Synthesis and characterisation of quantum dots coupled to mycolic acids as a water-soluble fluorescent probe for potential lateral flow detection of antibodies and diagnosis of tuberculosis

This work explores the potential use of cadmium-based quantum dots (QDs) coupled to mycolic acids (MAs) as a fluorescent probe to detect anti-MA antibodies which are biomarkers for tuberculosis (TB). The use of free MAs as antigens for the serodiagnosis of TB is known but has not been developed into a point of care test. This study focuses on the synthesis, solubility, and lateral flow of QDs coupled to MAs. Water-soluble CdSe/ZnS QDs capped with l -cysteine were synthesised and covalently coupled to MAs via amide linkages to form a water-soluble fluorescent probe: MA-CdSe/ZnS QDs. The MA-CdSe/ZnS QDs showed broad absorption bands and coupling, confirmed by the presence of amide bonds in the Fourier-transform infrared (FTIR) spectrum, resulting in a blue shift in fluorescence. Powder X-ray diffraction (XRD) revealed a shift and increase in the number of peaks for MA-CdSe/ZnS QDs relative to the L-cys-CdSe/ZnS QDs, suggesting that coupling changed the crystal structure. The average particle size of MA-CdSe/ZnS QDs was ~3.0 nm. Visual paper-based lateral flow of MA-CdSe/ZnS QDs was achieved on strips of nitrocellulose membrane with both water and membrane blocking solution eluents. The highly fluorescent MA-CdSe/ZnS QDs showed good water solubility and lateral flow, which are important properties for fluorescence sensing applications.     

Synthesis and characterization of water-soluble two-photon excited blue fluorescent chromophores for bioimaging.

We report here the synthesis and characterization of a new type of non-ionic blue fluorescent water-soluble chromophores specifically designed for two-photon absorption microscopy. The water solubility is induced by introduction of short oligo(ethylene glycol) monomethyl ether moieties. This work has led to low molecular weight dyes with efficient two-photon absorption cross sections and high fluorescence quantum yield in organic solvents as well as in aqueous solutions.     

Synthesis and carbohydrate binding studies of fluorescent α-amidoboronic acids and the corresponding bisboronic acids

Fluorescent boronic acids are very useful for the design and synthesis of carbohydrate sensors. In an earlier communication, we first described the effort of developing water soluble fluorescent α-amidoboronic acids, which change fluorescence upon sugar binding. In this report, we describe a general method of functionalizing such boronic acids and their applications in the preparation of bis-α-amidoboronic acids with significantly enhanced binding for oligosaccharides as compared to their monoboronic acid counterparts. The advantages of good water solubility, easy modification to generate diversity, and modularity in synthesis will make α-amidoboronic acids very useful building blocks for future synthesis of boronic acid-based fluorescent sensors.     

A water‐soluble fluorescent pH probe based on perylene dyes and its application to cell imaging

A fluorescent pH probe, N,N′‐bi( l ‐phenylalanine amine)‐perylene‐3,4;9,10‐dicarboxylic diimide (PDCDA) was synthesized and used for pH sensing in living cells. A significant fluorescence intensity change was observed over a pH range from 7.0 to 4.0. Electrostatic potential maps (MEP) suggested that the electronic repulsion between PDCDAs was increased by the high negative electrostatic potential which resulted in a high water solubility of PDCDA. PDCDA was successfully applied as a high‐performance fluorochrome for living HeLa cell imaging. The results demonstrate that the probe PDCDA is a good candidate for monitoring pH fluctuations in living cells with good water solubility, low cytotoxicity, high fluorescence quantum yield and photostability. Copyright © 2015 John Wiley & Sons, Ltd.     

Enhanced hydrolytic stability and water solubility of an aromatic nitrogen mustard by conjugation with molecular umbrellas

Chlorambucil, an aromatic nitrogen mustard, has been conjugated to putrescine- and spermidine-based scaffolds bearing one, two, and four persulfated cholic acid units. Those conjugates bearing two or four sterols show improved hydrolytic stability and water solubility relative to chlorambucil. A similar conjugate that contained only one sterol unit shows negligible improvement in hydrolytic stability but a significant increase in water solubility. Qualitatively, the hydrolytic stability within this series of conjugates parallels the shielding effects that have previously been found for related conjugates bearing a pendant, hydrophobic fluorescent probe. In vitro studies indicate that these conjugates possess modest to moderate activity against certain human lymphoblastic leukemia and human colon carcinoma cells.     

Novel water-soluble near-infrared cyanine dyes: synthesis, spectral properties, and use in the preparation of internally quenched fluorescent probes

In this paper, we describe the synthesis and the photophysical properties of two novel near-infrared (NIR) cyanine dyes (NIR5.5-2 and NIR7.0-2) which are water soluble potential substitutes of the commercially available Cy 5.5 and Cy 7.0 fluorescent labels respectively. For each one of these cyanine dyes, the synthetic strategy relies on the postsynthetic derivatization of a cyanine precursor in order to introduce the key functionalities required for bioconjugation of these NIR fluorophores. For NIR5.5-2, a reactive amino group was acylated with an original trisulfonated linker for water solubility. For NIR7.0-2, a vinylic chlorine atom was derivatized through a SRN1 reaction for the introduction of a monoreactive carboxyl group for labeling purposes. Unexpectedly, when these two fluorophores were closely associated within a peptidic architecture, mutual fluorescence quenching between NIR5.5-2 and NIR7.0-2 was observed both at 705 (NIR5.5-2) and 798 nm (NIR7.0-2). On the basis of this property, a novel internally quenched caspase-3-sensitive NIR fluorescent probe was prepared.     

Fluorescent rotors and their applications to the study of G-F transformation of actin.

New fluorescent rotor molecules having hydrophilic functional groups, which are derivatives of p-(N,N-dialkylamino)benzylidenemalononitrile, were synthesized. Their properties as fluorescent rotors were confirmed by an observation of solvent viscosity-dependent fluorescence. Incorporation of hydrophilic groups into the molecules increased the solubility of fluorescent rotors in aqueous media; the application of the compounds to biochemical systems became feasible as a consequence. To demonstrate this applicability, we attempted to monitor the G-F transformation of rabbit skeletal muscle actin with these newly synthesized compounds. All the compounds carrying a malononitrile moiety showed greater fluorescence in F-actin. Among them, 1-(2-hydroxyethyl)-6-[(2,2-dicyano)vinyl]-2,3,4-trihydroquinoli ne gave the best result by the criteria of the difference in fluorescence quantum yield for G- and F-actin, solubility, and stability of the compound. The method has the major advantage of not requiring covalent modification of actin.     

DNA-Ag nanoclusters as fluorescence probe for turn-on aptamer sensor of small molecules

As promising substitutes for organic dyes and quantum dots, few-atom fluorescent silver nanoclusters (Ag NCs) have recently gained much attention in a wide range from cellular imaging to chemical/biological detection applications owing to their ultrasmall size (<2 nm), excellent photostability, good biocompatibility and water solubility. Herein, we design an aptamer, guanine-rich (G-rich) DNA and Ag NCs nanocomplex to investigate its ability for the detection of small molecules. The design contains two DNA strands which are both chimeric conjugates of the DNA aptamer sequence fragment and G-rich sequence fragment. Using cocaine as a model molecule, the two DNA strands are in free state if there is no cocaine present, and the formed Ag NCs through the reduction of Ag(+) by NaBH(4) show weak fluorescence emission. In the presence of cocaine, however, the two aptamer fragments bind cocaine, which in turn puts the two G-rich sequence fragments in proximity and the fluorescent intensity of DNA-Ag NCs enhances greatly. As a result, DNA-Ag NCs are demonstrated as a novel, cost-effective and turn-on fluorescent probe for the analysis of cocaine, with a detection limit of 0.1 μM. Besides, successful detection of adenosine triphosphate (ATP) with detection limit of 0.2 μM demonstrates its potential to be a general method.     

Yield, solubility and conformational quality of soluble proteins are not simultaneously favored in recombinant Escherichia coli

Many enzymes or fluorescent proteins produced in Escherichia coli are enzymatically active or fluorescent respectively when deposited as inclusion bodies. The occurrence of insoluble but functional protein species with native-like secondary structure indicates that solubility and conformational quality of recombinant proteins are not coincident parameters, and suggests that both properties can be engineered independently. We have here proven this principle by producing elevated yields of a highly fluorescent but insoluble green fluorescent protein (GFP) in a DnaK- background, and further enhancing its solubility through adjusting the growth temperature and GFP gene expression rate. The success of such a two-step approach confirms the independent control of solubility and conformational quality, advocates for new routes towards high quality protein production and intriguingly, proves that high protein yields dramatically compromise the conformational quality of soluble versions.     

The acidity of protein fusion partners predominantly determines the efficacy to improve the solubility of the target proteins expressed in Escherichia coli

Maximization of the soluble protein expression in Escherichia coli (E. coli) via the fusion expression strategy is usually preferred for academic, industrial and pharmaceutical purposes. In this study, a set of distinct protein fusion partners were comparatively evaluated to promote the soluble expression of two target proteins including the bovine enterokinase largely prone to aggregation and the green fluorescent protein with moderate native solubility. Within protein attributes that are putatively involved in protein solubility, the protein acidity was of particular concern. Our results explicitly indicated the protein fusion partners with a stronger acidity remarkably exhibited a higher capacity to enhance the solubility of the target proteins. Among them, msyB, an E. coli acidic protein that suppresses the mutants lacking function of protein export, was revealed as an excellent protein fusion partner with the distinguished features including high potential to enhance protein solubility, efficient expression, relatively small size and the origin of E. coli itself. In principle, our results confirmed the modified solubility model of Wilkinson-Harrison and especially deepened understanding its essence. Meanwhile, the roles of other parameters such as protein hydrophilicity in solubility enhancement were discussed, a guideline to design or search an optimum protein solubility enhancer was also proposed.     

Physical and biological properties of fluorescent dansylated bile salt derivatives: the role of steroid ring hydroxylation

The hydroxyl groups of bile salts play a major role in determining their physical properties and physiologic behavior. To date, no fluorescent bile salt derivatives have been prepared which permit evaluation of the functional role of the steroid ring. We have prepared five fluorescent cholanoyl derivatives using a dansyl-ethylene diamine precursor linked to the sulfonyl group of taurine; N-(5-dimethylamino-1-naphthalenesulfonyl)-N'-(2-aminoethanesulf onyl)- ethylenediamine. The fluorescent dansyl-taurine was conjugated to the carboxyl group of free bile acids, enabling the labeling of the series: dehydrocholate, ursodeoxycholate, cholate, chenodeoxycholate and deoxycholate. Despite a systematic hydrophobic shift compared with the native bile salts (aqueous solubility and water:octanol partitioning), the influence of steroid ring hydroxylation was retained, with the dehydrocholate and cholate derivatives more water soluble than the dihydroxy derivatives. Similarly, the sequence of HPLC mobilities, reflecting relative hydrophilicity, was identical in the dansyl-taurine derivatives and the native taurine-conjugated bile salts. Cellular uptake of all five steroid derivatives was rapid, and partial inhibition of [3H]taurocholate uptake was observed in isolated hepatocytes. Rates of biliary excretion of the dansylated derivatives by the isolated perfused rat liver correlated closely with hydrophilicity. Collectively, these findings indicate that the influence of the hydroxyl groups is retained in this series of dansylated steroids, and that hydroxylation is a key determinant of their hepatocellular transport and biliary excretion. These fluorescent bile salt derivatives may thus serve as unique probes for investigating structure-function relationships in hepatic processing of steroid-based compounds.     

A deep-red xanthene-based highly sensitive fluorescent probe for detection of hypochlorite

As an oxidant, deodorant and bleaching agent, the hypochlorous acid (HClO) and hypochlorite (ClO⁻) are widely used in corrosion inhibitors, textile dyes, pharmaceutical intermediates and in our daily lives. However, excess usage or aberrant accumulation of ClO⁻ leads to tissue damage or some diseases and even cancer. Therefore, it is necessary to develop a fluorescent probe that specifically identifies ClO⁻. In this article, we synthesized a deep-red xanthene-based fluorescent probe (XA-CN). The strong electron deficient group dicyano endows the probe XA-CN deep-red fluorescent emission with high solubility, selectivity and sensitivity for ClO⁻ detection. Studies showed that the probe demonstrated turn-off fluorescence (643 nm) at the presence of ClO⁻ in dimethylsulfoxide/phosphate-buffered saline 1:1 (pH 9) solution with a limit of detection of 1.64 μM. Detection mechanism investigation revealed that the electron deficient group -CN and the hydroxyl group was oxidized into aldehyde or carbonyl groups at the presence of ClO⁻, resulting ultraviolet-visible absorption of the probe blue shifted and turned-off fluorescence. Furthermore, XA-CN was successfully used for the detection of ClO⁻ in tap water samples.     

New Drosophila transgenic reporters: insulated P-element vectors expressing fast-maturing RFP

In vivo green fluorescent protein (GFP)/red fluorescent protein (RFP) double-labeling studies have been hampered by several inconvenient properties of DsRed, the first described RFP. These disadvantages include a very slow (> 24 h) maturation time, emission of contaminating green light, and low solubility. A recently developed variant of DsRed, called DsRed.T4, has a much shorter maturation time, no significant green emission, and improved solubility. We have constructed Drosophila P-element transformation vectors encoding DsRed.T4 for promoter/enhancer analysis, labeling of living cells, or RFP tagging of proteins. These new vectors have all of the features of the widely used Pelican/Stinger GFP vectors, including insulator sequences to reduce position effects, an extensive polylinker, and both cytoplasmic and nuclear-localized forms of the reporter. We have also constructed an upstream activating sequence (UAS)-DsRed.T4 vector, for GAL4 activation of the reporter. We find that DsRed.T4 is very easily detected in transgenic flies without contamination of the GFP signal and that it matures to its fluorescent form nearly simultaneously with GFP. This advance in Drosophila reporter technology makes timed double-labeling experiments in developing transgenic animals possible for the first time.     

Synthesis,spectroscopic properties and protein labeling of water soluble 3,5-disubstituted boron dipyrromethenes

Sulfur-containing 3,5-disubstituted boron dipyrromethene (Bodipy^®) fluorescent probes with improved water solubility were synthesized. A dicarboxylic acid derivative that can be excited by the 543 nm HeNe laser line is very soluble in aqueous solution and retains high fluorescence quantum yield of the unionizable parent molecule. Conversion of the dicarboxylic acid to the succimidyl or sulfosuccinimidyl diester produces molecules capable of labeling proteins with a bright and stable fluorescence signal.     

The solvation study of carbon, silicon and their mixed nanotubes in water solution

Nanotubes are believed to open the road toward different modern fields, either technological or biological. However, the applications of nanotubes have been badly impeded for the poor solubility in water which is especially essential for studies in the presence of living cells. Therefore, water soluble samples are in demand. Herein, the outcomes of Monte Carlo simulations of different sets of multiwall nanotubes immersed in water are reported. A number of multi wall nanotube samples, comprised of pure carbon, pure silicon and several mixtures of carbon and silicon are the subjects of study. The simulations are carried out in an (N,V,T) ensemble. The purpose of this report is to look at the effects of nanotube size (diameter) and nanotube type (pure carbon, pure silicon or a mixture of carbon and silicon) variation on solubility of multiwall nanotubes in terms of number of water molecules in shell volume. It is found that the solubility of the multi wall carbon nanotube samples is size independent, whereas multi wall silicon nanotube samples solubility varies with diameter of the inner tube. The higher solubility of samples containing silicon can be attributed to the larger atomic size of silicon atom which provides more direct contact with the water molecules. The other affecting factor is the bigger inter space (the space between inner and outer tube) in the case of silicon samples. Carbon type multi wall nanotubes appeared as better candidates for transporting water molecules through a multi wall nanotube structure, while in the case of water adsorption problems it is better to use multi wall silicon nanotubes or a mixture of multi wall carbon/ silicon nanotubes.     

定点突变改善红色荧光指示蛋白玉米尿卟啉原Ⅲ甲基化酶的水溶性研究

尿卟啉原Ⅲ甲基化酶是一种新型的红色荧光指示蛋白,但是,在大肠杆菌重组表达的SUMT水溶性相对较低,限制了它的应用范围,而且对于结合在蛋白的色素组分尚不清楚。利用定点突变产生玉米尿卟啉原Ⅲ甲基化酶L88R/L89G双突变体和L166A突变体,两种突变体分别在大肠杆菌中重组表达,Ni-NTA一步纯化。紫外可见光谱扫描和质谱分析确定从纯化的L88R/L89G双突变体蛋白分离的色素组分。L88R/L89G双突变体在大肠杆菌细胞内有酶活,而L166A突变体胞内酶活丧失。结合蛋白的主要组分为三甲基化咕啉。纯化的双突变体蛋白水溶性增加,为提高它作为荧光指示蛋白检测外源融合蛋白的水溶性打下基础。     

Highly luminescent nitrogen‐doped carbon dots for simultaneous determination of chlortetracycline and sulfasalazine

Here, we have presented a green and facile strategy to fabricate nitrogen‐doped carbon dots (N‐CDs) and their applications for determination of chlortetracycline (CTC) and sulfasalazine (SSZ). The fluorescent N‐CDs, prepared by one‐step hydrothermal reaction of citric acid and l ‐arginine, manifested numerous excellent features containing strong blue fluorescence, good water‐solubility, narrow size distribution, and a high fluorescence quantum yield (QY) of 38.8%. Based on the fluorescence quenching effects, the as‐synthesized N‐CDs as a fluorescent nanosensor exhibited superior analytical performances for quantifying CTC and SSZ. The linear range for CTC was calculated to be from 0.85 to 20.38 μg ml^?1 with a low detection limit of 0.078 μg ml^?1. Meanwhile, the linear range for SSZ was estimated to be from 0.34 to 6.76 μg ml^?1 with a low detection limit of 0.032 μg ml^?1. Therefore, the N‐CDs hold admirable application potential for constructing a fluorescent sensor for pharmaceutical analysis.     

Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein

Existing protein tagging and detection methods are powerful but have drawbacks. Split protein tags can perturb protein solubility or may not work in living cells. Green fluorescent protein (GFP) fusions can misfold or exhibit altered processing. Fluorogenic biarsenical FLaSH or ReASH substrates overcome many of these limitations but require a polycysteine tag motif, a reducing environment and cell transfection or permeabilization. An ideal protein tag would be genetically encoded, would work both in vivo and in vitro, would provide a sensitive analytical signal and would not require external chemical reagents or substrates. One way to accomplish this might be with a split GFP, but the GFP fragments reported thus far are large and fold poorly, require chemical ligation or fused interacting partners to force their association, or require coexpression or co-refolding to produce detectable folded and fluorescent GFP. We have engineered soluble, self-associating fragments of GFP that can be used to tag and detect either soluble or insoluble proteins in living cells or cell lysates. The split GFP system is simple and does not change fusion protein solubility.     

The 5-substituted piperazine as a novel secondary pharmacophore greatly improving the physical properties of urea-based inhibitors of soluble epoxide hydrolase

The inhibition of the mammalian soluble epoxide hydrolase (sEH) is a promising new therapy in the treatment of hypertention and inflammation. The problems of limited water solubility and high melting points commonly displayed by the active 1,3-disubstituted ureas prevent the further development of potent urea-based sEH inhibitors. Therefore, a new class of potent inhibitors of sEH were designed and synthesized by the introduction of a polar constrained piperazino group in the right side of adasmantyl urea to increase the water solubility. A facile and general synthesis was established to prepare a series of 1-adamantan-1-yl-3-(2-piperazin-2-yl-ethyl)-ureas (1a-d) with various 5-substitutions on the 2-piperazino ring, which will advance the SAR study by the efficient making of structurally diverse analogs. The effect of the 5-substitution on the activity and the water solubility was examined. The best potency was exhibited by the 5-benzyl-substituted-piperazine-containing urea with an IC50 value of 1.37 microM against human sEH and good water solubility (S=7.46 mg/mL) and low melting point, in which the 5-substituted piperazine serves as a favorable secondary pharmacophore and a water-solubility enhancing group. Our present work provides a promising new template for the design of orally available therapeutic agents for the disorders that can be addressed by changing the in vivo concentration of the chemical mediators that contain an epoxide.     

Facile hydrothermal synthesis of nitrogen rich blue fluorescent carbon dots for cell bio-imaging of Candida albicans

Nitrogen doped carbon dots (N-CDs) are well documented as an outstanding fluorogenic material for protein tags, live cell imaging and protein-receptor based fluorescence sensors owing to its good optical features with less cytotoxicity and better water solubility. In this regard, the present work describes the synthesis of nitrogen rich blue fluorescent carbon dots (NR-CDs) through hydrothermal treatment of citric acid monohydrate (CA) and 2-aminopyridine (2-AP). The optical properties of NR-CDs are further analyzed by common analytical methods viz., Fourier transform infrared (FT-IR), UV–visible (UV–vis) and Fluorescence spectroscopies. The surface chemical composition and morphology of NR-CDs are acquired by X-ray photo electron spectroscopy (XPS) and high resolution transmission electron microscopy (HR-TEM), respectively. The NR-CDs produce blue fluorescent at 421 nm at the excitation wavelength of 310 nm, the calculated quantum yield is about 18% with respect to standard quinine sulfate. The synthesized NR-CDs contains 15.03 wt % of N revealed by XPS results. Further, the NR-CDs are used as a fluorescence staining agent for cell imaging of Candida albicans (C. albicans) and the cytotoxicity are also measured. All the outcomes proposed that the NR-CDs act as good staining agent for C. albicans with less cytotoxicity.