Fluorescence Resonance Energy Transfer System Research Articles

Binomial distribution-based quantitative measurement of multiple-acceptors fluorescence resonance energy transfer by partially photobleaching acceptor

We report that binomial distribution depending on acceptor photobleaching degree can be used to characterize the proportions of various kinds of FRET (Fluorescence Resonance Energy Transfer) constructs resulted from partial acceptor photobleaching of multiple-acceptors FRET system. On this basis, we set up a rigorous quantitation theory for multiple-acceptors FRET construct named as Mb-PbFRET which is not affected by the imaging conditions and fluorophore properties. We experimentally validate Mb-PbFRET with FRET constructs consisted of one donor and two or three acceptors inside living cells on confocal and wide-field microscopes.

Study on the fluorescence resonance energy transfer between CdS quantum dots and Eosin Y.

Water-soluble CdS quantum dots (QDs) were prepared using mercaptoacetic acid (TGA) as the stabilizer in an aqueous system. A fluorescence resonance energy transfer (FRET) system was constructed between water-soluble CdS QDs (donor) and Eosin Y (acceptor). Several factors that impacted the fluorescence spectra of the FRET system, such as pH (3.05-10.10), concentration of Eosin Y (2-80 mg/L) and concentration of CdS QDs (2-80 mg/L), were investigated and refined. Donor-to-acceptor ratios, the energy transfer efficiency (E) and the distance (r) between CdS QDs and Eosin Y were obtained. The results showed that a FRET system could be established between water-soluble CdS QDs and Eosin Y at pH 5.0; donor-to-acceptor ratios demonstrated a 1: 8 proportion of complexes; the energy transfer efficiency (E) and the distance (r) between the QDs and Eosin Y were 20.07% and 4.36 nm,respectively.

A carbon dot based biosensor for melamine detection by fluorescence resonance energy transfer

In this paper, we constructed a fluorescence resonance energy transfer (FRET) system between amino-functionalized carbon dots (C-dots) and gold nanoparticles (AuNPs). In this system, C-dots were treated as energy donors, while AuNPs were treated as energy acceptors. We optimized some important factors including incubation time, AuNPs concentration and media pH, which would affect the efficiency of the FRET system. Under the optimized experimental conditions, melamine could be detected based on fluorescence intensity of C-dots. We could get a linear relationship between 50nM and 500nM and the detection limit was 36nM. The proposed method was applied to the determination of melamine in milk samples with satisfactory results. Compared with previous reports, the proposed method manifested great advantages including high sensitivity, short analysis time, low cost and ease of operation.

Development of Ultrasound-switchable Fluorescence Imaging Contrast Agents based on Thermosensitive Polymers and Nanoparticles.

In this work we first introduced a recently developed high-resolution, deep-tissue imaging technique, ultrasound-switchable fluorescence (USF). The imaging principles based on two types of USF contrast agents were reviewed. To improve USF imaging techniques further, excellent USF contrast agents were developed based on high-performance thermoresponsive polymers and environment-sensitive fluorophores. Herein, such contrast agents were synthesized and characterized with five key parameters: (1) peak excitation and emission wavelengths (λex and λem), (2) the fluorescence intensity ratio between on and off states (IOn/IOff), (3) the fluorescence lifetime ratio between on and off states (τOn/τOff), (4) the temperature threshold to switch on fluorophores (Tth), and (5) the temperature transition bandwidth (TBW). We mainly investigated fluorescence intensity and lifetime changes of four environment-sensitive dyes [7-(2-Aminoethylamino)-N,N-dimethyl-4-benzofurazansulfonamide (DBD-ED), St633, Sq660, and St700] as a function of temperature, while the dye was attached to poly(N-isopropylacrylamide) linear polymers or encapsulated in nanoparticles. Six fluorescence resonance energy transfer systems were invented in which both the donor (DBD-ED or ST425) and the acceptor (Sq660) were adopted. Our results indicate that three Förster resonance energy transfer systems, where both IOn/IOff and τOn/τOff are larger than 2.5, are promising for application in future surface tissue bioimaging by USF technique.

Dual fluorescence resonance energy transfer assay between tunable upconversion nanoparticles and controlled gold nanoparticles for the simultaneous detection of Pb2+ and Hg2+

In this work, we presented a novel dual fluorescence resonance energy transfer (FRET) system for the simultaneous detection of Pb2+ and Hg2+. This system employed two color upconversion nanoparticles (UCNPs) as the donors, and controlled gold nanoparticles (AuNPs) as the acceptors. The two donor–acceptor pairs were fabricated by hybridizing the aptamers and their corresponding complementary DNA. Thus, the green and red upconversion fluorescence could be quenched because of a good overlap between the UCNPs fluorescence emission and the AuNPs absorption spectrum. In the presence of Pb2+ and Hg2+, the aptamers preferred to bind to their corresponding analytes and formed a G-quadruplexes structure for Pb2+ and the hairpin-like structure for Hg2+. As a result, the dual FRET was disrupted, and the green and red upconversion fluorescence was restored. Under optimized experimental conditions, the relative fluorescence intensity increased as the metal ion concentrations were increased, allowing for the quantification of Pb2+ and Hg2+. The relationships between the fluorescence intensity and plotting logarithms of ion concentrations were linear in the range from 0.1 to 100nM for Pb2+ and 0.5 to 500nM for Hg2+, and the detection limits of Pb2+ and Hg2+ were 50pM and 150pM, respectively. As a practical application, the aptasensor was used to monitor Pb2+ and Hg2+ levels in naturally contaminated samples and human serum samples. Ultimately, this type of dual FRET could be used to detect other metal ions or contaminants in food safety analysis and environment monitoring.

Artificial Light‐Harvesting Material Based on Self‐Assembly of Coordination Polymer Nanoparticles

Artificial light‐harvesting antenna materials as potential mimics for photosynthetic systems have attracted intense attention recently. Herein, a new modular approach to construct light‐harvesting material, which involves the self‐assembly of coordination polymer nanoparticles (CPNs) at room temperature, is presented. Fluorescence resonance energy transfer (FRET) occurs between donor and acceptor molecules encapsulated in the CPNs, and the emission signal of acceptor is amplified significantly. To the best of our knowledge, this is the first example of artificial light‐harvesting material generated from biomolecule‐based coordination polymer nanoparticles. The modularity of the material makes it convenient to manipulate the system by changing the composite of CPNs and the type and amount of dyes confined, implying it is a general strategy. The material functions not only in fluid medium, but also in the form of solid state, which extends its application areas greatly. Furthermore, photocurrent generation can be realized by the dye‐encapsulated CPNs system upon irradiation with visible light, implying the potential usefulness in light‐energy conversion and photoelectronic applications. Besides, the creation of FRET system provides a platform to mimic dual‐channel logic gate at nanoscale level, which is beneficial to the construction of integrated logic devices with multiple functions.

Clustering protease domains for use in directed evolution of specificity (967.6)

The objective of this study is to determine the most successful protease domains for use in directed evolution experiments against substrates with novel amino acid sequences. Building on our previous work that created an in vivo expressible fluorescence resonance energy transfer (FRET) system for protease specificity determination, we have initiated a study of protease domain clustering for in vivo protease evolution against a single recognition sequence. Using backbone root‐mean‐square deviation (RMSD), buried active‐site surface area, secondary structure analysis, and hydrophobicity scores, we have clustered protease domains into groups with a likelihood of potential success for in vivo evolution against specific amino acid sequences of maximum length. Our results demonstrate that many protease domains are not amenable to evolution studies due to energetic concerns related to instability of secondary structure interactions around the active site. Additionally, many proteases exhibit active site recognition regions that are relatively small, which eliminate the potential to target these domains against longer, and more specific, amino acid sequences. Encouraging results were obtained for two types of protease domains that have the maximum likelihood for successful evolution studies. These protease domains will be used in fluorescence‐activated cell sorting (FACS) experiments and evolved against loop regions of signal transduction receptors using our FRET protease specificity substrates. Antibody conjugates of these domains will then be constructed to target these evolved proteases to specific cell types based on antigen availability on the cell surface.Grant Funding Source: Supported by NIH GM080691

Protein A Detection Based on Quantum Dots-Antibody Bioprobe Using Fluorescence Coupled Capillary Electrophoresis

In this report, fluorescence detection coupled capillary electrophoresis (CE-FL) was used to detect Protein A. Antibody was first labeled with Cy5 and then mixed with quantum dots (QDs) to form QDs-antibody bioprobe. Further, we observed fluorescence resonance energy transfer (FRET) from QDs donor to Cy5 acceptor. The bioprobe was formed and brought QDs and Cy5 close enough to allow FRET to occur. After adding protein A, the FRET system was broken and caused the FRET signal to decrease. Thus, a new method for the determination of protein A was proposed based on the FRET signal changes. This study provides a new trail of thought for the detection of protein.

Reversible photoswitching aggregation and dissolution of spiropyran-functionalized copolymer and light-responsive FRET process

Abstract Well-defined, reversibly light-responsive amphiphilic diblock copolymer grafted with spiropyran, was prepared by reversible addition–fragmentation chain transfer (RAFT) polymerization. The copolymer self-assembles into polymeric micelles in water and exhibits reversible dissolution and re-aggregation characteristics upon ultraviolet (UV) and visible (Vis)-light irradiation. The fluorescence response of spiropyran immobilized onto the copolymer was light switchable. When nitrobenzoxadiazolyl derivative (NBD) dyes are encapsulated into the core of the micelles, a reversible, light-responsive, dual-color fluorescence resonance energy transfer (FRET) system is constructed and processed, which is well regulated by alternatively UV/vis irradiation. We anticipate these photoswitchable and FRET lighting up nanoparticles will be useful in drug delivery and cell imaging or tracking synchronously.

Sensing of Hg+2 and Ag+ through a pH dependent FRET system: Fabrication of molecular logic gates

The fluorescence resonance energy transfer (FRET) from the donor 6,7-dimethoxy-2,4[1H,3H]-quinazolinedione (Q) to the acceptor lumichrome (L) exhibits strong pH dependency and in aqueous solution at pH 9 it is used to sense Hg+2 or Ag+ ions, selectively. QL hybrid forms organic nanoparticles through self-assembly at pH 9 as evident from TEM micrographs. The QL system can sense Hg+2 or Ag+ ion in aqueous medium at pH9 at a nanomolar concentration range using FRET pathway via complexation and nanoparticle formation, respectively. In the QL system, the ions can be easily distinguished with the help of UV–vis spectroscopic study or through the naked eye. We have successfully utilized the above metal ions for the fabrication of NOR and INHIBIT molecular logic gates using FRET of QL system as a signal transducer and Hg+2, Ag+ ion and pH as chemical activators.

Rhodamine B-Coated Gold Nanoparticles as Effective “Turn-on” Fluorescent Sensors for Detection of Zinc II Ions in Water

ABSTRACT We designed a fluorescence resonance energy transfer system consisting of fluorophore Rhodamine B and gold nanoparticles for sensing of zinc ions in aqueous solution. The electrostatic attraction between positively charged N-atoms in Rhodamine molecules and negatively charged citrate corona of gold nanoparticles led to substantial fluorescence quenching. However, the quenching is switched off in the presence of zinc ions and therefore the system can be used as an effective “turn-on” fluorescence sensor. UV-Vis absorption, fluorescence spectroscopy, and transmission electron microscopy were used for sensor evaluation. The approach of “turn-on” fluorescence has real potential for sensing metallic ions in water.

Selective turn‐on fluorescence assay of 6–thioguanine by using harmine‐modified silver nanoparticles

This article reports on a novel fluorescence resonance energy transfer (FRET) system between harmine and silver nanoparticles (AgNPs), in which harmine acts as the donor and AgNPs act as the acceptor. As a result of FRET, harmine fluorescence is quenched efficiently with a corresponding Stern-Volmer constant of 3.61 × 10(11) L/mol. It was found that upon addition of the anticancer drug, 6-thioguanine (6-TG), the fluorescence was recovered due to the competitive adsorption of this compound onto AgNPs. Based on this effect, a selective turn-on fluorescence sensor was developed for the determination of 6-TG. Under optimum conditions, the enhanced fluorescence intensity displays a linear relationship with the concentration of 6-TG in the range 1.5 × 10(-8) -7.5 × 10(-7) M with a detection limit of 9.7 nM. The developed method was applied to the determination of this drug in a pharmaceutical preparation and human plasma samples.

Fluorescence resonance energy transfer between NaYF4:Yb,Tm upconversion nanoparticles and gold nanorods: Near-infrared responsive biosensor for streptavidin

We represent a fluorescence resonance energy transfer (FRET) system using upconversion nanoparticles (UCNPs) and the gold nanorods (GNRs) as the energy donor–acceptor pair for directly determining streptavidin in near-infrared (NIR) region. NaYF4:Yb,Tm UCNPs, which had a strong emission at 800nm under 980-nm excitation, were adopted as the energy donor. The GNRs, which demonstrated strong surface plasmon absorption around 800nm, were chosen as acceptor to quench the 800nm emissions of the UCNPs. There had the spectral overlap between the emission of the donor nanoparticles (UCNPs) and the absorption of the acceptor nanoparticles (GNRs). This UCNP-based FRET system was then used to determine the amount of streptavidin. In this system, NaYF4:Yb,Tm UCNPs conjugated with biotin, while GNRs conjugated with streptavidin. When added GNRs into UCNPs, the streptavidin were preferred to bind with biotin and decreased spacing between the donor and acceptor NPs. Consequently, FRET occurred and a linear relationship between the luminescence quenching efficiency and the concentration of streptavidin was obtained. Owing to the aforementioned merits of UCNPs as an energy donor and the strong quenching ability of GNRs, satisfactory analytical performances have been acquired.

基于钙黄绿素-臧红T荧光共振能量转移检测六偏磷酸钠

In a medium of tris-HCl buffer (pH 8.5), calcein (donor) can react with safranine T (acceptor) efficiently to produce fluorescence resonance energy transfer (FRET). However, the addition of sodium hexametaphosphate (SHMP)destroyed the FRET system because of the electrostatic interaction between SHMP and the acceptor, which makes fluorescence change ratio between the increased fluorescence intensity of calcein and the reduced fluorescence of ST ( FD/ FA) has a good linear correlated with the concentration of SHMP. Based on the phenomena, a novel rapid FRET method for determination of sodium hexametaphosphate was developed. Under the optimized conditions, the linear range is 3.0×10 6–1.0×10 5mol/L, and the relative standard deviation (RSD) is 3.1% for determination of sodium hexametaphosphat (c = 6.0×10 6, n=11). The proposed method is of high selectivity, simple to operate and rapid detection, and is successfully applied to determine the concentration of SHMP in drink samples.

Fluorescence and fluorescence resonance energy transfer of N-(aminobutyl)-N-(ethylisoluminol) functionalized gold nanoparticles

Abstract The synthesis of N-(aminobutyl)-N-(ethylisoluminol) (ABEI) functionalized gold nanoparticles (ABEI-AuNPs) and their chemiluminescence properties were described in our previous work. Since the ABEI molecules coated on the surface of ABEI-AuNPs have the fluorescence (FL) activity, the FL property of these ABEI-AuNPs was studied in the present work. It was observed that the FL intensity of a single gold nanoparticle was 634 times brighter than that of one free ABEI molecule, even though 86% of the FL emission of ABEI molecules on the surface of ABEI-AuNPs was inhibited by gold cores through intra- and inter-particle quenching effects. Moreover, the photostability of ABEI-AuNPs was studied and 96% FL intensity of as-prepared ABEI-AuNPs solution remained after 5 min continuous excitation, almost 70% FL intensity remained after 45 min. ABEI-AuNPs exhibited much better resistance to photobleaching property than luminol-AuNPs. Furthermore, ABEI-AuNPs and acriflavine were designed as energy donor and energy acceptor, respectively, to construct a novel fluorescence resonance energy transfer (FRET) system. The efficient energy transfer from ABEI-AuNPs to acriflavine was observed. Taking advantage of excellent labeling property of gold nanomaterials, the FRET behavior between ABEI-AuNPs and acriflavine may find future applications in bioassays based on the FL detection.

A pH sensitive and selective ratiometric PAMAM wavelength-shifting bichromophoric system based on PET, FRET and ICT

A fluorescent poly(amidoamine)-based wavelength-shifting bichromophore was synthesized using divergent strategy and characterized photophysically. Novel compound was configured as a light-harvesting antenna where the systems surface is labelled with yellow-green emitting 4-(N-piperazinyl)-1,8-naphthalimide “donor” units capable of absorbing light and efficiently transferring the energy to a focal Rhodamine 6G “acceptor”. The energy transfer was calculated to 91%. Moreover, the 1,8-naphthalimide periphery of the system was designed on the “fluorophore–spacer–receptor” format able to act as a molecular fluorescence photoinduced electron transfer based probe. Due to the both effects, photoinduced electron transfer in the periphery and pH dependent spirolactam to ring-opening amide equilibrium of rhodamine core, novel bichromophoric system acts as a selective ratiometric pH fluorescence probe. Thus, the distinguishing features of light-harvesting (fluorescence resonance energy transfer) systems were successfully combined with the properties of classical photoinduced electron transfer and ring-opening sensing systems.

Determination of chromium(III) in aqueous solution using CePO4:Tb3+ nanocrystals in a fluorescence resonance energy transfer system

Trivalent chromium is an essential element required for normal carbohydrate, lipid and protein metabolism in humans and animals. This article describes an efficient fluorescence resonance energy transfer (FRET) system between CePO4 :Tb(3+) nanocrystals as the donor and chromium(III) as the acceptor. CePO4 :Tb(3+) nanocrystals were synthesized in aqueous solution, and characterized by transmission electron microscopy. Under optimum conditions, a linear calibration graph was obtained (R(2) = 0.996). The linear range and detection limit of chromium(III) were 0.01-2.2 μM, and 9.1 nM, respectively. The proposed method had a wide linear range and proved to be very sensitive, rapid and simple. Moreover, the method was applied successfully to the determination of chromium(III) in synthetic samples and tap water.

Homogenous detection of fumonisin B1 with a molecular beacon based on fluorescence resonance energy transfer between NaYF4: Yb, Ho upconversion nanoparticles and gold nanoparticles

In this work, we presented a new aptasensor for fumonisin B1 (FB1) based on fluorescence resonance energy transfer (FRET) between NaYF4: Yb, Ho upconversion fluorescent nanoparticles (UCNPs) and gold nanoparticles (AuNPs). The quenchers (AuNPs) were attached to the 5′ end of the molecular beacon (MB), and the donors (UCNPs) were attached to the 3′ end of the MB. In the absence of target DNA (DNA complementary to FB1 aptamers), the energy donors and acceptors were placed in close proximity, leading to quenching of the fluorescence of the UCNPs. Due to the combination of FB1 and FB1-specific aptamers, this caused some complementary DNA dissociating from the magnetic nanoparticles (MNPs). In the presence of the complementary DNA, the MBs underwent spontaneous conformational change and caused the UCNPs and AuNPs to detach from each other, resulting in restoration of the upconversion fluorescence. Therefore, the fluorescence of UCNPs was restored in a FB1 concentration-dependent manner, which was the basis of the FB1 quantification. The aptasensors showed a linear relationship from 0.01 to 100ngmL−1 for FB1 with a detection limit of 0.01ngmL−1 in an aqueous buffer. As a practical application, the aptasensor was used to monitor FB1 levels in naturally contaminated maize samples. The results were consistent with that of a classic ELISA method, indicating that the UCNPs–FRET aptasensor, which benefited from the near infrared excitation of NaYF4: Yb, Ho UCNPs, was effective for directly sensing FB1 in foodstuff samples without optical interference. This work also created the opportunity to develop aptasensors for other targets using this FRET system.

A ratiometric lysosomal pH chemosensor based on fluorescence resonance energy transfer

In this work, we presented a naphthalimide–rhodamine based fluorescence resonance energy transfer system (FRET) NR1 as a ratiometric and intracellular pH probe, in which 1,2,3-triazole was identified as an ideal bridge and biocompatibility. It could selectively monitor pH variations in methanol/HEPES solution at room temperature. When the pH changed from 6.20 to 2.00, both the fluorescence intensities at 580 nm and the intensity ratios, R (I580 nm/I538 nm) increased, which allowed the detection of pH changes by both normal fluorescence and ratiometric fluorescence methods. The observation is consistent with the increased FRET from the 1,8-naphthalimide (donor) to the ring-opened, colored form of rhodamine (acceptor). Moreover, as NR1 is lysosomal with low cytotoxicity, it will be helpful for investigating the pivotal role of H+ in a biological context, especially in lysosomes through direct intracellular imaging.

Detection of melamine based on the fluorescence resonance energy transfer between CdTe QDs and Rhodamine B

In this paper, we constructed the fluorescence resonance energy transfer (FRET) system between MPA-capped CdTe quantum dots (QDs) and Rhodamine B (RB) in the environment of cetyltrimethylammonium bromide (CTAB). In this system, CdTe QDs were treated as energy donors, whilst RB was treated as energy acceptor. CTAB was used to make the distance between the particles of CdTe QDs and RB closer to improve the efficiency of FRET system. We optimized some important factors which would affect the efficiency of FRET system. Under the optimized experimental conditions, melamine (MB) could be detected based on the fluorescence intensity changes of RB in the FRET system. We could get a linear relationship between the fluorescence intensity of RB and the concentration of MB in the range of 0.05–4.0μmol/L and the detection limit was 0.01μmol/L. The proposed method was applied for the determination of melamine in milk samples with satisfactory results.