Photoinduced Electron Transfer Probes Research Articles

Simple and efficient PET and AIEE mechanism-based fluorescent probes for sensing Tabun mimic DCNP

The highly sensitive, selective, easy-to-prepare, aqueous media based on two novel probes 2-(pyren-1-yl)imidazo[1,2-a]pyridine (IMP-Py) and (2-(pyren-1-yl)imidazo[1,2-a]pyridin-3-yl)methanol (IMP-Py-OH) are synthesized for the detection of toxic chemical warfare nerve agent mimic diethylcyanochlorophosphonate (DCNP). Both probes are found effective in the detection of DCNP but comparatively, IMP-Py shows better properties in terms of instantaneous response, specificity, selectivity and a low detection limit of 16.9 nM. A significant enhancement of fluorescence intensity of IMP-Py due to aggregation-induced emission enhancement (AIEE) and photoinduced electron transfer (PET) phenomenon was inhibited due to phosphorylation of the hydroxy group of IMP-Py-OH in presence of DCNP has been observed. Taking the advantages of good sensitivity and fast response, probe IMP-Py has been fabricated into a viable paper strips portable product, tested for its potential for the detection of DCNP in tap water as well as with its vapor and response is visible under a UV lamp of 365 nm wavelength.

Crystalline H-aggregate nanoparticles for detecting dopamine release from M17 human neuroblastoma cells.

Dopamine (DA) is an important neurotransmitter, which is essential for transmitting signals in neuronal communications. The deficiency of DA release from neurons is implicated in neurological disorders. There has been great interest in developing new optical probes for monitoring the release behavior of DA from neurons. H-aggregates of organic dyes represent an ordered supramolecular structure with delocalized excitons. In this paper, we use the self-assembly of 3,3'-diethylthiadicarbocyanine iodide (DiSC2(5)) in ammonia solution to develop crystalline H-aggregate nanoparticles, in which DiSC2(5) molecules show long-range π-π stacking. The crystalline H-aggregate nanoparticles are stable in cell culture medium and can serve as an efficient photo-induced electron transfer (PET) probe for the detection of DA with the concentration as low as 0.1 nM in cell culture medium. Furthermore, the crystalline H-aggregate nanoparticle-based PET probe is used to detect the release behavior of DA from the M17 human neuroblastoma cells. We find that the DA release from the cells is enhanced by nicotine stimulations. Our results highlight the potential of crystalline H-aggregate nanoparticle-based PET probes for diagnosing nervous system diseases and verifying therapies.

Detection Sensitivity Enhancement of Naphthalimide PET Fluorescent Probes by 4-Methoxy-Substitution.

Naphthalimide photoinduced electron transfer (PET) fluorescent probes are widely used in fluorescence imaging. Thereinto, detection sensitivity is the vital parameter of PET probes. However, the modulation of detection sensitivity is yet to be reported for naphthalimide PET probes. Herein, the detection sensitivity enhancement of naphthalimide PET fluorescent probes through 4-methoxy-substitution is proposed in this work. Taking Zn2+ detection an example, 4-methoxy-naphthalimide PET probe 2-(2-(bis(pyridin-2-ylmethyl)amino)ethyl)-6-methoxy-1H-benzo[de]isoquinoline-1,3(2H)-dione (BPNM) and control PET probe 2-(2-(bis(pyridin-2-ylmethyl)amino)ethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (BPN) are separately synthesized. The addition of 4-methoxy group with ability of strong electron donating to naphthalimide facilitates the construction of electronic push-pull system in the fluorophore resulting in the bathochromic shift of absorption and fluorescence emission spectra of BPNM and is further conducive to the enhancement of molar extinction coefficient ε and fluorescence quantum yield Φf of BPNM. Compared with BPN, BPNM shows lower Zn2+ detection limit in titration assays. Meanwhile, the fluorescence signal change (off-on) before and after Zn2+ addition of intracellular BPNM is more obvious and easier to control in confocal laser scanning imaging. Therefore, 4-methoxy-substitution improves the detection sensitivity of naphthalimide PET probe, which is favorable for the precise sensing of analyte, and further lays a good foundation for the synthesis of PET probe with high sensitivity.

A General Descriptor ΔE Enables the Quantitative Development of Luminescent Materials Based on Photoinduced Electron Transfer.

Photoinduced electron transfer (PET) is one of the most important mechanisms for developing fluorescent probes and biosensors. Quantitative prediction of the quantum yields of these probes and sensors is crucial to accelerate the rational development of novel PET-based functional materials. Herein, we developed a general descriptor (ΔE) for predicting the quantum yield of PET probes, with a threshold value of ∼0.6 eV. When ΔE < ∼0.6 eV, the quantum yield is low (mostly <2%) due to the substantial activation of PET in polar environments; when ΔE > ∼0.6 eV, the quantum yield is high because of the inhibition of PET. This simple yet effective descriptor is applicable to a wide range of fluorophores, such as BODIPY, fluorescein, rhodamine, and Si-rhodamine. This ΔE descriptor enables us not only to establish new applications for existing PET probes but also to quantitatively design novel PET-based fluorophores for wash-free bioimaging and AIEgen development.

Activity-Based Sensing and Theranostic Probes Based on Photoinduced Electron Transfer.

Fluorescent probes have become powerful tools in detection, imaging and disease diagnosis due to their high sensitivity, specificity, fast response, and technical simplicity. In the last decades, researchers have made remarkable progress in developing signaling mechanisms to design fluorescent probes such as photoinduced electron transfer (PET), intramolecular charge transfer (ICT), and fluorescence resonance energy transfer (FRET). Typical PET is composed of a multicomponent system in which a fluorophore (electron acceptor) is separately linked with a recognition group (electron donor) via a short spacer. PET probes normally feature a low fluorescence background and significant fluorescence enhancement in response to targets. Recent research revealed that PET probes have also been used as theranostic agents, whose fluorescence and toxicity can be simultaneously activated by cancer-specific parameters. In this Account, we highlight the recent advances of rational design and applications of PET probes, focusing primarily on studies from our research group. For example, different from the case of the traditional single-atom electron donor (O, S, N, Se, Te, etc.) in typical PET, we used more a electron-rich pyrrole ring to "switch off" the fluorescence of the fluorophore more efficiently through an "enhanced PET" effect which provided a lower background fluorescence and higher signal-to-noise ratio. Furthermore, normal PET represents the main principle behind the design of small molecule "off-on" fluorescent sensors. We developed new PET platform through intramolecular space folding (folding PET) to overcome the difficulty of designing PET enzyme-targeting probes. Therefore, based on typical PET and these new PET concepts, we, for instance, reported PET probes for the detection of Zn2+ without proton interference, a BODIPY-based d-PET probe for reporting local hydrophilicity within lysosomes, and an "enhanced PET" fluorescent probe for imaging HClO in cancer cells. We also developed COX-2-specific probe for identifying cancer cells and quantifying cancer-related events, and a KIAA1363-sensitive probe for tracking solid tumors in living mice. Furthermore, we first applied an aminopeptidase N (APN)-sensitive probe based on PET for cancer diagnosis and therapy. We anticipate that further development of PET fluorescent probes providing more sensitivity and selectivity to analytes of interest will be equipped with more functions and play indispensable roles in the studies of pathology, diagnostics, and cancer therapies.

Fluorescence pH Probes Based on Photoinduced Electron Transfer Quenching of Long Fluorescence Lifetime Triangulenium Dyes

AbstractAzaoxatriangulenium dyes with long fluorescence lifetimes offer advantages in fluorescence lifetime imaging (FLIM), time‐gated detection and polarization assays. However, the long excited state lifetimes are also expected to increase sensitivity to photoinduced electron transfer (PET) quenching, potentially increasing the on‐off ratio of PET probes. We report the synthesis and investigation of the optical properties of azaoxatriangulenium salts substituted with phenol substituents for pH sensing. Two series of pH probes with the phenol hydroxy group placed in the ortho, meta, or para positions to azadioxatriangulenium (ADOTA) and diazaoxatriangulenium (DAOTA) chromophores are investigated. All the phenol‐substituted dyes possess pH‐responsive fluorescence signals in the biologically relevant pH window (pH 6–9) due to efficient intramolecular PET from the phenolate form of the pendant arm. While the PET mechanism is partly suppressed in the phenol form of the ADOTA derivatives, it is completely suppressed in the DAOTA derivatives. The long excited state lifetimes of the triangulenium dyes ensure efficient PET quenching leading to very high on‐off ratios of the pH probes. The study clearly demonstrates effects of the position of the phenol/phenolate group relative to the chromophore on both PET quenching rates and pKa values.

Novel luminescent paper based calix[4]arene chelation enhanced fluorescence- photoinduced electron transfer probe for Mn2+, Cr3+ and F-

A novel structurally simple calix[4]arene attached 1-aminoanthraquinone associated lower rim calix[4]arene conjugate was synthesized and has been used as turn on/off/on fluorescence probe for Mn2+, Cr3+ and F-. This chelation enhanced fluorescence - photoinduced electron transfer (CHEF-PET) based TAAC probe has been applied for its analytical application in real samples such as Mn2+ from blood serum, Cr3+ and F- from industrial effluent with 94–99% recovery. The limit of detection of this sensor is found to be 11 nM for Mn2+, 4 nM for Cr3+ and 19 nM for F- with the concentration range of 0–120 nM. Further, we report an easy-to-use, low cost and disposable paper-based sensing device for rapid chemical screening of Mn2+, Cr3+ and F-. The device comprises fluorescent sensing probes embedded into a nitrocellulose matrix where the resonance energy transfer phenomenon seems to be the sensing mechanism. It opens up new opportunities for simple and fast screening in remote settings where sophisticated instrumentation is not always available. The MOPAC-2016 software package has been used to optimize the TAAC using PM7 well established method and calculates the HOMO-LUMO energy band gap for structure TAAC and TAAC with Mn2+, Cr3+ and F- ion based structures.

Benzothiazole‐Based Sensors for Protons and Chromium(III) ions

AbstractTwo benzothiazole based Schiff bases 5‐(diethylamino)‐2‐(((6‐methoxybenzo[d]thiazol‐2‐yl)imino)methyl)phenol (probe 1) and N‐(4‐(dimethylamino)benzylidene)‐6‐methoxybenzo[d]thiazol‐2‐amine (probe 2) were efficiently synthesized and structurally characterized. Molecular structure of probe 1 is obtained by single crystal X‐ray analysis. The presence of hydroxyl substituent on phenyl moiety makes probe 1 as ESIPT (Excited State Intramolecular Proton Transfer) probe and the absence of hydroxyl on phenyl moiety differs probe 2 as PET (Photo Induced Electron Transfer) probe towards sensing of H+ ions. Probe 1 selectively detects H+ ions colorimetrically with reversible changes on adding a base like TEA. Besides H+ ions, probe 2 detects Cr(III) ions with a colorimetric response. The addition of TEA leads to reversible changes with primary protonation and these changes were irreversible with second protonation.

Significant expansion of fluorescent protein sensing ability through the genetic incorporation of superior photo-induced electron-transfer quenchers.

Photo-induced electron transfer (PET) is ubiquitous for photosynthesis and fluorescent sensor design. However, genetically coded PET sensors are underdeveloped, due to the lack of methods to site-specifically install PET probes on proteins. Here we describe a family of acid and Mn(III) turn-on fluorescent protein (FP) sensors, named iLovU, based on PET and the genetic incorporation of superior PET quenchers in the fluorescent flavoprotein iLov. Using the iLovU PET sensors, we monitored the cytoplasmic acidification process, and achieved Mn(III) fluorescence sensing for the first time. The iLovU sensors should be applicable for studying pH changes in living cells, monitoring biogentic Mn(III) in the environment, and screening for efficient manganese peroxidase, which is highly desirable for lignin degradation and biomass conversion. Our work establishes a platform for many more protein PET sensors, facilitates the de novo design of metalloenzymes harboring redox active residues, and expands our ability to probe protein conformational dynamics.

An acridinium-based sensor as a fluorescent photoinduced electron transfer probe for proton detection modulated by anionic micelles.

A water-soluble fluorescent pH sensor of 9-amino-10-methylacridinium chromophore with the 2-(diethylamine)ethyl chain as a receptor shows an "off-on" response going from basic to acidic solution. Photoinduced electron transfer has been directly demonstrated to be the quenching mechanism by the observation of the long-lived acridinyl radical. The interaction of the protonated sensor with anionic micelles causes a significant increase in the detection sensitivity of pH.

Reusable optical sensing microplate for hydrogen peroxide using a fluorescent photoinduced electron transfer probe (HP Green)

The first optical sensing microplate is described for repetitive high-throughput screening of hydrogen peroxide (H2O2) in solutions of physiological pH. The fluorescent probe HP Green which shows a photoinduced electron transfer (PET) was incorporated in a polyurethane polymer matrix (type D4) on the bottom of microplate wells. Enzymatic oxidation of H2O2 Green in the presence of horseradish peroxidase (HRP) enables the determination of H2O2 concentrations up to 300μM with a LOD as low as 8.0μM. The calibration plots show high correlation coefficients (R2=0.996). The new sensing microplate (V-bottom) enables parallel and rapid (96 samples in 20min) detection of H2O2 with a standard fluorescence microplate reader. The response time is in the range of 10–20min, and the response is reversible. Finally, the H2O2 sensing microplate has successfully been applied to the determination of H2O2 in colored cell nutrition medium as real biological sample with a strong matrix effect.

Intramolecular Distances and Dynamics from the Combined Photon Statistics of Single-Molecule FRET and Photoinduced Electron Transfer

Single-molecule Förster resonance energy transfer (FRET) and photoinduced electron transfer (PET) have developed into versatile and complementary methods for probing distances and dynamics in biomolecules. Here we show that the two methods can be combined in one molecule to obtain both accurate distance information and the kinetics of intramolecular contact formation. In a first step, we show that the fluorescent dyes Alexa 488 and Alexa 594, which are frequently used as a donor and acceptor for single-molecule FRET, are also suitable as PET probes with tryptophan as a fluorescence quencher. We then performed combined FRET/PET experiments with FRET donor- and acceptor-labeled polyproline peptides. The placement of a tryptophan residue into the polyglycylserine tail incorporated in the peptides allowed us to measure both FRET efficiencies and the nanosecond dynamics of contact formation between one of the fluorescent dyes and the quencher. Variation of the linker length between the polyproline and the Alexa dyes and in the position of the tryptophan residue demonstrates the sensitivity of this approach. Modeling of the combined photon statistics underlying the combined FRET and PET process enables the accurate analysis of both the resulting transfer efficiency histograms and the nanosecond fluorescence correlation functions. This approach opens up new possibilities for investigating single biomolecules with high spatial and temporal resolution.

Systematic evaluation of fluorescence correlation spectroscopy data analysis on the nanosecond time scale

Signal fluctuations in a fluorescence time trace on nanosecond time scales can be induced by specific quenching interactions that report on the dynamics of biomolecules. Fluorescence correlation spectroscopy is an analysis tool to investigate dynamic processes on time scales from pico- to milliseconds or longer. Under certain conditions, e.g. in a solvent of high viscosity, a fluorescence labeled dynamic biomolecule yields multiple independent correlation decays due to rotational and translational diffusion, fluorescence quenching interactions, and fluorophore photophysics. We compared parameter estimation for FCS data with multiple correlation decays by dynamical fingerprint analysis and by the non-linear Levenberg-Marquardt fitting procedure and identified conditions for which dynamical fingerprint analysis can be of advantage. In this context we identified a previously unrecognized photophysical process in ATTO655 that introduces fluorescence intermittency on nanosecond time scales that is absent in MR121. The optimized fitting procedure is used to resolve the viscosity dependence of fluorescence quenching for photoinduced electron transfer probes.

A Theoretical Investigation of Two Typical Two‐Photon pH Fluorescent Probes

Intracellular pH plays an important role in many cellular events, such as cell growth, endocytosis, cell adhesion and so on. Some pH fluorescent probes have been reported, but most of them are one-photon fluorescent probes, studies about two-photon fluorescent probes are very rare. In this work, the geometrical structure, electronic structure and one-photon properties of a series of two-photon pH fluorescent probes have been theoretically studied by using density functional theory (DFT) method. Their two-photon absorption (TPA) properties are calculated using the method of ZINDO/sum-over-states method. Two types of two-photon pH fluorescent probes have been investigated by theoretical methods. The mechanisms of the Photoinduced Charge Transfer (PCT) probes and the Photoinduced Electron Transfer (PET) probes are verified specifically. Some designed strategies of good two-photon pH fluorescent probes are suggested on the basis of the investigated results of two mechanisms. For the PCT probes, substituting a stronger electron-donating group for the terminal methoxyl group is an advisable choice to increase the TPA cross section. For the PET probes, the TPA cross sections increase upon protonation.

A New Fluorescent PET Probe for Hydrogen Peroxide and its Use in Enzymatic Assays for L‐Lactate and D‐Glucose

We present a new probe for the determination of hydrogen peroxide (HP). It is based on the yellow fluorophore 4-amino-1,8-napththalimide, coupled to p-anisidine (as a redox-active group) to form a probe that is based on photoinduced electron transfer (PET). The preparation of the probe (which we refer to as "HP Green") was accomplished in four steps with good yield. Its fluorescence is independent of pH in the physiological range and quenched by a PET process that occurs between the p-anisidine redox moiety and the naphthalimide luminophore. If the p-anisidine group is oxidized by HP, PET is suppressed and fluorescence intensity is strongly increased. Addition of horseradish peroxidase (HRP) enhances the oxidation of HP Green and further improves the detection limit of HP. The use of HRP and HP Green enables the determination of HP concentration in a range of 0.1 to 5 μM, with a limit of detection (LOD) as low as 64 nM (16 pmol per well in microtiter plates). HP Green and HRP also enable sensitive enzymatic assays of oxidase substrates in a kinetic format, as shown for L-lactate and D-glucose. L-Lactate concentration can be rapidly determined between 0.5 and 10 μM after 6 minutes of incubation at 30 °C, with an LOD of 164 nm (41 pmol per well). This LOD is more than sixfold lower than that of the best commercial assays for lactate. The detection range for D-glucose is 2 to 30 μm, and the LOD is 644 nM (161 pmol per well). These are among the lowest concentrations detectable for oxidase-based assays. The hexanoic acid moiety in HP Green may be further used to immobilize the probe in order to obtain sensor layers for continuous assays.

Electronically tuned 1,3,5-triarylpyrazolines as Cu(i)-selectivefluorescent probes

We have prepared and characterized a Cu(i)-responsive fluorescent probe, constructed using a large tetradentate, 16-membered thiazacrown ligand ([16]aneNS(3)) and 1,3,5-triaryl-substituted pyrazoline fluorophores. The fluorescence contrast ratio upon analyte binding, which is mainly governed by changes of the photoinduced electron transfer (PET) driving force between the ligand and fluorophore, was systematically optimized by increasing the electron withdrawing character of the 1-aryl-ring, yielding a maximum 50-fold fluorescence enhancement upon saturation with Cu(i) in methanol and a greater than 300-fold enhancement upon protonation with trifluoroacetic acid. The observed fluorescence increase was selective towards Cu(i) over a broad range of mono- and divalent transition metal cations. Previously established Hammett LFERs proved to be a valuable tool to predict two of the PET key parameters, the acceptor potential (E(A/A(-)) and the excited state energy DeltaE(00), and thus to identify a set of pyrazolines that would best match the thermodynamic requirements imposed by the donor potential E(D(+)/D) of the thiazacrown receptor. The described approach should be applicable for rationally designing high-contrast pyrazoline-based PET probes selective towards other metal cations.