Resonance Energy Transfer System Research Articles

Electrochemiluminescent aptasensor based on resonance energy transfer system between CdTe quantum dots and cyanine dyes for the sensitive detection of Ochratoxin A

In this work, an innovative aptasensor based on electrochemiluminescence resonance energy transfer (ECL-RET) from CdTe quantum dots (QDs) to a cyanine dye (Cy5) fluorophore for the determination of Ochratoxin A (OTA) was fabricated. A strong cathodic ECL emission was obtained by the CdTe QDs modified glassy carbon electrode (GCE). After the immobilization with the capture DNA (cDNA) and the sequential hybridization with the probe DNA-modified Cy5 (pDNA, the aptamer of OTA), the ECL signal enhanced obviously through the ECL-RET. Meanwhile, the spectrum- and distance-related ECL enhancement effect was investigated. When the target OTA was in the presence, the pDNA-Cy5 molecules were released from the electrode surface owing to the specific interaction between OTA and aptamer, resulting in an evident decrease of ECL signal. Under optimal conditions, the developed aptasensor displayed the linear response toward OTA in the wide range of 0.0005–50 ng/mL with a low detection limit of 0.17 pg/mL. With the excellent selectivity, stability and repeatability, the strategy provided an efficient and universal method for the sensitive detection of target in practical application.

Analysis of Protein-Protein Interaction in a Single Live Cell by Using a FRET System Based on Genetic Code Expansion Technology.

Hsp70 is known to directly bind to Bax for suppression of apoptosis. However, mechanisms on how Bax is dissociated from its complex with Hsp70 during apoptosis remain largely unknown. In the current study, we developed the efficient fluorescence resonance energy transfer (FRET) system which consisted of Hsp70-YFP and fluorescent amino acid (ANAP)-incorporated Bax, which was generated by using genetic code expansion technology, and applied the FRET system to elucidate mechanisms on how apoptosis-inducing substances dissociate Bax from Hsp70. Time-dependent analysis of single live cell images showed that Bax activators binding to Bax trigger sites inhibited the Bax-Hsp70 interaction but a Bax activator, which blocks phosphorylation of S184 via binding to the C-terminal S184 site, did not affect this interaction. Additionally, an inhibitor for Hsp70-Hsp40 interaction blocked the Bax-Hsp70 interaction. Furthermore, p53 activators promoted the dissociation of Bax from Hsp70 by reactivating p53 which disrupted the Bax-Hsp70 interaction. We also found that death ligands and a Bcl-2 inhibitor enhanced the dissociation of Bax from Hsp70 by activating activator BH3-only proteins. Results from this effort suggest that FRET systems consisting of the ANAP-incorporated protein and the YFP fusion protein will be valuable tools to gain an understanding of other types of protein-protein interactions.

Intermolecular distance measurement with TNT suppressor on the M13 bacteriophage-based F\xf6rster resonance energy transfer system

An M13 bacteriophage-based Förster resonance energy transfer (FRET) system is developed to estimate intermolecular distance at the nanoscale using a complex of CdSSe/ZnS nanocrystal quantum dots, genetically engineered M13 bacteriophages labeled with fluorescein isothiocyanate and trinitrotoluene (TNT) as an inhibitor. In the absence of trinitrotoluene, it is observed that a significant spectral shift from blue to green occur, which represents efficient energy transfer through dipole-dipole coupling between donor and acceptor, or FRET-on mode. On the other hand, in the presence of trinitrotoluene, the energy transfer is suppressed, since the donor-to-acceptor intermolecular distance is detuned by the specific capturing of TNT by the M13 bacteriophage, denoted as FRET-off mode. These noble features are confirmed by changes in the fluorescence intensity and the fluorescence decay curve. TNT addition to our system results in reducing the total energy transfer efficiency considerably from 16.1% to 7.6% compared to that in the non-TNT condition, while the exciton decay rate is significantly enhanced. In particular, we confirm that the energy transfer efficiency satisfies the original intermolecular distance dependence of FRET. The relative donor-to-acceptor distance is changed from 70.03 Å to 80.61 Å by inclusion of TNT.

Lignosulfonate: A Convenient Fluorescence Resonance Energy Transfer Platform for the Construction of a Ratiometric Fluorescence pH-Sensing Probe.

Lignin is a kind of natural fluorescent polymer material. However, the application based on the fluorescent property of lignin was rarely reported. Herein, a noncovalent lignin-based fluorescence resonance energy transfer (FRET) system was readily constructed by physical blending method with spirolactam rhodamine B (SRhB) and lignosulfonate (LS) as the acceptor and donor groups, respectively. The FRET behavior, self-assembly, and energy transfer mechanism of SRhB/LS composite were systematically studied. It was demonstrated that LS could be used as a convenient aptamer as energy donor to construct water-soluble ratiometric sensors because of its inherent property of intramicelle energy transfer cascades. Our results not only present a facile and general strategy for producing lignin-based functional material but also provide a fundamental understanding about lignin fluorescence to promote the functional and high-valued applications of lignin fluorescence characteristic.

A novel electrochemiluminescence resonance energy transfer system for simultaneous determination of two acute myocardial infarction markers using versatile gold nanorods as energy acceptors.

A novel electrochemiluminescence resonance energy transfer (ECL-RET) system using versatile gold nanorods as energy acceptors was introduced into the ECL biochemical analysis. A spatial- and potential-resolved platform coupled with the ECL-RET strategy was developed for simultaneous determination of two acute myocardial infarction markers.

An efficient fluorescence resonance energy transfer system from quantum dots to graphene oxide nano sheets: Application in a photoluminescence aptasensing probe for the sensitive detection of diazinon

In this paper a new aptasensor with high-sensitivity, and high-specificity for detection of diazinon, was discovered, based on fluorescence resonance energy transfer (FRET) between quantum dot (QD) as a donor and graphene oxide (GO) as an acceptor. l-cysteine capped CdS QDs/DF20 aptamer bioconjugates were successfully synthesized. GO was also attached to aptamers and photoluminescence quenching was obtained through FRET. By adding target diazinon to the bioconjugates containing GO, photoluminescence recovery was detected due to detachment of GO from the aptamer as a result of the difference in affinity for the aptamer. The detection limit of the biosensor was 0.13 nM and the linearity was maintained from 1.05 to 206 nM. Other pesticides and herbicides did not contribute to photoluminescence recovery due to lack of binding affinity for the aptamers, which demonstrates the selectivity of the biosensor. The results show the applicability of the aptasensor for monitoring diazinon in environmental and agricultural samples.

Achieving highly sensitive detection of Cu2+ based on AIE and FRET strategy in aqueous solution.

The aggregation-induced emission (AIE) luminogens are now showing strong potential in mimicking the energy donor of fluorescence resonance energy transfer (FRET) system. Herein, one highly efficient FRET system 1-NiR is successfully fabricated in aqueous solution based on an AIE active compound 1 and fluorescence dyes (Nile red (NiR)). 1 acts as the energy donor and NiR acts as the acceptor in the FRET system with the optimum concentrations ratio [1]/[NiR] = 100. Besides, the AIE(1) itself displays excellent selectivity for Cu2+ ions at 525 nm with the detection limit of 1.32 × 10-7 M. While through the FRET system of 1-NiR system, the detection limit of Cu2+ can be further decreased to 9.12 nM by monitoring the fluorescence at 630 nm. As a result, using an AIE probe to detect Cu2+ based on FRET mechanism is a promising strategy.

RNA Structure and Cellular Applications of Fluorescent Light‐Up Aptamers

AbstractThe cellular functions of RNA are not limited to their role as blueprints for protein synthesis. In particular, noncoding RNA, such as, snRNAs, lncRNAs, miRNAs, play important roles. With increasing numbers of RNAs being identified, it is well known that the transcriptome outnumbers the proteome by far. This emphasizes the great importance of functional RNA characterization and the need to further develop tools for these investigations, many of which are still in their infancy. Fluorescent light‐up aptamers (FLAPs) are RNA sequences that can bind nontoxic, cell‐permeable small‐molecule fluorogens and enhance their fluorescence over many orders of magnitude upon binding. FLAPs can be encoded on the DNA level using standard molecular biology tools and are subsequently transcribed into RNA by the cellular machinery, so that they can be used as fluorescent RNA tags (FLAP‐tags). In this Minireview, we give a brief overview of the fluorogens that have been developed and their binding RNA aptamers, with a special focus on published crystal structures. A summary of current and future cellular FLAP applications with an emphasis on the study of RNA–RNA and RNA–protein interactions using split‐FLAP and Förster resonance energy transfer (FRET) systems is given.

RNA Structure and Cellular Applications of Fluorescent Light-Up Aptamers.

The cellular functions of RNA are not limited to their role as blueprints for protein synthesis. In particular, noncoding RNA, such as, snRNAs, lncRNAs, miRNAs, play important roles. With increasing numbers of RNAs being identified, it is well known that the transcriptome outnumbers the proteome by far. This emphasizes the great importance of functional RNA characterization and the need to further develop tools for these investigations, many of which are still in their infancy. Fluorescent light‐up aptamers (FLAPs) are RNA sequences that can bind nontoxic, cell‐permeable small‐molecule fluorogens and enhance their fluorescence over many orders of magnitude upon binding. FLAPs can be encoded on the DNA level using standard molecular biology tools and are subsequently transcribed into RNA by the cellular machinery, so that they can be used as fluorescent RNA tags (FLAP‐tags). In this Minireview, we give a brief overview of the fluorogens that have been developed and their binding RNA aptamers, with a special focus on published crystal structures. A summary of current and future cellular FLAP applications with an emphasis on the study of RNA–RNA and RNA–protein interactions using split‐FLAP and Förster resonance energy transfer (FRET) systems is given.

A Ratiometric Two-Photon Fluorescent Cysteine Probe with Well-Resolved Dual Emissions Based on Intramolecular Charge Transfer-Mediated Two-Photon-FRET Integration Mechanism.

The development of an efficient ratiometric two-photon fluorescence imaging probe is crucial for in situ monitoring of biothiol cysteine (Cys) in biosystems, but the current reported intramolecular charge transfer (ICT)-based one suffers from serious overlap between the shifted emission bands. To address this issue, we herein for the first time constructed an ICT-mediated two-photon excited fluorescence resonance energy transfer (TP-FRET) system consisting of a two-photon fluorogen benzo[ h]chromene and a Cys-responsive benzoxadiazole-analogue dye. Different from a previous mechanism that utilized single two-photon fluorogen to acquire a ratiometric signal, ICT was used to switch on the TP-FRET process of the energy transfer dyad by eliciting an absorption shift of benzoxadiazole with Cys to modulate the spectral overlap level between benzo[ h]chromene emission and benzoxadiazole absorption, resulting in two well-separated emission signal changes with large emission wavelength shift (120 nm), fixed two-photon excitation maximum (750 nm), and significant variation in fluorescence ratio (over 36-fold). Therefore, it can be successfully employed to ratiometrically visualize Cys in HeLa cells and liver tissues. Importantly, this new ICT-mediated TP-FRET integration mechanism would be convenient for designing ratiometric two-photon fluorescent probes with two well-resolved emission spectra suitable for high resolution two-photon fluorescence bioimaging.

The DNA Duplex as an Aqueous One-Dimensional Soft Crystal Scaffold for Photochemistry

Abstract In this account, we demonstrate that DNA duplex is an ideal scaffold for photochemistry, particularly for comparison of photochemical theory with experiments. The well-defined structure of a DNA duplex can be regarded as an aqueous one-dimensional soft crystal composed of a chromophore-like base-pair assembly. When any base pair in the duplex is replaced with a chromophore, orientation, distance, and association number of chromophores can be precisely controlled. We have developed a new methodology for introduction of chromophores into DNA duplexes using d-threoninol. By using the DNA duplex as a scaffold, experiments on exciton interactions of chromophore assemblies can be compared with molecular exciton theory. A fluorescent resonance energy transfer (FRET) system was also constructed by introducing donor pyrene and acceptor perylene into the DNA duplex using d-threoninol monomers. Using this system, we demonstrated orientation-dependent FRET. We found that theories on both exciton interaction and FRET qualitatively coincide with experimental data and revealed the limitation of the point-dipole approximation. We also evaluated the intrinsic quantum yield of photodimerization of stilbene derivatives by suppressing a side reaction. We propose that there is a correlation of quantum yield of photodimerization with the energy gap of HOMO or LUMO, a hypothesis that deserves theoretical investigation.

A dual-functional probe for quantification and imaging of intracellular telomerase

The analysis of telomerase is of great significance for the study and diagnosis of cancer. In this work, a dual-functional Au NP probe for the quantification and imaging of intracellular telomerase was prepared. The probe contained an elemental tag (1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid (Tb-DOTA)) for accurate quantification using inductively coupled plasma mass spectrometry (ICP-MS) and a fluorescent tag (FAM) which constructed the fluorescence resonance energy transfer (FRET) system with Au NPs for fluorescence imaging. After the probe entered into the Hela cells, the telomerase primer was extended with TTAGGG repeats in the presence of the intracellular telomerase, resulting in turning on the fluorescence of FAM and releasing the Tb-DOTA. Thus, the intracellular telomerase could be in situ visualized by the released FAM with fluorescence imaging and quantified by the released Tb-DOTA with ICP-MS detection. The average amount of telomerase in one Hela cell was quantified to be 3.1 × 10−11 IU. The proposed method was demonstrated with good potential in the study of telomerase and the cancer diagnosis, providing a new strategy for the analysis of intracellular biomarkers by ICP-MS.

Highly Sensitive Detection of Melamine Based on the Fluorescence Resonance Energy Transfer between Conjugated Polymer Nanoparticles and Gold Nanoparticles.

Adding melamine as additives in food products will lead to many diseases and even death. However, the present techniques of melamine detection require time-consuming steps, complicated procedures and expensive analytical apparatus. The fluorescent assay method was facile and highly sensitive. In this work, a fluorescence resonance energy transfer (FRET) system for melamine detection was constructed based on conjugated polymer nanoparticles (CPNs) and gold nanoparticles (AuNPs). The energy transfer efficiency is up to 82.1%, and the system is highly selective and sensitive to melamine detection with a lower detection limit of 1.7 nmol/L. Moreover, the interaction mechanism was explored. The results showed that the fluorescence of CPNs were firstly quenched by AuNPs, and then restored after adding melamine because of reducing FRET between CPNs and AuNPs. Lastly, the proposed method was carried out for melamine detection in powdered infant formula with satisfactory results.

Electrochemiluminescence Energy Resonance Transfer System between RuSi Nanoparticles and Hollow Au Nanocages for Nucleic Acid Detection.

This paper describes an electrochemiluminescence resonance energy transfer (ECL-RET) system using Ru(bpy)32+-doped silica nanoparticles (RuSi NPs) as the ECL donor and hollow Au nanocages as the ECL acceptor. Tetrahedron DNA (TD) was used to construct the biosensing interface and control the distance (4.8 nm) between the ECL donor-acceptor pairs. The surface plasmon resonance (SPR) nanostructures, Au nanocages were assembled via the hairpin based sandwich assay. Due to the well overlap between the plasmon absorption spectrum of Au nanocages (628 nm) and the ECL emission spectrum of RuSi NPs (620 nm), high efficient energy transfer could occur. Subsequent cyclic DNA amplification further increased the binding amount of Au nanocages. Since the ECL inhibition is closely related with the binding amount of Au nanocages, a general "signal-off" ECL bioassay could thus be tailored with high sensitivity. At the optimized conditions, this ECL-RET system performed well with great stability and repeatability for nucleic acid detection in the range from 1.0 fM to 10 pM. This work manifested the great promise of hollow Au nanocages for an ECL-RET biosensor that to the best of our knowledge has not been reported. We believe that it could inspire more interest in the design and development of numerous other SPR nanostructures for advanced ECL-RET biosensors.

Construction of a Cytosine-Adjusted Electrochemiluminescence Resonance Energy Transfer System for MicroRNA Detection.

The cytosines in cluster-nucleation sequences play a vital role in the formation of silver nanoclusters (Ag NCs). Here, an innovative electrochemiluminescence (ECL) resonance energy transfer (RET) sensing system was developed using CdS quantum dots (QDs) as ECL donor and Ag NCs as ECL acceptor. Modulation of the number of cytosines in the cluster-nucleation sequences allowed tuning of Ag NCs absorption bands to match with the ECL emission spectrum of CdS QDs, yielding effective ECL-RET. The sensitivity of detection was improved by dual-target recycling amplification based on duplex-specific nuclease (DSN) and catalytic hairpin assembly. In the presence of target microRNA-21 (miRNA-21), DSN selectively cleaved the complementary DNA section (S1), resulting in the release of the transduction section (S2) and the reuse of miRNA-21 in the next recycling amplification. Interaction of the stem-loop structure of the DNA1 segment (H1) on CdS QDs-modified electrode with S2 led to the opening of the hairpin structure of H1 and the formation of H1:S2 duplex. Then, hairpin DNA2 encapsulated Ag NCs hybridized with the remaining single-stranded DNA segment of H1, and the S2 strand was replaced. Finally, the dissociated S2 participated in subsequent reaction cycles, introducing Ag NCs to the electrode surface and leading to ECL signal quenching of the CdS QDs. The proposed sensor showed excellent performance in detecting miRNA-21 at a wide linear range from 1 fM to 100 pM. The practical application ability of the strategy was tested in HeLa cells with acceptable results, suggesting that the detection platform is a promising approach for disease diagnosis and molecular biology research.

Fluorometric lateral flow immunochromatographic zearalenone assay by exploiting a quencher system composed of carbon dots and silver nanoparticles

It is found that the fluorescence of carbon dots (CD) with an emission peak at 459nm is strongly quenched by silver nanoparticles (AgNPs) with their absorption peak at 430nm. The finding was applied in a fluorescence quenchometric lateral flow immunochromatographic assay for detection of zearalenone (ZEN) with CDs conjugated to ovalbumin (OVA) as donor signal probe and AgNP-Ab as acceptor signal probe. The assay has an LOD of 0.1μg·L-1 for ZEN. This is 10 times better than the respective "turn-off" AgNP-based LFIA. In case of cereal samples and their products, the LODs range from 1 to 2.5μg·kg-1. Only minor cross reactivity is found for fusarium toxins, and no cross-sensitivity for aflatoxin B1, T-2 mycotoxin, ochratoxin A, deoxynivalenol, and fumonisin B1. Theassay represents a simple, sensitive, and rapid tool for determination of ZEN in cereal samples and their products. Graphical abstract Schematic presentation of fluorescence quenching lateral flow immunochromatographic assay (FLFIA) based on carbon dots (CD) and silver nanoparticle (AgNP) fluorescence resonance energy transfer (FRET) system for the rapid high sensitive detection of zearalenone (ZEN) in cereal samples.

Fluorescent resonance energy transfer of organic fluorescent dyes with gold nanoparticles and their analytical application

We report five effective fluorescence resonance energy transfer (FRET) systems based on gold nanoparticles (AuNPs) and organic fluorescent dyes, including ionic [fluorescein sodium (FS) and Eosin B (EB)] and cationic [rhodamine 6G (Rh6G), acridine orange (AO), and safranine T (ST)] fluorescent dyes. The fluorescence intensity of the five FRET systems demonstrates that efficient quenching is possible. The quenching efficiencies of Rh6G and FS by FRET were nearly 100%, 89% for AO, 60% for EB, and 55% for ST. A series of UV absorbance spectra and fluorescence emission spectra were used to explain the mechanism of fluorescence quenching. We found that there were different degrees of overlap between the absorption spectrum of the AuNPs and the emission spectrum of fluorescence dyes. This outcome indicates that highly efficient FRET is the possible mechanism of fluorescence quenching. We applied the FRET system to establish a sensitive and simple strategy for the determination of mercury (Hg2+). The maximum excitation was at 523 nm (λex = 523 nm). The enhanced fluorescence intensity at 551 nm was proportional to the concentration of Hg2+ with a range of 0.44–100 nmol L−1. The detection limit was 0.13 nM. The linear regression equation was ΔF = 27.05c (nmol L−1) − 79.88, and the regression coefficient was 0.9954. The proposed method has high sensitivity and convenience and does not require complex and expensive instruments.

Engineering Persistent Luminescence Nanoparticles for Biological Applications: From Biosensing/Bioimaging to Theranostics.

Persistent luminescence nanoparticles (PLNPs) are unique optical materials emitting long-lasting luminescence after ceasing excitation. Such a unique optical feature allows luminescence detection without constant external illumination to avoid the interferences of autofluorescence and scattering light from biological fluids and tissues. Besides, near-infrared (NIR) PLNPs have advantages of deep penetration and the reactivation of the persistent luminescence (PL) by red or NIR light. These features make the application of NIR-emitting PLNPs in long-term bioimaging no longer limited by the lifetime of PL. To take full advantage of PLNPs for biological applications, the versatile strategies for bridging PLNPs and biological system become increasingly significant for the design of PLNPs-based nanoprobes. In this Account, we summarize our systematic achievements in the biological applications of PLNPs from biosensing/bioimaging to theranostics with emphasizing the engineering strategies for fabricating specific PLNPs-based nanoprobes. We take surface engineering and manipulating energy transfer as the major principles to design various PLNPs-based nanoprobes based on the nature of interactions between nanoprobes and targets. We have developed target-induced formation or interruption of fluorescence resonance energy transfer systems for autofluorescence-free biosensing and imaging of cancer biomarkers. We have decorated single or dual targeting ligands on PLNPs for tumor-targeted imaging, and integrated other modal imaging agents into PLNPs for multimodal imaging. We have also employed specific functionalization for various biomedical applications including chemotherapy, photodynamic therapy, photothermal therapy, stem cells tracking and PL imaging-guided gene therapy. Besides, we have modified PLNPs with multiple functional units to achieve challenging metastatic tumor theranostics. The proposed design principle and comprehensive strategies show great potential in guiding the design of PLNPs nanoprobes and promoting further development of PLNPs in the fields of biological science and medicine. We conclude this Account by outlining the future directions to further promote the practical application of PLNPs. The novel protocols for the synthesis of small-size, monodisperse, and water-soluble PLNPs with high NIR PL intensity and superlong afterglow are the vibrant directions for the biomedical applications of PLNPs. In-depth theories and evidence on luminescence mechanism of PLNPs are highly desired for further improvement of their luminescence performance. Furthermore, other irradiations without tissue penetrating depth limit, such as X-ray, are encouraged for use in energy storage and re-excitation of PLNPs, enabling imaging in deep tissue in vivo and integrating other X-ray sensitized theranostic techniques such as computed tomography imaging and radiotherapy. Last but not least, PLNPs-based nanoprobes and the brand new hybrids of PLNPs with other nanomaterials show a bright prospect for accurate diagnosis and efficient treatment of diseases besides tumors.

A Bodipy-bearing pillar[5]arene for mimicking photosynthesis: Multi-fluorophoric light harvesting system

Herein, we submitted to the original synthesis, characterization, energy transfer mechanism of the Bodipy-bearing pillar[5]arene Bodipy and its reactants by employing of infrared, 1H, 11B, 13C, 19F-NMRs, UV–vis, fluorescence spectroscopy, melting point apparatus, CHN elemental analysis and mass spectroscopy. Preliminary UV–vis, fluorescence and excitation measurements were carried out in CH2Cl2 and the results revealed an effective fluorescence resonance energy transfer (FRET) system based on the interaction of pillar[5]arene and Bodipy derivative. εmax of target molecule reached to a maximum value and it was found as 955000 M−1 cm−1. This fluorescent macromolecule worked well for mimicking a light harvesting system with an energy transfer efficiency up to 92%.

A novel electrochemiluminescence resonance energy transfer system of luminol-graphene quantum dot composite and its application in H2O2 detection

Luminol-nitrogen doped graphene quantum dot (luminol-NGQDs) nanocomposite was synthesized and a novel electrochemiluminescence resonance energy transfer (ECL-RET) process occurred between luminol as the donor and NGQDs as the acceptor in the composite. This ECL-RET effect helped luminol-NGQDs composite produced an anodic ECL signal without coreactants. The ECL-RET mechanism was also studied based on the fluorescence spectra, the ultraviolet–visible absorption spectra and the electrochemiluminescence (ECL) spectra. Based on the significant sensitization effect of hydrogen peroxide on luminol-NGQDs ECL signal, an ECL method for the sensitive determination of hydrogen peroxide was established and then applied to the detection of hydrogen peroxide in water samples.