Approximately 80 fluorescent dyes from different chemical classes were investigated, including monomethine and trimethine cyanines and dioxaborine adducts of merocyanines. Patterns in the changes of their spectral and luminescent properties upon interaction with nucleic acids and proteins were established, and the influence of the dyes’ structural features on their fluorescence response was determined.
Among the compounds studied, fb128 proved the most promising for fluorescent DNA detection in real-time PCR. Upon interaction with DNA, it showed a substantial fluorescence enhancement (ΔQ ≈ 160) and high signal intensity (I ≈ 2000 a.u.). An optimal working concentration range of 0.2–2 µM was established for fb128, providing a sufficient fluorescence signal without significant inhibition of amplification. Within this range, the dye had only a minimal effect on amplicon melting temperature, with a shift of no more than 0.5 °C.
Standard curves for different amplicons demonstrated high PCR efficiency (≈102–103%) and good linearity for DNA quantification. DNA concentrations determined using fb128 were in good agreement with results obtained with the commercial dyes SYBR Green and EvaGreen, confirming the suitability of fb128 for quantitative real-time PCR.
The results identify fb128 as a promising fluorescent reporter for DNA detection and quantitative qPCR analysis and provide a basis for the development of new fluorescent reagents for molecular diagnostics.
Publications
- Kazakov-Kravchenko O. S., Balanda A., Losytskyy M., Yarmoluk S. “Effect of DNA, RNA and HSA on the spectral-luminescent properties of several monomethine cyanine dyes.” Biopolymers and Cell. 2025;41(2):121–129.
- Kazakov-Kravchenko O. S., Losytskyy M., Polishchuk V. M., Kulinich A. V., Shandura M. P., Yarmoluk S. “Spectral-fluorescent response of dioxaborine dyes with varying chromophore charge in the presence of serum albumins.” Biopolymers and Cell. 2025;41(3):222–230. DOI
- Kazakov-Kravchenko O. S., Kravchenko S. A., Yarmoluk S. M. “Evaluation of the monomethine cyanine dye FB128 for real-time PCR applications.” Biopolymers and Cell. 2025;41(4):300–308.
- Kazakov-Kravchenko O. S., Losytskyy M. Yu., Derevyanko N. A., Kulinich A. V., Shandura M. P., Yarmoluk S. M. “Substituent effects in thiacarbocyanine dyes on their spectral-fluorescent response in the presence of nucleic acids and serum albumins.” Biopolymers & Cell. 2026;42(2):139–149.