Water-soluble Probe Research Articles

Hemicyanine-Based Highly Water-Soluble Probe for Extracellular Nitroreductase.

Nitroreductase (NTR) has long been a target of interest for its important role involved in the nitro compounds metabolism. Various probes have been reported for NTR analysis, but rarely able to distinguish the extracellular NTR from intracellular ones. Herein we reported a new NTR sensor, HCyS-NO2, which was a hemicyanine molecule with one nitro and two sulfo groups attached. The nitro group acted as the reporting group to respond NTR reduction. Direct linkage of nitro group into the hemicyanine π conjugate system facilitated the intramolecular electron transfer (IET) process and thus quenched the fluorescence of hemicyanine core. Upon reduction with NTR, the nitro group was rapidly converted into the hydroxylamino and then the amino group, eliminating IET process and thus restoring the fluorescence. The sulfo groups installed significantly increased the hydrophilicity of the molecule, and introduced negative charges at physiological pH, preventing the diffusion into bacteria. Both gram-negative and gram-positive bacteria were able to turn on the fluorescence of HCyS-NO2, without detectable diffusion into cells, providing a useful tool to probe the extracellular reduction process.

Exploiting G-Quadruplex-DNA Damage as a Tool to Quantify Singlet Oxygen Production.

G-Quadruplexes (G4s) are highly dynamic and polymorphic nucleic acid structures that can adopt a variety of conformations. When exposed to oxidative conditions, more specifically singlet oxygen, the guanosine nucleobases can be oxidized, which in turn can affect the conformation and folding of the G4. Based on this peculiar phenomenon, it is rationalized that G4s can serve as quantification sensors for the production of singlet oxygen. Here, a method for determining the quantum yield of singlet oxygen generation for visible as well as UV-light excited photosensitizers, using a short G4 DNA sequence, readily available from common DNA companies, as a biological and water-soluble probe, is presented.

Development of a rhodamine-based fluorescent probe for ATP detection for potential applications in meat freshness assessment

Adenosine triphosphate (ATP) plays a vital role in physiological processes and is an essential indicator of microbial content in food. Herein, a new sensitive, rapid and water-soluble probe for ATP detection was developed. Rhodamine B and pentaethylenehexamine were employed to design and synthesise the probe rhodamine-pentaethylenehexamine (RP) for selective ATP detection. The synthesised probe RP was characterized using Fourier transform infrared, NMR and dynamic light scattering size distributions. Upon the addition of ATP, the probe exhibited a distinct change in fluorescence intensity, with fluorescence emission at 580 nm. A linear relationship was observed between fluorescence intensity and ATP concentrations at 0–50 μmol/L, with a limit of detection of 10.97 × 10−9 mol/L. The results of the zeta potential and molecular dynamics simulation demonstrated that the detection mechanism of the probe RP is associated with the electrostatic adsorption interaction between the multi-positively charged sites of RP and the negatively charged triphosphate structure of ATP. Our study provides new insights into improving charge site identification in small molecule detection. Furthermore, the successful detection of ATP on meat surfaces indicates that RP has the potential to assess meat freshness.

Chromo-Fluorogenic Rhodamine-Based Amphiphilic Probe as a Selective and Sensitive Sensor for Intracellular Cu(I) in Living Cells.

Fluorescent probes are widely studied for metal ion detection because of their multiple favorable properties such as high sensitivity and selectivity, quick response, naked eye detection, and in situ monitoring. However, optical probes that can effectively detect the Cu(I) level in cell interiors are rare due to the difficulty associated with selectively and sensitively detecting this metal ion in a cell environment. Therefore, we designed and synthesized three water-soluble probes (1-3) with a 1,3,5-triazine core decorated by three substituents: a hydrophobic alkyl chain, a hydrophilic maltose, and a rhodamine B hydrazine fluorophore. Among the probes, probe 1, which has an octyl chain and a branched maltose group, was the most effective at sensing Cu+ in aqueous solution. Upon addition of Cu+, this probe showed a dramatic color change from colorless to pink in daylight and displayed an intense yellow fluorescence emission under 365 nm light. The limit of detection and dissociation constant (Kd) of this probe were 20 nM and 1.1 × 10-12 M, respectively, which are the lowest values reported to date. The two metal ion-binding sites and the aggregation-induced emission enhancement effect, endowed by the branched maltose group and the octyl chain, respectively, are responsible for the high sensitivity and selectivity of this probe for Cu+ detection, as demonstrated by 1H NMR, dynamic light scattering, and transmission electron microscopy studies. Furthermore, the probe successfully differentiated the Cu(I) level of cancer cells from that of the normal cells. Thus, the probe holds potential for real-time monitoring of Cu(I) level in biological samples and bioimaging of cancer cells.

Iron-based compounds coordinated with phospho-polymers as biocompatible probes for dual 31P/1H magnetic resonance imaging and spectroscopy

In this work, we present the synthesis and evaluation of magnetic resonance (MR) properties of novel phosphorus/iron-containing probes for dual 31P and 1H MR imaging and spectroscopy (MRI and MRS). The presented probes are composed of biocompatible semitelechelic and multivalent phospho-polymers based on poly(2-methacryloyloxyethyl phosphorylcholine) (pMPC) coordinated with small paramagnetic Fe3+ ions or superparamagnetic maghemite (γ-Fe2O3) nanoparticles via deferoxamine group linked to the end or along the polymer chains. All probes provided very short 1H T1 and T2 relaxation times even at low iron concentrations. The presence of iron had a significant impact on the shortening of 31P relaxation, with the effect being more pronounced for probes based on γ-Fe2O3 and multivalent polymer. While the water-soluble probe having one Fe3+ ion per polymer chain was satisfactorily visualized by both 31P-MRS and 31P-MRI, the probe with multiple Fe3+ ions could only be detected by 31P-MRS, and the probes consisting of γ-Fe2O3 nanoparticles could not be imaged by either technique due to their ultra-short 31P relaxations. In this proof-of-principle study performed on phantoms at a clinically relevant magnetic fields, we demonstrated how the different forms and concentrations of iron affect both the 1H MR signal of the surrounding water molecules and the 31P MR signal of the phospho-polymer probe. Thus, this double contrast can be exploited to simultaneously visualize body anatomy and monitor probe biodistribution.

Reversible Recognition-Based Boronic Acid Probes for Glucose Detection in Live Cells and Zebrafish.

Glucose, a critical source of energy, directly determines the homeostasis of the human body. However, due to the lack of robust imaging probes, the mechanism underlying the changes of glucose homeostasis in the human body remains unclear. Herein, diboronic acid probes with good biocompatibility and high sensitivity were synthesized based on an ortho-aminomethylphenylboronic acid probe, phenyl(di)boronic acid (PDBA). Significantly, by introducing the water-solubilizing group -CN directly opposite the boronic acid group and -COOCH3 or -COOH groups to the β site of the anthracene in PDBA, we obtained the water-soluble probe Mc-CDBA with sensitive response (F/F0 = 47.8, detection limit (LOD) = 1.37 μM) and Ca-CDBA with the highest affinity for glucose (Ka = 4.5 × 103 M-1). On this basis, Mc-CDBA was used to identify glucose heterogeneity between normal and tumor cells. Finally, Mc-CDBA and Ca-CDBA were used for imaging glucose in zebrafish. Our research provides a new strategy for designing efficient boronic acid glucose probes and powerful new tools for the evaluation of glucose-related diseases.

Chiral amino acid recognition in water: A BINOL-based fluorescent probe

A novel BINOL-based fluorescence probe (S)-6 featuring a sodium sulfonate fragment at the 2′-position was designed and synthesized via simple synthetic procedures under mild reaction conditions. The water-soluble probe (S)-6 displays excellent enantioselective recognition toward 15 common amino acids, and it can be used for enantiomeric excess determination of amino acids. The fluorescence intensity of (S)-6 treated with amino acids reaches the maximum after standing for only 30 min at room temperature and remains stable in the following 5.5 h, which has great potential in the application of chiral fluorescence analysis due to its timeliness and outstanding fluorescent stability.

Fusion modifies the material properties of membranes.

Membrane fusion is a ubiquitous process in biology and potentially an alternative to endocytic internalization pathways in drug delivery. Due to its high complexity, assays have been developed to disentangle its key aspects, although many of its molecular mechanisms remain elusive. Here, we use a combination of multicolour and time-resolved confocal fluorescence microscopy to study the effects of membrane mechanics on fusion and conversely, how fusion alters membrane mechanics. As a minimal fusion system, we used cationic large unilamellar fusogenic liposomes (LUVs) that rapidly fuse with giant unilamellar vesicles (GUVs) as evidenced by the probes being delivered into the GUVs. Fusion is favoured in fluid membranes and hampered upon an increase in packing. Extensive fusion of a large number of LUVs leads to the creation of area leaflet asymmetry in GUVs, resulting in budding formation driven by spontaneous curvature. In these conditions, the membrane becomes perforated as a result of a decrease in edge tension and the size of the formed pores seems to scale with LUV concentration. Here, the GUVs also display a range of permeability states. In live human embryonic kidney (HEK) cells, lipid mixing and intracellular delivery of LUV encapsulated water-soluble probes are observed, consistent with fusion of the liposomes with the plasma membrane. Furthermore, when exposed to liposomes, cell membrane permeabilization and cell death is observed at liposomal concentrations an order of magnitude higher than pore formation in the GUVs, suggesting an increased resilience of cells compared to GUVs. Fusion also seems to fluidize the membrane and effects are observed within minutes. Overall, the results shed new light onto the effects of liposome-membrane interactions.

Breaching Bacterial Biofilm Barriers: Efficient Combinatorial Theranostics for Multidrug-Resistant Bacterial Biofilms with a Novel Penetration-Enhanced AIEgen Probe

The formation of microbial biofilms is acknowledged as a major virulence factor in a range of persistent local infections. Failures to remove biofilms with antibiotics foster the emergence of antibiotic-resistant bacteria and result in chronic infections. As a result, the construction of effective biofilm-inhibiting and biofilm-eradicating chemicals is urgently required. Herein, we designed a water-soluble probe APDIS for membrane-active fluorescence and broad-spectrum antimicrobial actions, particularly against methicillin-resistant Staphylococcus aureus (MRSA), which shows multidrug resistance. In vitro and in vivo experiments demonstrate its high antibacterial effects comparable to vancomycin. Furthermore, it inhibits biofilm formation by effectively killing planktonic bacteria at low inhibitory concentrations, without toxicity to mammalian cells. More importantly, this probe can efficiently penetrate through biofilm barriers and exterminate bacteria that are enclosed within biofilms and startle existing biofilms. In the mouse model of implant-related biofilm infections, this probe exhibits strong antibiofilm activity against MRSA biofilms, thus providing a novel theranostic strategy to disrupt biofilms in vivo effectively. Our results indicate that this probe has the potential to be used for the development of a combinatorial theranostic platform with synergistic enhanced effects for the treatment of multidrug-resistant bacterial infections and antibiofilm medications.

Colorimetric probe for sequential chemosensing of mercury(II) and cyanide ions in aqueous media, based on a benzoxadiazole-pyrazolin-5-one glycoconjugate with INHIBIT logic gate response

A new and efficient water-soluble probe L for the sequential detection of Hg2+ and CN− ions has been developed by the condensation of benzoxadiazole and glycoconjugated pyrazoline-5-one. The resulting probe has a water solubility of 45 g/L at room temperature and interacts selectively with Hg2+ ions in water over other cations by changing its color from yellow to red and forming the [L/Hg2+: 2:1] complex. On the other hand, the formed complex [L/Hg2+: 2:1] reacts selectively with CN− anions in an aqueous solution. This reaction is accompanied by a blue shift in wavelength from 545 nm to 472 nm with a color change from red to yellow visible to the naked eye. These reversible and repeatable colorimetric responses of probe L towards Hg2+ and CN− ions can be represented as a molecular logic gate 'IMPLICATION', where Hg2+ and CN− ions are considered as inputs and the absorbance at 472 nm as output. The detection limit for Hg2+ and CN− ions was found to be 2.31 ppb and 4.34 ppb, respectively. From a practical point of view, probe L was used to prepare a test strip based on filter paper treated with L. The resulting test strip selectively changes color after the sequential addition of Hg2+ and CN− ions in water at pH7. These high performances in terms of selectivity and sensitivity allow the probe L to be used for the sequential detection of Hg2+ and CN− ions in aqueous media.

A unique water soluble probe for measuring the cardiac marker homocysteine and its clinical validation.

A series of copper(II) compounds 1-4 were synthesized and developed as fluorogenic probes to measure the cardiac marker homocysteine (Hcy) without any interference from other bioanalytes prevalent in human blood plasma including, cysteine and glutathione. UV-vis and EPR studies have provided confirmatory evidence for reduction-induced-emission-enhancement of the probe, which is responsible for the observed "off-to-on" behaviour towards Hcy. Water solubility, remarkable fluorescence enhancement (55-111 fold), and low detection ability (nearly 2.5 μM) make the probe suitable for clinical testing of cardiac samples. Investigation of 1 against a few reductive interferents testifies its specificity for Hcy. Results from clinical examination of cardiac samples by 1 when combined with the outcome of the reliability testing involving a clinically approved commercial immunoassay kit, validates the prospect of the molecular probe for direct measurement of Hcy in human plasma, which is unprecedented.

A water-soluble polymer fluorescent probeviaRAFT polymerization for dynamic monitoring of cellular lipid droplet levels and zebrafish imaging

A coumarin derivative was chosen for co-polymerizing with terminal hydroxy polyethylene glycol to obtain a water-soluble probe P(Cou-PEG-LD). The probe was successfully applied to discrimination of starved and normal cells and zebrafish bioimaging.

A ratiometric fluorescent probe for hydrogen sulfide in neat aqueous solution and its application in lysosome-targetable cell imaging

Hydrogen sulfide (H2S) has been recently regarded as one of the most important gasotransmitters in the metabolic system, while abnormal H2S concentration is associated with various diseases. Although numerous fluorescent probes have been developed for the detection of cellular H2S, only a few of them can monitor lysosomal H2S with ratiometric fluorescent output. Here, we developed a water-soluble probe 1 toward H2S by introducing 2,4-dinitrophenyl ether into a novel merocyanine-based dye. As expected, H2S induced an obvious red-shift of the probe from 520 nm to 580 nm in neat aqueous solution, and this fluorescent ratiometric response is highly selective and sensitive (with the detection limit of 0.81 nM), rapid (within 10 s), and effective in a wide pH range (2.0–10.0). In particular, the probe was successfully applied for tracing H2S in the lysosomes of living cells and in zebrafish.

A water-soluble turn-on fluorescent probe for rapid discrimination and imaging of Cys/Hcy and GSH in cells and zebrafish through different fluorescent channels

Biothiols are a class of amino acids containing mercaptan (thiol, SH) groups that have similar structures but play different roles in physiological activities, making it important to effectively distinguish them. Here, a simple water-soluble probe, PMCI, was developed for the first time to rapidly discriminate Cys/Hcy and GSH by dual excitation and emission wavelengths. The introduction of an N–Me pyridinium salt thioether group into the fluorophore structure not only makes the probe highly water-soluble, but also serves as a potential reactive site for biothiols, which together with an aldehyde group allows the simultaneous differential detection of Cys/Hcy and GSH. The probe itself has no background fluorescence due to the intramolecular photoinduced electron transfer (PET) effect. After reaction with biothiols, the probe solution showed different strong fluorescent signals (green: Cys and Hcy, red: GSH). It can respond quickly to biothiols (Cys: 190 s; Hcy: 155 s; GSH: 80 s) with high selectivity and low detection limit. Moreover, the probe has been successfully used for the rapid discrimination imaging of endogenous and exogenous biothiols in cells and zebrafish through dual fluorescent channels.

Ratiometric Probe for Rapid Naked Eye Detection of Toxic Hydrazine: Real Time Application in Strip Test, Spray Test and Soil Analysis.

Striking colorimetric probe (CynH) for abrupt detection of hydrazine under complete aqueous solution was achieved. The water soluble probe was designed with electron "push-pull" strategy by coupling of 4-hydroxy benzaldehyde and 2, 3, 3-trimethylindolinine. The positively charged N-propylated indolinine make the probe completely soluble in water. The probe yields eye catching selective detection of hydrazine over other competing analytes with high sensitivity. Obvious colour change was observed from colourless to appearance of bright pink colour with hydrazine. It reacts quickly with hydrazine within 2min and makes the probe an effective candidate for practical application. The real time application was demonstrated using paper strip to detect hydrazine vapour. This probe is superior to earlier reported probes because of its effective sensing of hydrazine displayed with various applications including real-time strip based sensing, spray test and soil analysis. In all the examinations, the probe yields distinct response with rapid naked eye colour change which overcomes the drawbacks of previous reports.

CDs/ZnO composite material with solid state fluorescence performance for quantitative determination of methyl red content and antibacterial properties

A water-soluble probe composite material (CDs/ZnO) was synthesized by microwave pyrolysis methods of citric acid, urea and zinc oxide (ZnO) with aggregation-induced emission activity. We creatively realized the composite of CDs and nano-ZnO in the form of foam, then used as bactericide for the first time and showed excellent antibacterial properties. Also, the composite materials overcame the problem of aggregation-induced quenching (ACQ) and realized solid-state emission. The materials had two different emission centers, and the two emission peaks were varied with the change of excitation wavelength. Furthermore, Methyl red (MR) showed fluorescence quenching for CDs/ZnO with obvious linear correlation, which demonstrated potential in the measurement of MR in dyeing wastewater. The CDs/ZnO we obtained perfectly inherited the advantages of CDs and nano ZnO, it has not just no cytotoxicity to cells, but also has a good killing effect on bacteria. Hence, this work shows significant adaptable fluorescence-based protocol with enormous potential applications in biology.

Sensitive detection of DMSO/DMF in water, human urine and blood plasma using novel 1,8-naphthalimide-based amphiphilic spectroscopic probes

Dimethyl sulfoxide (DMSO) and dimethylformamide (DMF) are industrial toxins that need to be sensed. We synthesized 1,8-naphthalimide-m-benzenesulfonamide (NBS)-bearing probes (1–5) with different pendant chains for sensitive detection of DMSO and DMF. These probes exhibited aggregation-induced emission enhancement with increasing water concentration in ethanol. Among these, monopod probes with a single NBS unit (1–4) selectively showed large fluorescence quenching upon addition of DMSO/DMF in water. Based on this result, we prepared two water-soluble probes (8 and 9) bearing the NBS unit. These probes displayed enhanced sensitivity in detecting DMSO/DMF in water compared to probe 1, showing an extraordinary low LOD of 0.0025% for DMSO and DMF detection in water. Probe 9 was similarly effective at sensitively sensing DMSO/DMF when tap or lake water was used as the medium, and the LOD of this probe was 0.05% when biological fluids such as diluted human urine and blood plasma were used instead of water. According to DLS, FE-SEM and zeta potential studies as well as DFT calculations, disaggregation of probe aggregates and electron transfer from the photo-excited probe to DMSO/DMF are likely both responsible for the high selectivity and sensitivity of this probe in detecting the organic solvents containing a sulfoxide/amide group.

Spectroscopic behavior and intracellular application of a highly sensitive UV-fluorescence double ratio probe based on water-soluble indole for detection acid pH

Acidic pH is an important physiological parameter in environment and biological systems. Thus, designing a probe (Hcy-F) with good characters to monitor acidic pH is really important. Herein, a small molecular water-soluble probe is designed based on indole with high selectivity and sensitivity for detection the acidic pH in aqueous solution and living cells. When pH variation of aqueous is detected by probe, an obvious ratiometric and good linear response behavior are found in UV–vis absorption spectrum (R2 = 0.9973, A434 nm/A371 nm = 6.9929–4.2231 pH) and fluorescence emission spectrum (R2 = 0.9987, I571 nm/I514 nm = 7.2935–2.981 pH), which further proved the feasibility of Hcy-F to monitor the change of acidic pH in aqueous, respectively. Furthermore, Hcy-F is successfully applied for monitoring pH variation in living cells in dual channels (green and red) with higher cell survival rate, indicating the probe has great potential for pH imaging in biological applications.

Dual-modal fullerenol probe containing glypican-3 monoclonal antibody for electron paramagnetic resonance/fluorescence imaging

The multi-modal imaging contrast agent has been attracted an increased interest to the tumor detecting and diagnosis in these recent years since the multi-modal imaging technology combines the advantages of several medical imaging modalities. A water-soluble fullerenol probe containing glypican-3 monoclonal antibody (GPC3-mAb) group mAb-GPC3-C60(OH)10-(TMIO)3-(NI)3 was synthesized by the incorporation of 5-formyl-1,1,3,3-tetramethylisoindolin-2-yloxyl (FTMIO), naphthalimide (NI) and GPC3-mAb as a tumor-targeting group into fullerenol C60(OH)11. This nanomaterial was further characterized by FTIR, UV, MS, fluorescence, etc., and then its properties including electron paramagnetic resonance (EPR), particle size, electrophoresis, electrochemical assay, cell cytotoxicity, cell uptake and fluorescent imaging of HeLa tumor cells in vitro were also evaluated. Experiment results indicated that mAb-GPC3-C60(OH)10-(TMIO)3-(NI)3 had similar fluorescent properties as NI and similar electrochemical and redox properties as 5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl (CTMIO). Moreover, mAb-GPC3-C60(OH)10-(TMIO)3-(NI)3 possessed low cell cytotoxicity, good fluorescence and good tumor-targeting property. Meanwhile, mAb-GPC3-C60(OH)10-(TMIO)3-(NI)3 can be taken up highly by HeLa tumor cells, achieve good green fluorescent imaging in tumor cells and highly enhance the contrast of fluorescent images. Therefore, mAb-GPC3-C60(OH)10-(TMIO)3-(NI)3 can disperse homogeneously in aqueous solution and be used as a potential tumor-targeting dual-modal contrast agent for EPR imaging/fluorescence imaging (EPRI/FI).

A water-soluble probe with p-hydroxybenzyl quaternary ammonium linker for selective imaging in senescent cells

A water-soluble probe with p-hydroxybenzyl quaternary ammonium linker, FR-2a, for selective imaging in senescent cells is reported. Probe FR-2a integrated water-soluble fluorophore (HT-4a) and β-galactosidase (β-gal) trigger into one entity by a p-hydroxybenzyl quaternary ammonium linker. HT-4a is a styryl-based push–pull benzothiazole fluorophore with attractive properties, including excellent water-solubility, intense fluorescence emission and a large Stokes shift (161 nm), characterized by an intramolecular charge transfer (ICT) excited state. The formation of quaternary ammonium deactivated the ICT state, resulting in fluorescence quenching of FR-2a. In the presence of β-gal, the glycosidic bond was hydrolyzed and fluorophore HT-4a was released through self-immolative process, resulting in effective fluorescence recovery. FR-2a shows high affinity to β-gal (Km = 1.33 μM), exhibiting good sensitivity, selectivity and stability for imaging in senescent cells.