compared to both SBFI and CoroNa Red. It can be employed to measure changes in sodium concentration in living cells and other biological samples. Compared to the most common SBFI, SoNa™ 520 is much more sensitive, with a much larger fluorescence response under the same conditions. In addition, SoNa™ 520 can be well excited with the visible 488 nm laser or similar visible light to avoid the UV excitation that is required for exciting SBFI. The two ING dyes as well as SoNa™ 520 are also single-channel (non-ratiometric) indicators. This means that as binding with Na+ occurs, fluorescence increases significantly and when unbound, it is drastically reduced or entirely nonfluorescent.



Drug discovery research focused primarily on NA+-permeable channels and Na+ transporters are highly involved and of interest to continue to create more data and serve and provide improved, quality care. With the knowledge gained in sodium ion detection analysis, researchers and doctors can better treat various mental health disorders, pain, epilepsy and more. The downstream applications and use of this data will only prove to be more crucial in our understanding of cellular and electrical functions and as more fluorescent indicators are established.

Products

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References

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Fluorescent Na+ and K+ Indicators
Overview of Sodium's Role in the Body
In vivo sodium concentration continuously monitored with fluorescent sensors
Sodium Ion
Sodium (Na⁺) in physiology