Building‐Block Approach for the Straightforward Incorporation of a New FRET (Fluorescence‐Resonance‐Energy Transfer) System into Synthetic DNA

The synthesis of the two new phosphoramidites 5 and 8 bearing a carbostyril (=quinolin‐2(1H)‐one) chromophore used as donor entity in our recently developed new FRET (fluorescence‐resonance‐energy transfer) system is described (Schemes 1 and 2) The high stability of the chromophore to basic conditions enables the incorporation of the phosphoramidites directly into DNA during solid‐phase synthesis (Schemes 3 and 4). Since this is also possible for the (bathophenanthroline)ruthenium(II) complex used as acceptor (Scheme 4, Steps d and e), the whole labelling procedure to insert the FRET system into synthetic DNA is straightforward and represents a major improvement to our previous strategy.     

Comparative Studies of Different Quinolinone Derivatives as Donors in Fluorescence‐Resonance‐Energy Transfer (FRET) – Systems in Combination with a (Bathophenanthroline)ruthenium(II) Complex as Acceptor

We describe the preparation as well as a detailed photophysical study of Fmoc‐amino acid building blocks carrying different carbostyril (=quinolin‐2(1H)‐one) heterocycles as donors in a FRET (fluorescence‐resonance‐energy transfer) system in combination with a [Ru^II(bathophenanthroline)] complex (bathophenanthroline=4,7‐diphenyl‐1,10‐phenanthroline). The efforts resulted in a clear preference for building block 16 due to its ease of synthesis (Scheme 2), its chemical robustness, and the FRET efficiency when incorporated into peptides.     

Application of a Highly Robust and Efficient Fluorescence‐Resonance‐Energy‐Transfer (FRET) System in DNA

We report a feasibility study for the application of our newly developed highly efficient and robust fluorescence‐resonance‐energy‐transfer (FRET) system to DNA. A 2′‐oligodeoxynucleotide, 12 , equipped with a quinolinone derivative as donor and a (bathophenanthroline)ruthenium(II) complex as acceptor and having a single uridine as potential cleavage site under basic conditions revealed an intensive FRET, which almost vanished after cleavage of the oligonucleotide under basic conditions (Fig. 7). Furthermore, in the arrangement of a molecular beacon (MB) DNA (see 13 ), a significant decrease of the FRET was observed after hybridization to a target sequence (Fig. 9). Due to the long decay times of the fluorescence of the Ru‐complex, the system allows for highly sensitive time‐gated measurements.     

Verification and Biophysical Characterization of a New Three‐Color Förster Resonance‐Energy‐Transfer (FRET) System in DNA

We report on a new three‐color FRET system consisting of three fluorescent dyes, i.e., of a carbostyril (=quinolin‐2(1H)‐one)‐derived donor D, a (bathophenanthroline)ruthenium complex as a relay chromophore A₁, and a Cy dye as A₂ (FRET=Förster resonance‐energy‐transfer) (cf. Fig. 1). With their widely matching spectroscopic properties (cf. Fig. 2), the combination of these dyes yielded excellent FRET efficiencies. Furthermore, fluorescence lifetime measurements revealed that the long fluorescence lifetime of the Ru complex was transferred to the Cy dye offering the possibility to measure the whole system in a time‐resolved mode. The FRET system was established on double‐stranded DNA (cf. Fig. 3) but it should also be generally applicable to other biomolecules.     

A Highly Selective and Sensitive Ratiometric Fluorescent Probe for pH Measurement Based on Fluorescence Resonance Energy Transfer

Fluorescein-rhodamine 6G(Flu-Rh) was synthesized and used as the fluorescence probe for pH measurement based on fluorescence resonance energy transfer(FRET). In the probe, fluorescein fluorophore and pH-sensitive rhodamine 6G hydrazide were used as FRET donor and acceptor, respectively. The values of acidity constant(pK_a) and fluorescence quantum yield(Φ) of Flu-Rh were 3.71 and 0.72, respectively. The fluorescence efficiency of Flu-Rh remains almost constant when the pH value of the sample solution changed 10 times in a range of 4.78-7.03 continuously. The present probe is simple and easy-to-use for the pH measurement in acidic media.     

A Carbon Nano‐Onion–Ferrocene Donor–Acceptor System: Synthesis,Characterization and Properties

Don't cry! The attachment of ferrocene moieties on the surface of carbon nano‐onions influences the electrochemical properties of these moieties and the photophysical properties of the carbon nano‐onions (see figure). Quantum chemical calculations confirm that the spectral properties of carbon nano‐onions depend on their size and the degree of functionalisation.

     

Fluorescence resonance energy transfer (FRET) in chemistry and biology: Non-F?rster distance dependence of the FRET rate

Fluorescence resonance energy transfer (FRET) is a popular tool to study equilibrium and dynamical properties of polymers and biopolymers in condensed phases and is now widely used in conjunction with single molecule spectroscopy. In the data analysis, one usually employs the F?rster expression which predicts (l/R ⁶) distance dependence of the energy transfer rate. However, critical analysis shows that this e_xpression can be of rather limited validity in many cases. We demonstrate this by explicitly considering a donor-acceptor system, polyfluorene (PF₆)-tetraphenylporphyrin (TPP), where the size of both donor and acceptor is comparable to the distance separating them. In such cases, one may expect much weaker distance (as l/R ² or even weaker) dependence. We have also considered the case of energy transfer from a dye to a nanoparticle. Here we find l/R ⁴ distance dependence at large separations, completely different from F?rster. We also discuss recent application of FRET to study polymer conformational dynamics. Dedicated to Prof J Gopalakrishnan on his 62nd birthday.     

Aptamer-quantum dots and teicoplanin-gold nanoparticles constructed FRET sensor for sensitive detection of Staphylococcus aureus

The detection of bacterial pathogen such as Staphylococcus aureus(S.aureus) is essential for the regulation of food hygiene and disease diagnosis.Herein,we developed a simple one-step fluorescence resonance energy transfer(FRET)-based sensor for specific and sensitive detection of S.aureus in food and serum samples,in which aptamer-modified quantum dots(aptamer-QDs) was employed as the energy donor and antibiotic of teicoplanin functionalized-gold nanoparticles(Teico-AuNPs) was chosen as the energy acceptor.Within 1 h,the FRET-based sensor showed a linear range of from 10 cfu/mL to 5 × 10~8 cfu/mL,with the low limit of detection(LOD,2 cfu/mL) for S.aureus in buffer.When further applied to assay S.aureus in real samples,the FRET-based sensor showed good recoveries ranging from 84.5% to 110.0%,with relative standard derivations(RSDs) of 0.01%-0.44% and a LOD of 100 cfu/mL in milk,orange juice and human serum.     

Synthesis and Spectroscopic Characterization of Fluorophore‐Labeled Oligospiroketal Rods

Fluorescence probes consisting of well‐established fluorophores in combination with rigid molecular rods based on spirane‐type structures were investigated with respect to their fluorescence properties under different solvent conditions. The attachment of the dyes was accomplished by 1,3‐dipolar cycloaddition between alkynes and azides (‘click’ reaction) and is a prime example for a novel class of sensor constructs. Especially, the attachment of two (different) fluorophores on opposite sides of the molecular rods paves the way to new sensor systems with less bulky (compared to the conventional DNA‐ or protein‐based concepts), nevertheless rigid spacer constructs, e.g., for FRET‐based sensing applications. A detailed photophysical characterization was performed in MeOH (and in basic H₂O/MeOH mixtures) for i) rod constructs containing carboxyfluorescein, ii) rod constructs containing carboxyrhodamine, iii) rod constructs containing both carboxyfluorescein and carboxyrhodamine, and iv) rod constructs containing both pyrene and perylene parts. For each dye (pair), two rod lengths with different numbers of spirane units were synthesized and investigated. The rod constructs were characterized in ensemble as well as single‐molecule fluorescence experiments with respect to i) specific rod? dye and ii) dye? dye interactions. In addition to MeOH and MeOH/NaOH, the rod constructs were also investigated in micellar systems, which were chosen as a simplified model for membranes.     

A new nanobiosensor for glucose with high sensitivity and selectivity in serum based on fluorescence resonance Energy transfer (FRET) between CdTe quantum dots and Au nanoparticles

A novel assembled nanobiosensor QDs-ConA-beta-CDs-AuNPs was designed for the direct determination of glucose in serum with high sensitivity and selectivity. The sensing approach is based on fluorescence resonance energy transfer (FRET) between CdTe quantum dots (QDs) as an energy donor and gold nanoparticles (AuNPs) as an energy acceptor. The specific combination of concanavalin A (ConA)-conjugated QDs and thiolated beta-cyclodextrins (beta-SH-CDs)-modified AuNPs assembles a hyperefficient FRET nanobiosensor. In the presence of glucose, the AuNPs-beta-CDs segment of the nanobiosensor is displaced by glucose which competes with beta-CDs on the binding sites of ConA, resulting in the fluorescence recovery of the quenched QDs. Experimental results show that the increase in fluorescence intensity is proportional to the concentration of glucose within the range of 0.10-50 muM under the optimized experimental conditions. In addition, the nanobiosensor has high sensitivity with a detection limit as low as 50 nM, and has excellent selectivity for glucose over other sugars and most biological species present in serum. The nanobiosensor was applied directly to determine glucose in normal adult human serum, and the recovery and precision of the method were satisfactory. The unique combination of high sensitivity and good selectivity of this biosensor indicates its potential for the clinical determination of glucose directly and simply in serum, and provides the possibility to detect low levels of glucose in single cells or bacterial cultures. Moreover, the designed nanobiosensor achieves direct detection in biological samples, suggesting the use of nanobiotechnology-based assembled sensors for direct analytical applications in vivo or in vitro.     

A New and Sensitive Catalytic Resonance Scattering Spectral Assay for the Detection of Laccase Activity Using H2O2‐I−‐TDMAC System

In pH 3.8 acetic acid‐sodium acetate (HAC‐NaAC) buffer solution, laccase exhibited a strong catalytic effect on the H₂O₂ oxidation of I ^? to form I₂, and I₂ combined with excess I ^? to form I₃^? that reacted with cationic surfactants of tetradecyl dimethylbenzyl ammonium chloride (TDMAC) to produce the (TDMAC‐I₃)_n association complex particles, which exhibited a strong resonance scattering (RS) peak at 468 nm. Under the chosen conditions, as the concentration of laccase activity increased, the RS intensity at 468 nm (I_{468 nm}) increased linearly. The increased RS intensity ΔI_{468 nm} was linear to laccase activity in the range of 0.08–0.96 U/mL, with a regression equation of ΔI_{468 nm}?88.8U?1.9, and a detection limit of 0.02 U/mL laccase. This proposed method was applied to detect laccase activity in waste water, with satisfactory results.     

Two‐Photon Photochromism of a Diarylethene Enhanced by Förster Resonance Energy Transfer from Two‐Photon Absorbing Fluorenes

Resonance energy transfer from two-photon absorbing fluorene derivatives to the photochromic compound 3,4-bis-(2,4,5-trimethyl-thiophen-3-yl)furan-2,5-dione (PC 1) is investigated in hexane under one- and two-photon excitation. The quenching of the steady-state fluorescence of donor molecules in the presence of the diarylethene acceptor is used to study the nature of resonance energy transfer. The F?rster distances and critical acceptor concentrations are determined for nonbound donor-acceptor pairs in homogeneous molecular ensembles. Quite significantly, up to a two-fold enhancement in the velocity of the photochromic transformation of 1, in the presence of two-photon absorbing fluorene derivatives, is demonstrated.     

From Dark to Light to Fluorescence Resonance Energy Transfer (FRET): Polarity‐Sensitive Aggregation‐Induced Emission (AIE)‐Active Tetraphenylethene‐Fused BODIPY Dyes with a Very Large Pseudo‐Stokes Shift

The work presented herein is devoted to the fabrication of large Stokes shift dyes in both organic and aqueous media by combining dark resonance energy transfer (DRET) and fluorescence resonance energy transfer (FRET) in one donor–acceptor system. In this respect, a series of donor–acceptor architectures of 4,4‐difluoro‐4‐bora‐3a,4a‐diaza‐s‐indacene (BODIPY) dyes substituted by one, two, or three tetraphenylethene (TPE) luminogens were designed and synthesised. The photophysical properties of these three chromophore systems were studied to provide insight into the nature of donor–acceptor interactions in both THF and aqueous media. Because the generation of emissive TPE donor(s) is strongly polarity dependent, due to its aggregation‐induced emission (AIE) feature, one might expect the formation of appreciable fluorescence emission intensity with a very large pseudo‐Stokes shift in aqueous media when considering FRET process. Interestingly, similar results were also recorded in THF for the chromophore systems, although the TPE fragment(s) of the dyes are non‐emissive. The explanation for this photophysical behaviour lies in the DRET. This is the first report on combining two energy‐transfer processes, namely, FRET and DRET, in one polarity‐sensitive donor–acceptor pair system. The accuracy of the dark‐emissive donor property of the TPE luminogen is also presented for the first time as a new feature for AIE phenomena.     

Synthesis, characterization, and application of cationic water-soluble oligofluorenes in DNA-hybridization detection

A simple and efficient approach was developed for the synthesis of a series of cationic water-soluble oligofluorenes up to a chain length of a heptamer. Bromoalkyl-substituted fluorenyl boronic esters as the key intermediates were synthesized by using a modified Miyaura reaction. With an increasing number of repeat units (trimer to hexamer), the size-specific oligomers have shown redshifts in both the absorption and emission maxima. The emission maximum reaches the limit for the hexamer in both water and buffer solution. The quantum yields of the oligomers decreased with increased oligomer size in water. Both fluorescence quenching of the oligomers by 9,10-anthraquinone-2,6-disulfonate and the fluorescence resonance energy transfer experiments with the oligomers as the donor and fluorescein (Fl)-labeled double-stranded DNA (dsDNA-Fl) as the acceptor revealed the chain-length-dependent behavior. The Stern-Volmer quenching constant increased with the molecular size, whereas the highest donor-sensitized Fl emission was observed for the hexamer. These size-specific oligomers also served as a model to study the structure-property relationships for cationic polyfluorenes.     

Characterization of the Mode of Incorporation of Lipophilic Compounds in Solid Dispersions at the Nanoscale Using Fluorescence Resonance Energy Transfer (FRET)

Summary: Efficient engineering of solid dispersions stagnates by the current inability to establish the mode of drug distribution on a molecular level at a low drug load. This study describes the application of fluorescence resonance energy transfer (FRET) to characterize the mode of incorporation of dispersed lipophilic molecules in a solid matrix. Two different lipophilic fluorophores (donor and acceptor) were used as model substances and were incorporated in polyvinylpyrrolidone to form solid dispersions using two different production processes: lyophilization and fusion. The efficiency of the resonance energy transfer from donor to acceptor was measured by confocal microscopy. We show that the method can be used to compare the modes of drug incorporation of solid dispersions at the nanoscale.

Absorption and emission spectra of Bodipy R6G (donor) and Bodipy 650/665 (acceptor).     

Energy Transfer at the Single‐Molecule Level: Synthesis of a Donor–Acceptor Dyad from Perylene and Terrylene Diimides

In 2004, we reported single‐pair fluorescence resonance energy transfer (spFRET), based on a perylene diimide (PDI) and terrylene diimide (TDI) dyad ( 1 ) that was bridged by a rigid substituted para‐terphenyl spacer. Since then, several further single‐molecule‐level investigations on this specific compound have been performed. Herein, we focus on the synthesis of this dyad and the different approaches that can be employed. An optimized reaction pathway was chosen, considering the solubilities, reactivities, and accessibilities of the building blocks for each individual reaction whilst still using established synthetic techniques, including imidization, Suzuki coupling, and cyclization reactions. The key differentiating consideration in this approach to the synthesis of dyad 1 is the introduction of functional groups in a nonsymmetrical manner onto either the perylene diimide or the terrylene diimide by using imidization reactions. Combined with well‐defined purification conditions, this modified approach allows dyad 1 to be obtained in reasonable quantities in good yield.     

银纳米粒子簇的设计、 制备和表面增强拉曼光谱

通过匹配激光光斑直径与胶体微球的尺寸, 设计制备了银纳米粒子的表面增强拉曼散射(SERS)基底, 并将其用于研究单个银纳米粒子簇的表面增强拉曼光谱. 在制备纳米粒子的过程中, 考察了等离子体刻蚀时间与银沉积厚度对“单”银纳米粒子结构与形貌的影响. 将吡啶、 巯基苯和罗丹明R6G作为SERS探针分子, 研究了其SERS效应, 通过荧光共振能量转移(FRET)机理, 实现了染料分子在单银纳米粒子簇上的SERS效应. SERS光谱测试与相关计算结果表明, 单个银纳米粒子簇的拉曼增强因子能够达到约10⁶.     

Synthesis of phosphatidylcholine analogues derived from glyceric acid: a new class of biologically active phospholipid compounds

Synthesis of a new class of phosphatidylcholine analogues derived from glyceric acid is reported for spectroscopic studies of phospholipases and the conformation of phospholipid side-chains in biological membranes, using fluorescence resonance energy transfer (FRET) techniques.