Design Of Fluorescent Probes Research Articles

Sulfonyl-semicarbazide group as a recognition site: A novel ratiometric fluorescent probe for hypochlorous acid and imaging in living cells

In this work a sulfonyl-semicarbazide moiety was developed initially to be a selective recognition site for the design of HClO fluorescent probe. The 1,8-naphthalimide fluorophore platform was chosen to construct the probe by making use of its intramolecular charge transfer (ICT) nature. By modifying the electron-donating 4-amino group on the 1,8-naphthalimide molecular with the more electron-deficient sulfonyl-semicarbazide group, probe 1 was synthesized and showed ratiometric fluorescent responses towards HClO exclusively. This was ascribed to the specific cleavage of HClO towards the substituent resulting in the release of 4-amino-1,8-naphthalimide. Also, the probe showed excellent performance in high sensitivity and good selectivity towards HClO over other reactive oxygen species and a wide variety of coexist species in biological pH condition. The probe was successfully applied to detect hypochlorous acid in real water samples and image HClO in living U-373MG cells.

Metal Nanoclusters–Based Ratiometric Fluorescent Probes from Design to Sensing Applications

AbstractRatiometric fluorescent probes have many advantages including improved sensitivity, high reliability and accuracy, visualization sensing, etc. Herein, the development of metal nanoclusters (NCs)‐based ratiometric fluorescent probes in the recent years is summarized. NCs, an emerging new class of fluorescent nanomaterials, have demonstrated excellent optical properties, good biocompatibility, great aqueous solubility, low cost, and simple synthesis, and NCs‐based ratiometric fluorescent probes have attracted much attention. In this progress report, the preparation and properties of NCs and the design of ratiometric fluorescent probes are summarized. Sensing of a broad range of analytes including cations, gas, small molecules, macromolecules, temperature, and pH is discussed. In addition, the challenges and future directions for NCs‐based ratiometric fluorescent probes are also presented.

Rational design of a lysosomal-targeted ratiometric two-photon fluorescent probe for imaging hydrogen polysulfides in live cells

Hydrogen polysulfides (H2Sn, n > 1) play important roles in H2S associated physiological functions and pathological processes and H2S2 is regarded to be a newfound potent signal transducer. But fluorescent probes for rapid, sensitive, and selective detecting of H2Sn are still lacking, above all the lysosomal-targeted ones. In this work, by integrating the alkylmorpholine group and 2-fluoro-5-nitrobenzoyl- group into the molecular structure of 1,8-naphthalimide based fluorophore, a new lysosome-targetable ratiometric two-photon fluorescent probe for H2Sn has been designed and synthesized. The probe showed good stability, short response time (5 min), high selectivity and sensitivity (LOD = 10 nM), broad linear range (0–25 μM), admirable two-photon absorption cross sections (Φmax = 324 GM), fine membrane permeability and biocompatibility, and as well as the lysosome targeting ability. Moreover, this work affords a promising approach for detecting and imaging of lysosomal H2Sn, and therefore potentially applied in the study of H2Sn -related biology and pathology.

Structure-based rational design of staurosporine-based fluorescent probe with broad-ranging kinase affinity for kinase panel application.

Selectivity profiling of compounds is important for kinase drug discovery. To this end, we aimed to develop a broad-range protein kinase assay by synthesizing a novel staurosporine-derived fluorescent probe based on staurosporine and kinase-binding related structural information. Upon structural analysis of staurosporine with kinases, a 4'-methylamine moiety of staurosporine was found to be located on the solvent side of the kinases, to which several linker units can be conjugated by either alkylation or acylation. However, such conjugation was suggested to reduce the binding affinities of the modified compound for several kinases, owing to the elimination of hydrogen bond donor moiety of NH-group from 4'-methylamine and/or steric hindrance by acyl moiety. Based on this structural information, we designed and synthesized a novel staurosporine-based probe without methyl group in order to retain the hydrogen bond donor, similar to unmodified staurosporine. The broad range of the kinase binding assay demonstrated that our novel fluorescent probe is an excellent tool for developing broad-ranging kinase binding assay.

High selective distinguishable detection GSH and H2S based on steric configuration of molecular in Vivo

Due to the intimate relationship of glutathione (GSH) and H2S in redox biology, it is crucial to establish a convenient synchronous detection method for them. A modified coumarin-based dye with two reaction sites was designed. The α,β-unsaturated ketone was adjacent to benzaldehyde, which constructed a steric - resistive reaction model. GSH with long chain could react with both the α,β-unsaturated ketone and the aldehyde group of the molecule, thus induced a green fluorescent emission. H2S with compact structure firstly reacted with α, β-unsaturated ketone, then the sulfhydryl group triggered secondary nucleophilic-cyclization with aldehyde to form a five-membered ring conjugating the whole molecule, which made the system give out a yellow fluorescent emission. Thus, high selective distinguishable detection GSH and H2S by two emission channels was realized. Cysteine (Cys) or homocysteine (Hcy) had no positive response to the probe compared with GSH. Also, biological imaging verified that the probe can be used in cell and mice. The result provided a clear idea for the design of multifunctional fluorescent probe.

Design and characterization of pH-sensitive spirorhodamine 6G probes with aliphatic substituents

We report synthesis and photophysical studies of spirorhodamines (SRA) with amide N-aliphatic substituents, including measurements of single-crystal X-ray structures, fluorescence emission, and quantum mechanical calculations. Variations in the equilibrium position between closed and open states, a transition that is catalysed under acidic conditions, of several SRAs sharing the same central chromophore (Rhodamine 6G) were measured. For the different substituents in the lactam position, the pH-dependent lactam ring opening reaction displays a strong correlation with the volume of the functional group. The open form shows similar absorption and emission spectrum, fluorescence efficiency and emission lifetime, regardless of the nature of the substituents. The molecular volume of the substituent is a simple and straightforward molecular design feature to control the equilibrium between the “dark” and “bright” states of the probe, without any further changes in the emission properties of the fluorescent form. These results allow rational design of pH-sensitive aliphatic SRAs fluorescent probes.

Design of fluorescent probes, Tb3+-dtpa-2A, Tb3+-dtpa-2C and Tb3+-dtpa-AC, based on DNA single strand base sorting principle for xanthine detection

Three novel fluorescent probes were prepared based on the DNA single strand base sorting principle for selective detection of xanthine. The fluorescence response signals of three probes to xanthine under different influencing factors were compared and corresponding mechanism was proposed. Finally, they can successfully be applied to the detection of xanthine in real urine samples.

Design of a Nile red-based NIR fluorescent probe for the detection of hydrogen peroxide in living cells.

In this article, a novel fluorescent probe (NRBE) for detecting H2O2 was developed using benzyl boronic ester as the H2O2-recognized group and Nile red as the matrix. The probe has several advantages, such as good selectivity, high sensitivity (LOD = 75 nM), good water solubility and emission in the near-infrared region (ex/em:585/670 nm). With the NRBE probe, the endogenous H2O2 in human hepatocellular carcinoma cells BEL-7402, was detected, and the H2O2 generated during the ischemia-reperfusion of the cells was imaged. These results show that NRBE can be applied for real-time detection of H2O2 in biological systems.

Design and synthesis of nitrogen-fused pyridazinone fluorescent probes and their application in biological imaging

Design and synthesis of nitrogen-fused pyridazinone fluorescent probes and their application in biological imaging

The first small fluorescent probe as Tyrosyl-DNA phosphodiesterase 1 (TDP1) substrate

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a recently discovered enzyme specifically repairing DNA topoisomerase IB-mediated DNA damage, and is a rational anticancer target. Herein, we report the design and synthesis of the first small fluorescent probe 1 as TDP1 substrate. Probe 1 is a specific substrate of TDP1 and shows good spectroscopic properties with more than 140-fold fluorescence enhancement. A novel TDP1 inhibition assay was established using 1 as substrate. The possible cleavage pathway of 1 by TDP1 was also studied.

A Doubly-Quenched Fluorescent Probe for Low-Background Detection of Mitochondrial H2O2.

Hydrogen peroxide (H2O2) is an important product of oxygen metabolism and plays a crucial role in regulating a variety of cellular functions. Fluorescent probes have made a great contribution to our understanding of the biological role of endogenous H2O2. However, fluorescent probes for H2O2 featuring aryl boronates can suffer from moderate turn-on fluorescence responses. Strategies that can reduce the background fluorescence of these boronate-masked probes would significantly improve the sensitivity of endogenous H2O2 detection. In this work, we propose a general and reliable double-quenching concept for the design of fluorescent probes with low background fluorescence. A new fluorescent probe was developed for the detection of endogenous H2O2 in mitochondria of live cancer cells. This probe exploits a boronate-driven lactam formation and an eliminable quenching moiety simultaneously (i.e., the double-quenching effect) to reduce the background fluorescence, which ultimately results in the achievement of a >50-fold fluorescence turn-on. A linear concentration range of response between 1 and 60 μM and a detection limit of 0.025 μM can be obtained. This study not only presents a highly sensitive fluorescent probe for the detection of H2O2 but also provides a new concept for the design of fluorescent probes with a previously unachievable fluorescence off-on response ratio for other types of ROS and many other biologically relevant analytes.

3,5-dinitropyridin-2-yl as a fast thiol-responsive capping moiety in the development of fluorescent probes of biothiols

Biological thiols, such as glutathione, cysteine, and homocysteine, are closely associated with human health and disease. In this work, we demonstrated a general strategy for the design of fluorescent probes for the detection of various biological related thiols. The preferred strategy we employed was to introduce a 3,5-dinitropyridyl moiety to a selected fluorophore via ether linkage to afford fluorescent probe. Once the ether bond was thiolyzed by biothiol, the hydroxy-functionalized fluorescent dye would be released and fluorescence emission restored. Herein, some novel fluorescent probes based on this strategy have been developed for sensing biothiols in living cells. All these probes presented here showed desirable properties, such as high sensitivity, high selectivity and preferred reaction kinetics. Moreover, the probes have been used for imaging of intracellular biothiols with emission colors ranging from green to near-infrared (NIR).

Design, synthesis and evaluation of a novel hepatocyte-targeting fluorescent probe to accurately detect H2S in mitochondria

A novel hepatocyte-targeting and mitochondria-targeting fluorescent probe Dye-H2S-Gal was designed and synthesized. Dye-H2S-Gal with low toxicity and short response time (within 1 min) shows good selectivity toward H2S, which is not disturbed by anions (CO32−, SO42−, HCO32−, T-BuO-), reactive oxygen and biothiols (Cys, GSH, H2O2, T-BuO-) and the representative cations (Ag+, Ba2+, Ca2+, Cd2+, Ce2+, Co2+, Cr2+, Cu2+, Eu3+, Fe2+, Fe3+, Hg2+, K+, Li+, Mg2+, Mn2+, Ni2+, NH4+, Na+). The OFF-ON switch of Dye-H2S-Gal is triggered by H2S via removing the thiophenecarboxylate group. Good mitochondria-targeting capacity of Dye-H2S-Gal is driven by the electrostatic interaction between a quaternary amine group of Dye-H2S-Gal with positive charge and the mitochondria membrane with negative charges, and its good hepatocyte-targeting capacity is driven by the specific recognition of the galactose group on the asialoglycoprotein receptor (ASGPR) overexpressed on liver cells. On the whole, Dye-H2S-Gal can precisely monitor mitochondria H2S level in liver cells and is of great significance for the early diagnosis and precise treatment of liver diseases.

Survey of relevant taxonomic groups for the design of qPCR primers and internal fluorescent probes for whole characterization of subaerial biofilm

A deep survey of biodeteriogen microorganisms reported on stone monuments in Europe has been performed based on the available literature dating back to over 30 years. The obtained lists for eukaryotic algae, phototrophic and non-phototrophic bacteria, and fungi were sorted by Genera, and corresponding sequences in triplicate were downloaded by nucleotide database Genbank for a number of selected barcoding markers. On the basis of collected bibliometric diversity, multiple nucleotide alignments were produced and primers were designed for a qPCR assay. The aim of the present study was to obtain accurate oligos for the characterization of subaerial biofilms on the basis of the most comprehensive collection of reports and case studies regarding subaerial biofilms, with particular regard to phototrophic and non-phototrophic bacteria, eukaryotic algae, and molds. Primers were designed on conserved regions flanking a variable region, specific for each of the studied groups of microorganisms. Standard curve for absolute quantification relative to each group were determined for four markers. Then, variable regions in the alignments were used to design fluorescent internal probes for qPCR aimed for a multiplex reaction in which relative abundance can be determined. The authors propose this kind of cost-effective approach in the study of biofilms for the estimation of algae, molds, and bacteria both for direct in situ analysis and in vitro simulation.

Design of fluorescent probes with optimized responsiveness and selectivity to GSH

We designed and synthesized a series of BODIPY based probes with fast and distinct ratiometric responsiveness for discriminative detection of GSH from Cys and Hcy. The discriminative detection is based on the different products obtained by the SNAr between probes and thiol-containing amino acids. The amino group of the obtained thioether from the reaction with Cys or Hcy but not GSH would trigger an intramolecular nucleophilic substitution through five- or six-membered cyclic transition state, finally yielding an amino substituted derivative. To achieve highly discriminative detection and fast response, a series of structure modifications and improvements have been made by elongating the π-conjugation and introducing electron withdrawing groups, finally affording probe BOD-DBNPF with optimized responsiveness and selectivity. Importantly, BOD-DBNPF was successfully used for the selective detection of GSH from Cys with distinct fluorescent ratiometric responses in living HeLa cells.

Design and synthesis of a new fluorescent probe for cascade detection of Zn2+ and H2 PO4 - in water and targeted imaging of living cells.

Design and synthesis of new fluorescence probes with good water-solubility is of great importance to better understanding the significant role of ions which are related to biology and the environment. As important ions, zinc ion (Zn2+ ) and dihydrogen phosphate ion (H2 PO4 - ) display essential roles in living systems, and quantitative detection of these ions in water is still a challenge. In order to consider the significant role of the galactose moiety in the design of a water-soluble fluorescence sensor, herein, we have developed a novel probe, Gal-AQTF, for the cascade detection of Zn2+ and H2 PO4 - with excellent selectivity in water. Through the introduction of the galactose moiety onto the sensor AQTF, which has been reported earlier by us, the water-solubility, cell compatibility and targeting ability were enhanced. Gal-AQTF has been successfully applied in the imaging of the living cells of HepG2 and A549, and illustrated good selectivity for the HepG2 cells which overly express the asialoglycoprotein (ASGP) receptor.

Rational Structure-Based Design of Fluorescent Probes for Amyloid Folds.

Amyloid fibrils are pathological hallmarks of various human diseases, including Parkinson's, Alzheimer's, amyotrophic lateral sclerosis (ALS or motor neurone disease), and prion diseases. Treatment of the amyloid diseases are hindered, among other factors, by timely detection and therefore, early detection of the amyloid fibrils would be beneficial for treatment against these disorders. Here, a small molecular fluorescent probe is reported that selectively recognize the fibrillar form of amyloid beta(1-42), α-synuclein, and HET-s(218-289) protein over their monomeric conformation. The rational design of the reporters relies on the well-known cross-β-sheet repetition motif, the key structural feature of amyloids.

Cyano substitution effect on the emission quantum efficiency in stilbene derivatives: A computational study

Abstract A series of cyano-substituted stilbene derivatives exhibit quenched fluorescence in solution, in sharp contrast to their highly emissive parent counterparts. By employing the polarizable continuum model (PCM) method coupled with thermal vibration correlation function (TVCF) formalism, we comparatively investigated the electronic structures and excited-state dynamics of cyano-substituted para-distyrylbenzene (CN-DSB), 1,2-bis(pyridylphenyl)ethane (CN-BPPE) and the corresponding non-substituted DSB and BPPE lack of the cyano groups at their vinylene moieties in solution. The results show that the non-radiative decay rate constants of the CN-substituents are greatly enhanced by more than 4 orders of magnitude, while the radiative decay rate constants experience slight reduction, resulting in the very tiny fluorescence quantum efficiency. By analyzing the reorganization energies projected onto both normal modes and internal coordinates, the non-radiative consumption ways via molecular torsional motions of the single even double bonds adjacent to the CN group are active upon CN-substitution. Our study presents a rational explanation for the experimental CN-substitution induced quenching phenomena in stilbene derivatives, which is beneficial for the design of new fluorescent emitters and probes.

A near-infrared ratiometric fluorescent probe with large stokes based on isophorone for rapid detection of ClO− and its bioimaging in cell and mice

The reactive oxygen species (ROS) including hypochlorous acid (HOCl), on the one hand they can regulate physiological function in our living organisms, on the other hand abnormal concentration of ROS would induce a series of biomacromolecules damage and further endanger virtually all aspects of human health. Especially it can be directly related to cancer. Thus to elucidate and visualize their concentration is very imminent. Before many fluorescent probes were used to detect HOCl. In recent years, near-infrared or ratiometric fluorescent developed prosperously. The strategy of combining near-infrared and ratiometric fluorescent probe is overwhelming in the design of fluorescent probes. In this work, we successfully designed and obtained a near-infrared and ratiometric fluorescent probe with large stokes by reacting isophorone with coumarin, which can monitor HOCl high efficiently with quick response and low detection limit. In addition, cell imaging experiments show that the probe can identify endogenous and exogenous ClO− successfully, and nude mouse imaging experiments show that the probe can detect exogenous ClO−. It is possible that the probe can be applied in early clinical diagnosis.

Rational design of NIR fluorescence probes for sensitive detection of viscosity in living cells

Developing near-infrared (NIR) fluorescence probes for detection of intracellular viscosity is still sufficiently challenging. In this work, three kinds of D-A-D type naphthyl and 2,1,3‑benzoxadiazol hybrid NIR dyes functionalized with amino (NY1), N‑methylamino (NY2) and N,N‑dimethylamino (NY3) groups for intracellular micro-viscosity detection were designed and synthesized. All the probes exhibited very weak NIR emission in low viscosity environment and obvious fluorescence enhancement with the increased viscosity. Different substituent groups had a high impact on the photophysical properties and response sensitive of the probes to viscosity. The structure-property relationships were systematic investigated. The results showed that stronger electron-donating ability and larger steric effect of N,N‑dimethylamino led to a narrower energy gap and more sensitive to viscosity environment. Therefore, NY3 exhibited higher signal noise ratio for viscosity detection and was successfully applied for imaging the changes of intracellular micro-viscosity. This work provides an efficient way to design powerful NIR fluorescence probes for viscosity detection.