Laboratory of fluorescence spectroscopy and characterization
We are a small group working on in-vitro fluorescence experiments in biology, development and characterization of fluorescent dyes for biophysical application. Our research is focused of fluorescence techniques for detection of amyloid fibril formation in vitro. However, we have good experience in collaboration with groups working on development of environment-sensitive and solvatochromic fluorophores. Namely, we work with two-band ratiometric compounds from 3-hydroxycromone family undergoing excited state intramolecular proton transfer (ESIPT) reaction, extended analogues of ThT, other environment-sensitive compounds with strong dipole moment.
We are also open for collaborations with researchers willing to study interaction of proteins using fluorescence methods and can help with development of assay, selection of proper labels and protein labeling.
Relevant publications:
Dye development and characterization
• Giordano, L., Shvadchak, V.V., Arrupe, N., Falomir Lockhart, L.J., Sánchez, V.M., and Jovin, T.M. (2023) Tuning of environment-sensitive 3-hydroxychromone fluorophores based on strong donor substituents in positions 2 or 7. Dyes and Pigments. 218, 10.1016/j.dyepig.2023.111479
• Gaur, P., Galkin, M., Kurochka, A., Ghosh, S., Yushchenko, D.A., and Shvadchak, V.V. (2021) Fluorescent Probe for Selective Imaging of α-Synuclein Fibrils in Living Cells. ACS Chem. Neurosci. 12, 1293-1298, 10.1021/acschemneuro.1c00090
Kinetics of amyloid fibril formation
• Galkin, M., Topcheva, O., Priss, A., Borisova, T., and Shvadchak, V.V. (2023) Dopamine-Induced Oligomers of α-Synuclein Inhibit Amyloid Fibril Growth and Show No Toxicity. ACS Chem. Neurosci. 14, 2027-2034, 10.1021/acschemneuro.2c00815
Fluorimeter Shimadzu RF-5301 PC. Classical monochromator-based fluorimeter. Fluorescence emission and excitation spectra, time-based experiments. 3D-scan excitation-emission-intensity. Measurements in standard cuvettes (12×12mm). Acceptable sample volume 100-2400 μL.
Spectrophotometer DeNovix DS11-FX+ with fluorescence block.
Measurements of absorption spectra 190-840nm (diode array, fast measurement of spectra).
Cuvette-based (100-2000μL) measurements. Measurements from microvolumes (0.5-2μL drop).
Single-point and kinetic measurements.
Specialized programs for determination of protein and RNA/DNA concentration, calibration curves, custom settings.
Fluorescence module allows filter-based measurements (3 excitation and emission regions) from 500μL microtube, suitable for DNA/RNA concentration determination in very diluted samples. (characteristics)
Plate reader Fluorostar Galaxy is suitable for measurements of fluorescence and absorption of many parallel samples using filter-based detection. Allows measurements in 96- and 384-well plates with top and bottom reading. Temperature control of samples. Suitable for long-term kinetic measurements and testing multiple samples.
Services and expertise
– Characterization of fluorescent dyes: Emission and absorption spectra; fluorescence quantum yield; photostability (using appropriate reference); solvatochromic properties; sensitivity to polarity and rigidity of environment; characterization of dye interaction with proteins, DNA, membranes.
– Testing of inhibitors of amyloid fibril formation (ThT-based kinetic experiment, up to 384 samples per plate), determination of inhibition mechanism, kinetics of amyloid fibril formation.