What is Monolith Omni?
Monolith Omni is the latest development of the Monolith benchtop biophysical platform designed for multi-parameter characterization of biomolecular interactions. It combines four complementary detection technologies in a single instrument, enabling measurement of binding affinity, kinetics, thermodynamics (via Van't Hoff analysis), and qualitative protein stability. This happens in solution, without surface immobilization, and from the same sample.
Technologies
Spectral Shift: the foundation of Monolith Omni measurements
Spectral Shift quantifies molecular interactions by measuring picometer-level shifts in fluorescence emission (at 650 nm and 670 nm) of a fluorescently labeled target in response to ligand binding. It detects changes in the hydrophobicity of the target's surface. As an isothermal measurement, it is non-destructive and compatible with fragile or suboptimal samples, including those containing aggregation or precipitation.
Additional analyses powered by Spectral Shift
Van’t Hoff - thermodynamics
The temperature dependence of binding affinity can be used to derive thermodynamic parameters through Van’t Hoff analysis. In Monolith Omni, temperature-dependent Spectral Shift data can quantify the enthalpy change, ΔH, and entropy change, ΔS, associated with an interaction.
This thermodynamic context complements affinity and kinetic measurements, supporting a more complete understanding of the molecular forces and interaction mechanisms contributing to binding.
nanoTAK - binding kinetics
nanoTAK (nano Temperature Alteration Kinetics) determines the binding rate constants, kon and koff, of an interaction in solution.
Pre-equilibrated samples in capillaries are subjected to a rapid temperature step. This perturbation temporarily drives the system out of equilibrium. The subsequent re-equilibration is then monitored in real time through Spectral Shift data.
Because the measurement is performed in solution without surface immobilization, nanoTAK avoids mass-transport limitations and potential surface-related artifacts. This provides a solution-based approach to characterizing binding kinetics over a broad temperature range from 25°C to 40°C.
nanoLISA - protein stability
nanoLISA (nano Laser Induced Stability Analysis) uses the integrated infrared laser to heat samples to temperatures of up to 95°C while continuously monitoring Spectral Shift.
As the protein unfolds, changes in the fluorophore’s environment produce a characteristic sigmoidal thermal unfolding profile. Comparing profiles recorded in the presence and absence of a ligand can reveal ligand-induced stabilization resulting from covalent or non-covalent interactions.
nanoLISA can also help detect unfolding and early aggregation, providing qualitative information about protein stability. Thermal melting profiles can serve as an orthogonal approach for confirming binding and gaining deeper insight into ligand effects.
TRIC: an orthogonal affinity readout
Temperature Related Intensity Change, or TRIC, measures changes in fluorescence intensity caused by a small temperature perturbation. These changes reflect alterations in the structural flexibility and local environment of the labeled target.
TRIC uses the same solution-based samples as Spectral Shift and provides an orthogonal measurement of binding affinity. It can also provide additional insights into sample quality, including the detection of aggregation or compound-induced precipitation.
What parameters can Monolith Omni measure?
| Parameter | Technology Used |
|---|---|
| Binding affinity (Kd) | Spectral Shift, TRIC |
| Binding kinetics (kon, koff) | nanoTAK |
| Thermodynamics (ΔH, ΔS) | Van't Hoff analysis |
| Qualitative protein stability | nanoLISA |