Electron transfer reactivity and the catalytic activity of horseradish peroxidase incorporated in dipalmitoylphosphatidic acid films

Horseradish peroxidase (HRP) was incorporated in dipalmitoylphosphatidic acid (DPPA) to form a film and the film was modified on pyrolytic graphite electrode. UV-Vis spectra suggested that HRP in the film could keep its secondary structure similar to the native state. A pair of stable, well-defined, and quasi-reversible cyclic voltammetric peaks was observed with the formal potential at -276.2 mV (vs. saturated calomel electrode), characteristic of heme Fe(III)/Fe(II) redox couple of HRP. The apparent heterogeneous electron transfer rate constant and other electrochemical parameters were presented. The catalytic activity of HRP in DPPA film toward oxygen, hydrogen peroxide and nitric oxide were also examined.     

马心血红蛋白在氧化铟电极上的直接电子传递反应

马心血红蛋白在氧化铟电极上的直接电子传递反应杨秀娟，菊，陆天虹（中国科学院长春应用化学研究所电分析化学开放实验室，长春，１３００２２）关键词血红蛋白，氧化铟电极，循环伏安法蛋白质与电极之间的电子转移在某种程度上类似于生物体内蛋白质分子之间的电子转移过...     

新型四硫富瓦烯-杯芳烃衍生物的合成及与四氯苯醌的分子间电子转移

设计合成了两个新型醚键连接的四硫富瓦烯-杯芳烃衍生物4a和4b,并对其进行了核磁、质谱、红外等结构表征.循环伏安法研究表明化合物4a和4b均呈两电子的可逆氧化还原行为.同时,利用紫外-可见光谱研究了金属离子(Na+, K+, Ba2+)对化合物4a和4b作为电子供体分别和电子接受体四氯苯醌分子间电子转移的影响.     

Star-shaped tripodal chemosensors for the detection of aliphatic amines

In this work, three new tripodal triphenylamine dyes are presented that are capable of reversibly binding amines and diamines to form hemiaminals through a covalent bond. The dyes were synthesized by the Heck reaction and possess stilbene units with one, two, or three trifluoroacetyl groups as receptor moieties. Their interaction with amines and diamines led to changes in their absorption and emission properties, which were detected by UV/Vis and fluorescence spectroscopy. The influence of the number of trifluoroacetyl receptor moieties on the selectivity and sensitivity of the dyes was studied. Enhanced sensitivity and selectivity for diaminoalkanes was found for the dye we have labeled Tripod-1, with three chemically reactive trifluoroacetyl groups, related to only one or two trifluoroacetyl groups in the dye molecule.     

Synthesis of Symmetrical Multichromophoric Bodipy Dyes and Their Facile Transformation into Energy Transfer Cassettes

Multichromophoric boron‐dipyrromethene (Bodipy) dyes synthesized on phenylene‐ethynylene platforms have been be converted to energy transfer cassettes in a one‐step chemical transformation. Excitation energy transfer processes in these highly symmetrical derivatives were studied in detail, including time‐resolved fluorescence spectroscopy techniques. Excitation spectra and the emission lifetimes suggest efficient energy transfer between the donor and acceptor chromophore. These novel energy transfer cassettes, while highlighting a short‐cut approach to similar energy transfer systems, could be useful as large pseudo‐Stokes shift multichromophoric dyes with potential applications in diverse applications.     

Electron transfer between cytochrome c and copper enzymes

The electron transfer between cytochrome c and ascorbate oxidase or laccase from Coriolus hirsutus was investigated using both an electrochemical and a spectrophotometric method. A quasi-reversible cyclic volammogram of cytochrome c was observed on a gold electrode modified with 4,4′-dithiodipyridine. The addition of laccase resulted in the appearance of a catalytic current due to the regeneration of ferricytochrome c by laccase in the presence of oxygen. The second-order rate constant of the reaction between cytochrome c and laccase is calculated to be 9.2 × 10³ M⁻¹ s⁻¹ in 50 mM phosphate buffer of pH 5.8. The reaction rate with ascorbate oxidase is almost three orders of magnitude slower. The difference in the redox potential is considered to be the driving force of the reaction between cytochrome c and the copper proteins investigated.     

A close look at fluorescence quenching of organic dyes by tryptophan.

Understanding fluorescence quenching processes of organic dyes by biomolecular compounds is of fundamental importance for in-vitro and in-vivo fluorescence studies. It has been reported that the excited singlet state of some oxazine and rhodamine derivatives is efficiently and almost exclusively quenched by the amino acid tryptophan (Trp) and the DNA base guanine via photoinduced electron transfer (PET). We present a detailed analysis of the quenching interactions between the oxazine dye MR121 and Trp in aqueous buffer. Steady-state and time-resolved fluorescence spectroscopy, together with fluorescence correlation spectroscopy (FCS), reveal three contributing quenching mechanisms: 1) diffusion-limited dynamic quenching with a bimolecular quenching rate constant k(d) of 4.0 x 10(9) s(-1) M(-1), 2) static quenching with a bimolecular association constant K(s) of 61 M(-1), and 3) a sphere-of-action contribution to static quenching described by an exponential factor with a quenching constant lambda of 22 M(-1). The latter two are characterized as nonfluorescent complexes, formed with approximately 30 % efficiency upon encounter, that are stable for tens of nanoseconds. The measured binding energy of 20-30 kJ mol(-1) is consistent with previous estimates from molecular dynamics simulations that proposed stacked complexes due to hydrophobic forces. We further evaluate the influence of glycerol and denaturant (guanidine hydrochloride) on the formation and stability of quenched complexes. Comparative measurements performed with two other dyes, ATTO 655 and Rhodamine 6G show similar results and thus demonstrate the general applicability of utilizing PET between organic dyes and Trp for the study of conformational dynamics of biopolymers on sub-nanometer length and nanosecond time-scales.     

X-ray fluorescence investigation of ordered intermetallic phases as electrocatalysts towards the oxidation of small organic molecules

The composition of ordered intermetallic nanoparticles (PtBi and PtPb) has been quantitatively studied by in situ X-ray fluorescence (XRF) during active electrochemical control in solutions of supporting electrolyte and small organic molecules (SOMs). Because the Pt L(β1,2) lines and the Bi L(α1,2) lines are only separated by 200 eV, an energy-dispersive detector and a multiple-channel analyzer (MCA) were used to record the major fluorescent emission lines from these two elements. The molar ratios of platinum to the less-noble elements (Bi, Pb) in the nanoparticles dramatically changed as a function of the applied upper limit potentials (E(ulp)) in cyclic voltammetric (CV) characterization. Similar to previous investigations for bulk intermetallic surfaces, the less-noble elements leached out from the surfaces of the intermetallic nanoparticles. For PtBi nanoparticles, the ratios of fluorescence intensities of Pt/Bi in the samples were 0.42, 0.96, and 1.36 for E(ulp)=+0.40, +0.80, and 1.20 V, respectively, while cycling the potential from -0.20 V to the E(ulp) value for 10 cycles. The leaching-out process of the less-noble elements occurred at more negative E(ulp) values than expected. After cycling to relatively positive E(ulp) values, nonuniform PtM (M=Bi of Pb) nanoparticles formed with a Pt-rich shell and intermetallic PtM core. When the supporting solutions contained active fuel molecules in addition to the intermetallic nanoparticles (formic acid for PtBi, formic acid and methanol for PtPb), kinetic stabilization effects were observed for E(ulp)=+0.80 V, in a way similar to the response of the bulk materials. It was of great importance to quantitatively explore the change in composition and structure of the intermetallic nanoparticles under active electrochemical control. More importantly, this approach represents a simple, universal, and multifunctional method for the study of multi-element nanoparticles as electrocatalysts. This is, to our knowledge, the first report of nondestructive, quantitative characterization of bimetallic or multi-elemental nanoparticles electrocatalysts under active electrochemical control.     

乳过氧化物酶与伴刀豆球蛋白A共修饰电极的电化学活性研究

用乳过氧化物酶(LPO)和伴刀豆球蛋白A(Con A)共修饰金电极,首次得到了乳过氧化物酶的直接电化学响应,在此基础上研究了乳过氧化物酶对过氧化氢(H2O2)的电催化活性,并研究了一氧化氮(NO)对LPO电催化活性的影响.在Con A的作用下,乳过氧化物酶在循环伏安图中显示1对准可逆的氧化还原峰,表现出薄层电化学行为.在pH 7.4的磷酸缓冲溶液中的表观氧化还原电位为 -190 mV.该共修饰电极对H2O2表现出电催化还原活性,由此构建的传感器对H2O2的检测范围是2.0×10-5 ～4.0×10-3 mol/L.实验发现,微摩尔量级的NO会抑制乳过氧化物酶对H2O2的催化活性.     

Electrochemistry and electrocatalysis of hemoglobin in Nafion/nano-CaCO3 film on a new ionic liquid BPPF6 modified carbon paste electrode

Room temperature ionic liquid N-butylpyridinium hexafluorophosphate (BPPF6) was used as a binder to construct a new carbon ionic liquid electrode (CILE), which exhibited enhanced electrochemical behavior as compared with the traditional carbon paste electrode with paraffin. By using the CILE as the basal electrode, hemoglobin (Hb) was immobilized on the surface of the CILE with nano-CaCO3 and Nafion film step by step. The Hb molecule in the film kept its native structure and showed good electrochemical behavior. In pH 7.0 Britton-Robinson (B-R) buffer solution, a pair of well-defined, quasi-reversible cyclic voltammetric peaks appeared with cathodic and anodic peak potentials located at -0.444 and -0.285 V (vs SCE), respectively, and the formal potential (E degrees') was at -0.365 V, which was the characteristic of Hb Fe(III)/Fe(II) redox couples. The formal potential of Hb shifted linearly to the increase of buffer pH with a slope of -50.6 mV pH-1, indicating that one electron transferred was accompanied with one proton transportation. Ultraviolet-visible (UV-vis) and Fourier transform infrared (FT-IR) spectroscopy studies showed that Hb immobilized in the Nafion/nano-CaCO3 film still remained its native arrangement. The Hb modified electrode showed an excellent electrocatalytic behavior to the reduction of H2O2, trichloroacetic acid (TCA), and NaNO2.     

Molecular and Spectroscopic Characterization of Green and Red Cyanine Fluorophores from the Alexa Fluor and AF Series**

The use of fluorescence techniques has an enormous impact on various research fields including imaging, biochemical assays, DNA-sequencing and medical technologies. This has been facilitated by the development of numerous commercial dyes with optimized photophysical and chemical properties. Often, however, information about the chemical structures of dyes and the attached linkers used for bioconjugation remain a well-kept secret. This can lead to problems for research applications where knowledge of the dye structure is necessary to predict or understand (unwanted) dye-target interactions, or to establish structural models of the dye-target complex. Using a combination of optical spectroscopy, mass spectrometry, NMR spectroscopy and molecular dynamics simulations, we here investigate the molecular structures and spectroscopic properties of dyes from the Alexa Fluor (Alexa Fluor 555 and 647) and AF series (AF555, AF647, AFD647). Based on available data and published structures of the AF and Cy dyes, we propose a structure for Alexa Fluor 555 and refine that of AF555. We also resolve conflicting reports on the linker composition of Alexa Fluor 647 maleimide. We also conducted a comprehensive comparison between Alexa Fluor and AF dyes by continuous-wave absorption and emission spectroscopy, quantum yield determination, fluorescence lifetime and anisotropy spectroscopy of free and protein-attached dyes. All these data support the idea that Alexa Fluor and AF dyes have a cyanine core and are a derivative of Cy3 and Cy5. In addition, we compared Alexa Fluor 555 and Alexa Fluor 647 to their structural homologs AF555 and AF(D)647 in single-molecule FRET applications. Both pairs showed excellent performance in solution-based smFRET experiments using alternating laser excitation. Minor differences in apparent dye-protein interactions were investigated by molecular dynamics simulations. Our findings clearly demonstrate that the AF-fluorophores are an attractive alternative to Alexa- and Cy-dyes in smFRET studies or other fluorescence applications.     

Design,synthesis and photoelectrical properties of diphenylamine-containing triphenylamine-based D-D-π-A-type fluorescence dyes

Available online A series of diphenylamine (DPA)-containing triarylamine-based D-D-π-A organic fluorescence dyes were designed under the guidance of theoretical calculations and then synthesized (cyanoacrylic acid, methylene malononitrile or isophorone served as the electron acceptor while ethylene served as the π-bridge). The results of theoretical calculations, electrochemical properties and optical properties tests indicated that the new dyes possessed high thermal stability, desirable HOMO levels and intramolecular charge transfer property. Some of them possessed high fluorescence quantum yields and performed very well as luminescent material as expected..     

Comparative Photostability Studies of BODIPY and Fluorescein Dyes by Using Fluorescence Correlation Spectroscopy

In single‐molecule applications, the photostability of fluorescent molecules is a key parameter. We apply fluorescence correlation spectroscopy to compare the photostability of four fluorescein and four borondipyrromethene (BODIPY) dyes of similar structure but different triplet yields. The latter class of dyes are more stable. In the kinetic analysis the, diffusion and photobleaching are treated as competitive processes. Corrections, which account for saturation and for experimental artefacts, are achieved solely by using experimental data. Photobleaching is found to occur mainly through the first excited singlet state S₁, in contrast to previous findings.     

A Versatile Approach for the Site‐Specific Modification of Recombinant Antibodies Using a Combination of Enzyme‐Mediated Bioconjugation and Click Chemistry

A unique two‐step modular system for site‐specific antibody modification and conjugation is reported. The first step of this approach uses enzymatic bioconjugation with the transpeptidase Sortase A for incorporation of strained cyclooctyne functional groups. The second step of this modular approach involves the azide–alkyne cycloaddition click reaction. The versatility of the two‐step approach has been exemplified by the selective incorporation of fluorescent dyes and a positron‐emitting copper‐64 radiotracer for fluorescence and positron‐emission tomography imaging of activated platelets, platelet aggregates, and thrombi, respectively. This flexible and versatile approach could be readily adapted to incorporate a large array of tailor‐made functional groups using reliable click chemistry whilst preserving the activity of the antibody or other sensitive biological macromolecules.     

Fluorescene Difference Decay Curves: Resolution Of Complex Decays

Abstract

The use of fluorescence difference decay curves was explored as a way to isolate the decays of components in a complex system. Time-correlated single photon methodology allows one to subtract a “reference” curve from a “sample” curve from a “sample” curve to yield a difference decay curve. To test the feasibility of this method, a model 3-component system (6-carboxyfluorescein, pyranine, and 1-dimethyl-aminoaphtalene-5-sulfonate) was examined. From its complex fluorescence decay curve, the individual decays were obtained by subtracting the appropriate binary solution decay curves. These difference curves coincided with those of the single component systems. Stringent requirements included: use of the same instrumental settings for all solutions, low counting efficiency, avoidance of inner filter effects, absence of energy transfer, and lack of chemical interaction between components. The difference decay method was applied to: 1. Sequential dansylation of serum albumin. Lifetimes of the first two dyes bound are longer than those of the third. 2. Dynamic quenching of quinine fluorescence by chloride ion. When the reference differs from the sample only by having a shorter lifetime, the difference decay curve has a characteristic shape. 3. Quenching of intrinsic protein fluorescence by acrylamide. Bovine serum albumin and liver alchol dehydrogenase were examined. Of the two tryptophans in each protein, one was preferentially quenched and its decay curve was obtained by difference.     

Molecular Insight into Long‐Wavelength Fluorogenic Dye Design: Hydrogen Bond Induces Activation of a Dormant Acceptor

The detection of chemical or biological analytes in response to molecular changes relies increasingly on fluorescence methods. Therefore, there is a substantial need for the development of improved fluorogenic dyes. In this study, we demonstrated how an intramolecular hydrogen bond activates a dormant acceptor through a charge induction between phenolic hydrogen and a heteroaryl nitrogen moiety. As a result, a new fluorochrome is produced, and the molecule exhibits a strong fluorescent emission. When the strength of the hydrogen bonding was increased by conformational locking, the obtained dye emitted at longer wavelengths and fluoresced under physiological conditions. The dye was implemented in a turn‐ON system responsive to hydrogen peroxide. The molecular insight provided by this study should assist in the design of fluorescent dyes that are suitable for in vitro and in vivo applications.     

新型双发色团染料荧光光谱及其寿命的研究

有效的染料激光操作需要较高的荧光量子效率,若丹明是在500~700 nm光谱区中一类最重要的激光染料.然而,染料的基态分子和三线态对辐射能量的吸收将会大大降低激光输出效率,再者,由于若丹明类染料在紫外区的吸收系数较小,为了有效吸收泵浦能量(如用XeCI准分子激光,308 nm),就必须使用高浓度染料溶液,在这种情况下,若丹明类染料较小的Stokes位移就势必造成基态分子更大的重复吸收,即造成更大的谐振腔损耗^[1].     

细胞色素C在离子液体修饰电极上的电化学行为

分别合成了疏水和亲水性咪唑类离子液体,并制备了相应的两种离子液体修饰的玻碳电极。循环伏安法测量结果显示,细胞色素C在离子液体修饰的玻碳电极上的电子传递过程为一扩散控制的准可逆反应,表明咪唑类离子液体也可用作细胞色素C电子传递的有效促进剂。电化学交流阻抗谱的测量结果得到了与循环伏安相同的结论。     

Methoxyperylene bisimides and perylene lactame imides: novel, red fluorescent dyes

The synthesis of methoxyperylene bisimides and perylene lactame imides with aliphatic N-substituents is described. Both classes of dyes exhibit fluorescence in the bathochromic region of visible light so that red light is obtained. The lightfastness of the dyes is very high, thus, there is special interest for diverse applications.