Modification of Extended Open Frameworks with Fluorescent Tags for Sensing Explosives: Competition between Size Selectivity and Electron Deficiency

Three new electron-rich metal-organic frameworks (MOF-1-MOF-3) have been synthesized by employing ligands bearing aromatic tags. The key role of the chosen aromatic tags is to enhance the -electron density of the luminescent MOFs. Single-crystal X-ray structures have revealed that these MOFs form three-dimensional porous networks with the aromatic tags projecting inwardly into the pores. These highly luminescent electron-rich MOFs have been successfully utilized for the detection of explosive nitroaromatic compounds (NACs) on the basis of fluorescence quenching. Although all of the prepared MOFs can serve as sensors for NACs, MOF-1 and MOF-2 exhibit superior sensitivity towards 4-nitrotoluene (4-NT) and 2,4-dinitrotoluene (DNT) compared to 2,4,6-trinitrotoluene (TNT) and 1,3,5-trinitrobenzene (TNB). MOF-3, on the other hand, shows an order of sensitivity in accordance with the electron deficiencies of the substrates. To understand such anomalous behavior, we have thoroughly analyzed both the steady-state and time-resolved fluorescence quenching associated with these interactions. Determination of static Stern-Volmer constants (K-S) as well as collisional constants (K-C) has revealed that MOF-1 and MOF-2 have higher K-S values with 4-NT than with TNT, whereas for MOF-3 the reverse order is observed. This apparently anomalous phenomenon was well corroborated by theoretical calculations. Moreover, recyclability and sensitivity studies have revealed that these MOFs can be reused several times and that their sensitivities towards TNT solution are at the parts per billion (ppb) level.

Optical detection of submicromolar levels of nitro explosives by a submicron sized metal-organic phosphor material

Two isomorphous submicron sized metal-organic network compounds, Y-2(PDA)(3)(H2O)1]center dot 2H(2)O (PDA = 1,4-phenylenediacetate), 1 and Y1.8Tb0.2(PDA)(3)(H2O)1]center dot 2H(2)O, Tb@1 have been synthesized by employing solvent assisted liquid grinding followed by heating at 180 degrees C for 1' min and washing with water. Single crystal X-ray data of bulk 1 confirmed a three dimensional porous structure. The structure and morphology of 1 and Tb@1 were systematically characterized by PXRD, TGA, DSC, IR, SEM and EDX analysis. Dehydrated Tb@1 Tb@1'] shows a high intense visible green emission upon exposure to UV light. The green emission of Tb@1' was used for the detection of nitro explosives, such as 2,4,6-trinitrophenol (TNP), 1,3-dinitro benzene (DNB), 2,4-dinitro toluene (DNT), nitro benzene (NB), and 4-nitro toluene (NT) in acetonitrile. The results show that the emission intensity of dehydrated Tb@1' can be quenched by all the nitro analytes used in the present work. Remarkably, Tb@1' exhibited a high efficiency for TNP, DNB and DNT detection with K-SV K-SV = quenching constant based on linear Stern-Volmer plot] values of 70 920, 44 000 and 35 430 M-1, respectively, which are the highest values amongst known metal-organic materials. Using this material submicromolar level (equivalent to 0.18 ppm), a detection of nitro explosives has been achieved.

Characterization of a dissolved oxygen sensor made of plastic optical fiber coated with ruthenium-incorporated solgel

A dissolved oxygen sensor made of plastic optical fiber as the substrate and dichlorotris (1, 10-phenanthroline) ruthenium as a fluorescence indicator is studied. Oxygen quenching characteristics of both intensity and phase were measured; the obtained characteristics showed deviation from the linear relation described by the Stern-Volmer equation. A two-layer model is proposed to explain the deviation, and main parameters can be deduced with the model. (C) 2009 Optical Society of America

Estudo da interação de antipsicóticos atípicos e albumina sérica com base em modelos matemáticos e espectrofluorimétricos

Os antipsicóticos são drogas utilizadas no tratamento de muitos transtornos psiquiátricos, sendo classificados em dois grupos: típicos e atípicos. Os típicos formam o grupo de drogas que bloqueiam especialmente os receptores de dopamina e, por isto, causam efeitos colaterais característicos, que se manifestam através de sintomas extrapiramidais e podem terminar em discinesia tardia. Os atípicos apresentam eficácia antipsicótica similar à dos antipsicóticos típicos, mas produzem menos efeitos colaterais extrapiramidais e não causam discinesia tardia. Os antipsicóticos se ligam às proteínas plasmáticas, principalmente a albumina, a qual representa cerca de 60% do total das proteínas no soro humano. Neste trabalho estudamos os processos de interação de duas drogas antipsicóticas atípicas, risperidona e sulpirida, com as albuminas séricas humana (HSA) e bovina (BSA), através da técnica de supressão da fluorescência intrínseca do triptofano. A partir dos espectros de fluorescência, a análise dos dados foi feita obtendo-se os gráficos e as constantes de Stern-Volmer. A análise da supressão da fluorescência foi feita a partir da média aritmética dos dados oriundos dos experimentos realizados em cada condição adotada. Como a molécula da sulpirida é fluorescente desenvolvemos uma modelagem matemática do processo de interação, que nos permitiu então obter os dados referentes à supressão da fluorescência da proteína. Os resultados mostraram que a risperidona e a sulpirida suprimem a fluorescência de ambas albuminas por um processo de quenching estático, formando complexos droga-albumina. A risperidona tem uma afinidade com a HSA cerca de 6,5 vezes maior do que a sulpirida, a 37 oC. As constantes de associação calculadas para a interação risperidona-HSA, através da Teoria de Stern-Volmer, foram 1,43 ( 0,05) x 105 M-1, a 37 C, e 2,56 ( 0,09) x 105 M-1, a 25 C1; e para a sulpirida, 2,20 ( 0,08) x 104 M-1, a 37 C, e 5,46 ( 0,20) x 104 M-1, a 25 C. Como a taxa de quenching da BSA foi maior do que a da HSA, sugerimos que o sítio primário para a risperidona nas albuminas esteja localizado mais próximo ao domínio do triptofano 134 da BSA do que do domínio do triptofano 212 da HSA. O mesmo sugerimos com relação ao sítio para a sulpirida a 37 C.

In vitro study on the binding of neutral red to bovine serum albumin by molecular spectroscopy

In this paper, the binding of neutral red (NR) to bovine serum albumin (BSA) under physiological conditions has been studied by spectroscopy method including fluorescence, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy. The Stern-Volmer fluorescence quenching constant (K-SV), binding constant (K-b) and the number of binding sites (It) were measured by fluorescence quenching method. Fluorescence experiments were also performed at different ionic strengths. It was found K-SV was ionic strength dependent, which indicated the electrostatic interactions were part of the binding forces. The distance r between donor (BSA) and acceptor (NR) was obtained according to Foster's non-radiative energy transfer theory. CD spectroscopy and FT-IR spectroscopy were used to investigate the structural information of BSA molecules on the binding of NR, and the results showed no change of BSA conformation in our experimental conditions.

Thin-film oxygen sensors using a luminescent polynuclear gold(I) complex

Robust thin-film oxygen sensors were fabricated by encapsulating a lipophilic, polynuclear gold(I) complex, bis{m-(bis(diphenylphosphino)octadecylamine-P,P')}dichlorodigold(I), in oxygen permeable polystyrene and ormosil matrices. Strong phosphorescence, which was quenched by gaseous and dissolved oxygen, was observed from both matrices. The polystyrene encapsulated dye exhibited downward-turning Stern-Volmer plots which were well fitted by a two-site model. The ormosil trapped complex showed linear Stern-Volmer plots for dissolved oxygen quenching but was downward turning for gaseous oxygen. No leaching was observed when the ormosil based sensors were immersed in flowing water over an 8 h period. Both films exhibited fully reversible response and recovery to changing oxygen concentration with rapid response times. (C) 2011 Elsevier B.V. All rights reserved.

Effect of plasticizer viscosity on the sensitivity of an [RU(bpy)(3)(2+)(Ph4B-)(2)]-based optical oxygen sensor

The quenching of the electronically-excited, lumophoric state of [Ru(bpy)(3)(2+)(Ph4B-)(2)] by oxygen is studied in a wide variety of neat plasticizers. The Stern-Volmer constant, K-SV, is found to be inversely dependent upon the viscosity of the quenching medium, although the natural lifetime of the electronically excited state of [RU(bPY)(3)(2+)(Ph4B-)(2)] is largely independent of medium. The least viscous of the plasticizers tested, triethyl phosphate, did not, however, produce highly sensitive optical oxygen sensors when used to plasticize [RU(bPY)(3)(2+)(Ph4B-)(2)]-containing cellulose acetate butyrate (CAB) and poly(methyl methacrylate) (PMMA) films, Instead, the compatibility of the polymer-plasticizer combination, as measured by the difference in the values of the solubility parameter of the two, appears to be a major factor in determining the overall oxygen sensitivity of the thin plastic films. For highly compatible polymer-plasticizer combinations, the plasticizer with the lowest viscosity produces films of the highest oxygen sensitivity. This situation arises because in the film the quenching process is partly diffusion-controlled and, as a result, the quenching rate constant is inversely proportional to the effective viscosity of the reaction medium.

Use of luminescent gold compounds in the design of thin-film oxygen sensors

The use of two gold compounds incorporated into thin plastic films as luminescence quenching oxygen sensors is described. The films are sensitive both to gaseous oxygen and to oxygen dissolved in nonaqueous media such as ethanol. The luminescence quenching of these sensors by oxygen obeys the Stern-Volmer equation and Stern-Volmer constants of 5.35 x 10(-3) and 0.9 x 10(-3) Torr(-1) are found, respectively, for the two dyes in a polystyrene polymer matrix. The sensitivity of the films is strongly influenced by the nature of the polymer matrix, and greatest sensitivity was found in systems based an the polymers polystyrene or cellulose acetate butyrate. Sensitivity was not found to be temperature dependent though raising the temperature hom 15 to 50 degrees C did result in a slight decrease in emission intensity and a hypsochromic shift in the emission wavelength. The rate of response and recovery of the sensors can be increased either by decreasing film thickness or by increasing the operating temperature. The operational and storage stability of these films is generally good though exposure to light should be avoided as one of the dyes tends to undergo photobleaching probably due to a photoinduced ligand substitution reaction.

On the localization of water-soluble porphyrins in micellar systems evaluated by static and time-resolved frequency-domain fluorescence techniques

Fluorescence quenching of meso-tetrakis-4-sulfonatophenyl (TPPS4) and meso-tetrakis-4-N-methylpyridil (TMPyP) porphyrins is studied in aqueous solution and upon addition of micelles of sodium dodecylsulfate (SDS), cetyltrimethylammonium chloride (CTAC), N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS) and t-octylphenoxypolyethoxyethanol (Triton X-100). Potassium iodide (KI) was used as quencher. Steady-state Stern-Volmer plots were best fitted by a quadratic equation, including dynamic (K-D) and static (K-s) quenching. Ks was significantly smaller than K-D. Frequency-domain fluorescence lifetimes allowed estimating bimolecular quenching constants, k(q). At 25 degrees C, in aqueous solution, TMPyP shows k(q), values a factor of 2-3 higher than the diffusional limit. TPPS4 shows collisional quenching with pH dependent k(q) values. For TMPyP quenching results are consistent with reported binding constants: a significant reduction of quenching takes place for SDS, a moderate reduction is observed for H PS and almost no change is seen for Triton X-100. Similar data were obtained at 50 C. For CTAC-TPPS4 system an enhancement of quenching was observed as compared to pure buffer. This is probably associated to accumulation of iodide at the cationic micellar interface. The attraction between CTAC headgroups and 1(-), and repulsion between SDS and 1(-), enhances and reduces the fluorescence quenching, respectively, of porphyrins located at the micellar interface. The small quenching of TPPS4 in Triton X-100 is consistent with strong binding as reported in the literature. (C) 2008 Elsevier B.V. All rights reserved.

O efeito do pH na interação da albumina sérica humana e os flavonoides quercetina e quercitrina

Pós-graduação em Biofísica Molecular - IBILCE

Intra- vs intermolecular photoinduced electron transfer reactions of a macrocyclic donor-acceptor dyad

The synthesis, structural characterization, and photophysical behavior of a 14-membered tetraazamacrocycle with pendant 4-dimethylaminobenzyl (DMAB) and 9-anthracenylmethyl groups is reported (L-3, 6-((9-anthracenylmethyl)amino)-trans-6,13-dimethyl-13-((4-dimethylaminobenzyl)amino)-1,4,8,11-tetraaza-cyclotetradecane). In its free base form, this compound displays rapid intramolecular photoinduced electron transfer (PET) quenching of the anthracene emission, with both the secondary amines and the DMAB group capable of acting as electron donors. When complexed with Zn(II), the characteristic fluorescence of the anthracene chromophore is restored as the former of these pathways is deactivated by coordination. Importantly, it is shown that the DMAB group, which remains uncoordinated and PET active, acts only very weakly to quench emission, by comparison to the behavior of a model Zn complex lacking the pendant DMAB group, [ZnL2](2+) (Chart 1). By contrast, Stern-Volmer analysis of intermolecular quenching of [ZnL2](2+) by N,N-dimethylaniline (DMA) has shown that this reaction is diffusion limited. Hence, the pivotal role of the bridge in influencing intramolecular PET is highlighted.

Transport in the blood of an anti-tumor water-soluble rutheniun cyclopentadienyl complex: a fluorescence study on albumin binding

Dissertação de mestrado, Qualidade em Análises, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014

Biochemical and structural characterization of recombinant hyoscyamine 6 beta-hydroxylase from Datura metel L.

Hyoscyamine 6 beta-hydroxylase (H6H; EC 1.14.11.11), an important enzyme in the biosynthesis of tropane alkaloids, catalyzes the hydroxylation of hyoscyamine to give 6 beta-hydroxyhyoscyamine and its epoxidation in the biosynthetic pathway leading to scopolamine. Datura metel produces scopolamine as the predominant tropane alkaloid. The cDNA encoding H6H from D. mete! (DmH6H) was cloned, heterologously expressed and biochemically characterized. The purified recombinant His-tagged H6H from D. mete! (DmrH6H) was capable of converting hyoscyamine to scopolamine. The functionally expressed DmrH6H was confirmed by HPLC and ESI-MS verification of the products, 6 beta-hydroxyhyoscyamine and its derivative, scopolamine; the DmrH6H epoxidase activity was low compared to the hydroxylase activity. The K-m values for both the substrates, hyoscyamine and 2-oxoglutarate, were 50 mu M each. The CD (circular dichroism) spectrum of the DmrH6H indicated a preponderance of alpha-helicity in the secondary structure. From the fluorescence studies, Stern-Volmer constants for hyoscyamine and 2-oxoglutarate were found to be 0.14 M-1 and 0.56 M-1, respectively. These data suggested that the binding of the substrates, hyoscyamine and 2-oxoglutarate, to the enzyme induced significant conformational changes. (C) 2010 Elsevier Masson SAS. All rights reserved.

基于U型塑料光纤和邻啡咯啉钌的溶解氧传感器

本文提出了一种基于U型塑料光纤和邻啡咯啉钌的溶解氧传感器。光纤传感头放在样品池中，测量了不同氮氧体积比情况下荧光强度和寿命。研究了邻啡咯啉钌的浓度、传感头的退火时间和U型光纤的弯曲半径对系统灵敏度的影响。我们提出了双层模型来解释实验结果不符合S-V方程的实验现象，并用双层模型分析了弯曲半径对系统灵敏度的影响。通过对溶解氧浓度和荧光寿命亚线性曲线的拟合，我们得出敏感层的厚度和S-V系数分别为0.59和0.61。

高温胁迫诱导光系统Ⅱ及其异质性的变化

近年来有证据证明，光系统II(PS II)反应中心在结构与功能上存在着异质性，它与光舍原初过程、激发能的分配和调节、胁迫因子导致的光合单位的损伤与修复等密切相关。本论文主要研究了高温胁迫诱导PS II及其异质性的变化，以及人工电子受体与PS II还原侧异质性电子传递的关系．根据研究需要，建立了精确测定无活性PS II中心相时含量的软件和方法，圆满完成了本研究任务。此外，也参加了新的非调制式动力学荧光计的研制及其软件的编写． 以下是本论文的主要结果： 1．用N80-BASIC语言结合汇编语言重新编写了本室快速（ms级）叶绿素动力学荧光计的测控程序，使快速荧光上升曲线的采样速度提高了一个数量级（达到100μS/点），可对Fo、Fi等关键荧光参数进行精确测定，为无活性PS II中心相对含量的准确测定奠定了基础．新研制的荧光计的软件用C语言编写，可在IBM PC兼容机上运行，采样速度最快可达25μs/点，对Fo和Fi等参数的测定更加可靠和精确．新荧光计从软、硬件两方面进行了彻底地更新，具有高信噪比、高响应、高精度、低功耗等优点，其性能己达到国际同类产品的先进水平． 2．高温胁迫诱导小麦类囊体膜吸收光谱的变化，结果显示40℃-50℃20分钟以内的高温胁迫导致681nm的吸收峰下降，同时引起663nm的吸收峰增加，表明高温胁迫引起部分叶绿素蛋白复合体的破坏和游离的叶绿素分子的增多．在更严重的高温胁迫下（55℃5分钟以上），体内游离的叶绿素分子(△A663)本身也遭到进一步的降解． 3．小麦类囊体膜低温( 77K)荧光光谱的分析。结果证实温和的高温胁迫(40℃20分钟以内)可导致激发能更多地从PS II向光系统IcPsi)分配，而更严重的高温胁迫（45℃- 55℃20分钟以内）对PS II和PS I的叶绿素蛋白复合体（F684和F736）均有破坏作用． 4．高温胁迫诱导小麦叶片荧光诱导动力学、荧光猝灭及其荧光参数的变化的研究．结果表明，高温胁迫首先导致有效量子产量(E．Y．)的下降，胁迫作用进一步加强导致最适量子产量(0．Y．)下降，而对光化学猝灭qP的影响较晚．这说明和PS II电子受体侧的电子传递和与二氧化碳固定有关的酶系统对高温胁迫极为敏感．其次，PS II放氧系统的损伤也早于PS II原初反应中心的失活．同时，在自然界条件下，存在着高温和高光强对植物的加强协同的光抑制和破坏作用． 5．在研究高温胁迫诱导荧光动力学及其参数变化的基础上，提出测定和计算高温胁迫的植物样品中无活性PS II中心相对含量的合理方法．认为在高温胁迫导致可变荧光( Fv)猝灭的情况下，应以Fvi(Fvi=Fi-Fo)对室温对照的可变荧光(FVCK)的比值作为计算无活性PS II中心相对含量的指标(Fvi/FVCK)．我们在弱激发光下测得正常的小麦和菠菜的无活性PS II中心的相对含量分别为0.155±0.011和0.094士0.010． 6．高温胁迫诱导有活性和无活性PS II中心异质性的相互转化的研究。结果发现50℃以下小于10分钟的处理，对PS II有活性和无活性中心的比值无明显影响：而经过50℃和55℃高温处理5-10分钟，有活性PS II中心才明显向无活性中心转化并发现这一转化过程发生在Fo己明显增加和Fv明显猝灭之后，也就是说它迟于高温胁迫对PS II天线色素蛋白复合体( LHCII)与PS II反应中心结合的破坏以及对放氧侧的损伤． 7．高温胁迫后的室温恢复期中有活性和无活性PS II中心相互转化的研究．发现在高温胁迫不太严重时（如50℃1分钟），无活性PS II中心的含量降至对照的70%，在随后室温60分钟恢复过程中继续降为50%。而Psn氧化侧的活性在此过程中可以得到部分恢复。高温胁迫进一步加强（如55℃5分钟和55℃10分钟）后，无活性PS II中心数目在随后的60分钟室温恢复期中，从恢复开始时为对照的130%和150%继续增加到240%和290%，且有加速转化的趋势。这说明高温胁迫诱导PS II还原侧异质性中心的转化除包含一个快速、直接的机制外，还启动了某种间接转化的机制． 8．对DMQ和DCBQ两种人工电子受体对有活性和无活性PS II中心的作用提出了不同见解。Cao和Govindjee(1990)认为DMQ(>20μmoI.L-1)只接受有活性PS II中心的电子，而DCBQ（>15, μmoLL-1）可完全接受有活性和无活性两种PS II中心的电子。但Lavergne等(1993)认为DCBQ不能接受无活性Psn中心的电子．我们用Stern-Volmer猝灭公式对我们的实验结果进行了分析，结果表明DMQ在较高浓度下（如120μmoI.L-1）才可完全接受有活性PS II中心的电子．但DCBQ的浓度在比Cao等几乎高出一个数量级( 120μmoI.L-1)的情况下，也只接受部分无活性PS II中心的电子( 40%)。另外我们发现，DMQ和DCBQ对Fm的猝灭不是随猝灭剂浓度的增加呈线性关系，而是一条近似饱和曲线，说明它至少包括两种以上不同的猝灭机制． 9．Mg2+诱导PS II异质性(Cα/Cβ)的研究。我们小组发现Mg2+诱导的chl a荧光增强动力学曲线包含Cα和Cβ两个指数成分，说明Mg2+在抑制激发能满溢，调节激发能向有利于PS I1分配的过程中存在异质性。其中Cβ比Cα具有更长的迁移寿命、更低的活化能和Mg2+半饱和浓度．这些说明Cβ比Cα更有可能在体内生理条件下发生迁移，从而在两个光系统之间起调节激发能分配的作用． 10.提出了高温胁迫诱导PS II异质性中心相互转换的可能模型．高温胁迫导致PS II异质性的转化包括几个步骤：有活性的α型PS II专荧光猝灭态的PS II专有活性的β型PS II专无活性的β型PS II专破坏了的PSⅡ．前两种转化一般具有可逆性．当高温胁迫进一步加强后，转化失去可逆性，在胁迫去除后，有活性PS II中心可继续向无活性中心转化，后者还有可能进一步受到破坏。