Förster Resonance Energy Transfer Research Articles

Next‐Generation Metabolic Glycosylation Reporters Enable Detection of Protein O−GlcNAcylation in Living Cells without S‐Glyco Modification

AbstractProtein O−GlcNAcylation is a ubiquitous posttranslational modification of cytosolic and nuclear proteins involved in numerous fundamental regulation processes. Investigation of O−GlcNAcylation by metabolic glycoengineering (MGE) has been carried out for two decades with peracetylated N‐acetylglucosamine (GlcNAc) and N‐acetylgalactosamine derivatives modified with varying reporter groups. Recently, it has been shown that these derivatives can result in non‐specific protein labeling termed S‐glyco modification. Here, we report norbornene‐modified GlcNAc derivatives with a protected phosphate at the anomeric position and their application in MGE. These derivatives overcome two limitations of previously used O−GlcNAc reporters. They do not lead to detectable S‐glyco modification, and they efficiently react in the inverse‐electron‐demand Diels–Alder (IEDDA) reaction, which can be carried out even within living cells. Using a derivative with an S‐acetyl‐2‐thioethyl‐protected phosphate, we demonstrate the protein‐specific detection of O−GlcNAcylation of several proteins and the protein‐specific imaging of O−GlcNAcylation inside living cells by Förster resonance energy transfer (FRET) visualized by confocal fluorescence lifetime imaging microscopy (FLIM).

Luminescent metal–organic frameworks for ultrasensitive detection of pesticide and biomarker: In situ imaging and real field application

Frequent uses of pesticides for agricultural led to the environmental pollution and ultimately threaten the human body. On the other hand, the monitoring of the biomarker at the optimum level is also very imperative for human health. So the investigation of smart materials is very essential for visual and on‐site monitoring of pesticide and biomarker. In this regard, we have synthesized highly fluorescent MOF (ZnATA, 1) oriented with free‐NH2 functionality as a guest functional moiety. Framework 1 showed the selective and sensitive ratiometric detection of biomarker riboflavin (RBF). Further, sensor 1 exhibited the discriminating recognition of pesticide 2,6‐dichloro‐4‐nitroaniline (DCNA). The detection limit for both RBF and DCNA is very excellent, and the values are 0.13 and 0.07 ppm, respectively. The detection of RBF and DCNA is very fast (within 3 s) and has been realized through colorimetric response and by cost‐effective paper strip method. Interestingly, the sensor displayed the visual and on‐site (in situ imaging by using real fruit and vegetable) recognition of “DCNA” within 3 s. Sensor 1 further demonstrated the recognition of DCNA in different real field sample (environmental water sample) and RBF in food samples (milk and vitamin B2 tablet). The mechanism of sensing for DCNA and RBF has been comprehended through the occurrence of photo‐induced electron transfer, forster resonance energy transfer, and inert filter effect. Further, the recognition mechanism is justified by the theoretical study. The presence of free‐NH2 moiety in the linker of MOF is possibly to establish a strong supramolecular interaction with the analytes.

A molecular dynamics simulation study of glycine/serine octapeptides labeled with 2,3-diazabicyclo[2.2.2]oct-2-ene fluorophore.

The compound 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) is a versatile fluorophore widely used in Förster resonance energy transfer (FRET) spectroscopy studies due to its remarkable sensitivity, enabling precise donor-acceptor distance measurements, even for short peptides. Integrating time-resolved and FRET spectroscopies with molecular dynamics simulations provides a robust approach to unravel the structure and dynamics of biopolymers in a solution. This study investigates the structural behavior of three octapeptide variants: Trp-(Gly-Ser)3-Dbo, Trp-(GlyGly)3-Dbo, and Trp-(SerSer)3-Dbo, where Dbo represents the DBO-containing modified aspartic acid, using molecular dynamics simulations. Glycine- and serine-rich amino acid fragments, common in flexible protein regions, play essential roles in functional properties. Results show excellent agreement between end-to-end distances, orientational factors from simulations, and the available experimental and theoretical data, validating the reliability of the GROMOS force field model. The end-to-end distribution, modeled using three Gaussian distributions, reveals a complex shape, confirmed by cluster analysis highlighting a limited number of significant conformations dominating the peptide landscape. All peptides predominantly adopt a disordered state in the solvent, yet exhibit a compact shape, aligning with the model of disordered polypeptide chains in poor solvents. Conformations show marginal dependence on chain composition, with Ser-only chains exhibiting slightly more elongation. This study enhances our understanding of peptide behavior, providing valuable insights into their structural dynamics in solution.

Evaluation of miR-155 silencing using a molecular beacon in human lung adenocarcinoma cell line

Lung cancer (LC) is a leading cause of global cancer-related deaths, highlighting the development of innovative methods for biomarker detection improving the early diagnostics. microRNAs (miRs) alterations are known to be involved in the initiation and progression of human cancers and can act as biomarkers for diagnostics and treatment.Herein, we develop the application of molecular beacon (MB) technology to monitor miR-155-3p expression in human lung adenocarcinoma A549 cells without complementary DNA synthesis, amplification, or expensive reagents. Furthermore, we produced gold nanoparticles (AuNPs) for delivering antisense oligonucleotides into A549 cells to reduce miR-155-3p expression, which was subsequently detectable using the MB. The MB was designed and structural characterized by Förster Resonance Energy Transfer (FRET)-melting, Circular Dichroism (CD), Nuclear magnetic resonance (NMR), and fluorometric experiments, and then the hybridization conditions were optimized for an in vitro approach involving the detection of miR-155-3p in total RNA extracted from A549 cell line. The expression profile of miR-155-3p was obtained by RT-qPCR.The results demonstrated that MB was properly designed and showed efficacy in targeting miR-155-3p. Furthermore, a limit of detection down to nanomolar concentration was achieved and the specificity of the biosensor was proved. Moreover, the self-assembly of ASOs with AuNPs exhibited exceptional target specificity, effectively silencing miR-155-3p. Notably, compared to lipid-based transfection agent, AuNPs displayed superior silencing efficiency.We highlighted the ability of MB to detect changes in the target gene expression after gene silencing. Overall, this innovative approach represents a promising tool for detecting various biomarkers at the same time, with potential applications in clinical settings.

Engineering Metal Halide Perovskite Nanocrystals with BODIPY Dyes for Photosensitization and Photocatalytic Applications.

The sensitization of surface-anchored organic dyes on semiconductor nanocrystals through energy transfer mechanisms has received increasing attention owing to their potential applications in photodynamic therapy, photocatalysis, and photon upconversion. Here, we investigate the sensitization mechanisms through visible-light excitation of two nanohybrids based on CsPbBr3 perovskite nanocrystals (NC) functionalized with borondipyrromethene (BODIPY) dyes, specifically 8-(4-carboxyphenyl)-1,3,5,7-tetramethyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BDP) and 8-(4-carboxyphenyl)-2,6-diiodo-1,3,5,7-tetramethyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (I2-BDP), named as NC@BDP and NC@I2-BDP, respectively. The ability of I2-BDP dyes to extract hot hole carriers from the perovskite nanocrystals is comprehensively investigated by combining steady-state and time-resolved fluorescence as well as femtosecond transient absorption spectroscopy with spectroelectrochemistry and quantum chemical theoretical calculations, which together provide a complete overview of the phenomena that take place in the nanohybrid. Förster resonance energy transfer (FRET) dominates (82%) the photosensitization of the singlet excited state of BDP in the NC@BDP nanohybrid with a rate constant of 3.8 ± 0.2 × 1010 s-1, while charge transfer (64%) mediated by an ultrafast charge transfer rate constant of 1.00 ± 0.08 × 1012 s-1 from hot states and hole transfer from the band edge is found to be mainly responsible for the photosensitization of the triplet excited state of I2-BDP in the NC@I2-BDP nanohybrid. These findings suggest that the NC@I2-BDP nanohybrid is a unique energy transfer photocatalyst for oxidizing α-terpinene to ascaridole through singlet oxygen formation.

Water‐Soluble Fluorescent Polymer Dyes with Tunable Emission Spectra for Flow Cytometry Applications

AbstractThe application of spectrally unique, bright, and water‐soluble fluorescent dyes is indispensable for the analysis of biological systems. Multiparameter flow cytometry is a powerful tool for characterization of mixed cell populations. To discriminate the different cell populations, they are typically stained by a set of fluorescent reagents, e.g., antibody‐fluorophore conjugates. The number of parameters which can be studied simultaneously strongly depends on the availability of reagents which can be differentiated by their spectral properties. In this study a series of fluorescent polymer dyes was developed, that can be excited with a single violet laser (405 nm) but distinguished by their unique emission spectra. The polyfluorene‐based polymers can be used on their own, or in combination with covalently bound small‐molecule dyes to generate energy transfer constructs to red‐shift the emission wavelength based on Förster resonance energy transfer (FRET). The polymer dyes were utilized in a biological flow cytometry assay by conjugating several of them to antibodies, demonstrating their effectiveness as reagents. This report represents the first systematic investigation of structure–property relationships for this type of fluorescent dyes.

Water-Soluble Fluorescent Polymer Dyes with Tunable Emission Spectra for Flow Cytometry Applications.

The application of spectrally unique, bright, and water-soluble fluorescent dyes is indispensable for the analysis of biological systems. Multiparameter flow cytometry is a powerful tool for characterization of mixed cell populations. To discriminate the different cell populations, they are typically stained by a set of fluorescent reagents, e.g., antibody-fluorophore conjugates. The number of parameters which can be studied simultaneously strongly depends on the availability of reagents which can be differentiated by their spectral properties. In this study a series of fluorescent polymer dyes was developed, that can be excited with a single violet laser (405 nm) but distinguished by their unique emission spectra. The polyfluorene-based polymers can be used on their own, or in combination with covalently bound small-molecule dyes to generate energy transfer constructs to red-shift the emission wavelength based on Förster resonance energy transfer (FRET). The polymer dyes were utilized in a biological flow cytometry assay by conjugating several of them to antibodies, demonstrating their effectiveness as reagents. This report represents the first systematic investigation of structure-property relationships for this type of fluorescent dyes.

Energy transfer enhanced photocatalytic hydrogen evolution in organic heterostructure nanoparticles via flash nanoprecipitation processing

Organic nanophotocatalysts are promising candidates for solar fuels production, but they still face the challenge of unfavorable geminate recombination due to the limited exciton diffusion lengths. Here, we introduce a binary nanophotocatalyst fabricated by blending two polymers, PS-PEG5 (PS) and PBT-PEG5 (PBT), with matched absorption and emission spectra, enabling a Förster resonance energy transfer (FRET) process for enhanced photocatalysis. These heterostructure nanophotocatalysts are processed using a facile and scalable flash nanoprecipitation (FNP) technique with precious kinetic control over binary nanoparticle formation. The resulting nanoparticles exhibit an exceptional photocatalytic hydrogen evolution rate up to 65 mmol g−1 h−1, 2.5 times higher than that single component nanoparticle. Characterizations through fluorescence spectra and transient absorption spectra confirm the hetero-energy transfer within the binary nanoparticles, which prolongs the excited-state lifetime and extends the namely “effective exciton diffusion length”. Our finding opens new avenues for designing efficient organic photocatalysts by improving exciton migration.

A combined experimental and theoretical approach for doxycycline sensing using simple fluorescent probe with distinct fluorescence change in wide range of interferences

Overuse of doxycycline (DOXY) can cause serious problems to human health, environment and food quality. So, it is essential to develop a new sensing methodology that is both sensitive and selective for the quantitative detection of DOXY. In our current research, we synthesized a simple fluorescent probe 4,4′-bis(benzyloxy)-1,1′-biphenyl (BBP) for the highly selective detection of doxycycline by through fluorescence spectroscopy. The probe BBP displayed ultra-sensitivity towards doxycycline due to Forster resonance energy transfer (FRET). Fluorescence spectroscopy, density functional theory (DFT), 1H NMR titration, UV–Vis, and Job's plot were used to confirm the sensing mechanism. The charge transfer between the probe and analyte was further examined qualitatively by electron density differences (EDD) and quantitively by natural bond orbital (NBO) analyses. Whereas the non-covalent nature of probe BBP towards DOXY was verified by theoretical non-covalent interaction (NCI) analysis as along with Bader's quantum theory of atoms in molecules (QTAIM) analysis. Furthermore, probe BBP was also practically employed for the detection of doxycycline in fish samples, pharmaceutical wastewater and blood samples.

Simulation and design of dual-wavelength all-optical semiconductor optical amplifier with solution-processed quantum dots

All-optical semiconductor optical amplifiers (SOAs) present a cost-effective and highly efficient solution for long-distance WDM networks. In this study, the investigation of a dual-wavelength all-optical SOA incorporating two distinct sizes of solution-processed InAs/AlAs core/shell quantum dots (QDs), operating at 1.31 and 1.55 μm wavelengths, both simultaneously and individually, has been proposed. To comprehensively evaluate the optical performance of our proposed dual-wavelength all-optical SOA, the modified coupled rate equations governing carriers dynamics and the optical photon propagation equations have been solved numerically while taking into account both homogeneous and inhomogeneous broadening, as well as fluorescence or förster resonance energy transfer (FRET) effects. This computational approach provides valuable insights into the optical behavior of dual-wavelength all-optical SOA. In our proposed structure, cost-effective solution-based methods for implementing QDs of varying sizes offer flexibility to adjust QD size across a broad range, from as small as 0.1 nm to meet specific requirements. This adjustment is made with meticulous consideration of appropriate synthesis techniques.

Semi-Transparent Luminescent Solar Concentrators Based on Intramolecular Energy Transfer in Polyurethane Matrices.

Luminescent solar concentrators (LSCs) are spectral conversion devices offering interesting opportunities for the integration of photovoltaics into the built environment and portable systems. The Förster-resonance energy transfer (FRET) process can boost the optical response of LSCs by reducing energy losses typically associated to non-radiative processes occurring within the device under operation. In this work, a new class of FRET-based thin-film LSC devices is presented, in which the synthetic versatility of linear polyurethanes (PU) is exploited to control the photophysical properties and the device performance of the resulting LSCs. A series of luminescent linear PUs are synthesized in the presence of two novel bis-hydroxyl-functionalized luminophores of suitable optical properties, used as chain extenders during the step-growth polyaddition reaction for the formation of the linear macromolecular network. By synthetically tuning their composition, the obtained luminescent PUs can achieve a high energy transfer efficiency (≈90%) between the covalently linked luminophores. The corresponding LSC devices exhibit excellent photonic response, with external and internal photon efficiencies as high as ≈4% and ≈37%, respectively. Furthermore, their optimized power conversion efficiency combined with their enhanced average visible-light transmittance highlight their suitability for potential use as transparent solar energy devices.

Ultra-broadband triple-peak white light-emitting carbonized polymer dots materials based on Förster resonance energy transfer

Carbonized polymer dots (CPDs) with white-light-emitting property have greatly promising application in next generation of lighting and display technologies. However, most of the reported CPD materials exhibit single peak emission and narrow emission band, resulting in difficulty to obtain white-light emission. In this work, the ultra-broadband triple-peak emission (red, green, and blue) CD-based materials with high-efficiency white light-emitting property are realized for the first time by the Förster resonance energy transfer (FRET). Blue and green emissions are derived from the fluorescence and phosphorescence of donor CPDs, while the red light is derived from receptor by FRET. Remarkably, the fabricated CPD materials show bright pure white-light emission with high overall quantum yield (QY) of 36% and the full width at half maximum of 235 nm. Besides, afterglow colors of CPD-based materials can be tuned from green to red by adjusting the ratio of donor and acceptor. Based on the high-efficiency and wide-spectrum pure white light emission characteristics of CPD-based composites, white light emitting diodes were fabricated and they exhibit bright warm white light with CIE and CCT of (0.35, 0.31) and 4041 K.

Improvement of the Green-Red Förster Resonance Energy Transfer-Based Ca2+ Indicator by Using the Green Fluorescent Protein, Gamillus, with a Trans Chromophore as the Donor.

To monitor the Ca2+ dynamics in cells, various genetically encoded Ca2+ indicators (GECIs) based on Förster resonance energy transfer (FRET) between fluorescent proteins are widely used for live imaging. Conventionally, cyan and yellow fluorescent proteins have been often used as FRET pairs. Meanwhile, bathochromically shifted indicators with green and red fluorescent protein pairs have various advantages, such as low toxicity and autofluorescence in cells. However, it remains difficult to develop them with a similar level of dynamic range as cyan and yellow fluorescent protein pairs. To improve this, we used Gamillus, which has a unique trans-configuration chromophore, as a green fluorescent protein. Based on one of the best high-dynamic-range GECIs, Twitch-NR, we developed a GECI with 1.5-times higher dynamic range (253%), Twitch-GmRR, using RRvT as a red fluorescent protein. Twitch-GmRR had high brightness and photostability and was successfully applied for imaging the Ca2+ dynamics in live cells. Our results suggest that Gamillus with trans-type chromophores contributes to improving the dynamic range of GECIs. Therefore, selection of the cis-trans isomer of the chromophore may be a fundamental approach to improve the dynamic range of green-red FRET indicators, unlimited by GECIs.

An optofluidic antenna for enhancing the sensitivity of single-emitter measurements

Many single-molecule investigations are performed in fluidic environments, for example, to avoid unwanted consequences of contact with surfaces. Diffusion of molecules in this arrangement limits the observation time and the number of collected photons, thus, compromising studies of processes with fast or slow dynamics. Here, we introduce a planar optofluidic antenna (OFA), which enhances the fluorescence signal from molecules by about 5 times per passage, leads to about 7-fold more frequent returns to the observation volume, and significantly lengthens the diffusion time within one passage. We use single-molecule multi-parameter fluorescence detection (sm-MFD), fluorescence correlation spectroscopy (FCS) and Förster resonance energy transfer (FRET) measurements to characterize our OFAs. The antenna advantages are showcased by examining both the slow (ms) and fast (50 μs) dynamics of DNA four-way (Holliday) junctions with real-time resolution. The FRET trajectories provide evidence for the absence of an intermediate conformational state and introduce an upper bound for its lifetime. The ease of implementation and compatibility with various microscopy modalities make OFAs broadly applicable to a diverse range of studies.

DFT, real sample and smartphone studies of fluorescent probe, N-doped carbon quantum dots for sensitive and selective detection of bilirubin

In this investigation, nitrogen-doped carbon quantum dots (NCQDs) were successfully synthesized, showcasing robust fluorescence characteristics and photostability. These NCQDs demonstrated biocompatibility and were utilized as effective sensing agents for bilirubin (BR) detection. Their remarkable Stern-Volmer constant, attributed to exceptional molar absorptivity and photoluminescence quantum yield, rendered them highly proficient in BR sensing. Various analytical techniques such as X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDS), transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS) were employed to characterize the synthesized NCQDs, providing comprehensive insights into their structural, elemental, chemical composition, particle size, morphology, and optical properties. For sensing purposes, UV–Vis, steady-state, and time-resolved fluorescence spectroscopic techniques were utilized. The average particle size of the NCQDs was determined to be approximately 4.5 ± 0.3 nm. Notably, the Stern-Volmer constant (Ksv) for BR sensing from these quantum dots surpassed previously reported values in the literature. A linear relationship between emission intensity ratio and BR concentration within a range of 1.35–14.20 μM was observed. Accurate quantification of BR was achieved with a high Stern-Volmer constant (Ksv) S11 of approximately 7.48 × 105 M-1 and a limit of detection (LOD) of 4.86 μM. Leveraging the NCQDs as fluorescent probes, a highly sensitive and selective BR detection method based on the Förster resonance energy transfer (FRET) mechanism was established, with emission occurring at 360 nm. Density functional theory (DFT) studies supported FRET as the primary mechanism for fluorescence quenching, indicating electron transfer from NCQDs to BR, as evidenced by the energies of the lowest unoccupied molecular orbitals (LUMOs) of both NCQDs and BR. Real sample analyses of BR levels in human serum yielded a recovery range of 94.8 % to 104.21 %, validating the practical applicability of the method. Furthermore, smartphone-based BR detection using NCQDs achieved an LOD of 11.74 µM.

Förster Resonance Energy Transfer: Stimulus-Responsive Purely Organic Room Temperature Phosphorescence through Dynamic B-N bond.

Recently, stimulus-responsive organic materials with room-temperature phosphorescence (RTP) properties have attracted significant attention owing to their potential applications in chemical sensing, anticounterfeiting, and displays. However, molecular design currently lacks systematicity and effectiveness. Herein, we report a capture-release strategy for the construction of reversible RTP via B/N Lewis pairs. Specifically, the RTP of the Lewis acid of 7-bromo-5,9-dioxa-13b-boranaphtho[3,2,1-de]anthracene (BrBA) can be deactivated through capturing by the Lewis base, N,N-diphenyl-4-(pyridin-4-yl)aniline (TPAPy), and reactivated by dissociation of B-N bonds to release BrBA. Reversible RTP is attributed to the exceptional self-assembly capability of BrBA, whereas the tunable RTP colors are derived from distinct Förster resonance energy transfer (FRET) processes. The potential applications of RTP materials in information storage and anti-counterfeiting were also experimentally validated. The capture-release approach proposed in this study offers an effective strategy for designing stimulus-responsive materials.

Enhanced upconversion luminescence of NaYF4:Er nanocrystals based on the synergy between whispering gallery mode excitation and förster resonant energy transfer

The upconversion luminescence at the visible band was excited by the high power density whispering gallery mode (WGM) on the surface of the microsphere. Under the WGM excitation, Er3+ of NaYF4:Er3+ directly absorbed three photons at 1525 nm, followed by the generation of upconversion luminescence. We used WS2 quantum dots (QDs) as donors and NaYF4:Er3+ upconversion nanoparticles (UCNPs) as acceptors to enhance upconversion luminescence by Förster resonant energy transfer (FRET). The physical mixture composed of monolayer WS2 and NaYF4:Er3+ nanoparticles was coated on SiO2 microsphere by immersion method. Excited by high power density WGM, three photons were absorbed by WS2-QDs based on the quantum size effect to generate excited electrons as donors and UCNPs as acceptors based on FRET, subsequently, the energy of the electrons were transferred to the luminescence center Er3+ to improve the upconversion luminescence efficiency. The mechanism model of upconversion luminescence enhancement based on FRET of WS2–NaYF4:Er3+ was proposed, and the enhancement effect was experimentally studied and demonstrated. To study the distance dependence of energy transfer between QD and UCNP pairs, we covered the surface of the silica microsphere with a controlled concentration of WS2 mixed with NaYF4:Er3+ nano-particles. When the mixed concentration of WS2-QDs was 0.15%, the upconversion luminescence reached the highest efficiency. The results showed that a twofold upconversion luminescence enhancement can be experimentally achieved. The proposed approach for enhancing the upconversion luminescence could benefit the improvement of imaging signal-to-noise ratio (SNR) using UCNPs for fluorescent labels in life sciences.

Förster Resonance Energy Transfer: Stimulus‐Responsive Purely Organic Room Temperature Phosphorescence through Dynamic B−N bond

AbstractRecently, stimulus‐responsive organic materials with room‐temperature phosphorescence (RTP) properties have attracted significant attention owing to their potential applications in chemical sensing, anticounterfeiting, and displays. However, molecular design currently lacks systematicity and effectiveness. Herein, we report a capture‐release strategy for the construction of reversible RTP via B/N Lewis pairs. Specifically, the RTP of the Lewis acid of 7‐bromo‐5,9‐dioxa‐13b‐boranaphtho[3,2,1‐de]anthracene (BrBA) can be deactivated through capturing by the Lewis base, N,N‐diphenyl‐4‐(pyridin‐4‐yl)aniline (TPAPy), and reactivated by dissociation of B−N bonds to release BrBA. Reversible RTP is attributed to the exceptional self‐assembly capability of BrBA, whereas the tunable RTP colors are derived from distinct Förster resonance energy transfer (FRET) processes. The potential applications of RTP materials in information storage and anti‐counterfeiting were also experimentally validated. The capture‐release approach proposed in this study offers an effective strategy for designing stimulus‐responsive materials.

Rapid FRET Assay for the Early Detection of Alpha-Synuclein Aggregation in Parkinson's Disease.

Alpha-synuclein (α-Syn) is a key protein of Parkinson's disease (PD). Oligomers formed by misfolding and aggregation of α-Syn can cause many pathological phenomena and aggravate the development of PD. Therefore, sensitive and accurate detection of oligomers is essential to understanding the pathology of PD and beneficial to screening and developing new drugs against PD. Here, we demonstrated a simple and sensitive method to detect the early aggregation of α-Syn via Förster resonance energy transfer (FRET) technology. We performed systematic investigations of the FRET sensitizations, efficiencies, and donor-to-acceptor distances during α-Syn aggregation, which was proved to be more sensitive to reflect small distance changes in the early stage of α-Syn aggregation, especially for α-Syn oligomers. The FRET assays were also applied to study the influence of Ser129 phosphorylation (pS129) on the aggregation rate of α-Syn. Our results showed that pS129 modification promotes α-Syn aggregation and enhances the ability of preformed fibrils to induce monomer aggregation. pS129 also increased the cytotoxicity of α-Syn. These results are of great significance for a better understanding of the pathological mechanisms of PD and future PD drug development.

Real-time measurements of ATP dynamics via ATeams in Plasmodium falciparum reveal drug-class-specific response patterns.

Malaria tropica, caused by the parasite Plasmodium falciparum (P. falciparum), remains one of the greatest public health burdens for humankind. Due to its pivotal role in parasite survival, the energy metabolism of P. falciparum is an interesting target for drug design. To this end, analysis of the central metabolite adenosine triphosphate (ATP) is of great interest. So far, only cell-disruptive or intensiometric ATP assays have been available in this system, with various drawbacks for mechanistic interpretation and partly inconsistent results. To address this, we have established fluorescent probes, based on Förster resonance energy transfer (FRET) and known as ATeam, for use in blood-stage parasites. ATeams are capable of measuring MgATP2- levels in a ratiometric manner, thereby facilitating in cellulo measurements of ATP dynamics in real-time using fluorescence microscopy and plate reader detection and overcoming many of the obstacles of established ATP analysis methods. Additionally, we established a superfolder variant of the ratiometric pH sensor pHluorin (sfpHluorin) in P. falciparum to monitor pH homeostasis and control for pH fluctuations, which may affect ATeam measurements. We characterized recombinant ATeam and sfpHluorin protein in vitro and stably integrated the sensors into the genome of the P. falciparum NF54attB cell line. Using these new tools, we found distinct sensor response patterns caused by several different drug classes. Arylamino alcohols increased and redox cyclers decreased ATP; doxycycline caused first-cycle cytosol alkalization; and 4-aminoquinolines caused aberrant proteolysis. Our results open up a completely new perspective on drugs' mode of action, with possible implications for target identification and drug development.