Synthesis of Ethynylated Phenothiazine Based Fluorescent Boronic Acid Probes

Ethynylated phenothiazine based fluorescent boronic acid probes were prepared. Sonogashira coupling reaction was used to introduce substituted phenylethynylene fragments to the phenothiazine fluorophore to extend the π-conjugation and to enhance the emission property. The photophysical properties and the binding properties of these probes with hydroxyl acids were investigated. We found that the probes with significant ICT effect show emissions which are sensitive to solvent polarity. The phenothiazine moiety is proved to be electron-donating. We found the substitution profile imparts significant effect on the photophysical properties of the probes. For example, one of the probes shows d-PeT effect, whereas the regioisomer probe with similar π-conjugation fragment but different substitution profile shows the a-PeT effect. The easy derivatization of phenothiazine fluorophore, the structure-photophysical property relation and the novel d-PeT fluorescence transduction profile of the phenothiazine based probes described herein may inspire more investigation into this fascinating research area.     

Advances in fluorescence diagnosis to track footprints of cancer progression in vivo

Fluorescence spectroscopy and imaging have been widely used for in vivo cancer diagnosis and therapy monitoring in preclinical models, as well as clinical translation. Great attempts have been made to develop novel fluorescence techniques and improve on existing ones, which can now be used in conjunction with newly developed fluorescent probes for specific cancer imaging. In this review, a broad overview of fluorescence techniques is provided, including photodynamic diagnosis, laser confocal endomicroscopy and fluorescence lifetime imaging, coupled with endogenous and exogenous fluorophores. In particular, endogenous fluorophores, such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), are highlighted as they are linked to cellular metabolism in precancer growth. The use of near‐infrared dyes, such as indocynanine green (ICG), for imaging deep‐tissue regions is also reviewed. In addition, diagnostic algorithms used for tissue classification and cancer detection will be discussed. Lastly, emerging technologies in fluorescence diagnosis will also be included.     

Modification of single molecule fluorescence using external fields

Controlling and manipulating the fluorescence of single fluorophores is of great interest in recent years for its potential uses in improving the performance of molecular photonics and molecular electronics, such as in organic light-emitting devices, single photon sources, organic field-effect transistors, and probes or sensors based on single molecules. This review shows how the fluorescence emission of single organic molecules can be modified using local electromagnetic fields of metallic nanostructures and electric-field-induced electron transfer. Electric-field-induced fluorescence modulation, hysteresis, and the achievement of fluorescence switch are discussed in detail.     

Computational design of ratiometric two-photon fluorescent Zn2+ probes based on quinoline and di-2-picolylamine moieties

To improve two-photon absorption (TPA) response of a newly synthesized probe, a series of ratiometric two-photon fluorescent Zn²⁺ sensors based on quinoline and DPA moieties have been designed. The one-photon absorption, TPA, and emission properties of the experimental and designed probes before and after coordination with Zn²⁺ are investigated employing the density functional theory in combination with response functions. The design consists of two levels. In the first level of design, five probes are constructed through using several electron acceptors or donors to increase accepting or donating ability of the fluorophores. It shows that all the designed probes have stronger TPA intensities at longer wavelengths with respect to the experimental probe because of the increased intra-molecular charge transfer. Moreover, it is found that the probe 4 built by adding an acyl unit has the largest TPA cross section among the designed structures due to the form of longer conjugated length and more linear backbone. One dimethylamino terminal attached along the skeleton can improve TPA intensity more efficiently than two side amino groups. Therefore, in the second level of design, a new probe 7 is formed by both an acyl unit and a dimethylamino terminal. It exhibits that the TPA cross sections of probe 7 and its zinc complex increase dramatically. Furthermore, the fluorescence quantum yields of the designed probes 4 and 7 are calculated in a new way, which makes use of the relation between the computed difference of dipole moment and the measured fluorescence quantum yield. The result shows that our design also improves the fluorescence quantum yield considerably. All in all, the designed probes 4 and 7 not only possess enhanced TPA intensities but also have large differences of emission wavelength upon Zn²⁺ coordination and strong fluorescence intensity, which demonstrates that they are potential ratiometric two-photon fluorescent probes.     

Ophthalmic Glucose Monitoring Using Disposable Contact Lenses—A Review

We have developed a range of disposable and colorless tear glucose sensing contact lenses, using off-the-shelf lenses embedded with new water soluble, highly fluorescent and glucose sensitive boronic acid containing fluorophores. The new lenses are readily able to track tear glucose levels and therefore blood glucose levels, which are ideally suited for potential use by diabetics. The fluorescence responses from the lenses can be monitored using simple excitation and emission detection devices. The novelty of our approach is two fold. Firstly, the notion of sensing extremely low glucose concentrations in tears, which track blood levels, by our contact lens approach, and secondly, the unique compatibility of our new glucose signaling probes with the internal mildly acidic contact lens environment. The new lenses are therefore ideal for the non-invasive and continuous monitoring of tear glucose, with about 15-min response time, and a measured shelf life in excess of 3 months. In this review article, we show that fluorescence based signaling using plastic disposable lenses, which have already been industrially optimized with regard to vision correction and oxygen/analyte permeability etc, may a notable alternative to invasive and random finger pricking, the most widely used glucose monitoring technology by diabetics.     

Fluorescence Sensor Design for Transition Metal Ions: The Role of the PIET Interaction Efficiency

Multicomponent systems 1, 2, and 3 having fluorophore-spacer-receptor architecture have been prepared with a view to understand the role of the photoinduced intramolecular electron transfer (PIET) interactions in transition metal ion sensing efficiency of these systems. Structurally similar compounds 4-amino-1,8-naphthalimide (4), 4-aminophthalimide (5), and 4-methoxy-1,8-naphthalimide (6) were used as the fluorophore moieties. Dimethylamino group (as in the case of 1a, 2a, and 3a, series A) and an aniline moiety (like in 1b, 2b, and 3b, series B) have been employed as the receptor components. A two-carbon ethylene chain serves as a spacer unit. The absorption and fluorescence spectral features of the systems have been studied in the absence and presence of various transition metal ions. All the multicomponent systems (except 1a) show weak fluorescence intensities compared to that of their constituent fluorophores (4, 5, and 6) in any given solvent. The reason for this low fluorescence quantum yield could be ascribed to the efficient PIET interaction between receptor moiety and the electron deficient fluorophore component of the systems. This has been corroborated with the estimated thermodynamic driving force (delta G*) for the PIET process in the multicomponent systems, calculated using electrochemical and spectral properties of individual components, is more negative for 2 and 3 than for 1 having electron deficient fluorophores 5, and 6, respectively. Especially, as evidenced by their low fluorescence quantum yield values, the PIET interaction is found to be more significant in the systems of series B (1b, 2b, and 3b) than the respective system of series A (1a, 2a, and 3a, respectively). The sensing capability of the systems is directly related to the efficiency of the PIET interaction in the unbound state. Accordingly, all these systems (except 1a) show significant fluorescence enhancement in the presence of transition metal ions, well known for their high fluorescence quenching behavior. The present paper describes, the feasibility of optimizing the PIET interaction in the multicomponent sensor system in unbound state, and thus transition metal ion signaling capability of the system.     

Fluorescent Photoinduced Electron Transfer (PET) Sensors for Anions; From Design to Potential Application

This mini review highlights the synthesis and photophysical evaluation of anion sensors, for nonaqueous solutions, that have been developed in our laboratories over the last few years. We have focused our research mainly on developing fluorescent photoinduced electron transfer (PET) sensors based on the fluorophore-spacer-anion receptor principle using several anthracene (emitting in the blue) and 1,8-naphthalimide (emitting in the green) fluorophores, with the aim of targeting biologically and industrially relevant anions such as acetates, phosphate and amino acids, as well as halides such as fluoride. The receptors and the fluorophore are separated by a short methyl or ethyl spacer, where the charge neutral anion receptors are either aliphatic or aromatic urea (or thiourea) moieties. For these, the anion recognition is through hydrogen bonding, yielding anion:receptor complexes. Such bonding gives rise to enhanced reduction potential in the receptor moieties which causes enhancement in the rate of PET quenching of the fluorophore excited state from the anion:receptor moiety. This design can be further elaborated on by incorporating either two fluorophores, or urea/thiourea receptors into the sensor structures, using anthracene as a fluorophore. For the latter design, the sensors were designed to achieve sensing of bis-anions, such as di-carboxylates or pyrophosphate, where the anion bridged the anthracene moiety. In the case of the naphthalimide based mono-receptor based PET sensors, it was discovered that in DMSO the sensors were also susceptible to deprotonation by anions such as F⁻ at high concentrations. This led to substantial changes in the absorption spectra of these sensors, where the solution changed colour from yellow/green to deep blue, which was clearly visible to the naked eye. Hence, some of the examples presented can act as dual fluorescent-colorimetric sensors for anions. Further investigations into this phenomenon led to the development of simple colorimetric sensors for fluorides, which upon exposure to air, were shown to fix carbon dioxide as bicarbonate.     

Fluorophore tagged bio-molecules and their applications: A brief review

Bio-molecules are principal building blocks of living species and their inter-play is the cause of bio-activities. Various sophisticated experimental techniques as well as theoretical studies have been carried out for decades to understand the functions of bio-molecules. Fluorescence, one of the popular spectroscopic techniques has been used rigorously to follow various bio-events. However, fluorescence suffers from certain limitations due to the insufficient availability of fluorescent bio-molecules. Covalent attachment of fluorophores with different bio-molecules provides a way of overcoming this problem. Lipid, steroid, sugar, nucleic, and amino acid are the 5 main classes of bio-molecules although there are many other sub-classes. This review demonstrates the applications of such conjugated fluorescent molecular probes in different domains of biological activities. This also brings out the advantages and disadvantages of this particular type of fluorophores with the insight to the future perspectives.     

Effect of a protein on the light energy conversion in dual fluorophore-nitroxide probes studied by ESR and fluorescence spectroscopy

Probing biological environment with dual fluorophore-nitroxide (FN) molecules in which fluorophore is tethered with nitroxide, a fluorescence quencher, opens unique opportunities to study molecular dynamics and micropolarity of the medium which affect intramolecular fluorescence quenching (IFQ), electron transfer, photoreduction and light energy conversion. In such molecules, the excited fragment of the chromophore can serve as an electron donor, and the nitroxide fragment as an electron acceptor. The same groups allow monitoring of molecular dynamics and also make it possible to measure micropolarity of the medium in the vicinity of the donor (by fluorescence technique) and acceptor (by electron spin resonance [ESR]) moieties. In the present work, two dual dansyl-nitroxide probes were incorporated in a binding site of bovine serum albumin. Their interactions with the protein, mobility, and photoreduction, as well as micropolarity of the media, have been studied by ESR and fluorescence methods. IFQ and spectral relaxation shift of the dansyl fragment have been monitored by time-resolved fluorescence technique. In parallel, computational studies on intramolecular dynamics of the FN probe were performed. On the basis of the Marcus model of the electron transfer between the excited dansyl fluorophore (donor) and nitroxide group (acceptor) and our experimental data, the mechanism of the electron transfer in the dual molecules incorporated into bovine serum albumin was established. It was shown that dual FN molecules in the protein meet main requirements for an efficient light energy conversion system.     

Preparation of two-photon fluorescent probe and biological imaging application in cells

We report on a novel dibenzothiophene-based two-photon fluorescent probe for selective nuclear bioimaging,which contains bilaterally symmetrical pyridine rings connected by a central conjugated-bridge dibenzothiophene. This probe possesses a large two-photon absorption cross-section of 471 GM, yields a 25-fold enhancement of the fluorescence titration, and a stronger photostability for nuclei labeling than existing probes. The real-time observation period is a minimum of 1800 s under a femtosecond laser excitation, which is significantly longer than that of 40,6-diamidino-2-phenylindole. The above results confirm that this novel molecule is a suitable two-photon fluorescent probe for application to nuclear bioimaging in cells.     

有机发光材料中的三重激发态

有机发光材料有望广泛应用于新一代柔性光电子器件。由于自旋多重性,有机分子发光材料 中单重激发态和三重激发态转换较慢,限制有机发光器件特别是电注入荧光器件的效率。我们介绍 下近年来通过分子设计操控激发不同时间尺度三重态的动力学来突破这一限制的策略,通过控制激 发单重态和激发三线态之间的电子耦合,利用热激子系间窜越、反向系间窜越、激发三重态稳定化 等过程能够有效提高有机发光材料的发光效率。在此基础上实现的热活化延迟荧光、有机长余辉发 光等在有机发光二极管、传感器、生物成像等领域有重要潜在应用价值。     

A Zinc-dipicolylethylenediamine Modified Near Infrared Fluorophore for Sensing of ATP

The development of fluorescent probes for sensing of anions in biological environments is still a demanding task. Due to the structural versatility of biological active anions there are many challenges to cope with compared to fluoroionophores for the determination of metal cations. This concerns particularly the design of the recognition element, which has to provide a selective response, preferably unaffected by alterations of pH and ionic strength. Polyphosphate anions such as ATP are interesting targets in bioanalysis because they are involved in many enzymatic reactions and bear versatile biological functions. Zinc dipicolylamine complexes attached to fluorophores have been turned out to be promising candidates for ATP sensing with sufficient sensitivity and selectivity. We now report the first NIR probe that responds to ATP based on a zinc dipicolylethylenediamine receptor. It shows a “turn-on” fluorescence behavior which is selective to other polyphosphate species even at high ionic strength of the sample solution.     

Simultaneous fluorometry and phosphorometry of Langendorff perfused rat heart: ex vivo animal studies

Fluorescence imaging of intrinsic fluorophores of tissue is a powerful method to assess metabolic changes at the cellular and intracellular levels. At the same time, exogenous phosphorescent probes can be used to accurately measure intravascular tissue oxygenation. Heart failure is the leading cause of death in America. A rat heart can potentially model the human heart to study failures or other abnormalities optically. We report simultaneous fluorescence and phosphorescence measurements performed on a rat heart. We have used two different optical systems to acquire fluorescence signals of flavoprotein and nicotinamide adenine dinucleotide--the two intrinsic fluorophores of mitochondria--and the phosphorescence signal of an intravascular oxygen probe to extract intracellular and intravascular metabolism loads, respectively.     

近红外H2S荧光探针研究进展

硫化氢(H₂S)是一种具有臭鸡蛋气味的无色气体,其不但存在于外界环境中,而且是继一氧化氮(NO)和一氧化碳(CO)之后生物系统中第三种重要的内源性气体信号分子,近年来因其对人类健康和疾病的影响而越来越受关注。H₂S可以作为抗氧化剂清除人体内的活性氧(ROS)和活性氮(RNS),参与多种细胞的生理反应,包括细胞凋亡、血管舒张、神经调节等,是多种组织中的细胞保护剂和气体传递剂。现代医学研究已证实硫化氢含量与糖尿病、阿尔茨海默病、帕金森病等特定疾病密切相关,但人们尚不清楚H₂S影响细胞信号传导和其他生理事件的具体分子机制,因此开发用于可视化内源性H₂S的方法、研究其在细胞和生物体中的动态分布将具有非常重要的意义。目前检测H₂S浓度的近红外(NIR)荧光探针是一个研究的热点,这是因为在生物样本分析时近红外荧光探针具有许多显著的优点：光损伤更小、能穿透更深的组织、背景自荧光干扰低。在分子水平上,H₂S表现出独特的化学特性,既是良好的还原剂,又是良好的亲核试剂,用于H...     

Review on Recent Advances in Metal Ions Sensing Using Different Fluorescent Probes

Fluorescence probes serves as unique detection methods for its simplicity and low detection limit (LOD) and especially bioimaging ability. Research on the probes has already sprouted during the last decade with the help of its molecular recognition properties. This review spotlights recent progress in sensing and bioimaging biologically, environmentally and industrially important metal ions e.g. Zn²⁺, Cu²⁺, Hg²⁺, Ag⁺ etc. using suitable fluorescent chemosensors including carbon quantum dots (CQD).     

Prism-based Spectral Imaging of Four Species of Single-molecule Fluorophores by Using One Excitation Laser

A prism-based imaging system for simultaneously detecting four species of single-molecule (SM) fluorophores was developed. As for the detection method, four spectrally distinct species of BigDye fluorophores were bound to 50-nm-diameter gold nanoparticles (AuNPs) to form AuNP/BigDye complexes. Four species of complexes were randomly immobilized on different fused-silica slides. BigDyes were excited by an argon-ion-laser (excitation wavelengths: 488 and 514.5 nm) beam through total internal reflection on the slide surface. SM fluorescence emitted from a complex was spectrally dispersed through a prism to form an SM spot elongated in the spectral direction on a charge-coupled device. A scattered light spot generated by the AuNP of the same complex under 594-nm laser illumination was used as a wavelength reference, and the SM fluorescence spectrum was obtained from the pixel-intensity pattern of the elongated SM spot. Peak locations of fluorescence spectra of all the observed SM spots were obtained, and their histograms were distinctly separated according to species. SM spots can thus be classified as one of four species according to their peak locations. By statistically analyzing the histograms, the classification accuracy was estimated to be above 93.8 %. The number of pixels in the spectral direction required for classifying four species of SM fluorophores was estimated to be 10. As for the conventional system (which uses two excitation lasers), 15 pixels are required. Using BigDyes as the four fluorophores (which consist of donors linked to acceptors and can be excited by just an argon-ion laser) is the reason that such a small number of pixels was achieved. The developed system can thus detect 1.5 times more SM fluorophores per field of view; that is, its throughput is 1.5 times higher. The approach taken in this study, namely, using BigDye with a prism-type system, is effective for increasing the throughput of DNA microarray-chip analysis and SM real-time DNA sequencing.     

Confocal fluorescence polarization microscopy for linear unmixing of spectrally similar labels

Studies of biological samples often call for simultaneous identification of multiple molecular or structural components. Multiple labelling fluorescence techniques are a powerful way of achieving this. However, the ability to distinguish a number of fluorescent probes unambiguously can be restricted by the fact that fluorescence spectra are generally broad and overlapping. Recently a technique known as linear unmixing has been combined with spectral imaging to discriminate between multiple fluorophores. In this study a scheme is proposed whereby fluorescence polarization information is used to expand the capability of the linear unmixing technique to accommodate additional fluorescent probes. As a proof-of-concept, it is shown that this polarization-based technique can be used to divide the signals generated by two spectrally similar fluorescent probes into their separate components.     

4-Aminophthalimide Derivatives as Environment-Sensitive Probes

The potential of 4-aminophthalimide (AP) and its derivatives as fluorescence probes for organized media is highlighted. The fluorescence response of AP, as measured from the position of the fluorescence maximum, fluorescence intensity and lifetime, is highly sensitive to the polarity of the medium. The sensitivity of the fluorescence parameters is further enhanced due to the involvement of the emitting intramolecular charge transfer state in hydrogen bonding interaction with the solvent molecules containing hydroxyl groups. It is shown that the microheterogeneous environments of organized media such as cyclodextrins and micelles can be very conveniently monitored using this probe. The results of the investigations carried out employing AP and its derivatives as fluorescence probe molecules in these media clearly suggest that a combination of the hydrophobic interaction with the host media and hydrogen bonding interaction with the solvent molecules determine the location of the fluorophore, which in all cases is found to be the interfacial region separating the nonpolar core of the micelle or the cyclodextrin cavity and the polar aqueous environment. Guidelines for the design of probes of this class of systems for the nonpolar core region of the micelles are provided and possible ways to increase the sensitivity of the fluorescence response of the systems are suggested.     

Photoinduced electron transfer between anionic fluorophores and methyl viologen in homogeneous and microheterogeneous media

The rate and extent of photoinduced electron transfer change significantly as a result of confinement in nanovolumes. Study of such processes is an active area of research in physical chemistry. The effect is most interesting when the molecules that participate in PET are charged. In the present article, the modulation of PET has been studied for two anionic fluorophores: Lucifer Yellow CH and chlorin p₆ with Methylviologen dication. PET, manifested in the quenching of fluorescence of the fluorophores, has been modulated by incorporating the molecules in organized assemblies like micelles, reverse micelles and supramolecular hosts. The dynamics of the process has been monitored in the femtosecond to nanosecond timescale. The modulation of the electron transfer has been found to be occurring mainly due to the disruption of contact ion pairs formed between the fluorophores and the quencher.     

New Cross-Linking Quinoline and Quinolone Derivatives for Sensitive Fluorescent Labeling

A variety of contemporary analytical platforms, utilized in technical and biological applications, take advantage of labeling the objects of interest with fluorescent tracers-compounds that can be easily and sensitively detected. Here we describe the synthesis of new fluorescent quinoline and quinolone compounds, whose light emission can be conveniently tuned by simple structural modifications. Some of these compounds can be used as sensitizers for lanthanide emission in design of highly sensitive luminescent probes. In addition, we also describe simple efficient derivatization reactions that allow introduction of amine- or click-reactive cross-linking groups into the fluorophores. The reactivity of synthesized compounds was confirmed in reactions with low molecular weight nucleophiles, or alkynes, as well as with click-reactive DNA-oligonucleotide containing synthetically introduced alkyne groups. These reactive derivatives can be used for covalent attachment of the fluorophores to various biomolecules of interest including nucleic acids, proteins, living cells and small cellular metabolites. Obtained compounds are characterized using NMR, steady-state fluorescence spectroscopy as well as UV absorption spectroscopy.