Resonance Energy Transfer Efficiency Research Articles

Chemiexcitation-Triggered Photosensitizer Activation for Photooxidation of Aβ1-42 Aggregates.

Oxidative degradation of the pathogenic amyloid-β-peptide (Aβ) aggregation is an effective and promising method to treat Alzheimer's disease under light irradiation. However, the limited penetration of external light sources into deep tissues has hindered the development of this treatment. Therefore, we have designed an unprecedented chemiluminescence-initiated photodynamic therapy system to replace external laser irradiation, primarily composed of d-glucose-based polyoxalate (G-poly(oxalate)), the novel photosensitizer (BD-Se-QM), and bis [2,4,5-trichloro-6-(pentoxy-carbonyl) phenyl] ester. BD-Se-QM possesses excellent singlet oxygen (1O2) generation efficiency and the ability to photooxidize Aβ1-42 aggregates under white light. G-poly(oxalate) not only helps the nanosystem to cross the blood-brain barrier but also has sufficient oxalate ester groups to significantly enhance the efficiency of chemiluminescence resonance energy transfer. The oxalate ester groups in BD-Se-QM/NPs can chemically react with H2O2 to produce high-energy intermediates that activate BD-Se-QM, which can generate 1O2 to inhibit Aβ1-42 aggregates and also promote microglial uptake of Aβ1-42, reducing the Aβ1-42-induced neurotoxicity. The chemically stimulated nanoplatform not only solves the drug delivery problem but also eliminates the need for external light sources. We anticipate that this chemically excited nanosystem could also be used for targeted delivery of other small molecule drugs.

Enhanced chemiluminescence resonance energy transfer using surfactant-modified AIE carbon dots.

Chemiluminescence resonance energy transfer (CRET) efficiency can be enhanced by confining CRET donors and acceptors within nanoscale spaces. However, this enhanced efficiency is often affected by uncertainties stemming from the random distribution of CRET donors and acceptors in such confined environments. In this study, a novel confined nanospace was created through the surfactant modification of carbon dots (CDs) exhibiting aggregation-induced emission (AIE) characteristics. Hydrophobic CRET donors could be effectively confined within this nanospace. The distance between the CRET donors and acceptors could be controlled by anchoring the AIE-CDs as the CRET acceptors, resulting in significantly improved CRET efficiency. Furthermore, this AIE-CDs-based CRET system was successfully applied to the detection of hydrogen peroxide (H2O2) in rainwater, showcasing its potential for practical applications.

Unraveling Burst Selection Bias in Single-Molecule FRET of Species with Unequal Brightness and Diffusivity.

Single-molecule free diffusion experiments enable accurate quantification of coexisting species or states. However, unequal brightness and diffusivity introduce a burst selection bias and affect the interpretation of experimental results. We address this issue with a photon-by-photon maximum likelihood method, burstML, which explicitly considers burst selection criteria. BurstML accurately estimates parameters, including photon count rates, diffusion times, Förster resonance energy transfer (FRET) efficiencies, and population, even in cases where species are poorly distinguished in FRET efficiency histograms. We develop a quantitative theory that determines the fraction of photon bursts corresponding to each species and thus obtain accurate species populations from the measured burst fractions. In addition, we provide a simple approximate formula for burst fractions and establish the range of parameters where unequal brightness and diffusivity can significantly affect the results obtained by conventional methods. The performance of the burstML method is compared with that of a maximum likelihood method that assumes equal species brightness and diffusivity, as well as standard Gaussian fitting of FRET efficiency histograms, using both simulated and real single-molecule data for cold-shock protein, protein L, and protein G. The burstML method enhances the accuracy of parameter estimation in single-molecule fluorescence studies.

Catalytic NIR chemiluminescence sensor with enhanced persistence and intensity for in vivo imaging

Chemiluminescence (CL) is a self-illumination phenomenon that involves the emission of light from chemical reactions, and it provides favorable spatial and temporal information on biological processes. However, it is still a great challenge to construct effective CL sensors that equip strong CL intensity, long emission wavelength, and persistent luminescence for deep tissue imaging. Here, we report a liposome encapsulated polymer dots (Pdots)-based system using catalytic CL substrates (L-012) as energy donor and fluorescent polymers and dyes (NIR 695) as energy acceptors for efficient Near-infrared (NIR) CL in vivo imaging. Thanks to the modulation of paired donor and acceptor distance and the slow diffusion of biomarker by liposome, the Pdots show a NIR emission wavelength (λ em, max = 720 nm), long CL duration (>24 h), and a high chemiluminescence resonance energy transfer efficiency (46.5 %). Furthermore, the liposome encapsulated Pdots possess excellent biocompatibility, sensitive response to H2O2, and persistent whole-body NIR CL imaging in the drug-induced inflammation and the peritoneal metastatic tumor mouse model. In a word, this NIR-II CL nanoplatform with long-lasting emission and high spatial-temporal resolution will be a concise strategy in deep tissue imaging and clinical diagnostics.

Chemiluminescent Conjugated Polymer Nanoparticles for Deep-Tissue Inflammation Imaging and Photodynamic Therapy of Cancer.

Deep-tissue optical imaging and photodynamic therapy (PDT) remain a big challenge for the diagnosis and treatment of cancer. Chemiluminescence (CL) has emerged as a promising tool for biological imaging and in vivo therapy. The development of covalent-binding chemiluminescence agents with high stability and high chemiluminescence resonance energy transfer (CRET) efficiency is urgent. Herein, we design and synthesize an unprecedented chemiluminescent conjugated polymer PFV-Luminol, which consists of conjugated polyfluorene vinylene (PFV) main chains and isoluminol-modified side chains. Notably, isoluminol groups with chemiluminescent ability are covalently linked to main chains by amide bonds, which dramatically narrow their distance, greatly improving the CRET efficiency. In the presence of pathologically high levels of various reactive oxygen species (ROS), especially singlet oxygen (1O2), PFV-Luminol emits strong fluorescence and produces more ROS. Furthermore, we construct the PFV-L@PEG-NPs and PFV-L@PEG-FA-NPs nanoparticles by self-assembly of PFV-Luminol and amphiphilic copolymer DSPE-PEG/DSPE-PEG-FA. The chemiluminescent PFV-L@PEG-NPs nanoparticles exhibit excellent capabilities for in vivo imaging in different inflammatory animal models with great tissue penetration and resolution. In addition, PFV-L@PEG-FA-NPs nanoparticles show both sensitive in vivo chemiluminescence imaging and efficient chemiluminescence-mediated PDT for antitumors. This study paves the way for the design of chemiluminescent probes and their applications in the diagnosis and therapy of diseases.

Combination of Ternary Electrochemiluminescence System of BNQDs/AgMOG-K2S2O8 and Electrochemiluminescence Resonance Energy Transfer Strategy for Ultrasensitive Immunoassay of Amyloid-β Protein.

In this work, based on boron nitride quantum dots (BNQDs) as energy donors and MnO2@MWCNTs-COOH as energy receptors, we designed an efficient electrochemiluminescence resonance energy transfer (ECL-RET) immunosensor for the detection of amyloid-β (Aβ42) protein, a biomarker of Alzheimer's disease (AD). First, the signal amplification of a ternary ECL system composed of BNQDs (as the ECL emitter), K2S2O8 (as the coreactant), and silver metal-organic gels (AgMOG, as the coreaction accelerator) was realized, and PDDA as stabilizer was added, a strong and stable initial ECL signal was obtained. AgMOG could not only support a large amount of BNQDs and Aβ42 capture antibody (Ab1) through Ag-N bond but also exhibit excellent ECL catalytic performance and enhance the luminescent intensity of BNQDs@PDDA-K2S2O8 system. In addition, due to the broad absorption spectrum of MnO2@MWCNTs-COOH and the extensive overlap with the ECL emission spectrum of BNQDs, the quenching probe Ab2-MnO2@MWCNTs-COOH could be introduced into the ternary system through a sandwich immune response. On this basis, the signal on-off ECL immunosensor was constructed to achieve the ultrasensitive detection of Aβ42 through signal transformation. Under the optimal conditions, the prepared ECL biosensor manifested a wide linear range (10 fg/mL-100 ng/mL) with a detection limit of 2.89 fg/mL and showed excellent stability, selectivity, and repeatability, which provided an effective strategy for the ultrasensitive detection of biomarkers in clinical analysis.

Efficient resonance energy transfer study of BaClF: Ce3+ to Tb3+

Ce3+/Tb3+ co-doped BaClF luminescent materials with highly efficient green luminescent properties. The phosphor was synthesized by a co-precipitation hydrothermal method, and the structure and morphology of the phosphor were characterized using X-ray diffraction (XRD), field-emission scanning electron microscopy (FE-SEM), and X-ray photoelectron spectroscopy (XPS). The photoluminescence properties of the prepared products were investigated and the energy transfer efficiencies were calculated for BaClF:3.0 mol% Ce3+, ymol% Tb3+ ( y = 0–0.5). By adjusting the doping concentration of Tb3+ ions in BaClF: Ce3+, Tb3+ phosphors, the luminescent color of the phosphor can be easily changed from blue to green. The Ce3+ and Tb3+ co-doped BaClF phosphor shows strong PL emission in the green region due to the resonance energy transfer from Ce3+ to Tb3+. The energy transfer mechanism of Ce3+ and Tb3+ co-doped BaClF samples was analyzed based on the energy level relationship between Ce3+ and Tb3+. The energy transfer efficiency from Ce3+ to Tb3+ was calculated and found to be up to 89.63%. These results suggest that BaClF: Ce3+, Tb3+ fluorescent material can be a potential green luminescent material for optoelectronic applications.

Structure Engineered High Piezo-Photoelectronic Performance for Boosted Sono-Photodynamic Therapy.

Sono-photodynamic therapy is hindered by the limited tissue penetration depth of the external light source and the quick recombination of electron-hole owing to the random movement of charge carriers. In this study, orthorhombic ZnSnO3 quantum dots (QDs) with piezo-photoelectronic effects are successfully encapsulated in hexagonal upconversion nanoparticles (UCNPs) using a one-pot thermal decomposition method to form an all-in-one watermelon-like structured sono-photosensitizer (ZnSnO3 @UCNPs). The excited near-infrared light has high penetration depth, and the watermelon-like structure allows for full contact between the UCNPs and ZnSnO3 QDs, achieving ultrahigh Förster resonance energy transfer efficiency of up to 80.30%. Upon ultrasonic and near-infrared laser co-activation, the high temperature and pressure generated lead to the deformation of the UCNPs, thereby driving the deformation of all ZnSnO3 QDs inside the UCNPs, forming many small internal electric fields similar to isotropic electric domains. This piezoelectric effect not only increases the internal electric field intensity of the entire material but also prevents random movement and rapid recombination of charge carriers, thereby achieving satisfactory piezocatalytic performance. By combining the photodynamic effect arising from the energy transfer from UCNPs to ZnSnO3 , synergistic efficacy is realized. This study proposes a novel strategy for designing highly efficient sono-photosensitizers through structural design.

Ln-HOF Nanofiber Organogels with Time-Resolved Luminescence for Programmable and Reliable Encryption.

Developing tunable luminescent materials for high throughput information storage is highly desired following the explosive growth of global data. Although considerable success has been achieved, achieving programmable information encryption remains challenging due to current signal crosstalk problems. Here, we developed long-lived room-temperature phosphorescent organogels enabled by lanthanum-coordinated hydrogen-bonded organic framework nanofibers for time-resolved information programming. Via modulating coassembled lanthanum concentration and Förster resonance energy transfer efficiency, the lifetimes are prolonged and facilely manipulated (20-644 ms), realizing encoding space enlargement and multichannel data outputs. The aggregated strong interfacial supramolecular bonding endows organogels with excellent mechanical toughness (36.16 MJ m-2) and self-healing properties (95.7%), synergistically achieving photostability (97.6% lifetime retention in 10000 fatigue cycles) via suppressing nonradiative decays. This work presents a lifetime-gated information programmable strategy via lanthanum-coordination regulation that promisingly breaks through limitations of current responsive luminescent materials, opening unprecedented avenues for high-level information encryption and protection.

Bright and sensitive red voltage indicators for imaging action potentials in brain slices and pancreatic islets.

Genetically encoded voltage indicators (GEVIs) allow the direct visualization of cellular membrane potential at the millisecond time scale. Among these, red-emitting GEVIs have been reported to support multichannel recordings and manipulation of cellular activities with reduced autofluorescence background. However, the limited sensitivity and dimness of existing red GEVIs have restricted their applications in neuroscience. Here, we report a pair of red-shifted opsin-based GEVIs, Cepheid1b and Cepheid1s, with improved dynamic range, brightness, and photostability. The improved dynamic range is achieved by a rational design to raise the electrochromic Förster resonance energy transfer efficiency, and the higher brightness and photostability are approached with separately engineered red fluorescent proteins. With Cepheid1 indicators, we recorded complex firings and subthreshold activities of neurons on acute brain slices and observed heterogeneity in the voltage‑calcium coupling on pancreatic islets. Overall, Cepheid1 indicators provide a strong tool to investigate excitable cells in various sophisticated biological systems.

A nanozipper structured efficient FRET probe for ratiometric detection of ATP based on target-induced cycling amplification

In the realm of disease diagnostics and prognostics, accurate quantification of adenosine triphosphate (ATP) has become crucial, as the intricate interplay between ATP and cellular metabolism is a fundamental factor in the pathogenesis and progression of many pathological conditions. Herein, a ratiometric fluorescence DNA nanozipper is developed, which enables highly sensitive detection of ATP by employing catalytic hairpin assembly. This innovative approach utilizes target-induced conformational changes in the nanozipper structure to tune the distance between the carboxyfluorescein as donor and tetramethylrhodamine as acceptor around the Förster radius, thereby enhancing the efficiency of fluorescence resonance energy transfer (FRET). In the state of nanozipper tension, the rigid and steric hindrance Y-shaped configuration leads to a considerable distance between two fluorophores, resulting in FRET low efficiency. However, the presence of ATP triggers allosteric configuration of the DNA nanozipper, switching the nanozipper to a relaxed state, which results in a visible fluorescent ratiometric signal. The sensing strategy exhibits a linear response to ATP concentrations in the range of 30 − 600 nM and can detect concentrations as low as 9.6 nM. Additionally, this detection method introduces a novel idea for the design of efficient FRET probes and demonstrates promising application potential in biological samples.

LuMA-Functionalized Thermosensitive Hydrogel: A Versatile and Robust Dopamine-Triggered Platform for Diverse Biomolecules Sensing.

It is of great significance for the analysis of multiple biomarkers because a single biomarker is difficult to accurately achieve early diagnosis, disease course monitoring, and prognosis evaluation. Herein, a luminescence thermosensitive hydrogel was synthesized by radical polymerization using a methacrylic acid derivative monomer of luminol (LuMA) as luminescent, N-isopropylacrylamide (NIPAM) as thermosensitive monomer, and acrydite-oligonucleotides [dopamine (DA) aptamer, DNA C1, and DNA C2] as recognition elements. The combined DA based on the affinity interaction between the DA and the aptamer on the hydrogel polymer chain was electrochemically oxidized to dopamine quinone during the electrochemiluminescence (ECL) scanning, which effectively quenched the ECL signal of LuMA due to the resonance energy transfer (RET). In addition, the thermosensitive hydrogel showed swelling-collapse characteristics when the temperature was below and above the volume phase transition temperature. Undergoing the collapse process initiated by the temperature, the RET efficiency was further enhanced due to the shortened distance between the energy donor and acceptor, showing a 1.4 times signal amplification and achieving sensitive detection of DA with a limit of detection (LOD) of 1.7 × 10-10 M. For a proof of concept application, coupled with the target-induced release of DA from the DNA-magnetic beads bioconjugations based on duplex-specific nuclease (DSN)-assisted target recycling amplification strategy and DNAzyme cleavage reaction, this ECL-RET approach was successfully used to evaluate multiple targets including miRNA-141 and MUC1 with the LOD of 2.5 aM and 1.6 fg/mL, respectively. The excellent performances of the versatile and robust ECL-RET hydrogel in multiple target sensing showed potential applications in clinical diagnosis and disease therapeutic assay.

Giant Light‐Harvesting in Dye‐Loaded Nanoparticles Enhanced by Blank Hydrophobic Salts

AbstractLight‐harvesting is a fundamental process in nature, which inspires researchers to develop artificial systems for photocatalysis, photovoltaics, and biosensing. A previously introduced light‐harvesting nanoantenna, based on polymeric nanoparticles (NPs) loaded with rhodamine dyes and bulky hydrophobic counterions, provides a record‐breaking antenna effect ≈1000. However, the high dye cooperativity of its thousands of encapsulated dyes causes energy losses by traces of self‐quenched dye aggregates. Here, it is found that these imperfections can be suppressed by blank hydrophobic salts (BHS) formed by the same bulky counterion (fluorinated tetraphenylborate) with an optically inactive cation, analogs of ionic liquids. The presence of BHS increases twofold the fluorescence quantum yields and fluorescence lifetimes of NPs and suppresses their fluorescence blinking. This study assumes that BHS provides an excess of bulky counterions that excludes traces of dye aggregates. As a result, an efficient Forster resonance energy transfer (FRET) is achieved from 40 000 dye donors to a single acceptor within a 70 nm particle, leading to the antenna effect of 4800, which is by far the highest value reported to date. Using this nanoantenna, a single‐molecule detection of the FRET acceptor is realized at low excitation power using an RGB camera of a smartphone.

Aromatic Bridged Bis(triphenylamine) Cascade Assembly Achieved Tunable Nanosupramolecular Morphology and NIR Targeted Cell Imaging.

Possessing four cationic pyridium groups, phenyl-bridged bis(triphenylamine) derivatives (G1, G2) were encapsulated by cucurbit[8]uril (CB[8]) at a 1:2 stoichiometry to form the network-like organic two-dimensional nanosheet, which could efficiently enhance the near-infrared (NIR) luminescence and companies with a red-shift from 750 to 810 nm for G1. Benefiting from the supramolecular multivalent interaction, α-cyclodextrin modified hyaluronic acid (HACD) and G1/CB[8] formed nanoparticles to further enhance NIR luminescence behaviors. Compared with the short rigid aromatic bridged bis(triphenylamine) derivative (G2), the supramolecular assembly derived from G1 with long flexible cationic arms gives a larger Stokes shift, which further coassembles with the phosphorescent bromophenylpyridinium derivative/CB[8] pseudorotaxane, leading to efficient phosphorescent resonance energy transfer (PRET). Especially, the nanoparticle showed delayed NIR fluorescence under 308 nm light excitation with an ultralarge Stokes shift up to 502 nm, which was successfully applied in targeted NIR cell imaging.

Color‐Tunable Carbon Dots with Aggregation‐Induced Emission Constructed by FRET between Surface Luminescence Centers

AbstractAs a class of new fluorescent materials, carbon dots (CDs) have drawn widespread attention by virtue of their tunable photoluminescence (PL), excellent biocompatibility, and novel physicochemical properties. Unfortunately, in solid state, most CDs experience serious aggregation‐caused quenching (ACQ). In this work, color‐tunable CDs with aggregation‐induced emission (AIE) are prepared by simply changing the filling ratio of the autoclave. The obtained CDs show dual emission (green/red) in solid state, and the fluorescent color can be tuned from green to red. These CDs all exhibit blue fluorescence in dilute solution, and aggregation‐induced redshift behaviors are observed when the CDs form aggregates. Structural and optical characterizations prove that blue fluorescence is contributed by the carbon cores, whereas the green/red emission is attributed to the surface groups. Multicolor fluorescence is caused by increased fluorescence resonance energy transfer (FRET) efficiency among the dual emission (green/red) peaks from g‐CDs to r‐CDs. Surprisingly, delayed fluorescence (DF) of CDs are brightened by inserting the CDs into poly(vinyl alcohol) (PVA) films, which can be easily recognized by the naked eyes with a lifetime up to ms. Finally, owing to the unique AIE and DF properties, the four CDs can be applied to multimodal advanced anti‐counterfeiting and information encryption.

Active Control of Energy Transfer in Plasmonic Nanorod-Polyaniline Hybrids.

The hybridization of plasmonic energy and charge donors with polymeric acceptors is a possible means to overcome fast internal relaxation that limits potential photocatalytic applications for plasmonic nanomaterials. Polyaniline (PANI) readily hybridizes onto gold nanorods (AuNRs) and has been used for the sensitive monitoring of local refractive index changes. Here, we use single-particle spectroscopy to quantify a previously unreported plasmon damping mechanism in AuNR-PANI hybrids while actively tuning the PANI chemical structure. By eliminating contributions from heterogeneous line width broadening and refractive index changes, we identify efficient resonance energy transfer (RET) between AuNRs and PANI. We find that RET dominates the optical response in our AuNR-PANI hybrids during the dynamic tuning of the spectral overlap of the AuNR donor and PANI acceptor. Harnessing RET between plasmonic nanomaterials and an affordable and processable polymer such as PANI offers an alternate mechanism toward efficient photocatalysis with plasmonic nanoparticle antennas.

HLA DQ protein changes the cell surface distribution pattern of HLA proteins as monitored by Förster resonance energy transfer and high-resolution electron microscopy.

Peptide presentation by MHC class I and MHC class II molecules plays important roles in the regulation of the immune response. One factor in these displays is the density of antigen, which must exceed a critical threshold for the effective activation of T cells. Nonrandom distribution of MHC class I and class II has already been detected at the nanometer level and at higher hierarchical levels. It is not clear how the absence and reappearance of some protein molecules can influence the nonrandom distribution. Therefore, we performed experiments on HLA II-deficient bare lymphocyte syndrome (BLS1) cells: we created a stable transfected cell line, tDQ6-BLS-1, and were able to detect the effect of the appearance of HLA-DQ6 molecules on the homo and heteroassociation of different cell surface molecules by comparing Förster resonance energy transfer (FRET) efficiency on transfected cells to that on nontransfected BLS-1 and JY human B-cell lines. Our FRET results show a decrease in homoassociation FRET between HLA I chains in HLA-DQ6-transfected tDQ6-BLS-1 cells compared with the parent BLS-1 cell line and an increase in heteroassociation FRET between HLA I and HLA II (compared with JY cells), suggesting a similar pattern of antigen presentation by the HLA-DQ6 allele. Transmission electron microscopy (TEM) revealed that both HLA class I and class II molecules formed clusters at higher hierarchical levels on the tDQ6-BLS-1 cells, and the de novo synthesized HLA DQ molecules did not intersperse with HLA class I islands. These observations could be important in understanding the fine tuning of the immune response.

Heterodimerization of Human UDP-Glucuronosyltransferase 1A9 and UDP-Glucuronosyltransferase 2B7 Alters Their Glucuronidation Activities.

Human UDP-glucuronosyltransferases (UGTs) play a pivotal role as prominent phase II metabolic enzymes, mediating the glucuronidation of both endobiotics and xenobiotics. Dimerization greatly modulates the enzymatic activities of UGTs. In this study, we examined the influence of three mutations (H35A, H268Y, and N68A/N315A) and four truncations (signal peptide, single transmembrane helix, cytosolic tail, and di-lysine motif) in UGT2B7 on its heterodimerization with wild-type UGT1A9, using a Bac-to-Bac expression system. We employed quantitative fluorescence resonance energy transfer (FRET) techniques and co-immunoprecipitation assays to evaluate the formation of heterodimers between UGT1A9 and UGT2B7 allozymes. Furthermore, we evaluated the glucuronidation activities of the heterodimers using zidovudine and propofol as substrates for UGT2B7 and UGT1A9, respectively. Our findings revealed that the histidine residue at codon 35 was involved in the dimeric interaction, as evidenced by the FRET efficiencies and catalytic activities. Interestingly, the signal peptide and single transmembrane helix domain of UGT2B7 had no impact on the protein-protein interaction. These results provide valuable insights for a comprehensive understanding of UGT1A9/UGT2B7 heterodimer formation and its association with glucuronidation activity. SIGNIFICANCE STATEMENT: Our findings revealed that the H35A mutation in UGT2B7 affected the affinity of protein-protein interaction, leading to discernable variations in fluorescence resonance energy transfer efficiencies and catalytic activity. Furthermore, the signal peptide and single transmembrane helix domain of UGT2B7 did not influence heterodimer formation. These results provide valuable insights into the combined effects of polymorphisms and protein-protein interactions on the catalytic activity of UGT1A9 and UGT2B7, enhancing our understanding of UGT dimerization and its impact on metabolite formation.

A Curated Graphene Quantum Dot-Porphyrin-Based Photosensitizer for Effective Singlet Oxygen Generation through Energy Transfer.

Porphyrin-based photosensitizers are proven generators of reactive oxygen species (ROS), such as singlet oxygen, and used as anti-cancer therapeutic agents. However, most of these compounds suffer from potential drawbacks due to limited photostability, hydrophobicity, aggregation propensity, and low cellular uptake. Ultrasmall fluorescent graphene quantum dots (GQDs) have emerged as the next-generation carriers for drugs and have gained reputation in the pharmaceutical domain. Considering the various limiting factors in porphyrin-based ROS generation and cellular internalization, here, we have developed a method to generate tetrakis(m-nitrophenyl) porphyrin (TNPP)-GQD exciplexes. This allows resonance energy transfer (RET) from GQDs to TNPP. The calculated overlap integrals for GQD-TNPP and AGQD-TNPP (1.001 × 1018 and 1.257 × 1017 M-1 cm-1 nm4, respectively) assured 95 and 71% energy transfer. The optimum donor-acceptor distances in these couples are 59.82 and 62.65 Å, respectively, which yielded the rate constant of RET as 4.09 and 0.56 ns-1, respectively. The efficient RET helped in subsequent generation of singlet oxygen. The singlet oxygen quantum yields (SOQY) of around 0.435 and 0.464 for GQD-TNPP and AGQD-TNPP, respectively, are comparable to those of different porphyrin derivatives where the SOQY ranges from 0.55 to 0.70 when used with Triton X-100. The data show that non-conjugated amine- and amide-functionalized GQDs (AGQDs) are better candidates in this case because of the special properties of the amine groups. The systems could be excited at 450 nm for FRET, which favors biological usage.

Förster resonance energy transfer efficiency measurements on vinculin tension sensors at focal adhesions using a simple and cost-effective setup.

Forces inside cells play a fundamental role in tissue growth, affecting important processes such as cancer cell migration or tissue repair after injury. Förster resonance energy transfer (FRET)-based tension sensors are a remarkable tool for studying these forces and should be made easier to use. We prove that absolute FRET efficiency can be measured on a simple setup, an order of magnitude more cost-effective than a standard FRET microscopy setup, by applying it to vinculin tension sensors (VinTS) at the focal adhesions of live CHO-K1 cells. Our setup located at Université Paris-Saclay acquires donor and acceptor fluorescence in parallel on two low-cost CMOS cameras and uses two LEDs for rapid switching of the excitation wavelength at a reduced cost. The calibration required to extract FRET efficiency was achieved using a single construct (TSMod). FRET efficiencies were measured for VinTS and the tail-less control VinTL, lacking the actin-binding domain of vinculin. Measurements were confirmed on the same cell type using a more standard intensity-based setup located at Rutgers University. The average FRET efficiency of VinTS () over more than 10,000 focal adhesions is significantly lower () than that of VinTL (), our control that is insensitive to force, in agreement with the force exerted on vinculin at focal adhesions. Attachment of the CHO-K1 cells on fibronectin decreases FRET efficiency, thus increasing the force, compared with poly-lysine. FRET efficiency for the VinTL control is consistent with all measurements currently available in the literature, confirming the validity of our measurements and hence of our simpler setup. Force measurements, resolved spatially inside a cell, can be achieved using FRET-based tension sensors with a cost effective intensity-based setup. This will facilitate combining FRET with techniques for applying controlled forces such as optical tweezers.