Investigation on the temperature effect of a mixed vesicle composed of polydiacetylene and BODIPY 558

BODIPY 558 is an insensitive fluorescent reagent to temperature. But when it is inserted into polydiacetylene (PDA) vesicles, the resultant complex presents a considerable temperature effect. In this article, we reported the temperature-dependent fluorescence intensity of a vesicle-based sensor constructed by PDA and BODIPY fluorescent probe. The fluorescence of BODIPY was considerably quenched in the polymerized diacetylene lipid membrane, but recovered by increasing the temperature of vesicle solution. The mechanism of quenching was detailedly investigated, and we deduced that the fluorescence quenching and recovery were associated with the conjugated conformation of the PDA backbone.     

Manipulating FRET with polymeric vesicles: development of a "mix-and-detect" type fluorescence sensor for bacterial toxin

A simple "mix-and-detect" type of fluorescence sensor for cholera toxin (CT) is reported. The sensor consists of a BODIPY lipid dye and polydiacetylene (PDA) vesicles and utilizes the lipid insertion and FRET mechanism to offer a direct and fluorescence "turn-on" detection of the analyte. BODIPY conjugated GM1, dissolved in a Tris buffer through aggregate formation, demonstrated substantial fluorescence quenching with addition of PDA vesicle solution. The close proximity of the dye molecules to the conjugated chains as a result of lipid insertion enables energy transfer from dye to the polymer backbone, yielding the observed phenomenon. When CT is present, the binding of BO-GM1 to CT results in formation of a complex that prohibits it from membrane insertion, leading to the blocking of the quenching process. The fluorescence signal was found to be proportional to the CT concentration. The method is very simple and allows specific and sensitive detection of the protein toxin with just a few mixing steps. It can be further developed into a general sensing strategy for detection of other proteins with amplified FRET mechanism.     

Proton permeation into single vesicles occurs via a sequential two-step mechanism and is heterogeneous

This article describes the first single-vesicle study of proton permeability across the lipid membrane of small (approximately 100 nm) uni- and multilamellar vesicles, which were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). To follow proton permeation into the internal volume of each vesicle, we encapsulated carboxyfluorescein, a pH-sensitive dye whose fluorescence was quenched in the presence of excess protons. A microfluidic platform was used for easy exchange of high- and low-pH solutions, and fluorescence quenching of single vesicles was detected with single-molecule total internal reflection fluorescence (TIRF) microscopy. Upon solution exchange and acidification of the extravesicular solution (from pH 9 to 3.5), we observed for each vesicle a biphasic decay in fluorescence. Through single-vesicle analysis, we found that rate constants for the first decay followed a Poisson distribution, whereas rate constants for the second decay followed a normal distribution. We propose that proton permeation into each vesicle first arose from formation of transient pores and then transitioned into the second decay phase, which occurred by the solubility-diffusion mechanism. Furthermore, for the bulk population of vesicles, the decay rate constant and vesicle intensity (dependent on size) correlated to give an average permeability coefficient; however, for individual vesicles, we found little correlation, which suggested that proton permeability among single vesicles was heterogeneous in our experiments.     

Fluorescence lifetime correlation spectroscopy combined with lifetime tuning: new perspectives in supported phospholipid bilayer research

A new concept based on fluorescence lifetime correlation spectroscopy (FLCS) is presented allowing the simultaneous determination of diffusion coefficients of identical molecules located in different environments. The difference in fluorescence lifetimes, which is the main prerequisite for FLCS, is reached by locating one population of the dye close to a light-absorbing surface. Since such surfaces quench fluorescence, the fluorescence lifetime of chromophores located close to these surfaces can be tuned in a specific manner. This approach has been demonstrated for a BODIPY-tail-labeled lipid in supported phospholipid bilayers (SPBs) as well as in phospholipid multilayers adsorbed onto solid supports. In particular, the effect of the solid support type on the fluorescence lifetime as well as its dependence on the BODIPY-support distance has been characterized and verified by theoretical considerations based on precise determination of refractive indices of the used supports. While the fluorescence lifetime of BODIPY dye is 5.6 ns in small unilamellar vesicles (SUVs) composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS), the lifetime is 1.8 ns in DOPC/DOPS SPBs adsorbed onto ITO-covered glass or 3.0 ns in a DOPC/DOPS monolayer adsorbed onto seven 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) layers on oxidized silicon. Using these particular systems, we demonstrated that FLCS enables one to characterize simultaneously two-dimensional lipid diffusion in the planar lipid layers and three-dimensional vesicle diffusion in bulk above the lipid layers using single dye labeling. The autocorrelation functions obtained by this new approach do agree with those obtained by standard FCS on isolated SPBs or vesicles. Possible applications of this virtual two-channel measurement using single dye labeling as well as one detection channel are discussed.     

Fluorescence behavior of the pH-sensitive probe carboxy SNARF-1 in suspension of liposomes.

When exposed to the intracellular environment fluorescent probes sensitive to pH exhibit changes of photophysical characteristics as a result of an interaction of the dye molecule with cell constituents such as proteins, lipids or nucleic acids. This effect is reflected in calibration curves different from those found with the same dye in pure buffer solutions. To study an interaction of the probe 5'(and 6')-carboxy-10-dimethylamino-3-hydroxy- spiro[7H-benzo[c]xanthene-7,1'(3H)-isobenzofuran]-3'-one (carboxy SNARF-1) with membrane lipids, we measured its fluorescence in model systems of large unilamellar vesicles (LUV) prepared by extrusion. When the dye was removed from the bulk solution by gel filtration the relative fluorescence intensity of the lipid-bound dye form was enhanced, showing a strong interaction of the dye molecule with LUV membrane lipids. Surprisingly, the dye molecules seem to be bound predominantly to the outer surface of the lipid bilayer. The same situation was found with small unilamellar vesicles prepared by sonication. This effect makes it difficult to use carboxy SNARF-1 for measurements of the internal pH in suspensions of liposomes.     

A Novel Colorimetric and Fluorescent pH Sensor Derived from Iminocoumarin and Thiophene‐Carboxaldehyde

A novel colorimetric and fluorescent pH sensor derived from iminocoumarin and thiophene‐carboxaldehyde was designed and synthesized. The structures of the dye and related compounds were characterized by IR, ¹H NMR, ¹³C NMR, and mass spectra. Color change from green to yellow of the new sensor solution in ethanol–water with the decrease in the pH value from 7 to 2 was observed by the naked eye. Under acidic conditions, the intensity of the maximum fluorescence emission peak of the sensor increased gradually with the decrease in the acidity of the solution (the increase in the pH value from 2 to 7) and attained to the maximum value at about pH 6. Under basic conditions, the fluorescence intensity of the emission peak of the sensor did not exhibit a distinct change at pH ≤ 11.85, and the fluorescence was quenched at pH 13.36, concomitant with the green color of the solution turning pale. The sensor can be used as a fluorescent pH probe in the presence of common metal cations and anions without interference.     

新型含苯硼酸和萘双亲化合物的合成与囊泡荧光传感器的制备

合成了含有识别基团苯硼酸和荧光基团萘的新型对-[(5-十二烷氧基-1-氧基)萘]甲基苯硼酸{p-[(5-dodecyloxy-1-oxy) naphthalene] methyl-phenylboronic acid, DNMPBA}双亲化合物; 该化合物在THF/水选择性溶剂中自组织成囊泡, 囊泡的相变温度为56.8 ℃; 当向囊泡体系加糖时, DNMPBA囊泡中的萘生色基在345 nm的荧光峰强度急剧增强; 荧光强度随添加不同糖的变化趋势为果糖>葡萄糖>麦芽糖>乙二醇. 荧光强度增强可能归因于所形成的硼酸酯减弱了DNMPBA双亲化合物中一个氧原子孤对电子对萘生色基的猝灭作用而使荧光强度重新恢复. DNMPBA囊泡与糖的相互作用导致体系荧光强度变化, 使该体系有可能应用于检测生物物质如糖的化学传感器.     

新型“内标”式双重荧光自组装膜的制备和DNA的界面传感

为了有效降低依据单一荧光强度定量分析的误差, 充分发挥自组装膜设计灵活、制备简单且分析灵敏度高的优点, 提高荧光定量分析的准确度和灵敏度, 本文提出构建“内标”式自组装膜, 即于硅烷化石英表面分别组装吖啶橙(AO)和量子点CdTe. 以AO为内标, CdTe为荧光探针, 通过静电吸引作用于AO和CdTe之间, 依次组装聚苯乙烯磺酸钠(PSS)和壳聚糖(CS). PSS和CS的组装有效地“屏蔽”了AO, 使其荧光强度I1不随分析物种浓度的引入而变化, 这样既可发挥荧光内标作用, 又使膜外层量子点CdTe的荧光强度I2随分析物种浓度的改变而改变. 所构建的自组装膜双重荧光强度比I2/I1不随激发光强度的波动和传感器位置的移动等微环境变化而变化, 但与被分析物种的浓度呈良好的线性关系, 从而实现了直接利用I2/I1准确定量的“内标”式荧光分析, 显著提高了荧光分析的准确度.     

检测维生素C的囊泡荧光传感器的制备

利用合成的含有识别基团苯硼酸和荧光读出基团萘的新型双亲化合物(DNMPBA)在THF/水选择性溶剂中自组织成囊泡,囊泡的相变温度为56.8℃;当向囊泡体系加维生素C时,DNMPBA囊泡中的萘生色基在345nm的荧光峰强度急剧减弱.荧光强度减弱归于所形成的硼酸酯增强了DNMPBA双亲化合物中一个氧原子孤对电子对萘生色基的淬灭作用.DNMPBA囊泡与维生素C的相互作用而导致体系荧光强度变化,使该体系有可能应用于检测生物物质如维生素C的化学传感器.     

基于DNA双链取代策略免标记检测铅离子的研究

建立了一种基于DNA双链取代策略和SYBR GreenⅠ(SG)作为荧光染料插入剂进行免标记铅离子检测的荧光传感方法。SG作为一种染料分子,与单链DNA作用产生的荧光强度很弱,但可以插入双链DNA,使SG荧光强度明显增强。检测时铅离子适配体首先与其部分互补单链DNA杂交形成稳定的双链DNA结构,当溶液中存在铅离子时,铅离子与其适配体特异性结合,双链DNA的数量减少,加入SG可实现铅离子的免标记定量检测。此方法具有灵敏度高、特异性强、简便快速等优点。最低检测浓度为2 nmol/L,检出限(S/N=3)为1.6 nmol/L,实际样品检测结果良好。     

Polydiacetylene/Anti‐HBs Complexes for Visible and Fluorescent Detection of Hepatitis B Surface Antigen on a Nitrocellulose Membrane

The immunochromatographic assay (ICA) using a nitrocellulose (NC) membrane offers several advantages. This technique is a rapid and straightforward method in contrast to other immunoassays. Polydiacetylene (PDA) vesicles have unique optical properties, displaying red color and red fluorescence at the same time. In this system, red‐phase PDA vesicles are used as a fluorescent dye as well as a surface for immobilized hepatitis B surface antibody (HBsAb). PDA has a remarkable stability compared with other fluorescent dyes. In this study, the most suitable PDA/HBsAb complexes are introduced for detecting hepatitis B surface antigen (HBsAg). Then, the PDA/HBsAb complexes affixed antibody is attached to NC membrane, which has two lines to confirm detection of HBsAg. The main advantage of this system is that the detection of HBsAg can be observed in both visible and fluorescent images due to the optical properties of polydiacetylene. Detection of HBsAg is observed up to 0.1 ng mL⁻¹ by fluorescent analysis and confirmed by red line on the NC membrane up to 1 ng mL⁻¹ (HBsAg) using the naked eye. Consequently, these results show that PDA/HBsAb complexes were successfully applied to ICA for the diagnosis of hepatitis B.     

基于硼酸/二醇识别的新型糖囊泡荧光传感器

摘要 合成了含有识别基团苯硼酸、喹啉发色团的新型双亲化合物,N-硼苄基-8-16烷基溴化喹啉（N-(boronobenzyl)-8-hexadecyloxyquinolinium bromide (BHQB)）.该化合物在可选择性溶剂中自组织成囊泡,囊泡的相变温度为52.4℃;研究了BHQB囊泡的荧光性质,结果表明：当向囊泡体系加入糖时,喹啉在425nm 峰逐渐增强而508nm峰急剧减弱,变化趋势为葡萄糖>果糖.实验结果表明,BHQB囊泡可以作为可植入、连续检测血糖浓度的荧光囊泡传感器,可望用于临床实际应用.     

Monolayer-functionalized microfluidics devices for optical sensing of acidity

This paper describes the integration of opto-chemosensors in microfluidics networks. Our technique exploits the internal surface of the network as a platform to build a sensing system by coating the surface with a self-assembled monolayer and subsequently binding a fluorescent sensing molecule to the monolayer. Fluorescent molecules were used that can switch between a fluorescent and a non-fluorescent state, depending on the acidity of the surrounding solution. Two systems were investigated. The first employs surface confinement of a Rhodamine B dye in a glass micro channel that serves as a molecular switch in organic solutions. Upon rinsing the micro channels with acidic or basic solutions it was possible to switch between the fluorescent and non-fluorescent forms reversibly. Moreover, this system could be used to monitor the mixing of two solutions of different acidity along the micro channel. To widen the scope of optical sensing in micro channels an Oregon Green dye derivative was immobilized, which functions as a sensing molecule for pH differences in aqueous solutions. In this case, a hybrid system was used consisting of a glass slide and PDMS channels. The fluorescence intensity was found to be directly correlated to the pH of the solution in contact, indicating the possibility of using such a system as a pH sensor. These systems allow real-time measurements and can be easily implemented in micro- and nanofluidics systems thus enabling analysis of extremely small sample volumes in a fast and reproducible manner.     

A BODIPY‐based Fluorescent Probe for Thiophenol

Thiophenol has been listed as one of the main sources of pollution. Sensitive probes for thiophenol are very significant. Herein, a BODIPY‐based fluorescent probe, named BDP, for poisonous thiophenol detection has been reported. BDP shows rapid response (15 s) and clear fluorescence enhancement (30 folds) to thiophenol in solution. The intensity of fluorescence and concentration of thiophenol have a good linear relationship. The detection limit is as low as 13.6 nmol · L⁻¹. BDP is stable towards pH and light radiation. Cell experiments demonstrate that BDP has good cell membrane penetrability, low cell toxicity and excellent imaging properties in living cells. Therefore, BDP has significant value on the detection of thiophenol in solution and in living cell.     

A fluorescence ratiometric nano-pH sensor based on dual-fluorophore-doped silica nanoparticles

We have synthesized dual-fluorophore-doped core-shell silica nanoparticles used as ratiometric pH sensor. The nanoparticles were prepared with a reverse microemulsion technique by simultaneously encapsulating two different fluorophores, the pH-sensitive dye fluorescein as a pH indicator and the pH-insensitive dye phenosafranine as an internal reference for fluorescence ratiometric measurement, into silica shell. The nanoparticles prevent the fluorescence dyes leaching from the silica matrix when immersed inside water. The hydrophilic silica shells were made by hydrolysing and polymerizing tetraethoxysilane (TEOS) in water-in-oil microemulsion. The fluorescence intensity ratio of the two dyes varied linearly as a function of pH in the range from 4.0 to 8.0. The sensor was also applied to measure pH of real water samples. The results are in good agreements with that using the conventional glass electrode method. The as-prepared fluorescent nanoparticles showed rapid response, excellent stability and high reproducibility as pH sensors.     

Substituent Effects in BODIPY in Live Cell Imaging

Small‐molecule organoselenium‐based fluorescent probes possess great capacity in understanding biological processes through the detection of various analytes such as reactive oxygen/nitrogen species (ROS/RNS), biothiols (cysteine, homocysteine and glutathione), lipid droplets, etc. Herein, we present how substituents on the BODIPY system play a significant part in the detection of biologically important analytes for in vitro conditions and live cell imaging studies. The fluorescence of the probe was quenched by 2‐chloro and 6‐phenyl selenium groups; the probe shows high selectivity with NaOCl among other ROS/RNS, and gives a turn‐on response. The maximum fluorescence intensity is attained within ≈1–2 min with a low detection limit (19.6 nm ), and shows a ≈110‐fold fluorescence enhancement compared to signals generated for other ROS/RNS. Surprisingly, in live cell experiments, the probe specifically located and accumulated in lipid droplets, and showed a fluorescence turn‐on response. We believe this turn‐on response occurred because of aggregation‐induced emission (AIE), which surprisingly occurred only by introducing one lipophilic mesityl group at the meso position of the BODIPY.     

Nanometre-sized molecular oxygen sensors prepared from polymer stabilized phospholipid vesicles

Nanometre-sized, chemically-stabilized phospholipid vesicle sensors have been developed for detection of dissolved molecular oxygen. Sensors were prepared by forming 150 nm phospholipid vesicles from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or DOPC doped with small (<1%) mole percentages of 1,2-dioleoyl-sn-glycero-3-phosphoethanol amine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE). Sensors were stabilized via cross-linking polymerization of hydrophobic methacrylate monomers partitioned into the hydrophobic interior of the DOPC bilayer. The resultant unilamellar, nanometre-sized, polymer-lipid vesicles are spherical, biocompatible and protect sensing components that are loaded into the aqueous interior of the vesicle from interfering species in the exterior environment. For O(2) detection, the oxygen-sensitive fluorescent dye, tris(1,10-phenanthroline)ruthenium(II) chloride (Ru(phen)(3)) was encapsulated into the aqueous interior of the polymerized phospholipid vesicle. NBD-PE was introduced into the phospholipid bilayer of the sensor as a reference dye, allowing ratiometric sensors to be constructed. The resultant sensors show high sensitivity, excellent reversibility and excellent linearity over a physiological range of dissolved oxygen concentrations. These results suggest that polymerized phospholipid vesicle sensors can be used for monitoring intracellular O(2) dynamics.     

Fluorescence-quenching phenomenon by photoinduced electron transfer between a fluorescent dye and a nucleotide base.

Fluorescently labeled oligonucleotide probes have been widely used in biotechnology, and fluorescence quenching by the interaction between the dyes and a nucleobase has been pointed out. This quenching causes big problem in analytical methods, but is useful in some other cases. Therefore, it is necessary to estimate the fluorescence quenching intensity under various conditions. We focused on the redox properties of some commercially available fluorescent dyes, and investigated dye-nucleotide interactions between a free dye and a nucleotide in aqueous solution by electrochemical and spectroscopic techniques. Our results suggested that the quenching was accompanied by photoinduced electron transfer between a thermodynamically quenchable excited dye and a specific base. Several kinds of fluorescent dyes labeled to the 5'-end of oligonucleotide C10T6 were prepared, and their quenching ratios compared upon hybridization with the complementary oligonucleotide A6G10. The quenching was completely reversible and their efficiencies depended on the attached fluorophore types. The fluorescence of 5-FAM, BODIPY FL or TAMRA-modified probe was strongly quenched by hybridization.     

Proton diffusion across membranes of vesicles of poly(styrene-b-acrylic acid) diblock copolymers

A study of proton diffusion across membranes of block copolymer vesicles in dilute solution is described. The vesicles were formed by the self-assembly of a diblock copolymer of poly(styrene-b-acrylic acid) (PS(310)-b-PAA(36), where the numbers represent the degree of polymerization for individual blocks). A pH gradient was created across the vesicle membrane with the interior pH (pH(in)) of ca. 2.9 and the exterior pH (pH(out)) of ca. 8.5. The permeability of the polystyrene (PS) membrane was tuned by the addition of different amounts of dioxane (0-40 wt %) to the external aqueous solution. Proton concentrations in the solution outside of the vesicles were followed by monitoring the spectrum of a pH-sensitive fluorescent dye, namely 8-hydroxypyrene-1,3,6-trisulfonate. After the start of the experiment, the proton concentrations increase linearly with the square root of time, while the slopes of the lines increase with dioxane content. To calculate the diffusion coefficients of the protons across the vesicular membrane, the concentration data were fitted using a model, which describes the diffusion of species across the membrane of a reservoir. The apparent diffusion coefficient (D*, which equals the true diffusion coefficient multiplied by the partition coefficient of protons between PS and water) increases from 1.1 x 10(-18) cm(2)/s at 7 wt % dioxane in the external solution to 1.2 x 10(-14) cm(2)/s at 40 wt %. The increase of D* with dioxane content is related to its plasticization of the PS membrane, which can be used as a gating mechanism.     

Towards a fluorescent molecular switch for nucleic acid biosensing

A novel fluorescent molecular switch for the detection of nucleic acid hybridization has been explored in relation to the development of a structure that would be amenable for operation when immobilized for solid-phase analyses. The structure was prepared by self-assembly, and used Neutravidin as the central multivalent docking molecule, a newly synthesized biotinylated long-chain linker for intercalating dye that was modified with thiazole orange (TO) at one end, and a biotinylated probe oligonucleotide. Self-assembly of the biotinylated components on adjacent Neutravidin binding sites allowed for physical placement of an oligonucleotide probe molecule next to tethered TO. The TO located at the end of the flexible linker chain was available to intercalate, and could report if a duplex structure was formed by a probe–target interaction by means of fluorescence intensity. Subsequently, regeneration of the single-stranded probe was possible without loss of the intercalator to solution. The switch constructs were assembled in solution and subsequently immobilized onto biotin functionalized optical fibers to complete the sensor design. Solution-phase fluorescence lifetime data showed a biexponential behavior for switch constructs, suggesting intercalation as well as a significant secondary binding mode for the immobilized TO. It was found that the secondary binding mechanism for the dye to DNA could be decreased, thus shifting the dye to intercalative binding modes, by adjusting the solution conditions to a pH below the pI of Neutravidin, and by increasing the ionic strength of the buffer. Preliminary work demonstrated that it was possible to achieve up to a fivefold increase in fluorescence intensity on hybridization to the target.