In this review we will explore recent advances in the design and application of excited-state intramolecular proton-transfer (ESIPT) based fluorescent probes. Fluorescence based sensors and imaging agents (probes) are important in biology, physiology, pharmacology, and environmental science for the selective detection of biologically and/or environmentally important species. The development of ESIPT-based fluorescence probes is particularly attractive due to their unique properties, which include a large Stokes shift, environmental sensitivity and potential for ratiometric sensing.

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Excited-state intramolecular proton-transfer (ESIPT) based fluorescence sensors and imaging agents

Organic fluorescent bioprobes provide robust and powerful analytical techniques for direct detection and monitoring of important biological species and their related biological processes in live samples. In contrast to conventional fluorophores with aggregation-caused quenching effect, aggregation-induced emission (AIE) luminogens (AIEgens) exhibit remarkable photophysical properties such as bright fluorescence in aggregate or solid state, large Stokes shift, excellent photostability, and long retention time in live samples. Benefiting from these advantages, AIEgen-based bioprobes have been widely applied in diverse biomedical applications. In this review, we first introduce the AIE concept and working principles of AIE bioprobes. Then we briefly summarize their applications in biosensing and bioimaging with some representative examples recently reported mainly in our group. The challenge and future development of AIE bioprobes are also discussed and highlighted. Hopefully, this review can inspire more researchers to participate in this fascinating area and expand the versatile biomedical applications.

AIE luminogens as fluorescent bioprobes

Lysosomes, which can be easily targeted by molecules with abundant amino groups, play critical roles in endocytosis, autophagy, and phagocytosis; thus, it is important to accurately characterize lysosomes, including lysosomal pH, in living cells to understand their physiological and pathological functions Herein, a new type of highly photoluminescent (PL) emerald carbon dots (CDs) was easily prepared through a functional preservation strategy (FPS) by simply mixing p-benzoquinone and ethanediamine at room temperature The as-prepared CDs possessed abundant amino groups preserved from ethanediamine owing to FPS, and they exhibited excellent photostability as compared to the commercial LysoTracker probes Consequently, they actively targeted lysosomes to sensitively respond to lysosomal pH in vitro owing to their abundant amino groups and good hydrophilicity Thus, we could successfully monitor lysosomal pH dynamics during apoptosis in live cells

A functional preservation strategy for the production of highly photoluminescent emerald carbon dots for lysosome targeting and lysosomal pH imaging.

Measurements of two-photon fluorescence excitation (TPE) spectra are presented for 11 common molecular fluorophores in the excitation wavelength range 690 nm < λ < 1050 nm. Results of excitation by ∼100-fs pulses of a mode-locked Ti:sapphire laser are corroborated by single-mode cw Ti:sapphire excitation data in the range 710 nm < λ < 840 nm. Absolute values of the TPE cross section for Rhodamine B and Fluorescein are obtained by comparison with one-photon-excited fluorescence, assuming equal emission quantum efficiencies. TPE action cross sections for the other nine fluorophores are also determined. No differences between one-photon- and two-photon-excited fluorescence emission spectra are found. TPE emission spectra are independent of excitation wavelength. With both pulsed and cw excitation the fluorescence emission intensities are strictly proportional to the square of the excitation intensity to within ±4% for excitation intensities sufficiently below excited-state saturation.

Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm

Small-molecule fluorescent probes for specific detection and imaging of chemical species inside lysosomes

An NIR-emitting lysosome-targeting probe with large Stokes shift via coupling cyanine and excited-state intramolecular proton transfer

Bright red-emitting pyrene derivatives with a large Stokes shift for nucleus staining

In this review, we will summarize our recent progress in the design and application of novel organic sensors with emission in the near-infrared region (600-900 nm). By coupling different functional groups with excited-state intramolecular proton transfer (ESIPT) segments, new probes are developed to achieve a large Stokes shift, high sensitivity, and selectivity and to tune the emission toward the near-infrared region. The developed probes exhibit attractive optical properties for bioimaging and environmental science applications. In addition, we further discuss the photophysical properties of ESIPT dyes and how their fluorescence could be affected by structural/environmental factors, which should be considered during the development of robust ESIPT-based fluorescence probes. Their potential applications as imaging reagents are illustrated for intracellular membranes, mitochondria, lysosomes, and some biomolecules.

Chathura S. Abeywickrama

Papers

An NIR-emitting lysosome-targeting probe with large Stokes shift via coupling cyanine and excited-state intramolecular proton transfer

Bright red-emitting pyrene derivatives with a large Stokes shift for nucleus staining

Progress in Tuning Emission of the Excited-State Intramolecular Proton Transfer (ESIPT)-Based Fluorescent Probes

A bright red-emitting flavonoid for Al3+ detection in live cells without quenching ICT fluorescence.

Red-emitting pyrene-benzothiazolium: unexpected selectivity to lysosomes for real-time cell imaging without alkalinizing effect.