Fluorescence Resonance Energy Transfer System Research Articles

Development of a FRET aptasensor based on MoS2-doped Zn-MOF as luminophore for selective detection of cadmium in aqueous solutions.

A robust "on-off" fluorescent aptasensor was developed using nanohybrids of molybdenum sulfide (MoS2) quantum dot (QD)-doped zinc metal-organic frameworks (Zn-MOF) for selective and sensitive detection of cadmium ions (Cd2+) in water. This nanohybrid (MoS2@Zn-MOF), synthesized via "bottle around the ship" methodology, exhibited a high-intensity fluorescence emission centered at 430nm (λEm) (blue) on excitation at 320nm (λEx). Further, the conjugation of this fluorophore to phosphate-modified cadmium aptamer (Cd-2-2) was achieved through carbodiimide reaction. The hybridization of prepared sensing probe (MoS2@Zn-MOF/Cd-2-2 aptamer) was done with dabcyl-conjugated complementary DNA (cDNA), acting as energy donor-acceptor pair in the fluorescence resonance energy transfer (FRET) system. This hybridizationcauses the fluorescence quenching of the nanohybrid. In the presence of Cd2+, the aptamer from the fabricated nano-biosensing probe binds to these ions, resulting in release of dabcyl-cDNA oligomer. This release of dabcyl-cDNA oligomer from the sensing probes restores the fluorescence of the nanohybrid. Under optimized conditions (sensing probe/dabcyl-cDNA ratio 1/7, pH 7.4, and temp 28°C), the sensing probe showed a fast response time of 1min. The fluorescence intensity of the nanohybrid can be utilizedto determine the concentration of Cd2+. The proposed aptasensor achieved highly sensitive detection of Cd2+ with a limit of detection (LOD) of 0.24ppb over the range of 1 × 10-9 to 1 × 10-4M along with minimal effects of interferences (e.g., Hg2+, Pb2+, and Zn2+) and good reproducibility. The designed aptasensor based on MoS2@Zn-MOF nanofluorophore offers a highly sensitive and selective approach for rapid screening of metal ions in aqueous environments.

Multi-step FRET systems based on discrete supramolecular assemblies.

Fluorescence resonance energy transfer (FRET) from the excited state of the donor to the ground state of the acceptor is one of the most important fluorescence mechanisms and has wide applications in light-harvesting systems, light-mediated therapy, bioimaging, optoelectronic devices, and information security fields. The phenomenon of sequential energy transfer in natural photosynthetic systems provides great inspiration for scientists to make full use of light energy. In recent years, discrete supramolecular assemblies (DSAs) have been successively constructed to incorporate donor and multiple acceptors, and to achieve multi-step FRET between them. This perspective describes recent advances in the fabrication and application of DSAs with multi-step FRET. These DSAs are categorized based on the non-covalent scaffolds, such as amphiphilic nanoparticles, host-guest assemblies, metal-coordination scaffolds, and biomolecular scaffolds. This perspective will also outline opportunities and future challenges in this research area.

An ultrasensitive smartphone-assisted bicolor-ratiometric fluorescence sensing platform based on a “noise purifier” for point-of-care testing of pathogenic bacteria in food

Non-specific binding in fluorescence resonance energy transfer (FRET) remains a challenge in foodborne pathogen detection, resulting in interference of high background signals. Herein, we innovatively reported a dual-mode FRET sensor based on a “noise purifier” for the ultrasensitive quantification of Escherichia coli O157:H7 in food. An efficient FRET system was constructed with polymyxin B-modified nitrogen-sulfur co-doped graphene quantum dots (N, S-GQDs@PMB) as donors and aptamer-modified yellow carbon dots (Y-CDs@Apt) as acceptors. Magnetic multi-walled carbon nanotubes (Fe@MWCNTs) were employed as a “noise purifier” to reduce the interference of the fluorescence background. Under the background purification mode, the sensitivity of the dual-mode signals of the FRET sensor has increased by an order of magnitude. Additionally, smartphone-assisted colorimetric analysis enabled point-of-care detection of E. coli O157:H7 in real samples. The developed sensing platform based on a “noise purifier” provides a promising method for ultrasensitive on-site testing of trace pathogenic bacteria in various foodstuffs.

Copper Nanosheet-Based Wash-Free Fluorescence Imaging of Cancer Cells.

Near-infrared fluorescence (NIRF) probes greatly facilitate in vivo imaging of various biologically important species. However, there are several significant limitations such as consuming washing steps, photobleaching, and low signal intensity. Herein, we synthesized fluorescent copper nanosheets templated with DNA scaffolds (DNS/CuNSs). We employ them and Cy5.5 of the fluorescence resonance energy transfer (FRET) system, which have a larger Stokes shift (∼12-fold) than the traditional NIRF dye Cy5.5. Based on their excellent fluorescence properties, we employ DNS/CuNSs-Cy5.5 for fluorescence probes in cancer cell imaging. Compared with the free Cy5.5 fluorescence probe, the novel fluorescence imaging probe implements wash-free imaging and exhibits enhanced anti-photobleaching ability (∼5.5-fold). Moreover, the FRET system constructed by DNS/CuNSs has a higher signal amplification ability (∼4.17-fold), which is more similar to that of Cu nanoclusters prepared with DNA nanomonomers as a template. This work provides a new idea for cancer cell MCF-7 imaging and is expected to promote the development of cancer cell fluorescence imaging.

A comparative study on fluorescent performance of several heterocyclic dyes: In aqueous solution or immobilized on zirconia nanotube arrays

The solution pH values, cosolvent type, and immobilization of the fluorescent dyes affect their dispersion state and fluorescent performance significantly. Zirconia, with abundant resources, low cost and high refractive index, is a commonly used immobilization material for fluorescent dyes. In this paper, zirconia nanotube array films (ZNAF) prepared by anodic oxidation method were used as immobilization materials for common heterocyclic dyes (fluorescein, rhodamine B, acridine, acridine orange). A systematically comparative study on their fluorescent performance of aqueous solutions at different pH values with or without cosolvent sodium benzenesulfonate and of immobilization films with ZNAF as carriers was investigated. Moreover, the performances of acridine orange-rhodamine B fluorescence resonance energy transfer (FRET) system with different dispersion states were studied in detail. Results demonstrate that the solution pH values, immobilization and sodium benzenesulfonate have great influence on the performance of these fluorescent dyes, and the change rules of different fluorescent molecules are obviously different, which is closely related to their molecular structure and dispersion state. Compared with aqueous solutions, the fluorescence emission of acridine orange and rhodamine B is greatly improved by immobilization, which is 8.6 and 5.4 times higher than the original, respectively. Furthermore, energy transfer efficiency (E) and enhancement ratio of the acridine orange-rhodamine B system are significantly increased by loading it on ZNAF, with a maximum E value of 84.8% (pH = 13) and a maximum enhancement ratio of 5.27 (pH = 5).

Fluorescent Selectivity-Enhanced FRET Based on 3D Photonic Crystals for Multianalyte Sensing.

Fluorescence resonance energy transfer (FRET) finds widespread utility in biochemical sensing, single-molecule experiments, cell physiology, and various other domains due to its inherent simplicity and high sensitivity. Nevertheless, the efficiency of energy transfer between the FRET donor and acceptor is significantly contingent on the local photonic environment, a factor that limits its application in complex systems or multianalyte detections. Here, a fluorescent selectivity-enhanced acridine orange (AO)-aflatoxins (AFs) FRET system based on a range of 3D topological photonic crystals (PCs) was developed with the aim of enhancing the selectivity and discrimination capabilities of FRET. By exploring the angle-dependent characteristics of the photonic stopband, the stopband distribution across different 3D topological PCs pixels was investigated. This approach led to selective fluorescence enhancement in PCs that matched the stopbands, enabling the successful discrimination of six distinct aflatoxins and facilitating complex multianalysis of moldy food samples. In particular, the stopband, which was strategically positioned within the blue-purple structural color range, exhibited a strong alignment with the fluorescence peaks of both the FRET donor and acceptor. This alignment allowed the 3D three-pointed star PCs to be effectively employed for the identification of mixed samples containing six distinct aflatoxins as well as the detection of real aflatoxin samples present in moldy potatoes, bread, oats, and peanuts. Impressively, this approach achieved a remarkable accuracy rate of 100%. This innovative strategy not only presents a novel avenue for developing a multitarget discrimination analysis system but also offers a convenient pretreatment method for the quantitative detection of various aflatoxins.

A point-of-care aptasensor based on the upconversion nanoparticles/MoS2 FRET system for the detection of Pseudomonas aeruginosa infection.

The rapid detection of Pseudomonas aeruginosa (P. aeruginosa) is of great significance for the diagnosis of medical infection. In view of the above, a novel aptasensor based on fluorescence resonance energy transfer (FRET) was developed. It contained aptamer-coupled upconversion nanoparticles (UCNPs-apt) as a donor (excitation 980nm) and molybdenum disulfide (MoS2) nanosheets as an acceptor. The upconversion fluorescence aptamer system was investigated to obtain the optimal parameters of MoS2 concentration, the incubation time of UCNPs-apt/MoS2 and P. aeruginosa, and pH. Based on the optimal parameters, a linear calibrationequation (emission 654nm) with a wide detection range 8.7 × 10 ~ 8.7 × 107cfu/mL, a high coefficient of determination R2 0.9941, and a low limit of determination (LOD) 15.5cfu/mL were established. The method was validated with P. aeruginosa infected foci of mouse wound. The advantage of this aptasensor isthat analysisresults can be obtained within 1.5h, which was much faster than that ofthe standard method (18-24h). Furthermore, combined with a portable instrument, it can be used as a point-of-care testing for the early detection of P. aeruginosa infection, which is useful for selecting the correct antibiotics to achieve good therapeutic effects. Additionally, it also has a broad application prospect in food and environmental areas.

Highly sensitive fluorescent probe and portable test strip based on polyacrylic acid functionalized quantum dots for rapid visual detection of malachite green

Malachite green (MG) has been banned in aquaculture by many countries due to its high carcinogenicity, high teratogenicity, and easy residue. However, it is cheap and efficient characteristics have made it difficult to eliminate in recent decades, so it is essential to develop a rapid and accurate detection method for MG. Here, a highly Sensitive fluorescent probe based on polyacrylic acid (PAA) functionalized CdSe/CdxZn1-xS quantum dots (QDs) was prepared for the determination of MG. QDs functionalized by PAA (QDs@PAA) were used as energy donors, and MG was used as energy acceptor to construct fluorescence resonance energy transfer (FRET) system. The fluorescence of QDs@PAA could be linearly quenched by MG in the range of 0.05 ⁓ 2 μM, and the detection limit was 0.011 μM. In addition, a small amount of QDs@PAA (30 μL) was printed on the solid substrate by inkjet printing technology to prepare fluorescent test strips. When the concentration of MG was 2 μM, the fluorescent test strips were quenched and the detection process could be completed within 10 s, demonstrating significant potential for rapid visual detection of MG.

Carbon Dot‐Based Fluorescence Resonance Energy Transfer (FRET) Systems for Biomedical, Sensing, and Imaging Applications

AbstractCarbon dots (CDs) emerge as a potential group of photo‐luminescent nano‐materials due to their excellent optical, electrical, and chemical properties, as well as their competence in a wide range of environmental applications. CDs have unique and appealing properties such as excellent stability, low toxicity, water solubility, and derivability. When coupled with CDs, fluorescence resonance energy transfer (FRET) results in the development of highly sensitive ratiometric fluorescence sensor probes with potential applications in bio‐imaging, metal sensing, membrane dynamics, and environmental sensing. In this review, the progress and recent developments in CDs based FRET systems utilized for various environmental applications are conferred. An in‐depth description is provided regarding the numerous donor/acceptor systems which when integrated with CDs generate efficient FRET systems. The review enables researchers to identify and develop specific systems which can be utilized to generate a FRET pair with potential physico–chemical properties that aid the development of the same for various applications.

A Two-Step Fluorescence-Resonance Energy Transfer System Constructed by Platinum(II) Metallacycle Based Molecular Recognition.

Considerable progress in the construction of efficient fluorescence-resonance energy transfer (FRET) systems has promoted the development of artificial energy transfer materials. However, despite recent advances, the exploration of efficient and easy strategies to fabricate novel supramolecular systems with FRET activities is still a challenge. Here, we report that a two-step FRET system was successfully achieved, driven by platinum metallacycle based host-guest interactions. The two-step FRET system is used for the preparation of a white-light-emitting diode and serves as a nanoreactor for the photosynthetic process. This work offers a strategy for the fabrication of FRET systems and opens opportunities for functional materials constructed by platinum(II) metallacycle based host-guest interactions.

Rhodamine hydrazide-linked naphthalimide derivative: Selective naked eye detection of Cu2+, S2− and understanding the therapeutic potential of the copper complex as an anti-cervical cancer agent

A naphthalimide-labeled rhodamine hydrazone derivative HL has been synthesized, characterized and examined in metal ion recognition. It shows selective colorimetric detection of Cu2+ over a number of other metal ions with a detection limit of 1.66 × 10−7 M in CH3CN/HEPES buffer (v/v = 2:1, pH = 6.8). The spirolactam ring of rhodamine and the imino-phenol motif of naphthalimide in HL are involved in complexation of Cu2+ as shown by single crystal X-ray. Single crystal of the copper-complex is prepared by utilizing NaSCN and it is characterized as CuL(SCN). The emergence of new absorption at 550 nm in UV–vis and the pink color of the solution reveal the selective interaction toward Cu2+. HL is characterized as a fluorescence resonance energy transfer (FRET) system that remains ‘turned OFF’ while spirolactam ring exists. In the presence of Cu2+, FRET is ‘turned ON’ via the opening of spirolactam ring to give emission at 580 nm which is less intense due to the quenching effect of Cu2+ ion. The complexation is reversible and the ensemble of Cu2+.HL selectively recognizes S2− over a series of different anions involving a color change from pink to colorless via the formation of spirolactam ring. The copper complex CuL(SCN) is further employed to understand its efficacy as a therapeutic agent. The complex is cytotoxic to high-risk HPV positive cervical cancer cell lines like SiHa and HeLa and is efficient in the generation and accumulation of reactive oxygen species (ROS). The complex also initiates nuclear blebbing and shows DNA degradation as understood by DNA laddering assay.

Highly selective and sensitive biological probe for cysteine detection based on fluorescence resonance energy transfer between lanthanide oxysulfide nanoparticles and Au nanoparticles

Biothiols are biologically important targets because their transport and metabolism are closely related to the activities of many proteins and biological enzymes. In this paper, a kind of biological probe based on the fluorescence resonance energy transfer between lanthanide oxysulfide and gold nanoparticles was developed for the detection of cysteine. Ln2O2S:Tb3+ and Ln2O2S:Eu3+ nanoparticles were prepared by a thermolysis method. The as-prepared fluorescent nanoparticles were modified by polyethylenimine ligand exchange to increase their hydrophilicity. Then, Au nanoparticles were added to form fluorescence resonance energy transfer systems in which Au NPs and La2O2S:Ln3+ act as the acceptor and donor, respectively. Among the Ln2O2S:Tb3+ and Ln2O2S:Eu3+ systems, the cysteine content and fluorescence intensity showed good proportional proportionality, with detection limits of 6.75 nM and 14.09 nM in the range of 50∼1500 nM, respectively. The results of anti-interference experiments showed that this biological probe is highly selective for cysteine detection.

Ultrafast Dynamics of the Medicinal Pigment Curcumin inside the Imidazolium Surface Active Ionic Liquid Containing Giant Vesicles and White Light Generation via Triple-FRET Technique.

The naturally occurring yellow polyphenolic medicinal pigment curcumin shows ultrafast dynamics in the excited states. These ultrafast dynamics are strongly influenced by the rigidity of the environments of the systems. The present investigation unveils the ultrafast excited-state intramolecular hydrogen atom transfer (ESIHT) (which is involved in the antioxidant mechanism) and the solvation dynamics of curcumin inside the imidazolium surface active ionic liquid (SAIL), 1-hexadecyl-3-methylimidazolium chloride ([C16mim]Cl) micelle, and giant vesicles after introducing sorbitan monoesters (Span 20 and Span 80) in the aqueous medium. Interestingly, the short hydrocarbon chain containing Span 20 forms smaller, less rigid vesicles, and the long hydrocarbon chain containing Span 80 forms larger, more rigid giant vesicles after being assembled with [C16mim]Cl. The ESIHT and the solvation dynamics are slower in Span 80, containing rigid vesicles, than that in Span 20, comprising less rigid vesicles. Finally, we have established a three-component fluorescence resonance energy transfer (Triple-FRET) system to generate white light (WL) in the micelle and giant vesicles. Here the hydrophobic dye 1,6-diphenyl-1,3,5-hexatriene (DPH) acts as the donor, and the hydrophilic anticancer drug doxorubicin hydrochloride (DOX) serves as the acceptor along with the intermediate donor, curcumin. At a specific combination of the concentrations of these dyes in a particular self-assembled system, WL is generated due to the triple-FRET phenomena.

Surfactant regulated aggregation-induced emission of tetraphenylethene-based tetracationic salt for the construction of light harvesting supramolecular system

Fluorescence resonance energy transfer (FRET) is an efficient optical molecular scale that has been widely used in many fields, and aggregation-induced emission (AIE) luminescent materials show great potential in constructing FRET systems. In this paper, an efficient supramolecular polymer of AIE was prepared in aqueous solution based on tetraphenylethene-based tetracationic salt molecule (1) with AIE properties, using the hydrophobic effect and electrostatic interaction with sodium dodecyl sulfonate (AS). Then, based on supramolecular aggregate (1-AS) and Rhodamine B (RB) dye, the efficient FRET system was prepared in aqueous solution with 1-AS as energy donor and RB as energy acceptor. The Stokes shift of the system can reach 208 nm, and the fluorescence intensity of RB aqueous solution with the same concentration increased 11.80 times. Moreover, the energy transfer efficiency and the antenna effect value reached 77.5%, 14.3, respectively. The method based on simple AIE donor to achieve efficient luminescence of dye molecules is of great significance for the realization of efficient light-trapping system of aqueous solution in practical applications.

Simple design of upconversion nanoparticles-based aptasensor integrated microfluidic chip for ultra-sensitive detection of bisphenol A in aquatic products

Bisphenol A (BPA) is an endocrine disrupting chemical having estrogenic properties that can enter the body through leaking from food containers or the food chain. An upconversion nanoparticle-based aptasensor integrated with microfluidic chip was simply designed to achieve the determination of BPA in foodstuff. Core-shell structured upconversion nanoparticles with 6.41-fold luminescence enhancement were used as fluorescence donors and gold nanoparticles as fluorescence acceptors to construct fluorescence resonance energy transfer (FRET) system. The BPA aptamer serves as a link between the building of the FRET system and the BPA-specific recognition component. The fishbone-structured microfluidic chip only needs 0.5 μL sample solution and 4 min to determine the presence of BPA. The proposed method has good linearity for BPA in the range of 0.02–100 ng/mL with calibration curve of y = 0.0918x + 0.6477 and limit of detection of 0.014 ng/mL. It is tremendously competitive to perform on-chip analysis that enables microsampling and rapid determination.

Enhanced Energy Transfer in the Co‐assembly of Donor‐Receptor Polymer Nanoparticles: An Artificial Light Capture System to Trigger Effective Photodynamic Antibacterial Processes

AbstractLight‐harvesting system is an important way for capturing, transferring and utilizing light energy. It remains a key challenge to develop highly efficient artificial light‐harvesting systems. Herein, we report a supramolecular co‐assembly based on 5,10,15,20‐tetrakis (4‐hydroxyphenyl) porphyrin (THPP) and fluorescein derivatives as an artificial light‐harvesting systems. The assemblies use fluorescein derivatives as a donor and porphyrin (THPP) as an acceptor unit by covalent linkage and achieved fluorescence resonance energy transfer (FRET) owing to their strong spectral overlap and short distance. Firstly, three linkers of different chain lengths were synthesized through bromoalkane, and three fluorescein derivatives were obtained by connecting the linker and fluorescein through covalent bonds according to the principle of substitution reaction. Then, FRET systems between fluorescein derivatives and THPP were developed. As a result, a nanocapsule with an ultrathin shell designed for mimicking natural light‐harvesting systems was successfully constructed based on the covalent assembly of donor molecule called fluorescein and acceptor molecule called THPP. To our delight, the covalent FRET probes achieved enhanced energy transfer and a longer fluorescence lifetime. The covalent FRET probes also showed the obvious growth inhibition of Staphylococcus aureus cells in photodynamic antibacterial. Therefore, this study provides a new and efficient strategy to construct artificial light‐harvesting systems and they hold great promise for photophysical and photochemical applications.

Updates in Laboratory Identification of Invasive Fungal Infection in Neonates.

Invasive fungal infection (IFI) in immunocompromised neonates is significantly associated with high morbidity and mortality and has become the third most common infection in Neonatal Intensive Care Units. The early diagnosis of IFI for neonatal patients is difficult because of the lack of specific symptoms. The traditional blood culture remains the gold standard in clinical diagnosis for neonatal patients but it requires a long duration, which delays treatment initiation. Detections of fungal cell-wall components are developed for early diagnosis but the diagnostic accuracy in neonates needs to be improved. PCR-based laboratory methods, such as real-time PCR, droplet digital PCR, and the cationic conjugated polymer fluorescence resonance energy transfer (CCP-FRET) system, distinguish the infected fungal species by their specific nucleic acids and show a high sensitivity and specificity. Particularly, the CCP-FRET system, which contains a cationic conjugated polymer (CCP) fluorescent probe and pathogen-specific DNA labeled with fluorescent dyes, could identify multiple infections simultaneously. In the CCP-FRET system, the CCP and fungal DNA fragments can self-assemble into a complex with an electrostatic interaction and the CCP triggers the FRET effect under ultraviolet light to make the infection visible. Here, we summarize the recent laboratory methods for neonatal IFI identification and provide a new perspective for early clinical fungal diagnosis.

Development of FRET and Stress Granule Dual-Based System to Screen for Viral 3C Protease Inhibitors

3C proteases (3Cpros) of picornaviruses and 3C-like proteases (3CLpros) of coronaviruses and caliciviruses represent a group of structurally and functionally related viral proteases that play pleiotropic roles in supporting the viral life cycle and subverting host antiviral responses. The design and screening for 3C/3CLpro inhibitors may contribute to the development broad-spectrum antiviral therapeutics against viral diseases related to these three families. However, current screening strategies cannot simultaneously assess a compound's cytotoxicity and its impact on enzymatic activity and protease-mediated physiological processes. The viral induction of stress granules (SGs) in host cells acts as an important antiviral stress response by blocking viral translation and stimulating the host immune response. Most of these viruses have evolved 3C/3CLpro-mediated cleavage of SG core protein G3BP1 to counteract SG formation and disrupt the host defense. Yet, there are no SG-based strategies screening for 3C/3CLpro inhibitors. Here, we developed a fluorescence resonance energy transfer (FRET) and SG dual-based system to screen for 3C/3CLpro inhibitors in living cells. We took advantage of FRET to evaluate the protease activity of poliovirus (PV) 3Cpro and live-monitor cellular SG dynamics to cross-verify its effect on the host antiviral response. Our drug screen uncovered a novel role of Telaprevir and Trifluridine as inhibitors of PV 3Cpro. Moreover, Telaprevir and Trifluridine also modulated 3Cpro-mediated physiological processes, including the cleavage of host proteins, inhibition of the innate immune response, and consequent facilitation of viral replication. Taken together, the FRET and SG dual-based system exhibits a promising potential in the screening for inhibitors of viral proteases that cleave G3BP1.

Construction and application of thrombin-activated fluorescence-SERS dual-mode optical nanoprobes

Thrombin (TB) plays a key role in the pathological and physiological coagulation of diseases. In this work, a TB-activated fluorescence-surface-enhanced Raman spectroscopy (SERS) dual-mode optical nanoprobe (MRAu) was constructed by linking rhodamine B (RB)-modified magnetic fluorescent nanospheres with AuNPs through TB-specific recognition peptides. In the presence of TB, the polypeptide substrate could specifically be cleaved by TB, resulting in the weakening of SERS hotspot effect and the reduction of Raman signal. Meanwhile, the fluorescence resonance energy transfer (FRET) system was destroyed, and the RB fluorescence signal originally quenched by AuNPs was recovered. Using MRAu, SERS and fluorescence methods were combined to extend the TB detection range to 1–150 pM, and the detection limit was as low as 0.35 pM. In addition, the ability to detect TB in human serum also verified the effectiveness and practicality of the nanoprobe. The probe was also successfully employed to evaluate the inhibitory effect against TB of active components in Panax notoginseng. This study provides a new technical means for the diagnosis and drug development of abnormal TB-related diseases.

Conformational dynamics of eukaryotic translation.

The ribosome is a molecular machine that adopts multiple conformations during translation. The body of the small ribosomal subunit rotates relatively to the large ribosomal subunit by about 9 degrees. This movement reconfigures tRNA binding sites, which results in a partial movement of tRNAs on the ribosome. At the beginning of the elongation cycle, the ribosome awaits A-site decoding and occupies the non-rotated conformation. Upon A-site tRNA binding, the ribosome rapidly catalyzes peptidyl transfer. It is followed by counterclockwise rotations of the small ribosomal subunit that puts the ribosome into the rotated state. The resultant pre-translocation complex is dynamic and exchanges between rotated and non-rotated conformations. Eukaryotic elongation factor 2 (eEF2) catalyzes translocation, which moves tRNA-mRNA and resets the ribosome into the non-rotated state. Despite extensive biochemical and structural studies, the underlying mechanism of translation elongation remains elusive. We developed a single-molecule fluorescence resonance energy transfer (smFRET) system that allows the observation of intersubunit conformational change of yeast ribosomes in real-time. We followed the ribosome conformational change in pre- and post-translocation ribosome complexes. To interrogate the mechanism of translocation, we applied antifungal sordarin. Sordarin stabilizes eEF2 on the ribosome after GTP hydrolysis and phosphate release. Our results suggest that in the presence of sordarin, the mid and late stages of translocation are thermally driven.