De novo strategy with engineering anti-Kasha/Kasha fluorophores enables reliable ratiometric quantification of biomolecules

Abstract

Fluorescence-based technologies have revolutionized in vivo monitoring of biomolecules. However, significant technical hurdles in both probe chemistry and complex cellular environments have limited the accuracy of quantifying these biomolecules. Herein, we report a generalizable engineering strategy for dual-emission anti-Kasha-active fluorophores, which combine an integrated fluorescein with chromene (IFC) building block with donor-π-acceptor structural modification. These fluorophores exhibit an invariant near-infrared Kasha emission from the S₁ state, while their anti-Kasha emission from the S₂ state at around 520 nm can be finely regulated via a spirolactone open/closed switch. We introduce bio-recognition moieties to IFC structures, and demonstrate ratiometric quantification of cysteine and glutathione in living cells and animals, using the ratio (S₂/S₁) with the S₁ emission as a reliable internal reference signal. This de novo strategy of tuning anti-Kasha-active properties expands the in vivo ratiometric quantification toolbox for highly accurate analysis in both basic life science research and clinical applications.

Fig. 1. Strategies for ratiometric probes with characteristic analysis.

a FRET ratiometric probes: excitation/emission cross-talk. b Unimolecular ratiometric probes: different photobleaching rates and concentration-dependent diffusion between reactant and product fluorophores in dynamic cellular environment. c In this work, anti-Kasha-active ratiometric probes enabling accurate and reliable ratiometric quantification: an invariant NIR emission from the Kasha-based S₁ state serve as an internal reference; a green emission from the anti-Kasha-active S₂ state linearly increase with the concentration of targeted analyte, due to the rapid radiative process from S₂ to S₀ state, competing with the internal conversion (IC) from S₂ to S₁ state.

Fig. 2. Characterization of anti-Kasha mechanism in open-form versus Kasha mechanism in closed-form.

a Chemical structure of DCM-IFC in its open form. b Excitation spectra (dotted line, monitored at λ_em = 520 and 710 nm) and emission spectra (solid line, excited at λ_ex = 480 and 560 nm) of open-form DCM-IFC. c Electron–hole analysis involved during the photoexcitation of the open form, based on the molecular structure at the Franck–Condon state. Femtosecond time-resolved transient absorption spectra (d) and kinetics (e) of open-form DCM-IFC (excited at λ_ex = 480 nm). f Jablonski diagram illustrating the anti-Kasha mechanism. g Chemical structure of DCM-IFC in its closed form. h Excitation spectra (dotted line, monitored at λ_em = 710 nm) and emission spectra (solid line, excited at λ_ex = 480 and 560 nm) of closed-form DCM-IFC. i Electron–hole analysis involved during the photoexcitation of the closed form, based on the molecular structure at the Franck–Condon state. Femtosecond time-resolved transient absorption spectra (j) and kinetics (k) of closed-form DCM-IFC (excited at λ_ex = 480 nm). Note: open-form DCM-IFC was investigated at pH 11.3 and closed-form DCM-IFC was investigated at pH 2.0. l Two structurally related reference compounds that are locked into open form or closed form. Note: all fluorescence spectra were measured in a mixture solution of acetonitrile (MeCN)/Britton–Robinson buffer.

Fig. 3. A generalizable molecule engineering strategy for developing anti-Kasha/Kasha fluorophores.

a Schematic illustration of designing anti-Kasha/Kasha fluorophores. b Molecular and crystal structures of BI-IFC-ester (locked closed form). c Quantum chemical calculations and experimental data of anti-Kasha/Kasha fluorophores. ΔE(S₂−S₁) and oscillator strength were obtained from quantum chemical calculations. Emission was obtained from experimental data.

Fig. 4. Anti-Kasha/Kasha switch-induced dual-emission response with an internal intensity reference signal.

a Schematic illustration of spirolactone-switch-controlled molecules for tuning anti-Kasha/Kasha properties. Absorption (b) and fluorescence spectra (c) of DCM-IFC in the range of pH 2.2–8.0. Dual-emission response with the internal intensity reference signal of DCM-IFC (d), TCB-IFC (e), and BI-IFC (f) at different pH values, λ_ex = 480 nm. Source data are provided as a Source Data file. Concentration-dependent emission ratios (S₂/S₁) detected for DMC-IFC at pH 7.0 (g) and pH 6.0 (h). Source data are provided as a Source Data file. Dual-channel ratiometric imaging (i), fluorescence intensity (j) and ratio (k) of DCM-IFC in A549 cells across different pH values. Data with error bars are expressed as mean ± s.d., n = 3. Source data are provided as a Source Data file. Note: all fluorescence spectra were measured in a mixture solution of MeCN/Britton-Robinson buffer.

Fig. 5. A generalizable method for quantitative analysis with anti-Kasha-active chromophores.

a Quantitative sensing platform. Chemical structures of DCM-IFC probes with different biomolecular-recognition groups: (b) acrylate group towards Cys; (f) 2,4-dinitrobenzenesulfonyl towards GSH; (j) disulfide bond towards GSH, sense-and-release. Fluorescence spectra (c, g, k), ratiometric response with internal reference signal (d, h, l, Source data are provided as a Source Data file.) and dual-channel linear ratio analysis (e, i, m, Source data are provided as a Source Data file.) of DCM-IFC-1, DCM-IFC-2, DCM-IFC-3, and DCM-IFC-4 for various analytes. Note: all fluorescence spectra were measured in a mixture solution of MeCN/phosphate-buffered saline (PBS, pH = 7.4), λ_ex = 480 nm.

Fig. 6. Quantitative sensing in live cells and dual-emission imaging in vivo.

Dual-channel and ratiometric imaging of QSG-7701 cells (a–d, pretreated with various concentrations of GSH); A549 and Hep-G2 cells incubated with DCM-IFC-4 (30 µM) and either untreated (e, g) or treated with (f, h) NEM (a derivatization agent which covalently sequesters GSH). Note: The green channel was 520 ± 20 nm, the red channel was 700 ± 20 nm, and ratiometric images were generated from the 520 and 700 nm channels, λ_ex = 488 nm. i Dual-channel response with internal reference signal in cells. Data with error bars are expressed as mean ± s.d., n = 3. Source data are provided as a Source Data file. j Standard curve of the I_{520 nm}: I_{700 nm} ratio as a function of GSH concentration. Data with error bars are expressed as mean ± s.d., n = 3. Source data are provided as a Source Data file. k Ratio value in A549 and Hep-G2 cells with and without NEM. Data with error bars are expressed as mean ± s.d., n = 3. Source data are provided as a Source Data file. (l, m) In vivo dual-channel fluorescence imaging of xenograft tumor (A549 cell) bearing mice at various times (0.5, 2, 4, 6, 12, 24 h) after the intravenous injection of DCM-IFC-4 (2.44 mg kg⁻¹), the tumor site is circled in red. n, o Ex vivo dual-channel fluorescence imaging of the excised organs (tumor, heart, liver, spleen, lung, and kidney) at 24 h after the intravenous injection of DCM-IFC-4. Note: fluorescence signals at 600 nm (rainbow scale) and 700 nm (yellow-red scale).