Fluorescence Resonance Energy Transfer Efficiency Research Articles

Ultrafast synthesized monometallic nanohybrids as an efficient quencher and recognition antenna of upconversion nanoparticles for the detection of xanthine with enhanced sensitivity and selectivity

Upconversion nanoparticles (UCNPs) have shown great promise in bioanalytical applications owing to their excellent optical properties. Generally, most analytical applications are based on the fluorescence resonance energy transfer (FRET) principle to quench the fluorescence of UCNPs. However, each UCNP contains thousands of emission center ions, and most of them exceed the FRET critical distance, which hinders FRET efficiency and leads to a low signal-to-background ratio (SBR). Herein, a novel nanoprobe for the detection of Xanthine (XA) based on inner filter effects (IFE) and cascade signal amplification strategy was constructed by decorating UCNP with trypsin-chymotrypsin-stabilized gold nanoparticles-gold nanoclusters (Try-chy-AuNPs-AuNCs) monometallic nanohybrids. The Try-chy-AuNPs-AuNCs prepared by ultrafast (3 min) and green synthesis method have efficient upconversion fluorescence quenching ability (the quenching efficiency up to 90.9%), which can effectively improve the SBR of the probe, so as to improve the sensitivity. In addition, the Try-chy-AuNPs-AuNCs have a unique spatial structure, which can effectively prevent the interaction between large-size biothiol (glutathione) and the probe, thus improving its selectivity. Besides, combined with the excellent optical performance of UCNPs and cascaded signal amplification strategy, the sensitivity of the probe can be further improved. Under the optimized conditions, the linear response range of the probe was obtained from 0.05 to 50 μM, 0.06–80 μM and with the low detection limit of 22.6 nM and 26.3 nM for H2O2 and XA, respectively. Meanwhile, the developed method has been further applied to the detection of XA in human serum with satisfactory results.

Photoswitchable Thermally Activated Delayed Fluorescence Nanoparticles for “Double‐Check” Confocal and Time‐Resolved Luminescence Bioimaging

AbstractThermally activated delayed fluorescence (TADF) materials have attracted increasing attention due to their great potential in time‐resolved luminescence imaging (TRLI) in bioimaging. However, the triplet state (T1) of delay fluorescence is quenched readily by oxygen and the autofluorescence of biological systems remains inevitable interference in luminescence imaging, which often reduces signal‐to‐noise ratio during biodetection. To address this issue, the authors design a two‐component photoswitchable TADF polymeric nanoparticle (PDFPNs) by covalently incorporating a well‐designed TADF molecule (PDC‐DA) and a photochromic spiropyran derivative (SPMA). The as‐prepared PDFPNs with a unique luminescent “double‐check” strategy exhibit dual‐color fluorescence and variable fluorescence lifetime upon fluorescence resonance energy transfer (FRET) process through external lights irradiation. The obtained PDFPNs display the advantages of negligible oxygen‐sensitivity, high FRET efficiency, rapid photoresponsiveness, outstanding photoreversibility, and prominent long‐term fluorescence stability. The established PDFPNs show promising application in the high‐contrast dual‐color confocal and time‐resolved luminescence bioimaging in oxygenic living cells.

Probing functional conformation-state fluctuation dynamics in recognition binding between calmodulin and target peptide.

Conformational dynamics play a crucial role in protein functions. A molecular-level understanding of the conformational transition dynamics of proteins is fundamental for studying protein functions. Here, we report a study of real-time conformational dynamic interaction between calcium-activated calmodulin (CaM) and C28W peptide using single-molecule fluorescence resonance energy transfer (FRET) spectroscopy and imaging. Plasma membrane Ca-ATPase protein interacts with CaM by its peptide segment that contains 28 amino acids (C28W). The interaction between CaM and the Ca-ATPase is essential for cell signaling. However, details about its dynamic interaction are still not clear. In our current study, we used Cyanine3 labeled CaM (N-domain) and Dylight 649 labeled C28W peptide (N-domain) to study the conformational dynamics during their interaction. In this study, the FRET can be measured when the CaM-C28W complex is formed and only be observed when such a complex is formed. By using single-molecule FRET efficiency trajectory and unique statistical approaches, we were able to observe multiple binding steps with detailed dynamic features of loosely bound and tightly bound state fluctuations. The C-domain of CaM tends to bind with C28W first with a higher affinity, followed by the binding of the CaM N-domain. Due to the comparatively high flexibility and low affinity of the N-domain and the presence of multiple anchor hydrophobic residues on the peptide, the N-domain binding may switch between selective and non-selective binding states, while the C-domain remains strongly bound with C28W. The results provide a mechanistic understanding of the CaM signaling interaction and activation of the Ca-ATPase through multiple-state binding to the C28W. The new single-molecule spectroscopic analyses demonstrated in this work can be applied for broad studies of protein functional conformation fluctuation and protein-protein interaction dynamics.

Separated Micelles Formation of pH-Responsive Random and Block Copolymers Containing Phosphorylcholine Groups.

The self-assembly of pH-responsive random and block copolymers composed of 2-(N,N-diisopropylamino)ethyl methacrylate and 2-methacryloyloxyethyl phosphorylcholine was investigated in aqueous media. Their pH-responsive behaviors were investigated in aqueous media by dynamic light scattering (DLS) and fluorescence measurements using a pyrene hydrophobic fluorescence probe. In an acidic environment, these copolymers existed as single polymer chains that did not interact with each other. In contrast, upon increasing the pH of the solution above the critical value of ~8, separated micelles were formed in the mixture, which was indicated by bimodal distribution in DLS results with radius of 4.5 and 10.4 nm, corresponding to the random and block copolymer micelles, respectively. Fluorescence resonance energy transfer efficiencies were near to zero in the mixture of the donor labeled block and acceptor labeled random copolymers under both acidic and basic pH. These results demonstrated the coexistence of two distinct micelles.

A ratiometric fluorescent nanoprobe for signal amplification monitoring of intracellular telomerase activity.

Telomerase is considered a valuable diagnostic and prognostic cancer biomarker. Accurate and reliable detection of telomerase activity is of great value in clinical diagnosis, screening of inhibitors, and therapeutics. Here, we developed a novel amplified fluorescence resonance energy transfer (FRET) nanoprobe for highly sensitive and reliable monitoring of intracellular telomerase activity. The nanoprobe (QDSA@DNA) was composed of a streptavidin-modified quantum dot (QDSA) which was functionalized with a telomerase primer sequence (TP) and Cy5-tagged signal switching sequence (SS) through biotin-streptavidin interaction. When the nanoprobe was assembled, the Cy5 was in close proximity to the QDSA, resulting in high FRET efficiency from the QDSA to Cy5. In the presence of telomerase, the TP could be extended to produce telomeric repeat units, which was complementary to the loop of SS. Thus, the SS could hybridize with elongated sequences to form a rigid double-stranded structure, which forced the Cy5 away from the surface of the QDSA, causing low FRET efficiency. Furthermore, due to the production of multiple repeat units by telomerase, multiple hairpin structures could be opened, yielding significant fluorescence ratio (FQDsa/FCy5) enhancement for sensing of telomerase activity. In this way, the combination of a FRET and target-assisted strategy in a nanoprobe improved the detection accuracy and amplified the detection signal, respectively. The QDSA@DNA nanoprobe also showed high selectivity, excellent nuclease stability, and good biocompatibility. More importantly, this nanoprobe was found to be an excellent platform for efficient monitoring of intracellular telomerase activity, providing a potential platform in tumor diagnosis and screening of telomerase-related inhibitors.

Highly efficient fluorescence resonance energy transfer in co-encapsulated BODIPY nanoparticles

Fluorescence resonance energy transfer (FRET) is a powerful tool used in a wide range of applications due to its high sensitivity and many other advantages. Co-encapsulation of a donor and an acceptor in nanoparticles is a useful strategy to bring the donor-acceptor pair in proximity for FRET. A highly efficient FRET system based on BODIPY-BODIPY (BODIPY: boron-dipyrromethene) donor-acceptor pair in nanoparticles was synthesized. Nanoparticles were formed by co-encapsulating a green emitting BODIPY derivative (FRET donor, lmax = 501 nm) and a red emitting BODIPY derivative (FRET acceptor, lmax = 601 nm) in an amphiphilic polymer using the precipitation method. Fluorescence measurements of encapsulated BODIPY in water following 501 nm excitation caused a 3.6 fold enhancement of the acceptor BODIPY emission at 601 nm indicating efficient energy transfer between the green emitting donor BODIPY and the red emitting BODIPY acceptor with a 100 nm Stokes shift. The calculated FRET efficiency was 96.5%. Encapsulated BODIPY derivatives were highly stable under our experimental conditions.

Cover Feature: A DNA‐Encoded FRET Biosensor for Visualizing the Tension across Paxillin in Living Cells upon Shear Stress (Anal. Sens. 1/2022)

The cover shows a DNA-encoded biosensor based on a Fluorescence Resonance Energy Transfer (FRET) technique named PaxTS. The biosensor consists of an LD12 domain, an LIM1234 domain, a nano-spring and a pair of fluorescent proteins to convert the tension across paxillin to FRET efficiency in living cells, indicating that there exists a force transmission pathway described as ‘membrane-cytoskeleton-FAs’ and involves paxillin upon mechanical force. More information can be found in the Full Paper by Bo Liu and co-workers.

Fluorescence immunoassay rapid detection of 2019-nCoV antibody based on the fluorescence resonance energy transfer between graphene quantum dots and Ag@Au nanoparticle

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has dramatically changed the world, is a highly contagious virus. The timely and accurate diagnosis of SARS-CoV-2 infections is vital for disease control and prevention. Here in this work, a fluorescence immunoassay was developed to detect 2019 Novel Coronavirus antibodies (2019-nCoV mAb). Fluorescent graphene quantum dots (GQDs) and Ag@Au nanoparticles (Ag@AuNPs) were successfully synthesized and characterized. Fluorescence resonance energy transfer (FRET) enables effective quenching of GQDs fluorescence by Ag@AuNPs. With the presence of 2019-nCoV mAb, a steric hindrance was observed between the Ag@AuNPs-NCP (2019-nCoV antigen) complex and GQDs, which reduced the FRET efficiency and restored the fluorescence of GQDs. The fluorescence enhancement efficiency has a satisfactory linear relationship with the logarithm of the 2019-nCoV mAb in a concentration range of 0.1 pg mL−1–10 ng mL−1, and the limit of detection was 50 fg mL−1. The method has good selectivity. When the serum sample was spiked with 2019-nCoV mAb, the recovery rate was between 90.8% and 103.3%. The fluorescence immunosensor demonstrates the potential to complement the existing serological assays for COVID-19 diagnosis.

Supramolecular Catalysis: New Directions and Developments

Fluorescence Resonance Energy Transfer (FRET) between two organic dyes Fluorescein and Rhodamine 6G were successfully investigated in aqueous solution in presence and absence of 1,2-Dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC) at different pH. Spectroscopic studies suggest that both the dyes were present mainly as monomer in solution. FRET occurred from Fluorescein to Rhodamine 6G in solutions. Energy transfer efficiency increases in presence of DPPC and the maximum efficiency was 59.3% when the concentration of DPPC was 1.4×10−4 M at ambient condition. pH plays a crucial role in this investigation as the energy transfer efficiency was found to change in presence of DPPC at different pH. It has been demonstrated that with proper calibration it is possible to use the present system under investigation to realize various ionic states of DPPC by observing the change in FRET efficiency between these two dyes.

Monte Carlo Simulation of the Efficiency of Fluorescence Resonance Energy Transfer, FRET Phenomenon.

The article aimed to outline a statistical model based on nonlinear regressions for the efficiency of the Fluorescence Resonance Energy Transfer (FRET) phenomenon and to select a general simulation method of the FRET phenomenon, independent of the nature of the active substance used as a donor. For this purpose, several statistical models of Polynoal and nonlinear Gaussiene regressions were first tested, which would fit as well as possible the variation in time efficiency and its dependence on the concentrations of active substances. The regression models used were implemented and tested in the R language. In the second part of the study we aprooached the problem of simulating the efficiency of FRET by adapting a Monte Carlo method, using as a starting point the previously determined regressive models.

Conformational states and fluctuations in endothelial nitric oxide synthase under calmodulin regulation

Mechanisms that regulate nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions and is activated by calmodulin (CaM) binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two NOS electron transfer domains in an FRET dye-labeled endothelial NOS reductase domain (eNOSr) and to understand how CaM affects the dynamics to regulate catalysis by shaping the spatial and temporal conformational behaviors of eNOSr. In addition, we developed and applied a new imaging approach capable of recording three-dimensional FRET efficiency versus time images to characterize the impact on dynamic conformal states of the eNOSr enzyme by the binding of CaM, which identifies clearly that CaM binding generates an extra new open state of eNOSr, resolving more detailed NOS conformational states and their fluctuation dynamics. We identified a new output state that has an extra open conformation that is only populated in the CaM-bound eNOSr. This may reveal the critical role of CaM in triggering NOS activity as it gives conformational flexibility for eNOSr to assume the electron transfer output FMN-heme state. Our results provide a dynamic link to recently reported EM static structure analyses and demonstrate a capable approach in probing and simultaneously analyzing all of the conformational states, their fluctuations, and the fluctuation dynamics for understanding the mechanism of NOS electron transfer, involving electron transfer among FAD, FMN, and heme domains, during nitric oxide synthesis.

Photon upconversion of all-inorganic CsPbX3 quantum dots based on fluorescence resonance energy transfer in hetero-structured perovskite/upconversion nanocomposites

Photo upconversion that converts low-energy light into high-energy photons holds great promise for boosting the energy conversion efficiencies particularly for photovoltaic devices based on all-inorganic CsPbX3 (X = Cl, Br, and I) perovskite quantum dots. However, the realization of nonlinear upconversion luminescence (UCL) from these all-inorganic CsPbX3 perovskites has been met with very limited success until now. Herein, we propose a rational perovskite/upconversion hetero-structured design strategy to achieve excitonic UCL of all-inorganic CsPbX3 perovskites based on fluorescence resonance energy transfer (FRET), where all-inorganic CsPbX3 perovskites and upconverting CaF2:Ln (Ln = Yb3+/Er3+, Yb3+/Ho3+, and Yb3+/Tm3+) nanocrystals are closely integrated with each other as a whole by using a modified two-step epitaxial growth approach, forming the unique upconverting CsPbX3/CaF2:Ln hetero-structured nanocomposites (HSNCs). Benefitting from the extremely high FRET efficiency of ∼99.7%, excitonic UCL from CsPbX3 perovskites can be realized upon low-power density 980-nm diode laser irradiation. More importantly, all the HSNCs are found to outperform their pure CsPbX3 counterparts in terms of air-stability, thermal-stability, and excitonic downconversion luminescence intensity, clearly demonstrating the overwhelming advantages of upconverting HSNCs we prepared over the pure CsPbX3 perovskites for high-performance optoelectronic and photovoltaic applications.

A DNA‐Encoded FRET Biosensor for Visualizing the Tension across Paxillin in Living Cells upon Shear Stress

AbstractPaxillin is a potential participant in direct intracellular force transmission, which is considered as the foundation of cells sensing and responding to the extracellular environment. However, the detection of tension across paxillin has not been achieved due to lacking microsized tools. Herein, a paxillin tension sensor (PaxTs) based on the Fluorescence Resonance Energy Transfer (FRET) technique was constructed. PaxTs can be expressed and assembled to FA sites spontaneously to visualize the tension across paxillin with FRET efficiency of ∼62.4 % in living cells. The tension across paxillin was found to decrease upon shear stress, in which the membrane fluidity and contractility of actin acted as cushions. It is observed that paxillin participates in the pathway of cell membrane‐cytoskeleton‐FAs for force transmission upon mechanical force in real time visualization, which provides a promising new method to investigate the direct intracellular force transmission in biology and technology.

Open-Source Fluorescence Spectrometer for Noncontact Scientific Research and Education

Transforming fluorescence spectrometers into cost-effective, portable devices provides the potential for field-based applications in biological, environmental, and clinical research and education. However, the majority of developed spectroscopic technologies continue to require heavy, expensive equipment and trained personnel for operation or do not support multispectral analysis, thereby restricting their use in resource-limited environments. Herein, we report a wireless, portable, cost-effective, open-source fluorescence spectrometer (OpenFS) developed by compactly assembling optical and electronic elements in a 3D-printed housing. OpenFS outputs an accurate emission spectrum over a wide range of wavelengths and demonstrates greater sensitivity for fluorescence quantification compared to a conventional fluorometer. We demonstrate the functionality of OpenFS as a fluorescence resonance energy transfer (FRET)-based DNA sensor by detecting target DNA molecules with FRET efficiency and prove its utility as an Internet of Things device by performing wireless measurements and spectral analysis on a smartphone with a custom-developed Android application. This portable open-source spectrometer can lead to new opportunities in research and educational fields where fluorescence spectroscopy has not been available because of its cost and size and provides the potential for the development of mobile diagnostics platforms.

Laponite elementary sheets assisted fluorescence resonance energy transfer: A demonstration by Langmuir-Blodgett technique

The fluorescence resonance energy transfer (FRET) between donor-acceptor molecules of fluorescein (FL) and rhodamine B (RhB) was enhanced by clay sheets of laponite. This enhancement was implemented by the Langmuir-Blodgett (LB) technique. Hybrid films were prepared by incorporating elementary clay sheets into dye molecules (FL and RhB for different molar ratios) at the air/water interface. The spectroscopic measurements (UV–Vis absorption and photoluminescence) of these mixture systems (FL, RhB and clay mineral) were analyzed both in colloidal dispersion and LB films. A high efficiency (92%) of FRET from FL to RhB was achieved with presence of laponite. Characterizations such as surface pressure versus area (π-A) isotherms, atomic force microscopy (AFM) and contact angle measurements were used to confirm the successful incorporation of clay sheets in hybrid films with loaded dye molecules. The clay incorporation were indicated to shorten the distance between donor and acceptor chromophores (Förster radius), which was realized by the tightly bounded dye molecules on clay sheets and self-assembly of dye molecule pairs.

Label-free triplex DNA-based biosensing of transcription factor using fluorescence resonance energy transfer between N-doped carbon dot and gold nanoparticle

Herein, a new turn-on fluorescent assay was established as a platform for the sensing of transcription factor NF-kB p50 based on triplex DNA labeled with N-doped carbon dots (NCDs) and gold nanoparticles (AuNPs) as donors and acceptors, respectively in the fluorescence resonance energy transfer (FRET) system. The synthetized nanoparticles were studied by different characterization techniques. A labeled DNA molecule was designed to form a triplex when no target protein existence and reported its formation by the change in FRET efficiency. While the triplex DNA was formed, the fluorescence of carbon dots at 503 nm (excitation at 460 nm) was quenched by FRET between NCD and AuNP. However, presence of NF-kB p50 followed by the considerable enhancement in the fluorescence intensity caused by the release of AuNPs labeled single stranded DNA from the triplex DNA structure, used for sensitive determination of the transcription factor. This technique showed a linearity (R2 = 0.9943) in the range of 20–150 pM with a limit of detection of 9 pM for the determination of NF-kB p50. Moreover, the sequence-specific triplex-based biosensor could discriminate NF-kB p50 from the other proteins with high selectively. Our results suggest that the biosensor provides a generalizable platform for rapid detection of NF-kB p50 in synthetic medium, promising in prevention and early diagnosis of cancer.

Green Synthesis, Structural Characterization and Photocatalytic Applications of ZnO Nanoconjugates Using Heliotropium indicum

In recent years, biosynthesized zinc oxide nanoparticles (ZnO NPs) have been gaining importance due to their unique properties and tremendous applications. This study aimed to fabricate ZnO NPs by using extracts from various parts of the traditional medicinal plant Heliotropium indicum (H. indicum) and evaluate their photocatalytic activity. Further, their potential in photoluminescence and fluorescence resonance energy transfer (FRET) was assessed. The Ultraviolet-Visible spectrum exhibited a hypsochromic shifted absorption band between 350–380 nm. Transmission electron microscopy (TEM) analysis revealed spherical NPs, while X-ray diffraction (XRD) data revealed wurtzite, hexagonal and crystalline nature. The TEM and XRD consistently determined an average particle size range from 19 to 53 nm. The photocatalytic degradation reaches a maximum of 95% for biogenic ZnO NPs by monitoring spectrophotometrically the degradation of methylene blue dye (λmax = 662.8 nm) under solar irradiation. Photoluminescence analysis revealed differentiated spectra with high-intensity emission peaks for biogenic ZnO NPs compared with chemically synthesized ZnO NPs. Eventually, the highest efficiency of FRET (80%) was found in ZnO NPs synthesized from the leaves. This remains the first report highlighting the multifunctional ZnO NPs capabilities mediated by using H. indicum, which could lead to important potential environmental and biomedical applications.

Ultrasensitive Detection of Amyloid β Oligomers Based on the "DD-A" FRET Binary Probes and Quadrivalent Cruciform DNA Nanostructure-Mediated Cascaded Amplifier.

The reported donor donor-acceptor ("DD-A") fluorescence resonance energy transfer (FRET) was typically achieved through random collisions and interactions of DNA molecules in the bulk solution, which has inevitable defects, including weak biological stability, slow reaction kinetics, and low hybridization efficiency. In order to overcome these deficiencies, this work developed a quadrivalent cruciform DNA nanostructure (qCDN)-mediated cascaded catalyzed hairpin assembly (CHA) amplifier for the fluorescence detection of amyloid β oligomer species (AβOs). First, four H1 and four H2 hairpins were assembled on one qCDN to obtain qCDNH1 and qCDNH2, respectively. In the presence of AβOs, strand C was released from the P1-C hybrid hairpin and then alternately opened qCDNH1 and qCDNH2 to trigger the qCDN-mediated CHA. As a result, double donors in H1 and one acceptor in H2 were mutually closed, and the porous DNA nanonet with a high loading of "DD-A" FRET binary probes was formed. The FRET efficiency was approximately 78%, and the initial reaction rate was 25-fold faster than the conventional CHA. The detection limit of AβOs was as low as 0.69 pM. The combination of the "DD-A" FRET binary probes and qCDN-mediated cascaded amplifier exhibited great promise for detecting biomarkers with trace levels.

Aluminum plasmon-enhanced deep ultraviolet fluorescence resonance energy transfer in h-BN/graphene heterostructure

We theoretically investigate the fluorescence resonance energy transfer (FRET) in two dimensional (2D) graphene/h-BN heterostructure. We use plasmon to enhance the intensity of fluorescence and manipulate the direction of fluorescence to improve the efficiency of FRET. We calculated the plasmon-enhanced fluorescence resonance energy transfer of three different ratios of donor–acceptor systems and found that the ratio of donor and acceptor will both affect the intensity and efficiency of FRET. Our results contribute to the deep understanding of FRET in Graphene/h-BN donor–acceptor system and theoretically provide basis for design of emerging graphene/h-BN 2D light emission diode (2D-LED).

MASH-FRET: A Simplified Approach for Single-Molecule Multiplexing Using FRET.

Multiplexed detection has been a big motivation in biomarker analysis as it not only saves cost and labor but also improves the reliability of diagnosis. Among the many approaches for multiplexed detection, fluorescence resonance energy transfer (FRET)-based multiplexing is gaining popularity particularly due to its low background and quantitative nature. Although several FRET-based approaches have been developed for multiplexing, they require either multiple FRET pairs in combination with multiple excitation sources or complicated algorithms to accurately assign signals for individual FRET pairs. Therefore, the need for multiple FRET pairs and multiple excitation sources not only complicates the experimental design but also increases the cost and labor. In this regard, multiplexed sensing by tuning the interdye distance of a single FRET pair could be an ideal solution if identification of multiple FRET efficiencies in a single imaging is possible. Here, implementing a program called MASH-FRET, we evaluated the rigor and capability of this program in identifying seemingly overlapped FRET populations obtained from a multiplexed detection experiment using a single FRET pair. Through MASH-FRET-enabled bootstrap-based analysis of FRET data (also called BOBA-FRET), we demonstrated that the resolution and statistical confidence of the poorly resolved or even unresolved FRET populations can be readily determined. Using simulated FRET data, we further demonstrated that the program can easily identify FRET populations separated by ∼0.1 in mean FRET values, indicating an upper limit of ∼9-fold multiplexing without the need for complicated labeling schemes and multiexcitation sources. Therefore, this paper presents a data analysis approach on an existing platform that has a great potential to simplify the technological needs for multiplexing and to broaden the scope of FRET-based single-molecule analyses.