Luminescence Resonance Energy Transfer Research Articles

TrLRET microscopy: Ultrasensitive imaging of lanthanide luminophores.

In principle, the long emission lifetimes of lanthanide chelates should enable their ultrasensitive detection in biological systems by time-resolved optical microscopy. However, most lanthanide-imaging systems cannot achieve sensitivities that exceed those of conventional fluorescence microscopes, since they are limited by inefficient lanthanide excitation, the low photon flux of excited lanthanide luminophores, and optics-derived background photoluminescence. We recently reported a new lanthanide-imaging modality, trans-reflected illumination with luminescence resonance energy transfer (trLRET), which overcomes each of these constraints. Here we provide a detailed procedure for visualizing endogenous protein expression in zebrafish embryos, using lanthanide-labeled antibodies, Q-switched laser illumination, and trLRET microscopy. These methods allow ultrasensitive molecular imaging in cells and organisms, establishing a new paradigm for biological exploration and discovery.

A versatile luminescent resonance energy transfer (LRET)-based ratiometric upconversion nanoprobe for intracellular miRNA biosensing.

Rational design and fabrication of bio-nanoprobes for intracellular miRNA biosensing are highly desired for early clinical diagnosis and prognosis. Herein, we have developed a versatile LRET-based ratiometric (LBRU) nanoprobe of NaYF4:Yb,Er@NaYF4@NH2-mSiO2/rhodamine B/C-DNA sandwich-structured nanocomposites for intracellular miRNA biosensing. The nanoprobe was composed of NaYF4:Yb,Er@NaYF4 upconversion nanoparticles (energy donor) with an amino functionalized mesoporous silica shell (NH2-mSiO2), rhodamine B (acceptor) loaded into the mesopores of NH2-mSiO2, and the complementary sequences of target miRNA (denoted as C-DNA) acting as recognition species wrapped on the nanocomposite. Due to the LRET behavior between donors and acceptors, the loaded rhodamine B can quench the green upconversion emission of NaYF4:Yb,Er@NaYF4 at 540 nm completely. Moreover, it can be released from the nanocomposite in the presence of target miRNA, which blocked the LRET behavior to "turn on" the green upconversion luminescence. Besides, as the unaffected red upconversion luminescence (at 660 nm) can be used as an internal standard to provide built-in correction for environmental effects, the intensity ratio of upconversion luminescence at 540 and 660 nm (I540/I660) was employed as the output signal to afford an accurate detection of target miRNA. Due to the biocompatibility, high photostability and low auto-fluorescence background, the nanoprobe was successfully utilized to diagnose the intracellular miRNA-21 expression in MCF-7 cells via upconversion fluorescence imaging. We envision that the proposed LBRU nanoprobe has great potential applications in early cancer diagnosis.

Interaction of the motor protein SecA and the bacterial protein translocation channel SecYEG in the absence of ATP.

Translocation of many secretory proteins through the bacterial plasma membrane is facilitated by a complex of the SecYEG channel with the motor protein SecA. The ATP-free complex is unstable in detergent, raising the question how SecA may perform several rounds of ATP hydrolysis without being released from the membrane embedded SecYEG. Here we show that dual recognition of (i) SecYEG and (ii) vicinal acidic lipids confers an apparent nanomolar affinity. High-speed atomic force microscopy visualizes the complexes between monomeric SecA and SecYEG as being stable for tens of seconds. These long-lasting events and complementary shorter ones both give rise to single ion channel openings of equal duration. Furthermore, luminescence resonance energy transfer reveals two conformations of the SecYEG–SecA complex that differ in the protrusion depth of SecA's two-helix finger into SecYEG's aqueous channel. Such movement of the finger is in line with the power stroke mechanism of protein translocation.

Correction: A paper-supported sandwich immunosensor based on upconversion luminescence resonance energy transfer for the visual and quantitative determination of a cancer biomarker in human serum.

Correction for 'A paper-supported sandwich immunosensor based on upconversion luminescence resonance energy transfer for the visual and quantitative determination of a cancer biomarker in human serum' by Mengyuan He et al., Analyst, 2020, 145, 4181-4187, DOI: .

A paper-supported sandwich immunosensor based on upconversion luminescence resonance energy transfer for the visual and quantitative determination of a cancer biomarker in human serum.

In this paper, a paper-supported analytical device based on a sandwich immunoreaction and luminescence resonance energy transfer (LRET) was reported for the visual and quantitative determination of a cancer biomarker, in which upconversion nanoparticles (UCNPs) were located on the surface of the paper as energy donors and gold nanoparticles (AuNPs) were used as energy acceptors. Upon the recognition of the cancer biomarker by two rationally selected antibodies, the upconversion luminescence was quenched by the AuNPs in a biomarker concentration-dependent manner. As a model target, CEA was detected using this immunosensor, and a linear relationship within 0.5-30 ng mL-1 was obtained in buffer solution, with a detection limit of 0.21 ng mL-1. The immunosensor was also applicable in 20-fold diluted human serum with a linear range of 0.5-30 ng mL-1 and a detection limit of 0.36 ng mL-1. This technique also realized the qualitative judgment of the critical concentration of CEA in serum samples by the naked eye. This approach displays great application potential for point-of-care testing in clinical applications, as well as the potentiality to be extended to other kinds of disease-related biomolecules.

An efficient NIR-to-NIR signal-based LRET system for homogeneous competitive immunoassay

Upconversion nanoparticles (UCNPs) are promising materials for biological applications based on luminescence resonance energy transfer (LRET). In contrast to classical RET donors such as quantum dots, gold nanoparticles, UCNPs can emit near-infrared (NIR) upon the NIR irradiation, which provides enhanced signal-to-noise due to strong penetration and low autofluorescence in the NIR region known as the diagnostic window. Here we report the first efficient NIR-to-NIR signal-based LRET system for the detection of progesterone, chosen as a proof-of-concept target, via homogeneous competitive immunoassay. To enhance the efficiency of LRET, we constructed inert-core/active-shell/inert-ultrathin shell UCNPs (NaYF4@NaYF4:Yb,Tm@NaYF4) as an LRET donor and a compact progesterone/horseradish peroxidase/IRdyeQC-1 (P-HRP-dyes) complex as an LRET acceptor. The designed donor and acceptor showed significantly improved LRET efficiencies (95% and 85% for donor and acceptor, respectively) compared with conventional donor and acceptor (70% and 50%, respectively). Using the developed NIR-to-NIR LRET system, progesterone was successfully detected with a background-free signal and low limit of detection (1.36 pg/ml in ten-fold diluted human serum). We believe that the efficient NIR-to-NIR signal-based LRET system developed here has potential as a simple probe for homogeneous competitive immunoassay, with the ability to rapidly detect biomarkers.

A ratiometric fluorescence probe for melamine detection based on luminescence resonance energy transfer between the NaYF4:Yb, Er upconversion nanoparticles and gold nanoparticles

In this paper, a novel ratiometric fluorescence probe for melamine detection based on luminescence resonance energy transfer (LRET) between the NaYF4:Yb, Er upconversion nanoparticles (UCNPs) and gold nanoparticles (AuNPs) was developed. The detection mechanism could be interpreted to the AuNPs quenched the fluorescence intensity (F542 and F654) of the UCNPs under 980 nm excitation. after melamine addition, the fluorescence intensity of the UCNPs was recovered due to the aggregation of Au NPs. A good linear relationship between the ratio of F542 to F654 nm (F542/F654) was obtained in ranges of 20-200 μg/L of the melamine concentration and the limit of detection was 8.8 nM. Besides, The probe exhibited an excellent selectivity and good sensitivity to melamine, and achieved the melamine detection in real sample. This work paved a new avenue for the potential practice application for commercial instantaneous determination of the melamine.

A facile luminescence resonance energy transfer method for detecting cyano-containing pesticides in herbal medicines

It is urgently demanded to establish facile and rapid methods for screening pesticide residues in cultivated herbal medicines to guarantee drug safety. Herein a fluorescent (FL) method was constructed for detecting cyano-containing pesticides by employing the luminescence resonance energy transfer (LRET) behavior between upconversion nanoparticles (UCNPs) and gold nanoparticles (AuNPs). Negatively-charged citrate-capped AuNPs can be adsorbed onto the surface of positively-charged amino-modified UCNPs by an electrostatic interaction. It resulted in decrease of the FL signal of UCNPs through the LRET process. Cyano-containing pesticides can bind with AuNPs via AuNC metal-ligand coordination and remove citrate from the surface of AuNPs. The replacement process induced aggregation of AuNPs and inhibition of LRET. Thus the pesticides can be detected by a facile procedure within 6 min. Acetamiprid, fenpropathrin and chlorothalonil were adopted as model analytes to study the feasibility of the FL method for the detection of cyano-containing pesticides. With the optimum conditions, acetamiprid, fenpropathrin and chlorothalonil can be detected within the ranges of 0.10–100 ng mL−1, 1.0–100 ng mL−1 and 0.10–50 ng mL−1, with detection limits of 0.015 ng mL−1, 0.24 ng mL−1 and 0.011 ng mL−1, respectively. It has been successfully employed to detect acetamiprid in Lanceolata, Angelica dahurica and Astragalus with acceptable recovery values of 76.0%–120.0%, demonstrating its potential for practical application. The work provides a highly efficient methodologic platform for screening pesticide residues in herbal medicines.

Dual-Acceptor-Based Upconversion Luminescence Nanosensor with Enhanced Quenching Efficiency for in Situ Imaging and Quantification of MicroRNA in Living Cells.

Upconversion nanoparticles (UCNPs) have become competitive materials for bioanalysis, bioimaging, and early diagnosis of diseases, especially cancers. However, traditional upconversion luminescence (UCL) nanosensors are often challenged with complicated covalent modification and relatively poor stability. As efficient energy acceptors in the luminescence resonance energy-transfer (LRET) process, organic dyes exhibit unique advantages such as easy modification and stable property. Herein, a simple and universal bioplatform is constructed for in situ imaging and quantitation of intracellular microRNA-21 (miR-21) using dual-acceptor-based upconversion nanoprobes with enhanced quenching efficiency. In this assay, UCNPs with core-shell structures are synthesized, in which the emitting ions are confined in the shell to take the energy donors and acceptors in close proximity. The complementary DNA (cDNA) that can specifically recognize target miR-21 is labeled with organic dyes TAMRA and black hole quencher as dual acceptors and easily assembled on UCNPs via electrostatic adsorption. Compared with only one acceptor for LRET, two dyes quench more luminescence of UCNPs (>60%), which thus reduce the background and improve the sensitivity. With the enhanced quenching efficiency and simple assembly process, the proposed system is readily applied to in situ imaging of miR-21 in different cancer cells, which further achieves quantification of miR-21 in MCF-7 cells. Therefore, our proposed dual-acceptor-based upconversion nanoplatform opens up new opportunities for sensitive analysis of miRNA and provides potential applications in biomedical and clinical research.

Luminescence Lifetime-Based In Vivo Detection with Responsive Rare Earth-Dye Nanocomposite.

For years, luminescence lifetime imaging has served as a quantitative tool in indicating intracellular components and activities. However, very few studies involve the in vivo study of animals, especially in vivo stimuli-responsive activities of animals, as both excitation and emission wavelengths should fall into the near-infrared (NIR) optical transparent window (660-950 and 1000-1500 nm). Herein, this work reports a lifetime-responsive nanocomposite with both excitation and emission in the NIR I window (800 nm) and lifetime in the microsecond region. The incorporation of Tm3+ -doped rare-earth nanocrystals and NIR dye builds an efficient energy transfer pathway that enables a tunable luminescence lifetime range. The NaYF4 :Tm nanocrystal, which absorbs and emits photons at the same energy level, is found to be 33 times brighter than optimized core-shell upconversion nanocrystals, and proved to be an effective donor for NIR luminescence resonance energy transfer (LRET). The anti-interference capability of luminescence lifetime signals is further confirmed by luminescence and lifetime imaging. In vivo studies also verify the lifetime response upon stimulation generated in an arthritis mouse model. This work introduces an intriguing tool for luminescence lifetime-based sensing in the microsecond region.

Modulating near-infrared persistent luminescence of core-shell nanoplatform for imaging of glutathione in tumor mouse model

Developing near-infrared (NIR) persistent luminescence (PersL) bioprobes has attracted increasing attention due to their ultra-low autofluorescence and deep tissue penetration. Accurate imaging of glutathione (GSH) in vivo is of great significance to provide a real-time visualization of pathologies. Herein, manipulating metal ions as the switch of NIR afterglow was demonstrated to construct NIR PersL nanoprobes for the first time, which was highly efficient and much simpler than the luminescence resonance energy transfer (LRET) principle. As a proof-of-concept application, an activatable NIR PersL nanoprobe (Probe-1) built on core-shell structure was rationally fabricated for detection of GSH via employing Cu2+ as the quencher. In light of unique NIR PersL feature and flexible functionality, Probe-1 exhibited greatly enhanced analytical performances for GSH detection, and particularly imaging of tumor in living mouse. This approach without limits of spectral overlap from the LRET principle provided a new way to develop NIR PersL bioprobes for autofluorescence-free detection.

Single-Particle LRET Aptasensor for the Sensitive Detection of Aflatoxin B1 with Upconversion Nanoparticles.

Contamination of foods and feeds by aflatoxins is a universal yet serious problem all over the world. Particularly, aflatoxin B1 (AFB1) is the most primary form and readily leads to terrible damages to human health. In this work, we construct a sensitive aptasensor based on single-particle detection (SPD) to analyze AFB1 in peanut samples with luminescence resonance energy transfer (LRET) between the aptamer-modified upconversion nanoparticles (UCNPs-aptamer) and gold nanoparticles (GNPs). The UCNP-aptamer plays as the luminescence donor, while GNP acts as the energy acceptor. In the absence of AFB1, GNPs would adsorb onto the surface of UCNPs-aptamer because of the association between aptamers and GNPs, leading to luminescence quenching. However, the luminescence of UCNPs-aptamer is recovered gradually in the presence of AFB1, because the aptamers possess stronger affinity toward AFB1 than GNPs. Through statistically counting the number of luminescent particles on the glass slide surface, the concentration of AFB1 in solution is accurately determined. The linear dynamic range for AFB1 detection is from 3.13 to 125.00 ng/mL. The limit-of-detection (LOD) is 0.17 ng/mL, which is much lower than the allowable concentration in foods. As a result, this method would provide promising application for the sensitive detection of AFB1 in foods and feeds, which might make a meaningful contribution to food safety and public health in the future.

Upconversion Luminescence Sandwich Assay For Detection of Influenza H7 Subtype.

With large anti-Stokes shifts and background-free signals, upconversion luminescent (UCL) screening assays have been a promising method to reduce the transmission of influenza epidemic, which can critically alleviate the disease burden and extra annual deaths. In this work, a luminescent resonance energy transfer sandwich assay is developed, which utilizes core-shell upconversion nanoparticles and gold nanoparticles as the donor and acceptor, respectively. The influenza H7 gene of H7N9 virus is used as the target for optimization of the assay. Importantly, the hybridization time of the assay is ≈40 min and the specificity test indicates the probes are specific toward the H7 target. The limit of detection of the system is ≈134 × 10-12 m (≈3.22 × 1010 molecules). Moreover, the assay is tested with the use of polymerase chain reaction validated samples from human isolates. The results are promising for implanting future on-site rapid influenza screening application.

An aptasensor based on upconversion nanoparticles as LRET donors for the detection of exosomes

Exosomes may be used as a crucial medium for the early diagnosis and potential therapy of tumors because of containing a variety of biological information. Therefore, the accurate quantitative analysis of exosomes is especially important in the biomedical field. Herein, we proposed a washing-free aptasensor based on luminescence resonance energy transfer (LRET) between rare-earth doped upconversion nanoparticles (UCNPs) donor and tetramethyl rhodamine (TAMRA) acceptor for highly sensitive detection of exosomes. Therein, the combination of UCNPs and TAMRA can be achieved through the coupling of aptamers to the epithelial cell adhesion molecule (EpCAM), the highly expressed surface proteins of the exosomes. Therefore, when the system of UCNPs-TAMRA in the presence of exosomes is excited by near-infrared light at 980 nm, TAMRA emits yellow fluorescence at 585 nm due to LRET. As a result, the fluorescence intensity at 585 nm is linearly correlated to the concentration of exosomes, which is available for the detection and quantification of exosomes. Under optimal conditions, the proposed approach can reach a low limit of detection (LOD) of 80 particles/μL, and effectively reduce the background signal by using UCNPs as an energy donor. Moreover, this proposed aptasensor can be universally applied to the detection of other targets by strictly screening the aptamers.

Luminescence-Resonance-Energy-Transfer-Based Luminescence Nanoprobe for In Situ Imaging of CD36 Activation and CD36–oxLDL Binding in Atherogenesis

Macrophage foam cell formation mediated by CD36 receptor dependent internalization of oxidized low-density lipoprotein (oxLDL) is an important hallmark of early atherosclerosis. Activation of CD36 and its binding to oxLDL are the key points in foam cell formation. Herein, we develop a site-specific luminescence resonance energy transfer (LRET) system for the simultaneous imaging of CD36 activity and CD36-oxLDL binding on the cell surface. The system utilizes CD36-antibody-modified, SiO2-coated upconversion luminescent nanoparticles (UCNPs) as an energy donor to target the plasma membranes of macrophages, and DiI-oxLDL (energy acceptor) binds to CD36 and passes through the membrane during macrophage foam cell formation. Upon excitation at 980 nm, the LRET signal can be obtained because of the short distance between DiI-oxLDL and the nanoprobe. Additionally, the very specific fluorescence can be used to visualize distinct features of CD36. The nanoprobe also exhibits high sensitivity, good stability, simplicity, and low cost for the accurate detection and evaluation of macrophage foam cell formation. Moreover, using this novel nanoprobe, we also investigate the mechanism by which reactive oxygen species (ROS) signaling enhances the binding of oxLDL to CD36. ROS, especially O2·-, alter endothelial permeability and facilitate CD36 clustering, ultimately promoting the entry and internalization of oxLDL. Because of these advantages, this nanoprobe may provide a versatile platform for monitoring the progression of atherogenesis and elucidating atherogenesis signaling at the cellular level.

Detection of nitroaromatics in aqueous media based on luminescence resonance energy transfer using upconversion nanoparticles as energy donors

Upconversion nanoparticle (UCNP)-based luminescence resonance energy transfer (LRET) systems are a powerful tool widely used to detect organic molecules or metal ions because of their simplicity and high sensitivity. The sandwich structure NaYF4:Er3+,Yb3+@NaYF4@NH2 UCNPs, as a highly selective and sensitive aqueous probe for detecting nitroaromatics, has been designed and prepared by a cothermolysis method and modified with polyetherimide to acquire amine groups on the surface of the core/shell UCNPs. The detection principle of nitroaromatics is based on LRET, which forms the Meisnheimer complex between the electron-deficient cyclobenzene of nitroaromatics and the electron-rich amino group on the surface of the sandwich structure UCNPs. As a consequence, nitroaromatics can be brought into close proximity to the sandwich structure UCNPs. With the increase of nitroaromatics (2,4,6-trinitrophenol and 2,4,6-trinitrotoluene) concentrations, the sandwich structure NaYF4:Er3+,Yb3+@NaYF4@NH2 UCNPs display a dramatic luminescent quenching effect at 407 nm and 540 nm under 980 nm excitation. The luminescent quenching intensity of the sandwich structure UCNPs is linearly correlated to the concentration of the nitroaromatics. The detection limit of 2,4,6-trinitrophenol (TNP) and 2,4,6-trinitrotoluene (TNT) are 0.78 and 0.77 ng ml−1, respectively. Therefore, the sandwich structure of NaYF4:Er3+,Yb3+@NaYF4@NH2 UCNPs can act as a valuable probe to detect nitroaromatics in public safety and security conditions.

Breaking Through Bead-Supported Assay: Integration of Optical Tweezers Assisted Fluorescence Imaging and Luminescence Confined Upconversion Nanoparticles Triggered Luminescent Resonance Energy Transfer (LRET).

Herein, a conceptual approach for significantly enhancing a bead-supported assay is proposed. For the fluorescence imaging technology, optical tweezers are introduced to overcome the fluid viscosity interference and immobilize a single tested bead at the laser focus to guarantee a fairly precise imaging condition. For the selection of fluorescent materials and the signal acquisition means, a type of innovative luminescence confined upconversion nanoparticle with a unique sandwich structure is specially designed to act as an efficient energy donor to trigger the luminescent resonance energy transfer (LRET) process. By further combining the double breakthrough with a molecular beacon model, the newly developed detection strategy allows for achieving a pretty high LRET ratio (≈ 88%) to FAM molecules and offering sound assay performance toward miRNA analysis with a detection limit as low as the sub-fM level, and is capable of well identifying single-base mismatching. Besides, this approach not only is able to accurately qualify the low-abundance targets from as few as 30 cancer cells but also can be employed as a valid cancer early warning tool for performing liquid biopsy.

Evaluating spectral overlap with the degree of quenching in UCP luminescence energy transfer systems

The use of organic-based fluorophores has been firmly established as a key tool in the biological sciences, with many biological-sensing methods taking advantage of Förster Resonance Energy Transfer (FRET) between different fluorescent organic-based dyes following one photon excitation. Nevertheless, the employment of UV-visible absorbing dyes as fluorescent tags and markers typically suffer from several drawbacks including relatively high energy of excitation wavelength, photobleaching and competitive autofluorescence, which often limit their effectiveness and longevity both in vitro and in vivo. As an alternative, lanthanide-doped upconverting phosphors (UCP) have emerged as a new class of materials for use in optical imaging and FRET sensing; they exhibit high photo- and chemical stability and utilise near infrared (nIR) excitation. Approaches to sensing a given analyte target employing upconverting phosphors can be achieved by engineering the UCP to operate analogously to fluorescent dyes via Luminescence Resonance Energy Transfer (LRET) and such systems are now becoming central to optically sensing low concentrations of biologically important species and performing distance measurements. Similarly to FRET, the LRET process is distance dependent and requires spectral overlap between the absorption of the acceptor luminophore and the emission of the donor moiety, yet essential measures of the relationship between spectral overlap and the degree of quenching have not yet been established. To address this, we have investigated the Stern-Volmer relationship for a set of six commonly functionalised organic dyes and seven biomolecules that contain key chromophoric co-factors with Gd2SO4:Yb:Er (PTIR545) and Gd2SO4:Yb:Tm (PTIR475) UCPs under low power nIR excitation, and found that for the organic dyes a linear relationship between spectral overlap and degree of quenching is observed. However, this linear relationship is observed to break down for all the biomolecules investigated.

Ratiometric Upconversion Luminescence Nanoprobe with Near-Infrared Ag2S Nanodots as the Energy Acceptor for Sensing and Imaging of pH in Vivo.

A luminescence resonance energy transfer (LRET) system was successfully developed using near-infrared (NIR) Ag2S nanodots (NDs) as the energy acceptors and upconversion nanoparticles (UCNPs) as the energy donors. The system possessing the properties of NIR excitation (980 nm) and NIR emission (795 nm) was used for the ratiometric detection and bioimaging of pH in tumor cells and zebrafish. Glutathione and mercaptopropionic acid (MPA) co-modified Ag2S NDs (GM-Ag2S NDs) were prepared by ligand exchange with an excellent pH-responsive property over a pH range of 4.0 to 9.0. The NIR GM-Ag2S NDs were covalently grafted with silica coated UCNPs, and an efficient LRET platform was developed via modulation of the thickness of the silica coating. Due to the LRET process between UCNPs and GM-Ag2S NDs, a ratiometric luminescence nanoprobe with the properties of NIR excitation-NIR emission was constructed for pH biosensing and bioimaging. On the basis of high contrast bioimaging, the nanoplatform can distinguish between tumor and normal tissue in the zebrafish model.

Nicking enzyme-assisted signal-amplifiable Hg2+ detection using upconversion nanoparticles

A highly specific and sensitive isothermal method for mercury detection using DNA-conjugated upconversion nanoparticles is reported. A single-stranded DNA containing thymine bases, used as the Hg2+-capturing element through the formation of thymine-Hg2+-thymine complex, is covalently attached to the NaYF4: Yb3+, Tm3+ nanoparticles. Luminescence resonance energy transfer takes place between the NaYF4: Yb3+, Tm3+ nanoparticles as donor and DNA-intercalating SYBR Green I as the acceptor upon excitation of 980 nm. The sensitivity and selectivity toward Hg2+ are enhanced using the nicking enzyme, Nt. Alwl, which leads to signal amplification. By monitoring the ratio of acceptor emission to a reference peak, the presence of Hg2+ ions are quantitatively determined with a lower detection limit of 0.14 nM, which is much lower than the US Environmental Protection Agency (EPA) limit of Hg2+ in drinking water.