2
A P P L I C A T I O N N O T E
The Dianthus uHTS platform employs Spectral Shi technology to detect binding interactions in
solution, enabling ultra-high-throughput screening with minimal protein consumption. Spectral
Shi assays measure ligand-induced changes in the emission spectrum of a labelled protein's
environment, providing a highly sensitive readout of binding events without requiring secondary
labeling or immobilization. This approach allows for real-time monitoring of molecular interactions
in equilibrium, ensuring accurate affinity determination across a broad range of binding affinities.
Compared to traditional fluorescence-based assays, Spectral Shi technology minimizes interference
from autofluorescence and non-specific binding, improving assay robustness.
SPECTRAL SHIFT ASSAYS
Spectral Shi (SpS) [1] refers to the change in the maximum wavelength (λ
max
) of absorbance or
emission due to structural or environmental changes in a fluorescent molecule, such as the rotation
around a double bond or a change in solvent polarity. These shis are classified as red shis
(bathochromic), where λ
max
increases, and blue shis (hypsochromic), where λ
max
decreases. For
example, red shis oen occur in hydrophobic environments, while blue shis are typically observed
in polar environments.
The Lippert-Mataga equation mathematically describes the Spectral Shi phenomenon in context of
the interaction of a dye molecule with its environment (Equation 1). The magnitude of the Spectral
Shi depends on the difference in dipole moments between the excited state (μ
e
) and the ground
state (μ
g
) of the dye, the effective volume the dye can probe (a
3
), and changes in the dye's environment
(∆f).
Equation 1.
Introduction
