Functional Near‐Infrared Fluorescent Probes

Near‐infrared (NIR) fluorescent probes have attracted much attention, but despite the availability of various NIR fluorophores, only a few functional NIR probes, that is, probes whose absorption and/or fluorescence spectra change upon specific reaction with biomolecules, have been developed. However, functional probes operating in the NIR range that can be targeted to protons, metal ions, nitric oxide, β‐galactosidase, and cellular stress markers are expected to be effective for fluorescence imaging in vivo. This Focus Review concentrates on these functional NIR probes themselves, not their applications.     

Activatable Molecular Probes for Second Near‐Infrared Fluorescence,Chemiluminescence, and Photoacoustic Imaging

Optical imaging plays a crucial role in biomedicine. However, due to strong light scattering and autofluorescence in biological tissue between 650–900 nm, conventional optical imaging often has a poor signal‐to‐background ratio and shallow penetration depth, which limits its ability in deep‐tissue in vivo imaging. Second near‐infrared fluorescence, chemiluminescence, and photoacoustic imaging modalities mitigate these issues by their respective advantages of minimized light scattering, eliminated external excitation, and ultrasound detection. To enable disease detection, activatable molecular probes (AMPs) with the ability to change their second near‐infrared fluorescence, chemiluminescence, or photoacoustic signals in response to a biomarker have been developed. This Minireview summarizes the molecular design strategies, sensing mechanisms, and imaging applications of AMPs. The potential challenges and perspectives of AMPs in deep‐tissue imaging are also discussed.     

A New 3,5‐Bisporphyrinylpyridine Derivative as a Fluorescent Ratiometric Probe for Zinc Ions

A new 3,5‐disubstituted pyridine with two porphyrin moieties was prepared through an efficient synthetic approach involving 2‐formyl‐5,10,15,20‐tetraphenylporphyrin ( 1 ), piperidine, and catalytic amounts of [La(OTf)₃]. 3,5‐Bis(5,10,15,20‐tetraphenylporphyrin‐2‐ylmethyl)pyridine ( 2 ) was fully characterized and its sensing ability towards Zn²⁺, Cu²⁺, Hg²⁺, Cd²⁺, and Ag⁺ was evaluated in solution by absorption and fluorescence spectroscopy and in gas phase by using matrix‐assisted laser desorption/ionization (MALDI)‐TOF mass spectrometry. Strong changes in the ground and excited state were detected in the case of the soft metal ions Zn²⁺, Cd²⁺, Hg²⁺, and Cu²⁺. A three‐metal‐per‐ligand molar ratio was obtained in all cases and a significant ratiometric behavior was observed in the presence of Zn²⁺ with the appearance of a new band at 608 nm, which can be assigned to a metal‐to‐ligand charge transfer. The system was able to quantify 79 ppb of Zn²⁺ and the theoretical calculations are in accordance with the stoichiometry observed in solution. The gas‐phase sensorial ability of compound 2 towards all metal ions was confirmed by using MALDI‐TOF MS and in solid state by using polymeric films of polymethylmethacrylate (PMMA) doped with ligand 2 . The results showed that compound 2 can be analytically used to develop new colorimetric molecular devices that are able to discriminate between Hg²⁺ and Zn²⁺ in solid phase. The crystal structure of Zn^II complex of 3,5‐bisporphyrinylpyridine was unequivocally elucidated by using single‐crystal X‐ray diffraction studies.     

A Three‐Photon Active Organic Fluorophore for Deep Tissue Ratiometric Imaging of Intracellular Divalent Zinc

Deep tissue bioimaging with three‐photon (3P) excitation using near‐infrared (NIR) light in the second IR window (1.0–1.4 μm) could provide high resolution images with an improved signal‐to‐noise ratio. Herein, we report a photostable and nontoxic 3P excitable donor‐π‐acceptor system (GMP) having 3P cross‐section (σ₃) of 1.78×10^?80 cm⁶ s² photon^?2 and action cross‐section (σ₃η₃) of 2.31×10^?81 cm⁶ s² photon^?2, which provides ratiometric fluorescence response with divalent zinc ions in aqueous conditions. The probe signals the Zn²⁺ binding at 530 and 600 nm, respectively, upon 1150 nm excitation with enhanced σ₃ of 1.85×10^?80 cm⁶ s² photon^?2 and σ₃η₃ of 3.33×10^?81 cm⁶ s² photon^?2. The application of this probe is demonstrated for ratiometric 3P imaging of Zn²⁺ in vitro using HuH‐7 cell lines. Furthermore, the Zn²⁺ concentration in rat hippocampal slices was imaged at 1150 nm excitation after incubation with GMP, illustrating its potential as a 3P ratiometric probe for deep tissue Zn²⁺ ion imaging.     

A Pyrene‐functionalized Polynorbornene for Ratiometric Fluorescence Sensing of Pyrophosphate

A novel pyrene‐functionalized polynorbornene ( P1 ) bearing sulfonamide NH and triazolium donors has been synthesized for ratiometric fluorescence sensing of PPi in aqueous solution. In addition, P1 is also used to monitor intracellular PPi and to detect PPi released during polymerase chain reaction.     

New Chemodosimetric Reagents as Ratiometric Probes for Cysteine and Homocysteine and Possible Detection in Living Cells and in Blood Plasma

In this work, we have rationally designed and synthesized two new reagents ( L₁ and L₂ ), each bearing a pendant aldehyde functionality. This aldehyde group can take part in cyclization reactions with β‐ or γ‐amino thiols to yield the corresponding thiazolidine and thiazinane derivatives, respectively. The intramolecular charge‐transfer (ICT) bands of these thiazolidine and thiazinane derivatives are distinctly different from those of the molecular probes ( L₁ and L₂ ). Such changes could serve as a potential platform for using L₁ and L₂ as new colorimetric/fluorogenic as well as ratiometric sensors for cysteine (Cys) and homocysteine (Hcy) under physiological conditions. Both reagents proved to be specific towards Cys and Hcy even in the presence of various amino acids, glucose, and DNA. Importantly, these two chemodosimetric reagents could be used for the quantitative detection of Cys present in blood plasma by using a pre‐column HPLC technique. Such examples are not common in contemporary literature. MTT assay studies have revealed that these probes have low cytotoxicity. Confocal laser scanning micrographs of cells demonstrated that these probes could penetrate cell membranes and could be used to detect intracellular Cys/Hcy present within living cells. Thus, the results presented in this article not only demonstrate the efficiency and specificity of two ratiometric chemodosimeter molecules for the quantitative detection of Cys and Hcy, but also provide a strategy for developing reagents for analysis of these vital amino acids in biological samples.     

Ratiometric Single‐Nanoparticle Oxygen Sensors for Biological Imaging

It makes sense : Conjugated polymer nanoparticles doped with a platinum porphyrin dye exhibit bright phosphorescence that is highly sensitive to the concentration of molecular oxygen. The small size, extraordinary brightness, excellent sensitivity, and ratiometric emission, together with the demonstration of single‐particle sensing and cellular uptake, indicate the potential of the nanoparticle sensors for quantitative mapping of local molecular oxygen concentration.

     

Colorimetric and Fluorescent Bimodal Ratiometric Probes for pH Sensing of Living Cells

pH measurement is widely used in many fields. Ratiometric pH sensing is an important way to improve the detection accuracy. Herein, five water‐soluble cationic porphyrin derivatives were synthesized and their optical property changes with pH value were investigated. Their pH‐dependent assembly/disassembly behaviors caused significant changes in both absorption and fluorescence spectra, thus making them promising bimodal ratiometric probes for both colorimetric and fluorescent pH sensing. Different substituent identity and position confer these probes with different sensitive pH‐sensing ranges, and the substituent position gives a larger effect. By selecting different porphyrins, different signal intensity ratios and different fluorescence excitation wavelengths, sensitive pH sensing can be achieved in the range of 2.1–8.0. Having demonstrated the excellent reversibility, good accuracy and low cytotoxicity of the probes, they were successfully applied in pH sensing inside living cells.     

Near‐Infrared Fluorescent Molecular Probe for Sensitive Imaging of Keloid

Early detection of skin diseases is imperative for their effective treatment. However, fluorescence molecular probes that allow this are rare. The first activatable near‐infrared (NIR) fluorescent molecular probe is reported for sensitive imaging of keloid cells, skin cells from abnormal scar fibrous lesions. As keloid cells have high expression levels of fibroblast activation protein‐alpha (FAPα), the probe (FNP1) is designed to have a caged NIR dye and a FAPα‐cleavable peptide substrate linked by a self‐immolative segment. FNP1 can quickly and specifically turn on its fluorescence at 710 nm by 45‐fold in the presence of FAPα, allowing it to effectively recognize keloid cells from normal skin cells. Integration of FNP1 with a simple microneedle‐assisted topical application enables sensitive detection of keloid cells in metabolically‐active human skin tissue with a theoretical limit of detection down to 20 000 cells.     

Water‐Soluble Polymer Functionalized CdTe/ZnS Quantum Dots: A Facile Ratiometric Fluorescent Probe for Sensitive and Selective Detection of Nitroaromatic Explosives

A ratiometric fluorescent probe based on dual luminescence QD/CPL for selective sensing of the nitroaromatic explosive picric acid (PA) was constructed. The observed ratiometric fluorescence intensity change allows the quantitative detection of PA with a detection limit of 9 nM .     

A Ratiometric Fluorescent Probe for Gold and Mercury Ions

A fluorescent probe that displays a ratiometric fluorescence response towards gold and mercury ions has been devised. Emitting at a relatively longer wavelength, the conjugated form of the fluorescent dye transforms in the presence of the gold or mercury ions into a new dye, the molecular structure of which lacks the conjugation and consequently emits at a distinctly shorter wavelength.     

Phenoxazine‐based Near‐infrared Fluorescent Probes for the Specific Detection of Copper (II) Ions in Living Cells

Abstract : It is well known that copper ions play a critical role in various physiological processes. However, a variety of human diseases are tightly correlated with copper overload. Although there are numerous fluorescent probes capable of detecting copper ions, most of them are “turn‐off” probes owing to copper (II) ions fluorescence quenching effect, resulting in poor sensitivity. Herein, a novel “turn‐on” near‐infrared (NIR) fluorescent probe PZ‐N based on phenoxazine was designed and synthesized for the selective detection of copper (II) ions (Cu²⁺). Upon the addition of Cu²⁺, the probe could quickly react with Cu²⁺ and emit strong fluorescence, along with colour change from colourless to obvious blue. Moreover, the probe PZ‐N showed good water solubility, high selectivity, and excellent sensitivity with low limit of detection (1.93 nM) towards copper (II) ions. More importantly, PZ‐N was capable of effectively detecting Cu²⁺ in living cells.     

Near‐IR Core‐Substituted Naphthalenediimide Fluorescent Chemosensors for Zinc Ions: Ligand Effects on PET and ICT Channels

Near‐IR (NIR) emission can offer distinct advantages for both in vitro and in vivo biological applications. Two NIR fluorescent turn‐on sensors N,N′‐di‐n‐butyl‐2‐(N‐{2‐[bis(pyridin‐2‐ylmethyl)amino]ethyl})‐6‐(N‐piperidinyl)naphthalene‐1,4,5,8‐tetracarboxylic acid bisimide and N,N′‐di‐ n‐butyl‐2‐[N,N,N′‐tri(pyridin‐2‐ylmethyl)amino]ethyl‐6‐(N‐piperidinyl)naphthalene‐1,4,5,8‐tetracarboxylic acid bisimide (PND and PNT) for Zn²⁺ based on naphthalenediimide fluorophore are reported. Our strategy was to choose core‐substituted naphthalenediimide (NDI) as a novel NIR fluorophore and N,N‐di(pyridin‐2‐ylmethyl)ethane‐1,2‐diamine (DPEA) or N,N,N′‐tri(pyridin‐2‐ylmethyl)ethane‐1,2‐diamine (TPEA) as the receptor, respectively, so as to improve the selectivity to Zn²⁺. In the case of PND, the negligible shift in absorption and emission spectra is strongly suggestive that the secondary nitrogen atom (directly connected to the NDI moiety, N¹) is little disturbed with Zn²⁺. The fluorescence enhancement of PND with Zn²⁺ titration is dominated with a typical photoinduced electron‐transfer (PET) process. In contrast, the N¹ atom for PNT can participate in the coordination of Zn²⁺ ion, diminishing the electron delocalization of the NDI moiety and resulting in intramolecular charge‐transfer (ICT) disturbance. For PNT, the distinct blueshift in both absorbance and fluorescence is indicative of a combination of PET and ICT processes, which unexpectedly decreases the sensitivity to Zn²⁺. Due to the differential binding mode caused by the ligand effect, PND shows excellent selectivity to Zn²⁺ over other metal ions, with a larger fluorescent enhancement centered at 650 nm. Also both PND and PNT were successfully used to image intracellular Zn²⁺ ions in the living KB cells.     

A Triphenyl Amine‐Based Solvatofluorochromic Dye for the Selective and Ratiometric Sensing of OCl− in Human Blood Cells

A new visible‐light‐excitable fluorescence ratiometric probe for OCl^? has been developed based on a triphenylamine‐diamiomaleonitrile (TAM) moiety. The structure of the dye was confirmed by single‐crystal X‐ray analysis. It behaves as a highly selective and sensitive probe for OCl^? over other analytes with a fast response time (～100 s). OCl^? reacts with the probe leading to the formation of the corresponding aldehyde in a mixed‐aqueous system. The detection limit of the probe is in the 10^?8 M range. The probe (TAM) also exhibits solvatofluorochromism. Changing the solvent from non‐polar to polar, the emission band of TAM largely red‐shifted. Moreover, the probe shows an excellent performance in real‐life application in detecting OCl^? in human blood cells. The experimentally observed changes in the structure and electronic properties of the probe after reaction with OCl^? were studied by DFT and TDDFT computational calculations.     

Recent Progress of Small-Molecule Ratiometric Fluorescent Probes for Peroxynitrite in Biological Systems

Peroxynitrite (ONOO⁻) as a major reactive oxygen species plays important roles in cellular signal transduction and homeostatic regulation. Precise detection of ONOO⁻ in biological systems is vital for exploring its physiological and pathological function. Among numerous detection methods, fluorescence imaging technology using fluorescent probes offers some advantages, including simple operation, high sensitivity and selectivity, as well as real-time and nondestructive detection. In particular, ratiometric fluorescent probes, in which the built-in calibration of the two emission bands prevents interference from the biological environment, have been extensively employed to monitor the fluctuation of bioactive species. In this review, we will discuss small-molecule ratiometric fluorescent probes for ONOO⁻ in live cells or in vivo, which involves chemical structures, response mechanisms, and biological applications. Moreover, the challenges and future prospects of ONOO⁻-responsive ratiometric fluorescent probe are also proposed.     

Design of Dual‐Emission Chemosensors for Ratiometric Detection of ATP Derivatives

Nucleoside pyrophosphate (nucleoside PP) derivatives are widespread in living cells and play pivotal roles in various biological events. We report novel fluorescence chemosensors for nucleoside PPs that make use of coordination chemistry. The chemosensors, which contain two Zn^II–dipicolylamine units, bind strongly to nucleoside PPs (K_app>10⁶ M ^?1) in aqueous solution and sense them by a dual‐emission change. Detailed fluorescence and UV/Vis spectral studies revealed that the emission changes of the chemosensors upon binding to nucleoside PPs can be ascribed to the loss of coordination between Zn^II and the acridine fluorophore. This is a unique sensing system based on the anion‐induced rearrangement of the coordination. Furthermore, we demonstrated the utility of these chemosensors in real‐time monitoring of two important biological processes involving nucleoside PP conversion: the apyrase‐catalyzed hydrolysis of nucleoside PPs and the glycosyl transfer catalyzed by β‐1,4‐galactosyltransferase.     

Nanobody‐Conjugated Nanotubes for Targeted Near‐Infrared In Vivo Imaging and Sensing

Fluorescent nanomaterials such as single‐walled carbon nanotubes (SWCNTs) have many advantages in terms of their photophysics, but it is difficult to target them to specific locations in living systems. In contrast, the green fluorescent protein (GFP) has been genetically fused to proteins in many cells and organisms. Therefore, GFP can be seen not only as a fluorophore but as a universal target/handle. Here, we report the conjugation of GFP‐binding nanobodies to DNA‐wrapped SWCNTs. This approach combines the targeting capabilities of GFP‐binding nanobodies and the nonbleaching near‐infrared fluorescence (850–1700 nm) of SWCNTs. These conjugates allow us to track single Kinesin‐5‐GFP motor proteins in developing embryos of Drosophila melanogaster. Additionally, they are sensitive to the neurotransmitter dopamine and can be used for targeted sensing of dopamine in the nm regime.     

Design of Turn‐On Near‐Infrared Fluorescent Probes for Highly Sensitive and Selective Monitoring of Biopolymers

Simple, sensitive, and selective detection of specific biopolymers is critical in a broad range of biomedical and technological areas. We present a design of turn‐on near‐infrared (NIR) fluorescent probes with intrinsically high signal‐to‐background ratio. The fluorescent signal generation mechanism is based on the aggregation/de‐aggregation of phthalocyanine chromophores controlled by selective binding of small‐molecule “anchor” groups to a specific binding site of a target biopolymer. As a proof‐of‐concept, we demonstrate a design of a sensor for EGFR tyrosine kinase—an important target in cancer research. The universality of the fluorescent signal generation mechanism, as well as the dependence of the response selectivity on the choice of the small‐molecule “anchor” group, make it possible to use this approach to design reliable turn‐on NIR fluorescent sensors for detecting specific protein targets present in the low‐nanomolar concentration range.     

双光子荧光探针研究及其应用

双光子荧光显微成像兼具诸如近红外激发、暗场成像、避免荧光漂白和光致毒、定靶激发、高横向分辨率与纵向分辨率、降低生物组织吸光系数及降低组织自发荧光干扰等特点而显著地优于单光子荧光显微成像,为生命科学研究提供了更为锐利的工具。而用于像离子的含量及其对生理的影响、离子参与的生理活动机制、离子与分子的作用、特定分子的分布及其相互作用等方面研究的双光子荧光探针,是实现成像的关键。双光子荧光探针的研究旨在促进双光子荧光显微镜应用的发展,促进生命科学、医学科学的快速发展,同时也带动双光子荧光探针所隶属的化学这一学科的发展。因此对双光子荧光探针的研究具有重要的理论和实践意义。该文综述了双光子荧光显微成像的优点、双光子荧光探针设计的原理及双光子荧光探针在离子分析方面的应用,并展望了这类荧光探针的发展趋势与应用前景。