Enhanced conjugated polymer fluorescence quenching by dipyridinium-based quenchers in the presence of surfactant

Poly[(2-methoxy-5-propyloxysulfonate)phenylene vinylene] (MPS-PPV) was synthesized directly from its bischloromethylated monomer, considerably reducing the total number of steps involved in the polymer preparation. For the first time, a simple technique of ultracentrifugation was employed for final purification of the polymer. The interactions among the polymer, surfactant, and quencher molecules, as well as amplified fluorescence quenching and fluorescence enhancement associated with the interactions, were investigated and discussed. When compared with methyl viologen [MV]2+, higher values of Stern-Volmer constant K(SV) values on the order of > or =10(7) M(-1) were observed for the newly synthesized N-(2-carboxyhexadecanoyl)-N'-methyl-4,4'-bipyridinium iodide bromide ([CHMB]2+) quencher in the presence of 1,2-dioleoyl-3- trimethylammonium propane (DOTAP) surfactant. Comparisons of surfactants demonstrated that the K(SV) of [CHMB]2+ was 10-fold higher in the presence of dodecyltrimethylammonium bromide (DTAB) surfactant than with DOTAP. Polymer fluorescence was totally recovered upon addition of DOTAP surfactant to a MV-quenched polymer system, whereas only 50% of fluorescence was recovered upon addition of DOTAP surfactant to the CHMB-quenched polymer solution. In contrast, no fluorescence was recovered when DTAB was added to either the MV- or CHMB-quenched polymer systems. Thus, fluorescence enhancement was observed for the polymer complex with DOTAP, whereas fluorescence quenching was predominant in the polymer complex with DTAB. Such studies will not only help to better understand the intrinsic properties of the ionic conjugated polymer and amplified fluorescence quenching and enhancement but also provide guidelines to develop the next generation of ionic conjugated-polymer-based biosensors.     

pH-dependent optical properties of a poly(phenylene ethynylene) conjugated polyampholyte

A poly(phenylene ethynylene) conjugated polymer (PPE-NMe(3)(+)-COO(-)) containing tetraalkylammonium groups and carboxylate groups has been synthesized by Sonogashira coupling. Due to the presence of the strong cationic and weak anionic pendant units, the polymer undergoes a pH-induced transition from cationic polyelectrolyte to polyampholyte due to deprotonation of the carboxylic acid units in basic solution. Studies of the pH dependence of the polymers' optical properties reveal changes in absorption oscillator strength and fluorescence quantum efficiency that are triggered by the transition from cationic polyelectrolyte to polyampholyte nature. Stern-Volmer fluorescence quenching of PPE-NMe(3)(+)-COO(-) with a negatively charged quencher 1,4,5,8-naphthalenediimide-N,N-bis(methylsulfonate) (NDS) shows that the polymer fluorescence quenching is amplified at low pH where the polymer is a polycation, whereas the quenching efficiency is considerably less at high pH.     

Effects of polymer aggregation and quencher size on amplified fluorescence quenching of conjugated polyelectrolytes

The fluorescence of conjugated polyelectrolytes (CPEs) is efficiently quenched by low concentrations of quenchers with opposite charges. We have reported the close correlation between this amplified quenching phenomenon and CPE chain aggregation. In this paper, we further demonstrate the profound correlation between the fluorescence quenching efficiency, CPE chain aggregation, and quencher molecular size. Aggregation of a poly(phenylene ethynylene)-type CPE (PPE-CO2-) is induced by the addition of either water or Ca2+ to methanol solution, as indicated by absorption, fluorescence, dynamic light scattering, and fluorescence microscope measurements. For quencher ions with a small molecular size, such as methyl viologen (MV2+), either the loose (induced by the addition of Ca2+) or the compact (induced by the addition of water) CPE chain aggregates are beneficial to the fluorescence quenching. For quencher ions with large molecular size, such as tris(4,7-diphenyl-1,10-phenanthroline)ruthenium (Ru(dpp)32+), however, the loose chain aggregates are found to be favorable for quenching, while the quenching efficiency is lower for the compact polymer aggregates present in aqueous solution.     

Interaction of naphthalene‐labeled poly(hydrochloride quaternized 2‐norbornene‐5‐methyleneamine) with O/W microemulsion droplets

Interaction between naphthalene‐labeled poly(hydrochloride quaternized 2‐norbornene‐5‐methyleneamine), poly(HCQNBMA)/NA (luminophore), and quencher, emulsifier or coemulsifier in the aqueous or microemulsion media was studied by using steady‐state fluorescence measurements. Fluorescence experiments were carried out with poly(HCQNBMA)/NA dissolved in the aqueous solutions of ionic and nonionic emulsifiers, emulsifier/n‐hexane/water microemulsion (A) and emulsifier/n‐hexane/1‐pentanol/water microemulsion (B), respectively. The intensity of fluorescence emission of poly(HCQNBMA)/NA was much higher in the aqueous phase than in microemulsion. Furthermore, the aqueous solution of ionic emulsifier increased the monomer emission. The increase in the monomer emission can be ascribed to the shielding of the naphthalene (NA) groups by SDS micelles. This separates NA groups from each other, which depresses the deactivation of excited states. The strong decrease in monomer emission within the microemulsion media probably results from the elongated conformation structure of the polymer molecule, and higher conformation freedom of NA groups, which increases interaction between the probe and the quencher. The formation of nonfluorescence clusters is not ruled out. The quenching of NA emission by nitromethan (NM) is much stronger in microemulsion than in the aqueous phase, and the quenching is more pronounced for the low molecular weight 2‐norbornene‐5‐methoxylnaphthalene (NBMNA) than for its polymer. The extent of penetration of reactants into the interfacial layer governs quenching of hydrophobic NA probe by hydrophilic quencher. Furthermore, the quenching events are connected with the thickness and density of the interfacial layer, as well as its charge. The addition of coemulsifier (1‐pentanol) increases the total surface area of the microdroplets, the entry rate of reactants into the microdroplets, and the interaction of hydrophobic and hydrophilic reactants. The quenching events are more pronounced in the close packed o/w interfacial layer than in the loosely packed one. © 2000 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 39: 101–114, 2001     

Synthesis of ionic polyurethanes with pyrene rings: Spectral properties and fluorescence quenching study

Hydrophilic ionic polyurethanes with 4‐chloromethylphenylcarbamoyl‐1‐oxymethylpyrene located on the quaternary ammonium structure from a polymer based on poly(ethylene glycol), isophorone diisocyanate, and N‐methyldiethanolamine were prepared by a quaternization reaction, in which the amount of pyrene covalently attached to the polymeric backbone ranged from 1.14 to 19.82 mmol of fluorophore/100 g of polymer. It was interesting to compare the photoluminescence of the pyrene polyurethane carrying a few mole percent of pyrene moieties with that of a third polymer resulting from its subsequent quaternization with benzyl chloride up to a concentration of ionic groups as in the latter (quaternization degree = 14.15%). The process of excimer formation between the pyrene molecules attached to the ionic polyurethane was investigated in tetrahydrofuran (THF), dimethylformamide, film, and THF/H₂O to illustrate the expected differences in the polymer behavior compared with that of the starting pyrene derivative. The formation of aggregates or core–shell micelles was sustained by the fluorescence data, which indicated the existence of pyrene units in the ground state of the molecule, giving rise thus to an explanation for the high excimer‐to‐monomer intensity ratio. The fluorescence decay of pyrene polyurethanes in the presence of various concentrations of nitrobenzene used as a quencher was analyzed too when the fluorescence quenching in the polymer solution normally followed Stern–Volmer kinetics. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 3945–3956, 2005     

A study of molecular diffusion in alcohol—water solution of poly-4-vinylpyridine by fluorescence quenching

Intra- and intermolecular diffusion processes in alcohol—water solutions of poly-4-vinylpyridine (PVPy) were studied by the fluorescence quenching method. It was prepared fluorescent-labelled PVPy (PVPy-F) by copolymerizing 4-vinylpyridine with anthrylmethylmethacrylate (F) (one chromophore group per 500 chain links in the copolymer). PVPy carrying both the chromophore and quenching chain links (quaternization of the pyridine residue by dimethylsulphate) (PVPy-FQ) and PVPy carrying only the quenchers (PVPy-Q).Measurement of the mean lifetime of the excited state of the anthracene group for the polymers and for the monomer analogue (F) with increasing quencher concentration and use of the Stern-Volmer relation permit determination of the constants of bimolecular collisions between the monomer quencher (Q) (4-ethyl-N-methyl pyridinium sulphate) and F, between the monomer quencher and PVPy-F and between Q and F incorporated in the same polymer chain (PVPy-FQ). The values of the diffusion constant for the system of PVPy-F and Q and for the system of PVPy-FQ are approximately two and four times respectively less than that for the monomer pair (F + Q). For the latter system, the finding indicates some hindrance to the diffusion of the chain links with each other within the coil. Nevertheless, their mobility remains high and probably microdiffusion will not limit chemical reactions of functional groups within coils and with monomer molecules.In studying the fluorescence quenching of the chromophore for PVPy-F in the presence of the polymer quencher, it was found that mutual interpenetration of the polymer coils is intensified markedly when the solution volume is filled entirely by the swelling coils.     

Conjugated polyelectrolyte supported bead based assays for phospholipase A2 activity

A fluorescence based assay for human serum-derived phospholipase activity has been developed in which cationic conjugated polyelectrolytes are supported on silica microspheres. The polymer-coated beads are overcoated with an anionic phospholipid (1,2-dimyristoyl-sn-glycero-3-[phospho- rac-(1-glycerol)) (DMPG) to provide "lipobeads" that serve as a sensor for PLA2. The lipid serves a dual role as a substrate for PLA2 and an agent to attenuate quenching of the polymer fluorescence by the external electron transfer quencher 9,10-anthraquinone-2,6-disulfonic acid (AQS). In this case quenching of the polymer fluorescence by AQS increases as the PLA2 digests the lipid. The lipid can also be used itself as a quencher and substrate by employing a small amount of energy transfer quencher substituted lipid in the DMPG. In this case the fluorescence of the polymer is quenched when the lipid layer is intact; as the enzyme digests the lipid, the fluorescence of the polymer is restored. The sensing of PLA2 activity has been studied both by monitoring fluorescence changes in a multiwell plate reader and by flow cytometry. The assay exhibits good sensitivity with EC50 values in the nanomolar range.     

Quenching of the excited singlet state of acridine and 10-methylacridinium cation by thio-organic compounds in aqueous solution

In this work, the lowest excited singlet states of acridine (Acr), acridinium (AcrH⁺) and 10-methylacridinium (AcrMe⁺) are quenched by sulfur-containing amino acids and carboxylic acids in aqueous solution. Both steady-state and time-resolved fluorescence techniques were used to monitor the quenching of fluorescence. Stern–Volmer plots of the fluorescence intensity showed a static component (K_S) to the quenching. The experimental K_S values were compared to theoretical K_S values for outer-sphere complexes based on Debye–Hückel theory and the Fuoss equation. The general agreement between experimental and theoretical K_S values indicate that the static quenching can be attributed to non-fluorescing ion pairs associated as simple outer-sphere complexes. The computed values of the interionic distances of the ion pairs are consistent with the ion pairs of the Z_AZ_Q=−1 and −2 cases being solvent-separated ion pairs while those of the Z_AZ_Q=−3 case are contact ion pairs. The effect of the reactants’ charges on the quenching rate constants (dynamic component) was observed for the reactions of AcrMe⁺ with the anionic forms of the quenchers (having charges Z_Q=−1, −2 and −3). The rate constants (extrapolated to ionic strength, μ=0) for the quenching processes were determined to be 0.3–5.3×10¹⁰ M⁻¹ s⁻¹ depending on the ionic charge (Z_Q) of the quencher used. These trends in the quenching rate constants are rationalized with a quenching scheme for electron transfer. Analogous quenching rate constants for alanine and glycine were found to be at least an order of magnitude lower. Photoinduced electron transfer from the sulfur atom of the quencher molecule to the acridine excited singlet state is suggested to be the most likely mechanism of the process under discussion.     

荧光淬灭法研究疏水缔合共聚物(PAM-b-PPOEA)_n自组装聚集数

运用荧光淬灭技术,包括稳态荧光淬灭法(SSFQ)和时间分辨荧光淬灭法(TRFQ),研究了疏水缔合水溶性丙烯酰胺2苯氧基丙烯酸酯多嵌段共聚物[P(AM POEA)]在水溶液中自组装的聚集数.这类聚合物在水溶液中易形成胶束状聚集体,探针芘分子和淬灭剂二苯酮增溶于疏水微区,荧光测定结果很好地符合Poisson淬灭模型.实验结果表明聚合物链结构、聚合物浓度和无机盐对聚集体的尺寸具有重要影响.聚合物自组装聚集数NA随疏水单体含量的增加和疏水嵌段长度的减小而增大,同时也随聚合物浓度和NaCl浓度增加而增大.另外对聚合物链结构、聚集数和溶液粘度的相互关系进行了讨论.     

2-Methyl Naphthalene as Fluorescence Probe in Ionic and Nonionic Micelles: Fluorescence Quenching by Halides

The fluorescence characteristics of 2-methyl naphthalene have been studied in ionic micelles of sodium dodecyl sulphate (SDS) and cetyl trimethyl ammonium bromide (CTAB) and in nonionic micellar medium of p-t-octylphenyl polyethoxyethanol (Triton X-100). The fluorescence quenching of fluorophore by halides and pseudohalide obeys the Stern-Volmer Equation up to a certain concentration of quencher. A quenching sphere of action model has been considered to explain the deviations from Stern-Volmer behaviour. The distribution of quenchers in the micellar phase has been calculated.     

Fluorescence quenching of photoreaction products in the excited singlet state

The properties of steady-state spontaneous luminescence of a quantum system with a photoproduct with recordable fluorescence under the conditions of dynamic quenching of excited states by extraneous substances were considered. It was shown that the dependence of photoproduct fluorescence intensity and yield on quencher concentration was nontrivial and could not be conveniently used to determine the Stern-Volmer constant. At the same time, the initial form of the luminophore and its photoproduct produced in a kinetically controlled reaction are quenched in such a way that the ratio of their fluorescence intensities increases linearly as the quencher concentration grows. The corresponding equation was used to determine the constant of bimolecular quenching of reaction product excited states. The results were used in an analysis of the experimental fluorescence spectra of flavone (3-hydroxiflavone), whose fluorescence was excited under the conditions of dynamic quenching of the S ₁ state. Our analysis was shown to be applicable to a wide range of compounds with photoreactions accompanied by two-band fluorescence (charge transfer, proton transfer, phosphorescence, complex formation, etc.). It could be used to accurately determine bimolecular contact constants for excited states of photoreaction product molecules. Original Russian Text ? V.I. Tomin, 2009, published in Zhurnal Fizicheskoi Khimii, 2009, Vol. 83, No. 3, pp. 580–585.     

Study on the Interaction between Gatifloxacin Mesylate and Salmon Sperm DNA by Fluorescence Method

The interaction between gatifloxacin mesylate (GM) and salmon sperm DNA was studied by fluorescence spectrometry and ultra‐violet (UV) spectrometry. Additions of salmon sperm DNA to GM solution resulted in its strong fluorescence quenching and UV absorbance decrease due to the strong interaction between GM and salmon sperm DNA. Both the maximum fluorescence emission wavelength and the maximum UV absorbance wavelength of GM did not change with the increasing concentration of salmon sperm DNA, indicating no intercalative binding existed between them. The Stern‐Volmer plot indicated that the fluorescence‐quenching constant at different temperatures or different salmon sperm DNA concentration ranges was different. Effects of ionic strength and I^? on the fluorescence quenching of GM by salmon sperm DNA indicated that electrostatic interaction and groove binding coexisted between them.     

Superquenching in cyanine pendant poly(L-lysine) dyes: dependence on molecular weight, solvent, and aggregation.

A series of poly(L-lysines) ranging in number of repeat units (N(PRU)) from 6 to 900 has been synthesized and the photophysics of the series and monomer cyanine dye have been studied in solution. In water or aqueous dimethyl sulfoxide, the oligomers and polymers exhibit high sensitivity to fluorescence quenching by oppositely charged electron acceptors; in this study, 9,10-anthraquinone-2,6-disulfonate was used as a quencher for the cationic fluorescent polyelectrolytes. Quenching constants (K(SV)) measured in 50:50 (v/v) dimethyl sulfoxide-water increase monotonically with increase in N(PRU) ranging from 630 M(-1) for monomer to 1.2 x 10(9) M(-1) for dilute solutions of the polymer having N(PRU) approximately 900. The polymers having N(PRU) > 100 exhibit predominantly J-aggregate absorption and fluorescence and enhanced susceptibility to quenching. For the polymers exhibiting strong J-aggregation, the effective exciton domain quenched by a single quencher reaches approximately 100 PRU. The results of this study permit a semiquantitative analysis of superquenching of fluorescent polyelectrolytes in solution and the factors that control it.     

低频超声波照射下卟啉锰(HP-Mn)配合物对牛血清白蛋白(BSA)损伤的研究

应用紫外-可见(UV-vis)光谱和荧光光谱研究了卟啉-锰(HP-Mn)配合物与牛血清白蛋白(BSA)的相互作用及在超声波作用下HP-Mn对BSA的损伤作用,并探讨了超声波照射时间,HP-Mn浓度,酸度,离子种类和强度等因素对BSA损伤的影响。结果表明,HP-Mn对BSA荧光的猝灭属于静态猝灭,两者主要通过静电作用相互结合,同时也存在着配位作用,作用距离(r)为3.49 nm。另外,在超声波作用下,HP-Mn能够明显损伤BSA。损伤程度随着超声波照射时间的增长,酸度的升高和HP-Mn浓度的增大而增大,但离子种类和强度的影响较为复杂。这一结果为声动力学(SDT)疗法治疗肿瘤应用于临床具有重要的意义。     

Studies of dynamic interactions between a pyrene-labeled polyelectrolyte and an oppositely charged rodlike micelle by a fluorescence quenching technique with use of a hydrophobic quencher

Electrostatic interactions of poly(sodium 2-(acrylamido)-2-methylpropanesulfonate) (PyPAMPS) labeled with pyrene and a rodlike micelle of dimethyloleylamine oxide (DMOAO), an amine oxide type surfactant, mixed with varying mole fractions (Y) of hexadecyltrimethylammonium chloride (CTAC), a cationic surfactant, were investigated by a fluorescence quenching technique using 3,4'-dimethylbenzophenone (DBP), a hydrophobic quencher, that can only reside in the micellar phase. Fluorescence measurements were performed under homogeneous conditions in the region 0Yc, the fluorescence was efficiently quenched by DBP-carrying DMOAO/CTAC mixed micelles, both steady-state and time-dependent fluorescence data indicating that the degree of the quenching and hence the extent of the complex formation increased significantly with increasing Y. Applying a kinetic model to the steady-state and time-dependent fluorescence data, the residence time for PyPAMPS in the polymer-micelle complex was calculated. The residence time was found to depend on both Y and mu, e.g., when Y was increased from 0.01 to 0.03, the residence time increased from 4 to 80 mus at mu=0.05 whereas little or no increase in the residence time was observed in this range of Y at mu=0.20. At this higher ionic strength, the residence time increased only moderately from 3 to 10 mus when Y was increased from 0.01 to 0.09.     

Effect of surfactant on water-soluble conjugated polymer used in biosensor

The effect of nonionic surfactants on the cationic conjugated polymer (CCP), poly{9,9-bis[6-(N,N-trimethylammonium)hexyl]fluorene-co 1,4-phenylene} iodide 1, has been investigated. It is shown that the CCP in various solvents exists in three phases: isolated polymer chains, polymer aggregate, and variable size clusters (partially dissolved polymer). It is shown that nonionic surfactants enhance the photoluminescence (PL) quantum yield of the CCP in water by breakup of polymer aggregates, which eliminates the nonemissive interchain quenching with aggregates and increases surface-to-volume ratio of the CCP. Furthermore, the surfactants reduce quenching by incorporation of the CCP into aggregates or binary micelles. Surfactant also reduces the polar interaction strength between CCP and water and enhances CCP quenching by the counterions (iodine) by ion pairing effect. The dynamics of the interactions are complex and reveal that the surfactant induces rapid increase in the PL which imply that the main force that causes the aggregation is weak and may be due to hydrophobic interaction of the CCP in water rather than a solid, particulate-like state. Time-resolved fluorescence measurements at the exciton energy (420 nm) confirm that the CCP in water and in some organic solvents is a multiphase system in which three exponential decay terms are needed to fit the decay profile of the CCP. The change in the decay lifetime explains clearly the effect of surfactant and solvent polarity on the three CCP phases. The average lifetime of the CCP does not increase with surfactant, but the number of isolated polymer chains increases which leads to higher PL quantum yield. The association between the polymer and a quencher, single-strand deoxyribonucleic acid (ssDNA), was investigated. It indicated that CCP:ssDNA forms a weak electrostatic complex that does not alter the absorption spectra of the CCP but induces a strong CCP fluorescence quenching with association constant KS = 5 x 10(7) M(-1). At low ssDNA concentrations, the surfactant reduces quenching in the complex possibly by preventing charge-transfer processes. This may be due to an increase in the distance between the CCP and ssDNA through incorporation of the CCP into aggregates (micelles). However, at high ssDNA concentration, the quenching increases sharply which may be assigned to the increase in the electrostatic force destroying the micelles' structure around the CCP, leading to contact quenching as well as DNA induced CCP aggregation, which in turn leads to CCP-CCP quenching.     

The effects of formins on the conformation of subdomain 1 in actin filaments

In this study we investigated the effects of formins on the conformation of actin filaments by using the method of fluorescence quenching. Actin was labelled with IAEDANS at Cys³⁷⁴ and the quencher was acrylamide. The results showed that formin binding induced structural changes in the subdomain 1 of actin protomers which were reflected by greater quenching constants (K_SV). Simultaneously the fraction of the fluorophore population accessible for the quencher (α) decreased. These observations suggest that the conformational distribution characteristic for the actin protomers became broader after the binding of formins, for which the structural framework was provided by a more flexible protein matrix in the microenvironment of the label. The effects of formins depended on the formin:actin molar ratio, and also on the ionic strength of the medium. These observations are in agreement with previous results and underline the importance of the intramolecular conformational changes induced by formins in the structure of actin filaments.     

Studies on the fluorescence quenching of polysilane copolymers by chlorohydrocarbons

The room-temperature solution fluorescence quenching of polysilane copolymers by chlorohydrocarbons such as CCl₄, CHCl₃, C₂Cl₆, and Cl₂CHCHCl₂ was studied. The existence of dynamic quenching was preliminarily demonstrated by the experiment of fluorescence lifetime quenching. The fluorescence quenching data were in conformity with the equation: F₀/F = (1+K_SV[Q])exp(NV[Q]), where F and F₀ are the fluorescence intensity with and without the addition of quencher, K_SV is the Stern-Volmer constant, [Q] is the quencher concentration, N is the Avogadro constant, and V is the volume of the active sphere. The fluorescence quenching by the first three chlorohydrocarbons was attributed to the contemporaneous effect of dynamic quenching and static quenching. There exists, at least mathematically, a critical quencher concentration [Q]_C. When the quencher concentration [Q] < [Q]_C, the fluorescence quenching is dominated by the dynamic quenching part; when [Q] > [Q]_C, it is dominated by the static quenching part. However, the fluorescence quenching by Cl₂CHCHCl₂ was attributed to only static quenching. Furthermore, it was proposed that the dynamic quenching may be related with the electrical positivity of the central carbon nucleus of the quenching molecules while the static quenching may be caused by the “outside heavy atom effect” of the Cl element. © 1996 John Wiley & Sons, Inc.     

Enhancement in fluorescence response by a quencher for amiloride upon binding to thymine opposite an abasic site in a DNA duplex

By using iodide (I⁻) as a quencher, we successfully improve the fluorescence response of amiloride when binding to thymine opposite an AP site in a 21-meric DNA duplex. From fluorescence measurements, as compared to the NaCl solutions, the addition of NaI as a quencher as well as salt to adjust the ionic strength effectively suppresses the background fluorescence from unbound amiloride in a solution. The Stern-Volmer analysis shows that the bound amiloride to the nucleobase at the AP site is unexposed to NaI quencher. Therefore the high signal-to-background fluorescence response of amiloride is obtained. Such enhancement in fluorescence response of amiloride by using the quencher can provide the significant improvement of the detection limit for DNA duplexes carrying T target base. The method presented in this study is simple and effective. The present method could be applicable to other detection system where microenvironment of fluorophores changes at a recognition event.