Imaging hypoxia using fluorescence probes for nitroreductase (NTR) have attracted much attention in last decade. At least three different linkers have been commonly used to connect the recognition unit and reporting unit in reported probes for NTR. Meanwhile, the linker is known to be a key factor for achieving best sensing performance. In this work, three near-infrared fluorescence probes CyNP-1, CyNP-2 and CyNP-3 were designed and synthesized from an aminocyanine dye CyNP. The three probes have the same recognition unit and same fluorescence reporting unit, but different linkers. CyNP-1 was found to have the best sensing performance for NTR with 40-fold of fluorescence enhancement. It is well investigated how the difference of the linkers brings out the different sensing performance by HPLC, MS and docking calculations. In the end, CyNP-1 was found to have good selectivity for NTR and used to imaging hypoxia in Hela cells.Fine-tailoring the linker of nitroreductase fluorescence probes with a given recognition unit and reporting unit is found to be able to achieve the best sensing performance.Download high-res image (78KB)Download full-size image

Near-infrared (NIR) fluorescence imaging technology calls for highly bright and photostable emissive materials for long-term and real-time bioimaging and medical diagnosis. Herein, we report that four aminocyanine dyes were covalently encapsulated inside silica nanoparticles by reverse microemulsion using different linkage methods. The fluorescence brightness and photostability of the obtained fluorescent silica nanoparticles (FSNPs) were found to have direct correlation with the used covalent encapsulation methods, especially the number of anchoring sites of the encapsulated dyes. The aminocyanine dye 4-Si contains three anchoring sites for embedding into the silica nanoparticles, and provides FSNP-4 – the FSNP with the best brightness and photostability.

•A novel fluorescent dye was synthesized based on a BODIPY–hemicyanine dyad structure.•The probe exhibited a ‘turn-on’ fluorescence response to viscosity and hypochlorite.•The molecule can penetrate cell membrane easily and make fast fluorescence bioimaging.•The attack of hypochlorite caused the increase of intracellular viscosity.A novel fluorescent chemosensor was designed and synthesized based on a borondipyrromethene-hemicyanine dyad structure. This probe can be employed for the measurement of both viscosity and hypochlorite under different wavelengths. In non-viscous media, the dye had a very low fluorescence quantum yield. With the increase of viscosity, the fluorescence at 600 nm was enhanced significantly, which could be utilized for quantitative determination of viscosity. In addition, the probe exhibited a fast (within 1 min) ‘turn-on’ fluorescence response to hypochlorite with high selectivity. The fluorescence at 510 nm was directly proportional to hypochlorite concentration. Confocal fluorescence imaging experiments demonstrated the probe could permeate cell membranes and visualize viscosity and hypochlorite in living cells, where the action of hypochlorite caused the increase of intracellular viscosity.

A turn-on and colorimetric metal-free long lifetime fluorescent probe for sensing Cys has been synthesized. Utilizing the long emission-lifetime of DCF-MPYM, the time-resolved luminescent assay of DCF-MPYM-thiol for sensing Cys was realized successfully. DCF-MPYM-thiol can realize confocal and time-resolved microscopy bioimaging for Cys in living cells.

Compared with fluorescence imaging utilizing fluorophores whose lifetimes are in the order of nanoseconds, time-resolved fluorescence microscopy has more advantages in monitoring target fluorescence. In this work, compound DCF-MPYM, which is based on a fluorescein derivative, showed long-lived luminescence (22.11 μs in deaerated ethanol) and was used in time-resolved fluorescence imaging in living cells. Both nanosecond time-resolved transient difference absorption spectra and time-correlated single-photon counting (TCSPC) were employed to explain the long lifetime of the compound, which is rare in pure organic fluorophores without rare earth metals and heavy atoms. A mechanism of thermally activated delayed fluorescence (TADF) that considers the long wavelength fluorescence, large Stokes shift, and long-lived triplet state of DCF-MPYM was proposed. The energy gap (ΔEST) of DCF-MPYM between the singlet and triplet state was determined to be 28.36 meV by the decay rate of DF as a function of temperature. The ΔEST was small enough to allow efficient intersystem crossing (ISC) and reverse ISC, leading to efficient TADF at room temperature. The straightforward synthesis of DCF-MPYM and wide availability of its starting materials contribute to the excellent potential of the compound to replace luminescent lanthanide complexes in future time-resolved imaging technologies.

A novel red-emission boron-dipyrromethene(BODIPY) dye with a pyrrole ring was synthesized simply via one-pot reaction. The spectral properties of it were investigated under the conditions of different solvents. The results show that the as-prepared BODIPY dye is extremely sensitive to solvent polarity, and the fluorescent emission enhances with the decrease of solvent polarity. In aqueous buffer, the addition of bovine serum albumin leads to a ratiometric change in absorption spectra with an association constant of 1.16×106 L/mol. Meanwhile, the fluorescence emission increases greatly at 622 nm but changes slightly at 575 nm. The response time is very short(less than 3 min), and the changes of color can be noticed by naked eyes. Bovine serum albumin can be detected by this ratiometric fluorescence probe, but other proteins or enzymes cannot be detected by this method, which indicates that this novel dye has high selectivity towards bovine serum albumin. The reason is that bovine serum albumin has suitable hydrophobic cavities for binding with the dye. In addition, the dye molecule can penetrate cell membrane easily and make a fast fluorescent stain, which makes it a potential probe for living-cell fluorescence imaging.

A new fluorescent probe RY was synthesized for the detection of Au3+ ions based on a rhodamine B derivative. The fluorescent probe showed good selectivity and sensitivity to Au3+ ions. Obvious color and fluorescence changes could be observed with the naked eye while the fluorescent probe reacted with the Au3+ ions. The detection limit of the probe was determined to be 36 ppb by the fluorescence titration; the excellent linear relationship suggests that the probe is potentially useful for quantitative detection of Au3+in vitro. We also demonstrated its bioimaging application in both living cells and mice; this was the first time that a fluorescent probe was successfully applied to imaging Au3+ in living animals.

Fluorescence resonance energy transfer (FRET) has been widely used as a spectroscopic technique in various areas such as structural elucidation of biological molecules and their interactions, in vitro assays, in vivo monitoring in cellular research, nucleic acid analysis, signal transduction, light harvesting, and metallic nanomaterials. Meanwhile, based on the mechanism of FRET, a series of FRET nanomaterials systems have been recently developed as novel chemical sensors and biosensors. Compared with those based on small molecules traditional FRET systems, the surface chemistry of nanomaterial has encouraged the development of multiple probes based on linked recognition molecules such as peptides, nucleic acids, or small-molecule ligands. This critical review highlights the design and the applications of sensitive and selective ratiometric nanoprobes based on FRET. We focus on the benefits and limitations of nano-FRET systems and their applications as chemical sensors and biosensors.

A specific ratiometric nanoprobe for hypochlorite was constructed as a paradigm of FRET spectral unmixing. The separation of FRET pairs' emissions reaches 175 nm, which ensures that the FRET probing is more accurate. This new nanoprobe shows high selectivity and potential in biological systems.

Traditional organic fluorophores, like cyanine dyes, suffer from their poor stability and weak brightness of individual molecule. In this work, a novel cyanine dye with a large Stokes shift (∼75 nm) was encapsulated inside silica nanoparticles. The obtained small fluorescent silica nanoparticle (FSNP) exhibits more than ten times brightness than the free dye. The enhanced fluorescence brightness was assigned to the less homo Förster resonance energy transfer (HFRET) between multiple fluorophores, which was confirmed by the longer fluorescence lifetime of FSNP with a large Stokes shift than that with a normal Stokes shift. The FSNP’s photostability is much better than organic fluorophores and comparable with that of Quantum Dots. When used in bioimaging, the FSNP remained a stable fluorescence signal, while the control free dye faded within 12 h.Highlights► A new cyanine dye with a large Stokes shift (74 nm) was synthesized. ► The dye was encapsulated inside a silica nanoparticle in a core-shell format. ► The obtained nanoparticle exhibits brighter fluorescence than the free dye. ► The enhanced fluorescence brightness was assigned to the less homo-FRET. ► Better photostability and biostability are achieved.

Near-infrared (NIR) fluorescence imaging technology calls for highly bright and photostable emissive materials for long-term and real-time bioimaging and medical diagnosis. Herein, we report that four aminocyanine dyes were covalently encapsulated inside silica nanoparticles by reverse microemulsion using different linkage methods. The fluorescence brightness and photostability of the obtained fluorescent silica nanoparticles (FSNPs) were found to have direct correlation with the used covalent encapsulation methods, especially the number of anchoring sites of the encapsulated dyes. The aminocyanine dye 4-Si contains three anchoring sites for embedding into the silica nanoparticles, and provides FSNP-4 – the FSNP with the best brightness and photostability.

A specific ratiometric nanoprobe for hypochlorite was constructed as a paradigm of FRET spectral unmixing. The separation of FRET pairs' emissions reaches 175 nm, which ensures that the FRET probing is more accurate. This new nanoprobe shows high selectivity and potential in biological systems.