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What is Spectral shift?

Fluorescence Principles

NanoTemper’s newest technology, called spectral shift, harnesses fluorescence to detect small changes in emitted light during a biomolecular interaction, in solution, and under isothermal conditions.

In the most basic description, fluorescence is an event where a molecule in its resting state (S0) absorbs a photon of light (hνA) and enters an excited state (S0-->S1). Then, to return to the resting state, the molecule rapidly emits a photon (hνF) of lesser energy after vibrational relaxation; the amount of relaxation and subsequent energy loss strongly depends on the local environment, such as temperature, solvent (buffer), and steric hinderance (Figure 1).

 

Figure 1: Jablonski diagram describing the absorption and emission of a photon.

 

The amount of energy loss is varied over multiple relaxation pathways that specifies the bandwidth and the shape of the emission spectrum generated (Figure 2.A). The emission spectrum is therefore defined by its consistent local environment; some fluorescent molecules, that are hyper-sensitive to the smallest changes in their surroundings, can spectrally broaden or shift their emission spectrum to higher or lower wavelengths when an event occurs that alters the excited state relaxation pathways (Figure 2.B).

 

Figure 2: General fluorescence emission spectrum (A.) and how this emission spectrum can spectrally shift to higher or lower wavelengths due to environmental changes, as depicted in the red and blue dotted line traces (B.).

 

Spectral Shift Technology

NanoTemper’s ground-breaking spectral shift technology exploits the very principle discussed above by using precision optics to detect wavelength shifts of a fluorescently labeled target during a biomolecular interaction. When a ligand binds to a target, the solvation shell surrounding the bi-molecular event changes the local environment seen by the target through proximity and
conformational effects (Figure 3).

 

Figure 3: Ligands that bind close to the fluorophore, shown as a yellow dot, can directly influence the fluorophore's chemical environment. Ligands that bind in a distant position from the fluorophore can lead to ligand-induced conformational changes that affect the chemical environment of the fluorophore.

 

The spectral shift technology utilizes two precision optics to monitor the changes in fluorescence intensity at two distinct wavelengths with the ability to observe spectral shifts less than 0.05 nm, thus providing highly sensitive measurement of the biomolecular interaction. By plotting the ratio of the two fluorescence signals as a function of varied ligand concentration, a binding curve is generated that can be fit using Kd or EC50 models (Figure 4).

 

Figure 4: Ligands that bind close to the fluorophore, shown as a yellow dot, can directly influence the fluorophore's chemical environment. Ligands that bind in a distant position from the fluorophore can lead to ligand-induced conformational changes that affect the chemical environment of the fluorophore.

 

References

A New Spectral Shift-Based Method to Characterize Molecular Interactions, by Langer, et al. Assay Drug Dev Technol. 2022; 20, 83–94. doi: 10.1089/adt.2021.133

 

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