Selective Energy Transfer Between Quantum Dots and Gold Nanoparticles for Detection of Multiple Mutations in Epidermal Growth Factor Receptor

Selective energy transfer between quantum dots and gold nanoparticles was used to simultaneously detect mutations in the epidermal growth factor receptor (EGFR) gene. We functionalized the surface of gold nanoparticles and green and red-emitting quantum dots using four different probe DNAs that were designed to be a perfect complementary to an in-frame deletion mutation in exon 19 or L858 R point mutation in exon 21 of EGFR. We found that the presence of the deletion mutation in exon 19 in target oligonucleotides caused fluorescence quenching at 525 nm due to energy transfer from green-emitting quantum dots to gold nanoparticles, whereas point mutation in exon 21 resulted in quenching at 620 nm due to energy transfer from red-emitting quantum dots to gold nanoparticles. This method could successfully be used to simultaneously detect the presence of two types of mutations in EGFR. We also defined a parameter (i.e., the extent of quenching) to quantify fluorescence quenching phenomenon. By varying the fraction of mutant type DNA in target oligonucleotides, we showed that detection sensitivity based on the extent of quenching was about 5%, which is lower than the conventional direct sequencing method.     

Fluorescence studies on the interaction of ethidium bromide with duplex, triplex and quadruplex DNA structures

Under different conditions, oligonucleotides can form several alternative DNA structures such as duplex, triplex and quadruplex. All these structures can interact with ethidium bromide (EB) and make its fluorescence intensity change. The fluorescence spectra and other related parameters provided by static fluorescence techniques showed that the interaction mechanisms between EB and these structures were not always the same. Among them, B type duplex and triplex DNA adopt an intercalative mode when binding to the EB, which has a relatively high efficiency of energy transfer and the fluorescence of EB cannot be quenched easily. While for the parallel duplex DNA, the interaction mode is an outside binding in which energy transfer can hardly happen and its fluorescence intensity as well as Stern-Volmer constant is almost the same to the free EB. For the quadruplex, the binding mechanism to EB is more complex. Results from the energy transfer and quenching studies indicate that the two interaction modes note     

Self-assembled donor comprising quantum dots and fluorescent proteins for long-range fluorescence resonance energy transfer

We report on the development of a self-assembled donor for long-range fluorescence resonance energy transfer (FRET). To this end, a three-chromophore FRET (3Ch-FRET) system was constructed, which consists of a luminescent quantum dot (QD), enhanced yellow fluorescent proteins (EYFP), and Atto647-dye-modified oligonucleotides. The system was assembled by electrostatic binding of covalent EYFP-ssDNA conjugate to the QD and subsequent hybridization with complementary oligonucleotides labeled with Atto647-dye. The final conjugates comprise three different two-chromophore FRET (2Ch-FRET) subsystems, QD/EYFP, QD/Atto647, and EYFP/Atto647, respectively, which were studied in detail by steady-state and time-resolved photoluminescence measurements. The helicity of DNA allowed us to control donor/acceptor separations and thus enabled the detailed analysis of the various FRET processes. We found that the 2Ch-FRET and the 3Ch-FRET (QD/EYFP/Atto647) systems revealed FRET efficiencies and transfer rates that were affected by the availability of distinct FRET pathways. The derived energy-transfer efficiencies and F?rster radii indicated that within the 3Ch-FRET system, the 2Ch-FRET subsystem QD/EYFP showed highest FRET efficiencies ranging from 64 to 72%. Thus, it can be used as a powerful donor system that combines the intrinsic advantages of QDs (large and spectrally broad absorption cross section) and EYFP (high quantum yield) and enables long-distance FRET processes for donor-acceptor distances of up to 13 nm.     

Dissecting and reducing the heterogeneity of excited-state energy transport in DNA-based photonic wires

Molecular photonic wires are one-dimensional representatives of a family of nanoscale molecular devices that transport excited-state energy over considerable distances in analogy to optical waveguides in the far-field. In particular, the design and synthesis of such complex supramolecular devices is challenging concerning the desired homogeneity of energy transport. On the other hand, novel optical techniques are available that permit direct investigation of heterogeneity by studying one device at a time. In this article, we describe our efforts to synthesize and study DNA-based molecular photonic wires that carry several chromophores arranged in an energetic downhill cascade and exploit fluorescence resonance energy transfer to convey excited-state energy. The focus of this work is to understand and control the heterogeneity of such complex systems, applying single-molecule fluorescence spectroscopy (SMFS) to dissect the different sources of heterogeneity, i.e., chemical heterogeneity and inhomogeneous broadening induced by the nanoenvironment. We demonstrate that the homogeneity of excited-state energy transport in DNA-based photonic wires is dramatically improved by immobilizing photonic wires in aqueous solution without perturbation by the surface. In addition, our study shows that the in situ construction of wire molecules, i.e., the stepwise hybridization of differently labeled oligonucleotides on glass cover slides, further decreases the observed heterogeneity in overall energy-transfer efficiency. The developed strategy enables efficient energy transfer between up to five chromophores in the majority of molecules investigated along a distance of approximately 14 nm. Finally, we used multiparameter SMFS to analyze the energy flow in photonic wires in more detail and to assign residual heterogeneity under optimized conditions in solution to different leakages and competing energy-transfer processes.     

Exploring concerted effects of base pairing and stacking on the excited‐state nature of DNA oligonucleotides by DFT and TD‐DFT studies

We have taken (dA)₅, (dT)₅, and (dA)₅·(dT)₅ as model systems to study concerted effects of base pairing and stacking on excited‐state nature of DNA oligonucleotides using density functional theory (DFT) and time dependent DFT methods. The spectroscopic states are determined to be of a partial A → A charge‐transfer nature in the A·T oligonucleotides. The T → T charge‐transfer transitions produce dark states, which are hidden in the energy region of the steady‐state absorption spectra. This is different from the previous assignment that the T → T charge‐transfer transition is responsible for a shoulder at the red side of the first strong absorption band. The A → T charge‐transfer states were predicted to have relatively high energies in the A·T oligonucleotides. The present calculations predict that the T → A charge‐transfer states are not involved in the spectra and excited‐state dynamics of the A·T oligonucleotides. In addition, the influence of base pairing and stacking on the nature of the ¹nπ* and ¹ππ* states are discussed in detail. © 2010 Wiley Periodicals, Inc. Int J Quantum Chem, 2011     

Energy transfer processes in phycobilisome model complex at 77 K

Ultra time-resoived huorescence spectra were used to study the energy transter processes and mechanism of complex PEC/PC/APC at 77 K, which was reconstructed with phycobiliproteins (PEC, PC and APC) of Anabaena variabilis, and has intact light-harvesting system and single terminal emitter. The energy transfer relationships between different chromophores especially between rod and core were also discussed based on fluorescence decay kinetic under different detected wavelengths. As a result, we got the possible energy transfer pathways and transfer time constants to be 29 ps between two PEC trimers, 12 ps between PEC and C-PC, 51 ps between rod and core.     

Fundamentals of tandem mass spectrometry: a dynamics study of simple C−C bond cleavage in collision-activated dissociation of polyatomic ions at low energy

The loss of methyl radical in collision-activated dissociation (CAD) of acetone and propane molecular ions has been studied at low energy using a tandem hybrid mass spectrometer. Although the two processes are very similar chemically and energetically, very different dynamical features are observed. Acetyl ions from acetone ion are predominantly backward-scattered, with intensity maxima lying inside and outside the elastic scattering circle, confirming our previous observation that electronically excited states are important in low-energy acetone CAD. Ethyl ions from propane ion show a forward-scattered peak maximum at a nonzero scattering angle, which is consistent with generally accepted models for vibrational excitation and redistribution of energy before dissociation. Both processes demonstrate that CAD at low energy proceeds via small-impact-parameter collisions with momentum transfer. Comparison of the present results with higher energy CAD dynamics studies and earlier work leads to some tentative general conclusions about energy transfer in these processes.     

Kinetics of energy transfer processes in C-phycocyanin complexes

The antenna system of algae for photosynthesis is a functional entity composed of various phycobiliproteins and the linker polypeptides. Up to now, high-resolution crystal structure data have been available only for the isolated phycobiliproteins. To have an understanding of the functional connection between different phycobiliproteins, it is necessary to study the complexes composed of different phycobiliproteins. The energy transfer processes in C-phycocyanin complexes were studied through computer simulation because it is difficult to be studied by conventional experimental methods. The main pathways of energy flow and the dynamic property of the energy transfer were obtained. A fast transfer process between two neighboring disks was observed through analyzing the distribution curves of excitation energy over time. According to the definition of the time constants for energy transfer in time-resolved spectrum techniques, for a complex with three C-phycoeyanin hexamer disks, a fluorescence-rising comp     

Energy transfer in solution of lanthanide complexes

The lanthanides with their well-defined energy levels provide an excellent basis to study different Ln(III)-specific energy transfer processes in a variety of chemical environments. The studies concerning intramolecular and intermolecular energy transfer processes with participation of Ln(III) ions and a variety of ligand groups in solution are reviewed. Phenomena of energy transfer from ligands to Ln(III) ions, resulting consequently in a great enhancement of the Ln(III) ion luminescence (ligand sensitized luminescence), as well as from Ln(III) to other species and between Ln(III) ions are presented.     

电喷雾离子源中样品离子化能量转移理论的初探

电喷雾离子源（electrospray ionization,ESI）不仅可以用于小分子的检测,也能够用于蛋白质、多肽等大分子的研究。本文通过对离子化过程的系统分析,提出了基于能量最低原理的离子化过程能量转移理论。样品分子在由液相转移到气相形成离子化气体的过程中受到静电力、分子间的范德华力等多种力的作用。样品的离子化是多种力共同作用的结果,在不同的离子化阶段,不同形式的力的作用也不尽相同。电荷在样品表面蒸发和多电荷离子的形成之间存在竞争。对不同结构的分子,分子形态、构象改变导致的两相间转移Gibbs自由能变化不同,可能导致离子蒸发、大分子形成多电荷离子、产生链弹射等行为。离子化能量转移理论不仅能够对已有的3种理论加以简化统一,也可以说明溶剂、电解质离子等在离子化过程中的作用,为优化不同结构与形态样品的质谱检测、了解离子化的真实过程提供了一种可能的依据。     

Ultrafast processes in bimetallic dyads with extended aromatic bridges. Energy and electron transfer pathways in tetrapyridophenazine-bridged complexes

The energy and electron transfer processes taking place in binuclear polypyridine complexes of ruthenium and osmium based on the tetrapyrido[3,2-a:2',3'-c:3' ',2' '-h:2' "-3' "-j]phenazine bridging ligand (tpphz) have been investigated by ultrafast absorption spectroscopy. In the binuclear complexes, each chromophore is characterized by two spectrally distinguishable metal-to-ligand charge transfer (MLCT) excited states: MLCT1 (with promoted electron mainly localized on the bpy-like portion of tpphz, higher energy) and MLCT0 (with promoted electron mainly localized on the pyrazine-like portion of tpphz, lower energy). In the homodinuclear complexes Ru(II)-Ru(II) and Os(II)-Os(II), MLCT1 --> MLCT0 relaxation (intraligand electron transfer) is observed, with strongly solvent-dependent kinetics (ca. 10(-10) s in CH2Cl2, ca. 10(-12) s in CH3CN). In the heterodinuclear Ru(II)-Os(II) complex, *Ru(II)-Os(II) --> Ru(II)-Os(II) energy transfer takes place by two different sequences of time-resolved processes, depending on the solvent: (a) in CH2Cl2, ruthenium-to-osmium energy transfer at the MLCT1 level followed by MLCT1 --> MLCT0 relaxation in the osmium chromophore, (b) in CH3CN, MLCT1 --> MLCT0 relaxation in the ruthenium chromophore followed by osmium-to-ruthenium metal-to-metal electron transfer. In the mixed-valence Ru(II)-Os(III) species, the *Ru(II)-Os(III) --> Ru(III)-Os(II) electron transfer quenching is found to proceed by two consecutive steps in CH3CN: intraligand electron transfer followed by ligand-to-metal electron transfer. On a longer time scale, charge recombination leads back to the ground state. Altogether, the results show that the tpphz bridge plays an active mechanistic role in these systems, efficiently mediating the transfer processes with its electronic levels.     

Intramolecular energy hopping and energy trapping in polyphenylene dendrimers with multiple peryleneimide donor chromophores and a terryleneimide acceptor trap chromophore

Intramolecular F?rster-type excitation energy transfer (FRET) processes in a series of first-generation polyphenylene dendrimers substituted with spatially well-separated peryleneimide chromophores and a terryleneimide energy-trapping chromophore at the rim were investigated by steady-state and time-resolved fluorescence spectroscopy. Energy-hopping processes among the peryleneimide chromophores are revealed by anisotropy decay times of 50--80 ps consistent with a FRET rate constant of k(hopp) = 4.6 ns(-1). If a terryleneimide chromophore is present at the rim of the dendrimer together with three peryleneimide chromophores, more than 95% of the energy harvested by the peryleneimide chromophores is transferred and trapped in the terryleneimide. The two decay times (tau(1) = 52 ps and tau(2) = 175 ps) found for the peryleneimide emission band are recovered as rise times at the terryleneimide emission band proving that the energy trapping of peryleneimide excitation energy by the terryleneimide acceptor occurs via two different, efficient pathways. Molecular- modeling-based structures tentatively indicate that the rotation of the terryleneimide acceptor group can lead to a much smaller distance to a single donor chromophore, which could explain the occurrence of two energy-trapping rate constants. All energy-transfer processes are quantitatively describable with F?rster energy transfer theory, and the influence of the dipole orientation factor in the F?rster equation is discussed.     

DNA mismatch detection by resonance energy transfer between ruthenium(II) and osmium(II) tris(2,2'-bipyridyl) chromophores

Octahedral tris-chelate complexes [M(II)(bpy)(3)](2+) (M = Ru or Os, bpy = 2,2'-bipyridyl), covalently attached to the 3'- and 5'-phosphates of two oligonucleotides, are juxtaposed when hybridized contiguously to a fully complementary DNA target. Visible metal-to-ligand charge-transfer (MLCT) excitation of the [Ru(II)(bpy)(3)](2+) unit leads to resonance energy transfer to the MLCT state of the [Os(II)(bpy)(3)](2+) moiety, with the energy transfer efficiency depending on the degree of hybridization. The extent of attenuation of the intense red luminescence from the Ru(II) chromophore hence allows highly sensitive structural probing of the assembly and constitutes a novel approach to DNA sensing which is capable of detecting mutations.     

Freezing‐directed Stretching and Alignment of DNA Oligonucleotides

Most single‐stranded DNA oligonucleotides are random coils with a persistence length of below 1 nm. So far, no good methods are available to stretch oligonucleotides. Herein, it is shown that freezing can stretch DNA, as confirmed using fluorescence resonance energy transfer, thiazole‐orange staining, and surface‐enhanced Raman spectroscopy. Lateral inter‐strand interactions are critical, and the stretched DNA oligonucleotides are aligned. This work also provides a set of methods for studying frozen oligonucleotides. Upon freezing, DNA oligonucleotides are readily adsorbed onto various nanomaterials, including gold nanoparticles, graphene oxide, iron oxide, and WS₂ via the most thermodynamically stable conformation, leading to more stable conjugates.     

Porphyrin–Phthalocyanine/Pyridylfullerene Supramolecular Assemblies

The synthesis and photophysical properties of several porphyrin (P)–phthalocyanine (Pc) conjugates (P–Pc; 1 – 3 ) are described, in which the phthalocyanines are directly linked to the β‐pyrrolic position of a meso‐tetraphenylporphyrin. Photoinduced energy‐ and electron‐transfer processes were studied through the preparation of H₂P–ZnPc, ZnP–ZnPc, and PdP–ZnPc conjugates, and their assembly through metal coordination with two different pyridylfulleropyrrolidines ( 4 and 5 ). The resulting electron‐donor–acceptor hybrids, which were formed by axial coordination of compounds 4 and 5 with the corresponding phthalocyanines, mimicked the fundamental processes of photosynthesis; that is, light harvesting, the transduction of excited‐state energy, and unidirectional electron transfer. In particular, photophysical studies confirmed that intramolecular energy‐transfer resulted from the S₂ excited state as well as from the S₁ excited state of the porphyrins to the energetically lower‐lying phthalocyanines, followed by an intramolecular charge‐transfer to yield P–Pc^.+ ? C₆₀^.?. This unique sequence of processes opens the way for solar‐energy‐conversion processes.     

Capillary electrophoresis,a method for the determination of nucleic acid ligands covalently attached to quantum dots representing a donor of Förster resonance energy transfer

The synthesis and determination of the structure of a Förster resonance energy transfer probe intended for the detection of specific nucleic acid sequences are described here. The probe is based on the hybridization of oligonucleotide modified quantum dots with a fluorescently labeled nucleic acid sample resulting in changes of the fluorescence emission due to the energy transfer effect. The stoichiometry distribution of oligonucleotides conjugated to quantum dots was determined by capillary electrophoresis separation. The results indicate that one to four molecules of oligonucleotide are conjugated to the surface of a single nanoparticle. This conclusion is confirmed by the course of the dependence of Förster resonance energy transfer efficiency on the concentration of fluorescently labeled complementary single‐stranded nucleic acid, showing saturation. While the energy transfer efficiency of the probe hybridized with complementary nucleic acid strands was 30%, negligible efficiency was observed with a noncomplementary strand.     

Ultrafast Energy Transfer in Triptycene‐Grafted Bodipy Scaffoldings

A series of new compounds in which various Bodipy dyes are grafted logically on triptycene rigid structures are synthesized and characterized, and their absorption spectra and photophysical properties are studied, also by pump‐probe transient absorption spectroscopy. The studied compounds are: the mono‐Bodipy species TA, TB, and TC (where A, B, and C identify different Bodipy subunits absorbing and emitting at different wavelengths), the multichromophore species TA₃, which bears three identical A subunits, and the three multichromophoric species TAB, TBC, and TABC, all of them containing at least two different types of Bodipy subunits. The triptycene moiety plays the role of a rigid scaffold, keeping the various dyes at predetermined distances and allowing for a three‐dimensional structural arrangement of the multichromophoric species. The absorption spectra of the multichromophoric Bodipy species are essentially additive, indicating that negligible inter‐chromophoric interaction takes place at the ground state. Luminescence properties and transient absorption spectroscopy indicate that a very fast (on the picosecond time scale) and efficient photoinduced energy transfer occurs in all the multi‐Bodipy species, with the lower‐energy Bodipy subunits of each multi‐Bodipy compounds playing the role of an electronic energy collector. In TAB, an energy transfer from the A‐type Bodipy subunit to the B‐type one takes place with a rate constant of 1.6×10¹⁰ s^?1, whereas in TBC an energy transfer from the B‐type Bodipy subunit to the C‐type subunit is bi‐exponential, exhibiting rate constants of 1.7×10¹¹ and 1.9×10¹⁰ s^?1; the possible presence of different conformers with different donor–acceptor distances in this bichromophoric species is proposed to cause the bi‐exponential energy‐transfer process. Interpretation of the intricate energy‐transfer pathways occurring in TABC is made with the help of the processes identified in the bichromophoric compounds. In all cases, the measured energy‐transfer rate constants agree with a Förster mechanism for the energy‐transfer processes.     

On-chip multiplexed solid-phase nucleic acid hybridization assay using spatial profiles of immobilized quantum dots and fluorescence resonance energy transfer

A microfluidic based solid-phase assay for the multiplexed detection of nucleic acid hybridization using quantum dot (QD) mediated fluorescence resonance energy transfer (FRET) is described herein. The glass surface of hybrid glass-polydimethylsiloxane (PDMS) microfluidic channels was chemically modified to assemble the biorecognition interface. Multiplexing was demonstrated using a detection system that was comprised of two colors of immobilized semi-conductor QDs and two different oligonucleotide probe sequences. Green-emitting and red-emitting QDs were paired with Cy3 and Alexa Fluor 647 (A647) labeled oligonucleotides, respectively. The QDs served as energy donors for the transduction of dye labeled oligonucleotide targets. The in-channel assembly of the biorecognition interface and the subsequent introduction of oligonucleotide targets was accomplished within minutes using a combination of electroosmotic flow and electrophoretic force. The concurrent quantification of femtomole quantities of two target sequences was possible by measuring the spatial coverage of FRET sensitized emission along the length of the channel. In previous reports, multiplexed QD-FRET hybridization assays that employed a ratiometric method for quantification had challenges associated with lower analytical sensitivity arising from both donor and acceptor dilution that resulted in reduced energy transfer pathways as compared to single-color hybridization assays. Herein, a spatial method for quantification that is based on in-channel QD-FRET profiles provided higher analytical sensitivity in the multiplexed assay format as compared to single-color hybridization assays. The selectivity of the multiplexed hybridization assays was demonstrated by discrimination between a fully-complementary sequence and a 3 base pair sequence at a contrast ratio of 8 to 1.     

Electrochemical charge transfer mediated by metal nanoparticles and quantum dots

Electron transfer processes mediated by nanostructured materials assembled at electrode surfaces underpin fundamental processes in novel electrochemical sensors, light energy conversion systems and molecular electronics. Functionalisation of electrode surfaces with hierarchical architectures incorporating self-assembling molecular systems and materials, such as metal nanostructures, quantum dots, carbon nanotubes, graphene or biomolecules have been intensively studied over the last 20 years. Important steps have been made towards the rationalisation of the charge transfer dynamics from redox species in solution across molecular self-assembling systems to electrode surfaces. For instance, a unified picture has emerged describing the factors which determine the rate constant for electron transfer processes across rigid self-assembling molecular barriers. An increasing bulk of evidence has recently shown that the incorporation of nanomaterials into self-assembling monolayers leads to an entirely different electrochemical behaviour. This perspective rationalises some of the key observations associated with nanoparticle mediated charge transfer, such as the apparent distance independent charge transfer resistance observed for redox species in solution. This behaviour only manifests itself clearly in the case where the probability of direct charge transfer from the redox probe to the electrode is strongly attenuated by self-assembling molecular barriers. Here we will highlight specific issues concerning self-assembled monolayers as blocking barriers prior to discussing the effect of nanoparticles on the electrochemical response of the system. Selected examples will provide conclusive evidence that the extent of charge transfer mediation is determined by the overlap between the density of states of the nanostructures and the energy levels of redox species in solution. Only in the case where a strong overlap exists between the energy levels of the two components, the nanostructures behave as "electron launchers", allowing efficient charge transfer across insulating molecular layers.