Conjugated polydiacetylene (PDA) possessing stimuli-responsive properties has been intensively investigated for developing efficient sensors. We report here fluorescence resonance energy transfer (FRET) in liposomes synthesized using different molar ratios of dansyl-tagged diacetylene and diacetylene–carboxylic acid monomers. Photopolymerization of diacetylene resulted in cross-linked PDA liposomes. We used steady-state electronic absorption, emission, and fluorescence anisotropy (FA) analysis to characterize the thermal-induced FRET between dansyl fluorophores (donor) and PDA (acceptor). We found that the monomer ratio of acceptor to donor (Rad) and length of linkers (functional part that connects dansyl fluorophores to the diacetylene group in the monomer) strongly affected FRET. For Rad = 10 000, the acceptor emission intensity was amplified by more than 18 times when the liposome solution was heated from 298 to 338 K. A decrease in Rad resulted in diminished acceptor emission amplification. This was primarily attributed to lower FRET efficiency between donors and acceptors and a higher background signal. We also found that the FRET amplification of PDA emissions after heating the solution was much higher when dansyl was linked to diacetylene through longer and flexible linkers than through shorter linkers. We attributed this to insertion of dansyl in the bilayer of the liposomes, which led to an increased dansyl quantum yield and a higher interaction of multiple acceptors with limited available donors. This was not the case for shorter and more rigid linkers where PDA amplification was much smaller. The present studies aim at enhancing our understanding of FRET between fluorophores and PDA-based conjugated liposomes. Furthermore, receptor tagged onto PDA liposomes can interact with ligands present on proteins, enzymes, and cells, which will produce emission sensing signal. Therefore, using the present approach, there exist opportunities for designing FRET-based highly sensitive and selective chemical and biochemical sensors.
Charge transfer processes with semiconductor
quantum dots (QDs)
have generated much interest for potential utility in energy conversion.
Such configurations are generally nonbiological; however, recent studies
have shown that a redox-active ruthenium(II)–phenanthroline
complex (Ru2+-phen) is particularly efficient at quenching
the photoluminescence (PL) of QDs, and this mechanism demonstrates
good potential for application as a generalized biosensing detection
modality since it is aqueous compatible. Multiple possibilities for
charge transfer and/or energy transfer mechanisms exist within this
type of assembly, and there is currently a limited understanding of
the underlying photophysical processes in such biocomposite systems
where nanomaterials are directly interfaced with biomolecules such
as proteins. Here, we utilize
redox reactions, steady-state absorption, PL spectroscopy, time-resolved
PL spectroscopy, and femtosecond transient absorption spectroscopy
(FSTA) to investigate PL quenching in biological assemblies of CdSe/ZnS
QDs formed with peptide-linked Ru2+-phen. The results reveal
that QD quenching requires the Ru2+ oxidation state and
is not consistent with Förster resonance energy transfer, strongly
supporting a charge transfer mechanism. Further, two colors of CdSe/ZnS
core/shell QDs with similar macroscopic optical properties were found
to have very different rates of charge transfer quenching, by Ru2+-phen with the key difference between them appearing to be
the thickness of their ZnS outer shell. The effect of shell thickness
was found to be larger than the effect of increasing distance between
the QD and Ru2+-phen when using peptides of increasing
persistence length. FSTA and time-resolved upconversion PL results
further show that exciton quenching is a rather slow process consistent
with other QD conjugate materials that undergo hole transfer. An improved
understanding of the QD–Ru2+-phen system can allow
for the design of more sophisticated charge-transfer-based biosensors
using QD platforms.
Membrane fusion of a phospholipid vesicle with a planar lipid bilayer is preceded by an initial prefusion stage in which a region of the vesicle membrane adheres to the planar membrane. A resonance energy transfer (RET) imaging microscope, with measured spectral transfer functions and a pair of radiometrically calibrated video cameras, was used to determine both the area of the contact region and the distances between the membranes within this zone. Large vesicles (5-20 microns diam) were labeled with the donor fluorophore coumarin- phosphatidylethanolamine (PE), while the planar membrane was labeled with the acceptor rhodamine-PE. The donor was excited with 390 nm light, and separate images of donor and acceptor emission were formed by the microscope. Distances between the membranes at each location in the image were determined from the RET rate constant (kt) computed from the acceptor:donor emission intensity ratio. In the absence of an osmotic gradient, the vesicles stably adhered to the planar membrane, and the dyes did not migrate between membranes. The region of contact was detected as an area of planar membrane, coincident with the vesicle image, over which rhodamine fluorescence was sensitized by RET. The total area of the contact region depended biphasically on the Ca2+ concentration, but the distance between the bilayers in this zone decreased with increasing [Ca2+]. The changes in area and separation were probably related to divalent cation effects on electrostatic screening and binding to charged membranes. At each [Ca2+], the intermembrane separation varied between 1 and 6 nm within each contact region, indicating membrane undulation prior to adhesion. Intermembrane separation distances < or = 2 nm were localized to discrete sites that formed in an ordered arrangement throughout the contact region. The area of the contact region occupied by these punctate attachment sites was increased at high [Ca2+]. Membrane fusion may be initiated at these sites of closest membrane apposition.
We present a quantitative analysis of the electron transfer between single gold nanorods and monolayer graphene under no electrical bias. Using single particle dark-field scattering and photoluminescence spectroscopy to access the homogenous linewidth, we observe broadening of the surface plasmon resonance for gold nanorods on graphene compared to nanorods on a quartz substrate. Because of the absence of spectral plasmon shifts, dielectric interactions between the gold nanorods and graphene are not important and we instead assign the plasmon damping to charge transfer between plasmon-generated hot electrons and the graphene that acts as an efficient acceptor. Analysis of the plasmon linewidth yields an average electron transfer time of 160 ± 30 fs, which is otherwise difficult to measure directly in the time domain with single particle sensitivity. In comparison to intrinsic hot electron decay and radiative relaxation, we furthermore calculate from the plasmon linewidth that charge transfer between the gold nanorods and the graphene support occurs with an efficiency of ~ 10%. Our results are important for future applications of light harvesting with metal nanoparticle plasmons and efficient hot electron acceptors as well as for understanding hot electron transfer in plasmon-assisted chemical reactions.
Plasmon damping; hot electrons; one-photon photoluminescence; single particle spectroscopy; surface plasmon resonance; graphene; plasmon linewidth
A lipid transfer protein that facilitates the transfer of glycolipids between donor and acceptor membranes has been investigated using a fluorescence resonance energy transfer assay. The glycolipid transfer protein (23-24 kDa, pI 9.0) catalyzes the high specificity transfer of lipids that have sugars β-linked to either a ceramide or a diacylglycerol backbone, such as simple glycolipids and gangliosides, but not the transfer of phospholipids, cholesterol, or cholesterol esters. In this study, we examined the effect of different charged lipids on the rate of transfer of anthrylvinyl-labeled galactosylceramide (1 mol %) from a donor to acceptor vesicle population at neutral pH. Compared to neutral donor vesicle membranes, introduction of negatively charged lipid at 5 or 10 mol % into the donor vesicles significantly decreased the transfer rate. Introduction of the same amount of negative charge into the acceptor vesicle membrane did not impede the transfer rate as effectively. Also, positive charge in the donor vesicle membrane was not as effective at slowing the transfer rate as was negative charge in the donor vesicle. Increasing the ionic strength of the buffer with NaCl significantly reversed the charge effects. At neutral pH, the transfer protein (pI ≅ 9.0) is expected to be positively charged, which may promote association with the negatively charged donor membrane. Based on these and other experiments, we conclude that the transfer process follows first-order kinetics and that the off-rate of the transfer protein from the donor vesicle surface is the rate-limiting step in the transfer process.
In search of viable strategies to identify selective inhibitors of protein kinases, we have designed a binding assay to probe the interactions of human phosphoinositide-dependent protein kinase-1 (PDK1) with potential ligands. Our protocol is based on fluorescence resonance energy transfer (FRET) between semiconductor quantum dots (QDs) and organic dyes. Specifically, we have expressed and purified the catalytic kinase domain of PDK1 with an N-terminal histidine tag [His6-PDK1(ΔPH)]. We have conjugated this construct to CdSe-ZnS core-shell QDs coated with dihydrolipoic acid (DHLA) and tested the response of the resulting assembly to a molecular dyad incorporating an ATP ligand and a BODIPY chromophore. The supramolecular association of the BODIPY-ATP dyad with the His6-PDK1(ΔPH)-QD assembly encourages the transfer of energy from the QDs to the BODIPY dyes upon excitation. The addition of ATP results in the displacement of BODIPY-ATP from the binding domain of the His6-PDK1(ΔPH) conjugated to the nanoparticles. The competitive binding, however, does not prevent the energy transfer process. A control experiment with QDs, lacking the His6-PDK1(ΔPH), indicates that the BODIPY-ATP dyad adsorbs nonspecifically on the surface of the nanoparticles, promoting the transfer of energy from the CdSe core to the adsorbed BODIPY dyes. Thus, the implementation of FRET-based assays to probe the binding domain of PDK1 with luminescent QDs requires the identification of energy acceptors unable to interact nonspecifically with the surface of the nanoparticles.
This paper concerns the development of water-compatible fluorescent imaging-probes with tunable photonic properties that can be excited at a single wavelength. Bichromophoric cassettes 1a – 1c consisting of a BODIPY donor and a cyanine acceptor were prepared using a simple synthetic route, and their photophysical properties were investigated. Upon excitation of the BODIPY moiety at 488 nm the excitation energy is transferred through an acetylene bridge to the cyanine dye acceptor, which emits light at approximately 600, 700, and 800 nm, ie with remarkable dispersions. This effect is facilitated by efficient energy transfer that gives a ‘quasi-Stokes’ shift of between 86 – 290 nm opening a huge spectral window for imaging. The emissive properties of the cassettes depend on the energy transfer (ET) mechanism: the faster the transfer, the more efficient it is. Measurements of rates of energy transfer indicate that a through-bond energy transfer takes place in the cassettes 1a and 1b that is two orders of magnitude faster than the classical through-space, Förster, energy transfer (in the case of cassette 1c, however, both mechanisms are possible, and the rate measurements do not allow us to discern between them). Thus the cassettes 1a – 1c are well suited for multiplexing experiments in biotechnological methods that involve a single laser-excitation source. However, for widespread application of these probes their solubility in aqueous media must be improved. Consequently, the probes were encapsulated in calcium phosphate/silicate nanoparticles (diameter ca 22 nm) that are freely dispersible in water. This encapsulation process resulted in only minor changes in the photophysical properties of the cassettes. The system based on cassette 1a was chosen to probe how effectively these nanoparticles could be used to deliver the dyes into cells. Encapsulated cassette 1a permeated Clone 9 rat liver cells where it localized in the mitochondria and fluoresced through the acceptor part, ie red. Overall, this paper reports readily accessible, cyanine-based through-bond energy transfer cassettes that are lypophilic but can be encapsulated to form nanoparticles that disperse freely in water. These particles can be used to enter cells and to label organelles.
Multilayered epitaxial nanofibers
are exemplary model systems for
the study of exciton dynamics and lasing in organic materials because
of their well-defined morphology, high luminescence efficiencies,
and color tunability. We use temperature-dependent continuous wave
and picosecond photoluminescence (PL) spectroscopy to quantify exciton
diffusion and resonance-energy transfer (RET) processes in multilayered
nanofibers consisting of alternating layers of para-hexaphenyl (p6P)
and α-sexithiophene (6T) serving as exciton donor and acceptor
material, respectively. The high probability for RET processes is
confirmed by quantum chemical calculations. The activation energy
for exciton diffusion in p6P is determined to be as low as 19 meV,
proving p6P epitaxial layers also as a very suitable donor material
system. The small activation energy for exciton diffusion of the p6P
donor material, the inferred high p6P-to-6T resonance-energy-transfer
efficiency, and the observed weak PL temperature dependence of the
6T acceptor material together result in an exceptionally high optical
emission performance of this all-organic material system, thus making
it well suited, for example, for organic light-emitting devices.
Electron-transfer reactions are fundamental to many practical devices, but because of their complexity, it is often very difficult to interpret measurements done on the complete device. Therefore, studies of model systems are crucial. Here the rates of charge separation and recombination in donor–acceptor systems consisting of a series of butadiyne-linked porphyrin oligomers (n = 1–4, 6) appended to C60 were investigated. At room temperature, excitation of the porphyrin oligomer led to fast (5–25 ps) electron transfer to C60 followed by slower (200–650 ps) recombination. The temperature dependence of the charge-separation reaction revealed a complex process for the longer oligomers, in which a combination of (i) direct charge separation and (ii) migration of excitation energy along the oligomer followed by charge separation explained the observed fluorescence decay kinetics. The energy migration is controlled by the temperature-dependent conformational dynamics of the longer oligomers and thereby limits the quantum yield for charge separation. Charge recombination was also studied as a function of temperature through measurements of femtosecond transient absorption. The temperature dependence of the electron-transfer reactions could be successfully modeled using the Marcus equation through optimization of the electronic coupling (V) and the reorganization energy (λ). For the charge-separation rate, all of the donor–acceptor systems could be successfully described by a common electronic coupling, supporting a model in which energy migration is followed by charge separation. In this respect, the C60-appended porphyrin oligomers are suitable model systems for practical charge-separation devices such as bulk-heterojunction solar cells, where conformational disorder strongly influences the electron-transfer reactions and performance of the device.
The dissociation of photogenerated excitons and the subsequent spatial separation of the charges are of crucial importance to the design of efficient donor-acceptor heterojunction solar cells. While huge progress has been made in understanding charge generation at all-organic junctions, the process in hybrid organic:inorganic systems has barely been addressed. Here, we explore the influence of energetic driving force and local crystallinity on the efficiency of charge pair generation at hybrid organic:inorganic semiconductor heterojunctions. We use x-ray diffraction, photoluminescence quenching, transient absorption spectroscopy, photovoltaic device and electroluminescence measurements to demonstrate that the dissociation of photogenerated polaron pairs at hybrid heterojunctions is assisted by the presence of crystalline electron acceptor domains. We propose that such domains encourage delocalization of the geminate pair state. The present findings suggest that the requirement for a large driving energy for charge separation is relaxed when a more crystalline electron acceptor is used.
Aromatic triazoles have been frequently used as π-conjugated linkers in intramolecular electron transfer processes. To gain a deeper understanding of the electron mediating function of triazoles, we have synthesized a family of new triazole-based electron donor-acceptor conjugates. We have connected porphyrins and fullerenes through a central triazole moiety – (ZnP-Tri-C60) – each with a single change in their connection through the linker. An extensive photophysical and computational investigation reveals that the electron transfer dynamics – charge separation and charge recombination – in the different ZnP-Tri-C60 conjugates reflect a significant influence of the connectivity at the triazole linker. Except for m4m-ZnP-Tri-C60 17, the conjugates exhibit through-bond electron transfer with varying rate constants. Since the through-bond distance is nearly equal in the ZnP-Tri-C60 conjugates, the variation in charge separation and charge recombination dynamics is mainly associated with the electronic properties of the conjugates, including orbital energies, electron affinity, and the energies of the excited states. The changes of the electronic couplings are, in turn, a consequence of the different connectivity patterns at the triazole moieties.
In this work, we have demonstrated the structural and optoelectronic properties of the surface of ternary/quaternary (CISe/CIGSe/CZTSe) chalcopyrite nanocrystallites passivated by tri-n-octylphosphine-oxide (TOPO) and tri-n-octylphosphine (TOP) and compared their charge transfer characteristics in the respective polymer: chalcopyrite nanocomposites by dispersing them in poly(3-hexylthiophene) polymer. It has been found that CZTSe nanocrystallites due to their high crystallinity and well-ordered 3-dimensional network in its pristine form exhibit a higher steric- and photo-stability, resistance against coagulation and homogeneity compared to the CISe and CIGSe counterparts. Moreover, CZTSe nanocrystallites display efficient photoluminescence quenching as evident from the high value of the Stern–Volmer quenching constant (K
SV) and eventually higher charge transfer efficiency in their respective polymer P3HT:CZTSe composites. We modelled the dependency of the charge transfer from the donor and the charge separation mechanism across the donor–acceptor interface from the extent of crystallinity of the chalcopyrite semiconductors (CISe/CIGSe/CZTSe). Quaternary CZTSe chalcopyrites with their high crystallinity and controlled morphology in conjunction with regioregular P3HT polymer is an attractive candidate for hybrid solar cells applications.
chalcopyrites; charge-transfer; hybrid organic-inorganic; nanocomposites; P3HT
Detailed spectroscopic and computational studies of the low-spin iron complexes [FeIII(S2Me2N3(Pr,Pr))(N3)] (1) and [FeIII(S2Me2N3(Pr,Pr))]1+ (2) were performed to investigate the unique electronic features of these species and their relation to the low-spin ferric active sites of nitrile hydratases. Low-temperature UV/vis/NIR and MCD spectra of 1 and 2 reflect electronic structures that are dominated by antibonding interactions of the Fe 3d manifold and the equatorial thiolate S 3p orbitals. The six-coordinate complex 1 exhibits a low-energy Sπ → Fe 3dxy (~13000 cm−1) charge-transfer transition that results predominantly from the low energy of the singly occupied Fe 3dxy orbital, due to pure π interactions between this acceptor orbital and both thiolate donor ligands in the equatorial plane. The 3dπ → 3dσ ligand-field transitions in this species occur at higher energies (>15000 cm−1), reflecting its near-octahedral symmetry. The Fe 3dxz,yz → Fe 3dxy (dπ → dπ) transition occurs in the near-IR and probes the FeIII−S π-donor bond; this transition reveals vibronic structure that reflects the strength of this bond (νe ≈ 340 cm−1). In contrast, the ligand-field transitions of the five-coordinate complex 2 are generally at low energy, and the Sπ → Fe charge-transfer transitions occur at much higher energies relative to those in 1. This reflects changes in thiolate bonding in the equatorial plane involving the Fe 3dxy and
Fe3dx2−y2 orbitals. The spectroscopic data lead to a simple bonding model that focuses on the σ and π interactions between the ferric ion and the equatorial thiolate ligands, which depend on the S–Fe–S bond angle in each of the complexes. These electronic descriptions provide insight into the unusual S = ½ ground spin state of these complexes: the orientation of the thiolate ligands in these complexes restricts their π-donor interactions to the equatorial plane and enforces a low-spin state. These anisotropic orbital considerations provide some intriguing insights into the possible electronic interactions at the active site of nitrile hydratases and form the foundation for further studies into these low-spin ferric enzymes.
Förster resonance energy transfer (FRET) is a mechanism where energy is transferred from an excited donor fluorophore to adjacent chromophores via non-radiative dipole-dipole interactions. FRET theory primarily considers the interactions of a single donor-acceptor pair. Unfortunately, it is rarely known if only a single acceptor is present in a molecular complex. Thus, the use of FRET as a tool for measuring protein-protein interactions inside living cells requires an understanding of how FRET changes with multiple acceptors. When multiple FRET acceptors are present it is assumed that a quantum of energy is either released from the donor, or transferred in toto to only one of the acceptors present. The rate of energy transfer between the donor and a specific acceptor (kD→A) can be measured in the absence of other acceptors, and these individual FRET transfer rates can be used to predict the ensemble FRET efficiency using a simple kinetic model where the sum of all FRET transfer rates is divided by the sum of all radiative and non-radiative transfer rates.
The generality of this approach was tested by measuring the ensemble FRET efficiency in two constructs, each containing a single fluorescent-protein donor (Cerulean) and either two or three FRET acceptors (Venus). FRET transfer rates between individual donor-acceptor pairs within these constructs were calculated from FRET efficiencies measured after systematically introducing point mutations to eliminate all other acceptors. We find that the amount of energy transfer observed in constructs having multiple acceptors is significantly greater than the FRET efficiency predicted from the sum of the individual donor to acceptor transfer rates.
We conclude that either an additional energy transfer pathway exists when multiple acceptors are present, or that a theoretical assumption on which the kinetic model prediction is based is incorrect.
The effect of molecular topology, and conformation on the dynamics of photoinduced electron transfer (ET) processes has been studied in interlocked electron donor-acceptor systems, specifically rotaxanes with zinc(II)-tetraphenylporphyrin (ZnP) electron donor and fullerene (C60) as the electron acceptor. Formation or cleavage of coordinative bonds was used to induce major topological and conformational changes in the interlocked architecture. In the first approach, the tweezers-like structure created by the two ZnP stopper groups on the thread was used as a recognition site for complexation of 1,4-diazabicyclo[2.2.2]octane (DABCO), which creates a bridge between the two ZnP moieties on the rotaxane, generating a catenane structure. The photoinduced processes in the DABCO-complexed (ZnP)2-catenate-C60 system were compared with those of the (ZnP)2-rotaxane-C60 precursor and the previously reported ZnP-catenate-C60. Steady-state emission and transient absorption studies showed that a similar multistep ET pathway emerged for rotaxanes and catenanes upon photoexcitation at various wavelengths, ultimately resulting in a long-lived ZnP•+/C60•− charge separated radical pair state. However, the decay kinetics of the latter states clearly reflect the topological differences between the rotaxane, the catenate, and DABCO-complexed-catenate architectures. The lifetime of the long-distance ZnP•+–[Cu(I)phen2]+–C60•− charge separated state is more than four times longer in 3 (1.03 µs) than in 1 (0.24 µs) and approaches that in catenate 2 (1.1 µs). The results clearly showed that adoption of a catenane from a rotaxane topology inhibits the charge recombination process. In a second approach, the Cu(I) ion used as template to assemble the (ZnP)2–[Cu(I)phen2]+–C60 rotaxane was removed, and structural analysis suggested a major topographical change occurred, such that charge separation between the chromophores was no longer observed upon photoexcitation in nonpolar as well as polar solvents. Only ZnP and C60 triplet excited states were observed upon laser excitation. These results highlighted the critical importance of the central Cu(I) ion for long range ET processes in these large interlocked electron donor-acceptor systems.
Rotaxanes; Catenanes; Electron Transfer; Porphyrin; Fullerene
We report the synthesis, one- and two-photon absorption spectroscopy, fluorescence, and electrochemical properties of a series of quadrupolar molecules that feature proquinoidal π-aromatic acceptors. These quadrupolar molecules possess either donor-acceptor-donor (D–A–D) or acceptor-donor-acceptor (A–D–A) electronic motifs, and feature 4-N,N-dihexylaminophenyl, 4-dodecyloxyphenyl, 4-(N,N-dihexylamino)benzo[c][1,2,5]thiadiazolyl or 2,5-dioctyloxyphenyl electron donor moieties and benzo[c][1,2,5]thiadiazole (BTD) or 6,7-bis(3’,7’-dimethyloctyl)[1,2,5]thiadiazolo[3,4-g]quinoxaline (TDQ) electron acceptor units. These conjugated structures are highly emissive in nonpolar solvents and exhibit large spectral red-shifts of their respective lowest energy absorption bands relative to analogous reference compounds that incorporate phenylene components in place of BTD and TDQ moieties. BTD-based D-A-D and A-D-A chromophores exhibit increasing fluorescence emission red-shifts, and a concomitant decrease of the fluorescence quantum yield (Φf) with increasing solvent polarity; these data indicate that electronic excitation augments benzothiadiazole electron density via an internal charge transfer mechanism. The BTD- and TDQ-containing structures exhibit blue-shifted two-photon absorption (TPA) spectra relative to their corresponding one-photon absorption (OPA) spectra, and display high TPA cross-sections (>100 GM) within these spectral windows. D-A-D and A-D-A structures that feature more extensive conjugation within this series of compounds exhibit larger TPA cross-sections consistent with computational simulation. Factors governing TPA properties of these quadrupolar chromophores are discussed within the context of a three-state model.
A large class of cation-responsive fluorescent sensors utilizes a donor-spacer-acceptor (D-A) molecular framework that can modulate the fluorescence emission intensity through a fast photoinduced intramolecular electron transfer (PET) process. The emission enhancement upon binding of the analyte defines the contrast ratio of the probe, a key property that is particularly relevant in fluorescence microscopy imaging applications. Due to their unusual electronic structure, 1,3,5-triaryl-pyrazoline fluorophores allow for the differential tuning of the excited state energy ΔE00 and the fluorophore acceptor potential E(A/A−), both of which are critical parameters that define the ET thermodynamics and thus the contrast ratio. By systematically varying the number and attachment positions of fluoro-substituents on the fluorophore π-system, ΔE00 can be adjusted over a broad range (0.4 eV) without significantly altering the acceptor potential E(A/A−). Experimentally measured D-A coupling and reorganization energies were used to draw a potential map for identifying the optimal ET driving force that is expected to give a maximum fluorescence enhancement for a given change in donor potential upon binding of the analyte. The rational design strategy was tested by optimizing the fluorescence response of a pH sensitive probe, thus yielding a maximum emission enhancement factor of 400 upon acidification. Furthermore, quantum chemical calculations were used to reproduce the experimental trends of reduction potentials, excited state energies, and ET driving forces within the framework of linear free energy relationships (LFER). Such LFERs should be suitable to semi-empirically predict ET driving forces with an average unsigned error of 0.03 eV, consequently allowing for the computational prescreening of substituent combinations to best match the donor potential of a given cation receptor. Within the scaffold of the triarylpyrazoline platform, the outlined differential tuning of the electron transfer parameters should be applicable to a broad range of cation receptors for designing PET sensors with maximized contrast ratios.
Two nanocrystal-osmium(II) polypyridyl (NC-Os(II)PP) conjugates have been designed to detect oxygen in biological environments. Polypyridines appended with a single free amine were linked with facility to a carboxylic acid functionality of a semiconductor NC overlayer to afford a biologically stable amide bond. The Os(II)PP complexes possess broad absorptions that extend into the red spectral region; this absorption feature makes them desirable acceptors of energy from NC donors. Fluorescence resonance energy transfer (FRET) from the NC to the Os(II)PP causes an enhanced Os(II)PP emission with a concomitant quenching of the NC emission. Owing to the large two-photon absorption cross-section of the NCs, FRET from NC to the Os(II)PP can be established under two-photon excitation conditions. In this way, two-photon processes of metal polypyridyl complexes can be exploited for sensing. The emission of the NC is insensitive to oxygen, even at 1 atm, whereas excited states of both osmium complexes are quenched in the presence of oxygen. The NC emission may thus be used as an internal reference to correct for fluctuations in the photoluminescence intensity signal. These properties taken together establish NC-Os(II)PP conjugates as competent ratiometric, two-photon oxygen sensors for application in biological microenvironments.
The process of radiationless energy transfer from a chromophore in an excited electronic state (the “donor”) to another chromophore (an “acceptor”), in which the energy released by the donor effects an electronic transition, is known as “Förster Resonance Energy Transfer” (FRET). The rate of energy transfer is dependent on the sixth power of the distance between donor and acceptor. Determining FRET efficiencies is tantamount to measuring distances between molecules. A new method is proposed for determining FRET efficiencies rapidly, quantitatively, and non-destructively on ensembles containing donor acceptor pairs: at wavelengths suitable for mutually exclusive excitations of donors and acceptors, two laser beams are intensity-modulated in rectangular patterns at duty cycle ½ and frequencies f1 and f2 by electro-optic modulators. In an ensemble exposed to these laser beams, the donor excitation is modulated at f1, and the acceptor excitation, and therefore the degree of saturation of the excited electronic state of the acceptors, is modulated at f2. Since the ensemble contains donor acceptor pairs engaged in FRET, the released donor fluorescence is modulated not only at f1 but also at the beat frequency Δf: = |f1 − f2|. The depth of the latter modulation, detectable via a lock-in amplifier, quantitatively indicates the FRET efficiency.
FRET; LSM; dynamic; non-destructive; electro-optic modulator; beat; saturation; modulation
A lipid transfer protein, purified from bovine brain (23.7 kDa, 208 amino acids) and specific for glycolipids, has been used to develop a fluorescence resonance energy transfer assay (anthrylvinyl labeled lipids; energy donors and perylenoyl labeled lipids; energy acceptors) for monitoring the transfer of lipids between membranes. Small unilamellar vesicles composed of 1 mol% anthrylvinyl-galactosylceramide, 1.5 mol% perylenoyl-triglyceride, and 97.5% 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) served as donor membranes. Acceptor membranes were 100% POPC vesicles. Addition of glycolipid transfer protein to mixtures of donor and acceptor vesicles resulted in increasing emission intensity of anthrylvinyl-galactosylceramide and decreasing emission intensity of the nontransferable perylenoyl triglyceride as a function of time. The behavior was consistent with anthrylvinyl-galactosylceramide being transferred from donor to acceptor vesicles. The anthrylvinyl and perylenoyl energy transfer pair offers advantages over frequently used energy transfer pairs such as NBD and rhodamine. The anthrylvinyl emission overlaps effectively the perylenoyl excitation spectrum and the fluorescence parameters of the anthrylvinyl fluorophore are nearly independent of the medium polarity. The nonpolar fluorophores are localized in the hydrophobic region of the bilayer thus producing minimal disturbance of the bilayer polar region. Our results indicate that this method is suitable for assay of lipid transfer proteins including mechanistic studies of transfer protein function.
Glycosphingolipid; Lipid transfer protein; Phospholipid bilayers; Galactosylceramide; Anthrylvinyl; Perylenoyl
The fundamental limits of inorganic semiconductors for light emitting applications, such as holographic displays, biomedical imaging and ultrafast data processing and communication, might be overcome by hybridization with their organic counterparts, which feature enhanced frequency response and colour range. Innovative hybrid inorganic/organic structures exploit efficient electrical injection and high excitation density of inorganic semiconductors and subsequent energy transfer to the organic semiconductor, provided that the radiative emission yield is high. An inherent obstacle to that end is the unfavourable energy level offset at hybrid inorganic/organic structures, which rather facilitates charge transfer that quenches light emission. Here, we introduce a technologically relevant method to optimize the hybrid structure's energy levels, here comprising ZnO and a tailored ladder-type oligophenylene. The ZnO work function is substantially lowered with an organometallic donor monolayer, aligning the frontier levels of the inorganic and organic semiconductors. This increases the hybrid structure's radiative emission yield sevenfold, validating the relevance of our approach.
Hybrid inorganic-organic structures can overcome the limits of inorganic semiconductor light emitting devices but the energy level offset is an obstacle. Here, Schlesinger et al. lower the ZnO work function with an organometallic donor monolayer and enhance the radiative emission of the hybrid structure.
Electrostatically driven layer-by-layer (LbL) assembly is a simple and robust method for producing structurally tailored thin film biomaterials, of thickness ca. 10 nanometers, containing biofunctional ligands. We investigate the LbL formation of multilayer films composed of polymers of biological origin (poly(L-lysine) (PLL) and dextran sulfate (DS)), the adsorption of fibronectin (Fn) - a matrix protein known to promote cell adhesion - onto these films, and the subsequent spreading behavior of human umbilical vein endothelial cells (HUVEC). We employ optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microgravimetry with dissipation (QCMD) to characterize multilayer assembly in situ, and find adsorbed Fn mass on PLL terminated films to exceed that on DS terminated films by 40%, correlating with the positive charge and lower degree of hydration of PLL terminated films. The extent and initial rate of Fn adsorption to both PLL and DS terminated films exceed those onto the bare substrate, indicating the important role of electrostatic complexation between negatively charged protein and positively charged PLL at or near the film surface. We use phase contrast optical microscopy to investigate the time dependent morphological changes of HUVEC as a function of layer number, charge of terminal layer, and the presence of Fn. We observe HUVEC to attach, spread, and lose circularity on all surfaces. (Positively charged) PLL terminated films exhibit a greater extent of cell spreading than do (negatively charged) DS terminated films, and spreading is enhanced while circularity loss is suppressed by the presence of adsorbed Fn. The number of layers plays a significant role only for DS terminated films with Fn, where spreading on a bilayer greatly exceeds that on a multilayer, and PLL terminated films without Fn, where initial spreading is significantly higher on a monolayer. We observe initial cell spreading to be followed by retraction (i.e. decreased cell area and circularity with time) for films without Fn, and for DS terminated films with Fn. Overall, the Fn coated PLL monolayer and the Fn coated PLL terminated multilayer are the best performing films in promoting cell spreading. We conclude the presence of Fn to be an important factor (more so than film charge or layer number) in controlling the interaction between multilayer films and living cells, and thus to represent a promising strategy toward in vivo applications such as tissue engineering.
fibronectin; poly(L-lysine); dextran sulfate; protein adsorption; endothelial cell; layer-by-layer
Photosynthetic reaction centers convert excitation energy from absorbed sunlight into chemical potential energy in the form of a charge-separated state. The rates of the electron transfer reactions necessary to achieve long-lived, high-energy charge-separated states with high quantum yields are determined in part by precise control of the electronic coupling among the chromophores, donors and acceptors, and of the reaction energetics. Successful artificial photosynthetic reaction centers for solar energy conversion have similar requirements. Control of electronic coupling in particular necessitates chemical linkages between active component moieties that both mediate coupling and restrict conformational mobility so that only spatial arrangements that promote favorable coupling are populated. Toward this end, we report the synthesis, structure and photochemical properties of an artificial reaction center containing two porphyrin electron donor moieties and a fullerene electron acceptor in a macrocyclic arrangement involving a ring of 42 atoms. The two porphyrins are closely spaced, in an arrangement reminiscent of that of the special pair in bacterial reaction centers. The molecule is produced by an unusual cyclization reaction that yields mainly a product with C2 symmetry and trans-2 disubstitution at the fullerene. The macrocycle maintains a rigid, highly-constrained structure that was determined by UV-vis spectroscopy, NMR, mass spectrometry, and molecular modeling at the semi-empirical PM6 and DFT (B3LYP/6-31G**) levels. Transient absorption results for the macrocycle in 2-methyltetrahydrofuran reveal photoinduced electron transfer from the porphyrin first excited singlet state to the fullerene to form a P•+-C60•−-P charge separated state with a time constant of 1.1 ps. Photoinduced electron transfer to the fullerene excited singlet state to form the same charge-separated state has a time constant of 15 ps. The charge-separated state is formed with a quantum yield of essentially unity and has a lifetime of 2.7 ns. The ultrafast charge separation coupled with charge recombination that is over 2000 times slower is consistent with a very rigid molecular structure having a small reorganization energy for electron transfer, relative to related porphyrin-fullerene molecules.
Ultrafast photoinduced electron transfer preceding energy equilibration still poses many experimental and conceptual challenges to the optimization of photoconversion since an atomic-scale description has so far been beyond reach. Here we combine femtosecond transient optical absorption spectroscopy with ultrafast X-ray emission spectroscopy and diffuse X-ray scattering at the SACLA facility to track the non-equilibrated electronic and structural dynamics within a bimetallic donor–acceptor complex that contains an optically dark centre. Exploiting the 100-fold increase in temporal resolution as compared with storage ring facilities, these measurements constitute the first X-ray-based visualization of a non-equilibrated intramolecular electron transfer process over large interatomic distances. Experimental and theoretical results establish that mediation through electronically excited molecular states is a key mechanistic feature. The present study demonstrates the extensive potential of femtosecond X-ray techniques as diagnostics of non-adiabatic electron transfer processes in synthetic and biological systems, and some directions for future studies, are outlined.
Photoinduced electron transfer in solvated molecular assemblies occurs on the ultrafast timescale before full electronic and geometric relaxation take place. Here Canton et al. monitor this out-of-equilibrium process in a donor–acceptor bimetallic assembly using an X-ray free-electron laser.
Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research. This powerful tool can elucidate protein interactions in either a dynamic or steady state. We recently developed a series of FRET-based technologies to determine protein interaction dissociation constant and for use in high-throughput screening assays of SUMOylation. SUMO (small ubiquitin-like modifier) is conjugated to substrates through an enzymatic cascade. This important posttranslational protein modification is critical for multiple biological processes. Sentrin/SUMO-specific proteases (SENPs) act as endopeptidases to process the pre-SUMO or as isopeptidases to deconjugate SUMO from its substrate. Here, we describe a novel quantitative FRET-based protease assay for determining the kinetics of SENP1. Our strategy is based on the quantitative analysis and differentiation of fluorescent emission signals at the FRET acceptor emission wavelengths. Those fluorescent emission signals consist of three components: the FRET signal and the fluorescent emissions of donor (CyPet) and acceptor (YPet). Unlike our previous method in which donor and acceptor direct emissions were excluded by standard curves, the three fluorescent emissions were determined quantitatively during the SENP digestion process from onesample. New mathematical algorithms were developed to determine digested substrate concentrations directly from the FRET signal and donor/acceptor direct emissions. The kinetic parameters, kcat, KM, and catalytic efficiency (kcat/KM) of SENP1 catalytic domain for pre-SUMO1/2/3 were derived. Importantly, the general principles of this new quantitative methodology of FRET-based protease kinetic determinations can be applied to other proteases in a robust and systems biology approach.
quantitative FRET analysis; internal calibration; one-sample assay; protease kinetics; SENP