Fluorescent small organic probes for biosensing

Abstract

Small-molecule based fluorescent probes are increasingly important for the detection and imaging of biological signaling molecules due to their simplicity, high selectivity and sensitivity, whilst being non-invasive, and suitable for real-time analysis of living systems. With this perspective we highlight sensing mechanisms including Förster resonance energy transfer (FRET), intramolecular charge transfer (ICT), photoinduced electron transfer (PeT), excited state intramolecular proton transfer (ESIPT), aggregation induced emission (AIE) and multiple modality fluorescence approaches including dual/triple sensing mechanisms (DSM or TSM). Throughout the perspective we highlight the remaining challenges and suggest potential directions for development towards improved small-molecule fluorescent probes suitable for biosensing.

Scheme 1. Schematic representation of the fluorescent mechanisms discussed in this perspective.

Fig. 1. The mechanism of Förster resonance energy transfer (FRET). R is the distance between the energy donor and acceptor, J(λ) represents the degree of spectroscopic overlap between the donor emission and the acceptor absorption. Reproduced with permission from ref. . Copyright 2013, American Chemical Society.

Scheme 2. FRET-based ratiometric fluorescent probe ThioRB-FITC-MSN for the detection of HOCl.

Scheme 3. Cy3 and Cy5 (energy donor and acceptor)-based FRET fluorescent probe PNCy3Cy5, developed for the ratiometric detection of ONOO⁻. Also shown is the structure of its cleavage product PNCy3.

Scheme 4. FRET-based ratiometric fluorescent probe MITO-CC for the detection of ONOO⁻.

Scheme 5. FRET-based ratiometric fluorescent probe FIP-1 for the detection of Fe²⁺.

Scheme 6. FRET-based N₃-CR-PO₄ developed for the detection of H₂S and phosphatase.

Scheme 7. Illustration for the working principle of the designed TP ratiometric fluorescent probe AF633mCyd, for the determination of BACE1 in neurons and mouse brain tissue slice. Reproduced with permission from ref. . Copyright 2020, The Royal Society of Chemistry.

Scheme 8. Detection mechanism of probe DCM-βgal towards β-gal.

Scheme 9. Mechanism of detection of CYP2J2 by probe BnXPI, with initial test compound MXPI and fluorophore OXPI (deprotonated HXPI) also shown.

Scheme 10. Detection of ˙OH with ICT probe MD-B.

Scheme 11. Detection of ROS with TP ICT probe AzuFluor® 483-Bpin.

Scheme 12. High and low concentration of Cys detection pathways of probe CHMC-Thiol.

Scheme 13. Reversible detection of HCN of ICT probe MRP1.

Scheme 14. The mechanism of PeT-based fluorescent probes.

Scheme 15. Frontier orbital explanation for the PeT effect.

Scheme 16. The recognition mechanism of probe NI-OPD towards MGO.

Scheme 17. The structures of fluorescent probes 4, 5, 6, and 7.

Scheme 18. The recognition mechanism of probe LyNC towards H₂O₂.

Fig. 2. Schematic diagram of SP1 and in vivo fluorescence imaging of SP1 in a HT-29 tumour-bearing mouse model viaSP1 injection: (a) 0.1 mM, 100 μL; (b) 0.5 mM, 100 μL; (c) 1 mM, 100 μL; (d) 2 mM, 100 μL. The fluorescence signal was imaged at 500 to 720 nm under excitation with a 460 nm CW laser (power density of 1 mW cm⁻²). Reproduced with permission from ref. . Copyright 2018 American Chemical Society.

Scheme 19. Diagrammatic description of the ESIPT process. Reproduced with permission from ref. . Copyright 2018, The Royal Society of Chemistry.

Scheme 20. The structures of fluorescent probes NP1, NP2, NP3 and NP4.

Scheme 21. The structures of fluorescent probes ABAH-LW and TCBT-OMe.

Scheme 22. The structures of fluorescent probe HBTP-mito.

Scheme 23. The structures of fluorescent probes CORM3-green.

Scheme 24. ESIPT probes 3-HF-X (X = OMe, Me, H) for detecting ONOO⁻. The normal (N) and phototautomeric (T*) forms are shown.

Fig. 3. Fluorescence imaging of a brain section of a transgenic mouse treated with 3-HF-OMe (20 μM) (a) without and (b) with ONOO⁻ (30 μM). The excitation/emission wavelengths for the blue (N-state), green (T*-state), and red (anti-Aβ antibody) channels are 404/425−475, 404/500−550, and 561/640−730 nm, respectively. The white arrows indicate stained Aβ aggregates. Reprinted from ref. . Copyright 2018, American Chemical Society.

Scheme 25. Detection of CO by AIEgen BTCV–CO.

Fig. 4. Structures of AIEgens QM-3–QM-6.

Scheme 26. Nitroreductase detection mechanism by AIEgen Q-NO2.

Fig. 5. Structure of AIEgen QM-FN-SO3.

Scheme 27. DDP-1 detection of H₂S and H₂S_n using two distinct emission channels.

Scheme 28. ICT and FRET-based probe Mito-CM-BP developed to allow for the simultaneous detection of GSH and SO₂.

Scheme 29. A lysosome targetable fluorescent probe LysoFP-NO2 for detection of CO.

Scheme 30. The structure of probe AIE-mito-TPP and schematic representation of intracellular tracking and the therapeutic effect of AIE-mito-TPP in cancer cells. Reprinted from ref. . Copyright 2014, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Scheme 31. Detection of β-gal by combined AIE-ESIPT probe QM-HBT-βgal.

Scheme 32. Combined AIE-ICT probe CSMPP.